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Sample records for antibody mest-3 directed

  1. Waterverdamping uit dunne mest; resultaten van modelberekeningen

    NARCIS (Netherlands)

    Wagenberg, van A.V.; Verdoes, N.; Vranken, E.; Berckmans, D.

    1999-01-01

    In een geïntegreerd luchtbehandelingsysteem kunnen technische oplossingen voor de emissieproblematiek (ammoniak, geur) en de mestproblematiek worden gecombineerd. Door ventilatielucht uit de stal intensief in contact te brengen met mest kan water uit de mest verdampen. Daardoor droogt de mest. Het

  2. Historiens mest groteske pengepolitiske eksperiment?

    DEFF Research Database (Denmark)

    Hougaard Nielsen, Rasmus

    2017-01-01

    Er bitcoin en boble? Givetvis. Men føj den bare til rækken af bobler, som verdens mest magtfulde centralbankchefer har pustet op efter finanskrisen, skriver økonom.......Er bitcoin en boble? Givetvis. Men føj den bare til rækken af bobler, som verdens mest magtfulde centralbankchefer har pustet op efter finanskrisen, skriver økonom....

  3. Emissies uit opslag van vaste mest

    NARCIS (Netherlands)

    Ellen, H.H.; Groenestein, K.; Smits, M.

    2007-01-01

    Door de omschakeling naar meer welzijnsvriendelijke huisvestingssystemen produceert men in Nederland meer vaste mest. Om een indruk te krijgen wat de bijdrage zou kunnen zijn aan de emissies van ammoniak, geur en broeikasgassen is een inventarisatie gedaan naar de productie en opslag van deze mest

  4. A cluster randomized controlled platform trial comparing group MEmory specificity training (MEST) to group psychoeducation and supportive counselling (PSC) in the treatment of recurrent depression.

    Science.gov (United States)

    Werner-Seidler, Aliza; Hitchcock, Caitlin; Bevan, Anna; McKinnon, Anna; Gillard, Julia; Dahm, Theresa; Chadwick, Isobel; Panesar, Inderpal; Breakwell, Lauren; Mueller, Viola; Rodrigues, Evangeline; Rees, Catrin; Gormley, Siobhan; Schweizer, Susanne; Watson, Peter; Raes, Filip; Jobson, Laura; Dalgleish, Tim

    2018-06-01

    Impaired ability to recall specific autobiographical memories is characteristic of depression, which when reversed, may have therapeutic benefits. This cluster-randomized controlled pilot trial investigated efficacy and aspects of acceptability, and feasibility of MEmory Specificity Training (MEST) relative to Psychoeducation and Supportive Counselling (PSC) for Major Depressive Disorder (N = 62). A key aim of this study was to determine a range of effect size estimates to inform a later phase trial. Assessments were completed at baseline, post-treatment and 3-month follow-up. The cognitive process outcome was memory specificity. The primary clinical outcome was symptoms on the Beck Depression Inventory-II at 3-month follow-up. The MEST group demonstrated greater improvement in memory specificity relative to PSC at post-intervention (d = 0.88) and follow-up (d = 0.74), relative to PSC. Both groups experienced a reduction in depressive symptoms at 3-month follow-up (d = 0.67). However, there was no support for a greater improvement in depressive symptoms at 3 months following MEST relative to PSC (d = -0.04). Although MEST generated changes on memory specificity and improved depressive symptoms, results provide no indication that MEST is superior to PSC in the resolution of self-reported depressive symptoms. Implications for later-phase definitive trials of MEST are discussed. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

  5. Mest en urine: van afval naar waardevol product

    NARCIS (Netherlands)

    Bos, A.P.; Cornelissen, J.M.R.; Groot Koerkamp, P.W.G.

    2010-01-01

    Het gescheiden houden van mest en urine in de veehouderij belooft een reeks positieve effecten te geven, waarmee de veehouderij én de akkerbouw een duurzaamheidssprong kunnen maken. Maar dat lukt alleen als er veel praktijken tegelijkertijd veranderen.

  6. Drogen van mest onder het rooster geeft hoge kosten

    NARCIS (Netherlands)

    Ellen, H.H.; Haar, van der J.W.

    1999-01-01

    Voor de vleeskuikenouderdieren is in de Uitvoeringsregeling ammoniak en veehouderij (Uav) een systeem opgenomen waarbij de mest onder het rooster wordt belucht. De gebruikte lucht moet daarbij aan bepaalde eisen voldoen. Dit heeft onder andere gevolgen voor de energiekosten. In dit artikel wordt

  7. Hurtigste versus mest brændstoføkonomiske ruter

    DEFF Research Database (Denmark)

    Andersen, Ove; Krogh, Benjamin Bjerre; Lahrmann, Harry

    2013-01-01

    Der er stort fokus på at forbedre brændstoføkonomien i transportbranchen generelt. I denne artikel anvendes brændstoftallene fra Controller Area Network Bus (CANBus) kombineret med GPS data til at skabe et digitalt vejkort, hvor det er muligt at sammenligne de hurtigste ruter med de mest brændsto...

  8. Defective neuronal migration and inhibition of bipolar to multipolar transition of migrating neural cells by Mesoderm-Specific Transcript, Mest, in the developing mouse neocortex.

    Science.gov (United States)

    Ji, Liting; Bishayee, Kausik; Sadra, Ali; Choi, Seunghyuk; Choi, Wooyul; Moon, Sungho; Jho, Eek-Hoon; Huh, Sung-Oh

    2017-07-04

    Brain developmental disorders such as lissencephaly can result from faulty neuronal migration and differentiation during the formation of the mammalian neocortex. The cerebral cortex is a modular structure, where developmentally, newborn neurons are generated as a neuro-epithelial sheet and subsequently differentiate, migrate and organize into their final positions in the cerebral cortical plate via a process involving both tangential and radial migration. The specific role of Mest, an imprinted gene, in neuronal migration has not been previously studied. In this work, we reduced expression of Mest with in utero electroporation of neuronal progenitors in the developing embryonic mouse neocortex. Reduction of Mest levels by shRNA significantly reduced the number of neurons migrating to the cortical plate. Also, Mest-knockdown disrupted the transition of bipolar neurons into multipolar neurons migrating out of the sub-ventricular zone region. The migrating neurons also adopted a more tangential migration pattern upon knockdown of the Mest message, losing their potential to attach to radial glia cells, required for radial migration. The differentiation and migration properties of neurons via Wnt-Akt signaling were affected by Mest changes. In addition, miR-335, encoded in a Mest gene intron, was identified as being responsible for blocking the default tangential migration of the neurons. Our results suggest that Mest and its intron product, miR-335, play important roles in neuronal migration with Mest regulating the morphological transition of primary neurons required in the formation of the mammalian neocortex. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

  9. Subtask 4.27 - Evaluation of the Multielement Sorbent Trap (MEST) Method at an Illinois Coal-Fired Plant

    Energy Technology Data Exchange (ETDEWEB)

    Pavlish, John; Thompson, Jeffrey; Dunham, Grant

    2014-09-30

    Owners of fossil fuel-fired power plants face the challenge of measuring stack emissions of trace metals and acid gases at much lower levels than in the past as a result of increasingly stringent regulations. In the United States, the current reference methods for trace metals and halogens are wet-chemistry methods, U.S. Environmental Protection Agency (EPA) Methods 29 and 26 or 26A, respectively. As a possible alternative to the EPA methods, the Energy & Environmental Research Center (EERC) has developed a novel multielement sorbent trap (MEST) method to be used to sample for trace elements and/or halogens. Sorbent traps offer a potentially advantageous alternative to the existing sampling methods, as they are simpler to use and do not require expensive, breakable glassware or handling and shipping of hazardous reagents. Field tests comparing two sorbent trap applications (MEST-H for hydrochloric acid and MEST-M for trace metals) with the reference methods were conducted at two power plant units fueled by Illinois Basin bituminous coal. For hydrochloric acid, MEST measured concentrations comparable to EPA Method 26A at two power plant units, one with and one without a wet flue gas desulfurization scrubber. MEST-H provided lower detection limits for hydrochloric acid than the reference method. Results from a dry stack unit had better comparability between methods than results from a wet stack unit. This result was attributed to the very low emissions in the latter unit, as well as the difficulty of sampling in a saturated flue gas. Based on these results, the MEST-H sorbent traps appear to be a good candidate to serve as an alternative to Method 26A (or 26). For metals, the MEST trap gave lower detection limits compared to EPA Method 29 and produced comparable data for antimony, arsenic, beryllium, cobalt, manganese, selenium, and mercury for most test runs. However, the sorbent material produced elevated blanks for cadmium, nickel, lead, and chromium at levels

  10. Approaches to lung cancer treatment using the CD3E x GP-2-directed bispecific monoclonal antibody BIS-1

    NARCIS (Netherlands)

    Kroesen, BJ; Nieken, J; Sleijfer, DT; Molema, G; deVries, EGE; Groen, HJM; Helfrich, W; The, TH; Mulder, NH; deLeij, L

    1997-01-01

    The bispecific monoclonal antibody (bsAb) BIS-1 combines a monoclonal-antibody(mAb)-defined specificity for the CD3 complex, as present on all T lymphocytes, with a mAb-defined specificity for the pancarcinoma/epithelium associated glycoprotein EGP-2. In vitro studies indicate that BIS-1 can direct

  11. Mass, Energy, Space And Time System Theory---MEST A way to help our earth

    Science.gov (United States)

    Cao, Dayong

    2009-03-01

    There are two danger to our earth. The first, the sun will expand to devour our earth, for example, the ozonosphere of our earth is be broken; The second, the asteroid will impact near our earth. According to MEST, there is a interaction between Black hole (and Dark matter-energy) and Solar system. The orbit of Jupiter is a boundary of the interaction between Black hole (and Dark matter-energy) and Solar system. Because there are four terrestrial planets which is mass-energy center as solar system, and there are four or five Jovian planets which is gas (space-time) center as black hole system. According to MEST, dark matter-energy take the velocity of Jupiter gose up. So there are a lot of asteroids and dark matter-energy near the orbit of Jupiter-the boundary. Dark matter-energy can change the orbit of asteroid, and take it impacted near our earth. Because the Dark matter-energy will pressure the Solar system. It is a inverse process with sun's expandedness. So the ``two danger'' is from a new process of the balance system between Black hole (and Dark matter-energy) and Solar system. According to MEST, We need to find the right point for our earth in the ``new process of the balance system.''

  12. Direct immobilization of antibodies on Zn-doped Fe_3O_4 nanoclusters for detection of pathogenic bacteria

    International Nuclear Information System (INIS)

    Pal, Monalisa; Lee, Sanghee; Kwon, Donghoon; Hwang, Jeongin; Lee, Hyeonjeong; Hwang, Seokyung; Jeon, Sangmin

    2017-01-01

    Zinc-doped magnetic nanoclusters (Zn-MNCs) were synthesized and used to detect pathogenic bacteria in milk. Hydrothermally synthesized Zn-MNCs exhibited stronger magnetic properties than pure MNCs, which facilitated the magnetic separation from the sample using a permanent magnet. The presence of accessible Zn sites allows the direct immobilization of half-fragmented antibodies over Zn-MNCs through strong Zn−S bonds and prevents the tedious multiple steps of molecular functionalization or coating with costly noble metals prior to conjugation with an antibody. After the capture and magnetic separation of Salmonella in milk using the antibody-functionalized Zn-MNCs, the concentration of bacteria was determined with a portable ATP luminometer and the detection limit was found to be 10 CFU/mL. - Highlights: • Zn-doped Fe_3O_4 nanoclusters (Zn-MNCs) were synthesized by hydrothermal method. • Antibodies were directly immobilized over Zn-MNCs through strong Zn–S_t_h_i_o_l bonds. • Higher magnetization of Zn-MNCs than pure MNCs facilitates the magnetic separation. • Detection limit of pathogenic bacteria in milk was found to be 10 cfu/mL. • Cost effective, sensitive and selective detection of bacteria.

  13. Ammoniakemissie uit een vleesvarkensstal met opvang en verwijdering van mest via spoelgoten

    NARCIS (Netherlands)

    Huis in ‘t Veld, J.H.W.; Satter, I.H.G.

    1998-01-01

    Onderzoek werd verricht naar de ammoniakemissie uit een vleesvarkensstal met een zogenaamd spoelgotensysteem. Bij dit systeem bevonden zich onder de roosters, horizontaal gelegen, in V-vorm uitgevoerde goten van 26 cm diep. De mest van de varkens kwam in deze goten terecht en werd tweemaal daags uit

  14. Characterization of monoclonal antibodies directed against human thyroid stimulating hormone

    International Nuclear Information System (INIS)

    Soos, M.; Siddle, K.

    1982-01-01

    Monoclonal antibodies directed against human thyroid stimulating hormone (TSH) were obtained from hybrid myelomas, following fusion of mouse NSI myeloma cells with mouse spleen cells. Ten different antibodies were obtained from 4 separate fusions. Eight antibodies were of the IgG 1 subclass. Affinities of antibodies for TSH were in the range 2 x 10 8 -5 x 10 10 M -1 . Five of the antibodies were specific for TSH and did not react with LH, FSH or hCG. The remaining antibodies reacted with all these hormones and were assumed to recognise their common (α) subunit. The 5 specific antibodies fell into 3 subgroups recognising distinct antigenic determinants, whereas the 5 non-specific antibodies recognised a single determinant or closely related set of sites. It is concluded that these antibodies should be valuable reagents for use in sensitive and specific two-site immunoradiometric assays. (Auth.)

  15. Po sledeh postsocialističnega prestrukturiranja mest: spreminjajoča se središča v srednje- in vzhodnoevropskih prestolnicah

    Directory of Open Access Journals (Sweden)

    Jasna MARIOTTI

    2016-06-01

    Full Text Available Hitre politične in gospodarske spremembe po padcu socializma so povzročile dinamične procese urbanega prestrukturiranja v mestih, pri čemer so stare vzorce in modele rasti zamenjali tisti, pri katerih sta bila kapital in tržno gospodarstvo ključna za rast in razvoj. Preobrazba v tržno gospodarstvo in ponovna uvedba lastništva zemljišč sta zahtevali nov, decentralizirani sistem odločanja, kar je neposredno vplivalo na zgradbo in obliko mest. Med urbanimi preobrazbami so prišle jasno do izraza spremembe v lokacijskih vzorcih trgovskih objektov v mestih, ki so ponazarjale različne vzorce prostorske organizacije postsocialističnih mest. V članku avtorja preučujeta in predstavita nekatere posebnosti te preobrazbe v Bukarešti, Budimpešti, Pragi in Sofiji pred padcem socializma in po njem. Na podlagi analize izbranih mest predstavita procese postsocialističnega prostorskega prestrukturiranja, pri čemer preučujeta spremembe njihovih središč, ki so povzročile razdrobljenost mest in razpadanje urbanega tkiva. V primerjalni študiji je poleg tega prikazano, kako se v mestih s podobno preteklostjo lahko pojavijo različni razvojni vzorci.

  16. Primerjalna analiza platform za izdelavo spletnih mest google sites wix

    OpenAIRE

    Orešek, Mateja

    2017-01-01

    Tema diplomskega seminarja je izdelava spletnih mest s pomočjo brezplačnih orodij. V današnjem času podjetja nujno potrebujejo spletno mesto, saj večina ljudi pridobi informacije na svetovnem spletu in za nekatera podjetja je strošek izdelave spletnega mesta prevelik, zato smo v diplomskem projektu preverjali, ali lahko izdelamo spletno mesto brez znanja programiranja in ali je spletno mesto, izdelano z brezplačnimi orodji, vseeno lahko videti profesionalno. Namen diplomskega projekta je ...

  17. Direct immobilization of antibodies on Zn-doped Fe{sub 3}O{sub 4} nanoclusters for detection of pathogenic bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Pal, Monalisa; Lee, Sanghee; Kwon, Donghoon; Hwang, Jeongin; Lee, Hyeonjeong; Hwang, Seokyung; Jeon, Sangmin, E-mail: jeons@postech.ac.kr

    2017-02-01

    Zinc-doped magnetic nanoclusters (Zn-MNCs) were synthesized and used to detect pathogenic bacteria in milk. Hydrothermally synthesized Zn-MNCs exhibited stronger magnetic properties than pure MNCs, which facilitated the magnetic separation from the sample using a permanent magnet. The presence of accessible Zn sites allows the direct immobilization of half-fragmented antibodies over Zn-MNCs through strong Zn−S bonds and prevents the tedious multiple steps of molecular functionalization or coating with costly noble metals prior to conjugation with an antibody. After the capture and magnetic separation of Salmonella in milk using the antibody-functionalized Zn-MNCs, the concentration of bacteria was determined with a portable ATP luminometer and the detection limit was found to be 10 CFU/mL. - Highlights: • Zn-doped Fe{sub 3}O{sub 4} nanoclusters (Zn-MNCs) were synthesized by hydrothermal method. • Antibodies were directly immobilized over Zn-MNCs through strong Zn–S{sub thiol} bonds. • Higher magnetization of Zn-MNCs than pure MNCs facilitates the magnetic separation. • Detection limit of pathogenic bacteria in milk was found to be 10 cfu/mL. • Cost effective, sensitive and selective detection of bacteria.

  18. Monoclonal antibody against Porphyromonas (Bacteroides) endodontalis lipopolysaccharide and application of the antibody for direct identification of the species.

    Science.gov (United States)

    Hanazawa, S; Sagiya, T; Kitami, H; Ohta, K; Nishikawa, H; Kitano, S

    1991-01-01

    The aim of the present study was to develop a monoclonal antibody that recognizes the shared antigen of Porphyromonas endodontalis so that we could use the antibody in direct identification and detection of P. endodontalis in infectious material from apical periodontal patients. We established a hybridoma cell line producing monoclonal antibody (BEB5) specific for P. endodontalis. BEB5 antibody reacted with all of the P. endodontalis strains tested, but not with any of the other black-pigmented Porphyromonas and Bacteroides spp. The antibody reacted specifically with the lipopolysaccharide (LPS) of three P. endodontalis strains of different serotypes (O1K1, O1K2, and O1K-). Western blotting (immunoblotting) analysis confirmed the specificity of the antibody to these LPSs, because the antibody recognized the typical "repetitive ladder" pattern characteristic of LPS on sodium dodecyl sulfate-polyacrylamide electrophoretic gels. These observations demonstrate that P. endodontalis LPS is the shared antigen of this species. The antibody can specifically identify P. endodontalis on nitrocellulose membrane blots of bacterial colonies grown on agar. The antibody is also capable of directly detecting the presence of P. endodontalis in infectious material by immunoslot blot assay. These results indicate that LPS is the shared antigen of P. endodontalis and that BEB5 antibody against LPS is a useful one for direct identification and detection of the organisms in samples from apical periodontal patients. Images PMID:1774262

  19. "Når man sier helse, tenker jeg mest på fysisk helse"

    OpenAIRE

    Andresen, Håkon Boman

    2016-01-01

    Bakgrunn: Mange ungdommer trener i dag på treningssenter. Tidligere studier viser at det på treningssentrene finnes en sterk fysisk orientert helsediskurs. Dette til tross for at dagens mest brukte helsedefinisjoner anerkjenner helse som et komplekst begrep bestående av fysisk, mental og sosial helse. Det finnes en del forskning på ungdommers helse og selvopplevde helse. Likevel vites det lite om hva unge gutter forstår helse som. Formål: Formålet med studien er å undersøke hvordan gutte...

  20. Peripheral neuropathies associated with antibodies directed to intracellular neural antigens.

    Science.gov (United States)

    Antoine, J-C

    2014-10-01

    Antibodies directed to intracellular neural antigens have been mainly described in paraneoplastic peripheral neuropathies and mostly includes anti-Hu and anti-CV2/CRMP5 antibodies. These antibodies occur with different patterns of neuropathy. With anti-Hu antibody, the most frequent manifestation is sensory neuronopathy with frequent autonomic involvement. With anti-CV2/CRMP5 the neuropathy is more frequently sensory and motor with an axonal or mixed demyelinating and axonal electrophysiological pattern. The clinical pattern of these neuropathies is in keeping with the cellular distribution of HuD and CRMP5 in the peripheral nervous system. Although present in high titer, these antibodies are probably not directly responsible for the neuropathy. Pathological and experimental studies indicate that cytotoxic T-cells are probably the main effectors of the immune response. These disorders contrast with those in which antibodies recognize a cell surface antigen and are probably responsible for the disease. The neuronal cell death and axonal degeneration which result from T-cell mediated immunity explains why treating these disorders remains challenging. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  1. Isolation and characterization of monoclonal antibodies directed against two subunits of rabbit poxvirus-associated, DNA-directed RNA polymerase.

    OpenAIRE

    Morrison, D K; Carter, J K; Moyer, R W

    1985-01-01

    A library of monoclonal antibodies directed against individual proteins of the rabbit poxvirus (RPV) virion within a complex immunogenic mixture has been generated through the use of in vivo and in vitro immunization regimens. The relative efficacies of the two procedures were compared. Based on immunoblot analysis, the in vitro immunization regimen led both to a wider variety of monoclonal antibodies to different proteins and to a larger number of antibodies directed against proteins of high...

  2. Hvem er mest fornøyd med samlivet? : samlivskvalitet blant samboere og gifte

    OpenAIRE

    Wiik, Kenneth Aarskaug; Noack, Turid

    2011-01-01

    Artiklene i Samfunnsspeilet er tilgjengelige fra SSBs nettsider: http://www.ssb.no/ssp/ Norge og Sverige er land der samboerskap har en lang historie, og der denne samlivsformen er mest utbredt. I dag er et av fire samliv her til lands samboerskap. Men også her er det forskjeller mellom samboere og gifte. Hvis vi sammenlikner samlivskvaliteten blant samboere og gifte, finner vi at unge samboere i alderen 25 til 35 år er minst fornøyd med samlivet sitt. Samboere med planer om å gifte seg me...

  3. Monoclonal antibody against Porphyromonas (Bacteroides) endodontalis lipopolysaccharide and application of the antibody for direct identification of the species.

    OpenAIRE

    Hanazawa, S; Sagiya, T; Kitami, H; Ohta, K; Nishikawa, H; Kitano, S

    1991-01-01

    The aim of the present study was to develop a monoclonal antibody that recognizes the shared antigen of Porphyromonas endodontalis so that we could use the antibody in direct identification and detection of P. endodontalis in infectious material from apical periodontal patients. We established a hybridoma cell line producing monoclonal antibody (BEB5) specific for P. endodontalis. BEB5 antibody reacted with all of the P. endodontalis strains tested, but not with any of the other black-pigment...

  4. Technetium-99m direct radiolabeling of monoclonal antibody ior egf/r3

    Energy Technology Data Exchange (ETDEWEB)

    Morales, Alejo A. Morales; Crespo, Francisco Zayas; Gandolff, Gilda Nunez; Escobar, Normando Iznaga; Perez, Niuvis Perez; Hernandez, Juan C. Izquierdo

    1998-01-01

    Monoclonal antibodies (MAbs) are being widely used for imaging studies, coupled mainly with {sup 99m}Tc. The antibody ior egf/r3 is a MAb against human epidermal growth factor receptor (hEGF-r), and we have developed a method for optimum labeling of this MAb with {sup 99m}Tc. The reduction was performed with 2-mercaptoethanol (2-ME) at a molar ratio of 2000:1 (2-ME:MAb) and methylene diphosphonate as transchelant. The integrity of reduced MAb was checked by mean of native polyacrylamide gel electrophoresis (PAGE) and gel filtration chromatography on Superose 12 (purity >99%). Radio colloids remained lower than 2%, and the labeling efficiency was 98.5%. The number of sulfhydryl groups generated was quantified using Ellman's reagent and was found to be 6.65 {+-} 0.69 per antibody molecule. In vitro stability studies in several challenging conditions (DTPA, human serum albumin and human serum) were performed, and no significant loss in binding percentage was seen. Radio receptor assay was used to test immunoreactivity of the reduced MAb. Both labeled and unlabeled MAbs were able to compete for binding to the hEGF-r with radioiodinated EGF. Biodistribution studies in BALB/c mice are reported.

  5. Technetium-99m direct radiolabeling of monoclonal antibody ior egf/r3

    International Nuclear Information System (INIS)

    Morales, Alejo A. Morales; Crespo, Francisco Zayas; Gandolff, Gilda Nunez; Escobar, Normando Iznaga; Perez, Niuvis Perez; Hernandez, Juan C. Izquierdo

    1998-01-01

    Monoclonal antibodies (MAbs) are being widely used for imaging studies, coupled mainly with 99m Tc. The antibody ior egf/r3 is a MAb against human epidermal growth factor receptor (hEGF-r), and we have developed a method for optimum labeling of this MAb with 99m Tc. The reduction was performed with 2-mercaptoethanol (2-ME) at a molar ratio of 2000:1 (2-ME:MAb) and methylene diphosphonate as transchelant. The integrity of reduced MAb was checked by mean of native polyacrylamide gel electrophoresis (PAGE) and gel filtration chromatography on Superose 12 (purity >99%). Radio colloids remained lower than 2%, and the labeling efficiency was 98.5%. The number of sulfhydryl groups generated was quantified using Ellman's reagent and was found to be 6.65 ± 0.69 per antibody molecule. In vitro stability studies in several challenging conditions (DTPA, human serum albumin and human serum) were performed, and no significant loss in binding percentage was seen. Radio receptor assay was used to test immunoreactivity of the reduced MAb. Both labeled and unlabeled MAbs were able to compete for binding to the hEGF-r with radioiodinated EGF. Biodistribution studies in BALB/c mice are reported

  6. Monoclonal antibodies directed to E1 glycoprotein of rubella virus

    International Nuclear Information System (INIS)

    Umino, Y.; Sato, A.; Katow, S.; Matsuno, T.; Sugiura, A.

    1985-01-01

    We have prepared four monoclonal antibodies to rubella virus E1 glycoprotein. Three nonoverlapping antigenic sites were delineated on E1 protein by competitive binding assays. Antibodies binding to one site were characterized by high hemagglutination inhibition (HI) titer but poor neutralizing activity. The addition of antiglobulin conferred neutralizing activity. Antibodies directed to two other antigenic sites had modest hemolysis inhibition but little or no HI and neutralizing activities. The addition of antiglobulin markedly augmented HI activity but had little effect on neutralizing activity. Epitopes defined by three antibodies were conserved among four rubella virus strains examined. (Author)

  7. Methylation status of imprinted genes DLK1-GTL2, MEST (PEG1), ZAC (PLAGL1), and LINE-1 elements in spermatozoa of normozoospermic men, unlike H19 imprinting control regions, is not associated with idiopathic recurrent spontaneous miscarriages.

    Science.gov (United States)

    Ankolkar, Mandar; Salvi, Vinita; Warke, Himangi; Vundinti, Babu Rao; Balasinor, N H

    2013-05-01

    To study methylation aberrations in spermatozoa at developmentally important imprinted regions to ascertain their role in early embryo loss in idiopathic recurrent spontaneous miscarriages (RSM). Case-control study. Academic research setting at National Institute for Research in Reproductive Health, Parel, Mumbai. Male partners of couples with a history of RSM and male partners of couples with proven fertility (control group). None. DNA methylation levels at imprinting control regions of DLK1-GTL2, MEST (PEG1), and ZAC (PLAGL1) by Epityper Massarray and global methylation levels as measured by LINE-1 methylation and anti-5-methyl cytosine antibody in spermatozoa of 23 men in control group and 23 men in RSM group. We did not observe any aberration in the total methylation levels in any of the imprinted genes or global methylation analyzed. Our results indicate that paternal methylation aberrations at imprinting control regions of DLK1-GTL2, MEST (PEG1), and ZAC (PLAGL1) and global methylation levels are not associated with idiopathic RSM and may not be good epigenetic markers (unlike the H-19 imprinting control region) for diagnosis of idiopathic RSM. Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  8. Comparison of a direct and indirect ELISA for quantitating antisperm antibody in semen.

    Science.gov (United States)

    Lynch, D M; Howe, S E

    1987-01-01

    A direct and an indirect quantitative ELISA for antisperm antibody were compared using the spermatozoa and cell-free seminal fluid of 66 infertile males. The normal concentration of sperm binding immunoglobulin was less than or equal to 1.5 fg Ig per spermatozoon for the indirect seminal plasma assay and less than or equal to 1.5 fg Ig per spermatozoon by the direct assay. Of the 66 infertile males, 21% (14/66) had elevated levels of antisperm antibody in their seminal plasma and 26% (17/66) had elevated levels bound directly to their spermatozoa. The direct correlation between the results of these assays was 94%. A simple linear regression analysis between the indirect and direct measurements of antisperm antibody resulted in a correlation coefficient of r = 0.907. There was no statistically significant difference between results from the direct and indirect methods of the patients as a group. However, there was evidence of autospecificity in a small percentage of males who had elevated levels of antisperm antibody by the direct assay that was not detected by the indirect assay using pooled donor spermatozoa.

  9. Efficient one-step direct labelling of recombinant antibodies with technetium-99m

    International Nuclear Information System (INIS)

    Liberatore, M.; Neri, D.; Neri, G.; Pini, A.; Lurilli, A.P.; Ponzo, F.; Spampinato, G.; Padula, F.; Pala, A.; Colella, A.C.

    1995-01-01

    High-affinity bacterially expressed antibody fragments can nowadays be cloned from established hybridomas or, more conveniently, isolated directly from antibody libraries displayed on filamentous phage. Such antibodies can be tagged with C-terminal peptide tags containing one cysteine residue, which represents a convenient functionalisation site for a number of applications, including technetium-99m labelling. Here we describe a simple one-step method for 99m Tc labelling of cysteine-tagged recombinant antibodies with more than 50% radionuclide incorporation. The labelled antibodies displayed full retention of immuoreactivity and good stability. (orig.)

  10. Efficient one-step direct labelling of recombinant antibodies with technetium-99m

    Energy Technology Data Exchange (ETDEWEB)

    Liberatore, M. [Dipartimento di Medicina Sperimentale, Sezione di Medicina Nucleare, Policlinico Umberto I, Universita di Roma `La Sapienza` (Italy); Neri, D. [Cambridge Centre for Protein Engineering - MRC Centre (United Kingdom); Neri, G. [Dipartimento di Biologia Molecolare, Universita di Siena (Italy); Pini, A. [Dipartimento di Biologia Molecolare, Universita di Siena (Italy); Lurilli, A.P. [Dipartimento di Medicina Sperimentale, Sezione di Medicina Nucleare, Policlinico Umberto I, Universita di Roma `La Sapienza` (Italy); Ponzo, F. [Dipartimento di Medicina Sperimentale, Sezione di Medicina Nucleare, Policlinico Umberto I, Universita di Roma `La Sapienza` (Italy); Spampinato, G. [Laboratorio di Biochimica degli Ormoni Sessuali, Il Instituto di Clinica Ostetrica e Ginecologica, Universita di Roma `La Sapienza` (Italy); Padula, F. [Laboratorio di Biochimica degli Ormoni Sessuali, Il Instituto di Clinica Ostetrica e Ginecologica, Universita di Roma `La Sapienza` (Italy); Pala, A. [Laboratorio di Biochimica degli Ormoni Sessuali, Il Instituto di Clinica Ostetrica e Ginecologica, Universita di Roma `La Sapienza` (Italy); Colella, A.C. [Dipartimento di Medicina Sperimentale, Sezione di Medicina Nucleare, Policlinico Umberto I, Universita di Roma `La Sapienza` (Italy)

    1995-11-01

    High-affinity bacterially expressed antibody fragments can nowadays be cloned from established hybridomas or, more conveniently, isolated directly from antibody libraries displayed on filamentous phage. Such antibodies can be tagged with C-terminal peptide tags containing one cysteine residue, which represents a convenient functionalisation site for a number of applications, including technetium-99m labelling. Here we describe a simple one-step method for {sup 99m}Tc labelling of cysteine-tagged recombinant antibodies with more than 50% radionuclide incorporation. The labelled antibodies displayed full retention of immuoreactivity and good stability. (orig.)

  11. DIRECT AND INDIRECT FLUORESCENT-ANTIBODY TECHNIQUES FOR THE PSITTACOSIS-LYMPHOGRANULOMA VENEREUM-TRACHOMA GROUP OF AGENTS1

    Science.gov (United States)

    Ross, Martin R.; Borman, Earle K.

    1963-01-01

    Ross, Martin R. (Connecticut State Department of Health, Hartford) and Earle K. Borman. Direct and indirect fluorescent-antibody techniques for the psittacosis-lymphogranuloma venereum-trachoma group of agents. J. Bacteriol. 85:851–858. 1963.—Direct and indirect fluorescent-antibody (FA) techniques were developed for the detection of group antigen in infected tissue cultures and the titration of group antibody in human antiserum. The growth of the agent of meningopneumonitis (MP) in mouse embryo lung cell monolayers was followed by infectivity and complement-fixing (CF) antigen titrations, and cytological examination of FA stained cultures. Although infectivity and CF antigen reached a peak at 2 days and remained constant for an additional 3 days, only cells tested 2 to 3 days after infection were suitable for FA staining with labeled anti-MP serum because of excessive artifacts in the older cultures. Fluorescein isothiocyanate-labeled rooster and guinea pig anti-MP serums and human antipsittacosis serums were titrated in direct FA and hemagglutination-inhibition (HI) tests. The rooster conjugate showed brighter staining and higher antibody titers than the guinea pig or human conjugates and was more effective in detecting minimal amounts of virus antigen. FA staining reactions with 1 and 2 units of labeled rooster serum were inhibited by unlabeled rooster serum but clear-cut inhibition with human antipsittacosis serum could not be demonstrated. The indirect FA technique was successfully used for the titration of group antibody in human serum. A comparison of the indirect FA, HI, and CF tests showed the indirect FA technique to be intermediate in sensitivity between the HI and CF tests. None of the three tests showed significant cross reactions with human serums reactive for influenza A and B; parainfluenza 1, 2, and 3; respiratory syncytial virus; Q fever; or the primary atypical pneumonia agent. PMID:14044954

  12. Direct 99mTc labeling of monoclonal antibodies: radiolabeling and in vitro stability

    International Nuclear Information System (INIS)

    Garron, J.Y.; Moinereau, M.; Pasqualini, R.; Saccavini, J.C.

    1991-01-01

    Direct labeling involves 99m Tc binding to different donor groups on the protein, giving multiple binding sites of various affinities resulting in an in vivo instability. The stability has been considerably improved by activating the antibody using a controlled reduction reaction (using 2-aminoethanethiol). This reaction generates sulfhydryl groups, which are known to strongly bind 99m Tc. The direct 99m Tc antibody labeling method was explored using whole antibodies and fragments. Analytical methods were developed for routine evaluation of radiolabeling yield and in vitro stability. Stable direct antibody labeling with 99m Tc requires the generation of sulfhydryl groups, which show high affinity binding sites for 99m Tc. Such groups are obtained with 2-aminoethanethiol (AET), which induces the reduction of the intrachain or interchain disulfide bond, with no structural deterioration or any loss of immunobiological activity of the antibody. The development of fast, reliable analytical methods has made possible the qualitative and quantitative assessment of technetium species generated by the radiolabeling process. Labeling stability is determined by competition of the 99m Tc-antibody bond with three ligands, Chelex 100 (a metal chelate-type resin), free DTPA solution and 1% HSA solution. Very good 99m Tc-antibody stability is obtained with activated IgG (IgGa) and Fab' fragment, which makes these substances possible candidates for immunoscintigraphy use. (author)

  13. A synthetic glycan microarray enables epitope mapping of plant cell wall glycan-directed antibodies

    DEFF Research Database (Denmark)

    Ruprecht, Colin; Bartetzko, Max P; Senf, Deborah

    2017-01-01

    In the last three decades, more than 200 monoclonal antibodies have been raised against most classes of plant cell wall polysaccharides by different laboratories world-wide. These antibodies are widely used to identify differences in plant cell wall components in mutants, organ and tissue types......, and developmental stages. Despite their importance and broad use, the precise binding epitope for only a few of these antibodies has been determined. Here, we use a plant glycan microarray equipped with 88 synthetic oligosaccharides to comprehensively map the epitopes of plant cell wall glycan-directed antibodies....... Our results reveal the binding epitopes for 78 arabinogalactan-, rhamnogalacturonan-, xylan-, and xyloglucan-directed antibodies. We demonstrate that, with knowledge of the exact epitopes recognized by individual antibodies, specific glycosyl hydrolases can be implemented into immunological cell wall...

  14. Labeling and stability of radiolabeled antibody fragments by a direct 99mTc-labeling method

    International Nuclear Information System (INIS)

    Pak, K.Y.; Nedelman, M.A.; Tam, S.H.; Wilson, E.; Daddona, P.E.

    1992-01-01

    The in vitro labeling and stability of 99m Tc-labeled antibody Fab' fragments prepared by a direct labeling technique were evaluated. Eight antibody fragments derived from murine IgG1 (N = 5), IgG2a (N = 2) and IgG3 (N = 1) isotypes were labeled with a preformed 99m Tc-D-glucarate complex. No loss of radioactivity incorporation was observed for all the 99m Tc-labeled antibody fragments after 24 h incubation at 37 o C. 99m Tc-labeled antibody fragments (IgG1, N = 2; IgG2a, n = 2; IgG3, N = 1) were stable upon challenge with DTPA, EDTA or acidic pH. Using the affinity chromatography technique, two of the 99m Tc-labeled antibody fragments displayed no loss of immunoreactivity after prolonged incubation in phosphate buffer up to 24 h at 37 o C. Bonding between 99m Tc and antibody fragments was elucidated by challenging with a diamide ditholate (N 2 S 2 ) compound. The Fab' with IgG2a isotype displayed tighter binding to 99m Tc in comparison to Fab' from IgG1 and IgG3 isotype in N 2 S 2 challenge and incubation with human plasma. The in vivo biodistribution of five 99m Tc-labeled fragments were evaluated in normal mice. (Author)

  15. Synergistic cytotoxic effects of antibodies directed against different cell surface determinants

    Energy Technology Data Exchange (ETDEWEB)

    Elliott, E V; Pindar, A; Stevenson, F K; Stevenson, G T [Southampton General Hospital (UK). Tenovus Research Lab.

    1978-03-01

    Three antibody populations were raised in rabbits against surface antigens on guinea-pig L/sub 2/C leukaemic lymphocytes: against idiotypic determinants on the lambda chain of the surface immunoglobulin, against C region determinants on the lambda chain, and against the surface antigens recognised by conventional anti-lymphocyte sera. Complement and K-cell cytotoxicities effected by the antibodies on L/sub 2/C cells were studied in vitro. In both cytotoxic systems mixtures of the antibodies revealed synergy, in that the titres of the mixtures exceeded predicted additive titres of their components. The synergy was greater when the mixed antibodies were directed to determinants on the same molecule rather than to determinants on different molecules.

  16. Diagnosis of canine rabies by the direct fluorescent antibody ...

    African Journals Online (AJOL)

    Diagnosis of canine rabies by the direct fluorescent antibody technique in Plateau State, Nigeria. DO Ehizibolo, EA Ogunsan, MJ Muhammad, CI Nwosuh, S Olaleye, OOC Chuckwu, MY Sugun, NM Sati, NE Waziri, OK Egwu, J Kamani, CA Meseko, SE Idachaba, GI Dogo ...

  17. Human antibody responses to Schistosoma mansoni: does antigen directed, isotype restriction result in the production of blocking antibodies?

    Directory of Open Access Journals (Sweden)

    David W. Dunne

    1987-01-01

    Full Text Available After treatment young Kenyan schoolchildren are highly susceptible to reinfection with Schistosoma mansoni. Older children and adults are resistant to reinfection. There is no evidence that this age related resistance is due to a slow development of protective immunological mechanisms, rather, it appears that young children are susceptible because of the presence of blocking antibodies which decline with age, thus allowing the expression of protective responses. Correlations between antibody responses to different stages of the parasite life-cycle suggest that, in young children, antigen directed, isotype restriction of the response against cross-reactive polysaccharide egg antigens results in an ineffectual, or even blocking antibody response to the schistosomulum.

  18. Studies of a murine monoclonal antibody directed against DARC: reappraisal of its specificity.

    Directory of Open Access Journals (Sweden)

    Dorota Smolarek

    Full Text Available Duffy Antigen Receptor for Chemokines (DARC plays multiple roles in human health as a blood group antigen, a receptor for chemokines and the only known receptor for Plasmodium vivax merozoites. It is the target of the murine anti-Fy6 monoclonal antibody 2C3 which binds to the first extracellular domain (ECD1, but exact nature of the recognized epitope was a subject of contradictory reports. Here, using a set of complex experiments which include expression of DARC with amino acid substitutions within the Fy6 epitope in E. coli and K562 cells, ELISA, surface plasmon resonance (SPR and flow cytometry, we have resolved discrepancies between previously published reports and show that the basic epitope recognized by 2C3 antibody is 22FEDVW26, with 22F and 26W being the most important residues. In addition, we demonstrated that 30Y plays an auxiliary role in binding, particularly when the residue is sulfated. The STD-NMR studies performed using 2C3-derived Fab and synthetic peptide corroborated most of these results, and together with the molecular modelling suggested that 25V is not involved in direct interactions with the antibody, but determines folding of the epitope backbone.

  19. The Antitumor Effect of Single-domain Antibodies Directed Towards Membrane-associated Catalase and Superoxide Dismutase.

    Science.gov (United States)

    Bauer, Georg; Motz, Manfred

    2016-11-01

    Neutralizing single-domain antibodies directed towards catalase or superoxide dismutase (SOD) caused efficient reactivation of intercellular reactive oxygen species/reactive nitrogen species (ROS/RNS)-dependent apoptosis-inducing signaling specifically in human tumor cells. Single-domain antibodies targeted tumor cell-specific membrane-associated SOD and catalase, but not the corresponding intracellular enzymes. They were shown to be about 200-fold more effective than corresponding classical recombinant antigen-binding fragments and more than four log steps more efficient than monoclonal antibodies. Combined addition of single-domain antibodies against catalase and SOD caused a remarkable synergistic effect. Proof-of-concept experiments in immunocompromised mice using human tumor xenografts and single-domain antibodies directed towards SOD showed an inhibition of tumor growth. Neutralizing single-domain antibodies directed to catalase and SOD also caused a very strong synergistic effect with the established chemotherapeutic agent taxol, indicating an overlap of signaling pathways. This effect might also be useful in order to avoid unwanted side-effects and to drastically lower the costs for taxol-based therapy. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  20. Evaluation of iodovinyl antibody conjugates: Comparison with a p-iodobenzoyl conjugate and direct radioiodination

    International Nuclear Information System (INIS)

    Hadley, S.W.; Wilbur, D.S.

    1990-01-01

    The preparations and conjugations of 2,3,5,6-tetrafluorophenyl 5-[125I/131I]iodo-4-pentenoate (7a) and 2,3,5,6-tetrafluorophenyl 3,3-dimethyl-5-[125I/131I]iodo-4-pentenoate (7b) to monoclonal antibodies are reported. Reagents 7a and 7b were prepared in high radiochemical yield by iododestannylation of their corresponding 5-tri-n-butylstannyl precursors. Radioiodinated antibody conjugates were prepared by reaction of 7a or 7b with the protein at basic pH. Evaluation of these conjugates by several in vitro procedures demonstrated that the radiolabel was attached to the antibody in a stable manner and that the conjugates maintained immunoreactivity. Comparative dual-isotope biodistribution studies of a monoclonal antibody Fab fragment conjugate of 7a and 7b with the same Fab fragment labeled with N-succinimidyl p-[131I]iodobenzoate (PIB, p-iodobenzoate, 2) or directly radioiodinated have been carried out in tumor-bearing nude mice. Coinjection of the Fab conjugate of 7a with the Fab conjugate of 2 demonstrated that the biodistributions were similar in most organs, except the neck tissue (thyroid-containing) and the stomach, which contained substantially increased levels of the 7a label. Coinjection of the Fab conjugate of 7a with the Fab fragment radioiodinated by using the chloramine-T method demonstrated that the biodistributions were remarkably similar, suggesting roughly equivalent in vivo deiodination of these labeled antibody fragments. Coinjection of the Fab conjugate of 7a with the Fab conjugate of 7b indicated that there was ∼ a 2-fold reduction in the amount of in vivo deiodination of the 7b conjugate as compared to the 7a conjugate

  1. Site-specific conjugation and labelling of prostate antibody 7E11C5.3 (CYT-351) with technetium-99m

    International Nuclear Information System (INIS)

    Stalteri, M.A.; Mather, S.J.; Belinka, B.A.; Coughlin, D.J.; Chengazi, V.U.; Britton, K.E.

    1997-01-01

    Attachment of chelating agents to the sugar residues of antibodies for subsequent radiolabelling is an attractive approach since it may have less effect on the immunoreactivity than attachment through lysine residues, which are distributed throughout the antibody and may be present near the antigen binding site. We have attached a new hydrazide-linked chelator CYT-395 (Cytogen Corp., Princeton, N.J.) to the sugar residues of the anti-prostate monoclonal antibody 7E11C5.3 and optimised the conditions for labelling the conjugate with technetium-99m in order to compare the conjugate to 7E11C5.3 antibody labelled directly with technetium using a mercaptoethanol reduction technique. Labelling yields of 70%-90% were obtained at specific activities up to 2000 MBq/mg antibody. The stability of the technetium-labelled conjugate in plasma or to a challenge with 0.1 or 1.0 mM cysteine was similar to that of direct-labelled antibody. In nine patients with prostate cancer, the plasma clearance of the labelled conjugate followed a two-compartment model, with an average β-phase half-life of 31.4±3.9 h. The average urinary clearance at 24 h was 15.3±5.0% of the injected dose. In this group of patients there was no significant difference between the blood and urine clearance of the labelled conjugate, and the clearances of the direct-labelled antibody. (orig.). With 5 figs

  2. Serum transglutaminase 3 antibodies correlate with age at celiac disease diagnosis.

    Science.gov (United States)

    Salmi, Teea T; Kurppa, Kalle; Hervonen, Kaisa; Laurila, Kaija; Collin, Pekka; Huhtala, Heini; Saavalainen, Päivi; Sievänen, Harri; Reunala, Timo; Kaukinen, Katri

    2016-06-01

    Transglutaminase (TG)2 is the autoantigen in celiac disease, but also TG3 antibodies have been detected in the serum of celiac disease patients. To investigate the correlations between serum TG3 antibodies and clinical and histological manifestations of celiac disease and to assess gluten-dependency of TG3 antibodies. Correlations between serum TG3 antibody levels measured from 119 adults and children with untreated coeliac disease and the demographic data, clinical symptoms, celiac antibodies, histological data and results of laboratory tests and bone mineral densities were tested. TG3 antibodies were reinvestigated in 97 celiac disease patients after 12 months on a gluten-free diet (GFD). TG3 antibody titers were shown to correlate with the age at celiac disease diagnosis. Further, negative correlation with TG3 antibodies and intestinal γδ+ cells at diagnosis and on GFD was detected. Correlations were not detected with the clinical manifestation of celiac disease, TG2 or endomysial autoantibodies, laboratory values, severity of mucosal villous atrophy, associated diseases or complications. TG3 antibody titers decreased on GFD in 56% of the TG3 antibody positive patients. Serum TG3 antibody positivity in celiac disease increases as the diagnostic age rises. TG3 antibodies did not show similar gluten-dependency as TG2 antibodies. Copyright © 2016 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

  3. Direct binding of radioiodinated monoclonal antibody to tumor cells: significance of antibody purity and affinity for drug targeting or tumor imaging

    International Nuclear Information System (INIS)

    Kennel, S.J.; Foote, L.J.; Lankford, P.K.; Johnson, M.; Mitchell, T.; Braslawsky, G.R.

    1983-01-01

    For MoAb to be used efficiently for drug targeting and tumor imaging, the fraction of antibody binding to tumor cells must be maximized. The authors have studied the binding of 125 I MoAb in three different tumor systems. The fraction of antibody that could be bound to the cell surface was directly proportional to the antibody purity. The affinity constant also limits the fraction of antibody that can bind to cells at a given antigen concentration. Rearrangement of the standard expression for univalent equilibrium binding between two reactants shows that in antigen excess, the maximum fraction of antibody that can bind =Ka[Ag total]/1 + Ka[Ag total]. Binding data using four different MoAb with three cell systems confirm this relationship. Estimates for reasonable concentrations of tumor antigens in vivo indicate that antibodies with binding constants less than 10 8 M -1 are not likely to be useful for drug targeting or tumor imaging

  4. Direct labelling of monomeric antibody fragments Fab' with 99mTc

    International Nuclear Information System (INIS)

    Li Jun; Wang Shizhen; Yang Ziyi

    1994-01-01

    Direct labelling method and conditions of monomeric antibody Fab' with 99m Tc were investigated. Polyclonal antibody IgG was digested with ficin to produce dimeric fragments F(ab') 2 , which was subsequently reduced to monomeric fragments Fab' with 2-mercaptoethylamine. Finally, Fab' was incubated with sodium gluconate (Sn(II)) kit solution and 99m TcO 4 - eluted at room temperature to form 99m Tc-Fab'. The labelling efficiency was 85%-95%. The stability of labelled products was satisfactory and the elimination rate was faster than 99m Tc-IgG

  5. Identification of a novel myositis-associated antibody directed against cortactin.

    Science.gov (United States)

    Labrador-Horrillo, Moisés; Martínez, Maria Angeles; Selva-O'Callaghan, Albert; Trallero-Araguás, Ernesto; Grau-Junyent, Josep M; Vilardell-Tarrés, Miquel; Juarez, Candido

    2014-10-01

    The aim of this study is to describe a novel myositis-associated autoantibody (anti-cortactin antibody) and assess related clinical and immunological manifestations and its clinical significance. Adult patients with myositis (dermatomyositis, polymyositis, immune-mediated necrotizing myopathy, and inclusion body myositis), as well as patients with other autoimmune diseases and non-inflammatory myopathies were analyzed for the presence of anti-cortactin antibody using in-house developed ELISA and immunoblotting techniques with a commercial source of purified cortactin. The cut-off for positive status was determined in a group of healthy volunteers. Antibody against cortactin was positive in 7/34 (20%) polymyositis patients, 9/117 (7.6%) dermatomyositis, 2/7 (26%) immune-mediated necrotizing myopathy, and none of the 4 patients with inclusion body myositis. The antibody also tested positive in 3/101 patients with other autoimmune diseases (2 systemic sclerosis and 1 systemic lupus erythematosus), and in 1/29 patients with non-inflammatory myopathy. No relevant association with specific clinical features was found in patients with these antibodies. Anti-cortactin antibody was more frequently positive in patients with polymyositis and immune-mediated necrotizing myopathy than in the remaining myositis patients, and was the only myositis autoantibody found in sera of 3 patients from these groups. Our data indicate that cortactin is a novel target antigen in patients with autoimmune diseases, especially patients with polymyositis or immune-mediated necrotizing myopathy. Anti-cortactin can be considered a new myositis-associated antibody. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Quantum dot-linked immunosorbent assay (QLISA) using orientation-directed antibodies.

    Science.gov (United States)

    Suzuki, Miho; Udaka, Hikari; Fukuda, Takeshi

    2017-09-05

    An approach similar to the enzyme-linked immunosorbent assay (ELISA), with the advantage of saving time and effort but exhibiting high performance, was developed using orientation-directed half-part antibodies immobilized on CdSe/ZnS quantum dots. ELISA is a widely accepted assay used to detect the presence of a target substance. However, it takes time to quantify the target with specificity and sensitivity owing to signal amplification. In this study, CdSe/ZnS quantum dots are introduced as bright and photobleaching-tolerant fluorescent materials. Since hydrophilic surface coating of quantum dots rendered biocompatibility and functional groups for chemical reactions, the quantum dots were modified with half-sized antibodies after partial reduction. The half-sized antibody could be bound to a quantum dot through a unique thiol site to properly display the recognition domain for the core process of ELISA, which is an antigen-antibody interaction. The reducing conditions were investigated to generate efficient conjugates of quantum dots and half-sized antibodies. This was applied to IL-6 detection, as the quantification of IL-6 is significant owing to its close relationships with various biomedical phenomena that cause different diseases. An ELISA-like assay with CdSe/ZnS quantum dot institution (QLISA; Quantum dot-linked immunosorbent assay) was developed to detect 0.05ng/mL IL-6, which makes it sufficiently sensitive as an immunosorbent assay. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Proteolytic processing of poliovirus polypeptides: antibodies to polypeptide P3-7c inhibit cleavage at glutamine-glycine pairs

    International Nuclear Information System (INIS)

    Hanecak, R.; Semler, B.L.; Anderson, C.W.; Wimmer, E.

    1982-01-01

    Proteolytic processing of poliovirus polypeptides was examined by the addition of antibodies directed against the viral proteins P3-7c and P2-X to a cell-free translation extract prepared from infected HeLa cells. Antisera to P3-7c specifically inhibited in vitro processing at Gln-Gly pairs. Partial amino acid sequence analysis revealed a second Tyr-Gly pair that is utilized in protein processing. Neither Tyr-Gly cleavage is affected by antibody to P3-7C. Anti-P3-7c antibodies react not only with P3-7c but also with P3-6a and P3-2, two viral polypeptides NH 2 -coterminal with P3-7c. Preimmune and anti-P2-X antibodies had no effect on the processing of poliovirus proteins in vitro. The authors conclude that the activity responsible for processing poliovirus polypeptides at Gln-Gly pairs resides in the primary structure of P3-7c and not in P2-X

  8. Antibody Engineering and Therapeutics

    Science.gov (United States)

    Almagro, Juan Carlos; Gilliland, Gary L; Breden, Felix; Scott, Jamie K; Sok, Devin; Pauthner, Matthias; Reichert, Janice M; Helguera, Gustavo; Andrabi, Raiees; Mabry, Robert; Bléry, Mathieu; Voss, James E; Laurén, Juha; Abuqayyas, Lubna; Barghorn, Stefan; Ben-Jacob, Eshel; Crowe, James E; Huston, James S; Johnston, Stephen Albert; Krauland, Eric; Lund-Johansen, Fridtjof; Marasco, Wayne A; Parren, Paul WHI; Xu, Kai Y

    2014-01-01

    The 24th Antibody Engineering & Therapeutics meeting brought together a broad range of participants who were updated on the latest advances in antibody research and development. Organized by IBC Life Sciences, the gathering is the annual meeting of The Antibody Society, which serves as the scientific sponsor. Preconference workshops on 3D modeling and delineation of clonal lineages were featured, and the conference included sessions on a wide variety of topics relevant to researchers, including systems biology; antibody deep sequencing and repertoires; the effects of antibody gene variation and usage on antibody response; directed evolution; knowledge-based design; antibodies in a complex environment; polyreactive antibodies and polyspecificity; the interface between antibody therapy and cellular immunity in cancer; antibodies in cardiometabolic medicine; antibody pharmacokinetics, distribution and off-target toxicity; optimizing antibody formats for immunotherapy; polyclonals, oligoclonals and bispecifics; antibody discovery platforms; and antibody-drug conjugates. PMID:24589717

  9. Studies of monoclonal antibodies IOR-CEA-1 and IOR-EGF/R3 labelled with 99mTc

    International Nuclear Information System (INIS)

    Dias, Carla Roberta de Barros Rodrigues

    2005-01-01

    Nuclear Medicine is a speciality that uses radioisotopes for the diagnosis or treatment of diseases and it is considered one of the best tools among the diagnostic modalities for detection of cancer. 99m Tc is one of the main isotopes for labelling antibodies and in Nuclear Medicine in general, due to its adequate physical properties, availability and low cost. Labelled monoclonal antibodies have shown promising results for diagnosis and therapy of cancer and their use has brought great experimental and clinical advances in the field of oncology. The main clinical applications of immunoscintigraphy with monoclonal antibodies are staging and evaluation of tumoral reappearance. The antibodies employed in this work were: OIR-CEA-1, a murine monoclonal antibody that acts directly against CEA expressed in several neoplasia in particular those from the gastrointestinal tract (colorectal cancer) and IOR-EGF/R3, a murine monoclonal antibody that binds to the external domain of EGF-R and it has been used in the diagnosis of tumors of epithelial origin. The objectives of this work were the development and optimization of the reduction and purification processes, the radiolabelling techniques and quality control procedures (radiochemical, immunoreactivity and cystein challenge) and imaging studies of monoclonal antibodies OIR-CEA-1 and IOR-EGF/R3, using the simple, fast and efficient method of direct labelling of the antibody with 99m Tc. The final results was the definition of the best conditions for the preparation of lyophilized reactive kits of OIR-CEA-1 and IOR- EGF/R3 for an efficient diagnostic application in Nuclear Medicine. The most adequate conditions for the labelling of the antibodies were: 1.0 mg Ab, 29 μL MDP, 3.0 μg Sn 2+ , 1 mL of 99m Tc and 30 min. reaction time. With these conditions the labelling yield was always higher than 95% and the maximum activity of 99m Tc was about 2220 MBq (60 mCi). The evidences of the efficiency and quality of the methods here

  10. Analysis of Individuals from a Dengue-Endemic Region Helps Define the Footprint and Repertoire of Antibodies Targeting Dengue Virus 3 Type-Specific Epitopes.

    Science.gov (United States)

    Andrade, Daniela V; Katzelnick, Leah C; Widman, Doug G; Balmaseda, Angel; de Silva, Aravinda M; Baric, Ralph S; Harris, Eva

    2017-09-19

    The four dengue virus serotypes (DENV1 to 4) cause dengue, a major public health problem worldwide. Individuals exposed to primary DENV infections develop serotype-specific neutralizing antibodies, including strongly neutralizing antibodies targeting quaternary epitopes. To date, no studies have measured the levels and kinetics of serum antibodies directed to such epitopes among populations in regions where dengue is endemic. Here, we use a recombinant DENV4 (rDENV4/3-M14) displaying a major DENV3 type-specific quaternary epitope recognized by human monoclonal antibody 5J7 to measure the proportion, magnitude, and kinetics of DENV3 type-specific neutralizing antibody responses targeting this epitope. Primary DENV3 sera from 30 individuals in a dengue hospital-based study in Nicaragua were studied 3, 6, 12, and 18 months post-infection, alongside samples collected annually 1 to 4 years post-primary DENV3 infection from 10 individuals in a cohort study in Nicaragua. We found substantial individual variation in the proportion of DENV3 type-specific neutralizing antibody titers attributed to the 5J7 epitope (range, 0 to 100%), with the mean significantly increasing from 22.6% to 41.4% from 3 to 18 months. We extended the transplanted DENV3 5J7 epitope on the virion (rDENV4/3-M16), resulting in increased recognition in several individuals, helping define the footprint of the epitope. However, 37% and 13% of the subjects still showed little to no recognition of the 5J7 epitope at 3 and 18 months, respectively, indicating that one or more additional DENV3 type-specific epitopes exist. Overall, this study demonstrates how DENV-immune plasma from populations from areas of endemicity, when coupled with structurally guided recombinant viruses, can help characterize the epitope-specific neutralizing antibody response in natural DENV infections, with direct implications for design and evaluation of dengue vaccines. IMPORTANCE The four serotypes of dengue virus cause dengue

  11. Report on Project Action Sheet PP05 task 3 between the U.S. Department of Energy and the Republic of Korea Ministry of Education, Science, and Technology (MEST).

    Energy Technology Data Exchange (ETDEWEB)

    Snell, Mark Kamerer

    2013-01-01

    This report documents the results of Task 3 of Project Action Sheet PP05 between the United States Department of Energy (DOE) and the Republic of Korea (ROK) Ministry of Education, Science, and Technology (MEST) for Support with Review of an ROK Risk Evaluation Process. This task was to have Sandia National Laboratories collaborate with the Korea Institute of Nuclear Nonproliferation and Control (KINAC) on several activities concerning how to determine the Probability of Neutralization, PN, and the Probability of System Effectiveness, PE, to include: providing descriptions on how combat simulations are used to determine PN and PE; comparisons of the strengths and weaknesses of two neutralization models (the Neutralization.xls spreadsheet model versus the Brief Adversary Threat-Loss Estimator (BATLE) software); and demonstrating how computer simulations can be used to determine PN. Note that the computer simulation used for the demonstration was the Scenario Toolkit And Generation Environment (STAGE) simulation, which is a stand-alone synthetic tactical simulation sold by Presagis Canada Incorporated. The demonstration is provided in a separate Audio Video Interleave (.AVI) file.

  12. HER2 evaluation using the novel rabbit monoclonal antibody SP3 and CISH in tissue microarrays of invasive breast carcinomas.

    Science.gov (United States)

    Ricardo, Sara Alexandra Vinhas; Milanezi, Fernanda; Carvalho, Sílvia Teresa; Leitão, Dina Raquel Aguilera; Schmitt, Fernando Carlos Lander

    2007-09-01

    Laboratory methods for HER2 assessment currently include immunohistochemical (IHC) methods (measuring protein overexpression) and fluorescence in situ hybridisation (FISH) (measuring gene amplification). The measure of HER2 protein by IHC is usually assessed by the mouse monoclonal antibody CB11, and polyclonal antibodies (Herceptest) directed against the internal portion of the receptor. Recently, chromogenic in situ hybridisation (CISH), in which HER2 is detected by a peroxidase reaction and the gene amplification can be determined by regular bright-field microscopy, has emerged as an alternative to FISH. To evaluate the status of HER2 in tissue microarrays (TMAs) of invasive breast cancer using the novel rabbit monoclonal antibody SP3 directed against the external portion of HER2, and correlate the results with CB11 and CISH. IHC was performed with two antibodies (CB11 and SP3) and CISH for HER2 in 10 TMA blocks with 190 formalin-fixed paraffin-embedded cases of invasive breast carcinomas. The correlation between SP3 and CB11 was significant (pCISH (pCISH, shows that this novel antibody is a reliable candidate to evaluate the expression of HER2 in breast cancer.

  13. A four-step sandwich radioimmunoassay for direct selection of monoclonal antibodies to allergen molecules

    International Nuclear Information System (INIS)

    Ley, V.; Corbi, A.L.; Sanchez-Madrid, F.; Carreira, J.C.

    1985-01-01

    A 4-step radioimmunoassay has been devised for direct identification of monoclonal antibodies (MAb) directed to IgE-binding molecules. Polyvinyl chloride wells coated with purified anti-mouse kappa chain MAb (187-1) were successively incubated with: (1) MAb-containing hybridoma supernatants, (2) allergen extract, (3) allergic patients' serum pool, and (4) 125 I-labeled anti-human IgE antiserum, to detect MAb-allergen-IgE complexes. MAb to allergens from Parietaria judaica pollen and Dermatophagoides mites have been selected with this screening procedure. The affinity-purified allergen molecules competed the binding of IgE to allergen extracts coated to paper discs in a RAST inhibition assay, confirming the anti-allergen specificity of the selected MAb. This screening method is sensitive enough to allow detection of MAb directed to poorly represented allergens. (Auth.)

  14. IBC's 23rd Annual Antibody Engineering, 10th Annual Antibody Therapeutics international conferences and the 2012 Annual Meeting of The Antibody Society: December 3-6, 2012, San Diego, CA.

    Science.gov (United States)

    Klöhn, Peter-Christian; Wuellner, Ulrich; Zizlsperger, Nora; Zhou, Yu; Tavares, Daniel; Berger, Sven; Zettlitz, Kirstin A; Proetzel, Gabriele; Yong, May; Begent, Richard H J; Reichert, Janice M

    2013-01-01

    The 23rd Annual Antibody Engineering, 10th Annual Antibody Therapeutics international conferences, and the 2012 Annual Meeting of The Antibody Society, organized by IBC Life Sciences with contributions from The Antibody Society and two Scientific Advisory Boards, were held December 3-6, 2012 in San Diego, CA. The meeting drew over 800 participants who attended sessions on a wide variety of topics relevant to antibody research and development. As a prelude to the main events, a pre-conference workshop held on December 2, 2012 focused on intellectual property issues that impact antibody engineering. The Antibody Engineering Conference was composed of six sessions held December 3-5, 2012: (1) From Receptor Biology to Therapy; (2) Antibodies in a Complex Environment; (3) Antibody Targeted CNS Therapy: Beyond the Blood Brain Barrier; (4) Deep Sequencing in B Cell Biology and Antibody Libraries; (5) Systems Medicine in the Development of Antibody Therapies/Systematic Validation of Novel Antibody Targets; and (6) Antibody Activity and Animal Models. The Antibody Therapeutics conference comprised four sessions held December 4-5, 2012: (1) Clinical and Preclinical Updates of Antibody-Drug Conjugates; (2) Multifunctional Antibodies and Antibody Combinations: Clinical Focus; (3) Development Status of Immunomodulatory Therapeutic Antibodies; and (4) Modulating the Half-Life of Antibody Therapeutics. The Antibody Society's special session on applications for recording and sharing data based on GIATE was held on December 5, 2012, and the conferences concluded with two combined sessions on December 5-6, 2012: (1) Development Status of Early Stage Therapeutic Antibodies; and (2) Immunomodulatory Antibodies for Cancer Therapy.

  15. Tumor imaging with monoclonal antibodies

    International Nuclear Information System (INIS)

    Haisma, H.; Hilgers, J.

    1987-01-01

    Many monoclonal antibodies directed against tumor-associated antigens have been identified, but so far none of these are tumor specific. Polyclonal and monoclonal antibodies have been used for imaging of a wide variety of tumors with success. Radiolabeling of antibody is usually done with iodine isotopes of which 123 I is the best candidate for radioimmunodetection purposes. The labeling of antibodies through chelates makes it possible to use metal radioisotopes like 111 In, which is the best radioisotope for imaging with monoclonal antibodies due to its favorable half-life of 2.5 days. Usually imaging cannot be performed within 24 h after injection, but clearance of antibody can be increased by using F(ab) 2 of Fab. Another approach is to clear non-bound antibody by a second antibody, directed against the first. The detection limit of immunoimaging is about 2 cm, but will be improved by tomography or SPECT. There is still a high false positive and false negative rate, which makes it impossible to use radioimmunodetection as the only technique for diagnosis of tumors. In combination with other detection techniques, tumor imaging with monoclonal antibodies can improve diagnosis. 44 refs.; 3 tabs

  16. Direct Detection of Protein Biomarkers in Human Fluids Using Site-Specific Antibody Immobilization Strategies

    Directory of Open Access Journals (Sweden)

    Maria Soler

    2014-01-01

    Full Text Available Design of an optimal surface biofunctionalization still remains an important challenge for the application of biosensors in clinical practice and therapeutic follow-up. Optical biosensors offer real-time monitoring and highly sensitive label-free analysis, along with great potential to be transferred to portable devices. When applied in direct immunoassays, their analytical features depend strongly on the antibody immobilization strategy. A strategy for correct immobilization of antibodies based on the use of ProLinker™ has been evaluated and optimized in terms of sensitivity, selectivity, stability and reproducibility. Special effort has been focused on avoiding antibody manipulation, preventing nonspecific adsorption and obtaining a robust biosurface with regeneration capabilities. ProLinker™-based approach has demonstrated to fulfill those crucial requirements and, in combination with PEG-derivative compounds, has shown encouraging results for direct detection in biological fluids, such as pure urine or diluted serum. Furthermore, we have implemented the ProLinker™ strategy to a novel nanoplasmonic-based biosensor resulting in promising advantages for its application in clinical and biomedical diagnosis.

  17. Direct detection of protein biomarkers in human fluids using site-specific antibody immobilization strategies.

    Science.gov (United States)

    Soler, Maria; Estevez, M-Carmen; Alvarez, Mar; Otte, Marinus A; Sepulveda, Borja; Lechuga, Laura M

    2014-01-29

    Design of an optimal surface biofunctionalization still remains an important challenge for the application of biosensors in clinical practice and therapeutic follow-up. Optical biosensors offer real-time monitoring and highly sensitive label-free analysis, along with great potential to be transferred to portable devices. When applied in direct immunoassays, their analytical features depend strongly on the antibody immobilization strategy. A strategy for correct immobilization of antibodies based on the use of ProLinker™ has been evaluated and optimized in terms of sensitivity, selectivity, stability and reproducibility. Special effort has been focused on avoiding antibody manipulation, preventing nonspecific adsorption and obtaining a robust biosurface with regeneration capabilities. ProLinker™-based approach has demonstrated to fulfill those crucial requirements and, in combination with PEG-derivative compounds, has shown encouraging results for direct detection in biological fluids, such as pure urine or diluted serum. Furthermore, we have implemented the ProLinker™ strategy to a novel nanoplasmonic-based biosensor resulting in promising advantages for its application in clinical and biomedical diagnosis.

  18. Long-term clinical protection from falciparum malaria is strongly associated with IgG3 antibodies to merozoite surface protein 3.

    Directory of Open Access Journals (Sweden)

    Christian Roussilhon

    2007-11-01

    Full Text Available BACKGROUND: Surrogate markers of protective immunity to malaria in humans are needed to rationalize malaria vaccine discovery and development. In an effort to identify such markers, and thereby provide a clue to the complex equation malaria vaccine development is facing, we investigated the relationship between protection acquired through exposure in the field with naturally occurring immune responses (i.e., induced by the parasite to molecules that are considered as valuable vaccine candidates. METHODS AND FINDINGS: We analyzed, under comparative conditions, the antibody responses of each of six isotypes to five leading malaria vaccine candidates in relation to protection acquired by exposure to natural challenges in 217 of the 247 inhabitants of the African village of Dielmo, Senegal (96 children and 121 older adolescents and adults. The status of susceptibility or resistance to malaria was determined by active case detection performed daily by medical doctors over 6 y from a unique follow-up study of this village. Of the 30 immune responses measured, only one, antibodies of the IgG3 isotype directed to merozoite surface protein 3 (MSP3, was strongly associated with clinical protection against malaria in all age groups, i.e., independently of age. This immunological parameter had a higher statistical significance than the sickle cell trait, the strongest factor of protection known against Plasmodium falciparum. A single determination of antibody was significantly associated with the clinical outcome over six consecutive years in children submitted to massive natural parasite challenges by mosquitoes (over three parasite inoculations per week. Finally, the target epitopes of these antibodies were found to be fully conserved. CONCLUSIONS: Since anti-MSP3 IgG3 antibodies can naturally develop along with protection against P. falciparum infection in young children, our results provide the encouraging indication that these antibodies should be

  19. Onderzoek naar de ammoniak- en geuremissie van stallen LVI : scharrelstal voor leghennen met frequente mest- en strooiselverwijdering = Free range housing system for laying hens with frequent manure and litter removal

    NARCIS (Netherlands)

    Scheer, A.; Hol, J.G.M.; Mol, G.

    2002-01-01

    Door IMAG werd onderzoek verricht naar de ammoniak- en geuremissie van een scharrelstal voor leghennen met volledige ontmesting. Het ammoniakemissiereducerend principe berustte op het eenmaal per week verwijderen van de mest onder de beun en het strooisel in de stal. Hiervoor werden met behulp van

  20. Detection of antibodies in human serum using trimellityl-erythrocytes: direct and indirect haemagglutination and haemolysis.

    Science.gov (United States)

    Turner, E S; Pruzansky, J J; Patterson, R; Zeiss, C R; Roberts, M

    1980-02-01

    Utilizing trimellityl-erythrocytes (TM-E), antibodies were detected in sera of seven workers with trimellitic anhydride (TMA) induced airway syndromes by direct haemagglutination, indirect haemagglutination with anti-human IgG, IgA or IgM or by haemolysis. Detectable levels of antibody were obtained with all three methods. The most sensitive technique was indirect haemagglutination using anti-IgG. When added as an inhibitor, TM-human serum albumin produced a 10- to 800-fold reduction in titres. TM-ovalbumin of similar epitope density was less inhibitory and sodium trimellitate the least inhibitory on a molar basis. All of the assays using haptenized human red cells were also capable of detecting anti-TM antibodies in Rhesus monkeys whose airways had been exposed to TMA. These assays are useful for detecting anti-TM antibodies and may also be adapted to demonstrate antibodies induced against other inhaled haptens in sera of environmentally exposed individuals or in animal models of such exposure.

  1. CD3 directed bispecific antibodies induce increased lymphocyte-endothelial cell interactions in vitro

    NARCIS (Netherlands)

    Molema, G; Tervaert, JWC; Kroesen, BJ; Helfrich, W; Meijer, DKF; de Leij, LFMH

    Bispecific antibody (BsMAb) BIS-1 has been developed to redirect the cytolytic activity of cytotoxic T lymphocytes (CTL) to epithelial glycoprotein-2 (EGP-2) expressing tumour cells; intravenous administration of BIS-1 F(ab')(2) to carcinoma patients in a phase I/II clinical trial, caused

  2. Directed Selection of Recombinant Human Monoclonal Antibodies to Herpes Simplex Virus Glycoproteins from Phage Display Libraries

    Science.gov (United States)

    Sanna, Pietro Paolo; Williamson, R. Anthony; de Logu, Alessandro; Bloom, Floyd E.; Burton, Dennis R.

    1995-07-01

    Human monoclonal antibodies have considerable potential in the prophylaxis and treatment of viral disease. However, only a few such antibodies suitable for clinical use have been produced to date. We have previously shown that large panels of human recombinant monoclonal antibodies against a plethora of infectious agents, including herpes simplex virus types 1 and 2, can be established from phage display libraries. Here we demonstrate that facile cloning of recombinant Fab fragments against specific viral proteins in their native conformation can be accomplished by panning phage display libraries against viral glycoproteins "captured" from infected cell extracts by specific monoclonal antibodies immobilized on ELISA plates. We have tested this strategy by isolating six neutralizing recombinant antibodies specific for herpes simplex glycoprotein gD or gB, some of which are against conformationally sensitive epitopes. By using defined monoclonal antibodies for the antigen-capture step, this method can be used for the isolation of antibodies to specific regions and epitopes within the target viral protein. For instance, monoclonal antibodies to a nonneutralizing epitope can be used in the capture step to clone antibodies to neutralizing epitopes, or antibodies to a neutralizing epitope can be used to clone antibodies to a different neutralizing epitope. Furthermore, by using capturing antibodies to more immunodominant epitopes, one can direct the cloning to less immunogenic ones. This method should be of value in generating antibodies to be used both in the prophylaxis and treatment of viral infections and in the characterization of the mechanisms of antibody protective actions at the molecular level.

  3. Prihodnja osvetlitev in nočna podoba mest: študija primera

    Directory of Open Access Journals (Sweden)

    Simon Rankel

    2014-06-01

    Full Text Available Prihodnja osvetlitev na podlagi polprevodniških virov svetlobe bo spremenila nočno podobo mest. Možnosti njihove umestitve in uporabe v urbanem prostoru so številne. V članku zato proučujemo, ali bi njihova prihodnja uporaba v splošni zunanji razsvetljavi pozitivno vplivala na človekovo sprejemanje tako osvetljenega urbanega prostora. Na podlagi inovativne uporabe omenjenih virov svetlobe smo izoblikovali dva različna koncepta osvetlitve. Prvi koncept temelji na ploskovni osvetlitvi, drugi pa na detajlnem prikazu arhitekturno–urbanih elementov. Predlogi za osvetlitev so bili narejeni na petih različnih lokacijah mesta. Poleg izpolnjevanja funkcionalnih potreb upoštevajo tudi urbanistični in arhitekturni vidik osvetljevanja. Z ocenami afektivne kakovosti slikovnih prikazov smo predstavili in proučevali temeljna afektivna stanja anketiranih. Ugotovitve kažejo, da so se anketiranci pri večini predlogov strinjali pri ocenah spremenljivk »prijetnost« in »pozornost. Statistika pokaže med konceptoma značilne razlike pri ocenah obeh spremenljivk. Pri neposredni primerjavi obeh konceptov smo ugotovili, da je naklonjenost anketirancev do določenega osvetlitvenega koncepta odvisna tudi od mestne lokacije. S člankom želimo poudariti pomen nočne podobe mesta pri razvoju prihodnjih strategij osvetlitve, ki temeljijo na polprevodniških virih svetlobe.

  4. Induction of albuminuria in mice: synergistic effect of two monoclonal antibodies directed to different domains of aminopeptidase A.

    Science.gov (United States)

    Mentzel, S; van Son, J P; Dijkman, H B; Wetzels, J F; Assmann, K J

    1999-04-01

    Aminopeptidase A is an enzyme that is present on podocytes and is involved in the degradation of angiotensin II. In previous studies in mice, we administered single monoclonal antibodies directed against aminopeptidase A. We observed that only monoclonal antibodies that inhibited aminopeptidase A enzyme activity caused albuminuria. In this study, the effects of the combined injections of two monoclonal anti-aminopeptidase A antibodies (mAbs) were studied, using a combination of anti-aminopeptidase A mAbs that were directed against two different domains involved in the aminopeptidase A enzyme activity (ASD-3 or ASD-37) and an anti-aminopeptidase A mAb not related to the enzyme active site (ASD-41). An injection of the combinations ASD-3/37 (total 4 mg, 1:1 ratio) and ASD-37/41 (total 4 mg, 1:1 ratio) in doses that do not cause albuminuria when given alone (4 mg) induced massive albuminuria at day 1 after injection. The combination ASD-3/41 had no effect. This albuminuria was not dependent on systemic immune mediators of inflammation and could not merely be related to a blockade of aminopeptidase A enzyme activity. However, a correlation was observed between the induction of albuminuria and the aggregation of the mAbs injected and aminopeptidase A on the podocytes. An injection of the combinations ASD-3/37 or ASD-37/41 did not cause an increase in systemic blood pressure. The treatment with a combination of enalapril and losartan lowered blood pressure (53 +/- 10 vs. 90 +/- 3 mm Hg in untreated mice) and reduced the acute albuminuria by 55% (11,145 +/- 864 vs. 24,517 +/- 2448 micrograms albumin/18 hr in untreated mice). However, similar effects were observed using triple therapy. Therefore, the reduction of albuminuria by the combined treatment of enalapril/losartan seems to be the consequence of the reduction in the systemic blood pressure. These findings argue against a specific role for angiotensin II in this model. The combined injection of two mAbs directed

  5. Tc-99m direct radiolabeling of monoclonal antibody ior egf/r3: quality control and image studies in mice

    International Nuclear Information System (INIS)

    Dias, Carla Roberta; Marczewski, Barbara; Moraes, Vanessa; Barboza, Marycel Figols de; Osso Junior, Joao Alberto

    2005-01-01

    Monoclonal antibodies (Mabs) have been useful for immunoscintigraphic applications in clinical diagnosis since they were introduced in the practice of nuclear medicine. The ior egf/r3 (Centis, Cuba) is a murine monoclonal antibody against epidermal growth factor receptor (EGF-R) and has been widely used in the radioimmunodiagnosis of tumors of epithelial origin. Labeled with 99m Tc, its main application in Nuclear Medicine is the follow up, detection and evaluation of tumor recurrences. The objective of this work is to describe the preparation of a lyophilized formulation (kit) for radiolabeling the Mab ior egf/r3 with 99m Tc for immunoscintigraphic applications. Radiolabeling efficiency, effects on immunoreactivity, image studies and stability of the formulation are reported. The study demonstrated that the kit formulation can be labeled with 99m Tc at high yields and can be used to visualize in vivo human tumors of epithelial origin by immunoscintigraphy studies.(author)

  6. Evaluation of a direct immunofluorescent antibody (difma test using Leishmania genus - specific monoclonal antibody in the routine diagnosis of cutaneous leishmaniasis

    Directory of Open Access Journals (Sweden)

    Martha E. Chico

    1995-06-01

    Full Text Available A direct immunofluorescent antibody (DIFMA test using a Leishmania genus- specific monoclonal antibody was evaluated in the routine diagnosis of cutaneous leishmaniasis (CL in Ecuador. This test was compared with the standard diagnostic techniques of scrapings, culture and histology. Diagnostic samples were taken from a total of 90 active dermal ulcers from patients from areas of Ecuador known to be endemic for cutaneous leishmaniasis. DIFMA was positive in all lesions. It was shown to be significantly superior to standard diagnostic methods either alone or in combination. The sensitivity of DIFMA did not diminish with chronicity of lesions. This test proved to be extremely useful in the routine diagnosis of CL because it is highly sensitive, is easy to use and produces rapid results.

  7. The Impact of Therapeutic Antibodies on the Management of Digestive Diseases: History, Current Practice, and Future Directions.

    Science.gov (United States)

    Sofia, M Anthony; Rubin, David T

    2017-04-01

    The development of therapeutic antibodies represents a revolutionary change in medical therapy for digestive diseases. Beginning with the initial studies that confirmed the pathogenicity of cytokines in inflammatory bowel disease, the development and application of therapeutic antibodies brought challenges and insights into their potential and optimal use. Infliximab was the first biological drug approved for use in Crohn's disease and ulcerative colitis. The lessons learned from infliximab include the importance of immunogenicity and the influence of pharmacokinetics on disease response and outcomes. Building on this foundation, other therapeutic antibodies achieved approval for inflammatory bowel disease and many more are in development for several digestive diseases. In this review, we reflect on the history of therapeutic antibodies and discuss current practice and future directions for the field.

  8. Tc-{sup 99m} direct radiolabeling of monoclonal antibody ior egf/r3: quality control and image studies in mice

    Energy Technology Data Exchange (ETDEWEB)

    Dias, Carla Roberta; Marczewski, Barbara; Moraes, Vanessa; Barboza, Marycel Figols de; Osso Junior, Joao Alberto [Instituto de Pesquisas Energeticas e Nucleares (IPEN-CNEN/SP), SP (Brazil). Centro de Radiofarmacia]. E-mail: crdias@ipen.br

    2005-10-15

    Monoclonal antibodies (Mabs) have been useful for immunoscintigraphic applications in clinical diagnosis since they were introduced in the practice of nuclear medicine. The ior egf/r3 (Centis, Cuba) is a murine monoclonal antibody against epidermal growth factor receptor (EGF-R) and has been widely used in the radioimmunodiagnosis of tumors of epithelial origin. Labeled with 99m Tc, its main application in Nuclear Medicine is the follow up, detection and evaluation of tumor recurrences. The objective of this work is to describe the preparation of a lyophilized formulation (kit) for radiolabeling the Mab ior egf/r3 with 99m Tc for immunoscintigraphic applications. Radiolabeling efficiency, effects on immunoreactivity, image studies and stability of the formulation are reported. The study demonstrated that the kit formulation can be labeled with 99m Tc at high yields and can be used to visualize in vivo human tumors of epithelial origin by immunoscintigraphy studies.(author)

  9. Docking of Antibodies into Cavities in DNA Origami

    DEFF Research Database (Denmark)

    Quyang, X; Stefano, Mattia De; Krissanaprasit, Abhichart

    2017-01-01

    -selective immobilization of antibodies in designed cavities in 2D and 3D DNA origami structures. Two tris(NTA) modified strands are inserted into the cavity to form NTA-metal complexes with histidine clusters on the Fc domain. Subsequent covalent linkage to the antibody was achieved by coupling to lysines. Atomic force...... microscopy (AFM) and transmission electron microscopy (TEM) validated efficient antibody immobilization in the origami structures. The increased ability to control the orientation of antibodies in nanostructures and at surfaces has potential for directing the interactions of antibodies with targets...

  10. [Study of anti-idiotype antibodies to human monoclonal antibody].

    Science.gov (United States)

    Harada, R; Takahashi, N; Owaki, I; Kannagi, R; Endo, N; Morita, N; Inoue, M

    1992-02-01

    A human monoclonal antibody, ll-50 (IgM, lambda), was generated, which reacted specifically with a major of glycolipid present in LS174T colon cancer cells. The glycolipid antigen which reacted with the ll-50 antibody was expected to four sugar residues from its TLC mobility, and it was ascertained that the glycolipid antigen which reacted with ll-50 antibody might be Lc4 antigen [Gal beta 1----3 GLcNAc beta 1----3 Gal beta 1----4 Glc beta 1----1 Cer] judging from TLC immunostaining and ELISA when the reactivity of ll-50 antibody was tested using various pure glycolipids in 3-5 sugar residues as an antigen. Sera in patients with malignant disorders and healthy individuals were analyzed by Sandwich assay of immobilized and biotinylated ll-50 antibody. The serum of the Lc4 antigen recognized by ll-50 antibody was significantly higher in patients with malignant disorders than that in healthy individuals (p less than 0.05). Three mouse monoclonal anti-idiotype antibodies, G3, B3 and C5 (all IgG1), were generated by the immunization of BALB/c mice with ll-50 antibody. These anti-idiotype antibodies specifically bound to to human monoclonal antibody, ll-50 and had a significant inhibitory activity towards the binding of ll-50 antibody to the Lc4 antigen. This indicated that these anti-idiotype antibodies, G3, B3, and C5, were paratope-related anti-idiotype antibodies. G3, B3, and C5 were expected to define the nearest idiotope because they could mutually inhibit ll-50 antibody. Sera in patients with malignant disorders and healthy individuals were analyzed by Sandwich assay of immobilized and biotinylated anti-idiotype antibodies, G3, B3, and C5. As to the ll-50 like antibodies defined by C5 (Id-C5+), the mean serum level in patients with malignant disorders was significantly higher than that in healthy individuals (p less than 0.05). As to the ll-50 like antibodies defined by B3 (Id-B3+), the mean serum level in patients with malignant disorders was significantly higher

  11. [Preparation and characterization of mouse polyclonal antibody against conserved region of human FOXO3].

    Science.gov (United States)

    Li, Lei; Lyu, Dan

    2017-06-01

    Objective To purify the recombinant protein specific to conserved region of forkhead box O3 (FOXO3) and prepare mouse anti-human FOXO3 polyclonal antibody. Methods The DNA fragment (aa290-472) encoding conserved domain of FOXO3 was amplified by PCR, and subsequently cloned into pET28a vector. Following transformation into E.coli BL21, the soluble fusion protein His-FOXO3 was induced by IPTG and purified by Ni-NTA affinity chromatography. The purified protein was used to immunize BALB/c mice to generate polyclonal antibody. The characteristics of the polyclonal antibody were assessed by ELISA, Western blotting and immunoprecipitation assays. Results We successfully prepared the expression vector pET28a-FOXO3 (aa290-472) and expressed the purified fusion protein in a soluble form. By immunizing mice with the fusion protein, we obtained anti-human FOXO3 polyclonal antibody. ELISA and Western blotting showed that the mouse antibody could recognize specifically the endogenous FOXO3 protein. Conclusion The polyclonal antibody against conserved domain of FOXO3 can identify the endogenous FOXO3 protein. It can be used to analyze the endogenous FOXO3 expression level.

  12. A simplified radioimmunoassay for triiodothyronine (T3) using pre-incubated labelled antigen and antibody

    International Nuclear Information System (INIS)

    Pillai, M.R.A.; Nagvekar, U.H.; Desai, C.N.; Mani, R.S.

    1986-01-01

    The development of a simplified radioimmunoassay for triiodothyronine (T 3 ) using pre-incubated labelled T 3 and antibody is described. The assay is carried out by adding 50 μl of standard or sample to 0.4 ml of pre-incubated reagent dispensed in assay tubes. The reaction was allowed to proceed for about four hours and the antigen-antibody complex precipitated by the addition of 1 cm 3 of 22% polyethylene glycol solution. Due to the high dissociation constant of T 3 -antibody complex at 37 deg C, the labelled antigen-antibody complex dissociates and thereby the unlabelled antigen binds with the antibody. The sensitivity of this assay is comparable to an assay done by the equilibrium method using the same antibody. Sixty serum samples were analyzed using this method and were compared with the equilibrium assay. (author)

  13. In vitro stability of EDTA and DTPA immunoconjugates of monoclonal antibody 2G3 labeled with indium-111

    Energy Technology Data Exchange (ETDEWEB)

    Reilly, R.; Lee, N.; Houle, S. (The Toronto Hospital (Canada)); Law, J.; Marks, A. (Toronto Univ., ON (Canada))

    1992-08-01

    Monoclonal antibody 2G3 directed against a high molecular weight glycoprotein on breast and ovarian cancer cells was conjugated with bicyclic DTPA (or EDTA) anhydride or benzyl isothiocyanate DTPA (benzyl DTPA) and labeled with {sup 111}In. DTPA anhydride was more reactive with the antibody than benzyl DTPA, and kinetics of labeling with {sup 111}In were more rapid for DTPA substituted 2G3 than for benzyl DTPA substituted 2G3. On the other hand, {sup 111}In-2G3 conjugates prepared using DTPA anhydride were subject to more extensive dimerization and higher losses in immunoreactivity than those prepared using benzyl DTPA. On the basis of measurement of transchelation to transferrin, the stability of {sup 111}In-2G3 prepared using DTPA anhydride or benzyl DTPA did not differ during incubation in human plasma for 6 days at 37{sup o}C. These results suggest that an important advantage of benzyl DTPA over DTPA anhydride for preparing {sup 111}In-labeled antibodies is the prevention of intermolecular (and intramolecular) crosslinking during conjugation which ultimately leads to alterations in conformation and losses in immunoreactivity of the radioimmunoconjugate. (author).

  14. Regional and systemic distribution of anti-tumor x anti-CD3 heteroaggregate antibodies and cultured human peripheral blood lymphocytes in a human colon cancer xenograft

    International Nuclear Information System (INIS)

    Nelson, H.; Ramsey, P.S.; Kerr, L.A.; McKean, D.J.; Donohue, J.H.

    1990-01-01

    Anti-tumor antibody (317G5) covalently coupled to an anti-CD3 antibody (OKT3) produces a heteroaggregate (HA) antibody that can target PBL to lyse tumor cells expressing the appropriate tumor Ag. The i.v. and i.p. distribution of radiolabeled HA antibody 317G5 x OKT3 and of radiolabeled cultured human PBL were studied in athymic nude mice bearing solid intraperitoneal tumor established from the human colon tumor line, LS174T. Mice were injected with 125I-labeled HA antibody, 125I-labeled anti-tumor mAb, or 111In-labeled PBL, and at designated timepoints tissues were harvested and measured for radioactivity. 125I-317G5 x OKT3 localized specifically to tumor sites. Tumor radioactivity levels (percent injected dose/gram) were lower with 125I-317G5 x OKT3 HA antibody than with 125I-317G5 anti-tumor mAb, but were similar to levels reported for other anti-tumor mAb. The major difference in radioactivity levels observed between i.v. and i.p. administration of 125I-317G5 x OKT3 was an increase in hepatic radioactivity after i.v. HA antibody administration. HA antibodies produced from F(ab')2 fragments, which exhibit decreased m. w. and decreased Fc receptor-mediated binding, demonstrated improved tumor:tissue ratios as compared to intact antibody HA. 125I-317G5 F(ab')2 x OKT3 F(ab')2 antibody levels were equivalent to intact HA antibody levels in tumor, but were lower than intact HA antibody levels in the blood, bowel, and liver. Tumor:bowel ratios (20:1 at 48 h) were highest when 317G5 F(ab')2 x OKT3 F(ab')2 was injected i.p. Autoradiography confirmed that anti-tumor x anti-CD3 HA antibodies localized specifically to intraperitoneal tumor; that i.p. administered HA antibodies penetrated tumor directly; and that i.v. administered HA antibodies distributed along tumor vasculature

  15. Effect of kinase inhibitors on the therapeutic properties of monoclonal antibodies.

    Science.gov (United States)

    Duong, Minh Ngoc; Matera, Eva-Laure; Mathé, Doriane; Evesque, Anne; Valsesia-Wittmann, Sandrine; Clémenceau, Béatrice; Dumontet, Charles

    2015-01-01

    Targeted therapies of malignancies currently consist of therapeutic monoclonal antibodies and small molecule kinase inhibitors. The combination of these novel agents raises the issue of potential antagonisms. We evaluated the potential effect of 4 kinase inhibitors, including the Bruton tyrosine kinase inhibitor ibrutinib, and 3 PI3K inhibitors idelalisib, NVP-BEZ235 and LY294002, on the effects of the 3 monoclonal antibodies, rituximab and obinutuzumab (directed against CD20) and trastuzumab (directed against HER2). We found that ibrutinib potently inhibits antibody-dependent cell-mediated cytotoxicity exerted by all antibodies, with a 50% inhibitory concentration of 0.2 microM for trastuzumab, 0.5 microM for rituximab and 2 microM for obinutuzumab, suggesting a lesser effect in combination with obinutuzumab than with rituximab. The 4 kinase inhibitors were found to inhibit phagocytosis by fresh human neutrophils, as well as antibody-dependent cellular phagocytosis induced by the 3 antibodies. Conversely co-administration of ibrutinib with rituximab, obinutuzumab or trastuzumab did not demonstrate any inhibitory effect of ibrutinib in vivo in murine xenograft models. In conclusion, some kinase inhibitors, in particular, ibrutinib, are likely to exert inhibitory effects on innate immune cells. However, these effects do not compromise the antitumor activity of monoclonal antibodies in vivo in the models that were evaluated.

  16. Anti-pentraxin 3 auto-antibodies might be protective in lupus nephritis: a large cohort study.

    Science.gov (United States)

    Yuan, Mo; Tan, Ying; Pang, Yun; Li, Yong-Zhe; Song, Yan; Yu, Feng; Zhao, Ming-Hui

    2017-11-01

    Anti-pentraxin 3 (PTX3) auto-antibodies were found to be associated with the absence of renal involvement in systemic lupus erythematosus (SLE). This study is to investigate the prevalence of anti-PTX3 auto-antibodies and their clinical significance based on a large Chinese lupus nephritis cohort. One hundred and ninety-six active lupus nephritis patients, 150 SLE patients without clinical renal involvement, and 100 healthy controls were enrolled. Serum anti-PTX3 auto-antibodies and PTX3 levels were screened by enzyme-linked immunosorbent assay (ELISA). The associations between anti-PTX3 auto-antibodies and clinicopathological parameters in lupus nephritis were further analyzed. Anti-PTX3 auto-antibodies were less prevalent in active lupus nephritis patients compared with SLE without renal involvement (19.4% (38/196) versus 40.7% (61/150), p auto-antibodies were negatively correlated with proteinuria in lupus nephritis (r = -.143, p = .047). The levels of proteinuria, serum creatinine, and the prevalence of thrombotic microangiopathy were significantly higher in patients with higher PTX3 levels (≥3.207 ng/ml) and without anti-PTX3 auto-antibodies compared with patients with lower PTX3 levels (auto-antibodies (4.79 (3.39-8.28) versus 3.95 (1.78-7.0), p = .03; 168.84 ± 153.63 versus 101.44 ± 47.36, p = .01; 34.1% (14/41) versus 0% (0/9), p = .04; respectively). Anti-PTX3 auto-antibodies were less prevalent in active lupus nephritis patients compared with SLE without renal involvement and associated with less severe renal damage, especially with the combined evaluation of serum PTX3 levels.

  17. A novel antihuman C3d monoclonal antibody with specificity to the C3d complement split product

    DEFF Research Database (Denmark)

    Rasmussen, Karina Juhl; Skjødt, Mikkel-Ole; Vitved, Lars

    2017-01-01

    The complement component C3 and the cleavage products of C3b/iC3b, C3c and C3d are used as biomarkers in clinical diagnostics. Currently, no specific antibodies are able to differentiate C3d from other fragments, although such a distinction could be very valuable considering that they may reflect...... different pathophysiological mechanisms. We have developed a rat antihuman C3d monoclonal antibody with specificity to the end sequence of the N-terminal region of C3d. The antibody can therefore only bind to C3d when it manifests itself as the final end product of cleaved C3. We believe...

  18. Comparison of (/sup 125/I)beta-endorphin binding to rat brain and NG108-15 cells using a monoclonal antibody directed against the opioid receptor

    Energy Technology Data Exchange (ETDEWEB)

    Bidlack, J.M.; O' Malley, W.E.; Schulz, R.

    1988-02-01

    The properties of (/sup 125/I)beta h-endorphin-binding sites from rat brain membranes and membranes from the NG108-15 cell line were compared using a monoclonal antibody directed against the opioid receptor and opioid peptides as probes. The binding of (/sup 125/I)beta h-endorphin to both rat brain and NG108-15 membranes yielded linear Scatchard plots with Kd values of 1.2 nM and 1.5 nM, respectively, and Bmax values of 865 fmol/mg rat brain membrane protein and 1077 fmol/mg NG108-15 membrane protein. A monoclonal antibody, OR-689.2.4, capable of inhibiting mu and delta binding but not kappa binding to rat brain membranes, noncompetitively inhibited the binding of 1 nM (/sup 125/I)beta h-endorphin to rat brain and NG108-15 membranes with an IC50 value of 405 nM for rat brain membranes and 543 nM for NG108-15 membranes. The monoclonal antibody also inhibited the binding of 3 nM (/sup 3/H) (D-penicillamine2, D-penicillamine5) enkephalin to NG108-15 membranes with an IC50 value of 370 nM. In addition to blocking the binding of (/sup 125/I)beta h-endorphin to brain membranes, the antibody also displaced (/sup 125/I)beta h-endorphin from membranes. Site-specific opioid peptides had large variations in their IC50 values depending on whether they were inhibiting (/sup 125/I)beta h-endorphin binding to rat brain or the NG108-15 membranes. When the peptides were tested with the monoclonal antibody for their combined ability to inhibit (/sup 125/I)beta h-endorphin binding to both membrane preparations, the peptides and antibody blocked binding as though they were acting at allosterically coupled sites, not two totally independent sites. These studies suggest that mu-, delta-, and beta-endorphin-binding sites share some sequence homology with the 35,000-dalton protein that the antibody is directed against.

  19. Anti-SEMA3A Antibody: A Novel Therapeutic Agent to Suppress GBM Tumor Growth.

    Science.gov (United States)

    Lee, Jaehyun; Shin, Yong Jae; Lee, Kyoungmin; Cho, Hee Jin; Sa, Jason K; Lee, Sang-Yun; Kim, Seok-Hyung; Lee, Jeongwu; Yoon, Yeup; Nam, Do-Hyun

    2017-11-10

    Glioblastoma (GBM) is classified as one of the most aggressive and lethal brain tumor. Great strides have been made in understanding the genomic and molecular underpinnings of GBM, which translated into development of new therapeutic approaches to combat such deadly disease. However, there are only few therapeutic agents that can effectively inhibit GBM invasion in a clinical framework. In an effort to address such challenges, we have generated anti-SEMA3A monoclonal antibody as a potential therapeutic antibody against GBM progression. We employed public glioma datasets, Repository of Molecular Brain Neoplasia Data and The Cancer Genome Atlas, to analyze SEMA3A mRNA expression in human GBM specimens. We also evaluated for protein expression level of SEMA3A via tissue microarray (TMA) analysis. Cell migration and proliferation kinetics were assessed in various GBM patient-derived cells (PDCs) and U87-MG cell-line for SEMA3A antibody efficacy. GBM patient-derived xenograft (PDX) models were generated to evaluate tumor inhibitory effect of anti-SEMA3A antibody in vivo. By combining bioinformatics and TMA analysis, we discovered that SEMA3A is highly expressed in human GBM specimens compared to non-neoplastic tissues. We developed three different anti-SEMA3A antibodies, in fully human IgG form, through screening phage-displayed synthetic antibody library using a classical panning method. Neutralization of SEMA3A significantly reduced migration and proliferation capabilities of PDCs and U87-MG cell-line in vitro. In PDX models, treatment with anti-SEMA3A antibody exhibited notable tumor inhibitory effect through down-regulation of cellular proliferative kinetics and tumor-associated macrophages recruitment. In present study, we demonstrated tumor inhibitory effect of SEMA3A antibody in GBM progression and present its potential relevance as a therapeutic agent in a clinical framework.

  20. Strong antitumor activities of IgG3 antibodies to a human melanoma-associated ganglioside

    International Nuclear Information System (INIS)

    Hellstroem, I.; Brankovan, V.; Hellstroem, K.E.

    1985-01-01

    Three mouse monoclonal IgG3 antibodies, 2B2, IF4, and MG-21, recognize a G/sub D3/ ganglioside antigen that is expressed at the cell surface of most human melanomas. All three antibodies mediate antibody-dependent cellular cytotoxicity (ADCC) in vitro when tested with human lymphocytes or effector cells in a 2-hr or 4-hr 51 Cr-release test, and one antibody, MG-21, also gives strong complement-dependent cytotoxicity with human serum. Antibody 2B2, which gives ADDC also in the presence of mouse lymphocytes, inhibited the outgrowth of a human melanoma in nude mice, but antibody IF4, which showed no ADCC with mouse lymphocyte effectors, did not

  1. Resistance of Subtype C HIV-1 Strains to Anti-V3 Loop Antibodies

    Directory of Open Access Journals (Sweden)

    David Almond

    2012-01-01

    Full Text Available HIV-1’s subtype C V3 loop consensus sequence exhibits increased resistance to anti-V3 antibody-mediated neutralization as compared to the subtype B consensus sequence. The dynamic 3D structure of the consensus C V3 loop crown, visualized by ab initio folding, suggested that the resistance derives from structural rigidity and non-β-strand secondary protein structure in the N-terminal strand of the β-hairpin of the V3 loop crown, which is where most known anti-V3 loop antibodies bind. The observation of either rigidity or non-β-strand structure in this region correlated with observed resistance to antibody-mediated neutralization in a series of chimeric pseudovirus (psV mutants. The results suggest the presence of an epitope-independent, neutralization-relevant structural difference in the antibody-targeted region of the V3 loop crown between subtype C and subtype B, a difference that we hypothesize may contribute to the divergent pattern of global spread between these subtypes. As antibodies to a variable loop were recently identified as an inverse correlate of risk for HIV infection, the structure-function relationships discussed in this study may have relevance to HIV vaccine research.

  2. Novel anti-HER2 monoclonal antibodies: synergy and antagonism with tumor necrosis factor-α

    Directory of Open Access Journals (Sweden)

    Ceran Ceyhan

    2012-10-01

    Full Text Available Abstract Background One-third of breast cancers display amplifications of the ERBB2 gene encoding the HER2 kinase receptor. Trastuzumab, a humanized antibody directed against an epitope on subdomain IV of the extracellular domain of HER2 is used for therapy of HER2-overexpressing mammary tumors. However, many tumors are either natively resistant or acquire resistance against Trastuzumab. Antibodies directed to different epitopes on the extracellular domain of HER2 are promising candidates for replacement or combinatorial therapy. For example, Pertuzumab that binds to subdomain II of HER2 extracellular domain and inhibits receptor dimerization is under clinical trial. Alternative antibodies directed to novel HER2 epitopes may serve as additional tools for breast cancer therapy. Our aim was to generate novel anti-HER2 monoclonal antibodies inhibiting the growth of breast cancer cells, either alone or in combination with tumor necrosis factor-α (TNF-α. Methods Mice were immunized against SK-BR-3 cells and recombinant HER2 extracellular domain protein to produce monoclonal antibodies. Anti-HER2 antibodies were characterized with breast cancer cell lines using immunofluorescence, flow cytometry, immunoprecipitation, western blot techniques. Antibody epitopes were localized using plasmids encoding recombinant HER2 protein variants. Antibodies, either alone or in combination with TNF-α, were tested for their effects on breast cancer cell proliferation. Results We produced five new anti-HER2 monoclonal antibodies, all directed against conformational epitope or epitopes restricted to the native form of the extracellular domain. When tested alone, some antibodies inhibited modestly but significantly the growth of SK-BR-3, BT-474 and MDA-MB-361 cells displaying ERBB2 amplification. They had no detectable effect on MCF-7 and T47D cells lacking ERBB2 amplification. When tested in combination with TNF-α, antibodies acted synergistically on SK-BR-3 cells

  3. Routinely used immunoassays do not detect circulating anti-GBM antibodies against native NC1 hexamer and EA epitope of the α3 chain of type IV collagen.

    Science.gov (United States)

    Clavarino, Giovanna; Gauthier, Arnaud; Hellmark, Thomas; Carron, Pierre-Louis; Giovannini, Diane; Colliard, Sophie; Dragon-Durey, Marie-Agnès; Segelmark, Mårten; Cesbron, Jean-Yves; Dumestre-Pérard, Chantal

    2018-04-12

    Detection of circulating anti-GBM antibodies has a key role for the diagnosis of Goodpasture syndrome but immunoassays using purified or recombinant alpha3(IV)NC1 as antigen do not recognize all anti-GBM antibodies. We show that anti-GBM antibodies directed against epitopes in their native conformation or cryptic epitopes are detected by indirect immunofluorescence. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Antibody mimetics: promising complementary agents to animal-sourced antibodies.

    Science.gov (United States)

    Baloch, Abdul Rasheed; Baloch, Abdul Wahid; Sutton, Brian J; Zhang, Xiaoying

    2016-01-01

    Despite their wide use as therapeutic, diagnostic and detection agents, the limitations of polyclonal and monoclonal antibodies have inspired scientists to design the next generation biomedical agents, so-called antibody mimetics that offer many advantages over conventional antibodies. Antibody mimetics can be constructed by protein-directed evolution or fusion of complementarity-determining regions through intervening framework regions. Substantial progress in exploiting human, butterfly (Pieris brassicae) and bacterial systems to design and select mimetics using display technologies has been made in the past 10 years, and one of these mimetics [Kalbitor® (Dyax)] has made its way to market. Many challenges lie ahead to develop mimetics for various biomedical applications, especially those for which conventional antibodies are ineffective, and this review describes the current characteristics, construction and applications of antibody mimetics compared to animal-sourced antibodies. The possible limitations of mimetics and future perspectives are also discussed.

  5. Many Routes to an Antibody Heavy-Chain CDR3: Necessary, Yet Insufficient, for Specific Binding

    Science.gov (United States)

    D’Angelo, Sara; Ferrara, Fortunato; Naranjo, Leslie; Erasmus, M. Frank; Hraber, Peter; Bradbury, Andrew R. M.

    2018-01-01

    Because of its great potential for diversity, the immunoglobulin heavy-chain complementarity-determining region 3 (HCDR3) is taken as an antibody molecule’s most important component in conferring binding activity and specificity. For this reason, HCDR3s have been used as unique identifiers to investigate adaptive immune responses in vivo and to characterize in vitro selection outputs where display systems were employed. Here, we show that many different HCDR3s can be identified within a target-specific antibody population after in vitro selection. For each identified HCDR3, a number of different antibodies bearing differences elsewhere can be found. In such selected populations, all antibodies with the same HCDR3 recognize the target, albeit at different affinities. In contrast, within unselected populations, the majority of antibodies with the same HCDR3 sequence do not bind the target. In one HCDR3 examined in depth, all target-specific antibodies were derived from the same VDJ rearrangement, while non-binding antibodies with the same HCDR3 were derived from many different V and D gene rearrangements. Careful examination of previously published in vivo datasets reveals that HCDR3s shared between, and within, different individuals can also originate from rearrangements of different V and D genes, with up to 26 different rearrangements yielding the same identical HCDR3 sequence. On the basis of these observations, we conclude that the same HCDR3 can be generated by many different rearrangements, but that specific target binding is an outcome of unique rearrangements and VL pairing: the HCDR3 is necessary, albeit insufficient, for specific antibody binding. PMID:29568296

  6. Antibody conjugate radioimmunotherapy of superficial bladder cancer

    International Nuclear Information System (INIS)

    Perkins, Alan; Hopper, Melanie; Murray, Andrea; Frier, Malcolm; Bishop, Mike

    2002-01-01

    The administration of antibody conjugates for cancer therapy is now proving to be of clinical value. We are currently undertaking a programme of clinical studies using the monoclonal antibody C 595 (gG3) which reacts with the MUC1 glycoprotein antigen that is aberrantly expressed in a high proportion of bladder tumours. Radio immuno conjugates of the C 595 antibody have been produced with high radiolabelling efficiency and immuno reactivity using Tc-99 m and In-111 for diagnostic imaging, and disease staging and the cytotoxic radionuclides Cu-67 and Re-188 for therapy of superficial bladder cancer. A Phase I/II therapeutic trail involving the intravesical administration of antibody directly into the bladder has now begun. (author)

  7. A novel anti-GPC3 monoclonal antibody (YP7) | Center for Cancer Research

    Science.gov (United States)

    Glypican-3 (GPC3) is an emerging therapeutic target in hepatoma. A novel anti-GPC3 monoclonal antibody (YP7) has been generated through a combination of peptide immunization and high-throughput flow cytometry screening. YP7 binds cell-surface-associated GPC3 with high affinity and exhibits significant hepatoma xenograft growth inhibition in nude mice. The new antibody may have

  8. Direct labelling of monoclonal antibodies with 99Tcm. Assessment of labelling, stability, immunoreactivity and biodistribution

    International Nuclear Information System (INIS)

    Janoki, G.A.

    1998-01-01

    Reduction of disulfide bonds to sulfhydryl groups for direct radiolabelling of monoclonal antibodies for immunoscintigraphic application continues to be of significant interest. Reducing agents that have been used are the following: stannous ion, 2-mercaptoethanol, dithiothreitol, dithioerythriol, and ascorbic acid. The radiolabelling of the reduced and purified antibody is performed via Sn 2+ reduction of pertechnetate in the presence of an excess of a low-affinity chelating ligand. In a recent work the 2-mercaptoethanol (2-ME) reduction based method was studied by using different analytical and biological techniques. Human IgG (Sandoglobulin), anti-CEA MoAb (ior-1), and anti-granulocyte MoAb (MAK 47), were reduced with 2-ME at two different molar ratios. To determine the amount of contaminating mercaptoethanol which may have survived the gel-filtration step 14 C-ME was used. The number of the free endogenous sulfhydryl groups generated by reduction was determined by Ellman's reagent; absorbance was measured at 412 nm. Within the quality assurance procedure of the 3 freeze dried kits the labelling efficiency, stability, pH, sterility, apyrogenicity, vial yield, syringe retention, filterable activity, free SH determination and animal distribution were studied again. After receiving permission from local ethics committee pilot human studies were initiated. Study protocols were also approved

  9. An automated robotic platform for rapid profiling oligosaccharide analysis of monoclonal antibodies directly from cell culture.

    Science.gov (United States)

    Doherty, Margaret; Bones, Jonathan; McLoughlin, Niaobh; Telford, Jayne E; Harmon, Bryan; DeFelippis, Michael R; Rudd, Pauline M

    2013-11-01

    Oligosaccharides attached to Asn297 in each of the CH2 domains of monoclonal antibodies play an important role in antibody effector functions by modulating the affinity of interaction with Fc receptors displayed on cells of the innate immune system. Rapid, detailed, and quantitative N-glycan analysis is required at all stages of bioprocess development to ensure the safety and efficacy of the therapeutic. The high sample numbers generated during quality by design (QbD) and process analytical technology (PAT) create a demand for high-performance, high-throughput analytical technologies for comprehensive oligosaccharide analysis. We have developed an automated 96-well plate-based sample preparation platform for high-throughput N-glycan analysis using a liquid handling robotic system. Complete process automation includes monoclonal antibody (mAb) purification directly from bioreactor media, glycan release, fluorescent labeling, purification, and subsequent ultra-performance liquid chromatography (UPLC) analysis. The entire sample preparation and commencement of analysis is achieved within a 5-h timeframe. The automated sample preparation platform can easily be interfaced with other downstream analytical technologies, including mass spectrometry (MS) and capillary electrophoresis (CE), for rapid characterization of oligosaccharides present on therapeutic antibodies. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. 9 CFR 113.452 - Erysipelothrix Rhusiopathiae Antibody.

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Erysipelothrix Rhusiopathiae Antibody... REQUIREMENTS Antibody Products § 113.452 Erysipelothrix Rhusiopathiae Antibody. Erysipelothrix Rhusiopathiae Antibody is a specific antibody product containing antibodies directed against one or more somatic antigens...

  11. Antibody against Microbial Neuraminidases Recognizes Human Sialidase 3 (NEU3: the Neuraminidase/Sialidase Superfamily Revisited

    Directory of Open Access Journals (Sweden)

    Chiguang Feng

    2017-06-01

    Full Text Available Neuraminidases (NAs are critical virulence factors for several microbial pathogens. With a highly conserved catalytic domain, a microbial NA “superfamily” has been proposed. We previously reported that murine polymorphonuclear leukocyte (PMN sialidase activity was important in leukocyte trafficking to inflamed sites and that antibodies to Clostridium perfringens NA recognized a cell surface molecule(s, presumed to be a sialidase of eukaryotic origin on interleukin-8-stimulated human and murine PMNs. These antibodies also inhibited cell sialidase activity both in vitro and, in the latter instance, in vivo. We therefore hypothesized that mammalian sialidases share structural homology and epitopes with microbial NAs. We now report that antibodies to one of the isoforms of C. perfringens NA, as well as anti-influenza virus NA serum, recognize human NEU3 but not NEU1 and that antibodies to C. perfringens NA inhibit NEU3 enzymatic activity. We conclude that the previously described microbial NA superfamily extends to human sialidases. Strategies designed to therapeutically inhibit microbial NA may need to consider potential compromising effects on human sialidases, particularly those expressed in cells of the immune system.

  12. Direct radioimmunoassay of serum progesterone using heterologous bridge tracer and antibody

    International Nuclear Information System (INIS)

    Kothari, K.; Pillai, M.R.A.

    1998-01-01

    The standardisation of a direct radioimmunoassay for progesterone using an 125 I labeled progesterone prepared by iodinating the tyrosine methyl ester (TME) conjugated to a progesterone hemiphthalate derivative and an antibody prepared using a progesterone linked to bovine serum albumin through 11α hemisuccinate derivative is described. The hemiphthalate derivative of progesterone was prepared by reacting 11α-hydroxy progesterone with phthalic anhydride which was then conjugated to TME by using isobutyl chloroformate. The conjugate was iodinated with 125 I using chloramine-T as oxidising agent and purified by thin layer chromatography. Radiochemical purity of the tracer was >95% in all batches. The tracer gave 70-75% binding with excess antibody. Assays were optimised with 8-anilino-1-naphthalene sulphonic acid (ANS) and sodium salicylate as blocking agents to release the progesterone from binding proteins. The assays optimised with sodium salicylate as blocking agent has a sensitivity of 0.25 ng/ml and a working range of 0.25-50 ng/ml, whereas the assay with ANS has a sensitivity of 0.75 ng/ml and a working range of 0.75-100 ng/ml. Serum samples were analysed and compared with the values obtained with a homologous bridge assay. (author)

  13. Direct Thy-1/alphaVbeta3 integrin interaction mediates neuron to astrocyte communication.

    Science.gov (United States)

    Hermosilla, Tamara; Muñoz, Daniel; Herrera-Molina, Rodrigo; Valdivia, Alejandra; Muñoz, Nicolás; Nham, Sang-Uk; Schneider, Pascal; Burridge, Keith; Quest, Andrew F G; Leyton, Lisette

    2008-06-01

    Thy-1 is an abundant neuronal glycoprotein of poorly defined function. We recently provided evidence indicating that Thy-1 clusters a beta3-containing integrin in astrocytes to induce tyrosine phosphorylation, RhoA activation and the formation of focal adhesions and stress fibers. To date, the alpha subunit partner of beta3 integrin in DI TNC1 astrocytes is unknown. Similarly, the ability of neuronal, membrane-bound Thy-1 to trigger astrocyte signaling via integrin engagement remains speculation. Here, evidence that alphav forms an alphavbeta3 heterodimer in DI TNC1 astrocytes was obtained. In neuron-astrocyte association assays, the presence of either anti-alphav or anti-beta3 integrin antibodies reduced cell-cell interaction demonstrating the requirement of both integrin subunits for this association. Moreover, anti-Thy-1 antibodies blocked stimulation of astrocytes by neurons but not the binding of these two cell types. Thus, neuron-astrocyte association involved binding between molecular components in addition to the Thy-1-integrin; however, the signaling events leading to focal adhesion formation in astrocytes depended exclusively on the latter interaction. Additionally, wild-type (RLD) but not mutated (RLE) Thy-1 was shown to directly interact with alphavbeta3 integrin by Surface Plasmon Resonance analysis. This interaction was promoted by divalent cations and was species-independent. Together, these results demonstrate that the alphavbeta3 integrin heterodimer interacts directly with Thy-1 present on neuronal cells to stimulate astrocytes.

  14. Rabies direct fluorescent antibody test does not inactivate rabies or eastern equine encephalitis viruses.

    Science.gov (United States)

    Jarvis, Jodie A; Franke, Mary A; Davis, April D

    2016-08-01

    An examination using the routine rabies direct fluorescent antibody test was performed on rabies or Eastern equine encephalitis positive mammalian brain tissue to assess inactivation of the virus. Neither virus was inactivated with acetone fixation nor the routine test, thus laboratory employees should treat all samples as rabies and when appropriate Eastern equine encephalitis positive throughout the whole procedure. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Site-directed antibody immobilization using a protein A-gold binding domain fusion protein for enhanced SPR immunosensing.

    Science.gov (United States)

    de Juan-Franco, Elena; Caruz, Antonio; Pedrajas, J R; Lechuga, Laura M

    2013-04-07

    We have implemented a novel strategy for the oriented immobilization of antibodies onto a gold surface based on the use of a fusion protein, the protein A-gold binding domain (PAG). PAG consists of a gold binding peptide (GBP) coupled to the immunoglobulin-binding domains of staphylococcal protein A. This fusion protein provides an easy and fast oriented immobilization of antibodies preserving its native structure, while leaving the antigen binding sites (Fab) freely exposed. Using this immobilization strategy, we have demonstrated the performance of the immunosensing of the human Growth Hormone by SPR. A limit of detection of 90 ng mL(-1) was obtained with an inter-chip variability lower than 7%. The comparison of this method with other strategies for the direct immobilization of antibodies over gold surfaces has showed the enhanced sensitivity provided by the PAG approach.

  16. Immunoglobulin variable region sequences of two human monoclonal antibodies directed to an onco-developmental carbohydrate antigen, lactotetraosylceramide (LcOse4Cer).

    Science.gov (United States)

    Yago, K; Zenita, K; Ohwaki, I; Harada, R; Nozawa, S; Tsukazaki, K; Iwamori, M; Endo, N; Yasuda, N; Okuma, M

    1993-11-01

    A human monoclonal antibody, 11-50, was generated and was shown to recognize an onco-developmental carbohydrate antigen, LcOse4Cer. The isotype of this antibody was IgM, lambda, similar to the previously known human anti-LcOse4 antibodies, such as IgMWOO and HMST-1. We raised a murine anti-idiotypic antibody G3 (IgG1, kappa) against 11-50, and tested its reactivity towards the affinity purified human polyclonal anti-LcOse4 antibodies prepared from pooled human sera using a Gal beta 1-->3GlcNAc beta-immobilized column. The results indicated that at least a part of the human polyclonal anti-LcOse4 antibodies shared the G3 idiotype with 11-50. We further analyzed the sequence of variable regions of the two anti-LcOse4 antibodies, 11-50 and HMST-1. Sequence analysis of the heavy chain variable regions indicated that the VH regions of these two antibodies were highly homologous to each other (93.5% at the nucleic acid level), and these antibodies utilized the germline genes VH1.9III and hv3005f3 as the VH segments, which are closely related germline genes of the VHIII family. It was noted that these germline VH genes are frequently utilized in fetal B cells. The JH region of both antibodies was encoded by the JH4 gene. For the light chain, the V lambda segments of the two antibodies were 96.3% homologous to each other at the nucleic acid level. The V lambda segments of both antibodies showed the highest homology to the rearranged V lambda gene called V lambda II.DS among reported V lambda genes, while the exact germline V lambda genes encoding the two antibodies were not yet registered in available sequence databanks. The amino acid sequences of the J lambda segments of both antibodies were identical. These results indicate that the two human antibodies recognizing the onco-developmental carbohydrate antigen Lc4 are encoded by the same or very homologous germline genes.

  17. Factors determining anti-poliovirus type 3 antibodies among orally immunised Indian infants.

    Science.gov (United States)

    Kaliappan, Saravanakumar Puthupalayam; Venugopal, Srinivasan; Giri, Sidhartha; Praharaj, Ira; Karthikeyan, Arun S; Babji, Sudhir; John, Jacob; Muliyil, Jayaprakash; Grassly, Nicholas; Kang, Gagandeep

    2016-09-22

    Among the three poliovirus serotypes, the lowest responses after vaccination with trivalent oral polio vaccine (tOPV) are to serotype 3. Although improvements in routine immunisation and supplementary immunisation activities have greatly increased vaccine coverage, there are limited data on antibody prevalence in Indian infants. Children aged 5-11months with a history of not having received inactivated polio vaccine were screened for serum antibodies to poliovirus serotype 3 (PV3) by a micro-neutralisation assay according to a modified World Health Organization (WHO) protocol. Limited demographic information was collected to assess risk-factors for a lack of protective antibodies. Student's t-test, logistic regression and multilevel logistic regression (MLR) model were used to estimate model parameters. Of 8454 children screened at a mean age of 8.3 (standard deviation [SD]-1.8) months, 88.1% (95% confidence interval (CI): 87.4-88.8) had protective antibodies to PV3. The number of tOPV doses received was the main determinant of seroprevalence; the maximum likelihood estimate yields a 37.7% (95% CI: 36.2-38.3) increase in seroprevalence per dose of tOPV. In multivariable logistic regression analysis increasing age, male sex, and urban residence were also independently associated with seropositivity (Odds Ratios (OR): 1.17 (95% CI: 1.12-1.23) per month of age, 1.27 (1.11-1.46) and 1.24 (1.05-1.45) respectively). Seroprevalence of antibodies to PV3 is associated with age, gender and place of residence, in addition to the number of tOPV doses received. Ensuring high coverage and monitoring of response are essential as long as oral vaccines are used in polio eradication. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  18. Clinical prospective study with radioiodinated monoclonal antibodies directed against colorectal cancer

    International Nuclear Information System (INIS)

    Chatal, J.F.; Douillard, J.Y.; Kremer, M.; Curtet, C.; Le Mevel, B.; Saccavini, J.C.; Maurel, C.; Aubry, J.

    1985-01-01

    The diagnostic application of three monoclonal antibodies are studied: an anti-carcinoembryonic antigen (CEA) antibody designated as 202 and two monoclonal antibodies, designated as 17-1A and 19-9, which recognize different antigens associated with gastrointestinal carcinomas. The complementary specificity of these antibodies was determined by an immuno-histochemical study and the scintigraphic detection parameters by a radiopharmacokinetic study in colic-tumour-bearing nude mice. On the basis of a prospective study, the value of immunoscintigraphy was compared with conventional methods such as ultrasonography and computed tomography for localization of recurrences of colorectal cancers. (UK)

  19. Effect of antibody charge and concentration on deposition of antibody to glomerular basement membrane

    International Nuclear Information System (INIS)

    Madaio, M.P.; Salant, D.J.; Adler, S.; Darby, C.; Couser, W.G.

    1984-01-01

    Fixed anionic sites within the glomerular capillary wall influence the permeation of serum proteins, the localization of various antigens, and the deposition of antibody in the subepithelial space. In anti-GBM nephritis antibody deposition occurs very rapidly to antigenic sites located relatively proximal in the glomerular capillary wall. The authors examined the influence of the glomerular charge barrier on anti-GBM antibody deposition by comparing the rate of deposition of antibodies with cationic and anionic isoelectric points. Purified sheep anti-rat GBM IgG was isolated from acid eluates of kidneys obtained 24 hr after rats were injected with sheep antiserum to rat GBM. Anti-GBM IgG was separated into cationic (pI 6.4-8.5) and anionic (pI 4.2-6.8) fractions, which were radiolabelled with 131 I and 125 I, respectively, shown to have equal antibody contents measured by in vitro binding to normal glomeruli, mixed in equal amounts, and injected in incremental doses to ten rats. At 1 hr the glomerular antibody binding of each fraction was directly related to the blood level (r . 0.95, r . 0.97) and delivery of antibody (r . 0.98, r . 0.98). Glomerular binding of cationic antibody was four times greater than anionic antibody over the entire range of deliveries studied (P less than 0.001). The authors conclude that glomerular deposition of anti-GBM antibody is directly related to blood concentration and delivery of antibody. Furthermore, the deposition of cationic antibodies to GBM antigens was significantly greater than the deposition of anionic antibodies

  20. [VGKC-complex antibodies].

    Science.gov (United States)

    Watanabe, Osamu

    2013-04-01

    Various antibodies are associated with voltage-gated potassium channels (VGKCs). Representative antibodies to VGKCs were first identified by radioimmunoassays using radioisotope-labeled alpha-dendrotoxin-VGKCs solubilized from rabbit brain. These antibodies were detected only in a proportion of patients with acquired neuromyotonia (Isaacs' syndrome). VGKC antibodies were also detected in patients with Morvan's syndrome and in those with a form of autoimmune limbic encephalitis. Recent studies indicated that the "VGKC" antibodies are mainly directed toward associated proteins (for example LGI-1 and CASPR-2) that complex with the VGKCs themselves. The "VGKC" antibodies are now commonly known as VGKC-complex antibodies. In general, LGI-1 antibodies are most commonly detected in patients with limbic encephalitis with syndrome of inappropriate secretion of antidiuretic hormone. CASPR-2 antibodies are present in the majority of patients with Morvan's syndrome. These patients develop combinations of CNS symptoms, autonomic dysfunction, and peripheral nerve hyperexcitability. Furthermore, VGKC-complex antibodies are tightly associated with chronic idiopathic pain. Hyperexcitability of nociceptive pathways has also been implicated. These antibodies may be detected in sera of some patients with neurodegenerative diseases (for example, amyotrophic lateral sclerosis and Creutzfeldt-Jakob disease).

  1. Studies of monoclonal antibodies IOR-CEA-1 and IOR-EGF/R3 labelled with {sup 99m}Tc; Estudo de marcacao dos anticorpos monoclonais IOR-CEA-1 e IOR-EGF/R3 com {sup 99m}Tc

    Energy Technology Data Exchange (ETDEWEB)

    Dias, Carla Roberta de Barros Rodrigues

    2005-07-01

    Nuclear Medicine is a speciality that uses radioisotopes for the diagnosis or treatment of diseases and it is considered one of the best tools among the diagnostic modalities for detection of cancer. {sup 99m}Tc is one of the main isotopes for labelling antibodies and in Nuclear Medicine in general, due to its adequate physical properties, availability and low cost. Labelled monoclonal antibodies have shown promising results for diagnosis and therapy of cancer and their use has brought great experimental and clinical advances in the field of oncology. The main clinical applications of immunoscintigraphy with monoclonal antibodies are staging and evaluation of tumoral reappearance. The antibodies employed in this work were: OIR-CEA-1, a murine monoclonal antibody that acts directly against CEA expressed in several neoplasia in particular those from the gastrointestinal tract (colorectal cancer) and IOR-EGF/R3, a murine monoclonal antibody that binds to the external domain of EGF-R and it has been used in the diagnosis of tumors of epithelial origin. The objectives of this work were the development and optimization of the reduction and purification processes, the radiolabelling techniques and quality control procedures (radiochemical, immunoreactivity and cystein challenge) and imaging studies of monoclonal antibodies OIR-CEA-1 and IOR-EGF/R3, using the simple, fast and efficient method of direct labelling of the antibody with {sup 99m}Tc. The final results was the definition of the best conditions for the preparation of lyophilized reactive kits of OIR-CEA-1 and IOR- EGF/R3 for an efficient diagnostic application in Nuclear Medicine. The most adequate conditions for the labelling of the antibodies were: 1.0 mg Ab, 29 {mu}L MDP, 3.0 {mu}g Sn{sup 2+}, 1 mL of {sup 99m}Tc and 30 min. reaction time. With these conditions the labelling yield was always higher than 95% and the maximum activity of {sup 99m}Tc was about 2220 MBq (60 mCi). The evidences of the efficiency and

  2. Characterization of a Large Panel of Rabbit Monoclonal Antibodies against HIV-1 gp120 and Isolation of Novel Neutralizing Antibodies against the V3 Loop.

    Directory of Open Access Journals (Sweden)

    Yali Qin

    Full Text Available We recently reported the induction of potent, cross-clade neutralizing antibodies (nAbs against Human Immunodeficiency Virus type-1 (HIV-1 in rabbits using gp120 based on an M-group consensus sequence. To better characterize these antibodies, 93 hybridomas were generated, which represent the largest panel of monoclonal antibodies (mAbs ever generated from a vaccinated rabbit. The single most frequently recognized epitope of the isolated mAbs was at the very C-terminal end of the protein (APTKAKRRVVEREKR, followed by the V3 loop. A total of seven anti-V3 loop mAbs were isolated, two of which (10A3 and 10A37 exhibited neutralizing activity. In contrast to 10A3 and most other anti-V3 loop nAbs, 10A37 was atypical with its epitope positioned more towards the C-terminal half of the loop. To our knowledge, 10A37 is the most potent and broadly neutralizing anti-V3 loop mAb induced by vaccination. Interestingly, all seven anti-V3 loop mAbs competed with PGT121, suggesting a possibility that early induction of potent anti-V3 loop antibodies could prevent induction of more broadly neutralizing PGT121-like antibodies that target the conserved base of the V3 loop stem.

  3. Characterization of activating mutations of NOTCH3 in T-cell acute lymphoblastic leukemia and anti-leukemic activity of NOTCH3 inhibitory antibodies.

    Science.gov (United States)

    Bernasconi-Elias, P; Hu, T; Jenkins, D; Firestone, B; Gans, S; Kurth, E; Capodieci, P; Deplazes-Lauber, J; Petropoulos, K; Thiel, P; Ponsel, D; Hee Choi, S; LeMotte, P; London, A; Goetcshkes, M; Nolin, E; Jones, M D; Slocum, K; Kluk, M J; Weinstock, D M; Christodoulou, A; Weinberg, O; Jaehrling, J; Ettenberg, S A; Buckler, A; Blacklow, S C; Aster, J C; Fryer, C J

    2016-11-24

    Notch receptors have been implicated as oncogenic drivers in several cancers, the most notable example being NOTCH1 in T-cell acute lymphoblastic leukemia (T-ALL). To characterize the role of activated NOTCH3 in cancer, we generated an antibody that detects the neo-epitope created upon gamma-secretase cleavage of NOTCH3 to release its intracellular domain (ICD3), and sequenced the negative regulatory region (NRR) and PEST (proline, glutamate, serine, threonine) domain coding regions of NOTCH3 in a panel of cell lines. We also characterize NOTCH3 tumor-associated mutations that result in activation of signaling and report new inhibitory antibodies. We determined the structural basis for receptor inhibition by obtaining the first co-crystal structure of a NOTCH3 antibody with the NRR protein and defined two distinct epitopes for NRR antibodies. The antibodies exhibit potent anti-leukemic activity in cell lines and tumor xenografts harboring NOTCH3 activating mutations. Screening of primary T-ALL samples reveals that 2 of 40 tumors examined show active NOTCH3 signaling. We also identified evidence of NOTCH3 activation in 12 of 24 patient-derived orthotopic xenograft models, 2 of which exhibit activation of NOTCH3 without activation of NOTCH1. Our studies provide additional insights into NOTCH3 activation and offer a path forward for identification of cancers that are likely to respond to therapy with NOTCH3 selective inhibitory antibodies.

  4. Site-directed immobilization of a genetically engineered anti-methotrexate antibody via an enzymatically introduced biotin label significantly increases the binding capacity of immunoaffinity columns.

    Science.gov (United States)

    Davenport, Kaitlynn R; Smith, Christopher A; Hofstetter, Heike; Horn, James R; Hofstetter, Oliver

    2016-05-15

    In this study, the effect of random vs. site-directed immobilization techniques on the performance of antibody-based HPLC columns was investigated using a single-domain camelid antibody (VHH) directed against methotrexate (MTX) as a model system. First, the high flow-through support material POROS-OH was activated with disuccinimidyl carbonate (DSC), and the VHH was bound in a random manner via amines located on the protein's surface. The resulting column was characterized by Frontal Affinity Chromatography (FAC). Then, two site-directed techniques were explored to increase column efficiency by immobilizing the antibody via its C-terminus, i.e., away from the antigen-binding site. In one approach, a tetra-lysine tail was added, and the antibody was immobilized onto DSC-activated POROS. In the second site-directed approach, the VHH was modified with the AviTag peptide, and a biotin-residue was enzymatically incorporated at the C-terminus using the biotin ligase BirA. The biotinylated antibody was subsequently immobilized onto NeutrAvidin-derivatized POROS. A comparison of the FAC analyses, which for all three columns showed excellent linearity (R(2)>0.999), revealed that both site-directed approaches yield better results than the random immobilization; the by far highest efficiency, however, was determined for the immunoaffinity column based on AviTag-biotinylated antibody. As proof of concept, all three columns were evaluated for quantification of MTX dissolved in phosphate buffered saline (PBS). Validation using UV-detection showed excellent linearity in the range of 0.04-12μM (R(2)>0.993). The lower limit of detection (LOD) and lower limit of quantification (LLOQ) were found to be independent of the immobilization strategy and were 40nM and 132nM, respectively. The intra- and inter-day precision was below 11.6%, and accuracy was between 90.7% and 112%. To the best of our knowledge, this is the first report of the AviTag-system in chromatography, and the first

  5. Antithyroglobulin Antibodies and Antimicrosomal Antibodies in Various Thyroid Diseases

    International Nuclear Information System (INIS)

    Lee, Gwon Jun; Hong, Key Sak; Choi, Kang Won; Lee, Kyu; Koh, Chang Soon; Lee, Mun Ho; Park, Sung Hoe; Chi, Je Geun; Lee, Sang Kook

    1979-01-01

    The authors investigated the incidence of antithyroglobulin antibodies and antibodies and antimicrosomal antibodies measured by tanned red cell hemagglutination method in subjects suffering from various thyroid disorders. 1) In 15 normal patients, neither suffering from any thyroid diseases nor from any other autoimmune disorders, the antithyroglobulin antibodies were all negative, but the antimicrosomal antibody was positive only in one patient (6.7%). 2) The antithyroglobulin antibodies were positive in 31.5% (34 patients) of 108 patients with various thyroid diseases, and the antimicrosomal antibodies were positive in 37.0% (40 patients). 3) of the 25 patients with Graves' diseases, 7 patients (28.0%) showed positive for the antithyroglobulin antibodies, and 9 (36.0%) for the antimicrosomal antibodies. There was no definite differences in clinical and thyroid functions between the groups with positive and negative results. 4) Both antibodies were positive in 16 (88.9%) and 17 (94.4%) patients respectively among 18 patients with Hashimoto's thyroiditis, all of them were diagnosed histologically. 5) Three out of 33 patients with thyroid adenoma showed positive antibodies, and 3 of 16 patients with thyroid carcinoma revealed positive antibodies. 6) TRCH antibodies demonstrated negative results in 2 patients with subacute thyroiditis, but positive in one patient with idiopathic primary myxedema. 7) The number of patients with high titers(>l:802) was 16 for antithyroglobulin antibody, and 62.5% (10 patients) of which was Hashimoto's thyroiditis. Thirteen (65.0) of 20 patients with high titers (>l:802) for antimicrosomal antibody was Hashimoto's thyroiditis. TRCH test is a simple, sensitive method, and has high reliability and reproducibility. The incidences and titers of antithyroglobulin antibody and antimicrosomal antibody are especially high in Hashimoto's thyroiditis.

  6. Proteomic profiling of antibody-inducing immunogens in tumor tissue identifies PSMA1, LAP3, ANXA3, and maspin as colon cancer markers

    Science.gov (United States)

    Yang, Qian; Roehrl, Michael H.; Wang, Julia Y.

    2018-01-01

    We hypothesized that cancer tissue immunogens – antigens capable of inducing specific antibody production in patients – are promising targets for development of precision diagnostics and humoral immunotherapies. We developed an innovative immuno-proteomic strategy and identified new immunogenic markers of colon cancer. Proteins from cancers and matched normal tissues were separated by 2D gel electrophoresis and blotted with serum antibodies from the same patients. Antibody-reactive proteins were sequenced by mass spectrometry and validated by Western blotting and immunohistochemistry. 170 serum antibody-reactive proteins were identified only in cancerous but not matched normal. Among these, proteasome subunit alpha type 1 (PSA1), leucine aminopeptidase 3 (LAP3), annexin A3 (ANXA3), and maspin (serpin B5) were reproducibly found in tissues from three patients. Differential expression patterns were confirmed in samples from eight patients with various stages of colon adenocarcinoma and liver metastases. These tumor-resident proteins and/or their associated serum antibodies may be promising markers for colon cancer screening and early diagnosis. Furthermore, tumor tissue-specific antibodies could potentially be exploited as immunotherapeutic targets against cancer. More generally, proteomic profiling of antibody-inducing cancer-associated immunogens represents a powerful generic method for uncovering the tumor antigen-ome, i.e., the totality of immunogenic tumor-associated proteins. PMID:29423100

  7. Isolation of recombinant antibodies directed against surface proteins of Clostridium difficile.

    Science.gov (United States)

    Shirvan, Ali Nazari; Aitken, Robert

    2016-01-01

    Clostridium difficile has emerged as an increasingly important nosocomial pathogen and the prime causative agent of antibiotic-associated diarrhoea and pseudomembranous colitis in humans. In addition to toxins A and B, immunological studies using antisera from patients infected with C. difficile have shown that a number of other bacterial factors contribute to the pathogenesis, including surface proteins, which are responsible for adhesion, motility and other interactions with the human host. In this study, various clostridial targets, including FliC, FliD and cell wall protein 66, were expressed and purified. Phage antibody display yielded a large panel of specific recombinant antibodies, which were expressed, purified and characterised. Reactions of the recombinant antibodies with their targets were detected by enzyme-linked immunosorbent assay; and Western blotting suggested that linear rather than conformational epitopes were recognised. Binding of the recombinant antibodies to surface-layer proteins and their components showed strain specificity, with good recognition of proteins from C. difficile 630. However, no reaction was observed for strain R20291-a representative of the 027 ribotype. Binding of the recombinant antibodies to C. difficile M120 extracts indicated that a component of a surface-layer protein of this strain might possess immunoglobulin-binding activities. The recombinant antibodies against FliC and FliD proteins were able to inhibit bacterial motility. Copyright © 2016. Published by Elsevier Editora Ltda.

  8. Detection of thrombocytic antibodies with the direct and indirect haemolysis inhibition test and the radioimmuno-Coombs test

    International Nuclear Information System (INIS)

    Mettenboerger, D.; Vith, E.

    1982-01-01

    Methods of application of the direct and indirect haemolysis inhibition test were studied in order to optimise the test parameters: The ultimate aim was to standardize the test method and compare its sensitivity in detecting various platelet antibodies with platelet indirect radioactive Coombs-test and the platelet immunofluorescence test. (orig.) [de

  9. Focal CA3 hippocampal subfield atrophy following LGI1 VGKC-complex antibody limbic encephalitis.

    Science.gov (United States)

    Miller, Thomas D; Chong, Trevor T-J; Aimola Davies, Anne M; Ng, Tammy W C; Johnson, Michael R; Irani, Sarosh R; Vincent, Angela; Husain, Masud; Jacob, Saiju; Maddison, Paul; Kennard, Christopher; Gowland, Penny A; Rosenthal, Clive R

    2017-05-01

    Magnetic resonance imaging has linked chronic voltage-gated potassium channel (VGKC) complex antibody-mediated limbic encephalitis with generalized hippocampal atrophy. However, autoantibodies bind to specific rodent hippocampal subfields. Here, human hippocampal subfield (subiculum, cornu ammonis 1-3, and dentate gyrus) targets of immunomodulation-treated LGI1 VGKC-complex antibody-mediated limbic encephalitis were investigated using in vivo ultra-high resolution (0.39 × 0.39 × 1.0 mm3) 7.0 T magnetic resonance imaging [n = 18 patients, 17 patients (94%) positive for LGI1 antibody and one patient negative for LGI1/CASPR2 but positive for VGKC-complex antibodies, mean age: 64.0 ± 2.55 years, median 4 years post-limbic encephalitis onset; n = 18 controls]. First, hippocampal subfield quantitative morphometry indicated significant volume loss confined to bilateral CA3 [F(1,34) = 16.87, P 3 months from symptom onset) were associated with CA3 atrophy. Third, whole-brain voxel-by-voxel morphometry revealed no significant grey matter loss. Fourth, CA3 subfield atrophy was associated with severe episodic but not semantic amnesia for postmorbid autobiographical events that was predicted by variability in CA3 volume. The results raise important questions about the links with histopathology, the impact of the observed focal atrophy on other CA3-mediated reconstructive and episodic mechanisms, and the role of potential antibody-mediated pathogenicity as part of the pathophysiology cascade in humans. © The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain.

  10. Environmental burden by nitrogen from agriculture in the Netherlands. Outline of effects of manure and ammonia measures; Milieubelasting door stikstof uit de Nederlandse landbouw. Verkenning effecten mest- en ammoniakmaatregelen

    Energy Technology Data Exchange (ETDEWEB)

    Kros, J.; De Vries, W.; Oenema, O. [Divisie Bodem, Alterra, Wageningen Universiteit en Research Centrum, Wageningen (Netherlands)

    2004-03-01

    The nitrogen emission ceiling has been introduced to be able to assess the effectiveness of measures to control manure and ammonia. This integral method shows the effects of several policy options on the release of nitrogen from the agricultural sector into the environment on a regional scale. [Dutch] Het stikstofplafond is geintroduceerd om de effectiviteit van mest- en ammoniak-maatregelen te beoordelen. Deze integrale methodiek laat op een transparante en samenhangende wijze zien wat de effecten zijn van diverse mogelijke (beleids)maatregelen op de stikstofverliezen uit de landbouw naar het milieu op regionale schaal.

  11. Clinical implications of the detection of antibodies directed against domain 1 of β2-glycoprotein 1 in thrombotic antiphospholipid syndrome.

    Science.gov (United States)

    Montalvão, Silmara; Elídio, Priscila Soares; da Silva Saraiva, Sabrina; de Moraes Mazetto, Bruna; Colella, Marina Pereira; de Paula, Erich Vinícius; Appenzeller, Simone; Annichino-Bizzacchi, Joyce; Orsi, Fernanda Andrade

    2016-12-01

    Antibodies directed against domain 1 of β2 glycoprotein 1 (aβ2GP1-Dm1) have been involved in the immunopathogenesis of antiphospholipid syndrome (APS). However, the clinical relevance of aβ2GP1-Dm1 in thrombotic APS has not yet been fully explored. To determine the frequency of aβ2GP1-Dm1 in a cohort of patients with thrombotic APS, and to evaluate whether testing for aβ2GP1-Dm1 could have a clinical impact upon the risk assessment of the disease. Patients were tested for aβ2GP1-Dm1 antibodies by chemiluminescence (BioFlash/AcuStar®, ES). The presence of aβ2GP1-Dm1 was evaluated in different clinical presentations of the disease. Eight-four patients with a history of venous or arterial thrombosis were included. Forty-five (54%) patients had aβ2GP1 antibodies and 40% of them were positive for aβ2GP1-Dm1. Levels of aβ2GP1-Dm1 were higher in patients with systemic autoimmune disease (AUC=0.665; 95% CI=0.544-0.786; P=0.01), positive antinuclear antibody (AUC=0.654; 95% CI=0.535-0.772; P=0.01), triple antiphospholipid antibody (aPL) positivity (AUC=0.680; 95% CI=0.534-0.825; P=0.02) and positive lupus anticoagulant (AUC=0.639; 95% CI=0.502-0.776; P=0.07). In this cohort, aβ2GP1-Dm1 antibodies were not associated with the site of the first thrombosis (OR=0,62, 95% CI=0.20-1.94, P=0.42), thrombosis recurrence (OR=1.0, 95% CI=0.37-2.71, P=1.0) or pregnancy morbidity (OR=1.5, 95% CI=0.33-7.34, P=0.58). In multivariate analysis, positivity for aβ2GP1-Dm1 antibodies was associated with the diagnosis of systemic autoimmune disease (OR=4.01, 95% CI=1.14-14.2; P=0.03) and triple aPL positivity (OR=3.59, 95% CI=0.87-14.85; P=0.07). In the present cohort of thrombotic-APS patients, aβ2GP1-Dm1 antibodies were related to the diagnosis of systemic autoimmunity and complex serological profile of the disease, as triple aPL positivity and positive antinuclear antibody. Thus, our results suggest that testing for aβ2GP1-Dm1 antibodies may be useful for improving APS risk

  12. A novel merozoite surface antigen of Plasmodium falciparum (MSP-3 identified by cellular-antibody cooperative mechanism antigenicity and biological activity of antibodies

    Directory of Open Access Journals (Sweden)

    Claude Oeuvray

    1994-01-01

    Full Text Available We report the identification of a 48kDa antigen targeted by antibodies which inhibit Plasmodium falciparum in vitro growth by cooperation with blood monocytes in an ADCI assay correlated to the naturally acquired protection. This protein is located on the surface of the merozoite stage of P. falciparum, and is detectable in all isolates tested. Epidemiological studies demonstrated that peptides derived from the amino acid sequence of MSP-3 contain potent B and T-cell epitopes recognized by a majority of individuals living in endemic areas. Moreover human antibodies either purified on the recombinant protein, or on the synthetic peptide MSP-3b, as well as antibodies raised in mice, were all found to promote parasite killing mediated by monocytes.

  13. Antithyroglobulin Antibodies and Antimicrosomal Antibodies in Various Thyroid Diseases

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Gwon Jun; Hong, Key Sak; Choi, Kang Won; Lee, Kyu; Koh, Chang Soon; Lee, Mun Ho; Park, Sung Hoe; Chi, Je Geun; Lee, Sang Kook [Seoul National University College of Medicine, Seoul (Korea, Republic of)

    1979-03-15

    The authors investigated the incidence of antithyroglobulin antibodies and antibodies and antimicrosomal antibodies measured by tanned red cell hemagglutination method in subjects suffering from various thyroid disorders. 1) In 15 normal patients, neither suffering from any thyroid diseases nor from any other autoimmune disorders, the antithyroglobulin antibodies were all negative, but the antimicrosomal antibody was positive only in one patient (6.7%). 2) The antithyroglobulin antibodies were positive in 31.5% (34 patients) of 108 patients with various thyroid diseases, and the antimicrosomal antibodies were positive in 37.0% (40 patients). 3) of the 25 patients with Graves' diseases, 7 patients (28.0%) showed positive for the antithyroglobulin antibodies, and 9 (36.0%) for the antimicrosomal antibodies. There was no definite differences in clinical and thyroid functions between the groups with positive and negative results. 4) Both antibodies were positive in 16 (88.9%) and 17 (94.4%) patients respectively among 18 patients with Hashimoto's thyroiditis, all of them were diagnosed histologically. 5) Three out of 33 patients with thyroid adenoma showed positive antibodies, and 3 of 16 patients with thyroid carcinoma revealed positive antibodies. 6) TRCH antibodies demonstrated negative results in 2 patients with subacute thyroiditis, but positive in one patient with idiopathic primary myxedema. 7) The number of patients with high titers(>l:802) was 16 for antithyroglobulin antibody, and 62.5% (10 patients) of which was Hashimoto's thyroiditis. Thirteen (65.0) of 20 patients with high titers (>l:802) for antimicrosomal antibody was Hashimoto's thyroiditis. TRCH test is a simple, sensitive method, and has high reliability and reproducibility. The incidences and titers of antithyroglobulin antibody and antimicrosomal antibody are especially high in Hashimoto's thyroiditis.

  14. Pistachio (Pistacia vera L.) Detection and Quantification Using a Murine Monoclonal Antibody-Based Direct Sandwich Enzyme-Linked Immunosorbent Assay.

    Science.gov (United States)

    Liu, Changqi; Chhabra, Guneet S; Sathe, Shridhar K

    2015-10-21

    A commercially available direct sandwich enzyme-linked immunosorbent assay (ELISA) (BioFront Technologies, Tallahassee, FL, USA) using murine anti-pistachio monoclonal antibodies (mAbs) as capture and detection antibodies was evaluated. The assay was sensitive (limit of detection = 0.09 ± 0.02 ppm full fat pistachio, linear detection range = 0.5-36 ppm, 50% maximum signal concentration = 7.9 ± 0.7 ppm), reproducible (intra- and inter-assay variability pistachio seeds subjected to autoclaving (121 °C, 15 psi, 15, 30 min), blanching (100 °C, 5, 10 min), frying (191 °C, 1 min), microwaving (500, 1000 W, 3 min), and dry roasting (140 °C, 30 min; 168 °C, 12 min). No cross-reactivity was observed in 156 food matrices, each tested at 100,000 ppm, suggesting the ELISA to be pistachio specific. The pistachio recovery ranges for spiked (10 ppm) and incurred (10-50000 ppm) food matrices were 93.1-125.6% and 35.7-112.2%, respectively. The assay did not register any false-positive or -negative results among the tested commercial and laboratory prepared samples.

  15. Tetravalent anti-CD20/CD3 bispecific antibody for the treatment of B cell lymphoma

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Chia-Yen; Chen, Gregory J.; Tai, Pei-Han; Yang, Yu-Chen [Institute of Biologics, Development Center for Biotechnology, New Taipei City, Taiwan (China); Hsu, Yu-Shen, E-mail: yshsu@advagene.com.tw [Laboratory of Biopharmaceutical Research, Advagene Biopharma, Taipei, Taiwan (China); Chang, Mingi, E-mail: mingi.chang@advagene.com.tw [Laboratory of Biopharmaceutical Research, Advagene Biopharma, Taipei, Taiwan (China); Hsu, Chuan-Lung, E-mail: fabio@dcb.org.tw [Institute of Biologics, Development Center for Biotechnology, New Taipei City, Taiwan (China)

    2016-05-13

    Bispecific antibodies (bsAbs) are second generation antibodies for therapeutic application in immunotherapy. One of the major strategies of the bsAb platform is the recruitment of immune effector T cells by incorporating an anti-CD3 domain. A bispecific T-cell engager (BiTE), with one end having an affinity for CD3 and the other end with affinity for CD19, has been approved in the US and Europe for the treatment of acute lymphoblastic leukemia. However, due to their small size and lack of Fc region, these single-chain variable fragment (scFv) bsAbs have short half-lives in vivo. Additionally, poor solubility, structural instability, and low production yields have also become major challenges in the bulk production process. To overcome these challenges, we have engineered a tetravalent bsAb with bivalent binding specificity for the CD20 and CD3 antigen in an immunoglobulin G (IgG) format. The fusion of the anti-CD3 scFvs to the CD20 antibody via a linker-hinge domain (LHD) results in improved antibody stabilization and properties. Here we demonstrate this antibody's highly efficient cancer cell elimination in a dose-dependent manner in a CD20-expressing B lymphoblastoid cell line in vitro. Our data suggest the potential clinical application of this bsAb for the treatment of CD20-expressing B cell malignancies. - Highlights: • A bispecific antibody (bsAb) can increase immunotherapeutic efficacy. • A tetravalent bsAb with binding specificity for the CD20 and CD3 antigens is proposed. • A linker-hinge domain (LHD) within the bsAb results in improved antibody properties.

  16. Compositions, antibodies, asthma diagnosis methods, and methods for preparing antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Hongjun; Zangar, Richard C.

    2017-01-17

    Methods for preparing an antibody are provided with the method including incorporating 3-bromo-4-hydroxy-benzoic acid into a protein to form an antigen, immunizing a mammalian host with the antigen, and recovering an antibody having an affinity for the antigen from the host. Antibodies having a binding affinity for a monohalotyrosine are provided as well as composition comprising an antibody bound with monohalotyrosine. Compositions comprising a protein having a 3-bromo-4-hydroxy-benzoic acid moiety are also provided. Methods for evaluating the severity of asthma are provide with the methods including analyzing sputum of a patient using an antibody having a binding affinity for monohalotyrosine, and measuring the amount of antibody bound to protein. Methods for determining eosinophil activity in bodily fluid are also provided with the methods including exposing bodily fluid to an antibody having a binding affinity for monohalotyrosine, and measuring the amount of bound antibody to determine the eosinophil activity.

  17. THE HYDROGENOSOMAL ENZYME HYDROGENASE FROM THE ANAEROBIC FUNGUS NEOCALLIMASTIX SP L2 IS RECOGNIZED BY ANTIBODIES, DIRECTED AGAINST THE C-TERMINAL MICROBODY PROTEIN TARGETING SIGNAL SKL

    NARCIS (Netherlands)

    MARVINSIKKEMA, FD; KRAAK, MN; VEENHUIS, M; GOTTSCHAL, JC; PRINS, RA

    The question was addressed whether antibodies directed against the general microbody C-terminal protein targeting signal SKL recognized hydrogenosomal proteins from Neocallimastix sp. L2. Immunofluorescence, immunocytochemistry and Western blotting experiments using these antibodies indicated the

  18. Next Generation Antibody Therapeutics Using Bispecific Antibody Technology.

    Science.gov (United States)

    Igawa, Tomoyuki

    2017-01-01

    Nearly fifty monoclonal antibodies have been approved to date, and the market for monoclonal antibodies is expected to continue to grow. Since global competition in the field of antibody therapeutics is intense, we need to establish novel antibody engineering technologies to provide true benefit for patients, with differentiated product values. Bispecific antibodies are among the next generation of antibody therapeutics that can bind to two different target antigens by the two arms of immunoglobulin G (IgG) molecule, and are thus believed to be applicable to various therapeutic needs. Until recently, large scale manufacturing of human IgG bispecific antibody was impossible. We have established a technology, named asymmetric re-engineering technology (ART)-Ig, to enable large scale manufacturing of bispecific antibodies. Three examples of next generation antibody therapeutics using ART-Ig technology are described. Recent updates on bispecific antibodies against factor IXa and factor X for the treatment of hemophilia A, bispecific antibodies against a tumor specific antigen and T cell surface marker CD3 for cancer immunotherapy, and bispecific antibodies against two different epitopes of soluble antigen with pH-dependent binding property for the elimination of soluble antigen from plasma are also described.

  19. Purpose-Oriented Antibody Libraries Incorporating Tailored CDR3 Sequences

    OpenAIRE

    Bonvin, Pauline; Venet, Sophie; Kosco-Vilbois, Marie; Fischer, Nicolas

    2015-01-01

    The development of in vitro antibody selection technologies has allowed overcoming some limitations inherent to the hybridoma technology. In most cases, large repertoires of antibody genes have been assembled to create highly diversified libraries allowing the isolation of antibodies recognizing virtually any antigen. However, these universal libraries might not allow the isolation of antibodies with specific structural properties or particular amino acid contents that are rarely found in nat...

  20. Pretargeting vs. direct targeting of human betalox5 islet cells subcutaneously implanted in mice using an anti-human islet cell antibody

    International Nuclear Information System (INIS)

    Liu Guozheng; Dou Shuping; Akalin, Ali; Rusckowski, Mary; Streeter, Philip R.; Shultz, Leonard D.; Greiner, Dale L.

    2012-01-01

    Introduction: We previously demonstrated MORF/cMORF pretargeting of human islets and betalox 5 cells (a human beta cell line) transplanted subcutaneously in mice with the anti-human islet antibody, HPi1. We now compare pretargeting with direct targeting in the beta cell transplant model to evaluate the degree to which target/non-target (T/NT) ratios may be improved by pretargeting. Methods: Specific binding of an anti-human islet antibody HPi1 to the beta cells transplanted subcutaneously in mice was examined against a negative control antibody. We then compared pretargeting by MORF-HPi1 plus 111 In-labeled cMORF to direct targeting by 111 In-labeled HPi1. Results: HPi1 binding to betalox5 human cells in the transplant was shown by immunofluorescence. Normal organ 111 In backgrounds by pretargeting were always lower, although target accumulations were similar. More importantly, the transplant to pancreas and liver ratios was, respectively, 26 and 10 by pretargeting as compared to 9 and 0.6 by direct targeting. Conclusions: Pretargeting greatly improves the T/NT ratios, and based on the estimated endocrine to exocrine ratio within a pancreas, pretargeting may be approaching the sensitivity required for successful imaging of human islets within this organ.

  1. Reduced 99mTc labelled NCA-95/CEA-antibody uptake in liver due to gentle antibody reconstitution

    International Nuclear Information System (INIS)

    Reske, S.N.; Buell, U.

    1990-01-01

    The influence of reconstituting a murine monoclonal IgG 1 antibody kit with pertechnetate Tc99m on antibody distribution in the liver, spleen and sternal bone marrow of patients was examined. The 99m Tc-labelled antibody used is directed against non-specific cross-reacting antigen (NCA-95) and carcinoembryonic antigen (CEA) and has been successfully applied for imaging tissue inflammation and bone marrow scanning. Radioactivity uptake was determined in the liver, spleen, bone marrow and a precordial background region in a consecutive series of 25 patients, examined with an antibody preparation, routinely radiolabelled according to the manufacturer's recommendations and in 14 patients, in whom the antibody was reconstituted with special care, avoiding bubble formation and dropping of buffer into the antibody-containing vial. Gentle compared with routine antibody reconstitution caused a highly significant reduction of the antibody uptake in the liver, as determined by count densities, normalized to injected dose and acquisition time (13.2±5.5 vs 20.1±6.0 cpm per pixel, anti x±SD, P=0.008). The liver to background ratio was reduced from 3.4±1.4 to 1.9±0.5 (P<0.001). Spleen, sternal bone marrow and precordial background count rates were not significantly affected. These results clearly demonstrate that gentle antibody reconstitution can decrease non-specific antibody uptake in the liver by 34%±6.4% (anti x±SEM). Thus, scan quality is improved, and the potential deleterious camouflage of underlying structures is avoided. (orig.)

  2. Targeted delivery of immunotoxin by antibody to ganglioside GD3: a novel drug delivery route for tumor cells.

    Directory of Open Access Journals (Sweden)

    Vanina Torres Demichelis

    Full Text Available Gangliosides are sialic acid-containing glycolipids expressed on plasma membranes from nearly all vertebrate cells. The expression of ganglioside GD3, which plays essential roles in normal brain development, decreases in adults but is up regulated in neuroectodermal and epithelial derived cancers. R24 antibody, directed against ganglioside GD3, is a validated tumor target which is specifically endocytosed and accumulated in endosomes. Here, we exploit the internalization feature of the R24 antibody for the selective delivery of saporin, a ribosome-inactivating protein, to GD3-expressing cells [human (SK-Mel-28 and mouse (B16 melanoma cells and Chinese hamster ovary (CHO-K1 cells]. This immunotoxin showed a specific cytotoxicity on tumor cells grew on 2D monolayers, which was further evident by the lack of any effect on GD3-negative cells. To estimate the potential antitumor activity of R24-saporin complex, we also evaluated the effect of the immunotoxin on the clonogenic growth of SK-Mel-28 and CHO-K1(GD3+ cells cultured in attachment-free conditions. A drastic growth inhibition (>80-90% of the cell colonies was reached after 3 days of immunotoxin treatment. By the contrary, colonies continue to growth at the same concentration of the immuntoxin, but in the absence of R24 antibody, or in the absence of both immunotoxin and R24, undoubtedly indicating the specificity of the effect observed. Thus, the ganglioside GD3 emerge as a novel and attractive class of cell surface molecule for targeted delivery of cytotoxic agents and, therefore, provides a rationale for future therapeutic intervention in cancer.

  3. A technique to prepare boronated B72.3 monoclonal antibody for boron neutron capture therapy

    International Nuclear Information System (INIS)

    Ranadive, G.N.; Rosenzweig, H.S.; Epperly, M.W.

    1993-01-01

    B72.3 monoclonal antibody has been successfully boronated using mercaptoundecahydro-closo-dodecaborate (boron cage compound). The reagent was incorporated by first reacting the lysine residues of the antibody with m-maleimidobenzoyl succinimide ester (MBS), followed by Michael addition to the maleimido group by the mercapto boron cage compound to form a physiologically stable thioether linkage. Boron content of the antibody was determined by atomic absorption spectroscopy. For biodistribution studies, boronated antibody was radioiodinated with iodogen. 125 I-labeled and boronated B72.3 monoclonal antibody demonstrated clear tumor localization when administered via tail vein injections to athymic nude mice bearing LS174-T tumor xenografts. Boronated antibody was calculated to deliver 10 6 boron atoms per tumor cell. Although this falls short of the specific boron content originally proposed as necessary for boron neutron capture therapy (BNCT), recent calculations suggest that far fewer atoms of 10 B per tumor cell would be necessary to effect successful BNCT when the boron is targeted to the tumor cell membrane. (author)

  4. Screening for epitope specificity directly on culture supernatants in the early phase of monoclonal antibody production by an ELISA with biotin-labeled antigen.

    Science.gov (United States)

    Andersen, Ditte C; Jensen, Charlotte H; Gregersen, Annemette; Brandt, Jette; Kliem, Anette; Skjødt, Karsten; Koch, Claus; Teisner, Børge

    2004-01-01

    This report describes an assay for comparison of epitope specificity in groups of monoclonal antibodies against a given antigen. The only prerequisite is the biotin-labeled antigen. One of the monoclonal antibodies is captured onto a plastic surface via a rabbit anti-mouse Ig, and the other preincubated with biotinylated antigen. When the two antibodies react with the same epitope subsequent binding of the biotin-labeled antigen is abolished (inhibition). In the cases where no inhibition was observed, the two antibodies were considered to react with distinct, independent epitopes. The obvious advantages using this assay, are that it can be performed directly on culture supernatants in the early phase of monoclonal antibody production, and also works for antigens with repetitive epitopes. Moreover, the bonus effect, i.e., a signal in excess of the reference signal when sets of monoclonal antibodies with different epitope specificity are compared, gives a relative measure of affinity.

  5. The importance of Foxp3 antibody and fixation/permeabilization buffer combinations in identifying CD4+CD25+Foxp3+ regulatory T cells.

    Science.gov (United States)

    Law, Jacqueline P; Hirschkorn, Dale F; Owen, Rachel E; Biswas, Hope H; Norris, Philip J; Lanteri, Marion C

    2009-12-01

    Foxp3 is a key marker for CD4(+) regulatory T cells (T(regs)) and was used in developing a multiparameter flow cytometric panel to identify T(regs). Achieving reproducible staining and analysis first required optimization of Foxp3 staining. We present a comparative study of PCH101, 236A/E7, 3G3, 206D, 150D, and 259D/C7 clones of anti-human-Foxp3 antibodies used in combination with five different fixation/permeabilization buffers. Staining for CD25, CD152, and CD127 was also compared between fixation/permeabilization treatments. Promising antibody/buffer combinations were tested in a panel of peripheral blood mononuclear cells from 10 individuals, and then on fresh versus frozen cells from four individuals. Finally, different fluorochromes coupled to two representative antibodies were compared to optimize separation of Foxp3(+) from Foxp3(-) events. Foxp3 gates were set using two gating strategies based on CD127(+)CD25(-) "non-T(regs)" or based on isotype controls. For Foxp3 staining, the best conditions for fixation/permeabilization were obtained using the eBioscience Foxp3, Imgenex, BioLegend, and BD Foxp3 buffers. Comparing results from 10 subjects, 259D/C7, PCH101, 236A/E7, and 206D antibodies yielded statistically higher levels of Foxp3 cells than those by 150D and 3G3 antibodies (mean = 6.9, 5.1, 4.7, and 3.7% compared with 1.7, and 0.3% of CD25(+)Foxp3(+) events within CD4(+) cells, respectively). Importantly, the "nonspecificity" of some antibodies observed with a Foxp3 gate based on isotype controls could be eliminated by setting the Foxp3 gate on "non-T(regs)". Better separation of Foxp3(+) and Foxp3(-) populations was observed using the PCH101 clone coupled to Alexa647 compared with FITC or the 259D/C7 clone coupled to PE compared with Alexa488 fluorochrome. Foxp3 staining can be highly variable and depends on the choice of antibody/buffer pair and the fluorochrome used. Selecting the correct population for setting the Foxp3 gate is critical to avoid

  6. [Analysis of epitopes and function of anti-M3 muscarinic acetylcholine receptor antibodies in patients with Sjögren's syndrome].

    Science.gov (United States)

    Tsuboi, Hiroto; Matsuo, Naomi; Iizuka, Mana; Nakamura, Yumi; Matsumoto, Isao; Sumida, Takayuki

    2010-01-01

    Sjögren's syndrome (SS) is an autoimmune disease that affects exocrine glands including salivary and lacrimal glands. It is characterized by lymphocytic infiltration into exocrine glands, leading to dry mouth and eyes. A number of auto-antibodies, such as anti-SS-A and SS-B antibodies, are detected in patients with SS. However, no SS-specific pathologic auto-antibodies have yet been found in this condition. M3 muscarinic acetylcholine receptor (M3R) plays a crucial role in the secretion of saliva from salivary glands. It is reported that some patients with SS carried inhibitory auto-antibodies against M3R. To clarify the epitopes and function of anti-M3R antibodies in SS, we examined antibodies to the extracellular domains (N terminal region, the first, second, and third extracellular loop) of M3R by ELISA using synthesized peptide antigens encoding these domains in 42 SS and 42 healthy controls (HC). Titers and positivity of anti-M3R antibodies to every extracellular domain of M3R were significantly higher in SS than in HC. For functional analysis, human salivary gland (HSG) cells were pre-cultured with IgG from anti-M3R antibodies positive SS, negative SS, and HC. HSG cells were stimulated with cevimeline hydrochloride and intracellular calcium concentration ([Ca(2+)](i)) was measured. IgG from anti-M3R antibodies to the second loop positive SS inhibited the increase of [Ca(2+)](i), but IgG from antibodies to the N terminal or the first loop positive SS enhanced it, while IgG from antibodies to the third loop positive SS showed no effect on [Ca(2+)](i) as well as IgG from anti-M3R antibodies negative SS and HC. These findings indicated the presence of several B cell epitopes on M3R in SS and effect of anti-M3R antibodies on the salivary secretion might differ with these epitopes.

  7. Multiplex serology of paraneoplastic antineuronal antibodies.

    Science.gov (United States)

    Maat, Peter; Brouwer, Eric; Hulsenboom, Esther; VanDuijn, Martijn; Schreurs, Marco W J; Hooijkaas, Herbert; Smitt, Peter A E Sillevis

    2013-05-31

    Paraneoplastic neurological syndromes (PNS) are devastating neurological disorders secondary to cancer, associated with onconeural autoantibodies. Such antibodies are directed against neuronal antigens aberrantly expressed by the tumor. The detection of onconeural antibodies in a patient is extremely important in diagnosing a neurological syndrome as paraneoplastic (70% is not yet known to have cancer) and in directing the search for the underlying neoplasm. At present six onconeural antibodies are considered 'well characterized' and recognize the antigens HuD, CDR62 (Yo), amphiphysin, CRMP-5 (CV2), NOVA-1 (Ri), and Ma2. The gold standard of detection is the characteristic immunohistochemical staining pattern on brain tissue sections combined with confirmation by immunoblotting using recombinant purified proteins. Since all six onconeural antibodies are usually analyzed simultaneously and objective cut-off values for these analyses are warranted, we developed a multiplex assay based on Luminex technology. Reaction of serial dilutions of six onconeural standard sera with microsphere-bound antigens showed lower limits of detection than with Western blotting. Using the six standard sera at a dilution of 1:200, the average within-run coefficient of variation (CV) was 4% (range 1.9-7.3%). The average between-run within-day CV was 5.1% (range 2.9-6.7%) while the average between-day CV was 8.1% (range 2.8-11.6%). The shelf-life of the antigen coupled microspheres was at least two months. The sensitivity of the multiplex assay ranged from 83% (Ri) to 100% (Yo, amphiphysin, CV2) and the specificity from 96% (CV2) to 100% (Ri). In conclusion, Luminex-based multiplex serology is highly reproducible with high sensitivity and specificity for the detection of onconeural antibodies. Conventional immunoblotting for diagnosis of onconeural antibodies in the setting of a routine laboratory may be replaced by this novel, robust technology. Copyright © 2013 Elsevier B.V. All rights

  8. Direct solid-phase radioimmunoassay for the detection of Aujeszky's disease antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Doeller, G; Jakubik, J

    1980-06-01

    A direct solid-phase radioimmunoassy (dRIA) was developed in order to demonstrate antibodies against Aujeszky's disease virus (ADV) in sera obtained from pigs and rabbits. In the presence of guinea-pig complement the above test is 160-fold to 1500-fold more sensitive than the neutralization test (NT) and 320-fold to 150 000-fold more sensitive than sera obtained from an ADV-infected farm, which were found to be negative in the complement assisted NT. It is possible to test a single dilution of unknown serum by dRIA by comparing same with a standard curve and to make a statement regarding its ADV-specific binding capacity to /sup 125/I-labelled ADV antigen. The advantages of dRIA in comparison to the indirect RIA and the advantages and disadvantages with regard to ELISA were discussed.

  9. Synthesis-based approach toward direct sandwich immunoassay for ciguatoxin CTX3C.

    Science.gov (United States)

    Oguri, Hiroki; Hirama, Masahiro; Tsumuraya, Takeshi; Fujii, Ikuo; Maruyama, Megumi; Uehara, Hisatoshi; Nagumo, Yoko

    2003-06-25

    Ciguatoxins are the major causative toxins of ciguatera seafood poisoning. Limited availability of ciguatoxins has hampered the development of a reliable and specific immunoassay for detecting these toxins in contaminated fish. Monoclonal antibodies (mAbs) specific against both ends of ciguatoxin CTX3C were prepared by immunization of mice with protein conjugates of rationally designed synthetic haptens, 3 and 4, in place of the natural toxin. Haptenic groups that possess a surface area larger than 400 A(2) were required to produce mAbs that can bind strongly to CTX3C itself. A direct sandwich enzyme-linked immunosorbent assay (ELISA) using these mAbs was established to detect CTX3C at the ppb level with no cross-reactivity against other related marine toxins, including brevetoxin A, brevetoxin B, okadaic acid, or maitotoxin.

  10. Imaging of colorectal carcinoma with radiolabeled antibodies.

    Science.gov (United States)

    Goldenberg, D M; Goldenberg, H; Sharkey, R M; Lee, R E; Higgenbotham-Ford, E; Horowitz, J A; Hall, T C; Pinsky, C M; Hansen, H J

    1989-10-01

    Colorectal cancer has been the tumor type most frequently studied with radiolabeled antibodies. Among the various antibodies, a majority of patients with colorectal cancer have received xenogeneic polyclonal or monoclonal antibodies against carcino-embryonic antigen. This review summarizes the current status of colorectal cancer imaging with radiolabeled antibodies, ie, radioimmunodetection (RAID), and examines the published studies involving carcinoembryonic antigen (CEA) antibodies and 17-1A, 19-9, and B72.3, and other monoclonal antibodies. In order to better address the issue of the current and future clinical usefulness of this emerging technology, particular attention is given to the protocols, methods, and results of the published studies. Despite differences in study parameters, antibodies and forms, labels, administration routes and doses, and scanning instruments and methods, it has been found that (1) almost no adverse reactions have been evident; (2) antibody fragments are preferred over whole immunoglobulin G reagents because they achieve higher tumor-to-background ratios earlier, thus reducing or precluding the need for dual-isotope subtraction methods or long delays before imaging; (3) use of antibody fragments, including the monovalent Fab' form, permits imaging with short-lived radionuclides of excellent photon properties, such as 123I and 99mTc; (4) circulating antigens against which the imaging antibody is directed can complex with the injected antibody, but such complexes have not prevented successful RAID; (5) patients with high serum titers of the appropriate antigen target usually have higher rates of positive RAID; (6) patients who are seronegative for the tumor antigen being studied can have positive RAID findings, which can represent the detection of occult lesions; (7) single photon emission computed tomography appears to provide better image resolution than planar scanning; (8) regardless of the sensitivity reported in any particular

  11. Different Somatic Hypermutation Levels among Antibody Subclasses Disclosed by a New Next-Generation Sequencing-Based Antibody Repertoire Analysis

    Directory of Open Access Journals (Sweden)

    Kazutaka Kitaura

    2017-05-01

    Full Text Available A diverse antibody repertoire is primarily generated by the rearrangement of V, D, and J genes and subsequent somatic hypermutation (SHM. Class-switch recombination (CSR produces various isotypes and subclasses with different functional properties. Although antibody isotypes and subclasses are considered to be produced by both direct and sequential CSR, it is still not fully understood how SHMs accumulate during the process in which antibody subclasses are generated. Here, we developed a new next-generation sequencing (NGS-based antibody repertoire analysis capable of identifying all antibody isotype and subclass genes and used it to examine the peripheral blood mononuclear cells of 12 healthy individuals. Using a total of 5,480,040 sequences, we compared percentage frequency of variable (V, junctional (J sequence, and a combination of V and J, diversity, length, and amino acid compositions of CDR3, SHM, and shared clones in the IgM, IgD, IgG3, IgG1, IgG2, IgG4, IgA1, IgE, and IgA2 genes. The usage and diversity were similar among the immunoglobulin (Ig subclasses. Clonally related sequences sharing identical V, D, J, and CDR3 amino acid sequences were frequently found within multiple Ig subclasses, especially between IgG1 and IgG2 or IgA1 and IgA2. SHM occurred most frequently in IgG4, while IgG3 genes were the least mutated among all IgG subclasses. The shared clones had almost the same SHM levels among Ig subclasses, while subclass-specific clones had different levels of SHM dependent on the genomic location. Given the sequential CSR, these results suggest that CSR occurs sequentially over multiple subclasses in the order corresponding to the genomic location of IGHCs, but CSR is likely to occur more quickly than SHMs accumulate within Ig genes under physiological conditions. NGS-based antibody repertoire analysis should provide critical information on how various antibodies are generated in the immune system.

  12. Multiplex serology of paraneoplastic antineuronal antibodies

    NARCIS (Netherlands)

    P. Maat (Peter); E. Brouwer (Eric); E. Hulsenboom (Esther); M.M. van Duijn (Martijn); M.W.J. Schreurs (Marco); H. Hooijkaas (Herbert); P.A. Smitt (Peter)

    2013-01-01

    textabstractParaneoplastic neurological syndromes (PNS) are devastating neurological disorders secondary to cancer, associated with onconeural autoantibodies. Such antibodies are directed against neuronal antigens aberrantly expressed by the tumor. The detection of onconeural antibodies in a patient

  13. Direct evidence that the VEGF-specific antibody bevacizumab has antivascular effects in human rectal cancer

    Science.gov (United States)

    Willett, Christopher G; Boucher, Yves; di Tomaso, Emmanuelle; Duda, Dan G; Munn, Lance L; Tong, Ricky T; Chung, Daniel C; Sahani, Dushyant V; Kalva, Sanjeeva P; Kozin, Sergey V; Mino, Mari; Cohen, Kenneth S; Scadden, David T; Hartford, Alan C; Fischman, Alan J; Clark, Jeffrey W; Ryan, David P; Zhu, Andrew X; Blaszkowsky, Lawrence S; Chen, Helen X; Shellito, Paul C; Lauwers, Gregory Y; Jain, Rakesh K

    2009-01-01

    The effects of vascular endothelial growth factor (VEGF) blockade on the vascular biology of human tumors are not known. Here we show here that a single infusion of the VEGF-specific antibody bevacizumab decreases tumor perfusion, vascular volume, microvascular density, interstitial fluid pressure and the number of viable, circulating endothelial and progenitor cells, and increases the fraction of vessels with pericyte coverage in rectal carcinoma patients. These data indicate that VEGF blockade has a direct and rapid antivascular effect in human tumors. PMID:14745444

  14. Modular and aggregation resistant Vh antibodies from a phage display library

    DEFF Research Database (Denmark)

    Friis, Niels Anton; Mandrup, Ole Aalund; Lykkemark, Simon

    2012-01-01

    through immunisation of sharks or camels, or alternatively from recombinant libraries1. The domain antibodies have certain advantages, both pharmacologically and technically. Here we report the construction of a semi-synthetic and highly modular antibody library, based on a human framework (V3-23/D47......Directed evolution of antibodies through phage display is a powerful technique for producing binders of various biological targets. One of the recent innovations in the fi eld is the domain antibody, an antibody consisting only of a single variable domain. These anti bodies can be obtained either......). The antibody scaffold has been codon optimised to improve expression, and the CDR’s have been created using trinucleotide synthesis. These methods give a strict control over the randomisations, and the ability to design a library with minimal aggregation propensity. To facilitate further manipulation, unique...

  15. Homology of ab1 and ab3 monoclonal antibodies that neutralize Semliki Forest virus

    NARCIS (Netherlands)

    Fernandez, IM; Bos, NA; Harmsen, M; Verheul, AFM; Snippe, H; Kraaijeveld, CA

    2001-01-01

    A noninternal image monoclonal antiidiotypic antibody (ab2 mAb), designated 1,13A321, that had proved its efficacy as vaccine against infection with Semliki Forest virus (SFV) in BALB/c mice, was used as immunogen to generate a panel of SFV-neutralizing monoclonal anti-anti-idiotypic antibodies (ab3

  16. Docking of Antibodies into Cavities in DNA Origami

    DEFF Research Database (Denmark)

    Quyang, X; Stefano, Mattia De; Krissanaprasit, Abhichart

    2017-01-01

    microscopy (AFM) and transmission electron microscopy (TEM) validated efficient antibody immobilization in the origami structures. The increased ability to control the orientation of antibodies in nanostructures and at surfaces has potential for directing the interactions of antibodies with targets...

  17. Production of monoclonal antibodies for sandwich immunoassay detection of ciguatoxin 51-hydroxyCTX3C.

    Science.gov (United States)

    Tsumuraya, Takeshi; Fujii, Ikuo; Inoue, Masayuki; Tatami, Atsushi; Miyazaki, Keisuke; Hirama, Masahiro

    2006-09-01

    Every year, more than 50,000 people in subtropical and tropical regions suffer from ciguatera seafood poisoning. The extremely low level of the causative neurotoxins (ciguatoxins) in fish has hampered the preparation of antibodies for detection of the toxins. In this study, we produced a monoclonal antibody (8H4) against the right end of ciguatoxin CTX1B (1) and 51-hydroxyCTX3C (3) by immunizing mice with the keyhole limpet hemocyanin-conjugate of the synthetic HIJKLM ring fragment (10). We used 8H4 and another previously reported monoclonal antibody (10C9) that recognizes the left end of 3 to develop a sandwich enzyme-linked immunosorbent assay (ELISA) to detect 3. The assay could detect 3 down to the ppb level and lacked cross-reactivity with other related marine toxins, including brevetoxin A, brevetoxin B, okadaic acid, and maitotoxin.

  18. Dual Mechanism of Interleukin-3 Receptor Blockade by an Anti-Cancer Antibody

    Directory of Open Access Journals (Sweden)

    Sophie E. Broughton

    2014-07-01

    Full Text Available Interleukin-3 (IL-3 is an activated T cell product that bridges innate and adaptive immunity and contributes to several immunopathologies. Here, we report the crystal structure of the IL-3 receptor α chain (IL3Rα in complex with the anti-leukemia antibody CSL362 that reveals the N-terminal domain (NTD, a domain also present in the granulocyte-macrophage colony-stimulating factor (GM-CSF, IL-5, and IL-13 receptors, adopting unique “open” and classical “closed” conformations. Although extensive mutational analyses of the NTD epitope of CSL362 show minor overlap with the IL-3 binding site, CSL362 only inhibits IL-3 binding to the closed conformation, indicating alternative mechanisms for blocking IL-3 signaling. Significantly, whereas “open-like” IL3Rα mutants can simultaneously bind IL-3 and CSL362, CSL362 still prevents the assembly of a higher-order IL-3 receptor-signaling complex. The discovery of open forms of cytokine receptors provides the framework for development of potent antibodies that can achieve a “double hit” cytokine receptor blockade.

  19. Kotai Antibody Builder: automated high-resolution structural modeling of antibodies.

    Science.gov (United States)

    Yamashita, Kazuo; Ikeda, Kazuyoshi; Amada, Karlou; Liang, Shide; Tsuchiya, Yuko; Nakamura, Haruki; Shirai, Hiroki; Standley, Daron M

    2014-11-15

    Kotai Antibody Builder is a Web service for tertiary structural modeling of antibody variable regions. It consists of three main steps: hybrid template selection by sequence alignment and canonical rules, 3D rendering of alignments and CDR-H3 loop modeling. For the last step, in addition to rule-based heuristics used to build the initial model, a refinement option is available that uses fragment assembly followed by knowledge-based scoring. Using targets from the Second Antibody Modeling Assessment, we demonstrate that Kotai Antibody Builder generates models with an overall accuracy equal to that of the best-performing semi-automated predictors using expert knowledge. Kotai Antibody Builder is available at http://kotaiab.org standley@ifrec.osaka-u.ac.jp. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  20. Three-site sandwich radioimmunoassay with monoclonal antibodies for a sensitive determination of human alpha-fetoprotein

    International Nuclear Information System (INIS)

    Nomura, M.; Imai, M.; Takahashi, K.; Kumakura, T.; Tachibana, K.; Aoyagi, S.; Usuda, S.; Nakamura, T.; Miyakawa, Y.; Mayumi, M.

    1983-01-01

    Utilizing monoclonal antibodies against human alpha-fetoprotein, 3 distinct antigenic determinants were identified. These antigenic determinants, provisionally designated a, b and c, were arranged in such a manner that the binding of one determinant with the corresponding antibody did not inhibit, or only barely inhibited the binding of antibodies directed to the other 2 determinants. Monoclonal antibodies with 3 different specificities were, therefore, applied to develop a sandwich-type solid-phase radioimmunoassay of the antigen in which wells were coated with anti-a, and radiolabeled anti-b together with radiolabeled anti-c was employed to detect the bound antigen. The 3-site sandwich radioimmunoassay involving 3 different determinants gave a higher sensitivity than 2-site assays in which only anti-b or anti-c was employed as a radiolabeled reagent, because the radioactivity of the 2 labeled antibodies was added on the antigen bound to immobilized anti-a. (Auth.)

  1. Antibody immobilized on Fe3O4 particles and its application to RIAs

    International Nuclear Information System (INIS)

    Shen Rongsen; Xing Ruiyun

    1997-01-01

    A magnetic particle second antibody (MSA-I) was prepared by means of immobilizing donkey anti-rabbit antiserum on Fe 3 O 4 particles 10.8 nm +- 34% in diameter. Effects of some factors, such as pH of buffer used for immobilizing antiserum, amount of antiserum, time of immobilizing antiserum and blocking buffer on specific and nonspecific binding of MSA-I in RIAs were studied. The MSA-I was successfully applied to RIAs of T 3 , T 4 and TSH. The advantages of the magnetic second antibody were simplicity and time-saving in preparation and low cost

  2. New epitopes and function of anti-M3 muscarinic acetylcholine receptor antibodies in patients with Sjögren's syndrome.

    Science.gov (United States)

    Tsuboi, H; Matsumoto, I; Wakamatsu, E; Nakamura, Y; Iizuka, M; Hayashi, T; Goto, D; Ito, S; Sumida, T

    2010-10-01

    M3 muscarinic acetylcholine receptor (M3R) plays a crucial role in the secretion of saliva from salivary glands. It is reported that some patients with Sjögren's syndrome (SS) carried inhibitory autoantibodies against M3R. The purpose of this study is to clarify the epitopes and function of anti-M3R antibodies in SS. We synthesized peptides encoding the extracellular domains of human-M3R including the N-terminal region and the first, second and third extracellular loops. Antibodies against these regions were examined by enzyme-linked immunosorbent assay in sera from 42 SS and 42 healthy controls. For functional analysis, human salivary gland (HSG) cells were preincubated with immunoglobulin G (IgG) separated from sera of anti-M3R antibody-positive SS, -negative SS and controls for 12 h. After loading with Fluo-3, HSG cells were stimulated with cevimeline hydrochloride, and intracellular Ca(2+) concentrations [(Ca(2+) )i] were measured. Antibodies to the N-terminal, first, second and third loops were detected in 42·9% (18 of 42), 47·6% (20 of 42), 54·8% (23 of 42) and 45·2% (19 of 42) of SS, while in 4·8% (two of 42), 7·1% (three of 42), 2·4% (one of 42) and 2·4% (one of 42) of controls, respectively. Antibodies to the second loop positive SS-IgG inhibited the increase of (Ca(2+) )i induced by cevimeline hydrochloride. Antibodies to the N-terminal positive SS-IgG and antibodies to the first loop positive SS-IgG enhanced it, while antibodies to the third loop positive SS-IgG showed no effect on (Ca(2+) )i as well as anti-M3R antibody-negative SS-IgG. Our results indicated the presence of several B cell epitopes on M3R in SS. The influence of anti-M3R antibodies on salivary secretion might differ based on these epitopes. © 2010 The Authors. Clinical and Experimental Immunology © 2010 British Society for Immunology.

  3. Preparation of 188Re labelled antibodies

    International Nuclear Information System (INIS)

    Zhu Minghua; Cao Rongzhen; Li Wenxin; Sheng Rong; Yin Duanzhi; He Weiyu; Zhou Wei; Wang Yongxian

    1998-01-01

    A simple technique of directly labelling antibodies with 188 Re has been developed. The reduction of antibody disulfide groups was achieved by incubation of antibody with ascorbic acid (pH = 6.5) for an hour at room temperature and a solution of excess SnCl 2 in sodium gluconate was added to the AA-reduced antibody followed by the addition of perrhenate. Some factors that influence labelling efficiency, such as the pH of the reaction mixture, the labelling time, and the amount of antibodies and reductive agent, were studied experimentally and a better labelling method was established. The labelling yields, as determined by paper chromatography, were greater than 80%

  4. Membrane-associated precursor to poliovirus VPg identified by immunoprecipitation with antibodies directed against a synthetic heptapeptide

    International Nuclear Information System (INIS)

    Semelr, B.L.; Anderson, C.W.; Hanecak, R.; Dorner, L.F.; Wimmer, E.

    1982-01-01

    A synthetic heptapeptide corresponding to the C-terminal sequence of the poliovirus genome protein (VPg) has been linked to bovine serum albumin and used to raise antibodies in rabbits. These antibodies precipitate not only VPg but also at least two more virus-specific polypeptides. The smaller polypeptide, denoted P3-9 (12,000 daltons), has been mapped by Edman degradation and by fragmentation with cyanogen bromide and determined to be the N-terminal cleavage product of polypeptide P3-1b, a precursor to the RNA polymerase. P3-9 contains the sequence of the basic protein VPg (22 amino acids) at its C terminus. As predicted by the known RNA sequence of poliovirus, P3-9 also contains a hydrophobic region of 22 amino acids preceding VPg, an observation suggesting that P3-9 may be membrane-associated. This was confirmed by fractionation of infected cells in the presence or absence of detergent. We speculate that P3-9 may be the donor of VPg to RNA chains in the membrane-bound RNA replication complex

  5. Identitetshantering på Facebook : En kvalitativ studie om förhållningssättet till den egna identiteten hos två olika åldersgrupper på vår tids mest offentliga scen

    OpenAIRE

    Kåge, Isabelle

    2010-01-01

    Title: Identity management on Facebook: a qualitative study in approaches to personal identity between two different age groups on the most public stage of our time (Identitetshantering på Facebook: En kvalitativ studie om förhållningssättet till den egna identiteten hos två olika åldersgrupper på vår tids mest offentliga scen). Number of pages: 43. Author: Isabelle Kåge. Tutor: Amelie Hössjer. Course: Media and Communication Studies C. Period: Fall Semester 2010. University: Division of Medi...

  6. Higher cytotoxicity of divalent antibody-toxins than monovalent antibody-toxins

    International Nuclear Information System (INIS)

    Won, JaeSeon; Nam, PilWon; Lee, YongChan; Choe, MuHyeon

    2009-01-01

    Recombinant antibody-toxins are constructed via the fusion of a 'carcinoma-specific' antibody fragment to a toxin. Due to the high affinity and high selectivity of the antibody fragments, antibody-toxins can bind to surface antigens on cancer cells and kill them without harming normal cells [L.H. Pai, J.K. Batra, D.J. FitzGerald, M.C. Willingham, I. Pastan, Anti-tumor activities of immunotoxins made of monoclonal antibody B3 and various forms of Pseudomonas exotoxin, Proc. Natl. Acad. Sci. USA 88 (1991) 3358-3362]. In this study, we constructed the antibody-toxin, Fab-SWn-PE38, with SWn (n = 3, 6, 9) sequences containing n-time repeated (G 4 S) between the Fab fragment and PE38 (38 kDa truncated form of Pseudomonas exotoxin A). The SWn sequence also harbored one cysteine residue that could form a disulfide bridge between two Fab-SWn-PE38 monomers. We assessed the cytotoxicity of the monovalent (Fab-SWn-PE38), and divalent ([Fab-SWn-PE38] 2 ) antibody-toxins. The cytotoxicity of the dimer against the CRL1739 cell line was approximately 18.8-fold higher than that of the monomer on the ng/ml scale, which was approximately 37.6-fold higher on the pM scale. These results strongly indicate that divalency provides higher cytotoxicity for an antibody-toxin.

  7. Radiolabeled antibody imaging

    International Nuclear Information System (INIS)

    Wahl, R.L.

    1987-01-01

    Radiolabeled antibodies, in particular monoclonal antibodies, offer the potential for the specific nuclear imaging of malignant and benign diseases in man. If this imaging potential is realized, they may also have a large role in cancer treatment. This paper reviews: (1) what monoclonal antibodies are and how they differ from polyclonal antibodies, (2) how they are produced and radiolabeled, (3) the results of preclinical and clinical trials in cancer imaging, including the utility of SPECT and antibody fragments, (4) the role of antibodies in the diagnosis of benign diseases, (5) alternate routes of antibody delivery, (6) the role of these agents in therapy, and (7) whether this technology ''revolutionizes'' the practice of nuclear radiology, or has a more limited complementary role in the imaging department

  8. Development of Immunoassay Based on Monoclonal Antibody Reacted with the Neonicotinoid Insecticides Clothianidin and Dinotefuran

    OpenAIRE

    Uchigashima, Mikiko; Watanabe, Eiki; Ito, Shigekazu; Iwasa, Seiji; Miyake, Shiro

    2012-01-01

    Enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody (MoAb) was developed for the neonicotinoid insecticide clothianidin. A new clothianidin hapten (3-[5-(3-methyl-2-nitroguanidinomethyl)-1,3-thiazol-2-ylthio] propionic acid) was synthesized and conjugated to keyhole limpet hemocyanin, and was used for monoclonal antibody preparation. The resulting MoAb CTN-16A3-13 was characterized by a direct competitive ELISA (dc-ELISA). The 50% of inhibition concentration value with cl...

  9. Bispecific Antibody Pretargeting for Improving Cancer Imaging and Therapy

    Energy Technology Data Exchange (ETDEWEB)

    Sharkey, Robert M.

    2005-02-04

    The main objective of this project was to evaluate pretargeting systems that use a bispecific antibody (bsMAb) to improve the detection and treatment of cancer. A bsMAb has specificity to a tumor antigen, which is used to bind the tumor, while the other specificity is to a peptide that can be radiolabeled. Pretargeting is the process by which the unlabeled bsMAb is given first, and after a sufficient time (1-2 days) is given for it to localize in the tumor and clear from the blood, a small molecular weight radiolabeled peptide is given. According to a dynamic imaging study using a 99mTc-labeled peptide, the radiolabeled peptide localizes in the tumor in less than 1 hour, with > 80% of it clearing from the blood and body within this same time. Tumor/nontumor targeting ratios that are nearly 50 times better than that with a directly radiolabeled Fab fragment have been observed (Sharkey et al., ''Signal amplification in molecular imaging by a multivalent bispecific nanobody'' submitted). The bsMAbs used in this project have been composed of 3 antibodies that will target antigens found in colorectal and pancreatic cancers (CEA, CSAp, and MUC1). For the ''peptide binding moiety'' of the bsMAb, we initially examined an antibody directed to DOTA, but subsequently focused on another antibody directed against a novel compound, HSG (histamine-succinyl-glycine).

  10. Ex-vivo expanded human NK cells express activating receptors that mediate cytotoxicity of allogeneic and autologous cancer cell lines by direct recognition and antibody directed cellular cytotoxicity

    Directory of Open Access Journals (Sweden)

    Campana Dario

    2010-10-01

    Full Text Available Abstract Background The possibility that autologous NK cells could serve as an effective treatment modality for solid tumors has long been considered. However, implementation is hampered by (i the small number of NK cells in peripheral blood, (ii the difficulties associated with large-scale production of GMP compliant cytolytic NK cells, (iii the need to activate the NK cells in order to induce NK cell mediated killing and (iv the constraints imposed by autologous inhibitory receptor-ligand interactions. To address these issues, we determined (i if large numbers of NK cells could be expanded from PBMC and GMP compliant cell fractions derived by elutriation, (ii their ability to kill allogeneic and autologous tumor targets by direct cytotoxitiy and by antibody-mediated cellular cytotoxicity and (iii defined NK cell specific receptor-ligand interactions that mediate tumor target cell killing. Methods Human NK cells were expanded during 14 days. Expansion efficiency, NK receptor repertoire before and after expansion, expression of NK specific ligands, cytolytic activity against allogeneic and autologous tumor targets, with and without the addition of chimeric EGFR monoclonal antibody, were investigated. Results Cell expansion shifted the NK cell receptor repertoire towards activation and resulted in cytotoxicity against various allogeneic tumor cell lines and autologous gastric cancer cells, while sparing normal PBMC. Blocking studies confirmed that autologous cytotoxicity is established through multiple activating receptor-ligand interactions. Importantly, expanded NK cells also mediated ADCC in an autologous and allogeneic setting by antibodies that are currently being used to treat patients with select solid tumors. Conclusion These data demonstrate that large numbers of cytolytic NK cells can be generated from PBMC and lymphocyte-enriched fractions obtained by GMP compliant counter current elutriation from PBMC, establishing the preclinical

  11. Generation of a C3c specific monoclonal antibody and assessment of C3c as a putative inflammatory marker derived from complement factor C3

    DEFF Research Database (Denmark)

    Palarasah, Yaseelan; Skjodt, Karsten; Brandt, Jette

    2010-01-01

    complex (C5b-C9) and quantification of complement split products by precipitation-in-gel techniques (e.g. C3d). We have developed a mouse monoclonal antibody (mAb) that is able to detect fluid phase C3c without interference from other products generated from the complement component C3. The C3c specific m....... The C3c mAb was confirmed to be C3c specific, as it showed no cross-reactivity with native (un-cleaved) C3, with C3b, iC3b, or with C3d. Also, no significant reaction was observed with C3 fragments in factor I deficient sera or plasma. This antibody forms the basis for the generation of a robust ELISA...... that allows for a quick and reliable evaluation of complement activation and consumption as a marker for inflammatory processes. We established the C3c plasma range in 100 healthy Danish blood donors with a mean of 3.47 μg/ml and a range of 2.12-4.92 μg/ml. We believe that such an antibody might...

  12. Recombinant AAV-mediated in vivo long-term expression and antitumour activity of an anti-ganglioside GM3(Neu5Gc) antibody.

    Science.gov (United States)

    Piperno, G M; López-Requena, A; Predonzani, A; Dorvignit, D; Labrada, M; Zentilin, L; Burrone, O R; Cesco-Gaspere, M

    2015-12-01

    The ganglioside GM3(Neu5Gc) has gained increasing attention as therapeutic target because of its selective expression in various human tumours, such as melanoma, breast and lung cancer. 14F7 is a mouse IgG1 with specific reactivity to GM3(Neu5Gc)-positive tumours. The therapeutic activity of 14F7 has also been demonstrated in vivo, through its repetitive passive administration in tumour-bearing animals. In this work we used an alternative strategy to deliver recombinant 14F7 in vivo and analysed the therapeutic efficacy of this approach. We engineered a recombinant adeno-associated vector to direct the expression of secretable recombinant 14F7 in BALB/c animals. A single administration of the rAAV induced efficient production and secretion of the antibody in the bloodstream, with an expression level reaching plateau at ∼3 weeks after injection and persisting for almost a year. Strikingly, upon challenge with GM3(Neu5Gc)-positive X63-AG8.653 myeloma cells, tumour development was significantly delayed in animals treated with rAAV-14F7 with respect to animals treated with a control rAAV codifying for an irrelevant antibody. Finally, no significant differences in survival proportion were detected in animals injected with rAAV-14F7 or treated by standard administration of repetitive doses of purified monoclonal antibody 14F7.

  13. Glycoengineered Monoclonal Antibodies with Homogeneous Glycan (M3, G0, G2, and A2) Using a Chemoenzymatic Approach Have Different Affinities for FcγRIIIa and Variable Antibody-Dependent Cellular Cytotoxicity Activities.

    Science.gov (United States)

    Kurogochi, Masaki; Mori, Masako; Osumi, Kenji; Tojino, Mami; Sugawara, Shu-Ichi; Takashima, Shou; Hirose, Yuriko; Tsukimura, Wataru; Mizuno, Mamoru; Amano, Junko; Matsuda, Akio; Tomita, Masahiro; Takayanagi, Atsushi; Shoda, Shin-Ichiro; Shirai, Takashi

    2015-01-01

    Many therapeutic antibodies have been developed, and IgG antibodies have been extensively generated in various cell expression systems. IgG antibodies contain N-glycans at the constant region of the heavy chain (Fc domain), and their N-glycosylation patterns differ during various processes or among cell expression systems. The Fc N-glycan can modulate the effector functions of IgG antibodies, such as antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). To control Fc N-glycans, we performed a rearrangement of Fc N-glycans from a heterogeneous N-glycosylation pattern to homogeneous N-glycans using chemoenzymatic approaches with two types of endo-β-N-acetyl glucosaminidases (ENG'ases), one that works as a hydrolase to cleave all heterogeneous N-glycans, another that is used as a glycosynthase to generate homogeneous N-glycans. As starting materials, we used an anti-Her2 antibody produced in transgenic silkworm cocoon, which consists of non-fucosylated pauci-mannose type (Man2-3GlcNAc2), high-mannose type (Man4-9GlcNAc2), and complex type (Man3GlcNAc3-4) N-glycans. As a result of the cleavage of several ENG'ases (endoS, endoM, endoD, endoH, and endoLL), the heterogeneous glycans on antibodies were fully transformed into homogeneous-GlcNAc by a combination of endoS, endoD, and endoLL. Next, the desired N-glycans (M3; Man3GlcNAc1, G0; GlcNAc2Man3GlcNAc1, G2; Gal2GlcNAc2Man3GlcNAc1, A2; NeuAc2Gal2GlcNAc2Man3GlcNAc1) were transferred from the corresponding oxazolines to the GlcNAc residue on the intact anti-Her2 antibody with an ENG'ase mutant (endoS-D233Q), and the glycoengineered anti-Her2 antibody was obtained. The binding assay of anti-Her2 antibody with homogenous N-glycans with FcγRIIIa-V158 showed that the glycoform influenced the affinity for FcγRIIIa-V158. In addition, the ADCC assay for the glycoengineered anti-Her2 antibody (mAb-M3, mAb-G0, mAb-G2, and mAb-A2) was performed using SKBR-3 and BT-474 as target cells, and

  14. Glycoengineered Monoclonal Antibodies with Homogeneous Glycan (M3, G0, G2, and A2 Using a Chemoenzymatic Approach Have Different Affinities for FcγRIIIa and Variable Antibody-Dependent Cellular Cytotoxicity Activities.

    Directory of Open Access Journals (Sweden)

    Masaki Kurogochi

    Full Text Available Many therapeutic antibodies have been developed, and IgG antibodies have been extensively generated in various cell expression systems. IgG antibodies contain N-glycans at the constant region of the heavy chain (Fc domain, and their N-glycosylation patterns differ during various processes or among cell expression systems. The Fc N-glycan can modulate the effector functions of IgG antibodies, such as antibody-dependent cellular cytotoxicity (ADCC and complement dependent cytotoxicity (CDC. To control Fc N-glycans, we performed a rearrangement of Fc N-glycans from a heterogeneous N-glycosylation pattern to homogeneous N-glycans using chemoenzymatic approaches with two types of endo-β-N-acetyl glucosaminidases (ENG'ases, one that works as a hydrolase to cleave all heterogeneous N-glycans, another that is used as a glycosynthase to generate homogeneous N-glycans. As starting materials, we used an anti-Her2 antibody produced in transgenic silkworm cocoon, which consists of non-fucosylated pauci-mannose type (Man2-3GlcNAc2, high-mannose type (Man4-9GlcNAc2, and complex type (Man3GlcNAc3-4 N-glycans. As a result of the cleavage of several ENG'ases (endoS, endoM, endoD, endoH, and endoLL, the heterogeneous glycans on antibodies were fully transformed into homogeneous-GlcNAc by a combination of endoS, endoD, and endoLL. Next, the desired N-glycans (M3; Man3GlcNAc1, G0; GlcNAc2Man3GlcNAc1, G2; Gal2GlcNAc2Man3GlcNAc1, A2; NeuAc2Gal2GlcNAc2Man3GlcNAc1 were transferred from the corresponding oxazolines to the GlcNAc residue on the intact anti-Her2 antibody with an ENG'ase mutant (endoS-D233Q, and the glycoengineered anti-Her2 antibody was obtained. The binding assay of anti-Her2 antibody with homogenous N-glycans with FcγRIIIa-V158 showed that the glycoform influenced the affinity for FcγRIIIa-V158. In addition, the ADCC assay for the glycoengineered anti-Her2 antibody (mAb-M3, mAb-G0, mAb-G2, and mAb-A2 was performed using SKBR-3 and BT-474 as target

  15. From hybridomas to a robust microalgal-based production platform: molecular design of a diatom secreting monoclonal antibodies directed against the Marburg virus nucleoprotein.

    Science.gov (United States)

    Hempel, Franziska; Maurer, Michael; Brockmann, Björn; Mayer, Christian; Biedenkopf, Nadine; Kelterbaum, Anne; Becker, Stephan; Maier, Uwe G

    2017-07-27

    The ideal protein expression system should provide recombinant proteins in high quality and quantity involving low production costs only. However, especially for complex therapeutic proteins like monoclonal antibodies many challenges remain to meet this goal and up to now production of monoclonal antibodies is very costly and delicate. Particularly, emerging disease outbreaks like Ebola virus in Western Africa in 2014-2016 make it necessary to reevaluate existing production platforms and develop robust and cheap alternatives that are easy to handle. In this study, we engineered the microalga Phaeodactylum tricornutum to produce monoclonal IgG antibodies against the nucleoprotein of Marburg virus, a close relative of Ebola virus causing severe hemorrhagic fever with high fatality rates in humans. Sequences for both chains of a mouse IgG antibody were retrieved from a murine hybridoma cell line and implemented in the microalgal system. Fully assembled antibodies were shown to be secreted by the alga and antibodies were proven to be functional in western blot, ELISA as well as IFA studies just like the original hybridoma produced IgG. Furthermore, synthetic variants with constant regions of a rabbit IgG and human IgG with optimized codon usage were produced and characterized. This study highlights the potential of microalgae as robust and low cost expression platform for monoclonal antibodies secreting IgG antibodies directly into the culture medium. Microalgae possess rapid growth rates, need basically only water, air and sunlight for cultivation and are very easy to handle.

  16. Nitrous oxide emission from the agriculture in the Netherlands. Outline of effects of manure and ammonia measures; Lachgasemissie uit de Nederlandse landbouw. Verkenning effecten mest- en ammoniakmaatregelen

    Energy Technology Data Exchange (ETDEWEB)

    Kros, J.; De Vries, W.; Oenema, O.; Velthof, G.; Kuikman, P.J.; Van Hove, B. [Divisie Bodem, Alterra, Wageningen Universiteit en Research Centrum, Wageningen (Netherlands)

    2004-03-01

    By means of the model INITIATOR an integral outline was made of the effects of several measures within the framework of the manure and ammonia policy focusing on nitrous oxide emission. The results are compared with the IPCC method. The model INITIATOR is a mechanistic model by means of which the carbon cycle in the Netherlands can be differentiated on a regional scale as a function of land use, type of soil and hydrology. [Dutch] Met behulp van het model INITIATOR is een integrale verkenning gemaakt van de effecten van verschillende maatregelen in het kader van het mest- en ammoniakbeleid op de emissie van Iachgas.Voor de huidige situatie zijn de uitkomsten vergeleken met die van de gangbare IPCC-methode (International Panel on Climate Change)

  17. Development of magnetic microparticle antibodies and their application to RIA's for the evaluation of the thyroid function

    International Nuclear Information System (INIS)

    Shen Rongsen; Wang Renzhi; Xing Ruiyun; Li Yingqi; Zhou Fengqi; Jiang Shaohua; Lin Zhihao; Xu Banglei

    1996-01-01

    Three type of magnetic microparticle antibodies were developed: (1) magnetic second antibody I (MSA-I) where the antibody molecules were directly immobilized by physical adsorption on Fe 3 O 4 microparticles (magnetic nucleus, MN) 10 nm ± 34% in diameter; (2) magnetic second antibody II (MSA-II) where the antibody molecules were immobilized by chemical coupling on the MN coated with polyacrolein; and (3) magnetic first antibody (MFA-T 3 ) where the anti-T 3 antibody molecules were specifically immobilized by immunoadsorption to the second antibody molecules of the MSA-II. The optimal conditions for their preparation were elaborated. Some physical, chemical and immunological characteristics of these magnetic microparticles were described. The application of the MSA-I, MSAII and MFA-T 3 to RIAs for evaluation of thyroid function, such as triiodothyronine (T 3 ), reverse T 3 (rT 3 ), free T 3 (fT 3 ), thyroxine (T 4 ), free T 4 (fT 4 ), thyroid-stimulating hormone (TSH), thyroglobulin (TG) and TG-antibody (TG-Ab) etc., was reported. The preparation of MFA-T 3 was based on a new model of immobilized antibodies. It was shown that this immobilized model was successful but some technical problems related to the performance of antibody magnetic support remained to be solved. The MSA-I had an advantage of extremely simple preparation and could be prepared and used in anyone's own laboratory. The MSA-II exhibited more perfect performance and was therefore highly recommended. It is a universal immunoseparation reagent and could be successfully applied to different RIA items. (author). 14 refs, 10 figs, 6 tabs

  18. [Neuroimmunological diseases associated with VGKC complex antibodies].

    Science.gov (United States)

    Watanabe, Osamu

    2013-05-01

    Antibodies to voltage-gated potassium channels(VGKC) were first identified by radioimmunoassay of radioisotope labeled alpha-dendrotoxin-VGKCs solubilized from rabbit brain. These antibodies were found only in a proportion of patients with acquired neuromyotonia (Isaacs' syndrome). VGKC antibodies were also detected in Morvan's syndrome and in a form of autoimmune limbic encephalitis. Recent studies indicated that the "VGKC" antibodies are mainly directed toward associated proteins(for example LGI-1, Caspr-2) that complex with the VGKCs themselves. The "VGKC" antibodies are now usually known as VGKC-complex antibodies. In general, LGI-1 antibodies are most common in limbic encephalitis with SIADH. Caspr-2 antibodies are present in the majority of patients with Morvan's syndrome. These patients develop combinations of CNS symptoms, autonomic dysfunction, and peripheral nerve hyperexcitability.

  19. Monoclonal antibodies directed to human insulin-like growth factor I (IGF I)

    International Nuclear Information System (INIS)

    Laubli, U.K.; Baier, W.; Celio, M.R.; Binz, H.; Humbel, R.E.

    1982-01-01

    Mouse hybridomas secreting antibodies to human insulin-like growth factor I (IGF I) were produced by fusion of spleen cells of hyperimmunised mice with FO mouse-myeloma cells. Eight clones producing antibodies against human IGF I have been isolated, two of which have been characterised. One was used in a radioimmunoassay, the other for immunopurification of IGF. (Auth.)

  20. Repertoire Analysis of Antibody CDR-H3 Loops Suggests Affinity Maturation Does Not Typically Result in Rigidification

    Directory of Open Access Journals (Sweden)

    Jeliazko R. Jeliazkov

    2018-03-01

    Full Text Available Antibodies can rapidly evolve in specific response to antigens. Affinity maturation drives this evolution through cycles of mutation and selection leading to enhanced antibody specificity and affinity. Elucidating the biophysical mechanisms that underlie affinity maturation is fundamental to understanding B-cell immunity. An emergent hypothesis is that affinity maturation reduces the conformational flexibility of the antibody’s antigen-binding paratope to minimize entropic losses incurred upon binding. In recent years, computational and experimental approaches have tested this hypothesis on a small number of antibodies, often observing a decrease in the flexibility of the complementarity determining region (CDR loops that typically comprise the paratope and in particular the CDR-H3 loop, which contributes a plurality of antigen contacts. However, there were a few exceptions and previous studies were limited to a small handful of cases. Here, we determined the structural flexibility of the CDR-H3 loop for thousands of recent homology models of the human peripheral blood cell antibody repertoire using rigidity theory. We found no clear delineation in the flexibility of naïve and antigen-experienced antibodies. To account for possible sources of error, we additionally analyzed hundreds of human and mouse antibodies in the Protein Data Bank through both rigidity theory and B-factor analysis. By both metrics, we observed only a slight decrease in the CDR-H3 loop flexibility when comparing affinity matured antibodies to naïve antibodies, and the decrease was not as drastic as previously reported. Further analysis, incorporating molecular dynamics simulations, revealed a spectrum of changes in flexibility. Our results suggest that rigidification may be just one of many biophysical mechanisms for increasing affinity.

  1. Focal CA3 hippocampal subfield atrophy following LGI1 VGKC-complex antibody limbic encephalitis

    OpenAIRE

    Miller, T; Chong, T; Aimola Davies, A; Ng, T; Johnson, M; Irani, S; Vincent, A; Husain, M; Jacob, S; Maddison, P; Kennard, C; Gowland, P; Rosenthal, C

    2017-01-01

    Magnetic resonance imaging has linked chronic voltage-gated potassium channel (VGKC) complex antibody-mediated limbic encephalitis with generalized hippocampal atrophy. However, autoantibodies bind to specific rodent hippocampal subfields. Here, human hippocampal subfield (subiculum, cornu ammonis 1-3, and dentate gyrus) targets of immunomodulation-treated LGI1 VGKC-complex antibody-mediated limbic encephalitis were investigated using in vivo ultra-high resolution (0.39 × 0....

  2. A simple vector system to improve performance and utilisation of recombinant antibodies

    Directory of Open Access Journals (Sweden)

    Vincent Karen J

    2006-12-01

    Full Text Available Abstract Background Isolation of recombinant antibody fragments from antibody libraries is well established using technologies such as phage display. Phage display vectors are ideal for efficient display of antibody fragments on the surface of bacteriophage particles. However, they are often inefficient for expression of soluble antibody fragments, and sub-cloning of selected antibody populations into dedicated soluble antibody fragment expression vectors can enhance expression. Results We have developed a simple vector system for expression, dimerisation and detection of recombinant antibody fragments in the form of single chain Fvs (scFvs. Expression is driven by the T7 RNA polymerase promoter in conjunction with the inducible lysogen strain BL21 (DE3. The system is compatible with a simple auto-induction culture system for scFv production. As an alternative to periplasmic expression, expression directly in the cytoplasm of a mutant strain with a more oxidising cytoplasmic environment (Origami 2™ (DE3 was investigated and found to be inferior to periplasmic expression in BL21 (DE3 cells. The effect on yield and binding activity of fusing scFvs to the N terminus of maltose binding protein (a solubility enhancing partner, bacterial alkaline phosphatase (a naturally dimeric enzymatic reporter molecule, or the addition of a free C-terminal cysteine was determined. Fusion of scFvs to the N-terminus of maltose binding protein increased scFv yield but binding activity of the scFv was compromised. In contrast, fusion to the N-terminus of bacterial alkaline phosphatase led to an improved performance. Alkaline phosphatase provides a convenient tag allowing direct enzymatic detection of scFv fusions within crude extracts without the need for secondary reagents. Alkaline phosphatase also drives dimerisation of the scFv leading to an improvement in performance compared to monovalent constructs. This is illustrated by ELISA, western blot and

  3. Serologic aspects of IgG4 antibodies. II. IgG4 antibodies form small, nonprecipitating immune complexes due to functional monovalency

    NARCIS (Netherlands)

    van der Zee, J. S.; van Swieten, P.; Aalberse, R. C.

    1986-01-01

    Human IgG4 antibodies directed against phospholipase A, the P1 antigen from Dermatophagoïdes pteronyssinus extracts, and cat albumin were found unable to cross-link antigen. Previously, it was demonstrated that IgG4 antibodies, in contrast to IgG1 antibodies, did not cross-link Sepharose-bound

  4. IL-15 enhances cross-reactive antibody recall responses to seasonal H3 influenza viruses in vitro [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Junqiong Huang

    2017-11-01

    Full Text Available Background: Recently, several human monoclonal antibodies that target conserved epitopes on the stalk region of influenza hemagglutinin (HA have shown broad reactivity to influenza A subtypes. Also, vaccination with recombinant chimeric HA or stem fragments from H3 influenza viruses induce broad immune protection in mice and humans. However, it is unclear whether stalk-binding antibodies can be induced in human memory B cells by seasonal H3N2 viruses. Methods: In this study, we recruited 13 donors previously exposed to H3 viruses, the majority (12 of 13 of which had been immunized with seasonal influenza vaccines. We evaluated plasma baseline strain-specific and stalk-reactive anti-HA antibodies and B cell recall responses to inactivated H3N2 A/Victoria/361/2011 virus in vitro using a high throughput multiplex (mPlex-Flu assay. Results: Stalk-reactive IgG was detected in the plasma of 7 of the subjects. Inactivated H3 viral particles rapidly induced clade cross-reactive antibodies in B cell cultures derived from all 13 donors. In addition, H3 stalk-reactive antibodies were detected in culture supernatants from 7 of the 13 donors (53.8%.  H3 stalk-reactive antibodies were also induced by H1 and H7 subtypes. Interestingly, broadly cross-reactive antibody recall responses to H3 strains were also enhanced by stimulating B cells in vitro with CpG2006 ODN in the presence of IL-15. H3 stalk-reactive antibodies were detected in  CpG2006 ODN + IL-15 stimulated B cell cultures derived from 12 of the 13 donors (92.3%, with high levels detected in cultures from 7 of the 13 donors. Conclusions: Our results demonstrate that stalk-reactive antibody recall responses induced by seasonal H3 viruses and CpG2006 ODN can be enhanced by IL-15.

  5. Production and characterisation of monoclonal antibodies against RAI3 and its expression in human breast cancer

    International Nuclear Information System (INIS)

    Jörißen, Hannah; Klockenbring, Torsten; Bektas, Nuran; Dahl, Edgar; Hartmann, Arndt; Haaf, Anette ten; Di Fiore, Stefano; Kiefer, Hans; Thess, Andreas; Barth, Stefan

    2009-01-01

    RAI3 is an orphan G-protein coupled receptor (GPCR) that has been associated with malignancy and may play a role in the proliferation of breast cancer cells. Although its exact function in normal and malignant cells remains unclear and evidence supporting its role in oncogenesis is controversial, its abundant expression on the surface of cancer cells would make it an interesting target for the development of antibody-based therapeutics. To investigate the link with cancer and provide more evidence for its role, we carried out a systematic analysis of RAI3 expression in a large set of human breast cancer specimens. We expressed recombinant human RAI3 in bacteria and reconstituted the purified protein in liposomes to raise monoclonal antibodies using classical hybridoma techniques. The specific binding activity of the antibodies was confirmed by enzyme-linked immunosorbent assay (ELISA), western blot and immunocytochemistry. We carried out a systematic immunohistochemical analysis of RAI3 expression in human invasive breast carcinomas (n = 147) and normal breast tissues (n = 44) using a tissue microarray. In addition, a cDNA dot blot hybridisation assay was used to investigate a set of matched normal and cancerous breast tissue specimens (n = 50) as well as lymph node metastases (n = 3) for RAI3 mRNA expression. The anti-RAI3 monoclonal antibodies bound to recombinant human RAI3 protein with high specificity and affinity, as shown by ELISA, western blot and ICC. The cDNA dot blot and immunohistochemical experiments showed that both RAI3 mRNA and RAI3 protein were abundantly expressed in human breast carcinoma. However, there was no association between RAI3 protein expression and prognosis based on overall and recurrence-free survival. We have generated a novel, highly-specific monoclonal antibody that detects RAI3 in formaldehyde-fixed paraffin-embedded tissue. This is the first study to report a systematic analysis of RAI3 expression in normal and cancerous human

  6. Glutamate receptor antibodies directed against AMPA receptors subunit 3 peptide B (GluR3B) can be produced in DBA/2J mice, lower seizure threshold and induce abnormal behavior.

    Science.gov (United States)

    Ganor, Yonatan; Goldberg-Stern, Hadassa; Cohen, Ran; Teichberg, Vivian; Levite, Mia

    2014-04-01

    Anti-GluR3B antibodies (GluR3B Ab's), directed against peptide B/aa372-395 of GluR3 subunit of glutamate/AMPA receptors, are found in ∼35% of epilepsy patients, activate glutamate/AMPA receptors, evoke ion currents, kill neurons and damage the brain. We recently found that GluR3B Ab's also associate with neurological/psychiatric/behavioral abnormalities in epilepsy patients. Here we asked if GluR3B Ab's could be produced in DBA/2J mice, and also modulate seizure threshold and/or cause behavioral/motor impairments in these mice. DBA/2J mice were immunized with the GluR3B peptide in Complete Freund's Adjuvant (CFA), or with controls: ovalbumin (OVA), CFA, or phosphate-buffer saline (PBS). GluR3B Ab's and OVA Ab's were tested. Seizures were induced in all mice by the chemoconvulsant pentylenetetrazole (PTZ) at three time points, each time with less PTZ to avoid non-specific death. Behavior was examined in Open-Field, RotaRod and Grip tests. GluR3B Ab's were produced only in GluR3B-immunized mice, while OVA Ab's were produced only in OVA-immunized mice, showing high Ab's specificity. In GluR3B Ab's negative mice, seizure severity scores and percentages of animals developing generalized seizures declined in response to decreasing PTZ doses. In contrast, both parameters remained unchanged/high in the GluR3B Ab's positive mice, showing that these mice were more susceptible to seizures. The seizure scores associated significantly with the GluR3B Ab's levels. GluR3B Ab's positive mice were also more anxious in Open-Field test, fell faster in RotaRod test, and fell more in Grip test, compared to all the control mice. GluR3B Ab's are produced in DBA/2J mice, facilitate seizures and induce behavioral/motor impairments. This animal model can therefore serve for studying autoimmune epilepsy and abnormal behavior mediated by pathogenic anti-GluR3B Ab's. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Indium-111-labeled B72. 3 monoclonal antibody in the detection and staging of breast cancer: A phase 1 study

    Energy Technology Data Exchange (ETDEWEB)

    Lamki, L.M.; Buzdar, A.U.; Singletary, S.E.; Rosenblum, M.G.; Bhadkamkar, V.; Esparza, L.; Podoloff, D.A.; Zukiwski, A.; Hortobagyi, G.N.; Murray, J.L. (Department of Nuclear Medicine, University of Texas M.D. Anderson Cancer Center, Houston (USA))

    1991-07-01

    Sixteen patients with primary breast cancer were studied with a pancarcinoma monoclonal antibody B72.3, an IgG1 molecule directed against tumor-associated glycoprotein (TAG-72) present in several tumors. Five millicuries of 111In was used to label 0.2 mg (six patients), or 2 mg (six patients), or 20 mg using the site-directed bifunctional DTPA method (at carbohydrate moiety). Digital, planar, and SPECT images were obtained at 2, 48, 72 and 96 hr when possible. HAMA levels were obtained before the Mab infusion and at 1, 3, and 6 wk postinfusion. Fourteen of 14 known primary breast lesions were detected by imaging (100% sensitivity). Two fibrocystic lesions were negative. Seven of 14 patients had lymph node metastases by histologic methods, but all were missed by radioimmunoscintigraphy. Tumor uptake of Mab ranged 0.00054%-0.0038% of the ID/g. The tumor-to-normal breast tissue ratio was 4.3 {plus minus} 0.91 (mean {plus minus} s.e.m.). Lymph nodes localization of 111In-B72.3 by tissue analysis was similar for tumor-bearing and normal nodes (0.0039 {plus minus} 0.0023 versus 0.0025 {plus minus} 0.0019). Pharmacokinetics revealed mean plasma half-life of 33.3-41.2 hr for the different doses. There was no statistical difference between any of the pharmacokinetic parameters of different doses. HAMA was positive only in 17% of the patients. The study suggests that this antibody has 100% sensitivity for primary breast cancers, but very poor detection rate of metastatic lesions in axillary lymph nodes; thus making it of questionable value in the initial staging process of this disease.

  8. Generation of chimeric bispecific G250/anti-CD3 monoclonal antibody, a tool to combat renal cell carcinoma

    NARCIS (Netherlands)

    Luiten, R. M.; Coney, L. R.; Fleuren, G. J.; Warnaar, S. O.; Litvinov, S. V.

    1996-01-01

    The monoclonal antibody (MAb) G250 binds to a tumour-associated antigen, expressed in renal cell carcinoma (RCC), which has been demonstrated to be a suitable target for antibody-mediated immunotherapy. A bispecific antibody having both G250 and anti-CD3 specificity can cross-link G250

  9. Binding of monoclonal antibody to protein antigen in fluid phase or bound to solid supports

    Energy Technology Data Exchange (ETDEWEB)

    Kennel, S J

    1982-01-01

    Rat monoclonal antibody (MoAb) to fragment D (FgD) of human fibrinogen was used to characterize the direct binding of antibody to protein in solution or bound to solid supports. Purified IgG, F(ab')/sub 2/ and Fab' were prepared from ascites fluid of hybridoma 104-14B which is a fusion product of spleen cells from a rat immunized with FgD and the mouse myeloma cell line, P3-X63-Ag8. Two-dimensional electrophoresis of radioiodinated antibody preparations demonstrated the presence of hybrid immunoglobulin molecules, but only structures having rat heavy and rat light chains had active antibody combinig sites. The affinity constant for IgG as well as F(ab')/sub 2/ and Fab', 6x10/sup 9/ M/sup -1/, was identical when tested using fluid phase antigen (/sup 125/I-labeled FgD). Affinity constants determined for direct binding of iodinated IgG using FgD immobilized on solid supports showed a slight dependence on the antigen concentration used in the measurement. These values ranged from 0.5x10/sup 9/ M/sup -1/ at high antigen concentrations (1.3x10/sup -7/ M) to 9x10/sup 9/ M/sup -1/ at low antigen concentration (1.3x10/sup -10/ M). Binding constants for F(ab')/sub 2/ and Fab' gave similar results indicating that binding was homogeneous and univalent. The capacity of solid state antigen to bind antibody varied with the method used to bind FgD to the solid support. FgD bound directly to polystyrene plates was least efficient at binding labeled antibody; FgD bound to plates through intermediate carriers poly(L-lysine) was only slightly more efficient, while antigen bound to Sepharose beads by cyanogen bromide activation was the most active.

  10. Characterization of a monoclonal antibody with specificity for holo-transcobalamin

    Directory of Open Access Journals (Sweden)

    Fedosov Sergey N

    2006-01-01

    Full Text Available Abstract Background Holotranscobalamin, cobalamin-saturated transcobalamin, is the minor fraction of circulating cobalamin (vitamin B12, which is available for cellular uptake and hence is physiologically relevant. Currently, no method allows simple, direct quantification of holotranscobalamin. We now report on the identification and characterization of a monoclonal antibody with a unique specificity for holotranscobalamin. Methods The specificity and affinity of the monoclonal antibodies were determined using surface plasmon resonance and recombinant transcobalamin as well as by immobilizing the antibodies on magnetic microspheres and using native transcobalamin in serum. The epitope of the holotranscobalamin specific antibody was identified using phage display and comparison to a de novo generated three-dimensional model of transcobalamin using the program Rosetta. A direct assay for holotrnscobalamin in the ELISA format was developed using the specific antibody and compared to the commercial assay HoloTC RIA. Results An antibody exhibiting >100-fold specificity for holotranscobalamin over apotranscobalamin was identified. The affinity but not the specificity varied inversely with ionic strength and pH, indicating importance of electrostatic interactions. The epitope was discontinuous and epitope mapping of the antibody by phage display identified two similar motifs with no direct sequence similarity to transcobalamin. A comparison of the motifs with a de novo generated three-dimensional model of transcobalamin identified two structures in the N-terminal part of transcobalamin that resembled the motif. Using this antibody an ELISA based prototype assay was developed and compared to the only available commercial assay for measuring holotranscobalamin, HoloTC RIA. Conclusion The identified antibody possesses a unique specificity for holotranscobalamin and can be used to develop a direct assay for the quantification of holotranscobalamin.

  11. Characterization and expression of the human T cell receptor-T3 complex by monoclonal antibody F101.01

    DEFF Research Database (Denmark)

    Geisler, C; Plesner, T; Pallesen, G

    1988-01-01

    A murine monoclonal antibody (MoAb) F101.01 reacting with the T cell receptor (TCR)-T3 complex is presented. Immunohistological studies showed that F101.01 specifically stains T-zone lymphocytes in lymph nodes, tonsils, and splenic tissue. Two-colour immunofluorescence and flow cytometry...... demonstrated co-expression of the antigen defined by F101.01 and the pan-T cell antigens defined by CD2, CD3, CD5, and CD7 antibodies. Cells stained with CD4 and CD8 antibodies were both included in the F101.01-positive population, whereas CD16-positive natural killer cells (NK), B cells (CD19 and CD20......), and myeloid cells (CD13 and CD33) were excluded. The target antigen of F101.01 co-modulated with the CD3-defined antigen (T3) and the TCR recognized by the MoAb WT-31. CD3 antibody and WT-31 both blocked binding of F101.01. F101.01 precipitated the TCR-T3 complex from lysates of 125I-labelled peripheral blood...

  12. High affinity anti-TIM-3 and anti-KIR monoclonal antibodies cloned from healthy human individuals.

    Directory of Open Access Journals (Sweden)

    Stefan Ryser

    Full Text Available We report here the cloning of native high affinity anti-TIM-3 and anti-KIR IgG monoclonal antibodies (mAbs from peripheral blood mononuclear cells (PBMC of healthy human donors. The cells that express these mAbs are rare, present at a frequency of less than one per 105 memory B-cells. Using our proprietary multiplexed screening and cloning technology CellSpot™ we assessed the presence of memory B-cells reactive to foreign and endogenous disease-associated antigens within the same individual. When comparing the frequencies of antigen-specific memory B-cells analyzed in over 20 screening campaigns, we found a strong correlation of the presence of anti-TIM-3 memory B-cells with memory B-cells expressing mAbs against three disease-associated antigens: (i bacterial DNABII proteins that are a marker for Gram negative and Gram positive bacterial infections, (ii hemagglutinin (HA of influenza virus and (iii the extracellular domain of anaplastic lymphoma kinase (ALK. One of the native anti-KIR mAbs has similar characteristics as lirilumab, an anti-KIR mAb derived from immunization of humanized transgenic mice that is in ongoing clinical trials. It is interesting to speculate that these native anti-TIM-3 and anti-KIR antibodies may function as natural regulatory antibodies, analogous to the pharmacological use in cancer treatment of engineered antibodies against the same targets. Further characterization studies are needed to define the mechanisms through which these native antibodies may function in healthy and disease conditions.

  13. Endobronchial administration of iodine-131 B72.3 monoclonal antibody in patients with lung cancer

    International Nuclear Information System (INIS)

    Del Vecchio, S.; Mansi, L.; Petrillo, A.; Camera, L.; Salvatore, M.; Sofia, M.; Marra, A.; Carratu, L.

    1991-01-01

    We tested the feasibility of endobronchial administration of radiolabelled monoclonal antibodies (MoAbs) and the biodistribution of the radiotracer. Ten patients with histological confirmed adenocarcinoma or squamous cell carcinoma were studied. Nine received 470 μCi (103 μg) of 131 I-B72.3, a monoclonal antibody reacting against TAG 72 antigen, while one patient received 502 μCi (291 μg) of 131 I-4C4, an indifferent antibody used for comparison in a negative control study. The radiolabelled antibody was administered through a flexible fiberoptic bronchoscope and placed on the tumour mass under visual monitoring. Scans with a large field-of-view gamma-camera showed retention of 131 I-B72.3 at the tumour site up to 6-9 days in six of eight patients. No other organs were visualized with the exception of faint activity in the gastrointestinal tract, bladder and thyroid. On the contrary, the indifferent antibody 131 I-4C4 was not retained at the tumour site 6 days after MoAbs administration, and more prominent activity was found in the gastrointestinal tract. In one patient the study was not technically adequate because of failure of the delivery system. The vascular compartment contained less than 3% of the administered dose. We conclude that endobronchial administration is a feasible technique and allows stable and specific targetting of bronchial tumours. Furthermore, the low activity found in the plasma and other organs suggests that this approach may be used to deliver therapeutic doses of MoAbs to lung cancers. (orig.)

  14. Monoclonal antibodies for treating cancer

    International Nuclear Information System (INIS)

    Dillman, R.O.

    1989-01-01

    The purpose of this study is to assess the current status of in-vivo use of monoclonal antibodies for treating cancer. Publications appearing between 1980 and 1988 were identified by computer searches using MEDLINE and CANCERLIT, by reviewing the table of contents of recently published journals, and by searching bibliographies of identified books and articles. More than 700 articles, including peer-reviewed articles and book chapters, were identified and selected for analysis. The literature was reviewed and 235 articles were selected as relevant and representative of the current issues and future applications for in-vivo monoclonal antibodies for cancer therapy and of the toxicity and efficacy which has been associated with clinical trials. Approaches include using antibody alone (interacting with complement or effector cells or binding directly with certain cell receptors) and immunoconjugates (antibody coupled to radioisotopes, drugs, toxins, or other biologicals). Most experience has been with murine antibodies. Trials of antibody alone and radiolabeled antibodies have confirmed the feasibility of this approach and the in-vivo trafficking of antibodies to tumor cells. However, tumor cell heterogeneity, lack of cytotoxicity, and the development of human antimouse antibodies have limited clinical efficacy. Although the immunoconjugates are very promising, heterogeneity and the antimouse immune response have hampered this approach as has the additional challenge of chemically or genetically coupling antibody to cytotoxic agents. As a therapeutic modality, monoclonal antibodies are still promising but their general use will be delayed for several years. New approaches using human antibodies and reducing the human antiglobulin response should facilitate treatment. 235 references

  15. Solid phase radioimmunoassays using labelled antibodies: a conceptual framework for designing assays

    International Nuclear Information System (INIS)

    Kalmakoff, J.; Parkinson, A.J.; Crawford, A.M.; Williams, B.R.G.

    1977-01-01

    A simple theoretical model for the antigen-antibody reaction is presented and used to evaluate the optimum conditions for designing solid phase radioimmunoassays (RIA) using labelled antibodies. Both theoretical and experimental data are presented, using a wide variety of antigens and their corresponding antibodies. The types of RIA described include the direct, the indirect, the direct sandwich assays for detecting either antigen or antibody. The experimental results confirm in a semiquantitative manner that the greatest sensitivity of the RIA is achieved when the smallest amount of labelled antibody is used, that whenever possible the antigen/antibody ratio should be greater than unity(>1), and that the formation of the antigen-antibody complex is dependent on the mass action effect

  16. HIV antibodies for treatment of HIV infection.

    Science.gov (United States)

    Margolis, David M; Koup, Richard A; Ferrari, Guido

    2017-01-01

    The bar is high to improve on current combination antiretroviral therapy (ART), now highly effective, safe, and simple. However, antibodies that bind the HIV envelope are able to uniquely target the virus as it seeks to enter new target cells, or as it is expressed from previously infected cells. Furthermore, the use of antibodies against HIV as a therapeutic may offer advantages. Antibodies can have long half-lives, and are being considered as partners for long-acting antiretrovirals for use in therapy or prevention of HIV infection. Early studies in animal models and in clinical trials suggest that such antibodies can have antiviral activity but, as with small-molecule antiretrovirals, the issues of viral escape and resistance will have to be addressed. Most promising, however, are the unique properties of anti-HIV antibodies: the potential ability to opsonize viral particles, to direct antibody-dependent cellular cytotoxicity (ADCC) against actively infected cells, and ultimately the ability to direct the clearance of HIV-infected cells by effector cells of the immune system. These distinctive activities suggest that HIV antibodies and their derivatives may play an important role in the next frontier of HIV therapeutics, the effort to develop treatments that could lead to an HIV cure. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

  17. Antibody-Mediated Internalization of Infectious HIV-1 Virions Differs among Antibody Isotypes and Subclasses.

    Science.gov (United States)

    Tay, Matthew Zirui; Liu, Pinghuang; Williams, LaTonya D; McRaven, Michael D; Sawant, Sheetal; Gurley, Thaddeus C; Xu, Thomas T; Dennison, S Moses; Liao, Hua-Xin; Chenine, Agnès-Laurence; Alam, S Munir; Moody, M Anthony; Hope, Thomas J; Haynes, Barton F; Tomaras, Georgia D

    2016-08-01

    Emerging data support a role for antibody Fc-mediated antiviral activity in vaccine efficacy and in the control of HIV-1 replication by broadly neutralizing antibodies. Antibody-mediated virus internalization is an Fc-mediated function that may act at the portal of entry whereby effector cells may be triggered by pre-existing antibodies to prevent HIV-1 acquisition. Understanding the capacity of HIV-1 antibodies in mediating internalization of HIV-1 virions by primary monocytes is critical to understanding their full antiviral potency. Antibody isotypes/subclasses differ in functional profile, with consequences for their antiviral activity. For instance, in the RV144 vaccine trial that achieved partial efficacy, Env IgA correlated with increased risk of HIV-1 infection (i.e. decreased vaccine efficacy), whereas V1-V2 IgG3 correlated with decreased risk of HIV-1 infection (i.e. increased vaccine efficacy). Thus, understanding the different functional attributes of HIV-1 specific IgG1, IgG3 and IgA antibodies will help define the mechanisms of immune protection. Here, we utilized an in vitro flow cytometric method utilizing primary monocytes as phagocytes and infectious HIV-1 virions as targets to determine the capacity of Env IgA (IgA1, IgA2), IgG1 and IgG3 antibodies to mediate HIV-1 infectious virion internalization. Importantly, both broadly neutralizing antibodies (i.e. PG9, 2G12, CH31, VRC01 IgG) and non-broadly neutralizing antibodies (i.e. 7B2 mAb, mucosal HIV-1+ IgG) mediated internalization of HIV-1 virions. Furthermore, we found that Env IgG3 of multiple specificities (i.e. CD4bs, V1-V2 and gp41) mediated increased infectious virion internalization over Env IgG1 of the same specificity, while Env IgA mediated decreased infectious virion internalization compared to IgG1. These data demonstrate that antibody-mediated internalization of HIV-1 virions depends on antibody specificity and isotype. Evaluation of the phagocytic potency of vaccine

  18. A phase I, dose-escalation study of TB-403, a monoclonal antibody directed against PlGF, in patients with advanced solid tumours

    DEFF Research Database (Denmark)

    Lassen, U; Nielsen, D L; Sørensen, M

    2012-01-01

    BACKGROUND: TB-403 (RO 5323441), a humanised monoclonal antibody, is a novel antiangiogenesis agent directed against placental growth factor. The safety, pharmacokinetics (PK), and antitumour activity of TB-403 were assessed in a phase I, dose-escalation study in patients with advanced solid...

  19. Imaging thrombus with radiolabelled monoclonal antibody to platelets

    International Nuclear Information System (INIS)

    Loutfi, I.; Peters, A.M.; Lavender, J.P.; Epenetos, A.A.

    1988-01-01

    Indium-111-hydroxyquinoline labelled platelets, though useful in the detection of thrombus, have not gained widespread use owing to the time and technical skill required for their preparation. A study was therefore conducted evaluating a new method of imaging thrombus with platelets radiolabelled with a 111 In labelled monoclonal antibody, P 256 , directed to the platelet surface glycoprotein complex IIb/IIIa. When the number of receptors occupied by P 256 was less than 3% of the total available on the platelet surface platelet function, as assessed by platelet aggregometry, was undisturbed. P 256 was radiolabelled with 111 In using diethylenetriaminepenta-acetic acid, which achieved a specific activity of 185 MBq (5 mCi)/mg. No impairment of immunoreactivity was detected at this specific activity. Platelets were labelled with radiolabelled monoclonal antibody in vitro in two patients at a receptor occupancy of 6% and in vivo - that is, by direct intravenous injection of P 256 - in six patients at a receptor occupancy of 1%. In vivo recovery and biodistribution kinetics suggested that after in vitro labelling platelets were minimally activated. The 111 In kinetics recorded after intravenous P 256 suggested rapid and efficient radiolabelling of platelets and gave no indication of platelet activation. Of the six patients who received intravenous P 256 , three had documented thrombus, tow of whom gave positive results on P 256 platelet scintigraphy. The third subject had chromic deep venous thrombosis and was scintigraphically negative. Imaging thrombus using a radiolabelled monoclonal antibody directed to platelets appears to offer great potential as a simple, non-invasive approach to the diagnosis of thrombosis. 3 refs. (Author)

  20. Characterization of immobilization methods of antiviral antibodies in serum for electrochemical biosensors

    Energy Technology Data Exchange (ETDEWEB)

    Huy, Tran Quang, E-mail: huytq@nihe.org.vn [National Institute of Hygiene and Epidemiology (NIHE), No1 Yersin St., Hanoi (Viet Nam); International Training Institute for Materials Science (ITIMS), Hanoi University of Science and Technology (HUST), No1 Dai Co Viet, Hanoi (Viet Nam); Hanh, Nguyen Thi Hong; Van Chung, Pham; Anh, Dang Duc; Nga, Phan Thi [National Institute of Hygiene and Epidemiology (NIHE), No1 Yersin St., Hanoi (Viet Nam); Tuan, Mai Anh, E-mail: tuanma-itims@mail.hut.edu.vn [International Training Institute for Materials Science (ITIMS), Hanoi University of Science and Technology (HUST), No1 Dai Co Viet, Hanoi (Viet Nam)

    2011-06-01

    In this paper, we describes different methods to immobilize Japanese encephalitis virus (JEV) antibodies in human serum onto the interdigitated surface of a microelectrode sensor for optimizing electrochemical detection: (1) direct covalent binding to the silanized surface, (2) binding to the silanized surface via a cross-linker of glutaraldehyde (GA), (3) binding to glutaraldehyde/silanized surface via goat anti-human IgG polyclonal antibody and (4) binding to glutaraldehyde/silanized surface via protein A (PrA). Field emission scanning electron microscopy, Fourier transform infrared spectrometry, and fluorescence microscopy are used to verify the characteristics of antibodies on the interdigitated surface after the serum antibodies immobilization. The analyzed results indicate that the use of protein A is an effective choice for immobilization and orientation of antibodies in serum for electrochemical biosensors. This study provides an advantageous immobilization method of serum containing antiviral antibodies to develop electrochemical biosensors for preliminary screening of viruses in clinical samples from outbreaks.

  1. Heavy chain only antibodies

    DEFF Research Database (Denmark)

    Moghimi, Seyed Moein; Rahbarizadeh, Fatemeh; Ahmadvand, Davoud

    2013-01-01

    Unlike conventional antibodies, heavy chain only antibodies derived from camel contain a single variable domain (VHH) and two constant domains (CH2 and CH3). Cloned and isolated VHHs possess unique properties that enable them to excel conventional therapeutic antibodies and their smaller antigen...

  2. Ultrasensitive direct competitive FLISA using highly luminescent quantum dot beads for tuning affinity of competing antigens to antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Xiong, Sicheng; Zhou, Yaofeng; Huang, Xiaolin [State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047 (China); Yu, Ruijin [College of Science, Northwest A& F University, Yangling, Shaanxi 712100 (China); Lai, Weihua [State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047 (China); Xiong, Yonghua, E-mail: yhxiongchen@163.com [State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047 (China)

    2017-06-15

    Herein, for the first time we report a novel direct competitive fluorescence-linked immunosorbent assay (dcFLISA) for the ultrasensitive detection of ochratoxin A (OTA) by introducing a large size polymer beads loaded with quantum dots (QBs) as carrier of competing antigen for decreasing binding affinity to antibody and enhancing the fluorescent signal intensity. When using 255 nm QBs as carrier of competing antigen, the equilibrium dissociation constant of QB based competing antigen to antibodies can be tuned to 100 times higher than that of the horseradish peroxidase (HRP) based competing antigen by controlling labeled amounts of antigen on the surface of QBs. Various parameters that influenced the sensitivity of dcFLISA were investigated and optimized. Under optimum detection parameters, the dynamic linear range of developed dcFLISA for detecting OTA was established at 0.05 pg/mL to 1.56 pg/mL with a half maximal inhibitory concentration at 0.14 ± 0.04 pg/mL (n = 5), which is three orders of magnitude lower than that of conventional HRP-based dcELISA (0.24 ng/mL). The developed FLISA is also highly accurate, reliable, and shows no cross reaction to other mycotoxins. In summary, the proposed method offers a straightforward approach to improve the sensitivity of direct competitive immunoassay for trace small chemical molecule detection in food quality control, environmental monitoring, and clinical diagnosis. - Highlights: • Highly luminescent QBs were used as a carrier of competing antigen for ultrasensitive detection of OTA. • It is the first time to use a large size QBs as a carrier for tuning affinity of competing antigen to antibodies. • IC{sub 50} value of QB-based dcFLISA is three orders of magnitude lower than that of HRP-based dcELISA.

  3. Ultrasensitive direct competitive FLISA using highly luminescent quantum dot beads for tuning affinity of competing antigens to antibodies

    International Nuclear Information System (INIS)

    Xiong, Sicheng; Zhou, Yaofeng; Huang, Xiaolin; Yu, Ruijin; Lai, Weihua; Xiong, Yonghua

    2017-01-01

    Herein, for the first time we report a novel direct competitive fluorescence-linked immunosorbent assay (dcFLISA) for the ultrasensitive detection of ochratoxin A (OTA) by introducing a large size polymer beads loaded with quantum dots (QBs) as carrier of competing antigen for decreasing binding affinity to antibody and enhancing the fluorescent signal intensity. When using 255 nm QBs as carrier of competing antigen, the equilibrium dissociation constant of QB based competing antigen to antibodies can be tuned to 100 times higher than that of the horseradish peroxidase (HRP) based competing antigen by controlling labeled amounts of antigen on the surface of QBs. Various parameters that influenced the sensitivity of dcFLISA were investigated and optimized. Under optimum detection parameters, the dynamic linear range of developed dcFLISA for detecting OTA was established at 0.05 pg/mL to 1.56 pg/mL with a half maximal inhibitory concentration at 0.14 ± 0.04 pg/mL (n = 5), which is three orders of magnitude lower than that of conventional HRP-based dcELISA (0.24 ng/mL). The developed FLISA is also highly accurate, reliable, and shows no cross reaction to other mycotoxins. In summary, the proposed method offers a straightforward approach to improve the sensitivity of direct competitive immunoassay for trace small chemical molecule detection in food quality control, environmental monitoring, and clinical diagnosis. - Highlights: • Highly luminescent QBs were used as a carrier of competing antigen for ultrasensitive detection of OTA. • It is the first time to use a large size QBs as a carrier for tuning affinity of competing antigen to antibodies. • IC_5_0 value of QB-based dcFLISA is three orders of magnitude lower than that of HRP-based dcELISA.

  4. A novel polyclonal antibody against human cytomegalovirus ...

    African Journals Online (AJOL)

    Future research should be directed to epitope screening of synthetic HMCV peptides, which could help to understand HCMV infection and virus-neutralising antibodies more fully and to prepare HCMV vaccines and antiviral drugs. Key words: Human cytomegalovirus, AD169 strain, Towne strains, polyclonal antibody.

  5. Rapid screening of monoclonal antibodies: new 'microstick' radioimmunoassay

    International Nuclear Information System (INIS)

    Scheinberg, D.A.; Strand, M.; Wilsnack, R.

    1983-01-01

    A new system for assaying monoclonal antibodies consisting of an 8 x 12 array of sticks which fits into a 96-well microtiter plate is described. Tests using virus specific monoclonal antibodies and virus proteins demonstrated sensitivity equivalent to the conventional microtiter plate assay. Antibody production, antigen specific antibody, and immunoglobulin isotypes could be measured under sterile conditions directly in the original fusion mixture wells and much greater rapidity than with the microtiter plate assay. (Auth.)

  6. Investigation of antigen-antibody interactions of sulfonamides with a monoclonal antibody in a fluorescence polarization immunoassay using 3D-QSAR models

    Science.gov (United States)

    A three-dimensional quantitative structure-activity relationship (3D-QSAR) model of sulfonamide analogs binding a monoclonal antibody (MAbSMR) produced against sulfamerazine was carried out by Distance Comparison (DISCOtech), comparative molecular field analysis (CoMFA), and comparative molecular si...

  7. Purification of bovine thyroid-stimulating hormone by a monoclonal antibody

    International Nuclear Information System (INIS)

    Lock, A.J.; van Denderen, J.; Aarden, L.A.

    1988-01-01

    A monoclonal antibody directed against bovine TSH was obtained by hybridoma technology. This antibody was specific for TSH and did not react with bovine LH and FSH. Affinity chromatography of crude TSH was performed on anti-TSH Sepharose. Bovine TSH was purified in a single step to near homogeneity by this technique, as shown by cation exchange chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified TSH. The biological activity of the hormone was not affected during the purification, as determined by [ 3 H]thymidine incorporation of the TSH-dependent FRTL5 cell line. The results indicate that affinity purification of TSH by means of a monoclonal antibody is a simple one-step procedure for the production of biologically active, highly purified TSH

  8. Imaging of non-small cell lung cancers with a monoclonal antibody, KC-4G3, which recognizes a human milk fat globule antigen

    International Nuclear Information System (INIS)

    Dienhart, D.G.; Schmelter, R.F.; Lear, J.L.; Miller, G.J.; Glenn, S.D.; Bloedow, D.C.; Kasliwal, R.; Moran, P.; Seligman, P.; Murphy, J.R.

    1990-01-01

    To determine the role of lung cancer tumor imaging with monoclonal antibodies directed against high molecular weight human milk fat globule antigens, we administered i.v. 111In-KC-4G3 to 24 patients with advanced non-small cell lung cancer. One mg of 111In-KC-4G3 was mixed with 0, 9, 49, 99, or 499 mg of unlabeled KC-4G3 and infused i.v. over 1 to 5 h. The mean 111In-KC-4G3 radiochemical purity was greater than 97% and the resultant immunoreactivity averaged 62%. Successful imaging of cancer sites was accomplished in 92% of 24 patients, and 57% of 91 total lesions were visualized. Successful localization of tumor sites related to size (P less than 0.001), with 81% of lesions greater than 3.0 cm in diameter, 50% of lesions 1.5 to 3 cm, and 6% of lesions less than 1.5 cm successfully imaging, and to location (P less than 0.05), with 69% of pulmonary lesions, 80% of soft tissue lesions, and only 32% of bone metastases being visualized. Nonspecific reticulo-endothelial uptake of radioactivity was a major problem. Approximately 35% of 111In was chelated to serum transferrin by 24 and 48 h after infusion. The mean t 1/2 beta for plasma radioisotope and immunoreactive KC-4G3 was 29 and 27 h, respectively. There was no correlation between total infused antibody dose and imaging success or between total dose and effect on 111In and KC-4G3 kinetics. Circulating free KC-4 antigen was measurable in all but one patient before study. Tumor biopsy following infusion could demonstrate antibody presence but not saturable antigen binding. We conclude that (a) 111In-KC-4G3 demonstrates successful tumor localization in non-small cell lung cancers bearing generally high expression of its antigen and (b) further investigations to diminish nonspecific radioactivity for imaging and utilization of high dose radiolabeled antibody for therapeutic intent are warranted

  9. Library of monoclonal antibodies against brush border membrane epithelial antigens

    International Nuclear Information System (INIS)

    Behar, M.; Katz, A.; Silverman, M.

    1986-01-01

    A purified fraction of proximal tubule brush border membranes (BBM) was prepared from dog kidney and used to immunize mice. The standard technique of hybridoma production was followed as described by Kohler and Milstein. Production of antibodies was detected by indirect immunofluorescence on dog kidney slices and by immunodot against the purified fraction on nitrocellulose. Five hybrids exhibited anti BBM activity. These were cloned twice and yielded stable cell lines producing IgG type monoclonal antibodies against BBM. They were designated A 1 , C 7 , D 3 , D 7 and H 4 . As a family these five monoclonals have broad tissue specificity, i.e. positive staining of the surface mucosa of intestinal kidney proximal tubules. D 3 exhibits even broader specificity for epithelium reacting with bile canaliculi and choroid plexus. The authors have verified that at least 4/5 antibodies are directed against BBM protein as revealed by immunoprecipitation of solubilized BBM and detected by Coomassie blue staining or autoradiography of lactoperoxidase labelled BBM. Most interestingly all antibodies bind to the surface of LL CPK 1 cells, a continuous pig kidney cell line of undefined origin but exhibiting many characteristics of proximal tubule cells. The library of monoclonal antibodies obtained provide important probes with which to study membrane biogenesis and polarization in epithelial cells

  10. Antibody screening identifies 78 putative host proteins involved in Cyprinid herpesvirus 3 infection or propagation in common carp, Cyprinus carpio L.

    Science.gov (United States)

    Gotesman, M; Soliman, H; El-Matbouli, M

    2014-01-01

    Cyprinid herpesvirus 3 (CyHV-3) is the aetiological agent of a serious and notifiable disease afflicting common and koi carp, Cyprinus carpio L., termed koi herpesvirus disease (KHVD). Significant progress has been achieved in the last 15 years, since the initial reports surfaced from Germany, USA and Israel of the CyHV-3 virus, in terms of pathology and detection. However, relatively few studies have been carried out in understanding viral replication and propagation. Antibody-based affinity has been used for detection of CyHV-3 in enzyme-linked immunosorbent assay and PCR-based techniques, and immunohistological assays have been used to describe a CyHV-3 membrane protein, termed ORF81. In this study, monoclonal antibodies linked to N-hydroxysuccinimide (NHS)-activated spin columns were used to purify CyHV-3 and host proteins from tissue samples originating in either CyHV-3 symptomatic or asymptomatic fish. The samples were next analysed either by polyacrylamide gel electrophoresis (PAGE) and subsequently by electrospray ionization coupled to mass spectrometry (ESI-MS) or by ESI-MS analysis directly after purification. A total of 78 host proteins and five CyHV-3 proteins were identified in the two analyses. These data can be used to develop novel control methods for CyHV-3, based on pathways or proteins identified in this study. PMID:23347276

  11. Targeting to cells of fluorescent liposomes covalently coupled with monoclonal antibody or protein A

    Science.gov (United States)

    Leserman, Lee D.; Barbet, Jacques; Kourilsky, François; Weinstein, John N.

    1980-12-01

    Many applications envisioned for liposomes in cell biology and chemotherapy require their direction to specific cellular targets1-3. The ability to use antibody as a means of conferring specificity to liposomes would markedly increase their usefulness. We report here a method for covalently coupling soluble proteins, including monoclonal antibody and Staphylococcus aureus protein A (ref. 4), to small sonicated liposomes, by using the heterobifunctional cross-linking reagent N-hydroxysuccinimidyl 3-(2-pyridyldithio)propionate (SPDP, Pharmacia). Liposomes bearing covalently coupled mouse monoclonal antibody against human β2-microglobulin [antibody B1.1G6 (IgG2a, κ) (B. Malissen et al., in preparation)] bound specifically to human, but not to mouse cells. Liposomes bearing protein A became bound to human cells previously incubated with the B1.1G6 antibody, but not to cells incubated without antibody. The coupling method results in efficient binding of protein to the liposomes without aggregation and without denaturation of the coupled ligand; at least 60% of liposomes bound functional protein. Further, liposomes did not leak encapsulated carboxyfluorescein (CF) as a consequence of the reaction.

  12. VGKC antibodies in pediatric encephalitis presenting with status epilepticus.

    Science.gov (United States)

    Suleiman, J; Brenner, T; Gill, D; Brilot, F; Antony, J; Vincent, A; Lang, B; Dale, R C

    2011-04-05

    Voltage-gated potassium channel antibodies (VGKC Ab) are associated with limbic encephalitis and neuromyotonia in adults. There have been no systematic investigations in children to date. We looked for antibodies that are associated with CNS syndromes in adults including antibodies to VGKCs, NMDARs, glutamic acid decarboxylase (GAD), and glycine receptor (GlyR) in the stored acute serum from 10 children with unexplained encephalitis presenting with encephalopathy and status epilepticus. We also looked for antibodies to leucine-rich glioma-inactivated 1 (Lgi1) and contactin-associated protein-like 2 (Caspr2), which are now known to be tightly complexed with VGKCs in vivo. Sixty-nine pediatric controls were used for comparison. An elevated VGKC Ab (>100 pM) was detected in 4/10 patients with encephalitis compared to only 1/69 controls (p VGKC Ab-positive patients with encephalitis was variable including good recovery (n = 1), cognitive impairment (n = 3), temporal lobe epilepsy (n = 2), and mesial temporal sclerosis (n = 1). No other antibodies were detected, including those to Lgi1 and Caspr2. Encephalitis associated with VGKC Ab occurs in children and presents with status epilepticus and focal epilepsy. These antibodies are not directed against Lgi1 or Caspr2.

  13. Antibody Based Surgical Imaging and Photodynamic Therapy for Cancer

    NARCIS (Netherlands)

    de Boer, Esther

    2016-01-01

    In 1944 Albert Coons was the first to show that a fluorescent molecule could be conjugated directly to an antibody made against a target site of interest. This binding does not affect antibody specificity so that labeled antibodies can be used to visualize the location and distribution of the target

  14. Use of micro-emulsion technology for the directed evolution of antibodies.

    Science.gov (United States)

    Buhr, Diane L; Acca, Felicity E; Holland, Erika G; Johnson, Katie; Maksymiuk, Gail M; Vaill, Ada; Kay, Brian K; Weitz, David A; Weiner, Michael P; Kiss, Margaret M

    2012-09-01

    Affinity reagents, such as antibodies, are needed to study protein expression patterns, sub-cellular localization, and post-translational modifications in complex mixtures and tissues. Phage Emulsion, Secretion, and Capture (ESCape) is a novel micro-emulsion technology that utilizes water-in-oil (W/O) emulsions for the identification and isolation of cells secreting phage particles that display desirable antibodies. Using this method, a large library of antibody-displaying phage will bind to beads in individual compartments. Rather than using biopanning on a large mixed population, phage micro-emulsion technology allows us to individually query clonal populations of amplified phage against the antigen. The use of emulsions to generate microdroplets has the promise of accelerating phage selection experiments by permitting fine discrimination of kinetic parameters for binding to targets. In this study, we demonstrate the ability of phage micro-emulsion technology to distinguish two scFvs with a 300-fold difference in binding affinities (100nM and 300pM, respectively). In addition, we describe the application of phage micro-emulsion technology for the selection of scFvs that are resistant to elevated temperatures. Copyright © 2012. Published by Elsevier Inc.

  15. Technetium-99 labelling of DD-3B6/22 antifibrin monoclonal antibody fragmented Fab' for thrombus imaging

    International Nuclear Information System (INIS)

    Lee, F-T.; Boniface, G.R.; Lambrecht, R.M.; Rylatt, D.B.; Bundesen, P.G.

    1993-01-01

    The antifibrin DD-3B6/22 monoclonal antibody Fab' fragment, a murine immunoglobulin, IgG3, has been labelled with technetium-99m ( 99mTc ) via a transchelation reaction, to specific activity in excess of 30 mCi/mg protein. The radiolabelling of Fab' was dependent on time, temperature, pH, antibody concentrations and nature intermediary transchelation complex used. The resultant radioconjugate was stable in vitro and in vivo. Blood clearance of 99m Tc-Fab' in rat followed two compartment kinetics with the half time of the fast phase being 0.5 h. The main route of excretion was via the kidneys with little uptake indicated by other tissues. The results suggest that the inherent specificity of the antibody, small molecular size, rapid plasma clearance, high specific radioactivity, together with the physical properties of the 99m Tc label, combine to make this labelled monoclonal antibody (MoAb), potentially suitable as a radiopharmaceutical for the scintigraphic detection of thrombi in humans. 17 refs., 3 tabs., 5 figs

  16. DNA vaccines expressing soluble CD4-envelope proteins fused to C3d elicit cross-reactive neutralizing antibodies to HIV-1

    International Nuclear Information System (INIS)

    Bower, Joseph F.; Green, Thomas D.; Ross, Ted M.

    2004-01-01

    DNA vaccines expressing the envelope (Env) of the human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating high-titer, long-lasting, neutralizing antibodies in a variety of animal models. In this study, DNA vaccines were constructed to express a fusion protein of the soluble human CD4 (sCD4) and the gp120 subunit of the HIV-1 envelope. To enhance the immunogenicity of the expressed fusion protein, three copies of the murine C3d (mC3d 3 ) were added to the carboxyl terminus of the complex. Monoclonal antibodies that recognize CD4-induced epitopes on gp120 efficiently bound to sCD4-gp120 or sCD4-gp120-mC3d 3 . In addition, both sCD4-gp120 and sCD4-gp120-mC3d 3 bound to cells expressing appropriate coreceptors in the absence of cell surface hCD4. Mice (BALB/c) vaccinated with DNA vaccines expressing either gp120-mC3d 3 or sCD4-gp120-mC3d 3 elicited antibodies that neutralized homologous virus infection. However, the use of sCD4-gp120-mC3d 3 -DNA elicited the highest titers of neutralizing antibodies that persisted after depletion of anti-hCD4 antibodies. Interestingly, only mice vaccinated with DNA expressing sCD4-gp120-mC3d 3 had antibodies that elicited cross-protective neutralizing antibodies. The fusion of sCD4 to the HIV-1 envelope exposes neutralizing epitopes that elicit broad protective immunity when the fusion complex is coupled with the molecular adjuvant, C3d

  17. A novel polymorphism of human complement component C3 detected by means of a monoclonal antibody

    DEFF Research Database (Denmark)

    Koch, C; Behrendt, N

    1986-01-01

    A mouse monoclonal antibody, HAV 4-1, obtained after immunization of a BALB/c mouse with purified C3F, detected a novel genetic polymorphism of human complement component C3 in a simple immunoblotting system. The frequency of HAV 4-1-positive genes was 20.1%. Reactivity of HAV 4-1 was closely...... related to C3F, but certain individuals with the C3F allele did not react with HAV 4-1. Conversely, certain C3S homozygous individuals did react with HAV 4-1. The polymorphism detected by this monoclonal antibody is therefore different from the previously described polymorphism based on charge differences....

  18. Differentiation of infection from vaccination in foot-and-mouth disease by the detection of antibodies to the non-structural proteins 3D, 3AB and 3ABC in ELISA using antigens expressed in baculovirus

    DEFF Research Database (Denmark)

    Sørensen, K.J.; Madsen, K.G.; Madsen, E.S.

    1998-01-01

    The baculovirus expression system was found to be efficient at expressing the 3D, the 3AB and the 3ABC non-structural proteins (NSP) of foot-and-mouth disease virus (FMDV) as antigens recognised by immune sera in ELISA. ELISA's using 3D, 3AB and 3ABC detected antibodies from day 8 and 10 after...... experimental infection of susceptible cattle and sheep and cattle remained seropositive for more than 395 days. The ELISA's detected antibodies against any of the seven serotypes of FMDV. The 3D ELISA was specific and precise and as sensitive as established ELISA's which measure antibody to structural proteins....... The assay may be used as a resource saving alternative to established ELISA's for the detection of antibodies against any of the seven serotypes. The 3AB and the 3ABC ELISA were also specific and precise. FMDV infected cattle could be differentiated from those that had been merely vaccinated as they gave...

  19. High affinity human antibody fragments to dengue virus non-structural protein 3.

    Directory of Open Access Journals (Sweden)

    Nicole J Moreland

    Full Text Available BACKGROUND: The enzyme activities catalysed by flavivirus non-structural protein 3 (NS3 are essential for virus replication. They are distributed between the N-terminal protease domain in the first one-third and the C-terminal ATPase/helicase and nucleoside 5' triphosphatase domain which forms the remainder of the 618-aa long protein. METHODOLOGY/PRINCIPAL FINDINGS: In this study, dengue full-length NS3 protein with residues 49 to 66 of NS2B covalently attached via a flexible linker, was used as bait in biopanning with a naïve human Fab phage-display library. Using a range of truncated constructs spanning the NS2B cofactor region and the full-length NS3, 10 unique Fab were identified and characterized. Of these, monoclonal Fab 3F8 was shown to bind α3″ (residues 526 through 531 within subdomain III of the helicase domain. The antibody inhibits the ATPase and helicase activites of NS3 in biochemical assays and reduces DENV replication in HEK293 cells that were previously transfected with Fab 3F8 compared with mock transfected cells. CONCLUSIONS/SIGNIFICANCE: Antibodies such as 3F8 are valuable tools for studying the molecular mechanisms of flaviviral replication and for the monospecific detection of replicating dengue virus in vivo.

  20. Immunoassay of serum polypeptide hormones by using 125I-labelled anti(-immunoglobulin G) antibodies.

    Science.gov (United States)

    Beck, P; Nicholas, H

    1975-03-01

    1. A technique for indirectly labelling antibodies to polypeptide hormones, by combining them with radioactively labelled anti-(immunoglobulin G) is described. (a) 125I-labelled anti-(rabbit immunoglobulin G) and anti-(guinea-pig immunoglobulin G) antibodies with high specific radioactivity were prepared after purification of the antibodies on immunoadsorbents containing the respective antigens. (b) Rabbit immunoglobulin G antibodies to human growth hormone, porcine glucagon and guinea-pig immunoglobulin G antibodies to bovine insulin and bovine parathyroid hormone were combined with immunoadsorbents containing the respective polypeptide hormone antigen. (c) The immunoglobulin G antibodies to the polypeptide hormones were reacted with 125-I-labelled anti-(immunoglobulin G) antibodies directed against the appropriate species of immunoglobulin G,and the anti-hormone antibodies were combined with the hormone-containing immunoadsorbent. (d) 125I-labelled anti-(immunoglobulin G) antibodies and anti-hormone antibodies were simultaneously eluted from the hormone-containing immunoadsorbent by dilute HCl, pH 2.0. After elution the anti-(immunoglobulin G) antibodies and antihormone antibodies were allowed to recombine at pH 8.0 and 4 degrees C. 2. The resultant immunoglobulin G-anti-immunoglobulin G complex was used in immunoradiometric (labelled antibody) and two-site assays of the respective polypeptide hormone. 3. By using these immunoassays, concentrations down to 90pg of human growth hormone/ml, 100 pg of bovine insulin/ml, 80 pg of bovine parathyroid hormone/ml and 150 pg of glucagon/ml were readily detected. Assays of human plasma for growth hormone and insulin by these methods showed good agreement with results obtained by using a directly 125I-labelled anti-hormone antibody in an immunoradiometric assay of human growth hormone or by radioimmunoassay of human insulin. 4. The method described allows immunoradiometric or two-site assays to be performed starting with as

  1. Unexplained infertility: identification of anti sperm antibodies using radiometric immunoassay

    International Nuclear Information System (INIS)

    Megahed, Y.M.; Elnabarawy, F.; Hamada, T.; Ayiad, S.K.

    1992-01-01

    Several methods have been employed to measure anti sperm antibodies with variable sensitivity and specificity in serum and secretion of infertile patients. All of them are not precise means for identification of the presence of anti sperm antibodies for patients with unexplained infertility (Haas et al, 1980). Therefore, the modified radiolabelled anti globulin test, that has been used successfully to identify and quantitate the antibodies directed towards other human cell surfaces, was applied. A total number of 128 subjects in different groups were studied to quantitate the circulating anti sperm antibodies using the modified procedure. The present data revealed that the highest and the most significant incidence were found in the patients secretions (semen and cervical mucus) with unexplained infertility, as well as in the group of males with varicocele. Therefore it is greatly advisable to use the modified radiolabelled technique as a quantitative assay, which will be helpful in management of infertility in patients with unexplained and mediated infertility.3 tab., 4 fig

  2. Comprehensive Cross-Clade Characterization of Antibody-Mediated Recognition, Complement-Mediated Lysis, and Cell-Mediated Cytotoxicity of HIV-1 Envelope-Specific Antibodies toward Eradication of the HIV-1 Reservoir.

    Science.gov (United States)

    Mujib, Shariq; Liu, Jun; Rahman, A K M Nur-Ur; Schwartz, Jordan A; Bonner, Phil; Yue, Feng Yun; Ostrowski, Mario A

    2017-08-15

    Immunotherapy with passive administration of broadly neutralizing HIV-1 envelope-specific antibodies (bnAbs) in the setting of established infection in vivo has yielded mixed results. The contribution of different antibodies toward the direct elimination of infected cells is poorly understood. In this study, we determined the ability of 12 well-characterized anti-HIV-1 neutralizing antibodies to recognize and eliminate primary CD4 T cells infected with HIV-1 belonging to clades A, B, C, and D, via antibody-dependent complement-mediated lysis (ADCML) and antibody-dependent cell-mediated cytotoxicity (ADCC), in vitro We further tested unique combinations of these antibodies to determine the optimal antibody cocktails to be tested in future clinical trials. We report that antibody binding to infected CD4 T cells is highly variable and correlates with ADCML and ADCC processes. Particularly, antibodies targeting the envelope glycan shield (2G12) and V1/V2 site (PG9, PG16, and PGT145) are best at recognizing HIV-1-infected CD4 T cells. However, only PG9 and PG16 and their combinations with other bnAbs sufficiently induced the elimination of HIV-1-infected CD4 T cells by ADCML, ADCC, or both. Notably, CD4 binding site antibodies VRC01, 3BNC117, and NIH45-46 G54W did not exhibit recognition of infected cells and were unable to induce their killing. Future trials geared toward the development of a cure for HIV/AIDS should incorporate V1/V2 antibodies for maximal clearance of infected cells. With the use of only primary immune cells, we conducted a comprehensive cross-clade physiological analysis to aid the direction of antibodies as therapeutics toward the development of a cure for HIV/AIDS. IMPORTANCE Several antibodies capable of neutralizing the majority of circulating HIV-1 strains have been identified to date and have been shown to prevent infection in animal models. However, the use of combinations of such broadly neutralizing antibodies (bnAbs) for the treatment and

  3. Neutralisation of HIV-1 cell-cell spread by human and llama antibodies.

    Science.gov (United States)

    McCoy, Laura E; Groppelli, Elisabetta; Blanchetot, Christophe; de Haard, Hans; Verrips, Theo; Rutten, Lucy; Weiss, Robin A; Jolly, Clare

    2014-10-02

    Direct cell-cell spread of HIV-1 is a very efficient mode of viral dissemination, with increasing evidence suggesting that it may pose a considerable challenge to controlling viral replication in vivo. Much current vaccine research involves the study of broadly neutralising antibodies (bNabs) that arise during natural infection with the aims of eliciting such antibodies by vaccination or incorporating them into novel therapeutics. However, whether cell-cell spread of HIV-1 can be effectively targeted by bNabs remains unclear, and there is much interest in identifying antibodies capable of efficiently neutralising virus transmitted by cell-cell contact. In this study we have tested a panel of bNAbs for inhibition of cell-cell spread, including some not previously evaluated for inhibition of this mode of HIV-1 transmission. We found that three CD4 binding site antibodies, one from an immunised llama (J3) and two isolated from HIV-1-positive patients (VRC01 and HJ16) neutralised cell-cell spread between T cells, while antibodies specific for glycan moieties (2G12, PG9, PG16) and the MPER (2F5) displayed variable efficacy. Notably, while J3 displayed a high level of potency during cell-cell spread we found that the small size of the llama heavy chain-only variable region (VHH) J3 is not required for efficient neutralisation since recombinant J3 containing a full-length human heavy chain Fc domain was significantly more potent. J3 and J3-Fc also neutralised cell-cell spread of HIV-1 from primary macrophages to CD4+ T cells. In conclusion, while bNabs display variable efficacy at preventing cell-cell spread of HIV-1, we find that some CD4 binding site antibodies can inhibit this mode of HIV-1 dissemination and identify the recently described llama antibody J3 as a particularly potent inhibitor. Effective neutralisation of cell-cell spread between physiologically relevant cell types by J3 and J3-Fc supports the development of VHH J3 nanobodies for therapeutic or

  4. Cell-free synthesis of functional antibody fragments to provide a structural basis for antibody-antigen interaction.

    Directory of Open Access Journals (Sweden)

    Takayoshi Matsuda

    Full Text Available Growing numbers of therapeutic antibodies offer excellent treatment strategies for many diseases. Elucidation of the interaction between a potential therapeutic antibody and its target protein by structural analysis reveals the mechanism of action and offers useful information for developing rational antibody designs for improved affinity. Here, we developed a rapid, high-yield cell-free system using dialysis mode to synthesize antibody fragments for the structural analysis of antibody-antigen complexes. Optimal synthesis conditions of fragments (Fv and Fab of the anti-EGFR antibody 059-152 were rapidly determined in a day by using a 30-μl-scale unit. The concentration of supplemented disulfide isomerase, DsbC, was critical to obtaining soluble antibody fragments. The optimal conditions were directly applicable to a 9-ml-scale reaction, with linear scalable yields of more than 1 mg/ml. Analyses of purified 059-152-Fv and Fab showed that the cell-free synthesized antibody fragments were disulfide-bridged, with antigen binding activity comparable to that of clinical antibodies. Examination of the crystal structure of cell-free synthesized 059-152-Fv in complex with the extracellular domain of human EGFR revealed that the epitope of 059-152-Fv broadly covers the EGF binding surface on domain III, including residues that formed critical hydrogen bonds with EGF (Asp355EGFR, Gln384EGFR, H409EGFR, and Lys465EGFR, so that the antibody inhibited EGFR activation. We further demonstrated the application of the cell-free system to site-specific integration of non-natural amino acids for antibody engineering, which would expand the availability of therapeutic antibodies based on structural information and rational design. This cell-free system could be an ideal antibody-fragment production platform for functional and structural analysis of potential therapeutic antibodies and for engineered antibody development.

  5. Use of a solid-phase 3H-radioimmunoassay for the measurement of immunoglobulin produced in short-term cultures of antibody-secreting cells

    International Nuclear Information System (INIS)

    Mongini, P.K.A.; Heber-Katz, E.

    1982-01-01

    A solid-phase radioimmunoassay (RIA) for assaying immunoglobulin produced from antibody-secreting myeloma, hybridoma and immune spleen cells is described. Specific antibody is detected by culturing antibody-producing cells on antigen-coated flexible polyvinylchloride microtiter wells, washing away the cells, and measuring the bound specific antibody with tritiated affinity-purified anti-isotype reagents. Antibody produced from 10 3 myeloma cells can be detected with as little as 4 h of incubation. With 24-48 h of incubation, antibody from as few as approximately 3-15 myeloma, hybridoma or immune spleen plaque-forming cells (PFC) can be detected. This culture-well RIA has certain distinct advantages over the hemolytic PFC assay and RIA assays in which antibody in culture supernatants is measured. (Auth.)

  6. Antiphosphatidylserine/prothrombin antibodies (aPS/PT) as potential markers of antiphospholipid syndrome.

    Science.gov (United States)

    Vlagea, Alexandru; Gil, Antonio; Cuesta, Maria V; Arribas, Florencia; Diez, Jesús; Lavilla, Paz; Pascual-Salcedo, Dora

    2013-06-01

    The antiphospholipid antibodies present in antiphospholipid syndrome (APS) are directed at a number of phospholipid-binding proteins: β2 glycoprotein I (β2GPI), prothrombin, and so on. Antibodies directed at β2GPI are accepted as a classification criterion for APS, while the presence of antiprothrombin antibodies is not. In the present article, we investigated the possible role of antiphosphatidylserine/prothrombin antibodies (aPS/PT) as marker of APS on a cohort of 295 individuals with APS (95 primary APS and 45 secondary APS) and APS-related diseases. We found aPS/PT to be highly associated with venous thrombosis (immunoglobulin G [IgG] aPS/PT odds ratio [OR], 7.44; 95% confidence interval [CI], 3.97-13.92 and IgM aPS/PT OR, 2.54; 95% CI, 1.35-4.77) and obstetric abnormalities (IgG aPS/PT OR, 2.37; 95% CI, 1.04-5.43), but not with arterial thrombosis. A very high degree of concordance between the concentration of aPS/PT and lupus anticoagulant activity was demonstrated. Therefore, we support the inclusion of aPS/PT determination as second-level assay to confirm APS classification.

  7. Polyfunctional HIV-Specific Antibody Responses Are Associated with Spontaneous HIV Control.

    Directory of Open Access Journals (Sweden)

    Margaret E Ackerman

    2016-01-01

    Full Text Available Elite controllers (ECs represent a unique model of a functional cure for HIV-1 infection as these individuals develop HIV-specific immunity able to persistently suppress viremia. Because accumulating evidence suggests that HIV controllers generate antibodies with enhanced capacity to drive antibody-dependent cellular cytotoxicity (ADCC that may contribute to viral containment, we profiled an array of extra-neutralizing antibody effector functions across HIV-infected populations with varying degrees of viral control to define the characteristics of antibodies associated with spontaneous control. While neither the overall magnitude of antibody titer nor individual effector functions were increased in ECs, a more functionally coordinated innate immune-recruiting response was observed. Specifically, ECs demonstrated polyfunctional humoral immune responses able to coordinately recruit ADCC, other NK functions, monocyte and neutrophil phagocytosis, and complement. This functionally coordinated response was associated with qualitatively superior IgG3/IgG1 responses, whereas HIV-specific IgG2/IgG4 responses, prevalent among viremic subjects, were associated with poorer overall antibody activity. Rather than linking viral control to any single activity, this study highlights the critical nature of functionally coordinated antibodies in HIV control and associates this polyfunctionality with preferential induction of potent antibody subclasses, supporting coordinated antibody activity as a goal in strategies directed at an HIV-1 functional cure.

  8. Recombinant fragment of an antibody tailored for direct radioiodination

    Czech Academy of Sciences Publication Activity Database

    Sedláček, Juraj; Fábry, Milan; Sieglová, Irena; Král, Vlastimil; Uhnáková, Bronislava; Mudra, M.; Kronrád, L.; Sawicka, A.; Mikolajczak, R.; Řezáčová, Pavlína

    2012-01-01

    Roč. 55, č. 1 (2012), s. 52-56 ISSN 0362-4803 R&D Projects: GA MPO 2A-2TP1/076; GA MŠk 1M0505 Institutional research plan: CEZ:AV0Z50520514 Keywords : I125 labelling * single-chain antibody variable fragment * tyrosine-rich polypeptide segment * fusion protein Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.240, year: 2012

  9. Engineered antibodies for monitoring of polynuclear aromatic hydrocarbons

    International Nuclear Information System (INIS)

    Karu, A.E.; Li, Q.X.; Roberts, V.A.

    1998-01-01

    'The long-term goal of this project is to develop antibodies and antibody-based methods for detection and recovery of polynuclear aromatic hydrocarbons (PAHs) and PAH adducts that are potential biomarkers in environmental and biological samples. The inherent cross-reactivity will be exploited by pattern recognition methods. Dr. Karu''s laboratory uses new haptens representing key PAHs to derive recombinant Fab (rFab) and single-chain Fv (scFv) antibodies from hybridoma lines and combinatorial phage display libraries. Computational models of the haptens and combining sites made by Dr. Roberts''s group are used to guide antibody engineering by mutagenesis. Dr. Li''s laboratory develops enzyme immunoassays (EIAs), sensors, and immunoaffinity methods that make use of the novel haptens and antibodies for practical analytical applications in support of DOE''s mission. This report summarizes work completed in one and one-half years of a 3-year project, with close collaboration between the three research groups. Dr. Alexander Karu''s laboratory: the authors proceeded with the two strategies described in the original proposal. Site-directed mutagenesis was used to correct differences in the rFab N-terminal amino acids that were introduced by the degenerate PCR primers used for gene amplification. The binding constants of the rFabs with the corrected sequences will be compared with those of the parent MAbs, and should be very similar. The 4D5 and 10C10 heavy and light chain sequences are being moved to the pCOMB3H phagemid vector to facilitate selection of new engineered mutants.'

  10. Specificity of antibodies directed against the cytolethal distending toxin of Haemophilus ducreyi in patients with chancroid.

    Science.gov (United States)

    Mbwana, Judica; Ahmed, Hinda J; Ahlman, Karin; Sundaeus, Vivian; Dahlén, Gunnar; Lyamuya, Eligius; Lagergård, Teresa

    2003-09-01

    Antibodies specific for the cytolethal-distending toxin of Haemophilus ducreyi (HdCDT) complex and for the CdtA, CdtB, and CdtC components were measured by ELISA in the sera of 50 patients with culture and/or PCR proven chancroid, 42 patients with periodontitis, 50 blood donors from Tanzania, 50 blood donors from Sweden. In addition, the biological activity e.g. neutralization capacity of the sera were tested. Our results demonstrate that majority of chancroid patients and healthy individuals had detectable levels of serum antibodies to HdCDT complex and to separate toxin components. However, high levels (> or =100 units) of antibodies to HdCDT complex were significantly more prevalent in the sera of patients with both chancroid and periodontitis than in the sera of the corresponding controls (P=0.001 and P=0.04, respectively). In the sera of the 50 patients with chancroid, antibodies to CdtA, CdtB, and CdtC were detected in 50, 35, and 34 individuals, respectively. Antibodies to CdtC, being less frequently detected than the antibodies to other components, show a good correlation with the neutralizing capacity of sera. High levels of neutralizing antibodies (> or =160) were detected in only 22 and 2% of the patients with chancroid and periodontitis, respectively. The data suggest that the low levels of anti-HdCDT antibodies, which include neutralizing antibodies, may contribute to limited protection in chancroid and since anti-HdCDT antibodies, may be detected in healthy individuals and in patients with certain disease conditions (e.g. periodontitis), they may not be specific markers for chancroid infection.

  11. Dengue envelope-based 'four-in-one' virus-like particles produced using Pichia pastoris induce enhancement-lacking, domain III-directed tetravalent neutralising antibodies in mice.

    Science.gov (United States)

    Rajpoot, Ravi Kant; Shukla, Rahul; Arora, Upasana; Swaminathan, Sathyamangalam; Khanna, Navin

    2018-06-05

    Dengue is a significant public health problem worldwide, caused by four antigenically distinct mosquito-borne dengue virus (DENV) serotypes. Antibodies to any given DENV serotype which can afford protection against that serotype tend to enhance infection by other DENV serotypes, by a phenomenon termed antibody-dependent enhancement (ADE). Antibodies to the viral pre-membrane (prM) protein have been implicated in ADE. We show that co-expression of the envelope protein of all four DENV serotypes, in the yeast Pichia pastoris, leads to their co-assembly, in the absence of prM, into tetravalent mosaic VLPs (T-mVLPs), which retain the serotype-specific antigenic integrity and immunogenicity of all four types of their monomeric precursors. Following a three-dose immunisation schedule, the T-mVLPs elicited EDIII-directed antibodies in mice which could neutralise all four DENV serotypes. Importantly, anti-T-mVLP antibodies did not augment sub-lethal DENV-2 infection of dengue-sensitive AG129 mice, based on multiple parameters. The 'four-in-one' tetravalent T-mVLPs possess multiple desirable features which may potentially contribute to safety (non-viral, prM-lacking and ADE potential-lacking), immunogenicity (induction of virus-neutralising antibodies), and low cost (single tetravalent immunogen produced using P. pastoris, an expression system known for its high productivity using simple inexpensive media). These results strongly warrant further exploration of this vaccine candidate.

  12. [Immunohematologic study and transfusion approach to patients with public antibodies].

    Science.gov (United States)

    Solves, P; de la Rubia, J; Arriaga, F; Cervera, J; Arnao, M; Carpio, N; Marty, M L

    1997-02-01

    To analyze the different immunohematologic studies required to identify anti-red cell antibodies directed against high incidence antigens and comment the best tranfusion management. Five patients with suspected anti-red cell alloantibodies directed against high frequency antigens are reported. After a positive antibody screening test (AST), an agglutination test with a commercial panel of 24 red cells was performed. Red cells were treated with proteolytic enzymes and AET to try to identify the circulating antibody. However, it was necessary to send the samples to reference laboratories for definitive identification. In order to evaluate the haemolytic potential of the antibody serum samples were treated with DTT and immunoglobulin subtype was studied with the capillary agglutination test. Finally, we analyze the half life of Cr51 labelled red cells. To obtain compatible blood for transfusion, autologous transfusion and cross-match with blood from direct relatives were performed. AST was positive in every case. A decrease in the agglutination test was observed after ficin treatment in two patients, and an increase in the remaining. The treatment of red cells with ZZAP and AET resulted in a decrease of agglutination in three cases and an increase in the remaining two. Specificity of the antibodies was as follows: anti-Cellano (two cases), anti-Ku (one case) and anti-Yta (two cases). Anti-Kell antibodies were IgG1 and anti-Cartwright antibodies were IgG4. One patient was transfused with autologous blood alone, another patient received compatible blood from direct relatives. A third patient was transfused both with autologous and allogeneic compatible blood. The fourth patient did not need red cell transfusion and, finally the last patient had to be transfused with incompatible blood but no postransfusion haemolysis was observed. In patients with anti-red cell antibodies against high-frequency antigens, red blood cells treatment with proteolytic enzymes (ZZAP, ficin

  13. Monoclonal antibodies to carcino-embryonic antigen

    International Nuclear Information System (INIS)

    Teh, Jinghee; McKenzie, I.F.C.

    1990-01-01

    With the aim of producing new MoAb to colorectal carcinoma, immunization with cell suspensions of a fresh colonic tumour was performed and MoAb 17C4 was obtained. To produce other MoAb to colon cancer, an immunization protocol using fresh tumour, colonic cell lines and sera from patients with colonic tumours was employed and resulted in MoAb JGT-13, LK-4 and XPX-13. MoAb I-1 and O-1 were raised against sera from patients with colon cancer to produce MoAb directed against circulating tumour associated antigens. The six antibodies gave a range of reactions with normal and malignant tissues, indicating that they most likely reacted with different epitopes. Thus, apart from the reactions of 17C4, LK-4 and XPX-13 with fresh and formalin-fixed granulocytes, none of the antibodies reacted with formalin-fixed normal tissues. Despite the apparent specificity of these MoAb for colon cancer, serum testing using MoAb gave similar results to carcino-embryonic antigen polyclonal antibodies, that is the MoAb gave no obvious advantage. 9 refs., 1 tab., 3 figs

  14. An anti vimentin antibody promotes tube formation

    DEFF Research Database (Denmark)

    Jørgensen, Mathias Lindh; Møller, Carina Kjeldahl; Rasmussen, Lasse

    2017-01-01

    antibody technology, promotes tube formation of endothelial cells in a 2D matrigel assay. By binding vimentin, the antibody increases the tube formation by 21% after 5 hours of incubation. Addition of the antibody directly to cultured endothelial cells does not influence endothelial cell migration...... or proliferation. The enhanced tube formation can be seen for up to 10 hours where after the effect decreases. It is shown that the antibody-binding site is located on the coil 2 domain of vimentin. To our knowledge this is the first study that demonstrates an enhanced tube formation by binding vimentin in a 2D...

  15. Summary of workshop findings for porcine T-lymphocyte-specific monoclonal antibodies

    DEFF Research Database (Denmark)

    Saalmuller, A.; Kuebart, G.; Hollemweguer, E.

    2001-01-01

    antibodies, 37 data sets were used for the clustering of the respective mAb. Using the LTDB4 program, 19 preliminary clusters could be defined. Two clusters (C3 and C7) with 4 mAb showed no labelling of resting T-lymphocytes. Seven clusters (CI, C2, C4, C5, C6, C11, and C12) contain mAb (in total: 16 mAb...... to recognise CD45R. Cluster C17 is composed of different standards directed against CD2, CD3, CD5 and wCD6. Two additional mAb recognising the CD2a-epitope could be enclosed. CIS contains two mAb directed against SWC2.......Fifty-seven monoclonal antibodies (mAb) selected after the first round analyses in the Third International Swine CD workshop for their possible reactivity with T-lymphocyte specific antigens were further analysed in a second round. As target cells for flow cytometric analyses served peripheral...

  16. Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing.

    Science.gov (United States)

    Valdés-Alemán, Javier; Téllez-Sosa, Juan; Ovilla-Muñoz, Marbella; Godoy-Lozano, Elizabeth; Velázquez-Ramírez, Daniel; Valdovinos-Torres, Humberto; Gómez-Barreto, Rosa E; Martinez-Barnetche, Jesús

    2014-01-01

    High-throughput sequencing of the antibody repertoire is enabling a thorough analysis of B cell diversity and clonal selection, which may improve the novel antibody discovery process. Theoretically, an adequate bioinformatic analysis could allow identification of candidate antigen-specific antibodies, requiring their recombinant production for experimental validation of their specificity. Gene synthesis is commonly used for the generation of recombinant antibodies identified in silico. Novel strategies that bypass gene synthesis could offer more accessible antibody identification and validation alternatives. We developed a hybridization-based recovery strategy that targets the complementarity-determining region 3 (CDRH3) for the enrichment of cDNA of candidate antigen-specific antibody sequences. Ten clonal groups of interest were identified through bioinformatic analysis of the heavy chain antibody repertoire of mice immunized with hen egg white lysozyme (HEL). cDNA from eight of the targeted clonal groups was recovered efficiently, leading to the generation of recombinant antibodies. One representative heavy chain sequence from each clonal group recovered was paired with previously reported anti-HEL light chains to generate full antibodies, later tested for HEL-binding capacity. The recovery process proposed represents a simple and scalable molecular strategy that could enhance antibody identification and specificity assessment, enabling a more cost-efficient generation of recombinant antibodies.

  17. H3N2 influenza infection elicits more cross-reactive and less clonally expanded anti-hemagglutinin antibodies than influenza vaccination.

    Directory of Open Access Journals (Sweden)

    M Anthony Moody

    Full Text Available BACKGROUND: During the recent H1N1 influenza pandemic, excess morbidity and mortality was seen in young but not older adults suggesting that prior infection with influenza strains may have protected older subjects. In contrast, a history of recent seasonal trivalent vaccine in younger adults was not associated with protection. METHODS AND FINDINGS: To study hemagglutinin (HA antibody responses in influenza immunization and infection, we have studied the day 7 plasma cell repertoires of subjects immunized with seasonal trivalent inactivated influenza vaccine (TIV and compared them to the plasma cell repertoires of subjects experimentally infected (EI with influenza H3N2 A/Wisconsin/67/2005. The majority of circulating plasma cells after TIV produced influenza-specific antibodies, while most plasma cells after EI produced antibodies that did not react with influenza HA. While anti-HA antibodies from TIV subjects were primarily reactive with single or few HA strains, anti-HA antibodies from EI subjects were isolated that reacted with multiple HA strains. Plasma cell-derived anti-HA antibodies from TIV subjects showed more evidence of clonal expansion compared with antibodies from EI subjects. From an H3N2-infected subject, we isolated a 4-member clonal lineage of broadly cross-reactive antibodies that bound to multiple HA subtypes and neutralized both H1N1 and H3N2 viruses. This broad reactivity was not detected in post-infection plasma suggesting this broadly reactive clonal lineage was not immunodominant in this subject. CONCLUSION: The presence of broadly reactive subdominant antibody responses in some EI subjects suggests that improved vaccine designs that make broadly reactive antibody responses immunodominant could protect against novel influenza strains.

  18. Humanised IgG1 antibody variants targeting membrane-bound carcinoembryonic antigen by antibody-dependent cellular cytotoxicity and phagocytosis.

    Science.gov (United States)

    Ashraf, S Q; Umana, P; Mössner, E; Ntouroupi, T; Brünker, P; Schmidt, C; Wilding, J L; Mortensen, N J; Bodmer, W F

    2009-11-17

    The effect of glycoengineering a membrane specific anti-carcinoembryonic antigen (CEA) (this paper uses the original term CEA for the formally designated CEACAM5) antibody (PR1A3) on its ability to enhance killing of colorectal cancer (CRC) cell lines by human immune effector cells was assessed. In vivo efficacy of the antibody was also tested. The antibody was modified using EBNA cells cotransfected with beta-1,4-N-acetylglucosaminyltransferase III and the humanised hPR1A3 antibody genes. The resulting alteration of the Fc segment glycosylation pattern enhances the antibody's binding affinity to the FcgammaRIIIa receptor on human immune effector cells but does not alter the antibody's binding capacity. Antibody-dependent cellular cytotoxicity (ADCC) is inhibited in the presence of anti-FcgammaRIII blocking antibodies. This glycovariant of hPR1A3 enhances ADCC 10-fold relative to the parent unmodified antibody using either unfractionated peripheral blood mononuclear or natural killer (NK) cells and CEA-positive CRC cells as targets. NK cells are far more potent in eliciting ADCC than either freshly isolated monocytes or granulocytes. Flow cytometry and automated fluorescent microscopy have been used to show that both versions of hPR1A3 can induce antibody-dependent cellular phagocytosis (ADCP) by monocyte-derived macrophages. However, the glycovariant antibody did not mediate enhanced ADCP. This may be explained by the relatively low expression of FcgammaRIIIa on cultured macrophages. In vivo studies show the efficacy of glycoengineered humanised IgG1 PR1A3 in significantly improving survival in a CRC metastatic murine model. The greatly enhanced in vitro ADCC activity of the glycoengineered version of hPR1A3 is likely to be clinically beneficial.

  19. [Construction of human phage antibody library and screening for human monoclonal antibodies of amylin].

    Science.gov (United States)

    Gong, Qian; Li, Chang-ying; Chang, Ji-wu; Zhu, Tie-hong

    2012-06-01

    To screen monoclonal antibodies to amylin from a constructed human phage antibody library and identify their antigenic specificity and combining activities. The heavy chain Fd fragment and light chain of human immunoglobulin genes were amplified from peripheral blood lymphocytes of healthy donors using RT-PCR, and then inserted into phagemid pComb3XSS to generate a human phage antibody library. The insertion of light chain or heavy chain Fd genes were identified by PCR after the digestion of Sac I, Xba I, Xho Iand Spe I. One of positive clones was analyzed by DNA sequencing. The specific anti-amylin clones were screened from antibody library against human amylin antigens and then the positive clones were determined by Phage-ELISA analysis. A Fab phage antibody library with 0.8×10(8); members was constructed with the efficacy of about 70%. DNA sequence analysis indicated V(H); gene belonged to V(H);3 gene family and V(λ); gene belonged to the V(λ); gene family. Using human amylin as panning antigen, specific anti-amylin Fab antibodies were enriched by screening the library for three times. Phage-ELISA assay showed the positive clones had very good specificity to amylin antigen. The successful construction of a phage antibody library and the identification of anti-amylin Fab antibodies provide a basis for further study and preparation of human anti-amylin antibodies.

  20. Development of Broadly Neutralizing Antibody Mimitopes for Characterization of CRF01_AE HIV-1 Antibody Responses

    Directory of Open Access Journals (Sweden)

    Jesse V. Schoen

    2017-10-01

    Full Text Available Mapping humoral immune responses to HIV-1 over the course of natural infection is important in understanding epitope exposure in relation to elicitation of broadly neutralizing antibodies (bNAbs, which is considered imperative for effective vaccine design. When analyzing HIV-specific immune responses, the antibody binding profiles may be a correlate for functional antibody activity. In this study, we utilized phage display technology to identify novel mimitopes that may represent Env epitope structures bound by bNAbs directed at V1V2 and V3 domains, CD4 binding site (CD4bs and the membrane proximal external region (MPER of Env. Mimitope sequence motifs were determined for each bNAb epitope. Given the ongoing vaccine development efforts in Thailand, these mimitopes that represent CD4bs and MPER epitopes were used to map immune responses of HIV-1 CRF01_AE-infected individuals with known neutralizing responses from two distinct time periods, 1996-98 and 2012-15. The more contemporary cohort showed an increase in binding breadth with binding observed for all MPER and CD4bs mimitopes, while the older cohort showed only 75% recognition of the CD4bs mimitopes and no MPER mimotope binding. Furthermore, mimitope binding profiles correlated significantly with magnitude (p=0.0036 and breadth (p=0.0358 of neutralization of a multi-subtype Tier 1 panel of pseudoviruses. These results highlight the utility of this mimitope mapping approach for detecting human plasma IgG-specificities that target known neutralizing antibody epitopes, and may also provide an indication of the plasticity of antibody binding within HIV-1 Env neutralization determinants.

  1. Antibody response in vaccinated pregnant mares to recent G3BP[12] and G14P[12] equine rotaviruses

    Directory of Open Access Journals (Sweden)

    Nemoto Manabu

    2012-11-01

    Full Text Available Abstract Background Both the G3P[12] and the G14P[12] type of equine group A rotavirus (RVA have recently become predominant in many countries, including Japan. G3 types are classified further into G3A and G3B. The G3A viruses have been circulating in Europe, Australia, and Argentina, and the G3B viruses have been circulating in Japan. However, only an inactivated vaccine containing a single G3BP[12] strain is commercially available in Japan. To assess the efficacy of the current vaccine against recently circulating equine RVA strains, we examined antibody responses in pregnant mares to recent G3BP[12] and G14P[12] strains by virus neutralization test. Findings After vaccination in five pregnant mares, the geometric mean serum titers of virus-neutralizing antibody to recent G3BP[12] strains increased 5.3- to 7.0-fold and were similar to that against homologous vaccine strain. Moreover, antibody titers to recent G14P[12] strains were also increased 3.0- to 3.5-fold. Conclusions These results suggest that inoculation of mares with the current vaccine should provide foals with virus-neutralizing antibodies against not only the G3BP[12] but also the G14P[12] RVA strain via the colostrum.

  2. Biting back: BiTE antibodies as a promising therapy for acute myeloid leukemia.

    Science.gov (United States)

    Walter, Roland B

    2014-06-01

    The experience with gemtuzumab ozogamicin has highlighted both the potential value and limitations of antibodies in acute myeloid leukemia (AML). Recently, bispecific T-cell engager (BiTE) antibodies have emerged as a means to harness polyclonal cytotoxic T-cells and cause highly efficient lysis of targeted tumor cells. Promising early results have been obtained with the CD19-directed BiTE antibody, blinatumomab, in patients with acute lymphoblastic leukemia. A first candidate for AML is the CD33/CD3 molecule, AMG 330, for which several recent preclinical studies demonstrated high potency and efficacy in destroying CD33(+) human AML cells. Many questions remain to be addressed, but BiTE antibodies may offer an exciting new tool in a disease for which the outcomes in many patients remain unsatisfactory.

  3. Use of Re-188 labelled anti-EGFr humanized monoclonal antibody h-R3 for radioimmunotherapy of gliomas

    International Nuclear Information System (INIS)

    Perera, A.; Torres, L.A.; Lopez, G.; Casaco, A.; Batista, J.F.; Pena, Y.; Coca, M.A.; Leyva, R.; Garcia, I.

    2007-01-01

    Full text: Locally administered monoclonal antibodies labelled with radioisotopes like 90Y, 131I, 186Re, 212Bi, 211At, 177Lu and others, constitute a viable and promising alternative for management different kind of malignancies. The development of 188W/188Re generator has given the possibility of having a radionuclide showing satisfactory features for radioimmunotherapy (Eb2.12 MeV, Eg155 keV, T1/2=16.9 h and easy to make labelling approaches, similar to used for 99mTc). Neuroepithelial-derived tumours show an increased expression of the EGF receptor with regard to adjacent normal tissue. This overexpression could be related with the autocrine stimulation of the neoplasm by EGF and TGFb. Humanized monoclonal antibody h-R3 has shown a high affinity for this EGF receptor, blocking the binding of EGF to it receptor and inducing apoptosis. Thus, it could be a good candidate for radioimmunotherapy of neuroepithelial malignancies. The aim of the present work was to label monoclonal antibody h-R3 with 188Re, to assess it in an animal model and evaluate its internal dosimetry and toxicity in patients with grade III-IV gliomas through a phase I clinical trial. Schwarz's direct labelling method was employed. Briefly, a 2000-fold molar excess of 2-mercaptoethanol was used to reduce bisulfide bonds of the antibody. The amount of sodium glucoheptonate, ascorbic acid and stannous fluoride were varied to achieve optimum labelling yield. 188Re-labeling yield was proportional to the volume of stannous glucoheptonate solution added to the formulation. Radiochemical purity of 188Re-h-R3 was 98.0±0.4%. Challenge against 300-fold molar excess of L-cysteine was made to assess the stability of the tracer. There was no significant difference between stability of 188Re-h-R3 and 99mTc-h-R3 against cysteine challenge up to 24 h. Animal biodistribution study was performed at 3 and 24 h after intravenous administration of 188Re-h-R3 through tale vein of Male Wistar rats. The results were

  4. Expression of human mag-1 gene in E. coli and preparation of its antibody

    International Nuclear Information System (INIS)

    Lin Huiyun; Xu Yuanji; Wang Yan; Chen Huihua; Du Zhiyan; Tan Xiaogang; Lu Yinglin

    2006-01-01

    Objective: To further investigate the new metastasis associated gene, mag-1 expressed in E. coli and its anti-body was prepared in rabbit. Methods: mag-1 was amplified by PCR from pcDNA3-mag-1 and directly cloned into pET-28a vector. The fusion protein was expressed in BL21 and identified by Western blot using anti-His monoclonal antibody. Rabbit was immunized with partially purified fusion protein subcutaneously. Results: Sequence analysis revealed identity of the sequence obtained to the previous report. The recombinant His-mag-1 could be expressed in E. coli as a fusion protein of 18 x 10 3 . The recombinant protein was mostly expressed in the inclusion bodies on the induction by 0.1 mmol/L IPTG at 37 degree C for 6 hours. Western blot analysis showed that the recombinant protein could be recognized by His monoclonal anti-body. The titer of polyclonal antibody against mag-1 was 1:160000. Conclusion: The mag-1 gene is expressed in E. coli highly and its antibody is prepared successfully. (authors)

  5. Monoclonal antibodies and cancer

    International Nuclear Information System (INIS)

    Haisma, H.J.

    1987-01-01

    The usefulness of radiolabeled monoclonal antibodies for imaging and treatment of human (ovarian) cancer was investigated. A review of tumor imaging with monoclonal antibodies is presented. Special attention is given to factors that influence the localization of the antibodies in tumors, isotope choice and methods of radiolabeling of the monoclonal antibodies. Two monoclonal antibodies, OC125 and OV-TL3, with high specificity for human epithelial ovarian cancer are characterized. A simple radio-iodination technique was developed for clinical application of the monoclonal antibodies. The behavior of monoclonal antibodies in human tumor xenograft systems and in man are described. Imaging of tumors is complicated because of high background levels of radioactivity in other sites than the tumor, especially in the bloodpool. A technique was developed to improve imaging of human tumor xenographs in nude mice, using subtraction of a specific and a non-specific antibody, radiolabeled with 111 In, 67 Ga and 131 I. To investigate the capability of the two monoclonal antibodies, to specifically localize in human ovarian carcinomas, distribution studies in mice bearing human ovarian carcinoma xenografts were performed. One of the antibodies, OC125, was used for distribution studies in ovarian cancer patients. OC125 was used because of availability and approval to use this antibody in patients. The same antibody was used to investigate the usefulness of radioimmunoimaging in ovarian cancer patients. The interaction of injected radiolabeled antibody OC125 with circulating antigen and an assay to measure the antibody response in ovarian cancer patients after injection of the antibody is described. 265 refs.; 30 figs.; 19 tabs

  6. Recent Advances in Monoclonal Antibody Therapies for Multiple Sclerosis

    Science.gov (United States)

    Stavropoulos, Nikolaos; Wittenberg, Nathan J.; Dasari, Harika; Abdelrahim, Murtada A.; Henley, John R.; Oh, Sang-Hyun; Warrington, Arthur E.; Rodriguez, Moses

    2016-01-01

    Introduction Multiple sclerosis (MS) is the most common chronic inflammatory, demyelinating disease of the CNS and results in neurological disability. Existing immunomodulatory and immunosuppressive approaches lower the number of relapses but do not cure or reverse existing deficits nor improve long-term disability in MS patients. Areas Covered Monogenic antibodies were described as treatment options for MS, however the immunogenicity of mouse antibodies hampered the efficacy of potential therapeutics in humans. Availability of improved antibody production technologies resulted in a paradigm shift in MS treatment strategies. In this review, an overview of immunotherapies for MS that use conventional monoclonal antibodies reactive to immune system and their properties and mechanisms of action will be discussed, including recent advances in MS therapeutics and highlight natural autoantibodies (NAbs) that directly target CNS cells. Expert Opinion Recent challenges for MS therapy are the identification of relevant molecular and cellular targets, time frame of treatment, and antibody toxicity profiles to identify safe treatment options for MS patients. The application of monoclonal antibody therapies with better biological efficacy associated with minimum side effects possesses huge clinical potential. Advances in monoclonal antibody technologies that directly target cells of nervous system may promote the CNS regeneration field from bench to bedside. PMID:26914737

  7. Prevalence of antibodies against Neospora spp. and Sarcocystis neurona in donkeys from northeastern Brazil.

    Science.gov (United States)

    Gennari, Solange Maria; Pena, Hilda Fátima de Jesus; Lindsay, David Scott; Lopes, Marcos Gomes; Soares, Herbert Sousa; Cabral, Aline Diniz; Vitaliano, Sérgio Netto; Amaku, Marcos

    2016-01-01

    Sarcocystis neurona and Neospora hughesi are coccidian protozoa that can cause neurological illness in horses in America. In this study we report seroprevalence of Neospora spp. andS. neurona in sera of 333 donkeys from the northeastern region of Brazil. Antibodies to Neospora spp. were detected in 2% (7 donkeys) of 333 sera tested by the indirect fluorescent antibody test (IFAT) with a cut-off dilution of 1:40. Antibodies to S. neurona were found in 3% (10 donkeys) of the samples tested by IFAT (cut-off ≥50) and 21% (69 donkeys) by the direct agglutination test (SAT ≥50). The SAT and IFAT results for S. neurona showed a poor concordance (value of Kappa=0.051). This is the first report of Neospora spp. antibodies in Brazilian donkeys and the first detection of antibodies against S. neurona in this animal species.

  8. Radiolabelling of glycosylated MFE-23::CPG2 fusion protein (MFECP1) with 99mTc for quantitation of tumour antibody-enzyme localisation in antibody-directed enzyme pro-drug therapy (ADEPT).

    Science.gov (United States)

    Francis, R J; Mather, S J; Chester, K; Sharma, S K; Bhatia, J; Pedley, R B; Waibel, R; Green, A J; Begent, R H J

    2004-08-01

    MFECP1 is a glycosylated recombinant fusion protein composed of MFE-23, a high-affinity anti-carcinoembryonic antigen (CEA) single chain Fv (scFv), fused to the enzyme carboxypeptidase G2 (CPG2), and has been constructed for use in antibody-directed enzyme pro-drug therapy (ADEPT). Radiolabelling of glycosylated MFECP1 with technetium-99m was developed for the purpose of determining tumour localisation of MFECP1 in a phase I ADEPT clinical study. The method used was 99mTc-carbonyl [99mTc(H2O)3(CO)3]+ (abbreviated to TcCO) mediated labelling of 99mTc to the hexahistidine (His) tag of MFECP1. MFECP1 fusion protein was labelled with TcCO under a variety of conditions, and this was shown to be a relatively simple and robust method. Tissue biodistribution was assessed in a CEA-expressing LS174T (human colon carcinoma) nude mouse xenograft model. Tissues were taken at 1, 4 and 6 h for assessment of distribution of radioactivity and for measurement of CPG2 enzyme levels. The amount of radioactivity retained by the tumour proved to be an accurate estimation of actual measured enzyme activity, indicating that this radiolabelling method does not appear to damage the antibody-antigen binding or the enzyme activity of MFECP1. However, correlation between CPG2 enzyme activity and measured radioactivity in liver, spleen and kidney was poor, indicating retention of radioactivity in non-tumour sites but loss of enzyme activity. The high retention of technetium radioisotope in normal tissues may limit the clinical applicability of this radiolabelling method for MFECP1; however, these results suggest that this technique does have applicability for measuring the biodistribution of His-tagged recombinant proteins.

  9. Radiolabelling of glycosylated MFE-23::CPG2 fusion protein (MFECP1) with 99mTc for quantitation of tumour antibody-enzyme localisation in antibody-directed enzyme pro-drug therapy (ADEPT)

    International Nuclear Information System (INIS)

    Francis, R.J.; Chester, K.; Sharma, S.K.; Bhatia, J.; Pedley, R.B.; Green, A.J.; Begent, R.H.J.; Mather, S.J.; Waibel, R.

    2004-01-01

    MFECP1 is a glycosylated recombinant fusion protein composed of MFE-23, a high-affinity anti-carcinoembryonic antigen (CEA) single chain Fv (scFv), fused to the enzyme carboxypeptidase G2 (CPG2), and has been constructed for use in antibody-directed enzyme pro-drug therapy (ADEPT). Radiolabelling of glycosylated MFECP1 with technetium-99m was developed for the purpose of determining tumour localisation of MFECP1 in a phase I ADEPT clinical study. The method used was 99m Tc-carbonyl [ 99m Tc(H 2 O) 3 (CO) 3 ] + (abbreviated to TcCO) mediated labelling of 99m Tc to the hexahistidine (His) tag of MFECP1. MFECP1 fusion protein was labelled with TcCO under a variety of conditions, and this was shown to be a relatively simple and robust method. Tissue biodistribution was assessed in a CEA-expressing LS174T (human colon carcinoma) nude mouse xenograft model. Tissues were taken at 1, 4 and 6 h for assessment of distribution of radioactivity and for measurement of CPG2 enzyme levels. The amount of radioactivity retained by the tumour proved to be an accurate estimation of actual measured enzyme activity, indicating that this radiolabelling method does not appear to damage the antibody-antigen binding or the enzyme activity of MFECP1. However, correlation between CPG2 enzyme activity and measured radioactivity in liver, spleen and kidney was poor, indicating retention of radioactivity in non-tumour sites but loss of enzyme activity. The high retention of technetium radioisotope in normal tissues may limit the clinical applicability of this radiolabelling method for MFECP1; however, these results suggest that this technique does have applicability for measuring the biodistribution of His-tagged recombinant proteins. (orig.)

  10. The production of high affinity monoclonal antibodies to human growth hormone

    International Nuclear Information System (INIS)

    Stuart, M.C.; Walichnowski, C.M.; Hussain, S.; Underwood, P.A.; Harman, D.F.; Rathjen, D.A.; Sturmer, S.R. von

    1983-01-01

    The primary aim of this work was to produce specific monoclonal antibodies to human growth hormone (hGH) for use in a diagnostic RIA of hGH levels in serum. Three different schedules were used for immunization of BALB/c mice and the splenocytes from each mouse were fused with myeloma cells Sp 2/0 Ag 14. Each fusion resulted in the production of hundreds of hybridomas secreting hGH-directed antibodies. Six antibodies have been fully characterized and have been grouped into pairs which recognize 3 different epitopes on the hGH molecule. One pair exhibits no cross reaction with the structurally related placental hormone, human placental lactogen (hPL), a second pair has low cross reaction with hPL (1.6-3%) and a third pair reacts equally well with hGH and hPL indicating binding to a common epitope in the 2 molecules. The highest affinity antibody, 74/6, which has an affinity constant of 4.4x10 10 l/mol and 3% cross-reactivity with hPL, has been used to establish a RIA for serum hGH measurements. Evidence is provided that hGH levels measured in this assay correlate well with those obtained in a conventional rabbit antiserum assay. (Auth.)

  11. Immunization of rhesus macaques with Echinococcus multilocularis recombinant 14-3-3 antigen leads to specific antibody response.

    Science.gov (United States)

    Lampe, Karen; Gottstein, B; Becker, T; Stahl-Hennig, C; Kaup, F-J; Mätz-Rensing, K

    2017-01-01

    E. multilocularis (Em) is the etiologic agent of alveolar echinococcosis (AE), a severe and potentially fatal disease, primarily affecting the liver of and occurring in aberrant intermediate hosts, e.g., humans and non-human primates. Due to increasing numbers of spontaneous cases of AE in the Old World monkey colonies of the German Primate Center, the question arose as to whether vaccination of non-human primates may represent a useful prophylactic approach. In this pilot study, the recombinant antigen Em14-3-3, which has provided a 97 % protection against E. multilocularis challenge infection in rodent models, was used for the first time to immunize rhesus macaques. In order to increase immunogenicity, the antigen was formulated with different adjuvants including Quil A®, aluminum hydroxide (alum), and muramyl dipeptide (MDP). Also, different vaccination regimens were tested. All vaccinated animals developed antigen-specific antibodies. While Quil A® induced a local adverse reaction, alum proved to be the most potent adjuvant in terms of induced antibody levels, longevity as well as tolerability. In conclusion, our pilot study demonstrated that recombinant Em14-3-3 is safe and immunogenic in rhesus monkeys. As a next step, efficacy of the vaccination remains to be explored.

  12. A fully automated primary screening system for the discovery of therapeutic antibodies directly from B cells.

    Science.gov (United States)

    Tickle, Simon; Howells, Louise; O'Dowd, Victoria; Starkie, Dale; Whale, Kevin; Saunders, Mark; Lee, David; Lightwood, Daniel

    2015-04-01

    For a therapeutic antibody to succeed, it must meet a range of potency, stability, and specificity criteria. Many of these characteristics are conferred by the amino acid sequence of the heavy and light chain variable regions and, for this reason, can be screened for during antibody selection. However, it is important to consider that antibodies satisfying all these criteria may be of low frequency in an immunized animal; for this reason, it is essential to have a mechanism that allows for efficient sampling of the immune repertoire. UCB's core antibody discovery platform combines high-throughput B cell culture screening and the identification and isolation of single, antigen-specific IgG-secreting B cells through a proprietary technique called the "fluorescent foci" method. Using state-of-the-art automation to facilitate primary screening, extremely efficient interrogation of the natural antibody repertoire is made possible; more than 1 billion immune B cells can now be screened to provide a useful starting point from which to identify the rare therapeutic antibody. This article will describe the design, construction, and commissioning of a bespoke automated screening platform and two examples of how it was used to screen for antibodies against two targets. © 2014 Society for Laboratory Automation and Screening.

  13. The antibody approach of labeling blood cells

    International Nuclear Information System (INIS)

    Srivastava, S.C.

    1992-01-01

    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are critically assessed and evaluated

  14. The antibody approach of labeling blood cells

    International Nuclear Information System (INIS)

    Srivastava, S.C.

    1991-01-01

    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are criticality assessed and evaluated

  15. The antibody approach of labeling blood cells

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, S.C.

    1991-12-31

    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are criticality assessed and evaluated.

  16. The antibody approach of labeling blood cells

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, S.C.

    1991-01-01

    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are criticality assessed and evaluated.

  17. The antibody approach of labeling blood cells

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, S.C.

    1992-12-31

    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are critically assessed and evaluated.

  18. Characterization of SGN-CD123A, A Potent CD123-Directed Antibody-Drug Conjugate for Acute Myeloid Leukemia.

    Science.gov (United States)

    Li, Fu; Sutherland, May Kung; Yu, Changpu; Walter, Roland B; Westendorf, Lori; Valliere-Douglass, John; Pan, Lucy; Cronkite, Ashley; Sussman, Django; Klussman, Kerry; Ulrich, Michelle; Anderson, Martha E; Stone, Ivan J; Zeng, Weiping; Jonas, Mechthild; Lewis, Timothy S; Goswami, Maitrayee; Wang, Sa A; Senter, Peter D; Law, Che-Leung; Feldman, Eric J; Benjamin, Dennis R

    2018-02-01

    Treatment choices for acute myelogenous leukemia (AML) patients resistant to conventional chemotherapies are limited and novel therapeutic agents are needed. IL3 receptor alpha (IL3Rα, or CD123) is expressed on the majority of AML blasts, and there is evidence that its expression is increased on leukemic relative to normal hematopoietic stem cells, which makes it an attractive target for antibody-based therapy. Here, we report the generation and preclinical characterization of SGN-CD123A, an antibody-drug conjugate using the pyrrolobenzodiazepine dimer (PBD) linker and a humanized CD123 antibody with engineered cysteines for site-specific conjugation. Mechanistically, SGN-CD123A induces activation of DNA damage response pathways, cell-cycle changes, and apoptosis in AML cells. In vitro , SGN-CD123A-mediated potent cytotoxicity of 11/12 CD123 + AML cell lines and 20/23 primary samples from AML patients, including those with unfavorable cytogenetic profiles or FLT3 mutations. In vivo , SGN-CD123A treatment led to AML eradication in a disseminated disease model, remission in a subcutaneous xenograft model, and significant growth delay in a multidrug resistance xenograft model. Moreover, SGN-CD123A also resulted in durable complete remission of a patient-derived xenograft AML model. When combined with a FLT3 inhibitor quizartinib, SGN-CD123A enhanced the activity of quizartinib against two FLT3-mutated xenograft models. Overall, these data demonstrate that SGN-CD123A is a potent antileukemic agent, supporting an ongoing trial to evaluate its safety and efficacy in AML patients (NCT02848248). Mol Cancer Ther; 17(2); 554-64. ©2017 AACR . ©2017 American Association for Cancer Research.

  19. PCSK9 Antibodies for the Treatment of Hypercholesterolemia

    Directory of Open Access Journals (Sweden)

    Ioanna Gouni-Berthold

    2014-12-01

    Full Text Available The serine protease proprotein convertase subtilisin/kexin type 9 (PCSK9 binds to the low-density lipoprotein (LDL receptor (LDLR and directs it to lysosomes for intracellular degradation. This results in decreased numbers of LDLR available on the hepatic cell surface to bind LDL particles and remove them from the circulation and therefore to a subsequent increase in circulating LDL-cholesterol (LDL-C plasma levels. Since 2003, when the role of PCSK9 in LDL-C metabolism was discovered, there have been major efforts to develop efficient and safe methods to inhibit it. Amongst those, monoclonal antibodies against PCSK9 are the furthest in development, with multiple phase 3 trials already published and with cardiovascular endpoint trials currently underway. Two fully human monoclonal antibodies, evolocumab (AMG 145 and alirocumab (REGN727/SAR236553, have been extensively studied in a wide range of subjects, such as those with statin intolerance, as an add-on to statin therapy, as a monotherapy and in patients with familial hypercholesterolemia. PCSK9 antibodies result in a consistent and robust decrease in LDL-C plasma levels ranging from 40% to 70%, either on top of statins or as a monotherapy. If the safety data from the on-going phase 3 trials remain as reassuring as the data available till now, PCSK9 antibodies will offer a novel, powerful therapeutic option to decrease LDL-C plasma levels and, hopefully, cardiovascular risk.

  20. Current status of cancer immunodetection with radiolabeled human monoclonal antibodies.

    Science.gov (United States)

    De Jager, R; Abdel-Nabi, H; Serafini, A; Pecking, A; Klein, J L; Hanna, M G

    1993-04-01

    The use of radiolabeled murine monoclonal antibodies (MoAbs) for cancer immunodetection has been limited by the development of human antimouse antibodies (HAMA). Human monoclonal antibodies do not elicit a significant human antihuman (HAHA) response. The generation and production of human monoclonal antibodies met with technical difficulties that resulted in delaying their clinical testing. Human monoclonal antibodies of all isotypes have been obtained. Most were immunoglobulin (Ig) M directed against intracellular antigens. Two antibodies, 16.88 (IgM) and 88BV59 (IgG3k), recognize different epitopes on a tumor-associated antigen, CTA 16.88, homologous to cytokeratins 8, 18, and 19. CTA 16.88 is expressed by most epithelial-derived tumors including carcinomas of the colon, pancreas, breast, ovary, and lung. The in vivo targeting by these antibodies is related to their localization in nonnecrotic areas of tumors. Repeated administration of 16.88 over 5 weeks to a cumulative dose of 1,000 mg did not elicit a HAHA response. Two of 53 patients developed a low titer of HAHA 1 to 3 months after a single administration of 88BV59. Planar imaging of colorectal cancer with Iodine-131 (131I)-16.88 was positive in two studies in 9 of 12 and 16 of 20 patients preselected by immunohistochemistry. Tumors less than 2 cm in diameter are usually not detected. The lack of immunogenicity and long tumor residence time (average = 17 days) makes 16.88 a good candidate for therapy. Radioimmunlymphoscintigraphy with indium-111 (111In)-LiLo-16.88 administered by an intramammary route was used in the presurgical staging of primary breast cancer. The negative predictive value of lymph node metastases for tumors less than 3 cm was 90.5%. Planar and single photon emission computed tomography imaging of colorectal carcinoma with technetium-99m (99mTc) 88BV59 was compared with computed tomography (CT) scan in 36 surgical patients. The antibody scan was more sensitive than the CT scan in detecting

  1. Modification of Antibody Function by Mutagenesis.

    Science.gov (United States)

    Dasch, James R; Dasch, Amy L

    2017-09-01

    The ability to "fine-tune" recombinant antibodies by mutagenesis separates recombinant antibodies from hybridoma-derived antibodies because the latter are locked with respect to their properties. Recombinant antibodies can be modified to suit the application: Changes in isotype, format (e.g., scFv, Fab, bispecific antibodies), and specificity can be made once the heavy- and light-chain sequences are available. After immunoglobulin heavy and light chains for a particular antibody have been cloned, the binding site-namely, the complementarity determining regions (CDR)-can be manipulated by mutagenesis to obtain antibody variants with improved properties. The method described here is relatively simple, uses commercially available reagents, and is effective. Using the pComb3H vector, a commercial mutagenesis kit, PfuTurbo polymerase (Agilent), and two mutagenic primers, a library of phage with mutagenized heavy and light CDR3 can be obtained. © 2017 Cold Spring Harbor Laboratory Press.

  2. Clearance of 131I-labeled murine monoclonal antibody from patients' blood by intravenous human anti-murine immunoglobulin antibody

    International Nuclear Information System (INIS)

    Stewart, J.S.; Sivolapenko, G.B.; Hird, V.; Davies, K.A.; Walport, M.; Ritter, M.A.; Epenetos, A.A.

    1990-01-01

    Five patients treated with intraperitoneal 131I-labeled mouse monoclonal antibody for ovarian cancer also received i.v. exogenous polyclonal human anti-murine immunoglobulin antibody. The pharmacokinetics of 131I-labeled monoclonal antibody in these patients were compared with those of 28 other patients receiving i.p.-radiolabeled monoclonal antibody for the first time without exogenous human anti-murine immunoglobulin, and who had no preexisting endogenous human anti-murine immunoglobulin antibody. Patients receiving i.v. human anti-murine immunoglobulin antibody demonstrated a rapid clearance of 131I-labeled monoclonal antibody from their circulation. The (mean) maximum 131I blood content was 11.4% of the injected activity in patients receiving human anti-murine immunoglobulin antibody compared to 23.3% in patients not given human anti-murine immunoglobulin antibody. Intravenous human anti-murine immunoglobulin antibody decreased the radiation dose to bone marrow (from 131I-labeled monoclonal antibody in the vascular compartment) 4-fold. Following the injection of human anti-murine immunoglobulin antibody, 131I-monoclonal/human anti-murine immunoglobulin antibody immune complexes were rapidly transported to the liver. Antibody dehalogenation in the liver was rapid, with 87% of the injected 131I excreted in 5 days. Despite the efficient hepatic uptake of immune complexes, dehalogenation of monoclonal antibody was so rapid that the radiation dose to liver parenchyma from circulating 131I was decreased 4-fold rather than increased. All patients developed endogenous human anti-murine immunoglobulin antibody 2 to 3 weeks after treatment

  3. Antiphospholipid antibody: laboratory, pathogenesis and clinical manifestations

    Directory of Open Access Journals (Sweden)

    T. Ziglioli

    2011-06-01

    Full Text Available Antiphospholipid antibodies (aPL represent a heterogeneous group of antibodies that recognize various antigenic targets including beta2 glycoprotein I (β2GPI, prothrombin (PT, activated protein C, tissue plasminogen activator, plasmin and annexin A2. The most commonly used tests to detect aPL are: lupus anticoagulant (LAC, a functional coagulation assay, anticardiolipin antibody (aCL and anti-β2GPI antibody (anti-β2GPI, which are enzyme-linked immunoassay (ELISA. Clinically aPL are associated with thrombosis and/or with pregnancy morbidity. Apparently aPL alone are unable to induce thrombotic manifestations, but they increase the risk of vascular events that can occur in the presence of another thrombophilic condition; on the other hand obstetrical manifestations were shown to be associated not only to thrombosis but mainly to a direct antibody effect on the trophoblast.

  4. Freeze-dried formulation for direct 99mTc-labeling ior-egf/r3 MAb: additives, biodistribution, and stability

    International Nuclear Information System (INIS)

    Morales, Alejo A. Morales; Nunez-Gandolff, Gilda; Perez, Niuvis Perez; Veliz, Belkis Chico; Caballero-Torres, Idania; Duconge, Jorge; Fernandez, Eduardo; Crespo, Francisco Zayas; Veloso, Ana; Iznaga-Escobar, Normando

    1999-01-01

    Monoclonal antibodies (MAbs) have been useful for immunoscintigraphic applications in clinical diagnosis since they were introduced in nuclear medicine practice. The MAb ior egf/r3 developed at the Center of Molecular Immunology (Havana, Cuba) is a murine antibody that recognizes the human epidermal growth factor receptor (EGF-R) and has been used widely in the radioimmunodiagnosis of tumors of epithelial origin. Based on the direct Schwarz method, the present report describes the preparation of a freeze-dried formulation for radiolabeling the MAb ior egf/r3 with 99m Tc for immunoscintigraphic applications. Radiolabeling efficiency, effects on immunoreactivity, biodistribution, pharmacokinetic, and stability of the formulation are reported. The study demonstrated that the freeze-dried formulation can be labeled with 99m Tc at high yield. The resulting 99m Tc-labeled ior egf/r3 MAb can be used to visualize in vivo human tumors of epithelial origin by immunoscintigraphy studies. The kit does not need any other addition or purification at the time of tagging other than the requisite amount of pertechnetate (40-50 mCi). Because the contents of the kit are lyophilized, no special storage or transportation is required

  5. Prevalence of antibodies against Neospora spp. and Sarcocystis neurona in donkeys from northeastern Brazil

    Directory of Open Access Journals (Sweden)

    Solange Maria Gennari

    2016-03-01

    Full Text Available Abstract Sarcocystis neurona and Neospora hughesi are coccidian protozoa that can cause neurological illness in horses in America. In this study we report seroprevalence of Neospora spp. andS. neurona in sera of 333 donkeys from the northeastern region of Brazil. Antibodies to Neospora spp. were detected in 2% (7 donkeys of 333 sera tested by the indirect fluorescent antibody test (IFAT with a cut-off dilution of 1:40. Antibodies to S. neurona were found in 3% (10 donkeys of the samples tested by IFAT (cut-off ≥50 and 21% (69 donkeys by the direct agglutination test (SAT ≥50. The SAT and IFAT results for S. neurona showed a poor concordance (value of Kappa=0.051. This is the first report ofNeospora spp. antibodies in Brazilian donkeys and the first detection of antibodies against S. neurona in this animal species.

  6. Cloning, bacterial expression and crystallization of Fv antibody fragments

    Science.gov (United States)

    E´, Jean-Luc; Boulot, Ginette; Chitarra, V´ronique; Riottot, Marie-Madeleine; Souchon, H´le`ne; Houdusse, Anne; Bentley, Graham A.; Narayana Bhat, T.; Spinelli, Silvia; Poljak, Roberto J.

    1992-08-01

    The variable Fv fragments of antibodies, cloned in recombinant plasmids, can be expressed in bacteria as functional proteins having immunochemical properties which are very similar or identical with those of the corresponding parts of the parent eukaryotic antibodies. They offer new possibilities for the study of antibody-antigen interactions since the crystals of Fv fragments and of their complexes with antigen reported here diffract X-rays to a higher resolution that those obtained with the cognate Fab fragments. The Fv approach should facilitate the structural study of the combining site of antibodies and the further characterization of antigen-antibody interactions by site-directed mutagenesis experiments.

  7. Production and characterization of human anti-V3 monoclonal antibodies from the cells of HIV-1 infected Indian donors

    Directory of Open Access Journals (Sweden)

    Andrabi Raiees

    2012-09-01

    Full Text Available Abstract Background Analysis of human monoclonal antibodies (mAbs developed from HIV-1 infected donors have enormously contributed to the identification of neutralization sensitive epitopes on the HIV-1 envelope glycoprotein. The third variable region (V3 is a crucial target on gp120, primarily due to its involvement in co-receptor (CXCR4 or CCR5 binding and presence of epitopes recognized by broadly neutralizing antibodies. Methods Thirty-three HIV-1 seropositive drug naive patients (18 males and 15 females within the age range of 20–57 years (median = 33 years were recruited in this study for mAb production. The mAbs were selected from EBV transformed cultures with conformationally constrained Cholera-toxin-B containing V3C (V3C-CTB fusion protein. We tested the mAbs for their binding with HIV-1 derived proteins and peptides by ELISA and for neutralization against HIV-1 viruses by TZM-bl assays. Results We isolated three anti-V3 mAbs, 277, 903 and 904 from the cells of different individuals. The ELISA binding revealed a subtype-C and subtype-A specific binding of antibody 277 and 903 while mAb 904 exhibited cross reactivity also with subtype-B V3. Epitope mapping of mAbs with overlapping V3 peptides showed exclusive binding to V3 crown. The antibodies displayed high and low neutralizing activity against 2/5 tier 1 and 1/6 tier 2 viruses respectively. Overall, we observed a resistance of the tier 2 viruses to neutralization by the anti-V3 mAbs, despite the exposure of the epitopes recognized by these antibodies on two representative native viruses (Du156.12 and JRFL, suggesting that the affinity of mAb might equally be crucial for neutralization, as the epitope recognition. Conclusions Our study suggests that the anti-V3 antibodies derived from subtype-C infected Indian patients display neutralization potential against tier 1 viruses while such activity may be limited against more resistant tier 2 viruses. Defining the fine epitope

  8. Antigen-Dissociation from Antibody-Modified Nanotransistor Sensor Arrays as a Direct Biomarker Detection Method in Unprocessed Biosamples.

    Science.gov (United States)

    Krivitsky, Vadim; Zverzhinetsky, Marina; Patolsky, Fernando

    2016-10-12

    The detection of biomolecules is critical for a wide spectrum of applications in life sciences and medical diagnosis. Nonetheless, biosamples are highly complex solutions, which contain an enormous variety of biomolecules, cells, and chemical species. Consequently, the intrinsic chemical complexity of biosamples results in a significant analytical background noise and poses an immense challenge to any analytical measurement, especially when applied without prior efficient separation and purification steps. Here, we demonstrate the application of antigen-dissociation regime, from antibody-modified Si-nanowire sensors, as a simple and effective direct sensing mechanism of biomarkers of interest in complex biosamples, such as serum and untreated blood, which does not require ex situ time-consuming biosample manipulation steps, such as centrifugation, filtering, preconcentration, and desalting, thus overcoming the detrimental Debye screening limitation of nanowire-based biosensors. We found that two key parameters control the capability to perform quantitative biomarkers analysis in biosamples: (i) the affinity strength (k off rate) of the antibody-antigen recognition pair, which dictates the time length of the high-affinity slow dissociation subregime, and (ii) the "flow rate" applied during the solution exchange dissociation step, which controls the time width of the low-affinity fast-dissociation subregime. Undoubtedly, this is the simplest and most convenient approach for the SiNW FET-based detection of antigens in complex untreated biosamples. The lack of ex situ biosample manipulation time-consuming processes enhances the portability of the sensing platform and reduces to minimum the required volume of tested sample, as it allows the direct detection of untreated biosamples (5-10 μL blood or serum), while readily reducing the detection cycle duration to less than 5 min, factors of great importance in near-future point-of-care medical applications. We believe

  9. Antibodies to watch in 2014.

    Science.gov (United States)

    Reichert, Janice M

    2014-01-01

    Since 2010, mAbs has documented the biopharmaceutical industry's progress in transitioning antibody therapeutics to first Phase 3 clinical studies and regulatory review, and its success at gaining first marketing approvals for antibody-based products. This installment of the "Antibodies to watch" series outlines events anticipated to occur between December 2013 and the end of 2014, including first regulatory actions on marketing applications for vedolizumab, siltuximab, and ramucirumab, as well as the Fc fusion proteins Factor IX-Fc and Factor VIII-Fc; and the submission of first marketing applications for up to five therapeutics (secukinumab, ch14.18, onartuzumab, necitumumab, gevokizumab). Antibody therapeutics in Phase 3 studies are described, with an emphasis on those with study completion dates in 2014, including antibodies targeting interleukin-17a or the interleukin-17a receptor (secukinumab, ixekizumab, brodalumab), proprotein convertase subtilisin/kexin type 9 (alirocumab, evolocumab, bococizumab), and programmed death 1 receptor (lambrolizumab, nivolumab). Five antibodies with US Food and Drug Administration's Breakthrough Therapy designation (obinutuzumab, ofatumumab, lambrolizumab, bimagrumab, daratumumab) are also discussed.

  10. The future of antibody therapeutics: ADCs bi-specifics and RIT

    International Nuclear Information System (INIS)

    Reichert, J.

    2015-01-01

    Full text of publication follows. Antibodies are widely accepted as remarkably versatile therapeutic agents. As evidence of this, the ∼ 30 antibody products marketed worldwide had total global sales of more than 50 billion dollars in 2012, and the commercial clinical pipeline currently comprises over 350 antibody-based product candidates. In a testament to scientific ingenuity, the investigational molecules (clinical and preclinical) are notably diverse in their composition of matter and include antibodies conjugated to a variety of agents (drugs, radioisotopes), bi-specific antibodies, and fragments or domains of antibodies. The concepts that form the basis of these agents were established decades ago, but advances in technology are now allowing new opportunities for their development. In this presentation, future directions in antibody therapeutics development will be discussed, with a focus on antibody-drug conjugates, bi-specific antibodies and radioimmunotherapy. (author)

  11. Chloramine-T induced binding of monoclonal antibody B72. 3 to concanavalin-A

    Energy Technology Data Exchange (ETDEWEB)

    Cole, W.C.; Jhingran, S.G. (Methodist Hospital, Houston, TX (United States) Baylor Coll. of Medicine, Houston, TX (United States))

    1993-07-01

    The effects of chloramine-T (CT) on monoclonal antibody B72.3 were studied with particular reference to Con-A lectin binding. After exposure to chloramine-T concentrations from 0.8 to 4.0 mg/mL (115-574 mol CT/mol B72.3), B72.3 showed progressive binding to agarose-linked Con-A. This behavior was paralleled by decreasing immunoreactivity and increasing fragmentation and aggregation of B72.3 demonstrated by SDS-PAGE and size exclusion HPLC. (Author).

  12. Antithyroglobulin antibody

    Science.gov (United States)

    Thyroglobulin antibody; Thyroiditis - thyroglobulin antibody; Hypothyroidism - thyroglobulin antibody; Thyroiditis - thyroglobulin antibody; Graves disease - thyroglobulin antibody; Underactive thyroid - thyroglobulin antibody

  13. Use of monoclonal antibody B72.3 in the management of gynecologic malignancies

    International Nuclear Information System (INIS)

    Simpson, J.; Schlom, J.

    1988-01-01

    Monoclonal antibodies are currently used in the diagnosis of gynecologic malignancies by way of immunohistochemical assays, serum assays, and in situ radiolocalization of carcinoma lesions. Among them is MAb B72.3, generated against a human tumor-associated antigen (TAG-72). Using immunohistochemical techniques, MAb B72.3 has shown reactivity with 100 percent of common epithelial ovarian carcinomas and endometrial carcinomas and non-reactivity with normal adult tissues, with the exception of normal secretory endometrium. B72.3 appears to be a valuable immunocytologic adjunct, with greater than 90 percent of effusions and fine-needle aspiration biopsies from gynecologic carcinomas showing reactivity. Using a serum assay developed to detect the presence of the TAG-72 antigen, 48 percent of patients with ovarian carcinoma demonstrated TAG-72-positive sera versus 1 percent of control sera. 131 I-labeled MAb B72.3 IgG and gamma scanning have been used for the in situ detection of metastatic carcinoma. Twelve of 15 patients with ovarian carcinoma showed positive gamma scans, and approximately 80 percent of the lesions demonstrated specific localization of the antibody. These studies indicate the potential utility of MAb B72.3 in the diagnosis of gynecologic carcinoma. 57 references

  14. Antitumor and cytotoxic properties of a humanized antibody specific for the GM3(Neu5Gc) ganglioside.

    Science.gov (United States)

    Dorvignit, Denise; García-Martínez, Liliana; Rossin, Aurélie; Sosa, Katya; Viera, Justo; Hernández, Tays; Mateo, Cristina; Hueber, Anne-Odile; Mesa, Circe; López-Requena, Alejandro

    2015-12-01

    Gangliosides are sialic acid-bearing glycosphingolipids expressed on all mammalian cell membranes, and participate in several cellular processes. During malignant transformation their expression changes, both at the quantitative and qualitative levels. Of particular interest is the overexpression by tumor cells of Neu5Gc-gangliosides, which are absent, or detected in trace amounts, in human normal cells. The GM3(Neu5Gc) ganglioside in particular has been detected in many human tumors, and it is considered one of the few tumor specific antigen. We previously demonstrated that a humanized antibody specific for this molecule, named 14F7hT, retained the binding and cytotoxic properties of the mouse antibody. In this work, we confirm that 14F7hT exerts a non-apoptotic cell death mechanism in vitro and shows its potent in vivo antitumor activity on a solid mouse myeloma model. Also, we demonstrate, in contrast to the murine counterpart, the capacity of this antibody to induce antibody-dependent cell-mediated cytotoxicity using human effector cells, which increases its potential for the treatment of GM3(Neu5Gc)-expressing human tumors. Copyright © 2015 Elsevier GmbH. All rights reserved.

  15. Antibody recognition of Z-DNA

    International Nuclear Information System (INIS)

    Lafer, E.M.; Moeller, A.; Valle, R.P.C.; Nordheim, V.A.; Rich, A.; Stollar, B.D.; Massachusetts Inst. of Tech., Cambridge)

    1983-01-01

    To measure serological reactions under physiological ionic strength, we prepared a brominated (Bl) poly(dG-dC).poly(dG-dC), which forms a stable Z helix in solutions of low salt concentration. Mice and rabbits were immunized with this polymer complexed with the basic protein methylated bovine serum albumin (MBSA), and it was discovered that the Z-DNA helix is a strong immunogen. Various antibody populations were purified from the rabbit serum by quantitative immunoprecipitation. Spleen cells from the mice were used for the preparation of hybridoma cell lines secreting monoclonal antibodies. Anti-Z-DNA antibodies were also raised by immunizing animals with poly(dG-dm 5 C).poly(dG-dm 5 C) under conditions where it was reported to be in the left-handed Z conformation as well as unmodified poly(dG-dC).poly(dG-dC) that was in the right-handed B conformation: both were complexed with MBSA. Z-DNA reactive antibodies were found in both murine and human SLE. A Z-DNA-specific as well as a dDNA and Z-DNA cross-reactive antibody population were distinguished by affinity chromatography of the SLE sera. The specificities of the various anti-Z-DNA antibody populations were measured by direct-binding and competitive radioimmunoassays, using synthetic polymers of defined structure under various ionic strengths. These studies allow us to map the possible antigenic sites for these antibodies, which serve as a model for DNA-protein recognition. The findings also established the usefulness of the antibodies as biochemical probes for Z-DNA. 29 references, 6 figures, 1 table

  16. Potential application of labeled antibodies for thrombus detection

    Energy Technology Data Exchange (ETDEWEB)

    Ezekowitz, M.D.; Coller, B.S.; Srivastava, S.C.

    1986-01-01

    Labeling platelets with monoclonal antibodies in whole blood for imaging thrombi is less cumbersome than the established /sup 111/In-oxine method. 7E3, a murine monoclonal antibody directed against glycoprotein IIb and/or IIIa on both human and dog platelets was used to label canine platelets. Thrombi were induced by transcatheter placement of a copper coil followed by electrocoagulation. 7E3 was iodinated with /sup 131/I and labelled with /sup 111/In using 7E3-DTPA conjugate. Whole blood was incubated with 0.5 - 1.0 ..mu..g labeled 7E3/mL blood. In 4/4 dogs, experimental deep vein thrombi were identified using both /sup 131/I-and /sup 111/In-labeled 7E3 within 5-30 min after injection. For both isotopes, 1 h blood clearance was 54 + - 9%. In 1/3 dogs, experimental coronary thrombus could be identified ex vivo at 4 h. Clot to blood ratios ranged between 7 to 13:1. Using the /sup 111/In-oxine method, 0/3 coronary thrombi were seen. Thus, /sup 131/I-and /sup 111/In-labeled 7E3 may be used to readily identify peripheral venous thrombi. For reliable and prompt identification of coronary thrombi, more rapid clearance of the labeled platelets is required.

  17. Identification of antibody glycosylation structures that predict monoclonal antibody Fc-effector function.

    Science.gov (United States)

    Chung, Amy W; Crispin, Max; Pritchard, Laura; Robinson, Hannah; Gorny, Miroslaw K; Yu, Xiaojie; Bailey-Kellogg, Chris; Ackerman, Margaret E; Scanlan, Chris; Zolla-Pazner, Susan; Alter, Galit

    2014-11-13

    To determine monoclonal antibody (mAb) features that predict fragment crystalizable (Fc)-mediated effector functions against HIV. Monoclonal antibodies, derived from Chinese hamster ovary cells or Epstein-Barr virus-immortalized mouse heteromyelomas, with specificity to key regions of the HIV envelope including gp120-V2, gp120-V3 loop, gp120-CD4(+) binding site, and gp41-specific antibodies, were functionally profiled to determine the relative contribution of the variable and constant domain features of the antibodies in driving robust Fc-effector functions. Each mAb was assayed for antibody-binding affinity to gp140(SR162), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and for the ability to bind to FcγRIIa, FcγRIIb and FcγRIIIa receptors. Antibody glycan profiles were determined by HPLC. Neither the specificity nor the affinity of the mAbs determined the potency of Fc-effector function. FcγRIIIa binding strongly predicted ADCC and decreased galactose content inversely correlated with ADCP, whereas N-glycolylneuraminic acid-containing structures exhibited enhanced ADCP. Additionally, the bi-antenary glycan arm onto which galactose was added predicted enhanced binding to FcγRIIIa and ADCC activity, independent of the specificity of the mAb. Our studies point to the specific Fc-glycan structures that can selectively promote Fc-effector functions independently of the antibody specificity. Furthermore, we demonstrated antibody glycan structures associated with enhanced ADCP activity, an emerging Fc-effector function that may aid in the control and clearance of HIV infection.

  18. A comparison of labelled antibody methods for the detection of virus antigens in cell monolayers

    International Nuclear Information System (INIS)

    Oram, J.D.; Crooks, A.J.

    1979-01-01

    A number of labelled antibody methods have been applied to the detection of Semliki Forest virus antigens after replication of the virus in monolayers of host cells in multi-well polystyrene plates. The importance of several reaction variables has been investigated and the sensitivity of the methods compared for different periods of virus replication. Direct assays with radio-labelled antibody (RLA) and indirect assays peroxidase-antiperoxidase complexes (PAP) were equally sensitive. Direct and indirect assays using enzyme-linked antibodies (ELA) were slightly less sensitive than the direct RLA and PAP methods but were more sensitive than the indirect RLA or fluorescent antibody (FLA) methods. Direct assays using ELA were more rapid and easier to perform than the other assay methods. (Auth.)

  19. Differences in Allelic Frequency and CDRH3 Region Limit the Engagement of HIV Env Immunogens by Putative VRC01 Neutralizing Antibody Precursors

    Directory of Open Access Journals (Sweden)

    Christina Yacoob

    2016-11-01

    Full Text Available Elicitation of broadly neutralizing antibodies remains a long-standing goal of HIV vaccine research. Although such antibodies can arise during HIV-1 infection, gaps in our knowledge of their germline, pre-immune precursor forms, as well as on their interaction with viral Env, limit our ability to elicit them through vaccination. Studies of broadly neutralizing antibodies from the VRC01-class provide insight into progenitor B cell receptors (BCRs that could develop into this class of antibodies. Here, we employed high-throughput heavy chain variable region (VH/light chain variable region (VL deep sequencing, combined with biophysical, structural, and modeling antibody analyses, to interrogate circulating potential VRC01-progenitor BCRs in healthy individuals. Our study reveals that not all humans are equally predisposed to generate VRC01-class antibodies, not all predicted progenitor VRC01-expressing B cells can bind to Env, and the CDRH3 region of germline VRC01 antibodies influence their ability to recognize HIV-1. These findings will be critical to the design of optimized immunogens that should consider CDRH3 interactions.

  20. Antibody engineering using phage display with a coiled-coil heterodimeric Fv antibody fragment.

    Directory of Open Access Journals (Sweden)

    Xinwei Wang

    Full Text Available A Fab-like antibody binding unit, ccFv, in which a pair of heterodimeric coiled-coil domains was fused to V(H and V(L for Fv stabilization, was constructed for an anti-VEGF antibody. The anti-VEGF ccFv showed the same binding affinity as scFv but significantly improved stability and phage display level. Furthermore, phage display libraries in the ccFv format were constructed for humanization and affinity maturation of the anti-VEGF antibody. A panel of V(H frameworks and V(H-CDR3 variants, with a significant improvement in affinity and expressibility in both E. coli and yeast systems, was isolated from the ccFv phage libraries. These results demonstrate the potential application of the ccFv antibody format in antibody engineering.

  1. Identification of a linear epitope recognized by a monoclonal antibody directed to the heterogeneous nucleoriboprotein A2

    DEFF Research Database (Denmark)

    Tronstrøm, Julie; Dragborg, Anette H.; Hansen, Paul Robert

    2014-01-01

    to as RA33. In the absence of citrulline antibodies, RA33 antibodies have been suggested to be associated with a milder disease course. In this study we screened the reactivity of a monoclonal antibody to RA33-derived peptides by modified enzyme-linked immunosorbent assays (ELISA). Terminally truncated......Rheumatoid arthritis (RA) is a chronic autoimmune disorder, characterized by progressive joint destruction and disability. Classical autoantibodies of RA are rheumatoid factors and citrulline antibodies. Patients positive for these autoantibodies are usually associated with a progressive disease...... course. A subgroup of RA patients does not express citrulline antibodies, instead are approximately 35% of these anti-citrulline-negative patients reported to express autoantibodies to the heterogeneous nucleoriboprotein A2, a ribonucleoprotein involved in RNA transport and processing also referred...

  2. Avelumab: combining immune checkpoint inhibition and antibody-dependent cytotoxicity.

    Science.gov (United States)

    Hamilton, Gerhard; Rath, Barbara

    2017-04-01

    Immune checkpoint inhibition holds great promise for selected tumors. The human monoclonal antibody (mAB) avelumab is directed to programmed death ligand-1 (PD-L1) and is supposed to inhibit the immunosuppressive PD-L1/PD-1 interaction and, furthermore, effect antibody-dependent cytotoxicity (ADCC) lysis of tumor cells. Areas covered: This article presents an overview of the current means to activate the antitumor immune defense by targeting PD-1 or PD-L1 with mABs and their possible role in ADCC-mediated tumor cell elimination. Expert opinion: Avelumab contains a Fc region which can bind cognate receptors on immune effector cells and induce ADCC-mediated tumor cell lysis, in contrast to other mABs directed to PD-1/PD-L1 which lack the ability to trigger ADCC due to belonging to the IgG4 subclass or possessing a mutated Fc region. Preclinical and clinical data indicate that avelumab can be safely administered to cancer patients with a toxicity profile comparable to other mABs and without lysis of PD-L1-positive activated immune cells. This antibody yielded durable responses in a phase II trial in advanced Merkel cell carcinoma patients. Tumor cell lysis by avelumab prevents cells from resorting to alternative checkpoints as shown by targeting PD-1 and the upregulation of TIM-3.

  3. A Comparative Analysis of Polymerase Chain Reaction and Direct Fluorescent Antibody Test for Diagnosis of Genital Herpes.

    Science.gov (United States)

    Patwardhan, Vrushali; Bhalla, Preena; Rawat, Deepti; Garg, Vijay Kumar; Sardana, Kabir; Sethi, Sumit

    2017-01-01

    To compare laboratory tests that can simultaneously detect and type herpes simplex virus (HSV) directly from the genital ulcer specimens in clinically suspected cases of genital herpes. A study was conducted over 10 months and 44 adult male and female patients clinically suspected with genital herpes were recruited. Genital ulcer swab specimens were subjected to glycoprotein-G gene-based conventional polymerase chain reaction (PCR) and commercially available direct fluorescent antibody (DFA) test and the results were compared. PCR for HSV was positive in 82% (36/44) cases. DFA was positive in 68.2% (30/44) cases. There was 100% agreement between HSV types detected by DFA and PCR. The strength of agreement between the results was better in primary genital herpes than recurrent cases. PCR was found to be better in the detection of HSV in recurrent genital herpes patients. It is a better modality, especially when genital herpes clinically presents with ulcerative or crusted lesions, and is also a cheaper alternative as compared to DFA.

  4. Radiolabeled Humanized Anti-CD3 Monoclonal Antibody Visilizumab for Imaging Human T-Lymphocytes

    NARCIS (Netherlands)

    Malviya, Gaurav; D'Alessandria, Calogero; Bonanno, Elena; Vexler, Vladimir; Massari, Roberto; Trotta, Carlo; Scopinaro, Francesco; Dierckx, Rudi; Signore, Alberto

    2009-01-01

    Visilizumab is an IgG(2) humanized monoclonal antibody (mAb) characterized by non-Fc gamma R binding and specific to the CD3 antigen, expressed on more than 95% of circulating resting T-lymphocytes and on activated T-lymphocytes homing in inflamed tissues. We hypothesized that the use of a

  5. A novel antibody engineering strategy for making monovalent bispecific heterodimeric IgG antibodies by electrostatic steering mechanism.

    Science.gov (United States)

    Liu, Zhi; Leng, Esther C; Gunasekaran, Kannan; Pentony, Martin; Shen, Min; Howard, Monique; Stoops, Janelle; Manchulenko, Kathy; Razinkov, Vladimir; Liu, Hua; Fanslow, William; Hu, Zhonghua; Sun, Nancy; Hasegawa, Haruki; Clark, Rutilio; Foltz, Ian N; Yan, Wei

    2015-03-20

    Producing pure and well behaved bispecific antibodies (bsAbs) on a large scale for preclinical and clinical testing is a challenging task. Here, we describe a new strategy for making monovalent bispecific heterodimeric IgG antibodies in mammalian cells. We applied an electrostatic steering mechanism to engineer antibody light chain-heavy chain (LC-HC) interface residues in such a way that each LC strongly favors its cognate HC when two different HCs and two different LCs are co-expressed in the same cell to assemble a functional bispecific antibody. We produced heterodimeric IgGs from transiently and stably transfected mammalian cells. The engineered heterodimeric IgG molecules maintain the overall IgG structure with correct LC-HC pairings, bind to two different antigens with comparable affinity when compared with their parental antibodies, and retain the functionality of parental antibodies in biological assays. In addition, the bispecific heterodimeric IgG derived from anti-HER2 and anti-EGF receptor (EGFR) antibody was shown to induce a higher level of receptor internalization than the combination of two parental antibodies. Mouse xenograft BxPC-3, Panc-1, and Calu-3 human tumor models showed that the heterodimeric IgGs strongly inhibited tumor growth. The described approach can be used to generate tools from two pre-existent antibodies and explore the potential of bispecific antibodies. The asymmetrically engineered Fc variants for antibody-dependent cellular cytotoxicity enhancement could be embedded in monovalent bispecific heterodimeric IgG to make best-in-class therapeutic antibodies. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Freeze-dried formulation for direct {sup 99m}Tc-labeling ior-egf/r3 MAb: additives, biodistribution, and stability

    Energy Technology Data Exchange (ETDEWEB)

    Morales, Alejo A. Morales; Nunez-Gandolff, Gilda; Perez, Niuvis Perez; Veliz, Belkis Chico; Caballero-Torres, Idania; Duconge, Jorge; Fernandez, Eduardo; Crespo, Francisco Zayas; Veloso, Ana; Iznaga-Escobar, Normando E-mail: normando@ict.sld.cu

    1999-08-01

    Monoclonal antibodies (MAbs) have been useful for immunoscintigraphic applications in clinical diagnosis since they were introduced in nuclear medicine practice. The MAb ior egf/r3 developed at the Center of Molecular Immunology (Havana, Cuba) is a murine antibody that recognizes the human epidermal growth factor receptor (EGF-R) and has been used widely in the radioimmunodiagnosis of tumors of epithelial origin. Based on the direct Schwarz method, the present report describes the preparation of a freeze-dried formulation for radiolabeling the MAb ior egf/r3 with {sup 99m}Tc for immunoscintigraphic applications. Radiolabeling efficiency, effects on immunoreactivity, biodistribution, pharmacokinetic, and stability of the formulation are reported. The study demonstrated that the freeze-dried formulation can be labeled with {sup 99m}Tc at high yield. The resulting {sup 99m}Tc-labeled ior egf/r3 MAb can be used to visualize in vivo human tumors of epithelial origin by immunoscintigraphy studies. The kit does not need any other addition or purification at the time of tagging other than the requisite amount of pertechnetate (40-50 mCi). Because the contents of the kit are lyophilized, no special storage or transportation is required.

  7. High incidence of antibodies to HTLV-I tax in blood relatives of adult T cell leukemia patients.

    Science.gov (United States)

    Okayama, A; Chen, Y M; Tachibana, N; Shioiri, S; Lee, T H; Tsuda, K; Essex, M

    1991-01-01

    Adult T cell leukemia (ATL) is caused by the human T cell leukemia virus type I (HTLV-I). Although the mechanisms of the leukemogenic process are unknown, the tax gene may have a role in this process. Because clustering occurs with HTLV-I and ATL, members of ATL families were examined for antibodies to the tax protein and compared with matched HTLV-I-positive blood donors. To investigate the antibody response to this protein, a plasmid, pBHX-4, was constructed to express a recombinant tax protein (r-tax). For ATL patients and their HTLV-I antibody-positive blood relatives, the rate of seroreactivity with the r-tax protein was 67.3% (35/52), compared with 51.6% (97/188) for HTLV-I antibody-positive control blood donors (P less than .05). The difference between direct offspring of ATL patients and matched HTLV-I blood donors was even greater (84.2% [16/91] vs. 44.2% [42/95]; P less than .005). Thus, tax antibody positivity in direct offspring of ATL patients may reflect differences in time or route of HTLV-I infection. Alternatively, it might reflect genetic differences in host susceptibility or virus strain.

  8. Prevalence of antinuclear and anti-erythrocyte antibodies in healthy cats.

    Science.gov (United States)

    Abrams-Ogg, Anthony C G; Lim, Sophia; Kocmarek, Helen; Ho, Kim; Blois, Shauna L; Shewen, Patricia E; Wood, R Darren; Bienzle, Dorothee

    2018-03-01

    Positive antinuclear antibody and direct antiglobulin tests support diagnoses such as systemic lupus erythematosus and immune-mediated anemia, respectively. Positive tests may occur in cats, but the prevalence of positive results in healthy cats is not well known. The study's purpose was to determine prevalences of positive antinuclear antibody and direct antiglobulin tests in healthy cats. Antinuclear antibody titers were measured by indirect immunofluorescence, and anti-erythrocyte antibodies were measured by the microtitration direct antiglobulin test at 37, 23, and 4°C in 61 client-owned and 28 facility-owned cats. Differences between the 2 groups were examined using chi-squared tests. For the antinuclear antibody tests, 70% of client-owned cats were negative, 10% had weak titers (1:40-1:80), and 20% had strong titers (1:160-1:320). Facility-owned cats had significantly fewer positive titers with 96% negative and one positive (1:8). For the antiglobulin test at 37°C, 93% of all cats were negative, 2 cats in each group were positive at low dilutions (1:2), and 2 client-owned cats were transiently positive at high dilutions (≥ 1:2048). At 23°C, 90% of all cats were negative, and 2 client-owned and 5 facility-owned cats were positive at low dilutions (1:2-1:8). At 4°C, 67% of client-owned cats had invalid results (negative control well agglutination), and 33% had negative results, while of facility-owned cats 14% had invalid results, 14% had agglutination at low dilutions, and 72% were negative. Healthy cats may have positive antinuclear antibody and direct antiglobulin tests, but the prevalence of strong reactions is low. © 2018 American Society for Veterinary Clinical Pathology.

  9. Passive vaccination with a human monoclonal antibody: generation of antibodies and studies for efficacy in Bacillus anthracis infections.

    Science.gov (United States)

    vor dem Esche, Ulrich; Huber, Maria; Zgaga-Griesz, Andrea; Grunow, Roland; Beyer, Wolfgang; Hahn, Ulrike; Bessler, Wolfgang G

    2011-07-01

    A major difficulty in creating human monoclonal antibodies is the lack of a suitable myeloma cell line to be used for fusion experiments. In order to create fully human monoclonal antibodies for passive immunization, the human mouse heteromyeloma cell line CB-F7 was evaluated. Using this cell line, we generated human monoclonal antibodies against Bacillus anthracis toxin components. Antibodies against protective antigen (PA) and against lethal factor (LF) were obtained using peripheral blood lymphocytes (PBLs) from persons vaccinated with the UK anthrax vaccine. PBL were fused with the cell line CB-F7. We obtained several clones producing PA specific Ig and one clone (hLF1-SAN) producing a monoclonal antibody (hLF1) directed against LF. The LF binding antibody was able to neutralize Anthrax toxin activity in an in vitro neutralization assay, and preliminary in vivo studies in mice also indicated a trend towards protection. We mapped the epitope of the antibody binding to LF by dot blot analysis and ELIFA using 80 synthetic LF peptides of 20 amino acid lengths with an overlapping range of 10 amino acids. Our results suggest the binding of the monoclonal antibody to the peptide regions 121-150 or 451-470 of LF. The Fab-fragment of the antibody hLF1 was cloned in Escherichia coli and could be useful as part of a fully human monoclonal antibody for the treatment of Anthrax infections. In general, our studies show the applicability of the CB-F7 line to create fully human monoclonal antibodies for vaccination. Copyright © 2010 Elsevier GmbH. All rights reserved.

  10. Radioimmunoassay with heterologous antibody (hetero-antibody RIA)

    International Nuclear Information System (INIS)

    Iwasawa, Atsushi; Hayashi, Hiroaki; Itoh, Zen; Wakabayashi, Katsumi

    1991-01-01

    To develop a homologous radioimmunoassay (RIA) for a hormone of a small or rare animal often meets difficulty in collecting a large amount of purified antigen required for antibody production. On the other hand, to employ a heterologous RIA to estimate the hormone often gives poor sensitivity. To overcome this difficulty, a 'hetero-antibody' RIA was studied. In a hetero-antibody RIA system, a purified preparation of a hormone is used for radioiodination and standardization and a heterologous antibody to the hormone is used for the first antibody. Canine motilin and rat LH were selected as examples, and anti-porcine motilin and anti-hCG, anti-hCGβ or anti-ovine LHβ was used as the heterologous antibody. The sensitivities of the hetero-antibody RIAs were much higher than those of heterologous RIAs in any case, showing that these hetero-antibody RIA systems were suitable for practical use. To clarify the principle of hetero-antibody RIA, antiserum to porcine motilin was fractionated on an affinity column where canine motilin was immobilized. The fraction bound had greater constants of affinity with both porcine and canine motilins than the rest of the antibody fractions. This fraction also reacted with a synthetic peptide corresponding to the C-terminal sequence common to porcine and canine motilins in a competitive binding test with labeled canine motilin. These results suggest that an antibody population having high affinity and cross-reactivity is present in polyclonal antiserum and indicate that the population can be used in hetero-antibody RIA at an appropriate concentration. (author)

  11. Pre-vaccination evolution of antibodies among infants 0, 3 and 6months of age: A longitudinal analysis of measles, enterovirus 71 and coxsackievirus 16.

    Science.gov (United States)

    Fu, Chuanxi; Shen, Jichuan; Lu, Long; Li, Yajing; Cao, Yimin; Wang, Ming; Pei, Sen; Yang, Zhicong; Guo, Qing; Shaman, Jeffrey

    2017-07-05

    Due to waning levels of maternal antibodies (measles; enterovirus 71, EV71; and coxsackievirus A16, CoxA16), some infants may lose protection against infection prior to vaccination. Using a longitudinal design, we examine how maternal antibody levels evolve over time in infants prior to vaccination. In 2013-2014, we collected sera at ages 0, 3 and 6months from infants. We assayed for levels of measles IgG antibody (717, 233 and 75 sample sera tested at months 0, 3 and 6, respectively), and neutralizing antibodies for EV71 and CoxA16 (225, 217, and 72). Demographic and health information were collected, and a linear mixed model (LMM) was used to describe antibody levels over time. Pre-vaccination monotonic antibody decreases were observed for measles (1410, 195 and 22mIU/ml, p<0.001), EV71 (1:19.9, 6.3 and 4.5, p<0.001) and CoxA16 (1:16.3, 5.9, and 4.5, p<0.001). At 6months of age, only 2.7% (95%CI, 0.6-8.3), 6.8% (95%CI, 2.7-14.4) and 5.6% (95%CI, 1.9-12.7) of infants were antibody positive for measles, EV71 and CoxA16, respectively. LMM findings indicated that infants with higher antibody titers at birth experienced a greater loss of antibody level. An infection rate of 1.3% (95%CI, 0.1-6.1) was reported for both EV71 and CoxA16. Further modifications of vaccination strategies for measles, earlier vaccination for EV71 infection, and deployment of a CoxA16 vaccine need to be considered to limit infection among the very young. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. [Immunochemical Detection of Azospirilla in Soil with Genus-Specific Antibodies].

    Science.gov (United States)

    Shirokov, A A; Krasov, A I; Selivanov, N Yu; Burygin, G L; Shchegolev, S Yu; Matora, L Yu

    2015-01-01

    Immunoelectrophoresis and immunodiffusion analysis with antibodies to whole intact cells of the type strain of nitrogen-fixing soil bacteria Azospirillum brasilense Sp7 revealed at least three conservative surface immunogenic proteins of azospirilla. Cross-reactions with these proteins made it possible to use the above antibodies for detection of azospirilla as a genus-specific probe conjugated with horseradish peroxidase as an enzymatic label. Direct immune-enzyme analysis of soil suspensions (typical chernozem, Saratov oblast) confirmed applicability of the conjugates based on genus-specific antibodies to the surface proteins of azospirilla for direct detection of this bacterial genus in environmental samples. These results provide a basis for broad application of this method for analysis of Azospirillum occurrence in soil.

  13. Binding of HIV-1 gp41-directed neutralizing and non-neutralizing fragment antibody binding domain (Fab and single chain variable fragment (ScFv antibodies to the ectodomain of gp41 in the pre-hairpin and six-helix bundle conformations.

    Directory of Open Access Journals (Sweden)

    John M Louis

    Full Text Available We previously reported a series of antibodies, in fragment antigen binding domain (Fab formats, selected from a human non-immune phage library, directed against the internal trimeric coiled-coil of the N-heptad repeat (N-HR of HIV-1 gp41. Broadly neutralizing antibodies from that series bind to both the fully exposed N-HR trimer, representing the pre-hairpin intermediate state of gp41, and to partially-exposed N-HR helices within the context of the gp41 six-helix bundle. While the affinities of the Fabs for pre-hairpin intermediate mimetics vary by only 2 to 20-fold between neutralizing and non-neutralizing antibodies, differences in inhibition of viral entry exceed three orders of magnitude. Here we compare the binding of neutralizing (8066 and non-neutralizing (8062 antibodies, differing in only four positions within the CDR-H2 binding loop, in Fab and single chain variable fragment (ScFv formats, to several pre-hairpin intermediate and six-helix bundle constructs of gp41. Residues 56 and 58 of the mini-antibodies are shown to be crucial for neutralization activity. There is a large differential (≥ 150-fold in binding affinity between neutralizing and non-neutralizing antibodies to the six-helix bundle of gp41 and binding to the six-helix bundle does not involve displacement of the outer C-terminal helices of the bundle. The binding stoichiometry is one six-helix bundle to one Fab or three ScFvs. We postulate that neutralization by the 8066 antibody is achieved by binding to a continuum of states along the fusion pathway from the pre-hairpin intermediate all the way to the formation of the six-helix bundle, but prior to irreversible fusion between viral and cellular membranes.

  14. Arrayed antibody library technology for therapeutic biologic discovery.

    Science.gov (United States)

    Bentley, Cornelia A; Bazirgan, Omar A; Graziano, James J; Holmes, Evan M; Smider, Vaughn V

    2013-03-15

    Traditional immunization and display antibody discovery methods rely on competitive selection amongst a pool of antibodies to identify a lead. While this approach has led to many successful therapeutic antibodies, targets have been limited to proteins which are easily purified. In addition, selection driven discovery has produced a narrow range of antibody functionalities focused on high affinity antagonism. We review the current progress in developing arrayed protein libraries for screening-based, rather than selection-based, discovery. These single molecule per microtiter well libraries have been screened in multiplex formats against both purified antigens and directly against targets expressed on the cell surface. This facilitates the discovery of antibodies against therapeutically interesting targets (GPCRs, ion channels, and other multispanning membrane proteins) and epitopes that have been considered poorly accessible to conventional discovery methods. Copyright © 2013. Published by Elsevier Inc.

  15. Tumor detection using radiolabeled monoclonal antibodies

    International Nuclear Information System (INIS)

    Moldofsky, P.J.; Powe, J.; Hammond, N.D.

    1987-01-01

    Radioisotope conjugated to monoclonal antibody products has been used for imaging tumors targeted by the antibody. As imaging progresses, new sets of procedural and technical questions arise. In this chapter, we discuss several current problems in imaging tumor with radiolabeled monoclonal antibody. These include (1) methods for selection of specific antibody and, once the particular antibody is selected, which fragment form is to be used; (2) imaging procedures: what are the optimum imaging parameters, such as optimum time for imaging after administration of tracer and considerations regarding background subtraction; and (3) noninvasive quantitative techniques: quantitation of localization of antibody indirectly from quantitative information in the images.100 references

  16. Direct, Specific and Rapid Detection of Staphylococcal Proteins and Exotoxins Using a Multiplex Antibody Microarray.

    Directory of Open Access Journals (Sweden)

    Bettina Stieber

    Full Text Available S. aureus is a pathogen in humans and animals that harbors a wide variety of virulence factors and resistance genes. This bacterium can cause a wide range of mild to life-threatening diseases. In the latter case, fast diagnostic procedures are important. In routine diagnostic laboratories, several genotypic and phenotypic methods are available to identify S. aureus strains and determine their resistances. However, there is a demand for multiplex routine diagnostic tests to directly detect staphylococcal toxins and proteins.In this study, an antibody microarray based assay was established and validated for the rapid detection of staphylococcal markers and exotoxins. The following targets were included: staphylococcal protein A, penicillin binding protein 2a, alpha- and beta-hemolysins, Panton Valentine leukocidin, toxic shock syndrome toxin, enterotoxins A and B as well as staphylokinase. All were detected simultaneously within a single experiment, starting from a clonal culture on standard media. The detection of bound proteins was performed using a new fluorescence reading device for microarrays.110 reference strains and clinical isolates were analyzed using this assay, with a DNA microarray for genotypic characterization performed in parallel. The results showed a general high concordance of genotypic and phenotypic data. However, genotypic analysis found the hla gene present in all S. aureus isolates but its expression under given conditions depended on the clonal complex affiliation of the actual isolate.The multiplex antibody assay described herein allowed a rapid and reliable detection of clinically relevant staphylococcal toxins as well as resistance- and species-specific markers.

  17. Antibodies and Selection of Monoclonal Antibodies.

    Science.gov (United States)

    Hanack, Katja; Messerschmidt, Katrin; Listek, Martin

    Monoclonal antibodies are universal binding molecules with a high specificity for their target and are indispensable tools in research, diagnostics and therapy. The biotechnological generation of monoclonal antibodies was enabled by the hybridoma technology published in 1975 by Köhler and Milstein. Today monoclonal antibodies are used in a variety of applications as flow cytometry, magnetic cell sorting, immunoassays or therapeutic approaches. First step of the generation process is the immunization of the organism with appropriate antigen. After a positive immune response the spleen cells are isolated and fused with myeloma cells in order to generate stable, long-living antibody-producing cell lines - hybridoma cells. In the subsequent identification step the culture supernatants of all hybridoma cells are screened weekly for the production of the antibody of interest. Hybridoma cells producing the antibody of interest are cloned by limited dilution till a monoclonal hybridoma is found. This is a very time-consuming and laborious process and therefore different selection strategies were developed since 1975 in order to facilitate the generation of monoclonal antibodies. Apart from common automation of pipetting processes and ELISA testing there are some promising approaches to select the right monoclonal antibody very early in the process to reduce time and effort of the generation. In this chapter different selection strategies for antibody-producing hybridoma cells are presented and analysed regarding to their benefits compared to conventional limited dilution technology.

  18. Efficient generation of monoclonal antibodies from single rhesus macaque antibody secreting cells.

    Science.gov (United States)

    Meng, Weixu; Li, Leike; Xiong, Wei; Fan, Xuejun; Deng, Hui; Bett, Andrew J; Chen, Zhifeng; Tang, Aimin; Cox, Kara S; Joyce, Joseph G; Freed, Daniel C; Thoryk, Elizabeth; Fu, Tong-Ming; Casimiro, Danilo R; Zhang, Ningyan; A Vora, Kalpit; An, Zhiqiang

    2015-01-01

    Nonhuman primates (NHPs) are used as a preclinical model for vaccine development, and the antibody profiles to experimental vaccines in NHPs can provide critical information for both vaccine design and translation to clinical efficacy. However, an efficient protocol for generating monoclonal antibodies from single antibody secreting cells of NHPs is currently lacking. In this study we established a robust protocol for cloning immunoglobulin (IG) variable domain genes from single rhesus macaque (Macaca mulatta) antibody secreting cells. A sorting strategy was developed using a panel of molecular markers (CD3, CD19, CD20, surface IgG, intracellular IgG, CD27, Ki67 and CD38) to identify the kinetics of B cell response after vaccination. Specific primers for the rhesus macaque IG genes were designed and validated using cDNA isolated from macaque peripheral blood mononuclear cells. Cloning efficiency was averaged at 90% for variable heavy (VH) and light (VL) domains, and 78.5% of the clones (n = 335) were matched VH and VL pairs. Sequence analysis revealed that diverse IGHV subgroups (for VH) and IGKV and IGLV subgroups (for VL) were represented in the cloned antibodies. The protocol was tested in a study using an experimental dengue vaccine candidate. About 26.6% of the monoclonal antibodies cloned from the vaccinated rhesus macaques react with the dengue vaccine antigens. These results validate the protocol for cloning monoclonal antibodies in response to vaccination from single macaque antibody secreting cells, which have general applicability for determining monoclonal antibody profiles in response to other immunogens or vaccine studies of interest in NHPs.

  19. Fc receptors for mouse IgG1 on human monocytes: polymorphism and role in antibody-induced T cell proliferation.

    Science.gov (United States)

    Tax, W J; Hermes, F F; Willems, R W; Capel, P J; Koene, R A

    1984-09-01

    In previous studies, it was shown that there is polymorphism in the mitogenic effect of mouse IgG1 monoclonal antibodies against the T3 antigen of human T cells. This polymorphism implies that IgG1 anti-T3 antibodies are not mitogenic for T cells from 30% of healthy individuals. The present results demonstrate that this polymorphism is caused by polymorphism of an Fc receptor for mouse IgG1, present on human monocytes. The Fc receptor for murine IgG1 could be detected by a newly developed rosetting assay on monocytes from all individuals responsive to the mitogenic effect of IgG1 anti-T3 antibodies. This Fc receptor was not detectable on monocytes from those individuals exhibiting no mitogenic responses to IgG1 anti-T3 monoclonal antibodies. Cross-linking of T3 antigens appears to be essential for antibody-induced mitosis of T cells, because mononuclear cells that did not proliferate in response to WT 31 (an IgG1 antibody against T3 antigen) showed a proliferative response to Sepharose beads coated with WT 31. The Fc receptor--if functionally present--may be involved in the cross-linking of T3 antigens through anti-T3 antibodies. Further evidence for the involvement of this Fc receptor in antibody-induced T cell proliferation was provided by inhibition studies. Immune complexes containing IgG1 antibodies were able to inhibit the proliferative response to IgG1 anti-T3 antibodies. This inhibition by immune complexes appears to be mediated through the monocyte Fc receptor for mouse IgG1. These findings are important for the interpretation of previously described inhibitory effects of anti-T cell monoclonal antibodies on T cell proliferation, and show that such inhibitory effects may be monocyte-mediated (via immune complexes) rather than caused by a direct involvement of the respective T cell antigens in T cell mitosis. The Fc receptor for mouse IgG1 plays a role in antibody-induced T cell proliferation. Its polymorphism may have important implications for the

  20. Non-Neutralizing Antibodies Directed against HIV and Their Functions

    Directory of Open Access Journals (Sweden)

    Luzia M. Mayr

    2017-11-01

    Full Text Available B cells produce a plethora of anti-HIV antibodies (Abs but only few of them exhibit neutralizing activity. This was long considered a profound limitation for the enforcement of humoral immune responses against HIV-1 infection, especially since these neutralizing Abs (nAbs are extremely difficult to induce. However, increasing evidence shows that additional non-neutralizing Abs play a significant role in decreasing the viral load, leading to partial and sometimes even total protection. Mechanisms suspected to participate in protection are numerous. They involve the Fc domain of Abs as well as their Fab part, and consequently the induced Ab isotype will be determinant for their functions, as well as the quantity and quality of the Fc-receptors (FcRs expressed on immune cells. Fc-mediated inhibitory functions, such as Ab-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis, aggregation, and even immune activation have been proposed. However, as for nAbs, the non-neutralizing activities are limited to a subset of anti-HIV Abs. An improved in-depth characterization of the Abs displaying these functional responses is required for the development of new vaccination strategies, which aim to selectively trigger the B cells able to induce the right functional Ab combinations both at the right place and at the right time. This review summarizes our current knowledge on non-neutralizing functional inhibitory Abs and discusses the potential benefit of inducing them via vaccination. We also provide new insight into the roles of the FcγR-mediated Ab therapeutics in clinical trials for HIV diseases.

  1. Characterization and expression of the human T cell receptor-T3 complex by monoclonal antibody F101.01

    DEFF Research Database (Denmark)

    Geisler, C; Plesner, T; Pallesen, G

    1988-01-01

    demonstrated co-expression of the antigen defined by F101.01 and the pan-T cell antigens defined by CD2, CD3, CD5, and CD7 antibodies. Cells stained with CD4 and CD8 antibodies were both included in the F101.01-positive population, whereas CD16-positive natural killer cells (NK), B cells (CD19 and CD20...

  2. Specificity and polyreactivity of the antibody response during natural HIV-1 infection

    OpenAIRE

    Wang, Xin

    2006-01-01

    The specificity and polyreactivity of the antibody response in natural HIV-1 infection were studied. First, to investigate the overall antibody response, overlapping linear peptides were used to screen sera taken from HIV-1-infected individuals. The polyclonal antibody response was relatively stable during long-term infection, compared with acute infection, and mostly directed against immunodominant regions. Low level, transient antibody responses were detected against membrane proximal exter...

  3. Probable C4d-negative accelerated acute antibody-mediated rejection due to non-HLA antibodies.

    Science.gov (United States)

    Niikura, Takahito; Yamamoto, Izumi; Nakada, Yasuyuki; Kamejima, Sahoko; Katsumata, Haruki; Yamakawa, Takafumi; Furuya, Maiko; Mafune, Aki; Kobayashi, Akimitsu; Tanno, Yudo; Miki, Jun; Yamada, Hiroki; Ohkido, Ichiro; Tsuboi, Nobuo; Yamamoto, Hiroyasu; Yokoo, Takashi

    2015-07-01

    We report a case of probable C4d-negative accelerated acute antibody-mediated rejection due to non-HLA antibodies. A 44 year-old male was admitted to our hospital for a kidney transplant. The donor, his wife, was an ABO minor mismatch (blood type O to A) and had Gitelman syndrome. Graft function was delayed; his serum creatinine level was 10.1 mg/dL at 3 days after transplantation. Open biopsy was performed immediately; no venous thrombosis was observed during surgery. Histology revealed moderate peritubular capillaritis and mild glomerulitis without C4d immunoreactivity. Flow cytometric crossmatching was positive, but no panel-reactive antibodies against HLA or donor-specific antibodies (DSAbs) to major histocompatibility complex class I-related chain A (MICA) were detected. Taken together, we diagnosed him with probable C4d-negative accelerated antibody-mediated rejection due to non-HLA, non-MICA antibodies, the patient was treated with steroid pulse therapy (methylprednisolone 500 mg/day for 3 days), plasma exchange, intravenous immunoglobulin (40 g/body), and rituximab (200 mg/body) were performed. Biopsy at 58 days after transplantation, at which time S-Cr levels were 1.56 mg/dL, found no evidence of rejection. This case, presented with a review of relevant literature, demonstrates that probable C4d-negative accelerated acute AMR can result from non-HLA antibodies. © 2015 Asian Pacific Society of Nephrology.

  4. Bone marrow dosimetry in rats using direct tissue counting after injection of radio-iodinated intact monoclonal antibodies or F(ab')2 fragments

    International Nuclear Information System (INIS)

    Buchegger, F.; Chalandon, Y.; Pelegrin, A.; Hardman, N.; Mach, J.P.

    1991-01-01

    Normal rats were injected intravenously with 131I- and 125I-labeled intact murine and chimeric mouse-human monoclonal antibodies directed against carcinoembryonic antigen or with the corresponding F(ab')2 fragments. At different times after injection, individual animals were killed and radioactivity of blood and major organs, including bones and bone marrow, was determined. Ratios comparing radioactivity concentration in different tissues with that of bone marrow were calculated and found to remain stable during several effective half-lives of the antibodies. Mean bone marrow radioactivity was 35% (range, 29%-40%) of that of blood and 126% (range, 108%-147%) of that of liver after injection of intact Mabs or F(ab')2 fragments. In nude rats bearing human colon carcinoma xenografts producing carcinoembryonic antigen, relative bone marrow radioactivity was slightly lower than that in normal rats

  5. Decay of maternal antibodies in broiler chickens.

    Science.gov (United States)

    Gharaibeh, Saad; Mahmoud, Kamel

    2013-09-01

    The objective of this study was to determine the decay rate of maternal antibodies against major broiler chicken pathogens. A total of 30 one-day-old broiler chicks were obtained from a commercial hatchery and reared in isolation. These chicks were retrieved from a parent flock that received a routine vaccination program. Chicks were bled at hatch and sequentially thereafter every 5 d through 30 d of age. Maternal antibody titers were measured by ELISA for avian encephalomyelitis (AEV), avian influenza virus (AIV), chicken anemia virus (CAV), infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), Mycoplasma gallisepticum (MG), Mycoplasma synoviae (MS), and reovirus (Reo). Maternal antibody titers for Newcastle disease virus (NDV) were measured using a hemagglutination inhibition test. Half-life estimates of maternal antibody titers were 5.3, 4.2, 7, 5.1, 3.9, 3.8, 4.9, 4.1, 6.3, and 4.7 d for AEV, AIV, CAV, IBDV, IBV, ILTV, MG, MS, NDV, and Reo, respectively. The statistical analysis revealed significant differences among half-lives of maternal antibody titers against certain pathogens. Furthermore, all maternal antibody titers were depleted by 10 d of age except for IBDV.

  6. Chemoradiotherapy of cancer using boronated monoclonal antibodies. Progress report, December 1, 1982-November 30, 1983

    International Nuclear Information System (INIS)

    Soloway, A.H.

    1984-01-01

    The feasibility was established of using antibodies for the delivery of 10 B. Problems faced included 1) preservation of antibody activity following boronation, 2) antigenic receptor site density of the target cells, and 3) delivery of a critical number of 10 B atoms per cell. The linkage of a heavily boronated polymeric species to antibody by means of a single functional group allow for the delivery of a large number 10 B atoms per antibody molecule without a significant reduction in affinity. Both the polyclonally derived anti-thymocyte globulin (ATG) and the monoclonal anti-colorectal carcinoma antibody (17-1A) recognize antigens that are expressed with a density of approximately 10 6 epitopes per cell. The major concept that we advance is that just as effective cancer chemotherapy is based on the use of a combination of drugs, similarly a combination of compounds could be employed to deliver the requisite amount of 10 B to tumor target cells. This could include compounds such as Na 2 B 12 H 11 Sh together with boronated antibodies directed against tumor associated antigens. (DT)

  7. Molecular signatures of hemagglutinin stem-directed heterosubtypic human neutralizing antibodies against influenza A viruses.

    Directory of Open Access Journals (Sweden)

    Yuval Avnir

    2014-05-01

    Full Text Available Recent studies have shown high usage of the IGHV1-69 germline immunoglobulin gene for influenza hemagglutinin stem-directed broadly-neutralizing antibodies (HV1-69-sBnAbs. Here we show that a major structural solution for these HV1-69-sBnAbs is achieved through a critical triad comprising two CDR-H2 loop anchor residues (a hydrophobic residue at position 53 (Ile or Met and Phe54, and CDR-H3-Tyr at positions 98±1; together with distinctive V-segment CDR amino acid substitutions that occur in positions sparse in AID/polymerase-η recognition motifs. A semi-synthetic IGHV1-69 phage-display library screen designed to investigate AID/polη restrictions resulted in the isolation of HV1-69-sBnAbs that featured a distinctive Ile52Ser mutation in the CDR-H2 loop, a universal CDR-H3 Tyr at position 98 or 99, and required as little as two additional substitutions for heterosubtypic neutralizing activity. The functional importance of the Ile52Ser mutation was confirmed by mutagenesis and by BCR studies. Structural modeling suggests that substitution of a small amino acid at position 52 (or 52a facilitates the insertion of CDR-H2 Phe54 and CDR-H3-Tyr into adjacent pockets on the stem. These results support the concept that activation and expansion of a defined subset of IGHV1-69-encoded B cells to produce potent HV1-69-sBnAbs does not necessarily require a heavily diversified V-segment acquired through recycling/reentry into the germinal center; rather, the incorporation of distinctive amino acid substitutions by Phase 2 long-patch error-prone repair of AID-induced mutations or by random non-AID SHM events may be sufficient. We propose that these routes of B cell maturation should be further investigated and exploited as a pathway for HV1-69-sBnAb elicitation by vaccination.

  8. Characterization of Notch1 antibodies that inhibit signaling of both normal and mutated Notch1 receptors.

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    Miguel Aste-Amézaga

    2010-02-01

    Full Text Available Notch receptors normally play a key role in guiding a variety of cell fate decisions during development and differentiation of metazoan organisms. On the other hand, dysregulation of Notch1 signaling is associated with many different types of cancer as well as tumor angiogenesis, making Notch1 a potential therapeutic target.Here we report the in vitro activities of inhibitory Notch1 monoclonal antibodies derived from cell-based and solid-phase screening of a phage display library. Two classes of antibodies were found, one directed against the EGF-repeat region that encompasses the ligand-binding domain (LBD, and the second directed against the activation switch of the receptor, the Notch negative regulatory region (NRR. The antibodies are selective for Notch1, inhibiting Jag2-dependent signaling by Notch1 but not by Notch 2 and 3 in reporter gene assays, with EC(50 values as low as 5+/-3 nM and 0.13+/-0.09 nM for the LBD and NRR antibodies, respectively, and fail to recognize Notch4. While more potent, NRR antibodies are incomplete antagonists of Notch1 signaling. The antagonistic activity of LBD, but not NRR, antibodies is strongly dependent on the activating ligand. Both LBD and NRR antibodies bind to Notch1 on human tumor cell lines and inhibit the expression of sentinel Notch target genes, including HES1, HES5, and DTX1. NRR antibodies also strongly inhibit ligand-independent signaling in heterologous cells transiently expressing Notch1 receptors with diverse NRR "class I" point mutations, the most common type of mutation found in human T-cell acute lymphoblastic leukemia (T-ALL. In contrast, NRR antibodies failed to antagonize Notch1 receptors bearing rare "class II" or "class III" mutations, in which amino acid insertions generate a duplicated or constitutively sensitive metalloprotease cleavage site. Signaling in T-ALL cell lines bearing class I mutations is partially refractory to inhibitory antibodies as compared to cell

  9. Monoclonal antibody hapten radiopharmaceutical delivery

    International Nuclear Information System (INIS)

    Goodwin, D.A.; McTigue, M.

    1986-01-01

    One hundred μg of monoclonal antibody (MoAb) CHA255 with a binding constant Kb of 4 x 10 9 was complexed with indium-111 labelled BLEDTA II, BLEDTA IV, benzyl EDTA, and an EDTA conjugate of Fab. The 24-h tumour and organ distribution of BALB/c mice bearing KHJJ tumours was studied for each compound alone, the antibody complex, and 3 h following a chelate chase of the antibody complex. Whole body biological half-life was measured for 7 days with and without a chelate chase for each antibody complex. The 24-h whole body counts dropped 20 to 60% and blood concentration fell over 89% within 3 h of administering the chelate chase. Theoretical equivalent human organ doses were calculated from the 24-h organ concentrations, effective half-life, and MIRD 11 S values (absorbed dose per cumulated activity). Liver and spleen were the target organs, with the dose ranging from 0.50 to 3.91 rads mCi -1 . The reduction in organ radiation dose varied up to 95% following the chelate chase. Rapid selective renal clearance of chelate labelled radiopharmaceuticals by competitive inhibition (chelate chase) of their reversible binding to monoclonal antibodies enhances tumour imaging and improves the radiation dosimetry. (author)

  10. High-affinity uranyl-specific antibodies suitable for cellular imaging

    International Nuclear Information System (INIS)

    Reisser-Rubrecht, L.; Torne-Celer, C.; Renier, W.; Averseng, O.; Plantevin, S.; Quemeneur, E.; Bellanger, L.; Vidaud, C.

    2008-01-01

    Monoclonal antibodies (mAbs) have proved to be valuable models for the study of protein-metal interactions, and previous reports have described very specific antibodies to chelated metal ions, including uranyl. We raised specific mAbs against UO 2 2+ -DCP-BSA (DCP, 1, 10-phenanthroline-2,9-dicarboxylic acid) to generate new sets of antibodies that might cross-react with various complexed forms of uranyl in different environments for further application in the field of toxicology. Using counter-screening with UO 2 2+ -DCP-casein, we selected two highly specific mAbs against uranyl-DCP (K D = 10-100 pM): U04S and U08S. Competitive assays in the presence of different metal ions (UO 2 2+ , Fe 3+ , Zn 2+ , Cu 2+ , and Ca 2+ ) showed that uranyl in solution can act as a good competitor, suggesting some antibody ability to cross-react with chelating groups other than DCP in the UO 2 2+ equatorial coordination plane. Interestingly, one of the antibodies could be used for revealing uranyl cations in cell samples. Fluorescence activated cell sorting analyses after immuno-labeling revealed the interaction of uranyl with human kidney cells HK2. The intracellular accumulation of uranyl could be directly visualized by metal-immunostaining using fluorescent-labeled mAb. Our results suggest that U04S mAb epitopes mostly include the uranyl fraction and its para-topes can accommodate a wide variety of chelating groups. (authors)

  11. Inhibition of fibroblast growth factor receptor 3-dependent lung adenocarcinoma with a human monoclonal antibody

    Directory of Open Access Journals (Sweden)

    Yongjun Yin

    2016-05-01

    Full Text Available Activating mutations in fibroblast growth factor receptor 3 (FGFR3 have been identified in multiple types of human cancer and in congenital birth defects. In human lung cancer, fibroblast growth factor 9 (FGF9, a high-affinity ligand for FGFR3, is overexpressed in 10% of primary resected non-small cell lung cancer (NSCLC specimens. Furthermore, in a mouse model where FGF9 can be induced in lung epithelial cells, epithelial proliferation and ensuing tumorigenesis is dependent on FGFR3. To develop new customized therapies for cancers that are dependent on FGFR3 activation, we have used this mouse model to evaluate a human monoclonal antibody (D11 with specificity for the extracellular ligand-binding domain of FGFR3, that recognizes both human and mouse forms of the receptor. Here, we show that D11 effectively inhibits signaling through FGFR3 in vitro, inhibits the growth of FGFR3-dependent FGF9-induced lung adenocarcinoma in mice, and reduces tumor-associated morbidity. Given the potency of FGF9 in this mouse model and the absolute requirement for signaling through FGFR3, this study validates the D11 antibody as a potentially useful and effective reagent for treating human cancers or other pathologies that are dependent on activation of FGFR3.

  12. Relative susceptibility of Giardia muris trophozoites to killing by mouse antibodies of different isotypes.

    Science.gov (United States)

    Heyworth, M F

    1992-02-01

    The aim of this work was to examine the ability of mouse IgA, IgG, and IgM anti-Giardia antibodies to kill Giardia muris trophozoites in the presence and absence of complement. Using a 2-color flow cytometry assay, binding of antibody to trophozoites was assessed with fluorescein-conjugated anti-mouse immunoglobulin, and percentages of killed trophozoites were quantified by staining with propidium iodide. Trophozoites were killed in the presence of complement by IgG3 and IgM anti-trophozoite monoclonal antibodies. Anti-trophozoite IgA, obtained from the intestinal lumen of G. muris-infected BALB/c mice, became bound to trophozoites in vitro but did not kill these organisms in the presence or absence of complement. The results suggest that clearance of G. muris infection by intestinal IgA directed against G. muris trophozoites does not involve antibody-dependent killing of trophozoites in the intestinal lumen.

  13. Structure-based, targeted deglycosylation of HIV-1 gp120 and effects on neutralization sensitivity and antibody recognition

    International Nuclear Information System (INIS)

    Koch, Markus; Pancera, Marie; Kwong, Peter D.; Kolchinsky, Peter; Grundner, Christoph; Wang Liping; Hendrickson, Wayne A.; Sodroski, Joseph; Wyatt, Richard

    2003-01-01

    The human immunodeficiency virus (HIV-1) exterior envelope glycoprotein, gp120, mediates receptor binding and is the major target for neutralizing antibodies. Primary HIV-1 isolates are characteristically more resistant to broadly neutralizing antibodies, although the structural basis for this resistance remains obscure. Most broadly neutralizing antibodies are directed against functionally conserved gp120 regions involved in binding to either the primary virus receptor, CD4, or the viral coreceptor molecules that normally function as chemokine receptors. These antibodies are known as CD4 binding site (CD4BS) and CD4-induced (CD4i) antibodies, respectively. Inspection of the gp120 crystal structure reveals that although the receptor-binding regions lack glycosylation, sugar moieties lie proximal to both receptor-binding sites on gp120 and thus in proximity to both the CD4BS and the CD4i epitopes. In this study, guided by the X-ray crystal structure of gp120, we deleted four N-linked glycosylation sites that flank the receptor-binding regions. We examined the effects of selected changes on the sensitivity of two prototypic HIV-1 primary isolates to neutralization by antibodies. Surprisingly, removal of a single N-linked glycosylation site at the base of the gp120 third variable region (V3 loop) increased the sensitivity of the primary viruses to neutralization by CD4BS antibodies. Envelope glycoprotein oligomers on the cell surface derived from the V3 glycan-deficient virus were better recognized by a CD4BS antibody and a V3 loop antibody than were the wild-type glycoproteins. Absence of all four glycosylation sites rendered a primary isolate sensitive to CD4i antibody-mediated neutralization. Thus, carbohydrates that flank receptor-binding regions on gp120 protect primary HIV-1 isolates from antibody-mediated neutralization

  14. Functional single-walled carbon nanotubes based on an integrin αvβ3 monoclonal antibody for highly efficient cancer cell targeting

    International Nuclear Information System (INIS)

    Ou Zhongmin; Wu Baoyan; Xing Da; Zhou Feifan; Wang Huiying; Tang Yonghong

    2009-01-01

    The application of single-walled carbon nanotubes (SWNTs) in the field of biomedicine is becoming an entirely new and exciting topic. In this study, a novel functional SWNT based on an integrin α v β 3 monoclonal antibody was developed and was used for cancer cell targeting in vitro. SWNTs were first modified by phospholipid-bearing polyethylene glycol (PL-PEG). The PL-PEG functionalized SWNTs were then conjugated with protein A. A SWNT-integrin α v β 3 monoclonal antibody system (SWNT-PEG-mAb) was thus constructed by conjugating protein A with the fluorescein labeled integrin α v β 3 monoclonal antibody. In vitro study revealed that SWNT-PEG-mAb presented a high targeting efficiency on integrin α v β 3 -positive U87MG cells with low cellular toxicity, while for integrin α v β 3 -negative MCF-7 cells, the system had a low targeting efficiency, indicating that the high targeting to U87MG cells was due to the specific integrin targeting of the monoclonal antibody. In conclusion, SWNT-PEG-mAb developed in this research is a potential candidate for cancer imaging and drug delivery in cancer targeting therapy.

  15. Glutamate receptor antibodies in neurological diseases: anti-AMPA-GluR3 antibodies, anti-NMDA-NR1 antibodies, anti-NMDA-NR2A/B antibodies, anti-mGluR1 antibodies or anti-mGluR5 antibodies are present in subpopulations of patients with either: epilepsy, encephalitis, cerebellar ataxia, systemic lupus erythematosus (SLE) and neuropsychiatric SLE, Sjogren's syndrome, schizophrenia, mania or stroke. These autoimmune anti-glutamate receptor antibodies can bind neurons in few brain regions, activate glutamate receptors, decrease glutamate receptor's expression, impair glutamate-induced signaling and function, activate blood brain barrier endothelial cells, kill neurons, damage the brain, induce behavioral/psychiatric/cognitive abnormalities and ataxia in animal models, and can be removed or silenced in some patients by immunotherapy.

    Science.gov (United States)

    Levite, Mia

    2014-08-01

    Glutamate is the major excitatory neurotransmitter of the Central Nervous System (CNS), and it is crucially needed for numerous key neuronal functions. Yet, excess glutamate causes massive neuronal death and brain damage by excitotoxicity--detrimental over activation of glutamate receptors. Glutamate-mediated excitotoxicity is the main pathological process taking place in many types of acute and chronic CNS diseases and injuries. In recent years, it became clear that not only excess glutamate can cause massive brain damage, but that several types of anti-glutamate receptor antibodies, that are present in the serum and CSF of subpopulations of patients with a kaleidoscope of human neurological diseases, can undoubtedly do so too, by inducing several very potent pathological effects in the CNS. Collectively, the family of anti-glutamate receptor autoimmune antibodies seem to be the most widespread, potent, dangerous and interesting anti-brain autoimmune antibodies discovered up to now. This impression stems from taking together the presence of various types of anti-glutamate receptor antibodies in a kaleidoscope of human neurological and autoimmune diseases, their high levels in the CNS due to intrathecal production, their multiple pathological effects in the brain, and the unique and diverse mechanisms of action by which they can affect glutamate receptors, signaling and effects, and subsequently impair neuronal signaling and induce brain damage. The two main families of autoimmune anti-glutamate receptor antibodies that were already found in patients with neurological and/or autoimmune diseases, and that were already shown to be detrimental to the CNS, include the antibodies directed against ionotorpic glutamate receptors: the anti-AMPA-GluR3 antibodies, anti-NMDA-NR1 antibodies and anti-NMDA-NR2 antibodies, and the antibodies directed against Metabotropic glutamate receptors: the anti-mGluR1 antibodies and the anti-mGluR5 antibodies. Each type of these anti

  16. A monoclonal antibody to pestviruses in bovine and ovine sera

    International Nuclear Information System (INIS)

    Mweene, A.S.

    1991-01-01

    An enzyme-linked immunoabsorbent assay (ELISA) has been developed to defeat antibodies to pestviruses in bovine and ovine sera. Single sera from 211 cattle and 22 sheep from 7 different farms were tested using ELISA and Serum Neutralisation Test (SNT). 17 Monoclonal antibodies (Mabs) directed against P80, gp48 and gp53 were tested for ability to coat ELISA plates and capture the bovine viral diarrhea antigen. 5 mabs(WB 103, WB, 105, WB 112 against P80 kDa protein, WB 210 and WB 214 directed against gp48 and gp 53 kDa protein. Specific antibody to BVDV was detected by rabbit anti-bovine and anti-ovine IgG antisera. The quantitative correlation between two tests was good

  17. Functional characterization of two scFv-Fc antibodies from an HIV controller selected on soluble HIV-1 Env complexes: a neutralizing V3- and a trimer-specific gp41 antibody.

    Directory of Open Access Journals (Sweden)

    Maria Trott

    Full Text Available HIV neutralizing antibodies (nAbs represent an important tool in view of prophylactic and therapeutic applications for HIV-1 infection. Patients chronically infected by HIV-1 represent a valuable source for nAbs. HIV controllers, including long-term non-progressors (LTNP and elite controllers (EC, represent an interesting subgroup in this regard, as here nAbs can develop over time in a rather healthy immune system and in the absence of any therapeutic selection pressure. In this study, we characterized two particular antibodies that were selected as scFv antibody fragments from a phage immune library generated from an LTNP with HIV neutralizing antibodies in his plasma. The phage library was screened on recombinant soluble gp140 envelope (Env proteins. Sequencing the selected peptide inserts revealed two major classes of antibody sequences. Binding analysis of the corresponding scFv-Fc derivatives to various trimeric and monomeric Env constructs as well as to peptide arrays showed that one class, represented by monoclonal antibody (mAb A2, specifically recognizes an epitope localized in the pocket binding domain of the C heptad repeat (CHR in the ectodomain of gp41, but only in the trimeric context. Thus, this antibody represents an interesting tool for trimer identification. MAb A7, representing the second class, binds to structural elements of the third variable loop V3 and neutralizes tier 1 and tier 2 HIV-1 isolates of different subtypes with matching critical amino acids in the linear epitope sequence. In conclusion, HIV controllers are a valuable source for the selection of functionally interesting antibodies that can be selected on soluble gp140 proteins with properties from the native envelope spike.

  18. Endocytosis of ABCG2 drug transporter caused by binding of 5D3 antibody: trafficking mechanisms and intracellular fate.

    Science.gov (United States)

    Studzian, Maciej; Bartosz, Grzegorz; Pulaski, Lukasz

    2015-08-01

    ABCG2, a metabolite and xenobiotic transporter located at the plasma membrane (predominantly in barrier tissues and progenitor cells), undergoes a direct progressive endocytosis process from plasma membrane to intracellular compartments upon binding of 5D3 monoclonal antibody. This antibody is specific to an external epitope on the protein molecule and locks it in a discrete conformation within its activity cycle, presumably providing a structural trigger for the observed internalization phenomenon. Using routine and novel assays, we show that ABCG2 is endocytosed by a mixed mechanism: partially via a rapid, clathrin-dependent pathway and partially in a cholesterol-dependent, caveolin-independent manner. While the internalization process is entirely dynamin-dependent and converges initially at the early endosome, subsequent intracellular fate of ABCG2 is again twofold: endocytosis leads to only partial lysosomal degradation, while a significant fraction of the protein is retained in a post-endosomal compartment with the possibility of at least partial recycling back to the cell surface. This externally triggered, conformation-related trafficking pathway may serve as a general regulatory paradigm for membrane transporters, and its discovery was made possible thanks to consistent application of quantitative methods. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. P. falciparum infection and maternofetal antibody transfer in malaria-endemic settings of varying transmission.

    Directory of Open Access Journals (Sweden)

    Alistair R D McLean

    Full Text Available During pregnancy, immunoglobulin G (IgG is transferred from the mother to the fetus, providing protection from disease in early infancy. Plasmodium falciparum infections may reduce maternofetal antibody transfer efficiency, but mechanisms remain unclear.Mother-cord paired serum samples collected at delivery from Papua New Guinea (PNG and the Thailand-Myanmar Border Area (TMBA were tested for IgG1 and IgG3 to four P. falciparum antigens and measles antigen, as well as total serum IgG. Multivariable linear regression was conducted to assess the association of peripheral P. falciparum infection during pregnancy or placental P. falciparum infection assessed at delivery with maternofetal antibody transfer efficiency. Path analysis assessed the extent to which associations between P. falciparum infection and antibody transfer were mediated by gestational age at delivery or levels of maternal total serum IgG.Maternofetal antibody transfer efficiency of IgG1 and IgG3 was lower in PNG compared to TMBA (mean difference in cord antibody levels (controlling for maternal antibody levels ranged from -0.88 to 0.09, median of -0.20 log2 units. Placental P. falciparum infections were associated with substantially lower maternofetal antibody transfer efficiency in PNG primigravid women (mean difference in cord antibody levels (controlling for maternal antibody levels ranged from -0.62 to -0.10, median of -0.36 log2 units, but not multigravid women. The lower antibody transfer efficiency amongst primigravid women with placental infection was only partially mediated by gestational age at delivery (proportion indirect effect ranged from 0% to 18%, whereas no mediation effects of maternal total serum IgG were observed.Primigravid women may be at risk of impaired maternofetal antibody transport with placental P. falciparum infection. Direct effects of P. falciparum on the placenta, rather than earlier gestational age and elevated serum IgG, are likely responsible for

  20. Optimization and validation of a neutralizing antibody assay for HIV-1 in A3R5 cells.

    Science.gov (United States)

    Sarzotti-Kelsoe, Marcella; Daniell, Xiaoju; Todd, Christopher A; Bilska, Miroslawa; Martelli, Amanda; LaBranche, Celia; Perez, Lautaro G; Ochsenbauer, Christina; Kappes, John C; Rountree, Wes; Denny, Thomas N; Montefiori, David C

    2014-07-01

    A3R5 is a human CD4(+) lymphoblastoid cell line that was engineered to express CCR5 and is useful for the detection of weak neutralizing antibody responses against tier 2 strains of HIV-1. Here we describe the optimization and validation of the HIV-1 neutralizing antibody assay that utilizes A3R5 cells, performed in compliance with Good Clinical Laboratory Practice (GCLP) guidelines. The assay utilizes Renilla luciferase-expressing replication competent infectious molecular clones (IMC) encoding heterologous env genes from different HIV-1 clades. Key assay validation parameters tested included specificity, accuracy, precision, limit of detection and quantitation, specificity, linearity and range, and robustness. Plasma samples demonstrated higher non-specific activity than serum samples in the A3R5 assay. This assay can tolerate a wide range of virus input but is more sensitive to cell concentration. The higher sensitivity of the A3R5 assay in neutralization responses to tier 2 strains of HIV-1 makes it complementary to, but not a substitute for the TZM-bl assay. The validated A3R5 assay is employed as an endpoint immunogenicity test for vaccine-elicited neutralizing antibodies against tier 2 strains of HIV-1, and to identify correlates of protection in HIV-1 vaccine trials conducted globally. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Anti-IL-39 (IL-23p19/Ebi3) polyclonal antibodies ameliorate autoimmune symptoms in lupus-like mice

    Science.gov (United States)

    Wang, Xiaoqian; Zhang, Yu; Wang, Zhiding; Liu, Xiaoling; Zhu, Gaizhi; Han, Gencheng; Chen, Guojiang; Hou, Chunmei; Wang, Tianxiao; Shen, Beifen; Li, Yan; Xiao, He; Ma, Ning; Wang, Renxi

    2018-01-01

    The interleukin (IL)-12 family cytokines have been examined as therapeutic targets in the treatment of several autoimmune diseases. Our previous study showed that a novel IL-12 family cytokine, IL-39 (IL-23p19/Ebi3) mediates inflammation in lupus-like mice. In the present study, the effect of anti-mouse IL-39 polyclonal antibodies on autoimmune symptoms in lupus-like mice was investigated. Rabbit anti-mouse IL-39 polyclonal antibodies were produced by immunization with recombinant mouse IL-39, and purified using protein A chromatography. These antibodies were subsequently used to treat lupus-like mice. Flow cytometry, captured images, ELISA and H&E staining were used to determine the effect of anti-IL-39 polyclonal antibodies on inflammatory cells, autoantibody titers, proteinuria, infiltrating inflammatory cells and the structure of the glomerular region. The anti-IL-39 polyclonal antibodies effectively reduced the numbers of inflammatory cells, splenomegaly, autoantibody titers, proteinuria, infiltrating inflammatory cells, and restored the structure of the glomerular region in MRL/lpr mice. Taken together, these results suggested that anti-IL-39 polyclonal antibodies ameliorated autoimmune symptoms in lupus-like mice. Therefore, IL-39 may be used as a possible target for the treatment of systemic lupus erythematosus. PMID:29138852

  2. Are Onconeural Antibodies a Clinical Phenomenology in Paraneoplastic Limbic Encephalitis?

    Directory of Open Access Journals (Sweden)

    Hongliang Zhang

    2013-01-01

    Full Text Available Paraneoplastic neurological syndromes (PNSs occur in patients with cancer and can cause clinical symptoms and signs of dysfunction of the nervous system that are not due to a local effect of the tumor or its metastases. Most of these clinical syndromes in adults are associated with lung cancer, especially small cell lung cancer (SCLC, lymphoma, and gynecological tumors. The finding of highly specific antibodies directed against onconeural antigens has revolutionized the diagnosis and promoted the understanding of these syndromes and led to the current hypothesis of an autoimmune pathophysiology. Accumulating data strongly suggested direct pathogenicity of these antibodies. The field of PNS has expanded rapidly in the past few years with the discovery of limbic encephalitis associated with glutamic acid decarboxylase (GAD 65, the voltage (VGKC-gated potassium channel complex, the methyl (N-NMDA-D-aspartate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA, and gamma aminobutyric acid (GABA (B receptors, and so forth. Despite this, the clinical spectrum of these diseases has not yet been fully investigated. The clinical importance of these conditions lies in their frequent response to immunotherapies and, less commonly, their association with distinctive tumors. This review provides an overview on the pathogenesis and diagnosis of PNS, with emphasis on the role of antibodies in limbic encephalitis.

  3. Quantitative relationship between antibody affinity and antibody avidity

    International Nuclear Information System (INIS)

    Griswold, W.R.

    1987-01-01

    The relationship between antibody avidity, measured by the dissociation of the antigen-antibody bond in antigen excess, and antibody affinity was studied. Complexes of radiolabelled antigen and antibody of known affinity were prepared in vitro and allowed to stand for seven days to reach equilibrium. Then nonlabelled antigen in one hundred fold excess was added to dissociate the complexes. After an appropriate incubation the fraction of antigen bound to antibody was measured by the ammonium sulfate precipitation method. The dissociation index was the fraction bound in the experimental sample divided by the fraction bound in the control. The correlation coefficient between the dissociation index and the antibody binding constant was 0.92 for early dissociation and 0.98 for late dissociation. The regression equation relating the binding constant to the dissociation index was K = 6.4(DI) + 6.25, where DI is the late dissociation index and K is the logarithm to the base 10 of the binding constant. There is a high correlation between avidity and affinity of antibody. Antibody affinity can be estimated from avidity data. The stability of antigen-antibody complexes can be predicted from antibody affinity

  4. 3B11-N, a monoclonal antibody against MERS-CoV, reduces lung pathology in rhesus monkeys following intratracheal inoculation of MERS-CoV Jordan-n3/2012

    International Nuclear Information System (INIS)

    Johnson, Reed F.; Bagci, Ulas; Keith, Lauren; Tang, Xianchun; Mollura, Daniel J.; Zeitlin, Larry; Qin, Jing; Huzella, Louis; Bartos, Christopher J.; Bohorova, Natasha; Bohorov, Ognian; Goodman, Charles; Kim, Do H.; Paulty, Michael H.; Velasco, Jesus; Whaley, Kevin J.; Johnson, Joshua C.; Pettitt, James; Ork, Britini L.; Solomon, Jeffrey

    2016-01-01

    Middle East Respiratory Syndrome Coronavirus (MERS-CoV) was identified in 2012 as the causative agent of a severe, lethal respiratory disease occurring across several countries in the Middle East. To date there have been over 1600 laboratory confirmed cases of MERS-CoV in 26 countries with a case fatality rate of 36%. Given the endemic region, it is possible that MERS-CoV could spread during the annual Hajj pilgrimage, necessitating countermeasure development. In this report, we describe the clinical and radiographic changes of rhesus monkeys following infection with 5×10"6 PFU MERS-CoV Jordan-n3/2012. Two groups of NHPs were treated with either a human anti-MERS monoclonal antibody 3B11-N or E410-N, an anti-HIV antibody. MERS-CoV Jordan-n3/2012 infection resulted in quantifiable changes by computed tomography, but limited other clinical signs of disease. 3B11-N treated subjects developed significantly reduced lung pathology when compared to infected, untreated subjects, indicating that this antibody may be a suitable MERS-CoV treatment. - Highlights: • MERS-CoV Jordan-n3/2012 challenge of rhesus monkeys results in a mild disease. • CT can be used to monitor disease progression to aid models of human disease. • Treatment with the human monoclonal antibody 3B11-N resulted in decreased disease.

  5. 3B11-N, a monoclonal antibody against MERS-CoV, reduces lung pathology in rhesus monkeys following intratracheal inoculation of MERS-CoV Jordan-n3/2012

    Energy Technology Data Exchange (ETDEWEB)

    Johnson, Reed F., E-mail: johnsonreed@mail.nih.gov [Emerging Viral Pathogens Section National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, MD 21702 (United States); Bagci, Ulas [Center for Infectious Disease Imaging, National Institutes of Health Clinical Center, Bethesda MD 20892 (United States); Center for Research in Computer Vision (CRCV), Department of Electrics Electronics and Computer Science, University of Central Florida, Orlando, FL 32816, USA. (United States); Keith, Lauren [Integrated Research Facility, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, MD 21702 (United States); Tang, Xianchun [Department of Cancer Immunology & AIDS, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02215 (United States); Mollura, Daniel J. [Center for Infectious Disease Imaging, National Institutes of Health Clinical Center, Bethesda MD 20892 (United States); Zeitlin, Larry [Mapp Biopharmaceutical, Inc., San Diego CA 92121 (United States); Qin, Jing [Biostatistics Research Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 (United States); Huzella, Louis; Bartos, Christopher J. [Integrated Research Facility, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, MD 21702 (United States); Bohorova, Natasha; Bohorov, Ognian; Goodman, Charles; Kim, Do H.; Paulty, Michael H.; Velasco, Jesus; Whaley, Kevin J. [Mapp Biopharmaceutical, Inc., San Diego CA 92121 (United States); Johnson, Joshua C.; Pettitt, James; Ork, Britini L. [Integrated Research Facility, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, MD 21702 (United States); Solomon, Jeffrey [Clinical Research Directorate/Clinical Monitoring Research Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research,Frederick, MD 21702-USA. (United States); and others

    2016-03-15

    Middle East Respiratory Syndrome Coronavirus (MERS-CoV) was identified in 2012 as the causative agent of a severe, lethal respiratory disease occurring across several countries in the Middle East. To date there have been over 1600 laboratory confirmed cases of MERS-CoV in 26 countries with a case fatality rate of 36%. Given the endemic region, it is possible that MERS-CoV could spread during the annual Hajj pilgrimage, necessitating countermeasure development. In this report, we describe the clinical and radiographic changes of rhesus monkeys following infection with 5×10{sup 6} PFU MERS-CoV Jordan-n3/2012. Two groups of NHPs were treated with either a human anti-MERS monoclonal antibody 3B11-N or E410-N, an anti-HIV antibody. MERS-CoV Jordan-n3/2012 infection resulted in quantifiable changes by computed tomography, but limited other clinical signs of disease. 3B11-N treated subjects developed significantly reduced lung pathology when compared to infected, untreated subjects, indicating that this antibody may be a suitable MERS-CoV treatment. - Highlights: • MERS-CoV Jordan-n3/2012 challenge of rhesus monkeys results in a mild disease. • CT can be used to monitor disease progression to aid models of human disease. • Treatment with the human monoclonal antibody 3B11-N resulted in decreased disease.

  6. Antibodies directed against monomorphic and evolutionary conserved self epitopes may be generated in 'knock-out' mice. Development of monoclonal antibodies directed against monomorphic MHC class I determinants

    DEFF Research Database (Denmark)

    Claesson, M H; Endel, B; Ulrik, J

    1994-01-01

    Beta-2 microglobulin (beta 2m) gene 'knock-out' mice (C1D) were primed with purified H-2Kb and H-2Db molecules and spleen cells from immunized mice were used to generate monoclonal antibody secreting B-cell hybridomas. Approximately 0.2% of the Ig-secreting primary microcultures contained H-2b...

  7. Detection of human antibodies binding with smooth and rough LPSs from Proteus mirabilis O3 strains S1959, R110, R45.

    Science.gov (United States)

    Gleńska-Olender, J; Durlik, K; Konieczna, I; Kowalska, P; Gawęda, J; Kaca, W

    2017-11-01

    Bacteria of the genus Proteus of the family Enterobacteriaceae are facultative human pathogens responsible mainly for urinary tract and wound infections, bacteremia and the development of rheumatoid arthritis (RA). We have analyzed and compared by ELISA the titer of antibodies in plasmas of healthy individuals and in sera of rheumatoid arthritis patients recognizing a potential host cross-reactive epitope (lysine-galacturonic acid epitopes) present in Proteus lipopolysaccharide (LPS). In our experiments LPSs isolated from two mutants of smooth Proteus mirabilis 1959 (O3), i.e. strains R110 and R45, were used. R110 (Ra type mutant) is lacking the O-specific polysaccharide, but possesses a complete core oligosaccharide, while R45 (Re type) has a reduced core oligosaccharide and contains two 3-deoxy-D-manno-oct-2-ulosonic acid residues and one of 4-amino-4-deoxy-L-arabinopyranose residues. Titer of P. mirabilis S1959 LPS-specific-antibodies increased with the age of blood donors. RA and blood donors' sera contained antibodies against S and Ra and Re type of P. mirabilis O3 LPSs. Antibodies recognizing lysine-galacturonic acid epitopes of O3 LPS were detected by ELISA in some plasmas of healthy individuals and sera of rheumatoid arthritis patients. RA patients antibodies reacting with P. mirabilis S1959 S and R LPSs may indicate a potential role of anti-LPS antibodies in molecular mimicry in RA diseases.

  8. Tyrosine kinase inhibition increases the cell surface localization of FLT3-ITD and enhances FLT3-directed immunotherapy of acute myeloid leukemia.

    Science.gov (United States)

    Reiter, K; Polzer, H; Krupka, C; Maiser, A; Vick, B; Rothenberg-Thurley, M; Metzeler, K H; Dörfel, D; Salih, H R; Jung, G; Nößner, E; Jeremias, I; Hiddemann, W; Leonhardt, H; Spiekermann, K; Subklewe, M; Greif, P A

    2018-02-01

    The fms-related tyrosine kinase 3 (FLT3) receptor has been extensively studied over the past two decades with regard to oncogenic alterations that do not only serve as prognostic markers but also as therapeutic targets in acute myeloid leukemia (AML). Internal tandem duplications (ITDs) became of special interest in this setting as they are associated with unfavorable prognosis. Because of sequence-dependent protein conformational changes FLT3-ITD tends to autophosphorylate and displays a constitutive intracellular localization. Here, we analyzed the effect of tyrosine kinase inhibitors (TKIs) on the localization of the FLT3 receptor and its mutants. TKI treatment increased the surface expression through upregulation of FLT3 and glycosylation of FLT3-ITD and FLT3-D835Y mutants. In T cell-mediated cytotoxicity (TCMC) assays, using a bispecific FLT3 × CD3 antibody construct, the combination with TKI treatment increased TCMC in the FLT3-ITD-positive AML cell lines MOLM-13 and MV4-11, patient-derived xenograft cells and primary patient samples. Our findings provide the basis for rational combination of TKI and FLT3-directed immunotherapy with potential benefit for FLT3-ITD-positive AML patients.

  9. Generation of human scFvs antibodies recognizing a prion protein epitope expressed on the surface of human lymphoblastoid cells

    Directory of Open Access Journals (Sweden)

    Imperiale Valentina

    2007-07-01

    Full Text Available Abstract Background A hallmark of prion disease is the transformation of normal cellular prion protein (PrPc into an infectious disease-associated isoform, (PrPsc. Anti-prion protein monoclonal antibodies are invaluable for structure-function studies of PrP molecules. Furthermore recent in vitro and in vivo studies indicate that anti-PrP monoclonal antibodies can prevent the incorporation of PrPc into propagating prions. In the present article, we show two new human phage antibodies, isolated on recombinant hamster prion protein (rHaPrP. Results We adopted an antibody phage display strategy to isolate specific human antibodies directed towards rHaPrP which has been used as a bait for panning the synthetic ETH-2 antibody phage library. Two phage antibodies clones named MA3.B4 and MA3.G3 were isolated and characterized under genetic biochemical and immunocytochemical aspects. The clones were found to recognize the prion protein in ELISA studies. In flow-cytometry studies, these human single chain Fragment variable (scFv phage-antibodies show a well defined pattern of reactivity on human lymphoblastoid and myeloid cells. Conclusion Sequence analysis of the gene encoding for the antibody fragments and antigen recognition patterns determined by flow-cytometry analysis indicate that the isolated scFvs recognize novel epitopes in the PrPc molecule. These new anti PrPc human antibodies are unique reagents for prion protein detection and may represent a biologic platform to develop new reagents to treat PrPsc associated disease.

  10. Development and testing of monoclonal antibody-based rapid immunodiagnostic test kits for direct detection of Vibrio cholerae O139 synonym Bengal.

    Science.gov (United States)

    Hasan, J A; Huq, A; Nair, G B; Garg, S; Mukhopadhyay, A K; Loomis, L; Bernstein, D; Colwell, R R

    1995-11-01

    We report on the development and testing of two monoclonal antibody-based rapid immunodiagnostic test kits, BengalScreen, a coagglutination test, and Bengal DFA, a direct fluorescent-antibody test, for direct detection of Vibrio cholerae O139 synonym Bengal in clinical and environmental specimens. The BengalScreen test requires less than 5 min to complete and can be used in the field. Bengal DFA, being more sensitive than BengalScreen, requires only one reagent and less than 20 min for detection and enumeration of V. cholerae O139 synonym Bengal. In tests for specificity, all 40 strains of V. cholerae O139 reacted with both test kits, whereas 157 strains of heterologous species examined did not, yielding 100% specificity in this study. A field trial was conducted in with both BengalScreen and Bengal DFA, and the results were compared with those obtained by conventional culture methods. BengalScreen demonstrated a sensitivity of 95%, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 94%. Results obtained by Bengal DFA, on the other hand, were 100% sensitive and 100% specific and yielded 100% positive and negative predictive values compared with culture methods. In a second evaluation, 93 stool specimens from Mexico that were negative for V. cholerae O139 by culture were also tested with both the BengalScreen and Bengal DFA kits. None of the 93 specimens were positive for V. cholerae O139 by both tests. A concentration method was optimized for screening of environmental water samples for V. cholerae O139 synonym Bengal with rapid test kits. BengalScreen results were unequivocally positive when water samples contained at least 2.0 x 10(3) CFU/ml, whereas Bengal DFA demonstrated an unequivocally positive reaction when the water sample contained at least 1.5 x 10(2) CFU/ml. When Bengal DFA was compared with conventional culture methods for enumeration of V. cholerae O139 synonym Bengal organisms, no difference was observed.

  11. Cuban Monoclonal Antibodies for Radioimmunodiagnosis and Radioimmunotherapy of Cancer Diseases

    International Nuclear Information System (INIS)

    Casaco, A.

    2009-01-01

    tumours. Nevertheless, the lack of a direct evidence of this antigenic display in human cancers has kept the subject controversial. For the first time, we described herein the 'in vivo' detection of GM3(NeuGc) ganglioside in human breast primary tumours using a radioimmunoscintigraphic technique with 14F7, a highly specific anti-GM3(NeuGc) ganglioside monoclonal antibody, labelled with 99mTc. In an open, prospective Phase I/II clinical trial, including women diagnosed in stage II breast cancer, the 14F7 monoclonal antibody accumulation in tumours at doses of 0.3 (n=5), 1 (n=5) and 3 mg (n=4) was evaluated. Noteworthy, the immunoscintigraphic study showed antibody accumulation in 100% of patients' tumours for the 1 mg dose group. In turn, the radioimmunoconjugate injected at doses of 0.3 mg or 3 mg of the antibody, was uptaken by 60 and 33.3% of breast tumours, respectively. 'In vivo' immune recognition of GM3(NeuGc) in breast tumours reinforces the value of this peculiar target for cancer immunotherapy. In two phase II Clinical Trials including women with metastatic breast cancer (n=14) and patients with colon cancer (n=19) in all stages, the 14F7 monoclonal antibody (3 mg) labelled with 99mTc (30-40 mCi) was also able to detect distant metastasis over expressing the GM3(NeuGc) ganglioside. (author)

  12. How protein recognizes ladder-like polycyclic ethers. Interactions between ciguatoxin (CTX3C) fragments and its specific antibody 10C9.

    Science.gov (United States)

    Ui, Mihoko; Tanaka, Yoshikazu; Tsumuraya, Takeshi; Fujii, Ikuo; Inoue, Masayuki; Hirama, Masahiro; Tsumoto, Kouhei

    2008-07-11

    Ciguatoxins are a family of marine toxins composed of transfused polycyclic ethers. It has not yet been clarified at the atomic level on the pathogenic mechanism of these toxins or the interaction between a polycyclic ether compounds and a protein. Using the crystal structures of anti-ciguatoxin antibody 10C9 Fab in ligand-free form and in complexes with ABCD-ring (CTX3C-ABCD) and ABCDE-ring (CTX3C-ABCDE) fragments of the antigen CTX3C at resolutions of 2.6, 2.4, and 2.3 angstroms, respectively, we elucidated the mechanism of the interaction between the polycyclic ethers and the antibody. 10C9 Fab has an extraordinarily large and deep binding pocket at the center of the variable region, where CTX3C-ABCD or CTX3C-ABCDE binds longitudinally in the pocket via hydrogen bonds and van der Waals interactions. Upon antigen-antibody complexation, 10C9 Fab adjusts to the antigen fragments by means of rotational motion in the variable region. In addition, the antigen fragment lacking the E-ring induces a large motion in the constant region. Consequently, the thermostability of 10C9 Fab is enhanced by 10 degrees C upon complexation with CTX3C-ABCDE but not with CTX3C-ABCD. The crystal structures presented in this study also show that 10C9 Fab recoginition of CTX3C antigens requires molecular rearrangements over the entire antibody structure. These results further expand the fundamental understanding of the mechanism by which ladder-like polycyclic ethers are recognized and may be useful for the design of novel therapeutic agents by antibodies, marine toxins, or new diagnostic reagents for the detection and targeting of members of the polycyclic ether family.

  13. The interfacial character of antibody paratopes: analysis of antibody-antigen structures.

    Science.gov (United States)

    Nguyen, Minh N; Pradhan, Mohan R; Verma, Chandra; Zhong, Pingyu

    2017-10-01

    In this study, computational methods are applied to investigate the general properties of antigen engaging residues of a paratope from a non-redundant dataset of 403 antibody-antigen complexes to dissect the contribution of hydrogen bonds, hydrophobic, van der Waals contacts and ionic interactions, as well as role of water molecules in the antigen-antibody interface. Consistent with previous reports using smaller datasets, we found that Tyr, Trp, Ser, Asn, Asp, Thr, Arg, Gly, His contribute substantially to the interactions between antibody and antigen. Furthermore, antibody-antigen interactions can be mediated by interfacial waters. However, there is no reported comprehensive analysis for a large number of structured waters that engage in higher ordered structures at the antibody-antigen interface. From our dataset, we have found the presence of interfacial waters in 242 complexes. We present evidence that suggests a compelling role of these interfacial waters in interactions of antibodies with a range of antigens differing in shape complementarity. Finally, we carry out 296 835 pairwise 3D structure comparisons of 771 structures of contact residues of antibodies with their interfacial water molecules from our dataset using CLICK method. A heuristic clustering algorithm is used to obtain unique structural similarities, and found to separate into 368 different clusters. These clusters are used to identify structural motifs of contact residues of antibodies for epitope binding. This clustering database of contact residues is freely accessible at http://mspc.bii.a-star.edu.sg/minhn/pclick.html. minhn@bii.a-star.edu.sg, chandra@bii.a-star.edu.sg or zhong_pingyu@immunol.a-star.edu.sg. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

  14. HPA antibodies in Algerian multitransfused patients: Prevalence and involvement in platelet refractoriness.

    Science.gov (United States)

    Brouk, Hacene; Bertrand, Gérald; Zitouni, Selma; Djenouni, Amel; Martageix, Corinne; Griffi, Fatiha; Kaplan, Cecile; Ouelaa, Hanifa

    2015-06-01

    Patients receiving cellular blood components may form HLA or HPA antibodies. The frequency and the specificity of HPA antibodies after a series of blood transfusions have never been reported in the Algerian population which is ethnically diverse and runs a higher risk of platelet alloimmunization due to high b allelic frequencies observed for the HPA systems. 117 polytransfused patients were included in this study; the detection of HPA antibodies was performed by the Monoclonal Antibody-specific Immobilization of Platelet Antigens method (MAIPA). Post-transfusion platelet effectiveness was evaluated by the calculation of corrected count increment (CCI). The antibodies against platelets were detected in 10.26% of the patients. In this study, the platelet systems concerned by the alloimmunizations were specifically HPA-1, -3 and -5 with particular predominance of HPA-1. Twenty two patients were refractory to platelet transfusion, as assessed by a CCI; in which 64% have factors associated with increased platelet consumption. Platelet Immunization was found in 14% of platelet refractoriness (PTR) cases. 03 Anti-platelet antibodies were directed against GPIb-IX (n = 1), anti-HPA-1b (n = 1) and anti HPA-5b (n = 1) associated with anti-HLA antibodies in two cases. HLA and HPA alloimmunization is common among chronically transfused patients. PTR detection, identification of the underlying causes, and selection of the appropriate product for transfusion are fundamental to reduce the risk of major bleedings. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Antibody-mediated Prevention of Fusarium Mycotoxins in the Field

    Directory of Open Access Journals (Sweden)

    Yu-Cai Liao

    2008-10-01

    Full Text Available Fusarium mycotoxins directly accumulated in grains during the infection of wheat and other cereal crops by Fusarium head blight (FHB pathogens are detrimental to humans and domesticated animals. Prevention of the mycotoxins via the development of FHB-resistant varieties has been a challenge due to the scarcity of natural resistance against FHB pathogens. Various antibodies specific to Fusarium fungi and mycotoxins are widely used in immunoassays and antibody-mediated resistance in planta against Fusarium pathogens has been demonstrated. Antibodies fused to antifungal proteins have been shown to confer a very significantly enhanced Fusarium resistance in transgenic plants. Thus, antibody fusions hold great promise as an effective tool for the prevention of mycotoxin contaminations in cereal grains. This review highlights the utilization of protective antibodies derived from phage display to increase endogenous resistance of wheat to FHB pathogens and consequently to reduce mycotoxins in field. The role played by Fusarium-specific antibody in the resistance is also discussed.

  16. Rotavirus specific maternal antibodies and immune response to RV3-BB neonatal rotavirus vaccine in New Zealand

    OpenAIRE

    Chen, Mee-Yew; Kirkwood, Carl D.; Bines, Julie; Cowley, Daniel; Pavlic, Daniel; Lee, Katherine J.; Orsini, Francesca; Watts, Emma; Barnes, Graeme; Danchin, Margaret

    2017-01-01

    Background: Maternal antibodies, acquired passively via placenta and/or breast milk, may contribute to the reduced efficacy of oral rotavirus vaccines observed in children in developing countries. This study aimed to investigate the effect of rotavirus specific maternal antibodies on the serum IgA response or stool excretion of vaccine virus after any dose of an oral rotavirus vaccine, RV3-BB, in parallel to a Phase IIa clinical trial conducted at Dunedin Hospital, New Zealand. At the time o...

  17. High-efficiency astatination of antibodies using N-iodosuccinimide as the oxidising agent in labelling of N-succinimidyl 3-(trimethylstannyl)benzoate

    International Nuclear Information System (INIS)

    Lindegren, S.; Andersson, H.; Baeck, T.; Jacobsson, L.; Karlsson, B.; Skarnemark, G.

    2001-01-01

    Monoclonal antibodies C215, reactive with colorectal carcinomas, and MOv18, reactive with most of the ovarian carcinomas, were radiohalogenated with [ 211 At]astatine. The radiohalogen was conjugate coupled to antibodies via the intermediate labelling reagent N-succinimidyl-3-(trimethylstannyl)benzoate (m-MeATE) in a two-step, single-pot reaction. Optimisation of the labelling of the reagent was achieved using N-iodosuccinimide, NIS, as the oxidising agent. The yields ranged from 69-95% in the labelling of 0.1-1.0 nmole of the m-MeATE precursor. Subsequent conjugation to antibodies resulted in yields of 58±7%. In vitro binding to tumour cells showed that the immunoreactivity of both antibodies was retained after astatine labelling

  18. The HIV-1 V3 domain on field isolates: participation in generation of escape virus in vivo and accessibility to neutralizing antibodies

    DEFF Research Database (Denmark)

    Arendrup, M; Akerblom, L; Heegaard, P M

    1995-01-01

    The V3 domain is highly variable and induces HIV neutralizing antibodies (NA). Here we addressed the issues of 1) the participation of mutations in V3 in generation of neutralization resistant escape virus in vivo and 2) the applicability of synthetic V3 peptides corresponding to field isolates...... to induce neutralizing immune sera. Seven peptides corresponding to the V3 region of primary and escape virus from 3 HIV-1 infected patients were synthesized and used for antibody (Abs) studies and immunizations. The anti-V3 Abs titre in patient serum was generally low against peptides corresponding...... to autologous virus isolated later than the serum sample in contrast to the titre against peptides corresponding to virus isolated earlier than the serum sample. Furthermore, neutralizing anti-V3 monoclonal antibodies (MAbs) raised against V3 peptides from laboratory strains of HIV-1 showed distinct binding...

  19. Projecting human pharmacokinetics of therapeutic antibodies from nonclinical data: What have we learned?

    OpenAIRE

    Deng, Rong; Iyer, Suhasini; Theil, Frank-Peter; Mortensen, Deborah L; Fielder, Paul J; Prabhu, Saileta

    2011-01-01

    The pharmacokinetics (PK) of therapeutic antibodies is determined by target and non-target mediated mechanisms. These antibody-specific factors need to be considered during prediction of human PK based upon preclinical information. Principles of allometric scaling established for small molecules using data from multiple animal species cannot be directly applied to antibodies. Here, different methods for projecting human clearance (CL) from animal PK data for 13 therapeutic monoclonal antibodi...

  20. Complement-fixing antibodies against denatured HLA and MICA antigens are associated with antibody mediated rejection.

    Science.gov (United States)

    Cai, Junchao; Terasaki, Paul I; Zhu, Dong; Lachmann, Nils; Schönemann, Constanze; Everly, Matthew J; Qing, Xin

    2016-02-01

    We have found antibodies against denatured HLA class I antigens in the serum of allograft recipients which were not significantly associated with graft failure. It is unknown whether transplant recipients also have denatured HLA class II and MICA antibodies. The effects of denatured HLA class I, class II, and MICA antibodies on long-term graft outcome were further investigated based on their ability to fix complement c1q. In this 4-year retrospective cohort study, post-transplant sera from 975 kidney transplant recipients were tested for antibodies against denatured HLA/MICA antigens and these antibodies were further classified based on their ability to fix c1q. Thirty percent of patients had antibodies against denatured HLA class I, II, or MICA antigens. Among them, 8.5% and 21.5% of all patients had c1q-fixing and non c1q-fixing antibodies respectively. There was no significant difference on graft survival between patients with or without antibodies against denatured HLA/MICA. However, when these antibodies were further classified according to their ability to fix c1q, patients with c1q-fixing antibodies had a significantly lower graft survival rate than patients without antibodies or patients with non c1q-fixing antibodies (p=0.008). In 169 patients who lost renal grafts, 44% of them had c1q-fixing antibodies against denatured HLA/MICA antigens, which was significantly higher than that in patients with functioning renal transplants (25%, pantibodies were more significantly associated with graft failure caused by AMR (72.73%) or mixed AMR/CMR (61.9%) as compared to failure due to CMR (35.3%) or other causes (39.2%) (p=0.026). Transplant recipients had antibodies against denatured HLA class I, II, and MICA antigens. However, only c1q-fixing antibodies were associated with graft failure which was related to antibody mediated rejection. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Antibodies against major histocompatibility complex class II antigens directly inhibit the growth of T cells infected with Theileria parva without affecting their state of activation

    OpenAIRE

    Eichhorn, M; Prospero, T D; Heussler, Volker; Dobbelaere, D A

    1993-01-01

    We have analyzed the effect of antibodies (Abs) directed against major histocompatibility complex (MHC) class II Abs on the proliferation of Theileria parva-infected (Tpi) T cells. Anti-MHC class II Abs exert a direct effect on Tpi T cells causing an acute block in their proliferation. The inhibition does not involve apoptosis and is also entirely reversible. The rapid arrest of DNA synthesis caused by anti- MHC class II Abs is not due to interference with the state of activation of the T cel...

  2. Production and characterization of peptide antibodies

    DEFF Research Database (Denmark)

    Trier, Nicole Hartwig; Hansen, Paul Robert; Houen, Gunnar

    2012-01-01

    Proteins are effective immunogens for generation of antibodies. However, occasionally the native protein is known but not available for antibody production. In such cases synthetic peptides derived from the native protein are good alternatives for antibody production. These peptide antibodies...... are powerful tools in experimental biology and are easily produced to any peptide of choice. A widely used approach for production of peptide antibodies is to immunize animals with a synthetic peptide coupled to a carrier protein. Very important is the selection of the synthetic peptide, where factors......, including solid-phase peptide-carrier conjugation and peptide-carrier conjugation in solution. Upon immunization, adjuvants such as Al(OH)(3) are added together with the immunogenic peptide-carrier conjugate, which usually leads to high-titred antisera. Following immunization and peptide antibody...

  3. Monoclonal anti-melanoma antibodies and their possible clinical use

    International Nuclear Information System (INIS)

    Hellstroem, K.E.; Hellstroem, Ingegerd; Washington Univ., Seattle; Washington Univ., Seattle

    1985-01-01

    Cell surface antigens of human melanoma, as defined by monoclonal antibodies, are discussed and in particular the three antigens p97, a GD3 ganglioside and a proteoglycan. The potential diagnostic uses of antibodies to melanoma antigens are reviewed including in vitro diagnosis by immuno-histology, in vitro diagnosis by serum assays and in vivo diagnosis by tumour imaging using radioactively labelled antibodies. The potential therapeutic uses of monoclonal antibodies to melanoma antigens are also reviewed including targets for antibody therapy, the use of antibodies alone, radiolabelled antibodies, antibody-toxin conjugates, antibody-drug conjugates, anti-idiotypic antibodies and vaccines. (UK)

  4. Antibody- and TRIM21-dependent intracellular restriction of Salmonella enterica.

    Science.gov (United States)

    Rakebrandt, Nikolas; Lentes, Sabine; Neumann, Heinz; James, Leo C; Neumann-Staubitz, Petra

    2014-11-01

    TRIM21 ('tripartite motif-containing protein 21', Ro52) is a ubiquitously expressed cytosolic Fc receptor, which has a potent role in protective immunity against nonenveloped viruses. TRIM21 mediates intracellular neutralisation of antibody-coated viruses, a process called ADIN (antibody-dependent intracellular neutralisation). Our results reveal a similar mechanism to fight bacterial infections. TRIM21 is recruited to the intracellular pathogen Salmonella enterica in epithelial cells early in infection. TRIM21 does not bind directly to S. enterica, but to antibodies opsonising it. Most importantly, bacterial restriction is dependent on TRIM21 as well as on the opsonisation state of the bacteria. Finally, Salmonella and TRIM21 colocalise with the autophagosomal marker LC3, and intracellular defence is enhanced in starved cells suggesting an involvement of the autophagocytic pathway. Our data extend the protective role of TRIM21 from viruses to bacteria and thereby strengthening the general role of ADIN in cellular immunity. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  5. Template-directed covalent conjugation of DNA to native antibodies, transferrin and other metal-binding proteins

    Science.gov (United States)

    Rosen, Christian B.; Kodal, Anne L. B.; Nielsen, Jesper S.; Schaffert, David H.; Scavenius, Carsten; Okholm, Anders H.; Voigt, Niels V.; Enghild, Jan J.; Kjems, Jørgen; Tørring, Thomas; Gothelf, Kurt V.

    2014-09-01

    DNA-protein conjugates are important in bioanalytical chemistry, molecular diagnostics and bionanotechnology, as the DNA provides a unique handle to identify, functionalize or otherwise manipulate proteins. To maintain protein activity, conjugation of a single DNA handle to a specific location on the protein is often needed. However, preparing such high-quality site-specific conjugates often requires genetically engineered proteins, which is a laborious and technically challenging approach. Here we demonstrate a simpler method to create site-selective DNA-protein conjugates. Using a guiding DNA strand modified with a metal-binding functionality, we directed a second DNA strand to the vicinity of a metal-binding site of His6-tagged or wild-type metal-binding proteins, such as serotransferrin, where it subsequently reacted with lysine residues at that site. This method, DNA-templated protein conjugation, facilitates the production of site-selective protein conjugates, and also conjugation to IgG1 antibodies via a histidine cluster in the constant domain.

  6. Antibodies to actin in autoimmune haemolytic anaemia

    Directory of Open Access Journals (Sweden)

    Ritzmann Mathias

    2010-03-01

    Full Text Available Abstract Background In autoimmune haemolytic anaemia (AIHA, autoreactive antibodies directed against red blood cells are up-regulated, leading to erythrocyte death. Mycoplasma suis infections in pigs induce AIHA of both the warm and cold types. The aim of this study was to identify the target autoantigens of warm autoreactive IgG antibodies. Sera from experimentally M. suis-infected pigs were screened for autoreactivity. Results Actin-reactive antibodies were found in the sera of 95% of all animals tested. The reactivity was species-specific, i.e. reactivity with porcine actin was significantly higher than with rabbit actin. Sera of animals previously immunised with the M. suis adhesion protein MSG1 showed reactivity with actin prior to infection with M. suis indicating that molecular mimicry is involved in the specific autoreactive mechanism. A potentially cross-reactive epitope was detected. Conclusions This is the first report of autoreactive anti-actin antibodies involved in the pathogenesis of autoimmune haemolytic anaemia.

  7. High-affinity uranyl-specific antibodies suitable for cellular imaging

    Energy Technology Data Exchange (ETDEWEB)

    Reisser-Rubrecht, L.; Torne-Celer, C.; Renier, W.; Averseng, O.; Plantevin, S.; Quemeneur, E.; Bellanger, L.; Vidaud, C. [CEA Valrho, DSV, IBEB, Serv Biochim et Toxicol Nucl, F-30207 Bagnols Sur Ceze (France)

    2008-07-01

    Monoclonal antibodies (mAbs) have proved to be valuable models for the study of protein-metal interactions, and previous reports have described very specific antibodies to chelated metal ions, including uranyl. We raised specific mAbs against UO{sub 2}{sup 2+}-DCP-BSA (DCP, 1, 10-phenanthroline-2,9-dicarboxylic acid) to generate new sets of antibodies that might cross-react with various complexed forms of uranyl in different environments for further application in the field of toxicology. Using counter-screening with UO{sub 2}{sup 2+}-DCP-casein, we selected two highly specific mAbs against uranyl-DCP (K{sub D} = 10-100 pM): U04S and U08S. Competitive assays in the presence of different metal ions (UO{sub 2}{sup 2+}, Fe{sup 3+}, Zn{sup 2+}, Cu{sup 2+}, and Ca{sup 2+}) showed that uranyl in solution can act as a good competitor, suggesting some antibody ability to cross-react with chelating groups other than DCP in the UO{sub 2}{sup 2+} equatorial coordination plane. Interestingly, one of the antibodies could be used for revealing uranyl cations in cell samples. Fluorescence activated cell sorting analyses after immuno-labeling revealed the interaction of uranyl with human kidney cells HK2. The intracellular accumulation of uranyl could be directly visualized by metal-immunostaining using fluorescent-labeled mAb. Our results suggest that U04S mAb epitopes mostly include the uranyl fraction and its para-topes can accommodate a wide variety of chelating groups. (authors)

  8. Participation of L3T4 in T cell activation in the absence of class II major histocompatibility complex antigens. Inhibition by anti-L3T4 antibodies is a function both of epitope density and mode of presentation of anti-receptor antibody

    DEFF Research Database (Denmark)

    Owens, T; Fazekas de St Groth, B

    1987-01-01

    two monoclonal antibodies, KJ16-133.18 and F23.1, that recognize a determinant encoded by the T cell receptor V beta 8 gene family. These antibodies were used to select two clones of T cells with surface phenotype Thy-1.2+, L3T4+, Lyt-2-, KJ16-133.18+, F23.1+, IA-, IE-. One of these clones (E9.D4......The recognition of many class II major histocompatibility complex (MHC)-associated antigens by T cells requires the participation of the L3T4 molecule. It has been proposed that this molecule acts to stabilize low affinity binding to antigen in association with MHC and thereby increases the avidity...... of T cell/antigen interactions. By using antibodies against the T cell antigen receptor (TCR) to activate T cells, thereby circumventing the requirement for antigen presenting cells and MHC-associated antigen, we have been able to study the function of L3T4 in the absence of class II MHC. We have used...

  9. Development of recombinant antigen array for simultaneous detection of viral antibodies.

    Directory of Open Access Journals (Sweden)

    Yi Liu

    Full Text Available Protein microarrays have been developed to study antibody reactivity against a large number of antigens, demonstrating extensive perspective for clinical application. We developed a viral antigen array by spotting four recombinant antigens and synthetic peptide, including glycoprotein G of herpes simplex virus (HSV type 1 and 2, phosphoprotein 150 of cytomegalovirus (CMV, Rubella virus (RV core plus glycoprotein E1 and E2 as well as a E1 peptide with the optimal concentrations on activated glass slides to simultaneously detect IgG and IgM against HSV1, HSV2, CMV and RV in clinical specimens of sera and cerebrospinal fluids (CSFs. The positive reference sera were initially used to measure the sensitivity and specificity of the array with the optimal conditions. Then clinical specimens of 144 sera and 93 CSFs were tested for IgG and IgM antibodies directed against HSV1, HSV2, CMV and RV by the antigen array. Specificity of the antigen array for viral antibodies detection was satisfying compared to commercial ELISA kits but sensitivity of the array varied relying on quality and antigenic epitopes of the spotting antigens. In short, the recombinant antigen array has potential to simultaneous detect multiple viral antibodies using minute amount (3 µl of samples, which holds the particularly advantage to detect viral antibodies in clinical CSFs being suspicious of neonatal meningitis and encephalitis.

  10. A novel, colorimetric neutralization assay for measuring antibodies to influenza viruses.

    Science.gov (United States)

    Lehtoranta, Liisa; Villberg, Anja; Santanen, Riitta; Ziegler, Thedi

    2009-08-01

    A colorimetric cell proliferation assay for measuring neutralizing antibodies to influenza viruses in human sera is described. Following a 90-min incubation, the serum-virus mixture was transferred to Madin-Darby canine kidney cells cultured in 96-well plates. After further incubation for three days, a tetrazolium salt was added to the wells. Cellular mitochondrial dehydrogenases cleave the tetrazolium salt to formazan, and the resulting color change is read by a spectrophotometer. The absorbance values correlate directly to the number of viable cells in the assay well and thus also to the neutralizing activity of influenza-specific antibodies present in the serum. With the few hands-on manipulations required, this assay allows simultaneous testing of a considerable number of sera, offers opportunities for automation, and is suitable for use under biosafety level-3 conditions. The test was used to study the antibody response after the administration of seasonal, inactivated, trivalent influenza vaccine. Antibody titers determined by the neutralization test in pre- and post-vaccination serum pairs were compared with those obtained by the hemagglutination inhibition assay. The neutralization test yielded higher pre- and post-vaccination titers and a larger number of significant increases in post-vaccination antibody titer than the hemagglutination inhibition test. This new test format could serve as a valuable laboratory tool for influenza vaccine studies.

  11. Vurdering af omfang og konsekvenser af sprøjtning og gødskning af § 3-beskyttede naturarealer

    DEFF Research Database (Denmark)

    Strandberg, Morten Tune; Bak, Jesper Leth; Bladt, Jesper Stentoft

    2012-01-01

    Det er overvejende de ferske enge, der gødskes og sprøjtes, hvorimod dette kun undtagelsesvist er konstateret på andre naturtyper som overdrev og heder m.m. De ferske enge er samtidig den mest udbredte § 3-naturtype. Behandling i form af gødskning og sprøjtning foregår anslået på op til 20 % af a...

  12. Complement-Mediated Enhancement of Monocyte Adhesion to Endothelial Cells by HLA Antibodies, and Blockade by a Specific Inhibitor of the Classical Complement Cascade, TNT003

    Science.gov (United States)

    Valenzuela, Nicole M.; Thomas, Kimberly A.; Mulder, Arend; Parry, Graham C.; Panicker, Sandip; Reed, Elaine F.

    2017-01-01

    Background Antibody-mediated rejection (AMR) of most solid organs is characterized by evidence of complement activation and/or intragraft macrophages (C4d + and CD68+ biopsies). We previously demonstrated that crosslinking of HLA I by antibodies triggered endothelial activation and monocyte adhesion. We hypothesized that activation of the classical complement pathway at the endothelial cell surface by HLA antibodies would enhance monocyte adhesion through soluble split product generation, in parallel with direct endothelial activation downstream of HLA signaling. Methods Primary human aortic endothelial cells (HAEC) were stimulated with HLA class I antibodies in the presence of intact human serum complement. C3a and C5a generation, endothelial P-selectin expression, and adhesion of human primary and immortalized monocytes (Mono Mac 6) were measured. Alternatively, HAEC or monocytes were directly stimulated with purified C3a or C5a. Classical complement activation was inhibited by pretreatment of complement with an anti-C1s antibody (TNT003). Results Treatment of HAEC with HLA antibody and human complement increased the formation of C3a and C5a. Monocyte recruitment by human HLA antibodies was enhanced in the presence of intact human serum complement or purified C3a or C5a. Specific inhibition of the classical complement pathway using TNT003 or C1q-depleted serum significantly reduced adhesion of monocytes in the presence of human complement. Conclusions Despite persistent endothelial viability in the presence of HLA antibodies and complement, upstream complement anaphylatoxin production exacerbates endothelial exocytosis and leukocyte recruitment. Upstream inhibition of classical complement may be therapeutic to dampen mononuclear cell recruitment and endothelial activation characteristic of microvascular inflammation during AMR. PMID:28640789

  13. Amended Final Report - Antibodies to Radionuclides. Engineering by Surface Display for Immunosensors

    Energy Technology Data Exchange (ETDEWEB)

    Blake, Diane A. [Tulane Univ., New Orleans, LA (United States)

    2013-06-14

    The relatively new techniques of antibody display, which permit molecular engineering of antibody structure and function, have the potential to revolutionize the way scientists generate binding proteins for specific applications. However, the skills required to efficiently use antibody display techniques have proven difficult for other laboratories to acquire without hands-on training and exchange of laboratory personnel. This research project is designed bring important expertise in antibody display to the State of Louisiana while pursuing a project with direct relevance to the DOE’s EM program.

  14. Development of an EGFRvIII specific recombinant antibody

    Directory of Open Access Journals (Sweden)

    Li Gordon

    2010-10-01

    Full Text Available Abstract Background EGF receptor variant III (EGFRvIII is the most common variant of the EGF receptor observed in human tumors. It results from the in frame deletion of exons 2-7 and the generation of a novel glycine residue at the junction of exons 1 and 8. This novel juxtaposition of amino acids within the extra-cellular domain of the EGF receptor creates a tumor specific and immunogenic epitope. EGFRvIII expression has been seen in many tumor types including glioblastoma multiforme (GBM, breast adenocarcinoma, non-small cell lung carcinoma, ovarian adenocarcinoma and prostate cancer, but has been rarely observed in normal tissue. Because this variant is tumor specific and highly immunogenic, it can be used for both a diagnostic marker as well as a target for immunotherapy. Unfortunately many of the monoclonal and polyclonal antibodies directed against EGFRvIII have cross reactivity to wild type EGFR or other non-specific proteins. Furthermore, a monoclonal antibody to EGFRvIII is not readily available to the scientific community. Results In this study, we have developed a recombinant antibody that is specific for EGFRvIII, has little cross reactivity for the wild type receptor, and which can be easily produced. We initially designed a recombinant antibody with two anti-EGFRvIII single chain Fv's linked together and a human IgG1 Fc component. To enhance the specificity of this antibody for EGFRvIII, we mutated tyrosine H59 of the CDRH2 domain and tyrosine H105 of the CDRH3 domain to phenylalanine for both the anti-EGFRvIII sequence inserts. This mutated recombinant antibody, called RAbDMvIII, specifically detects EGFRvIII expression in EGFRvIII expressing cell lines as well as in EGFRvIII expressing GBM primary tissue by western blot, immunohistochemistry (IHC and immunofluorescence (IF and FACS analysis. It does not recognize wild type EGFR in any of these assays. The affinity of this antibody for EGFRvIII peptide is 1.7 × 107 M-1 as

  15. Direct Competitive Enzyme-Linked Immunosorbent Assay (ELISA).

    Science.gov (United States)

    Kohl, Thomas O; Ascoli, Carl A

    2017-07-05

    The competitive enzyme-linked immunosorbent assay (ELISA) (cELISA; also called an inhibition ELISA) is designed so that purified antigen competes with antigen in the test sample for binding to an antibody that has been immobilized in microtiter plate wells. The same concept works if the immobilized molecule is antigen and the competing molecules are purified labeled antibody versus antibody in a test sample. Direct cELISAs incorporate labeled antigen or antibody, whereas indirect assay configurations use reporter-labeled secondary antibodies. The cELISA is very useful for determining the concentration of small-molecule antigens in complex sample mixtures. In the direct cELISA, antigen-specific capture antibody is adsorbed onto the microtiter plate before incubation with either known standards or unknown test samples. Enzyme-linked antigen (i.e., labeled antigen) is also added, which can bind to the capture antibody only when the antibody's binding site is not occupied by either the antigen standard or antigen in the test samples. Unbound labeled and unlabeled antigens are washed away and substrate is added. The amount of antigen in the standard or the test sample determines the amount of reporter-labeled antigen bound to antibody, yielding a signal that is inversely proportional to antigen concentration within the sample. Thus, the higher the antigen concentration in the test sample, the less labeled antigen is bound to the capture antibody, and hence the weaker is the resultant signal. © 2017 Cold Spring Harbor Laboratory Press.

  16. The interaction of antibodies with lipid membranes unraveled by fluorescence methodologies

    Science.gov (United States)

    Figueira, Tiago N.; Veiga, Ana Salomé; Castanho, Miguel A. R. B.

    2014-12-01

    The interest and investment in antibody therapies has reached an overwhelming scale in the last decade. Yet, little concern has been noticed among the scientific community to unravel important interactions of antibodies with biological structures other than their respective epitopes. Lipid membranes are particularly relevant in this regard as they set the stage for protein-protein recognition, a concept potentially inclusive of antibody-antigen recognition. Fluorescence techniques allow experimental monitoring of protein partition between aqueous and lipid phases, deciphering events of adsorption, insertion and diffusion. This review focuses on the available fluorescence spectroscopy methodologies directed to the study of antibody-membrane interactions.

  17. Broadly-Reactive Neutralizing and Non-neutralizing Antibodies Directed against the H7 Influenza Virus Hemagglutinin Reveal Divergent Mechanisms of Protection.

    Directory of Open Access Journals (Sweden)

    Gene S Tan

    2016-04-01

    Full Text Available In the early spring of 2013, Chinese health authorities reported several cases of H7N9 influenza virus infections in humans. Since then the virus has established itself at the human-animal interface in Eastern China and continues to cause several hundred infections annually. In order to characterize the antibody response to the H7N9 virus we generated several mouse monoclonal antibodies against the hemagglutinin of the A/Shanghai/1/13 (H7N9 virus. Of particular note are two monoclonal antibodies, 1B2 and 1H5, that show broad reactivity to divergent H7 hemagglutinins. Monoclonal antibody 1B2 binds to viruses of the Eurasian and North American H7 lineages and monoclonal antibody 1H5 reacts broadly to virus isolates of the Eurasian lineage. Interestingly, 1B2 shows broad hemagglutination inhibiting and neutralizing activity, while 1H5 fails to inhibit hemagglutination and demonstrates no neutralizing activity in vitro. However, both monoclonal antibodies were highly protective in an in vivo passive transfer challenge model in mice, even at low doses. Experiments using mutant antibodies that lack the ability for Fc/Fc-receptor and Fc/complement interactions suggest that the protection provided by mAb 1H5 is, at least in part, mediated by the Fc-fragment of the mAb. These findings highlight that a protective response to a pathogen may not only be due to neutralizing antibodies, but can also be the result of highly efficacious non-neutralizing antibodies not readily detected by classical in vitro neutralization or hemagglutination inhibition assays. This is of interest because H7 influenza virus vaccines induce only low hemagglutination inhibiting antibody titers while eliciting robust antibody titers as measured by ELISA. Our data suggest that these binding but non-neutralizing antibodies contribute to protection in vivo.

  18. Development of two murine antibodies against Neospora caninum using phage display technology and application on the detection of N. caninum.

    Directory of Open Access Journals (Sweden)

    Jinhua Dong

    Full Text Available Neosporosis, caused by an intracellular parasite, Neospora caninum, is an infectious disease primarily of cattle and dogs. It occurs worldwide and causes huge damages to dairy farms. In this study, we immunized mice with recombinant surface-associated protein 1 of N. caninum (rNcSAG1 and developed two novel monoclonal antibodies, A10 and H3, against NcSAG1 using phage-display technology. Both clones bound to purified rNcSAG1 and the half maximal inhibitory concentrations of A10 and H3 are 50 and 72 nM of rNcSAG1, respectively. In immunofluorescence assays, both A10 and H3 Fabs bound to N. caninum parasites. Direct detection of N. caninum parasites was developed firstly using an enzyme-linked immunosorbent assay (ELISA with A10 and H3. Binding of A10 and H3 antibodies to rNcSAG1 was also inhibited by some certain anti-N. caninum antibodies in the neosporosis-positive cattle sera, suggesting they might bind to the same epitopes of NcSAG1 with those anti-N. caninum antibodies of bovine. These antibodies were demonstrated to have a potential for monitoring the N. caninum parasites in a dairy farm, which may lead to protect livestock from parasite-infection.

  19. Anti-pituitary antibodies against corticotrophs in IgG4-related hypophysitis.

    Science.gov (United States)

    Iwata, Naoko; Iwama, Shintaro; Sugimura, Yoshihisa; Yasuda, Yoshinori; Nakashima, Kohtaro; Takeuchi, Seiji; Hagiwara, Daisuke; Ito, Yoshihiro; Suga, Hidetaka; Goto, Motomitsu; Banno, Ryoichi; Caturegli, Patrizio; Koike, Teruhiko; Oshida, Yoshiharu; Arima, Hiroshi

    2017-06-01

    IgG4-related disease is a systemic inflammatory disease characterized by infiltration of IgG4-positive plasma cells into multiple organs, including the pituitary gland. Autoimmunity is thought to be involved in the pathogenesis of IgG4-related disease. The diagnosis of IgG4-related hypophysitis (IgG4-RH) is difficult because its clinical features, such as pituitary swelling and hypopituitarism, are similar to those of other pituitary diseases, including lymphocytic hypophysitis and sellar/suprasellar tumors. The presence and significance of anti-pituitary antibodies (APA) in IgG4-RH is unclear. In this case-control study, we used single indirect immunofluorescence on human pituitary substrates to assess the prevalence of serum APA in 17 patients with IgG4-RH, 8 control patients with other pituitary diseases (lymphocytic infundibulo-neurohypophysitis, 3; craniopharyngioma, 2; germinoma, 3), and 9 healthy subjects. We further analyzed the endocrine cells targeted by the antibodies using double indirect immunofluorescence. APA were found in 5 of 17 patients with IgG4-RH (29%), and in none of the pituitary controls or healthy subjects. The endocrine cells targeted by the antibodies in the 5 IgG4-RH cases were exclusively corticotrophs. Antibodies were of the IgG1 subclass, rather than IgG4, in all 5 cases, suggesting that IgG4 is not directly involved in the pathogenesis. Finally, antibodies recognized pro-opiomelanocortin in 2 of the cases. Our study suggests that autoimmunity is involved in the pathogenesis of IgG4-RH and that corticotrophs are the main antigenic target, highlighting a possible new diagnostic marker for this condition.

  20. Structural basis for diverse N-glycan recognition by HIV-1-neutralizing V1-V2-directed antibody PG16

    Energy Technology Data Exchange (ETDEWEB)

    Pancera, Marie; Shahzad-ul-Hussan, Syed; Doria-Rose, Nicole A.; McLellan, Jason S.; Bailer, Robert T.; Dai, Kaifan; Loesgen, Sandra; Louder, Mark K.; Staupe, Ryan P.; Yang, Yongping; Zhang, Baoshan; Parks, Robert; Eudailey, Joshua; Lloyd, Krissey E.; Blinn, Julie; Alam, S. Munir; Haynes, Barton F.; Amin, Mohammed N.; Wang, Lai-Xi; Burton, Dennis R.; Koff, Wayne C.; Nabel, Gary J.; Mascola, John R.; Bewley, Carole A; Kwong, Peter D. [NIH; (Scripps); (Duke); (Maryland-MED); (IAVI)

    2013-08-05

    HIV-1 uses a diverse N-linked-glycan shield to evade recognition by antibody. Select human antibodies, such as the clonally related PG9 and PG16, recognize glycopeptide epitopes in the HIV-1 V1–V2 region and penetrate this shield, but their ability to accommodate diverse glycans is unclear. Here we report the structure of antibody PG16 bound to a scaffolded V1–V2, showing an epitope comprising both high mannose–type and complex-type N-linked glycans. We combined structure, NMR and mutagenesis analyses to characterize glycan recognition by PG9 and PG16. Three PG16-specific residues, arginine, serine and histidine (RSH), were critical for binding sialic acid on complex-type glycans, and introduction of these residues into PG9 produced a chimeric antibody with enhanced HIV-1 neutralization. Although HIV-1–glycan diversity facilitates evasion, antibody somatic diversity can overcome this and can provide clues to guide the design of modified antibodies with enhanced neutralization.

  1. Anti sperm antibodies detection in infertile patients by radioimmunometry

    International Nuclear Information System (INIS)

    ELnabarawy, F.; Megahed, Y.M.; Tadrous, G.A.; Hamada, T.; Elbadry, A.

    1992-01-01

    Three different methods of testing for anti sperm antibodies were compared: complement cytotoxicity, sperm agglutination, and radiolabelled anti globulin antibody technique, for detection of anti sperm antibodies in serum and secretions (seminal plasma and cervical mucus). Sample from 120 patients with infertility were investigated by the previous three methods. The results of unexplained infertile patients revealed wide variations in figures, concerning the positivity of anti sperm antibody whether in their serum or secretions, by using the cytotoxicity or sperm agglutination tests. Using a specific radiolabelled anti globulin test, a subset of patients (44.9% in the serum of men and 50% in seminal plasma) with IgG anti sperm antibody was identified, and this antibody was present in 65.4% and 78,6% of infertile wives sera and cervical mucus, respectively. Therefore, this test has been used to identify and quantitate antibodies directed toward other human cell surfaces. It was concluded that this radiolabelled method is a clinically useful and a potentially versatile procedure that can be successfully applied to the diagnosis and management of patients with suspected immunologic infertility. 1 fig., 5 tab

  2. Binding-site analysis of opioid receptors using monoclonal anti-idiotypic antibodies

    International Nuclear Information System (INIS)

    Conroy, W.G.

    1988-01-01

    Structural relatedness between the variable region of anti-ligand antibodies and opioid binding sites allowed the generation of anti-idiotypic antibodies which recognized opioid receptors. The IgG 3 k antibodies which bound to opioid receptors were obtained when an anti-morphine antiserum was the idiotype. Both antibodies bound to opioid receptors, but only one of these blocked the binding of [ 3 H]naloxone. The antibody which did not inhibit the binding of [ 3 H]naloxone was itself displaced from the receptor by opioid ligands. The unique binding properties displayed by this antibody indicated that anti-idiotypic antibodies are not always a perfect image of the original ligand, and therefore may be more useful than typical ligands as probes for the receptor. An auto-anti-idiotypic technique was successfully used to obtain anti-opioid receptor antibodies. Another IgG 3 k antibody that blocked the binding of [ 3 H]naloxone to rat brain opioid receptors was obtained when a mouse was immunized with naloxone conjugated to bovine serum albumin. These data confirmed that an idiotype-anti-idiotype network which can generate an anti-receptor antibody normally functions when an opioid ligand is introduced into an animal in an immunogenic form

  3. Proteomic Identification of Non-Gal Antibody Targets After Pig-to-Primate Cardiac Xenotransplantation

    Science.gov (United States)

    Byrne, Guerard W.; Stalboerger, Paul G.; Davila, Eduardo; Heppelmann, Carrie J.; Gazi, Mozammel H.; McGregor, Hugh C. J.; LaBreche, Peter T.; Davies, William R.; Rao, Vinay P.; Oi, Keiji; Tazelaar, Henry D.; Logan, John S.; McGregor, Christopher G. A.

    2008-01-01

    Background Experience with non-antigenic galactose α1,3 galactose (αGal) polymers and development of αGal deficient pigs has reduced or eliminated the significance of this antigen in xenograft rejection. Despite these advances, delayed xenograft rejection (DXR) continues to occur most likely due to antibody responses to non-Gal endothelial cell (EC) antigens. Methods To gauge the diversity of the non-Gal antibody response we used antibody derived from CD46 transgenic heterotopic cardiac xenografts performed without T-cell immunosuppression, Group A (n = 4) and Gal knockout (GT-KO) heart transplants under tacrolimus and sirolimus immunosuppression, Group B (n = 8). Non-Gal antibody was measured by flow cytometry and by Western blots using GT-KO EC membrane antigens. A nanoLC/MS/MS analysis of proteins recovered from 2D gels was used to identify target antigens. Results Group A recipients exhibited a mixed cellular and humoral rejection. Group B recipients mainly exhibited classical DXR. Western blot analysis showed a non-Gal antibody response induced by GT+ and GT-KO hearts to an overlapping set of pig aortic EC membrane antigens. Proteomic analysis identified 14 potential target antigens but failed to define several immunodominant targets. Conclusions These experiments indicate that the non-Gal antibody response is directed to a number of stress response and inflammation related pig EC antigens and a few undefined targets. Further analysis of these antibody specificities using alternative methods is required to more fully define the repertoire of non-Gal antibody responses. PMID:18957049

  4. Monomeric CH3: A Small, Stable Antibody Domain with Therapeutic Promise | Poster

    Science.gov (United States)

    By Ashley DeVine, Staff Writer Antibody domains are emerging as promising biopharmaceuticals because of their relatively small size compared to full-sized antibodies, which are too large to effectively penetrate tumors and bind to sterically restricted therapeutic targets. In an article published in The Journal of Biological Chemistry, Tianlei Ying, Ph.D., Dimiter Dimitrov,

  5. Human recombinant antibodies against Plasmodium falciparum merozoite surface protein 3 cloned from peripheral blood leukocytes of individuals with immunity to malaria demonstrate antiparasitic properties

    DEFF Research Database (Denmark)

    Lundquist, Rasmus; Nielsen, Leif Kofoed; Jafarshad, Ali

    2006-01-01

    against MSP-3 residues 194 to 257 (MSP-3(194-257)) on the molecular level. mRNA from peripheral blood leukocytes from clinically immune individuals was used as a source of Fab (fragment antibody) genes. A Fab-phage display library was made, and three distinct antibodies designated RAM1, RAM2, and RAM3...

  6. Antithyroid microsomal antibody

    Science.gov (United States)

    Thyroid antimicrosomal antibody; Antimicrosomal antibody; Microsomal antibody; Thyroid peroxidase antibody; TPOAb ... Granulomatous thyroiditis Hashimoto thyroiditis High levels of these antibodies have also been linked with an increased risk ...

  7. Expression of recombinant multi-coloured fluorescent antibodies in gor -/trxB- E. coli cytoplasm

    Directory of Open Access Journals (Sweden)

    Markiv Anatoliy

    2011-11-01

    Full Text Available Abstract Background Antibody-fluorophore conjugates are invaluable reagents used in contemporary molecular cell biology for imaging, cell sorting and tracking intracellular events. However they suffer in some cases from batch to batch variation, partial loss of binding and susceptibility to photo-bleaching. In theory, these issues can all be addressed by using recombinant antibody fused directly to genetically encoded fluorescent reporters. However, single-chain fragment variable domains linked by long flexible linkers are themselves prone to disassociation and aggregation, and in some cases with isoelectric points incompatible with use in physiologically relevant milieu. Here we describe a general approach that permits fully functional intracellular production of a range of coloured fluorescent recombinant antibodies with optimally orientated VH/VL interfaces and isoelectric points compatible for use in physiological solutions at pH 7.4 with a binding site to fluorophore stoichiometry of 1:1. Results Here we report the design, assembly, intracellular bacterial production and purification of a panel of novel antibody fluorescent protein fusion constructs. The insertion of monomeric fluorescent protein derived from either Discosoma or Aequorea in-between the variable regions of anti-p185HER2-ECD antibody 4D5-8 resulted in optimal VH/VL interface interactions to create soluble coloured antibodies each with a single binding site, with isoelectric points of 6.5- 6. The fluorescent antibodies used in cell staining studies with SK-BR-3 cells retained the fluorophore properties and antibody specificity functions, whereas the conventional 4D5-8 single chain antibody with a (Gly4Ser3 linker precipitated at physiological pH 7.4. Conclusions This modular monomeric recombinant fluorescent antibody platform may be used to create a range of recombinant coloured antibody molecules for quantitative in situ, in vivo and ex vivo imaging, cell sorting and cell

  8. Antibody Characterization Process | Office of Cancer Clinical Proteomics Research

    Science.gov (United States)

    The goal of the NCI's Antibody Characterization Program (ACP) is to have three monoclonal antibodies produced for each successfully expressed/purified recombinant antigen and one antibody per peptide (1 to 3 peptides per protein). To date, over 4000 clones have been screened before selecting the current 393 antibodies. They are winnowed down based on the projected end use of the antibody.

  9. Novel immunoradiometric assay of thyroglobulin in serum with use of monoclonal antibodies selected for lack of cross-reactivity with autoantibodies

    International Nuclear Information System (INIS)

    Piechaczyk, M.; Baldet, L.; Pau, B.; Bastide, J.M.

    1989-01-01

    A multisite immunoradiometric assay for measurement of serum thyroglobulin (Tg), designated Magnogel-IRMA-Tg, has been developed, involving magnetic microbeads (Magnogel). This assay is based on the use of five anti-Tg monoclonal antibodies (MAbs) directed against three antigenic regions on the Tg molecule that are not recognized by anti-Tg autoantibodies (aAbs). Four of these MAbs, directed against two antigenic domains, were coupled to the magnetic beads and were used to trap the serum antigen. Another MAb, directed against the third region, was iodinated and served as the labeled second antibody. The Magnogel-IRMA-Tg technique is reproducible, rapid, and sensitive (lower detection limit, 3 micrograms/L). The assay reliably measures serum Tg in the presence of anti-Tg aAbs

  10. Structural basis for the recognition in an idiotype-anti-idiotype antibody complex related to celiac disease

    KAUST Repository

    Vangone, Anna

    2014-07-30

    Anti-idiotype antibodies have potential therapeutic applications in many fields, including autoimmune diseases. Herein we report the isolation and characterization of AIM2, an anti-idiotype antibody elicited in a mouse model upon expression of the celiac disease-specific autoantibody MB2.8 (directed against the main disease autoantigen type 2 transglutaminase, TG2). To characterize the interaction between the two antibodies, a 3D model of the MB2.8-AIM2 complex has been obtained by molecular docking. Analysis and selection of the different obtained docking solutions was based on the conservation within them of the inter-residue contacts. The selected model is very well representative of the different solutions found and its stability is confirmed by molecular dynamics simulations. Furthermore, the binding mode it adopts is very similar to that observed in most of the experimental structures available for idiotype-anti-idiotype antibody complexes. In the obtained model, AIM2 is directed against the MB2.8 CDR region, especially on its variable light chain. This makes the concurrent formation of the MB2.8-AIM2 complex and of the MB2.8-TG2 complex incompatible, thus explaining the experimentally observed inhibitory effect on the MB2.8 binding to TG2. © 2014 Vangone et al.

  11. Structural basis for the recognition in an idiotype-anti-idiotype antibody complex related to celiac disease

    KAUST Repository

    Vangone, Anna; Abdel-Azeim, Safwat; Caputo, Ivana; Sblattero, Daniele; Di Niro, Roberto; Cavallo, Luigi; Oliva, Romina

    2014-01-01

    Anti-idiotype antibodies have potential therapeutic applications in many fields, including autoimmune diseases. Herein we report the isolation and characterization of AIM2, an anti-idiotype antibody elicited in a mouse model upon expression of the celiac disease-specific autoantibody MB2.8 (directed against the main disease autoantigen type 2 transglutaminase, TG2). To characterize the interaction between the two antibodies, a 3D model of the MB2.8-AIM2 complex has been obtained by molecular docking. Analysis and selection of the different obtained docking solutions was based on the conservation within them of the inter-residue contacts. The selected model is very well representative of the different solutions found and its stability is confirmed by molecular dynamics simulations. Furthermore, the binding mode it adopts is very similar to that observed in most of the experimental structures available for idiotype-anti-idiotype antibody complexes. In the obtained model, AIM2 is directed against the MB2.8 CDR region, especially on its variable light chain. This makes the concurrent formation of the MB2.8-AIM2 complex and of the MB2.8-TG2 complex incompatible, thus explaining the experimentally observed inhibitory effect on the MB2.8 binding to TG2. © 2014 Vangone et al.

  12. Detection of anti-lactoferrin antibodies and anti-myeloperoxidase antibodies in autoimmune hepatitis: a retrospective study.

    Science.gov (United States)

    Tan, Liming; Zhang, Yuhong; Peng, Weihua; Chen, Juanjuan; Li, Hua; Ming, Feng

    2014-01-01

    Anti-lactoferrin antibodies (ALA) and anti-myeloperoxidase antibodies (AMPA) are specific serological markers for autoimmune hepatitis (AIH). The project aimed to detect ALA and AMPA and explore their clinical significances in AIH patients. 59 AIH patients, 217 non AIH patients, and 50 healthy controls were enrolled in this study. ALA and AMPA were detected by ELISA. Antineutropil cytoplasmic antibodies (ANCA) and anti-smooth muscle antibodies (ASMA) were examined by indirect immunofluorescence. Antimitochondrial antibody M2 subtype (AMA-M2), anti-liver kidney microsomal antibody Type 1 (LKM1), anti-liver cytosol antibody Type 1 (LC1), and anti-soluble liver antigen/liver-pancreas antibodies (SLA/LP) were tested by immunoblot. The positivity for ALA was 18.6% in AIH group, only one patient in non-AIH group was positive for ALA; the positivity for AMPA was 59.3% in AIH group, with significant differences (P < 0.01) compared with other groups. The specificities for ALA and AMPA were 99.63% and 97.75%; the sensitivities were 18.64% and 59.32%; and the accuracy rates were 84.97% and 90.80%, respectively. A certain correlation was observed between ALA and SLA/LP, AMPA and ANCA, ASMA in AIH group. ALA and AMPA were associated with AIH, and had high clinical diagnostic value. Co-detection with other relative autoantibodies could play an important role in differential diagnosis of AIH.

  13. Antiprothrombin Antibodies

    Directory of Open Access Journals (Sweden)

    Polona Žigon

    2015-05-01

    Full Text Available In patients with the antiphospholipid syndrome (APS, the presence of a group of pathogenic autoantibodies called antiphospholipid antibodies causes thrombosis and pregnancy complications. The most frequent antigenic target of antiphospholipid antibodies are phospholipid bound β2-glycoprotein 1 (β2GPI and prothrombin. The international classification criteria for APS connect the occurrence of thrombosis and/or obstetric complications together with the persistence of lupus anticoagulant, anti-cardiolipin antibodies (aCL and antibodies against β2GPI (anti-β2GPI into APS. Current trends for the diagnostic evaluation of APS patients propose determination of multiple antiphospholipid antibodies, among them also anti-prothrombin antibodies, to gain a common score which estimates the risk for thrombosis in APS patients. Antiprothrombin antibodies are common in APS patients and are sometimes the only antiphospholipid antibodies being elevated. Methods for their determination differ and have not yet been standardized. Many novel studies confirmed method using phosphatidylserine/prothrombin (aPS/PT ELISA as an antigen on solid phase encompass higher diagnostic accuracy compared to method using prothrombin alone (aPT ELISA. Our research group developed an in-house aPS/PT ELISA with increased analytical sensitivity which enables the determination of all clinically relevant antiprothrombin antibodies. aPS/PT exhibited the highest percentage of lupus anticoagulant activity compared to aCL and anti-β2GPI. aPS/PT antibodies measured with the in-house method associated with venous thrombosis and presented the strongest independent risk factor for the presence of obstetric complications among all tested antiphospholipid antibodies

  14. The therapeutic potential of plant-derived vaccines and antibodies.

    Science.gov (United States)

    Rodgers, P B; Hamilton, W D; Adair, J R

    1999-03-01

    The production of recombinant proteins in plants is reviewed with a particular focus on plant-derived vaccines and antibodies for human healthcare. Issues relating to foreign gene expression, such as protein yield, localisation and glycosylation are also considered. Emphasis is placed on reporting progress with preclinical and clinical evaluation of plant-derived vaccines and antibodies. An assessment is made of the likely future direction of research and development in this area.

  15. Should we ignore western blots when selecting antibodies for other applications?

    DEFF Research Database (Denmark)

    Uhlén, Mathias

    2017-01-01

    .In the Human Protein Atlas (HPA) program, we have validated more than 24,000 in-house-generated antibodies directed to 17,000 human target proteins2. Although there is often a correlation between performance in different applications, we have observed many examples of antibodies that show strong support...... applications and that this influences the epitopes exposed on the target protein, which might have profound consequences for the ability of a given antibody to bind specifically to its target. As an example, proteins that are analyzed by immunohistochemistry (IHC) are normally first cross-linked with formalin.......In conclusion, western blot and protein array analyses can indeed be useful tools when selecting specific antibodies for other applications. The use of these methods is encouraged both for antibody providers and users, and antibodies with signs of cross-reactivity in these applications should be treated...

  16. Anti‑livin antibodies in Hashimoto thyroiditis.

    Science.gov (United States)

    Baumann-Antczak, Aleksandra; Kosowicz, Jerzy; Zamysłowska, Hanna; Ruchała, Marek

    2012-01-01

    Livin belongs to the family of apoptosis inhibitors. High livin expression is observed in malignancies of the gastrointestinal tract, lungs, breast, and kidneys, but it is not present in differentiated adult tissues. In some malignant processes, anti‑livin antibodies are present. The aim of the study was to evaluate the prevalence of anti‑livin antibodies in Hashimoto thyroiditis, a disease characterized by rapid and widespread thyrocyte apoptosis. The study comprised 65 women with Hashimoto thyroiditis and the control group of 40 healthy women. In the majority of the patients, clinical manifestations of hypothyroidism were observed; all patients had high levels of serum antithyroid peroxidase antibodies. A solid‑phase radioimmunoassay in livin‑coated polyethylene tubes using 125I-labeled protein A was used to determine anti-livin antibodies. Significant amounts of anti-livin antibodies were reported in 18 patients (26.8%); 3 patients (4.6%) had borderline antibody levels; while in controls only 1 patient was positive (2.5%, P Hashimoto thyroiditis, an autoimmune process is more general and involves numerous autoantibodies including an antibody against apoptosis inhibitor - livin. Anti‑livin antibodies cannot serve only as a marker of malignancy because they are also present in autoimmune processes.

  17. Improved Lysosomal Trafficking Can Modulate the Potency of Antibody Drug Conjugates.

    Science.gov (United States)

    DeVay, Rachel M; Delaria, Kathy; Zhu, Guoyun; Holz, Charles; Foletti, Davide; Sutton, Janette; Bolton, Gary; Dushin, Russell; Bee, Christine; Pons, Jaume; Rajpal, Arvind; Liang, Hong; Shelton, David; Liu, Shu-Hui; Strop, Pavel

    2017-04-19

    Antibody drug conjugates (ADCs) provide an efficacious and relatively safe means by which chemotherapeutic agents can be specifically targeted to cancer cells. In addition to the selection of antibody targets, ADCs offer a modular design that allows selection of ADC characteristics through the choice of linker chemistries, toxins, and conjugation sites. Many studies have indicated that release of toxins bound to antibodies via noncleavable linker chemistries relies on the internalization and intracellular trafficking of the ADC. While this can make noncleavable ADCs more stable in the serum, it can also result in lower efficacy when their respective targets are not internalized efficiently or are recycled back to the cell surface following internalization. Here, we show that a lysosomally targeted ADC against the protein APLP2 mediates cell killing, both in vitro and in vivo, more effectively than an ADC against Trop2, a protein with less efficient lysosomal targeting. We also engineered a bispecific ADC with one arm targeting HER2 for the purpose of directing the ADC to tumors, and the other arm targeting APLP2, whose purpose is to direct the ADC to lysosomes for toxin release. This proof-of-concept bispecific ADC demonstrates that this technology can be used to shift the intracellular trafficking of a constitutively recycled target by directing one arm of the antibody against a lysosomally delivered protein. Our data also show limitations of this approach and potential future directions for development.

  18. Unique biological properties of catalytic domain directed human anti-CAIX antibodies discovered through phage-display technology.

    Directory of Open Access Journals (Sweden)

    Chen Xu

    2010-03-01

    Full Text Available Carbonic anhydrase IX (CAIX, gene G250/MN-encoded transmembrane protein is highly expressed in various human epithelial tumors such as renal clear cell carcinoma (RCC, but absent from the corresponding normal tissues. Besides the CA signal transduction activity, CAIX may serve as a biomarker in early stages of oncogenesis and also as a reliable marker of hypoxia, which is associated with tumor resistance to chemotherapy and radiotherapy. Although results from preclinical and clinical studies have shown CAIX as a promising target for detection and therapy for RCC, only a limited number of murine monoclonal antibodies (mAbs and one humanized mAb are available for clinical testing and development. In this study, paramagnetic proteoliposomes of CAIX (CAIX-PMPLs were constructed and used for anti-CAIX antibody selection from our 27 billion human single-chain antibody (scFv phage display libraries. A panel of thirteen human scFvs that specifically recognize CAIX expressed on cell surface was identified, epitope mapped primarily to the CA domain, and affinity-binding constants (KD determined. These human anti-CAIX mAbs are diverse in their functions including induction of surface CAIX internalization into endosomes and inhibition of the carbonic anhydrase activity, the latter being a unique feature that has not been previously reported for anti-CAIX antibodies. These human anti-CAIX antibodies are important reagents for development of new immunotherapies and diagnostic tools for RCC treatment as well as extending our knowledge on the basic structure-function relationships of the CAIX molecule.

  19. Uses of monoclonial antibody 8H9

    Science.gov (United States)

    Cheung, Nai-Kong V.

    2015-06-23

    This invention provides an antibody that binds the same antigen as that of monoclonal antibody 8H9, wherein the heavy chain CDR (Complementary Determining Region)1 comprises NYDIN, heavy chain CDR2 comprises WIFPGDGSTQY, heavy chain CDR3 comprises QTTATWFAY, and the light chain CDR1 comprises RASQSISDYLH, light chain CDR2 comprises YASQSIS, and light chain CDR3 comprises QNGHSFPLT. In another embodiment, there is provided a polypeptide that binds the same antigen as that of monoclonal antibody 8H9, wherein the polypeptide comprises NYDIN, WIFPGDGSTQY, QTTATWFAY, RASQSISDYLH, YASQSIS, and QNGHSFPLT.

  20. Development of Immunoassay Based on Monoclonal Antibody Reacted with the Neonicotinoid Insecticides Clothianidin and Dinotefuran

    Directory of Open Access Journals (Sweden)

    Seiji Iwasa

    2012-11-01

    Full Text Available Enzyme-linked immunosorbent assay (ELISA based on a monoclonal antibody (MoAb was developed for the neonicotinoid insecticide clothianidin. A new clothianidin hapten (3-[5-(3-methyl-2-nitroguanidinomethyl-1,3-thiazol-2-ylthio] propionic acid was synthesized and conjugated to keyhole limpet hemocyanin, and was used for monoclonal antibody preparation. The resulting MoAb CTN-16A3-13 was characterized by a direct competitive ELISA (dc-ELISA. The 50% of inhibition concentration value with clothianidin was 4.4 ng/mL, and the working range was 1.5–15 ng/mL. The antibody showed high cross-reactivity (64% to dinotefuran among the structurally related neonicotinoid insecticides. The recovery examinations of clothianidin for cucumber, tomato and apple showed highly agreement with the spiked concentrations; the recovery rate was between 104% and 124% and the coefficient of variation value was between 1.8% and 15%. Although the recovery rate of the dc-ELISA was slightly higher than that of HPLC analysis, the difference was small enough to accept the dc-ELISA as a useful method for residue analysis of clothianidin in garden crops.

  1. Development of immunoassay based on monoclonal antibody reacted with the neonicotinoid insecticides clothianidin and dinotefuran.

    Science.gov (United States)

    Uchigashima, Mikiko; Watanabe, Eiki; Ito, Shigekazu; Iwasa, Seiji; Miyake, Shiro

    2012-11-15

    Enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody (MoAb) was developed for the neonicotinoid insecticide clothianidin. A new clothianidin hapten (3-[5-(3-methyl-2-nitroguanidinomethyl)-1,3-thiazol-2-ylthio] propionic acid) was synthesized and conjugated to keyhole limpet hemocyanin, and was used for monoclonal antibody preparation. The resulting MoAb CTN-16A3-13 was characterized by a direct competitive ELISA (dc-ELISA). The 50% of inhibition concentration value with clothianidin was 4.4 ng/mL, and the working range was 1.5–15 ng/mL. The antibody showed high cross-reactivity (64%) to dinotefuran among the structurally related neonicotinoid insecticides. The recovery examinations of clothianidin for cucumber, tomato and apple showed highly agreement with the spiked concentrations; the recovery rate was between 104% and 124% and the coefficient of variation value was between 1.8% and 15%. Although the recovery rate of the dc-ELISA was slightly higher than that of HPLC analysis, the difference was small enough to accept the dc-ELISA as a useful method for residue analysis of clothianidin in garden crops.

  2. Study on quantification of HBs-antibody by immunoradiometric assay

    International Nuclear Information System (INIS)

    Kondo, Yuichi; Itoi, Yoshihiro; Kajiyama, Shizuo

    1989-01-01

    Quantification of HBs-antibody assay was carried out using a commercialized assay kit and standard solutions of HBs-antibody recognised as 1 st reference preparation of hepatitis B immunogloblin by WHO. Standard curve of HBs-antibody was drawn with the function of 3D-spline and the correlation factor was obtained as r = 0.999. Coefficient of intra-assay variance was 3.8 % and that of inter-assay variance was 7.8 %. Dilution tests showed satisfactory results in the range of 2-16 times. Correlation between value of cut-off indices and concentration of HBs-antibody was obtained as the formula of y = 2.599 x-3.894 (r = 0.992) and 2.1 of cut-off index corresponded to about 5 mIU/ml of HBs-antibody concentration. (author)

  3. The immunoglobulin class of anti-hapten antibody secreted during secondary responses in vitro and in vivo.

    Science.gov (United States)

    North, J R; Dresser, D W

    1977-05-01

    A comparison has been made of the in vitro and in vivo response of primed mouse spleen cells to the hapten DNP. The responses were analysed in terms of six classes (sub-classes) of humoral antibody directed against the cross-reacting hapten TNP. By comparison with the response in intact mice the adoptive secondary response is delayed by 3 days in addition to being somewhat lesser in magnitude. The timing of the response in vitro is similar to that observed in intact mice. The preponderant class in all three responses was gammaG1 with gammaA and gammaG3 secreting cells consistently comprising the smallest proportion of the total of antibody-secreting cells.

  4. Neonatal hyperbilirubinemia caused by anti-Jra antibodies - the first case report in Serbia

    Directory of Open Access Journals (Sweden)

    Radonjić Zorica

    2017-01-01

    Full Text Available Introduction. Jra is a high-frequency antigen belonging to the JR blood group system. Population studies have established that the Jr (a- phenotype is rare. The clinical significance of anti-Jra antibodies is controversial. This case report describes a newborn with prolonged jaundice due to alloimmunization against Jra antigen. Case Outline. A female Roma infant, 27 days of age, was admitted to hospital due to prolonged jaundice and failure to thrive. Immunohematological testing determined a blood group type A, D+ C+ E+ c+ e+, K-, and the presence of an antibody direct against a high-prevalence red blood cell antigens. On admission, total bilirubin was 199.6 μmol/l, direct bilirubin 10.3 μmol/l, hemoglobin concentration 132 g/l, hematocrit 41.1%, reticulocytes 1.08%. The newborn was the third child from a third routinely monitored pregnancy. Maternal sensitization to Jra antigen was detected during the second pregnancy. The titer of anti-Jra reached the highest value of 1,024 at the 28th week of gestation. Conclusion. This is the first description of neonatal hyperbilirubinemia caused by anti-Jra antibody in the Republic of Serbia. This case report provides new data about the clinical significance of anti-Jra in pregnancy and the newborn.

  5. A case of proteinase 3 anti-neutrophil cytoplasmic antibody (PR3-ANCA positive/IgG4-related lung disease

    Directory of Open Access Journals (Sweden)

    Hirokazu Touge

    2017-01-01

    Full Text Available IgG4-related lung disease (IgG4-RLD is a rare and chronic progressive autoimmune disease. We report a case of IgG4-related inflammatory pseudo-tumor of the lung that was seropositive for proteinase 3 anti-neutrophil cytoplasmic antibody (PR3-ANCA. A 61-year-old male had a mass lesion in the right lower lung field in chest X-ray. Transbronchial lung biopsy resulted in a pathological diagnosis of IgG4-RLD. The condition was improved by hormonal therapy.

  6. Comparison of multiple assays for detecting human antibodies directed against antigens on normal and malignant tissue culture cells

    International Nuclear Information System (INIS)

    Rosenberg, S.A.; Schwarz, S.; Anding, H.; Hyatt, C.; Williams, G.M.; Johns Hopkins Univ., Baltimore, Md.

    1977-01-01

    Four separate assays of human antibody reactivity to four separate normal and malignant human tissue culture cells lines from two patients have been evaluated using a single highly-reactive allogeneic serum. The visual end-point cytolysis assay and the chromium-51 release assay were equally sensitive in measuring complement mediated antibody cytotoxicity and both were far more sensitive than a trypan blue dye exclusion assay. The assay of antibody reactivity by hemadsorption technique was about 10 times more sensitive than any of the cytotoxicity assays. This latter assay measures only IgG antibody however. These assays showed that cell lines from different patients may differ greatly in 'reactivity' to an allogeneic serum and emphasized the importance of utilizing tumor and normal cells from the same patient when using tissue culture cells to search for tumor specific reactivity. These observations emphasize the importance of utilizing multiple assays against paired normal and malignant cells from the same patient to be certain of the specificity and magnitude of the measured antibody

  7. Human antibody technology and the development of antibodies against cytomegalovirus.

    Science.gov (United States)

    Ohlin, Mats; Söderberg-Nauclér, Cecilia

    2015-10-01

    Cytomegalovirus (CMV) is a virus that causes chronic infections in a large set of the population. It may cause severe disease in immunocompromised individuals, is linked to immunosenescence and implied to play an important role in the pathogenesis of cardiovascular diseases and cancer. Modulation of the immune system's abilities to manage the virus represent a highly viable therapeutic option and passive immunotherapy with polyclonal antibody preparations is already in clinical use. Defined monoclonal antibodies offer many advantages over polyclonal antibodies purified from serum. Human CMV-specific monoclonal antibodies have consequently been thoroughly investigated with respect to their potential in the treatment of diseases caused by CMV. Recent advances in human antibody technology have substantially expanded the breadth of antibodies for such applications. This review summarizes the fundamental basis for treating CMV disease by use of antibodies, the basic technologies to be used to develop such antibodies, and relevant human antibody specificities available to target this virus. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Radiobromination of humanized anti-HER2 monoclonal antibody trastuzumab using N-succinimidyl 5-bromo-3-pyridinecarboxylate, a potential label for immunoPET

    Energy Technology Data Exchange (ETDEWEB)

    Mume, Eskender [Organic Chemistry, Department of Chemistry, Uppsala University, S-751 24 Uppsala (Sweden); Orlova, Anna [Affibody AB, S-161 02 Bromma (Sweden); Malmstroem, Per-Uno [Division of Urology, Department of Surgical Sciences, Uppsala University, S-751 85 Uppsala (Sweden); Lundqvist, Hans [Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, S-751 85 Uppsala (Sweden); Sjoeberg, Stefan [Organic Chemistry, Department of Chemistry, Uppsala University, S-751 24 Uppsala (Sweden); Tolmachev, Vladimir [Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, S-751 85 Uppsala (Sweden)]. E-mail: vladimir.tolmachev@bms.uu.se

    2005-08-01

    Combining the specificity of radioimmunoscintigraphy and the high sensitivity of PET in an in vivo detection technique could improve the quality of nuclear diagnostics. Positron-emitting nuclide {sup 76}Br (T {sub 1/2}=16.2 h) might be a possible candidate for labeling monoclonal antibodies (mAbs) and their fragments, provided that the appropriate labeling chemistry has been established. For internalizing antibodies, such as the humanized anti-HER2 monoclonal antibody, trastuzumab, radiobromine label should be residualizing, i.e., ensuring that radiocatabolites are trapped intracellularly after the proteolytic degradation of antibody. This study evaluated the chemistry of indirect radiobromination of trastuzumab using N-succinimidyl 5-(tributylstannyl)-3-pyridinecarboxylate. Literature data indicated that the use of this method provided residualizing properties for iodine and astatine labels on some antibodies. An optimized 'one-pot' procedure produced an overall labeling efficiency of 45.5{+-}1.2% over 15 min. The bromine label was stable under physiological and denaturing conditions. The labeled trastuzumab retained its capacity to bind specifically to HER2-expressing SKOV-3 ovarian carcinoma cells in vitro (immunoreactivity more than 75%). However, in vitro cell test did not demonstrate that the radiobromination of trastuzumab using N-succinimidyl 5-bromo-3-pyridinecarboxylate improves cellular retention of radioactivity in comparison with the use of N-succinimidyl 4-bromobenzoate.

  9. Antiphospholipid antibodies in Brazilian hepatitis C virus carriers

    Directory of Open Access Journals (Sweden)

    A.M. Atta

    2008-06-01

    Full Text Available Hepatitis C, a worldwide viral infection, is an important health problem in Brazil. The virus causes chronic infection, provoking B lymphocyte dysfunction, as represented by cryoglobulinemia, non-organ-specific autoantibody production, and non-Hodgkin's lymphoma. The aim of this research was to screen for the presence of antiphospholipid autoantibodies in 109 Brazilian hepatitis C virus carriers without clinical history of antiphospholipid syndrome. Forty healthy individuals were used as the control group. IgA, IgG, and IgM antibodies against cardiolipin and β2-glycoprotein I were measured with an enzyme-linked immunosorbent assay, using a cut-off point of either 20 UPL or 20 SBU. While 24 (22.0% hepatitis C carriers had moderate titers of IgM anticardiolipin antibodies (median, 22.5 MPL; 95%CI: 21.5-25.4 MPL, only three carriers (<3% had IgG anticardiolipin antibodies (median, 23 GPL; 95%CI: 20.5-25.5 GPL. Furthermore, IgA anticardiolipin antibodies were not detected in these individuals. Male gender and IgM anticardiolipin seropositivity were associated in the hepatitis C group (P = 0.0004. IgA anti-β2-glycoprotein-I antibodies were detected in 29 of 109 (27.0% hepatitis C carriers (median, 41 SAU; 95%CI: 52.7-103.9 SAU. Twenty patients (18.0% had IgM anti-β2-glycoprotein I antibodies (median, 27.6 SMU; 95%CI: 23.3-70.3 SMU, while two patients had IgG antibodies against this protein (titers, 33 and 78 SGU. Antiphospholipid antibodies were detected in only one healthy individual, who was seropositive for IgM anticardiolipin. We concluded that Brazilian individuals chronically infected with hepatitis C virus present a significant production of antiphospholipid antibodies, mainly IgA anti-β2-glycoprotein I antibodies, which are not associated with clinical manifestations of antiphospholipid syndrome.

  10. Antimitochondrial antibody

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/003529.htm Antimitochondrial antibody To use the sharing features on this page, please enable JavaScript. Antimitochondrial antibodies (AMA) are substances ( antibodies ) that form against mitochondria. ...

  11. Purification of immunoreactive radiolabeled moniclonal antibodies with anti-iodiotypic moniclonal antibodies

    International Nuclear Information System (INIS)

    Temponi, M.; Pupa, S.; Ferrone, S.

    1990-01-01

    A method is described to purify immunoreactive moniclonal antibodies from radiolabeled monoclonal antibody preparations. The method is based on incubation of radiolabeled monoclonal antibodies with insolubilized anti-idiotypic monoclonal antibodies to idiotopes within the antigen-combining site of monoclonal antibodies to be purified an elution of bound monoclonal antibodies with a low pH buffer. The immunoreactive fraction of the purified monoclonal antibodies was at least 82%; the yeald was at least 73%. The purification procedure did not cause any detectable change in the affinity constant of the eluted monoclonal antibodies. The method is simple and rapid; the requirement for anti-idiotypic monoclonal antibodies to idiotopes within the antigen-combining site of the antibodies to be purified is not likely to represent a major limitation in the broad application of the present method, since the hybridoma technology has greatly facilitated the development of anti-idiotypic monoclonal antibodies. (author). 12 refs.; 4 figs.; 1 tab

  12. Ovarian carcinoma glyco-antigen targeted by human IgM antibody.

    Directory of Open Access Journals (Sweden)

    Yi Chen

    Full Text Available Epithelial Ovarian Cancer (EOC cells expression of a novel carbohydrate antigen was defined using a human VH4-34 encoded IgM monoclonal antibody (mAb216. MAb216 binds to a poly N-acetyllactosamine epitope expressed on B cells and kills normal and malignant B cells in vitro and in vivo. EOC patient ascites and EOC cell lines were used to study the anti tumor effect of mAb216. Various assays were used to characterize the epitope and demonstrate antibody-mediated binding and cytotoxicity in EOC. Drug and antibody combination effects were determined by calculating the combination index values using the Chou and Talalay method. MAb216 displays direct antibody mediated cytotoxicity on a population of human EOC tumor and ascites samples and EOC cell lines, which express high amounts of poly N-acetyllactosamine epitope, carried by CD147/CD98. Eighty four percent of patient samples, including platin resistant, had a tumor population that bound the monoclonal antibody. The binding pattern of mAb216 and mechanism of cytotoxicity was similar to that seen on normal and malignant B cells with unique general membrane disruption and "pore" formation. In vitro incubation with mAb216 and cisplatin enhanced killing of OVCAR3 cell line. In EOC cell lines percent cytotoxicity correlated with percent expression of epitope. Although in vitro data shows specific EOC cytotoxicity, for possible treatment of EOC MAb216 would need to be evaluated in a clinical trial with or without chemotherapy.

  13. Immunotherapy for the treatment of colorectal tumors: focus on approved and in-clinical-trial monoclonal antibodies

    Directory of Open Access Journals (Sweden)

    Françoso A

    2017-01-01

    Full Text Available Alex Françoso,1 Patricia Ucelli Simioni1–3 1Department of Biomedical Science, Faculty of Americana, Americana, 2Department of Genetics, Evolution and Bioagents, Institute of Biology, University of Campinas, Campinas, 3Department of Biochemistry and Microbiology, Institute of Biosciences, Universidade Estadual Paulista, Rio Claro, São Paulo, Brazil Abstract: Colorectal cancer is considered a disease of the elderly population. Since the number of geriatric patients continues to rise, monoclonal antibody therapy is the most promising therapy in the recent research. Presently, the monoclonal antibodies most frequently used in the treatment of colorectal tumors are bevacizumab, cetuximab, panitumumab, and ramucirumab. Bevacizumab is a monoclonal antibody that acts on VEGF. Cetuximab and panitumumab act on EGFR. Ramucirumab binds directly to the ligand-binding pocket of VEGFR-2 to block the binding of VEGF-A, VEGF-C, and VEGF-D. These monoclonal antibodies, alone or in association with radiotherapy or chemotherapy, are presenting good results and are increasing patient survival, despite the side effects. Due to the limited number of molecules available, several studies are trying to develop new monoclonal antibodies for the treatment of colorectal tumors. Among those being studied, some recent molecules are in phase I and/or II trials and are yielding advantageous results, such as anti-DR5, anti-Fn14, anti-IGF-1R, anti-EGFR, anti-NRP1, and anti-A33 antibodies. This has been successful in reducing side effects and in treating nonresponsive patients. Keywords: monoclonal antibodies, colorectal tumor, bevacizumab, cetuximab, panitumumab, ramucirumab

  14. Boronic acid-modified magnetic materials for antibody purification

    Science.gov (United States)

    Dhadge, Vijaykumar L.; Hussain, Abid; Azevedo, Ana M.; Aires-Barros, Raquel; Roque, Ana C. A.

    2014-01-01

    Aminophenyl boronic acids can form reversible covalent ester interactions with cis-diol-containing molecules, serving as a selective tool for binding glycoproteins as antibody molecules that possess oligosaccharides in both the Fv and Fc regions. In this study, amino phenyl boronic acid (APBA) magnetic particles (MPs) were applied for the magnetic separation of antibody molecules. Iron oxide MPs were firstly coated with dextran to avoid non-specific binding and then with 3-glycidyloxypropyl trimethoxysilane to allow further covalent coupling of APBA (APBA_MP). When contacted with pure protein solutions of human IgG (hIgG) and bovine serum albumin (BSA), APBA_MP bound 170 ± 10 mg hIgG g−1 MP and eluted 160 ± 5 mg hIgG g−1 MP, while binding only 15 ± 5 mg BSA g−1 MP. The affinity constant for the interaction between hIgG and APBA_MP was estimated as 4.9 × 105 M−1 (Ka) with a theoretical maximum capacity of 492 mg hIgG adsorbed g−1 MP (Qmax), whereas control particles bound a negligible amount of hIgG and presented an estimated theoretical maximum capacity of 3.1 mg hIgG adsorbed g−1 MP (Qmax). APBA_MPs were also tested for antibody purification directly from CHO cell supernatants. The particles were able to bind 98% of IgG loaded and to recover 95% of pure IgG (purity greater than 98%) at extremely mild conditions. PMID:24258155

  15. Antibody-Based Therapies in Multiple Myeloma

    Directory of Open Access Journals (Sweden)

    Yu-Tzu Tai

    2011-01-01

    Full Text Available The unmet need for improved multiple myeloma (MM therapy has stimulated clinical development of monoclonal antibodies (mAbs targeting either MM cells or cells of the bone marrow (BM microenvironment. In contrast to small-molecule inhibitors, therapeutic mAbs present the potential to specifically target tumor cells and directly induce an immune response to lyse tumor cells. Unique immune-effector mechanisms are only triggered by therapeutic mAbs but not by small molecule targeting agents. Although therapeutic murine mAbs or chimeric mAbs can cause immunogenicity, the advancement of genetic recombination for humanizing rodent mAbs has allowed large-scale production and designation of mAbs with better affinities, efficient selection, decreasing immunogenicity, and improved effector functions. These advancements of antibody engineering technologies have largely overcome the critical obstacle of antibody immunogenicity and enabled the development and subsequent Food and Drug Administration (FDA approval of therapeutic Abs for cancer and other diseases.

  16. Exploratory Study on Plasma Immunomodulator and Antibody Profiles in Tuberculosis Patients

    Science.gov (United States)

    Ravindran, Resmi; Krishnan, Viswanathan V.; Khanum, Azra; Luciw, Paul A.

    2013-01-01

    Host immune responses to Mycobacterium tuberculosis are generally able to contain infection and maintain a delicate balance between protection and immunopathology. A shift in this balance appears to underlie active disease observed in about 10% of infected individuals. Effects of local inflammation, combined with anti-M. tuberculosis systemic immune responses, are directly detectable in peripheral circulation, without ex vivo stimulation of blood cells or biopsy of the affected organs. We studied plasma immunomodulator and antibody biomarkers in patients with active pulmonary tuberculosis (TB) by a combination of multiplex microbead immunoassays and computational tools for data analysis. Plasma profiles of 10 immunomodulators and antibodies against eight M. tuberculosis antigens (previously reported by us) were examined in active pulmonary TB patients in a country where TB is endemic, Pakistan. Multiplex analyses were performed on samples from apparently healthy individuals without active TB from the same community as the TB patients to establish the assay baselines for all analytes. Over 3,000 data points were collected from patients (n = 135) and controls (n = 37). The data were analyzed by multivariate and computer-assisted cluster analyses to reveal patterns of plasma immunomodulators and antibodies. This study shows plasma profiles that in most patients represented either strong antibody or strong immunomodulator biomarkers. Profiling of a combination of both immunomodulators and antibodies described here may be valuable for the analysis of host immune responses in active TB in countries where the disease is endemic. PMID:23761664

  17. Limbic encephalitis and antibodies to Ma2: a paraneoplastic presentation of breast cancer.

    Science.gov (United States)

    Sutton, I; Winer, J; Rowlands, D; Dalmau, J

    2000-08-01

    A patient with atypical medullary breast cancer is described who presented with symptoms of limbic encephalitis. The patient's serum and CSF contained antibodies that reacted with the nervous system and the tumour. These antibodies recognised Ma2, a neuronal protein related to paraneoplastic limbic and brainstem encephalitis in men with testicular tumours. This report highlights the importance of testing for paraneoplastic antineuronal antibodies in cases of unexplained limbic encephalitis and suggests screening for breast cancer in women with antibodies predominantly directed to Ma2.

  18. Rhinovirus antibodies in an isolated Amazon Indian tribe.

    Science.gov (United States)

    Thwing, C J; Arruda, E; Vieira Filho, J P; Castelo Filho, A; Gwaltney, J M

    1993-06-01

    In early 1985, the Parakana-Apiterewa, a small, primitive Indian tribe, was contacted in the southern Amazon Basin. The tribe was thought to have been totally isolated from civilization until recent development of their land. Blood specimens were collected in 1985, shortly after the discovery of the tribe, and analyzed for the presence of rhinovirus-neutralizing antibody to nine different immunotypes. Six to forty-seven percent of the serum samples tested contained antibody to at least one immunotype of rhinovirus. The prevalence of rhinovirus antibody in the Parakana-Apiterewa Indians was similar to that reported in United States populations, suggesting that there had been considerable direct or indirect contact in the past between tribe members and persons in the outside world.

  19. Comparative study on antibody immobilization strategies for efficient circulating tumor cell capture.

    Science.gov (United States)

    Ates, Hatice Ceren; Ozgur, Ebru; Kulah, Haluk

    2018-03-23

    Methods for isolation and quantification of circulating tumor cells (CTCs) are attracting more attention every day, as the data for their unprecedented clinical utility continue to grow. However, the challenge is that CTCs are extremely rare (as low as 1 in a billion of blood cells) and a highly sensitive and specific technology is required to isolate CTCs from blood cells. Methods utilizing microfluidic systems for immunoaffinity-based CTC capture are preferred, especially when purity is the prime requirement. However, antibody immobilization strategy significantly affects the efficiency of such systems. In this study, two covalent and two bioaffinity antibody immobilization methods were assessed with respect to their CTC capture efficiency and selectivity, using an anti-epithelial cell adhesion molecule (EpCAM) as the capture antibody. Surface functionalization was realized on plain SiO 2 surfaces, as well as in microfluidic channels. Surfaces functionalized with different antibody immobilization methods are physically and chemically characterized at each step of functionalization. MCF-7 breast cancer and CCRF-CEM acute lymphoblastic leukemia cell lines were used as EpCAM positive and negative cell models, respectively, to assess CTC capture efficiency and selectivity. Comparisons reveal that bioaffinity based antibody immobilization involving streptavidin attachment with glutaraldehyde linker gave the highest cell capture efficiency. On the other hand, a covalent antibody immobilization method involving direct antibody binding by N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC)-N-hydroxysuccinimide (NHS) reaction was found to be more time and cost efficient with a similar cell capture efficiency. All methods provided very high selectivity for CTCs with EpCAM expression. It was also demonstrated that antibody immobilization via EDC-NHS reaction in a microfluidic channel leads to high capture efficiency and selectivity.

  20. A Recombinant Secondary Antibody Mimic as a Target-specific Signal Amplifier and an Antibody Immobilizer in Immunoassays.

    Science.gov (United States)

    Min, Junseon; Song, Eun Kyung; Kim, Hansol; Kim, Kyoung Taek; Park, Tae Joo; Kang, Sebyung

    2016-04-11

    We construct a novel recombinant secondary antibody mimic, GST-ABD, which can bind to the Fc regions of target-bound primary antibodies and acquire multiple HRPs simultaneously. We produce it in tenth of mg quantities with a bacterial overexpression system and simple purification procedures, significantly reducing the manufacturing cost and time without the use of animals. GST-ABD is effectively conjugated with 3 HRPs per molecule on an average and selectively bind to the Fc region of primary antibodies derived from three different species (mouse, rabbit, and rat). HRP-conjugated GST-ABD (HRP-GST-ABD) is successfully used as an alternative to secondary antibodies to amplify target-specific signals in both ELISA and immunohistochemistry regardless of the target molecules and origin of primary antibodies used. GST-ABD also successfully serves as an anchoring adaptor on the surface of GSH-coated plates for immobilizing antigen-capturing antibodies in an orientation-controlled manner for sandwich-type indirect ELISA through simple molecular recognition without any complicated chemical modification.

  1. Development and validation of an epitope-blocking ELISA using an anti-haemagglutinin monoclonal antibody for specific detection of antibodies in sheep and goat sera directed against peste des petits ruminants virus.

    Science.gov (United States)

    Bodjo, Sanne Charles; Baziki, Jean-de-Dieu; Nwankpa, Nick; Chitsungo, Ethel; Koffi, Yao Mathurin; Couacy-Hymann, Emmanuel; Diop, Mariame; Gizaw, Daniel; Tajelser, Idris Badri Adam; Lelenta, Mamadou; Diallo, Adama; Tounkara, Karim

    2018-07-01

    Peste des petits ruminants (PPR) is a contagious and economically important disease affecting production of small ruminants (i.e., sheep and goats). Taking into consideration the lessons learnt from the Global Rinderpest Eradication Programme (GREP), PPR is now targeted by the international veterinary community as the next animal disease to be eradicated. To support the African continental programme for the control of PPR, the Pan African Veterinary Vaccine Centre of the African Union (AU-PANVAC) is developing diagnostics tools. Here, we describe the development of a blocking enzyme-linked immunosorbent assay (bELISA) that allows testing of a large number of samples for specific detection of antibodies directed against PPR virus in sheep and goat sera. The PPR bELISA uses an anti-haemagglutinin (H) monoclonal antibody (MAb) as a competitor antibody, and tests results are interpreted using the percentage of inhibition (PI) of MAb binding generated by the serum sample. PI values below or equal to 18% (PI ≤ 18%) are negative, PI values greater than or equal to 25% (PI ≥ 25%) are positive, and PI values greater than 18% and below 25% are doubtful. The diagnostic specificity (DSp) and diagnostic sensitivity (DSe) were found to be 100% and 93.74%, respectively. The H-based PPR-bELISA showed good correlation with the virus neutralization test (VNT), the gold standard test, with a kappa value of 0.947. The H-based PPR-bELISA is more specific than the commercial kit ID Screen® PPR Competition (N-based PPR-cELISA) from IDvet (France), but the commercial kit is slightly more sensitive than the H-based PPR-bELISA. The validation process also indicated good repeatability and reproducibility of the H-based PPR-bELISA, making this new test a suitable tool for the surveillance and sero-monitoring of the vaccination campaign.

  2. Impact of Uniform Methods on Interlaboratory Antibody Titration Variability: Antibody Titration and Uniform Methods.

    Science.gov (United States)

    Bachegowda, Lohith S; Cheng, Yan H; Long, Thomas; Shaz, Beth H

    2017-01-01

    -Substantial variability between different antibody titration methods prompted development and introduction of uniform methods in 2008. -To determine whether uniform methods consistently decrease interlaboratory variation in proficiency testing. -Proficiency testing data for antibody titration between 2009 and 2013 were obtained from the College of American Pathologists. Each laboratory was supplied plasma and red cells to determine anti-A and anti-D antibody titers by their standard method: gel or tube by uniform or other methods at different testing phases (immediate spin and/or room temperature [anti-A], and/or anti-human globulin [AHG: anti-A and anti-D]) with different additives. Interlaboratory variations were compared by analyzing the distribution of titer results by method and phase. -A median of 574 and 1100 responses were reported for anti-A and anti-D antibody titers, respectively, during a 5-year period. The 3 most frequent (median) methods performed for anti-A antibody were uniform tube room temperature (147.5; range, 119-159), uniform tube AHG (143.5; range, 134-150), and other tube AHG (97; range, 82-116); for anti-D antibody, the methods were other tube (451; range, 431-465), uniform tube (404; range, 382-462), and uniform gel (137; range, 121-153). Of the larger reported methods, uniform gel AHG phase for anti-A and anti-D antibodies had the most participants with the same result (mode). For anti-A antibody, 0 of 8 (uniform versus other tube room temperature) and 1 of 8 (uniform versus other tube AHG), and for anti-D antibody, 0 of 8 (uniform versus other tube) and 0 of 8 (uniform versus other gel) proficiency tests showed significant titer variability reduction. -Uniform methods harmonize laboratory techniques but rarely reduce interlaboratory titer variance in comparison with other methods.

  3. Dengue-Immune Humans Have Higher Levels of Complement-Independent Enhancing Antibody than Complement-Dependent Neutralizing Antibody.

    Science.gov (United States)

    Yamanaka, Atsushi; Konishi, Eiji

    2017-09-25

    Dengue is the most important arboviral disease worldwide. We previously reported that most inhabitants of dengue-endemic countries who are naturally immune to the disease have infection-enhancing antibodies whose in vitro activity does not decrease in the presence of complement (complement-independent enhancing antibodies, or CiEAb). Here, we compared levels of CiEAb and complement-dependent neutralizing antibodies (CdNAb) in dengue-immune humans. A typical antibody dose-response pattern obtained in our assay system to measure the balance between neutralizing and enhancing antibodies showed both neutralizing and enhancing activities depending on serum dilution factor. The addition of complement to the assay system increased the activity of neutralizing antibodies at lower dilutions, indicating the presence of CdNAb. In contrast, similar dose-response curves were obtained with and without complement at higher dilutions, indicating higher levels of CiEAb than CdNAb. For experimental support for the higher CiEAb levels, a cocktail of mouse monoclonal antibodies against dengue virus type 1 was prepared. The antibody dose-response curves obtained in this assay, with or without complement, were similar to those obtained with human serum samples when a high proportion of D1-V-3H12 (an antibody exhibiting only enhancing activity and thus a model for CiEAb) was used in the cocktail. This study revealed higher-level induction of CiEAb than CdNAb in humans naturally infected with dengue viruses.

  4. Epitope mapping of monoclonal antibodies directed to aminopeptidase A and their relevance for albuminuria in mice.

    Science.gov (United States)

    Gerlofs-Nijland, Miriam E; Assmann, Karel J M; van Son, Jacco P H F; Dijkman, Henry B P M; te Loeke, Nathalie A J M; van der Zee, Ruurd; Wetzels, Jack F M; Groenen, Patricia J T A

    2003-01-01

    We have shown previously that injection of specific combinations of anti-aminopeptidase A monoclonal antibodies induces an acute massive albuminuria in mice. This albuminuria is neither dependent on systemic mediators of inflammation nor angiotensin II. In this study, we examined the contribution of two individual antibodies, the enzyme-inhibiting antibody ASD-37 and the non-enzyme-inhibiting antibody ASD-41, in the induction of albuminuria as well as the interactions between these two monoclonals. In addition, we have mapped the epitopes of both antibodies using in vitro coupled transcription/translation of specifically designed cDNA fragments followed by immunoprecipitation, and using peptide enzyme-linked immunosorbent assay in case of a continuous epitope. A single intravenous injection of 4 mg of either ASD-37 or ASD-41 did not induce albuminuria. This dose of ASD-37 did not completely inhibit enzyme activity. The combination of 4 mg ASD-37/41 (1:1 weight ratio) induced albuminuria and almost completely inhibited enzyme activity. Similar results were obtained with a combination of ASD-37/41 in a 1:39 or 39:1 weight ratio. Administration of 2 mg ASD-41 24 h before injection of 2 mg ASD-37 significantly enhanced albuminuria. The epitope of ASD-37 is located at the C-terminal end of aminopeptidase A, whereas the ASD-41 epitope is mapped near the enzyme active site. Our data suggest that ASD-41 modulates the binding of ASD-37 to its epitope and/or vice versa. As a consequence, ASD-37 and ASD-41 act synergistically, not only in inhibiting enzyme activity but also in inducing albuminuria. Copyright 2003 S. Karger AG, Basel

  5. Aligning physics and physiology: Engineering antibodies for radionuclide delivery.

    Science.gov (United States)

    Tsai, Wen-Ting K; Wu, Anna M

    2018-03-14

    The exquisite specificity of antibodies and antibody fragments renders them excellent agents for targeted delivery of radionuclides. Radiolabeled antibodies and fragments have been successfully used for molecular imaging and radioimmunotherapy (RIT) of cell surface targets in oncology and immunology. Protein engineering has been used for antibody humanization essential for clinical applications, as well as optimization of important characteristics including pharmacokinetics, biodistribution, and clearance. Although intact antibodies have high potential as imaging and therapeutic agents, challenges include long circulation time in blood, which leads to later imaging time points post-injection and higher blood absorbed dose that may be disadvantageous for RIT. Using engineered fragments may address these challenges, as size reduction and removal of Fc function decreases serum half-life. Radiolabeled fragments and pretargeting strategies can result in high contrast images within hours to days, and a reduction of RIT toxicity in normal tissues. Additionally, fragments can be engineered to direct hepatic or renal clearance, which may be chosen based on the application and disease setting. This review discusses aligning the physical properties of radionuclides (positron, gamma, beta, alpha, and Auger emitters) with antibodies and fragments and highlights recent advances of engineered antibodies and fragments in preclinical and clinical development for imaging and therapy. Copyright © 2018 John Wiley & Sons, Ltd.

  6. Use of polyclonal/monoclonal antibody therapies in transplantation.

    Science.gov (United States)

    Yeung, Melissa Y; Gabardi, Steven; Sayegh, Mohamed H

    2017-03-01

    For over thirty years, antibody (mAb)-based therapies have been a standard component of transplant immunosuppression, and yet much remains to be learned in order for us to truly harness their therapeutic capabilities. Current mAbs used in transplant directly target and destroy graft-destructive immune cells, interrupt cytokine and costimulation-dependent T and B cell activation, and prevent down-stream complement activation. Areas covered: This review summarizes our current approaches to using antibody-based therapies to prevent and treat allograft rejection. It also provides examples of promising novel mAb therapies, and discusses the potential for future mAb development in transplantation. Expert opinion: The broad capability of antibodies, in parallel with our growing ability to synthetically modulate them, offers exciting opportunities to develop better biologic therapeutics. In order to do so, we must further our understanding about the basic biology underlying allograft rejection, and gain better appreciation of how characteristics of therapeutic antibodies affect their efficacy.

  7. Ibrutinib interferes with the cell-mediated anti-tumor activities of therapeutic CD20 antibodies: implications for combination therapy

    DEFF Research Database (Denmark)

    Da Roit, F.; Engelberts, P. J.; Taylor, R. P.

    2015-01-01

    The novel Bruton tyrosine kinase inhibitor ibrutinib and phosphatidyl-4-5-biphosphate 3-kinase-delta inhibitor idelalisib are promising drugs for the treatment of chronic lymphocytic leukemia and B-cell non-Hodgkin lymphoma, either alone or in combination with anti-CD20 antibodies. We investigated...... the possible positive or negative impact of these drugs on all known mechanisms of action of both type I and type II anti-CD20 antibodies. Pretreatment with ibrutinib for 1 hour did not increase direct cell death of cell lines or chronic lymphocytic leukemia samples mediated by anti-CD20 antibodies. Pre......-treatment with ibrutinib did not inhibit complement activation or complement-mediated lysis. In contrast, ibrutinib strongly inhibited all cell-mediated mechanisms induced by anti-CD20 antibodies rituximab, ofatumumab or obinutuzumab, either in purified systems or whole blood assays. Activation of natural killer cells...

  8. Anti-Nuclear antibodies: Current concepts and future direction for diagnosing connective tissue disease

    Directory of Open Access Journals (Sweden)

    K Gautam

    2015-03-01

    Full Text Available Identification of antinuclear antibodies has been used for the diagnosis of connective tissue diseases for more than fifty years. Indirect immunofluorescence on human epithelial (HEp-2 cells is considered the gold standard screening method for the detection of antinuclear autoantibodies. As the demand of ANA testing increased, the need for automation and standardization has also come forth. A high level of false positive and false negative cases is seen in various populations making it difficult to take clinical decisions. Newer technologies were introduced for the antibody detection to ensure high sensitivity and specificity. This article intends to provide an overview of the concepts on ANA testing, the different diagnostic methods available, the various patterns and clinical utility, the clinical guidelines to be followed, the drawbacks and what lies ahead in the future of ANA testing.Journal of Pathology of Nepal (2015 Vol. 5, 766-773

  9. 9th annual European Antibody Congress, November 11-13, 2013, Geneva, Switzerland.

    Science.gov (United States)

    Reichert, Janice M; Beck, Alain; Lugovskoy, Alexey A; Wurch, Thierry; Coats, Steven; Brezski, Randall J

    2014-01-01

    The annual European Antibody Congress (EAC) has traditionally been the key event for updates on critical scientific advances in the antibody field, and 2013 was no exception. Organized by Terrapinn, the well-attended meeting featured presentations on considerations for developing antibodies and antibody-like therapeutics, with separate tracks for antibody-drug conjugates, naked antibodies, and multispecific antibodies or protein scaffolds. The overall focus of the EAC was current approaches to enhance the functionality of therapeutic antibodies or other targeted proteins, with the ultimate goal being improvement of the safety and efficacy of the molecules as treatments for cancer, immune-mediated disorders and other diseases. Roundtable discussion sessions gave participants opportunities to engage in group discussions with industry leaders from companies such as Genmab, Glenmark Pharmaceuticals, MedImmune, Merrimack Pharmaceuticals, and Pierre Fabre. As the 2013 EAC was co-located with the World Biosimilar Congress, participants also received an update on European Medicines Agency guidelines and thoughts on the future direction and development of biosimilar antibodies in the European Union.

  10. A malaria vaccine that elicits in humans antibodies able to kill Plasmodium falciparum.

    Directory of Open Access Journals (Sweden)

    Pierre Druilhe

    2005-11-01

    Full Text Available Plasmodium falciparum merozoite surface protein 3 is a malaria vaccine candidate that was identified, characterised, and developed based on a unique immuno-clinical approach. The vaccine construct was derived from regions fully conserved among various strains and containing B cell epitopes targeted by human antibodies (from malaria-immune adults that are able to mediate a monocyte-dependent parasite killing effect. The corresponding long synthetic peptide was administered to 36 volunteers, with either alum or Montanide ISA720 as adjuvant.Both formulations induced cellular and humoral immune responses. With alum, the responses lasted up to 12 mo. The vaccine-induced antibodies were predominantly of cytophilic classes, i.e., able to cooperate with effector cells. In vitro, the antibodies induced an inhibition of the P. falciparum erythrocytic growth in a monocyte-dependent manner, which was in most instances as high as or greater than that induced by natural antibodies from immune African adults. In vivo transfer of the volunteers' sera into P. falciparum-infected humanized SCID mice profoundly reduced or abrogated parasitaemia. These inhibitory effects were related to the antibody reactivity with the parasite native protein, which was seen in 60% of the volunteers, and remained in samples taken 12 mo postimmunisation.This is the first malaria vaccine clinical trial to clearly demonstrate antiparasitic activity by vaccine-induced antibodies by both in vitro and in vivo methods. The results, showing the induction of long-lasting antibodies directed to a fully conserved polypeptide, also challenge current concepts about malaria vaccines, such as unavoidable polymorphism, low antigenicity, and poor induction of immune memory.

  11. Label-free Fab and Fc affinity/avidity profiling of the antibody complex half-life for polyclonal and monoclonal efficacy screening.

    Science.gov (United States)

    Read, Thomas; Olkhov, Rouslan V; Williamson, E Diane; Shaw, Andrew M

    2015-09-01

    A unified approach to affinity screening for Fab and Fc interactions of an antibody for its antigen and FcγR receptor has been developed. An antigen array is used for the Fab affinity and cross-reactivity screening and protein A/G proxy is the FcγR receptor. The affinities are derived using a simple 1:1 binding model with a consistent error analysis. The association and dissociation kinetics are measured over optimised times for accurate determination. The Fab/Fc affinities are derived for ten antibodies: mAb-actin (mouse), pAb-BSA (sheep), pAb-collagen V (rabbit), pAb-CRP (goat), mAb-F1 (mouse), mAbs (mouse) 7.3, 12.3, 29.3, 36.3 and 46.3 raised against LcrV in Yersinia pestis. The rate of the dissociation of antigen-antibody complexes relates directly to their immunological function as does the Fc-FcγR complex and a new half-life plot has been defined with a Fab/Fc half-life range of 17-470 min. The upper half-life value points to surface avidity. Two antibodies that are protective as an immunotherapy define a Fab half-life >250 min and an Fc half-life >50 min as characteristics of ideal interactions which can form the basis of an antibody screen for immunotherapy.

  12. Immobilization of antibodies and enzyme-labeled antibodies by radiation polymerization

    International Nuclear Information System (INIS)

    Kumakura, M.; Kaetsu, I.; Suzuki, M.; Adachi, S.

    1983-01-01

    Immobilization of antibodies and enzyme-labeled antibodies by radiation polymerization at low temperatures was studied. The antibody activity of antibody was not affected by irradiation at an irradiation dose of below 8 MR and low temperatures. Immobilization of peroxidase-labeled anti-rabbit IgG goat IgG, anti-peroxidase, peroxidase, and anti-alpha-fetoprotein was carried out with hydrophilic and hydrophobic monomers. The activity of the immobilized enzyme-labeled antibody membranes varied with the thickness of the membranes and increased with decreasing membrane thickness. The activity of the immobilized antibody particles was varied by particle size. Immobilized anti-alpha-fetoprotein particles and membranes can be used for the assay of alpha-fetoprotein by the antigen-antibody reaction, such as a solid-phase sandwich method with high sensitivity

  13. Antibody guided diagnosis and therapy of brain gliomas using radiolabeled monoclonal antibodies against epidermal growth factor receptor and placental alkaline phosphatase

    International Nuclear Information System (INIS)

    Kalofonos, H.P.; Pawlikowska, T.R.; Hemingway, A.

    1989-01-01

    Twenty-seven patients with brain glioma were scanned using 123 I-labeled monoclonal antibodies against epidermal growth factor receptor (EGFR1) or placental alkaline phosphatase (H17E2). Successful localization was achieved in 18 out of 27 patients. Eleven out of 27 patients were also studied using a nonspecific control antibody (11.4.1) of the same immunoglobulin subclass and observable tumor localization was also achieved in five patients. The specificity of targeting was assessed by comparing images obtained with specific and nonspecific antibodies and by examining tumor and normal tissue biopsies after dual antibody administration. Ten patients with recurrent grade III or IV glioma who showed good localization of radiolabeled antibody were treated with 40-140 mCi of 131 I-labeled antibody delivered to the tumor area intravenously (n = 5) or by infusion into the internal carotid artery (n = 5). Six patients showed clinical improvement lasting from 6 mo to 3 yr. One patient continues in remission (3 yr after therapy), but the other five who responded initially relapsed 6-9 mo after therapy and died. No major toxicity was attributable to antibody-guided irradiation. Targeted irradiation by monoclonal antibody may be clinically useful and should be explored further in the treatment of brain gliomas resistant to conventional forms of treatment

  14. Omega-3 fiskeolie

    DEFF Research Database (Denmark)

    Lunde, Anita; Sørensen, Jan

    2009-01-01

    Rapport afgrænser sig til evidensbaserede helbredsmæssige gevinster ved et øget indtag af langkædede omega-3, som opnås ved en kost rig på fisk eller som et tilskud af fiskeolier. Der gennemføres en systematisk litteraturgennemgang, som baserer sig på et evidensniveau svarende til styrke A. Det...... betyder, at gennemgangen inkluderer metaanalyser/oversigtsartikler af enten eksperimentelle studier eller observationsstudier, endvidere indgår udvalgte større RCT, som er refereret i meta-analyserne. Sammenfattende findes på baggrund af litteraturgennemgang, at tilskud af omega-3 har effekt på...... hjertesygdom ved at nedsætte mortaliteten. Effekten er mest evident ved personer i særlig risiko for at udvikle hjerte-karsygdom, eller som sekundær/tertiær profylakse. Tilsvarende findes også ved tilskud af omega-3 en forebyggende effekt i forhold til iskæmisk apopleksi. Af mulige virkningsmekanismer viser...

  15. Ocaratuzumab, an Fc-engineered antibody demonstrates enhanced antibody-dependent cell-mediated cytotoxicity in chronic lymphocytic leukemia.

    Science.gov (United States)

    Cheney, Carolyn M; Stephens, Deborah M; Mo, Xiaokui; Rafiq, Sarwish; Butchar, Jonathan; Flynn, Joseph M; Jones, Jeffrey A; Maddocks, Kami; O'Reilly, Adrienne; Ramachandran, Abhijit; Tridandapani, Susheela; Muthusamy, Natarajan; Byrd, John C

    2014-01-01

    Chronic lymphocytic leukemia (CLL) is common in both developed and developing nations where the need for inexpensive and convenient administration of therapy is apparent. Ocaratuzumab is a novel Fc-engineered humanized IgG1 anti-CD20 monoclonal antibody (mAb) designed for effective antibody-dependent cell-mediated cytotoxicity (ADCC) at very low concentrations that may facilitate sub-cutaneous (vs. intravenous) dosing. Here, we report ocaratuzumab's potency against CLL cells. In vitro assessment of ocaratuzumab's direct cytotoxicity (DC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), and ADCC was performed on CLL cells. Ocaratuzumab induced DC, CDC, and ADCP similarly to rituximab or ofatumumab (anti-CD20 mAbs). However, ocaratuzumab showed an advantage in NK cell-mediated ADCC over these antibodies. In allogeneic ADCC, [E:T (effector:target) ratios = 25:1, 12:1, 6:1], ocaratuzumab (10 µg/mL) improved ADCC by ~3-fold compared with rituximab or ofatumumab (P<0.001 all tested E:T ratios). Notably, the superiority of ocaratuzumab-induced ADCC was observed at low concentrations (0.1-10 ug/ml; P<0.03; allogeneic assays). In extended allogeneic ADCC E:T titration, ocaratuzumab (0.1 µg/mL) demonstrated 19.4% more cytotoxicity than rituximab (E:T = 0.38:1; P = 0.0066) and 21.5% more cytotoxicity than ofatumumab (E:T = 1.5:1; P = 0.0015). In autologous ADCC, ocaratuzumab (10 µg/mL) demonstrated ~1.5-fold increase in cytotoxicity compared with rituximab or ofatumumab at all E:T ratios tested (E:Ts = 25:1,12:1,6:1; all P<0.001). Obinutuzumab, a glyco-engineered anti-CD20 mAb, showed no improvement in ADCC activity compared with ocaratuzumab. The enhanced ADCC of ocaratuzumab suggests that it may be effective at low concentrations. If supported by clinical investigation, this feature could potentially allow for subcutaneous dosing at low doses that could expand the potential of administering chemoimmunotherapy in developing

  16. Suppression of the immune response to ovalbumin in vivo by anti-idiotypic antibodies

    International Nuclear Information System (INIS)

    Grinevich, A.S.; Pinegin, B.V.

    1986-01-01

    Conditions of suppression of the immune response to a food allergin (ovalbumin) were studied with the aid of anti-idiotypic (AID) antibodies. Hen ovalbumin was used and the experiments were performed on mice. Antibodies were isolated from the resulting protein fractions and tested for inhibitor activity by the method of direct radioimmunologic analysis. The test system consisted of the reaction of binding the globulin fraction to the total preparation of antibodies to ovalbumin from mice and a 125 I-labeled total preparation of antibodies to ovalbumin of the same animals

  17. Anti-idiotypic antibodies directed against anti-HBs among the patients with chronic hepatitis B.

    Science.gov (United States)

    Kobayashi, K; Suzuki, H; Ueno, Y; Nagatomi, R; Kanno, A; Otsuki, M; Toyota, T

    1990-08-01

    Anti-idiotypic antibodies (anti-Id) against anti-HBs were found in the sera of patients with chronic hepatitis type B. Anti-idiotypic antibodies were detected by an enzyme-linked immunosorbent assay using horseradish peroxidase conjugated mouse monoclonal anti-HBs. Ten of 72 HBsAg positive sera contained anti-Id (13.9%). The prevalence of anti-Id did not appear to correlate with HBeAg/anti-HBe system. However, HB virus specific DNA polymerase activity was significantly higher in anti-Id positive sera. In the sera obtained from the patients treated with predonisolone before, anti-Id positive rate was higher than that in the patients without a history of predonisolone therapy. These results suggest that anti-Id may be related to the immunoregulatory mechanism of HB virus replication.

  18. Hematopoietic stem cell capture and directional differentiation into vascular endothelial cells for metal stent-coated chitosan/hyaluronic acid loading CD133 antibody.

    Science.gov (United States)

    Zhang, Shixuan; Zhang, Fan; Feng, Bo; Fan, Qingyu; Yang, Feng; Shang, Debin; Sui, Jinghan; Zhao, Hong

    2015-03-01

    A series of metal stents coated with chitosan/hyaluronic acid (CS/HA) loading antibodies by electrostatic self-assembled method were prepared, and the types of cells captured by antibodies and their differentiation in vascular endothelial cells (ECs) evaluated by molecular biology and scanning electron microscope. The results showed that CD133 stent can selectively capture hematopoietic stem cells (HSC),which directionally differentiate into vascular ECs in peripheral blood by (CS/HA) induction, and simultaneously inhibit migration and proliferation of immune cells and vascular smooth muscle cells (MCs). CD34 stent can capture HSC, hematopoietic progenitor cells that differentiate into vascular ECs and immune cells, promoting smooth MCs growth, leading to thrombosis, inflammation, and rejection. CD133 stent can be implanted into miniature pig heart coronary and can repair vascular damage by capturing own HSC, thus contributing to the rapid natural vascular repair, avoiding inflammation and rejection, thrombosis and restenosis. These studies demonstrated that CD133 stent of HSC capture will be an ideal coated metal stent providing a new therapeutic approach for cardiovascular and cerebrovascular disease.

  19. Facilitation of syngeneic stem cell engraftment by anti-class I monoclonal antibody pretreatment of unirradiated recipients

    International Nuclear Information System (INIS)

    Voralia, M.; Semeluk, A.; Wegmann, T.G.

    1987-01-01

    We have established a murine model of syngeneic bone marrow transplantation based on the use of monoclonal antibody as the sole conditioning regimen in unirradiated recipients. Administration of a single injection of monoclonal antibody directed against major histocompatibility complex-encoded class I determinants facilitated permanent hemopoietic stem cell engraftment without any apparent side-effects. Whereas untreated hosts exhibited a maximal chimerism of 15% at donor cell doses of up to 12 X 10(7) bone marrow cells, pretreatment by 2 mg of anti-class I antibody one week prior to transplantation of 3 X 10(7) syngeneic bone marrow cells resulted in a mean donor representation of about 80%. The antibody can be given up to four weeks prior to transplantation, and the degree of donor engraftment observed is a function of the dose of antibody administered. The fact that specific antibody enhanced engraftment in two strain combinations indicates that antibody is the active agent in facilitating engraftment and that facilitation is not strain-restricted. Anti-class I antibodies of the IgG2a, but not IgG1, isotype are effective in promoting engraftment. Although the isotype requirement suggests a role for antibody-mediated cytotoxicity in promoting stem cell engraftment, the extensive time-frame of facilitation suggests that other effects of the antibody may also be involved. The model of syngeneic bone marrow transplantation we describe here will be useful in studying the mechanisms regulating stem cell engraftment and may have potential clinical application as an approach to autologous marrow transplantation

  20. The in vivo mechanism of action of CD20 monoclonal antibodies depends on local tumor burden

    Science.gov (United States)

    Boross, Peter; Jansen, J.H. Marco; de Haij, Simone; Beurskens, Frank J.; van der Poel, Cees E.; Bevaart, Lisette; Nederend, Maaike; Golay, Josée; van de Winkel, Jan G.J.; Parren, Paul W.H.I.; Leusen, Jeanette H.W.

    2011-01-01

    Background CD20 monoclonal antibodies are widely used in clinical practice. Antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity and direct cell death have been suggested to be important effector functions for CD20 antibodies. However, their specific contributions to the in vivo mechanism of action of CD20 immunotherapy have not been well defined. Design and Methods Here we studied the in vivo mechanism of action of type I (rituximab and ofatumumab) and type II (HuMab-11B8) CD20 antibodies in a peritoneal, syngeneic, mouse model with EL4-CD20 cells using low and high tumor burden. Results Interestingly, we observed striking differences in the in vivo mechanism of action of CD20 antibodies dependent on tumor load. In conditions of low tumor burden, complement was sufficient for tumor killing both for type I and type II CD20 antibodies. In contrast, in conditions of high tumor burden, activating FcγR (specifically FcγRIII), active complement and complement receptor 3 were all essential for tumor killing. Our data suggest that complement-enhanced antibody-dependent cellular cytotoxicity may critically affect tumor killing by CD20 antibodies in vivo. The type II CD20 antibody 11B8, which is a poor inducer of complement activation, was ineffective against high tumor burden. Conclusions Tumor burden affects the in vivo mechanism of action of CD20 antibodies. Low tumor load can be eliminated by complement alone, whereas elimination of high tumor load requires multiple effector mechanisms. PMID:21880632

  1. Oral antibody to interleukin-10 reduces growth rate depression due to Eimeria spp. infection in broiler chickens.

    Science.gov (United States)

    Sand, Jordan M; Arendt, Maria K; Repasy, Alec; Deniz, Gűlay; Cook, Mark E

    2016-02-01

    Eimeria spp. must be controlled in floor-reared poultry to prevent the onset of coccidiosis. Here we use an oral antibody to chicken IL-10 to prevent growth depression due to Eimeria spp. infection. Egg antibody directed against an antigenic peptide of IL-10 was produced in laying hens and measured using an ELISA. In the first experiment, egg yolk powder containing antibody to chicken IL-10 (vlpramqt conjugate) (anti-IL-10 yolk powder) was fed at 3.4 g/kg feed to determine growth response following mixed Eimeria spp. challenge. Chicks were fed either anti-IL-10 antibodies or control antibodies and challenged (d3) with either sterile saline or a 10× attenuated Eimeria spp. vaccine. Control-fed and Eimeria-challenged chicks grew 8.8% slower than those challenged with saline (P < 0.04), whereas anti-IL-10-fed Eimeria challenged chicks were not different from untreated controls. In the second trial a dose response was performed with doses of either 0 (control antibody), 0.34-, or 3.4-g anti-IL-10 yolk powder/kg feed. Control-fed, Eimeria-challenged chicks grew 10.6% slower than control saline-challenged chicks (P < 0.05); however, anti-IL-10-fed chicks fed either dose of anti-IL-10 were not different from saline-challenged chicks. Finally, the effect of anti-IL-10 on acquired immunity was investigated. Chicks were fed control or anti-IL-10 yolk powder and vaccinated with a 1× dose of Eimeria vaccine at d 3. After 14 d, antibody was removed from the diet. Chicks were either saline or 10× Eimeria challenged at d 17. We found that the anti-IL-10-fed chickens did not show a reduction in growth due to challenge; hence anti-IL-10 does not appear to affect adaptive immunity during the primary immunization. Overall, use of an antibody to IL-10 is a novel method in preventing adverse effects of Eimeria spp. infection in poultry. © 2016 Poultry Science Association Inc.

  2. Heterogeneity of Polyneuropathy Associated with Anti-MAG Antibodies

    Directory of Open Access Journals (Sweden)

    Laurent Magy

    2015-01-01

    Full Text Available Polyneuropathy associated with IgM monoclonal gammopathy and anti-myelin associated glycoprotein (MAG antibodies is an immune-mediated demyelinating neuropathy. The pathophysiology of this condition is likely to involve anti-MAG antibody deposition on myelin sheaths of the peripheral nerves and it is supposed to be distinct from chronic inflammatory demyelinating neuropathy (CIDP, another immune-mediated demyelinating peripheral neuropathy. In this series, we have retrospectively reviewed clinical and laboratory findings from 60 patients with polyneuropathy, IgM gammopathy, and anti-MAG antibodies. We found that the clinical picture in these patients is highly variable suggesting a direct link between the monoclonal gammopathy and the neuropathy. Conversely, one-third of patients had a CIDP-like phenotype on electrodiagnostic testing and this was correlated with a low titer of anti-MAG antibodies and the absence of widening of myelin lamellae. Our data suggest that polyneuropathy associated with anti-MAG antibodies is less homogeneous than previously said and that the pathophysiology of the condition is likely to be heterogeneous as well with the self-antigen being MAG in most of the patients but possibly being another component of myelin in the others.

  3. [EIA-IgG antibody measles prevention level estimated from measles neutralizing, particle agglutination and hemagglutination-inhibition antibody titer].

    Science.gov (United States)

    Takayama, Naohide; Saika, Shizuko; Ichinohe, Sadato

    2009-09-01

    Measles hemagglutination inhibition (HI) antibody titer, widely used in clinical practice to simply and easily determine the measles immunity level has, in recent years, been increasingly replaced by measles IgG-antibody titer determined by enzyme-immunoassay (EIA). HI antibody titer appears to reflect this protective level, because HI measures the antibody against H protein required for the measles virus to adhere to host cells. EIA-IgG antibody titer does not correlate with the protective level, similar to particle agglutination (PA) titer, because EIA measures different antibodies, including those unrelated to measles protection. After determining HI, PA, neutralizing test (NT) results, and EIA-IgG antibody titer for individual specimens, we compared EIA-IgG antibody titer obtained using an EIA-Kit (Denka Seiken) to HI, PA, and NT titer with the following results: (1) Subjects with EIA-IgG titer of > or = 12.0 may be protected against measles: (2) Subjects with EIA-IgG titer of 4.0 to 8.0 appear to be protected insufficiently requiring a booster dose against measles: (3) Subjects with EIA-IgG titer of 8.0 to 12.0 may benefit from booster vaccination.

  4. Research Paper Polyclonal antibodies production against ...

    African Journals Online (AJOL)

    The main aim of this project is to produce polyclonal antibodies directed against the Staphylococcus aureus protein A and their use to appreciate bacteriological analysis of milk quality. In this context, an immunization produce was set up to test and detect in a batch of animals the convenient responder to the injected ...

  5. Prediction of antibody persistency from antibody titres to natalizumab

    DEFF Research Database (Denmark)

    Jensen, Poul Erik H; Koch-Henriksen, Nils; Sellebjerg, Finn

    2012-01-01

    In a subgroup of patients with multiple sclerosis natalizumab therapy causes generation of anti-natalizumab antibodies that may be transient or persistent. It is recommended to discontinue natalizumab therapy in persistently antibody-positive patients.......In a subgroup of patients with multiple sclerosis natalizumab therapy causes generation of anti-natalizumab antibodies that may be transient or persistent. It is recommended to discontinue natalizumab therapy in persistently antibody-positive patients....

  6. Antibodies against interferon-beta in neuromyelitis optica patients

    DEFF Research Database (Denmark)

    Asgari, Nasrin; Kyvik, Kirsten Ohm; Steenstrup, Troels

    2014-01-01

    of IFN-neutralizing antibodies (NAbs) in 15 IFN-ß treated NMO-patients from a population-based retrospective case series cohort. NMO patients not treated with IFN-ß acted as a reference group. IFN-ß antibody determinations included binding antibodies (BAbs) measured by immunoassay and NAbs measured...... by a neutralization bioassay. Antibodies were determined 6-36 months after initiation of IFN-β therapy and NAbs additionally 5-10 years post-therapy. BAbs were detected in 14/15 NMO patients; 6/15 were NAbs-positive (3 at 5-10 years post-therapy) two of those anti-AQP4 antibody-positive; seven of the nine NAbs......, at significantly higher frequencies than NMO reference group (pneutralizing antibody status....

  7. Amelioration of murine passive immune thrombocytopenia by IVIg and a therapeutic monoclonal CD44 antibody does not require the Myd88 signaling pathway.

    Directory of Open Access Journals (Sweden)

    Andrew R Crow

    Full Text Available Immune thrombocytopenia (ITP is an autoimmune bleeding disorder characterized by a low platelet count and the production of anti-platelet antibodies. The majority of ITP patients have antibodies to platelet integrin α(IIbβ₃ (GPIIbIIIa which can direct platelet phagocytosis by macrophages. One effective treatment for patients with ITP is intravenous immunoglobulin (IVIg which rapidly reverses thrombocytopenia. The exact mechanism of IVIg action in human patients is unclear, although in mouse models of passive ITP, IVIg can rapidly increase platelet counts in the absence of adaptive immunity. Another antibody therapeutic that can similarly increase platelet counts independent of adaptive immunity are CD44 antibodies. Toll-like receptors (TLRs are pattern recognition receptors which play a central role in helping direct the innate immune system. Dendritic cells, which are notable for their expression of TLRs, have been directly implicated in IVIg function as an initiator cell, while CD44 can associate with TLR2 and TLR4. We therefore questioned whether IVIg, or the therapeutic CD44 antibody KM114, mediate their ameliorative effects in a manner dependent upon normal TLR function. Here, we demonstrate that the TLR4 agonist LPS does not inhibit IVIg or KM114 amelioration of antibody-induced thrombocytopenia, and that these therapeutics do not ameliorate LPS-induced thrombocytopenia. IVIg was able to significantly ameliorate murine ITP in C3H/HeJ mice which have defective TLR4. All known murine TLRs except TLR3 utilize the Myd88 adapter protein to drive TLR signaling. Employing Myd88 deficient mice, we found that both IVIg and KM114 ameliorate murine ITP in Myd88 deficient mice to the same extent as normal mice. Thus both IVIg and anti-CD44 antibody can mediate their ameliorative effects in murine passive ITP independent of the Myd88 signaling pathway. These data help shed light on the mechanism of action of IVIg and KM114 in the amelioration of

  8. Ibrutinib interferes with the cell-mediated anti-tumor activities of therapeutic CD20 antibodies: implications for combination therapy

    Science.gov (United States)

    Roit, Fabio Da; Engelberts, Patrick J.; Taylor, Ronald P.; Breij, Esther C.W.; Gritti, Giuseppe; Rambaldi, Alessandro; Introna, Martino; Parren, Paul W.H.I.; Beurskens, Frank J.; Golay, Josée

    2015-01-01

    The novel Bruton tyrosine kinase inhibitor ibrutinib and phosphatidyl-4-5-biphosphate 3-kinase-δ inhibitor idelalisib are promising drugs for the treatment of chronic lymphocytic leukemia and B-cell non-Hodgkin lymphoma, either alone or in combination with anti-CD20 antibodies. We investigated the possible positive or negative impact of these drugs on all known mechanisms of action of both type I and type II anti-CD20 antibodies. Pretreatment with ibrutinib for 1 hour did not increase direct cell death of cell lines or chronic lymphocytic leukemia samples mediated by anti-CD20 antibodies. Pre-treatment with ibrutinib did not inhibit complement activation or complement-mediated lysis. In contrast, ibrutinib strongly inhibited all cell-mediated mechanisms induced by anti-CD20 antibodies rituximab, ofatumumab or obinutuzumab, either in purified systems or whole blood assays. Activation of natural killer cells, and antibody-dependent cellular cytotoxicity by these cells, as well as phagocytosis by macrophages or neutrophils were inhibited by ibrutinib with a half maximal effective concentration of 0.33 μM. Analysis of anti-CD20 mediated activation of natural killer cells isolated from patients on continued oral ibrutinib treatment suggested that repeated drug dosing inhibits these cells in vivo. Finally we show that the phosphatidyl-4-5-biphosphate 3-kinase-δ inhibitor idelalisib similarly inhibited the immune cell-mediated mechanisms induced by anti-CD20 antibodies, although the effects of this drug at 10 μM were weaker than those observed with ibrutinib at the same concentration. We conclude that the design of combined treatment schedules of anti-CD20 antibodies with these kinase inhibitors should consider the multiple negative interactions between these two classes of drugs. PMID:25344523

  9. Cation-exchange antibody labeling for simultaneous electrochemical detection of tumor markers CA15-3 and CA19-9

    International Nuclear Information System (INIS)

    Wang, Guangjie; Qing, Yi; Shan, Jinlu; Jin, Feng; Wang, Dong; Yuan, Ruo

    2013-01-01

    We report on a new kind of non-covalent multi-label electrochemical immunoassay that was applied to simultaneously quantify the tumor markers CA15-3 and CA19-9. The method employs a nanohybrid composed of an ionomer and conductive titanium dioxide nanoparticles that act as a matrix support for the antibodies. The two antibodies (anti-CA153 and anti-CA199) were labeled (a) with a cobaltous dipyridine complex, and (b) with methylene blue. Labeling is based on cation-exchange interaction rather than on covalent conjugation. The redox potentials of the two labels are separated by an interval of 0.3 V. The resulting sandwich-type immunosensor was read out by differential pulse voltammetry. The potential sites and currents of the two redox probes reflect the concentration of the two analytes. The two analytes were determined with a detection limit of 1.6 U mL −1 for CA19-9, and of 0.3 U mL −1 for CA15-3 (author)

  10. Detection of Antibodies in Blood Plasma Using Bioluminescent Sensor Proteins and a Smartphone.

    Science.gov (United States)

    Arts, Remco; den Hartog, Ilona; Zijlema, Stefan E; Thijssen, Vito; van der Beelen, Stan H E; Merkx, Maarten

    2016-04-19

    Antibody detection is of fundamental importance in many diagnostic and bioanalytical assays, yet current detection techniques tend to be laborious and/or expensive. We present a new sensor platform (LUMABS) based on bioluminescence resonance energy transfer (BRET) that allows detection of antibodies directly in solution using a smartphone as the sole piece of equipment. LUMABS are single-protein sensors that consist of the blue-light emitting luciferase NanoLuc connected via a semiflexible linker to the green fluorescent acceptor protein mNeonGreen, which are kept close together using helper domains. Binding of an antibody to epitope sequences flanking the linker disrupts the interaction between the helper domains, resulting in a large decrease in BRET efficiency. The resulting change in color of the emitted light from green-blue to blue can be detected directly in blood plasma, even at picomolar concentrations of antibody. Moreover, the modular architecture of LUMABS allows changing of target specificity by simple exchange of epitope sequences, as demonstrated here for antibodies against HIV1-p17, hemagglutinin (HA), and dengue virus type I. The combination of sensitive ratiometric bioluminescent detection and the intrinsic modularity of the LUMABS design provides an attractive generic platform for point-of-care antibody detection that avoids the complex liquid handling steps associated with conventional immunoassays.

  11. [Screening serum response special antibodies of U251 cell line from surface display phage antibody library].

    Science.gov (United States)

    Yu, Min; Tan, De-Yong; Qian, Wei; Lai, Jian-Hua; Sun, Gui-Lin

    2004-05-01

    U251 cell is a sensitive cell line to serum, which stops at G0 phase of cell cycle in no-serum medium, and recovers growth when the serum is added into no-serum medium. The cell can express corresponding proteins in different phase of cell cycle. Therefore it is very signification for the study of cell cycle regulation mechanism that explores these proteins. In this paper, the mouse antibody phage display library was added into the bottle in which the serum starvation U251 cells had been cultured, and the special antibody phages were absorbed. Then the absorbed antibody phages were amplified by adding E. coli TG1 and helper phage M13K07. Amplified antibody phages were added into bottle in which the serum cultured cell after serum starvation (follow named as serum recovered cells) were incubated, so that the cell absorbed the no-special antibody phages for the serum starvation cell and the special antibody phages were in supernatant. The remaining no-special antibody phages in the supernatant were discarded by repeating above program 3-4 times. The pure special antibody phages were gotten, and amplified by adding the host cell E. coli TG1 and helper phage M13K07. Then the host bacterium infected special antibody phage was spread on the plate medium with ampicillin, and the monoclonal antibody phages were gotten. Using same as above program, the monoclonal antibody phages absorbed specially for serum recovered U251 cells were obtained when the serum recovered cells instead of serum starvation cells and serum starvation cells instead of serum recovered cells. In this study, ninety-six positive monoclonal antibody phages that absorbed specially the serum starvation cells and eighty-two positive monoclonal antibody phages that absorbed specially the serum recovered cells were obtained. By using cell immunochemistry assay, two special signification antibodies were obtained. one (No.11) was the strong response in serum starvation cells, the other (No.2) was the strong

  12. Radiolabeled monoclonal antibodies for imaging and therapy: Potential, problems, and prospects: Scientific highlights

    International Nuclear Information System (INIS)

    Srivastava, S.C.; Buraggi, G.L.

    1986-01-01

    This meeting focused on areas of research on radiolabeled monoclonal antibodies. Topics covered included the production, purification, and fragmentation of monoclonal antibodies and immunochemistry of hybridomas; the production and the chemistry of radionuclides; the radiohalogenation and radiometal labeling techniques; the in-vivo pharmacokinetics of radiolabeled antibodies; the considerations of immunoreactivity of radiolabeled preparations; the instrumentation and imaging techniques as applied to radioimmunodetection; the radiation dosimetry in diagnostic and therapeutic use of labeled antibodies; the radioimmunoscintigraphy and radioimmunotherapy studies; and perspectives and directions for future research. Tutorial as well as scientific lectures describing the latest research data on the above topics were presented. Three workshop panels were convened on ''Methods for Determining Immunoreactivity of Radiolabeled Monoclonal Antibodies - Problems and Pitfalls,'' Radiobiological and Dosimetric Considerations for Immunotherapy with Labeled Antibodies,'' and ''The Human Anti-Mouse Antibody Response in Patients.''

  13. Dynamic interaction of 111indium-labeled monoclonal antibodies with surface antigens of solid tumors visualized in vivo by external scintigraphy

    International Nuclear Information System (INIS)

    Hwang, K.M.; Keenan, A.M.; Frincke, J.; David, G.; Pearson, J.; Oldham, R.K.; Morgan, A.C. Jr.

    1986-01-01

    Two 111 indium-labeled murine monoclonal antibodies (MoAb), D3 and 9.2.27, directed to tumor antigens of L-10 hepatocarcinoma and human melanoma, respectively, selectively localized antigen-positive target cells in guinea pigs and nude mice. The fate of MoAb differed in the two antigen-antibody systems after reacting with their corresponding tumor antigens in vivo as reflected by patterns of distribution and turnover in vivo. The 9.2.27 localized in melanoma xenograft in nude mice after intravenous administration with slow loss from tumor but more rapid loss from normal tissues and thus demonstrated optimal imaging of small tumors (approximately equal to 5 mm) between 3 and 6 days after injection of the radiolabeled antibody. In contrast, D3 demonstrated a biphasic localization in guinea pig L-10 hepatocarcinoma with a maximal activity on the 2d day after administration and showed rapid loss from both tumor and normal tissues. Nonspecific localization of antibodies in liver and in kidney was found both in syngeneic (nude mice) and xenogeneic (guinea pig) hosts but was more pronounced in the xenogeneic species. These results indicate that the nature of the antigen-antibody interaction may be of importance in selecting MoAb for both diagnosis and therapy of malignant diseases

  14. Surface immobilized antibody orientation determined using ToF-SIMS and multivariate analysis.

    Science.gov (United States)

    Welch, Nicholas G; Madiona, Robert M T; Payten, Thomas B; Easton, Christopher D; Pontes-Braz, Luisa; Brack, Narelle; Scoble, Judith A; Muir, Benjamin W; Pigram, Paul J

    2017-06-01

    Antibody orientation at solid phase interfaces plays a critical role in the sensitive detection of biomolecules during immunoassays. Correctly oriented antibodies with solution-facing antigen binding regions have improved antigen capture as compared to their randomly oriented counterparts. Direct characterization of oriented proteins with surface analysis methods still remains a challenge however surface sensitive techniques such as Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) provide information-rich data that can be used to probe antibody orientation. Diethylene glycol dimethyl ether plasma polymers (DGpp) functionalized with chromium (DGpp+Cr) have improved immunoassay performance that is indicative of preferential antibody orientation. Herein, ToF-SIMS data from proteolytic fragments of anti-EGFR antibody bound to DGpp and DGpp+Cr are used to construct artificial neural network (ANN) and principal component analysis (PCA) models indicative of correctly oriented systems. Whole antibody samples (IgG) test against each of the models indicated preferential antibody orientation on DGpp+Cr. Cross-reference between ANN and PCA models yield 20 mass fragments associated with F(ab') 2 region representing correct orientation, and 23 mass fragments associated with the Fc region representing incorrect orientation. Mass fragments were then compared to amino acid fragments and amino acid composition in F(ab') 2 and Fc regions. A ratio of the sum of the ToF-SIMS ion intensities from the F(ab') 2 fragments to the Fc fragments demonstrated a 50% increase in intensity for IgG on DGpp+Cr as compared to DGpp. The systematic data analysis methodology employed herein offers a new approach for the investigation of antibody orientation applicable to a range of substrates. Controlled orientation of antibodies at solid phases is critical for maximizing antigen detection in biosensors and immunoassays. Surface-sensitive techniques (such as ToF-SIMS), capable of direct

  15. The Perfect Storm: HLA Antibodies, Complement, FcγRs and Endothelium in Transplant Rejection

    OpenAIRE

    Thomas, Kimberly A.; Valenzuela, Nicole M.; Reed, Elaine F.

    2015-01-01

    The pathophysiology of antibody-mediated rejection (AMR) in solid organ transplants is multi-faceted and predominantly caused by antibodies directed against polymorphic donor human leukocyte antigens (HLA). Despite the clearly detrimental impact of HLA antibodies (HLA-Ab) on graft function and survival, the prevention, diagnosis and treatment of AMR remain a challenge. Histological manifestations of AMR reflect signatures of HLA-Ab-triggered injury, specifically endothelial changes, recipient...

  16. In vivo instability of reduction-mediated 99mTc-labeled monoclonal antibody

    International Nuclear Information System (INIS)

    Sakahara, Harumi; Saga, Tsuneo; Endo, Keigo

    1993-01-01

    A murine monoclonal antibody that reacts with human osteogenic sarcoma (OST7) was reduced and directly labelled with 99m Tc without any loss of immunoreactivity. No fragmentation of the antibody was detected by high performance liquid chromatography after the labelling. However, SDS-PAGE analysis of the labelled antibody demonstrated the presence of low molecular weight species. Although more than 95% of the radioactivity remained bound at the antibody after incubation with human serum for 24 h, 99m Tc-labelled OST7 was cleared faster from the circulation than 125 I-labelled OST7 or 111 In-labelled OST7 in mice. (author)

  17. Thyroid Antibodies

    Science.gov (United States)

    ... PF4 Antibody Hepatitis A Testing Hepatitis B Testing Hepatitis C Testing HER2/neu Herpes Testing High-sensitivity C-reactive Protein (hs-CRP) Histamine Histone Antibody HIV Antibody and HIV Antigen (p24) HIV Antiretroviral Drug Resistance Testing, Genotypic HIV Viral Load HLA Testing HLA- ...

  18. Ethylbenzene induces microsomal oxygen free radical generation: antibody-directed characterization of the responsible cytochrome P450 enzymes.

    Science.gov (United States)

    Serron, S C; Dwivedi, N; Backes, W L

    2000-05-01

    Small aromatic hydrocarbons cause changes in oxidative metabolism by modulating the levels of cytochrome P450 enzymes, with the changes in these enzymes being responsible for qualitative changes in aromatic hydrocarbon metabolism. The goal of this study was to determine if exposure to the small alkylbenzene ethylbenzene (EB) leads to an increase in hepatic free radical production. Male F344 rats were treated with ip injections of EB (10 mmol/kg) and compared to corn oil controls. Hepatic free radical production was examined by measuring the conversion of 2',7'-dichlorofluorescin diacetate (DCFH-DA) to its fluorescent product 2',7'-dichlorofluorescein (DCF). A significant elevation of fluorescent DCF production was observed after treatment with EB, despite the lack of effect on overall cytochrome P450 levels. This process was shown to be inhibitable by metyrapone, an inhibitor of P450. DCF production was also inhibited by catalase, suggesting that hydrogen peroxide (H(2)O(2)) is one of the reactive oxygen intermediates involved in EB-mediated reactive oxygen species (ROS) formation. Interestingly, superoxide dismutase (SOD) did not inhibit DCF production in corn oil-treated rats but was an effective inhibitor in the EB-treated groups. In an effort to determine if the increase in ROS production was related to changes in specific P450 enzymes, DCF production was measured in the presence of anti-CYP2B, anti-CYP2C11, anti-CYP2E1, and anti-CYP3A2 inhibitory antibodies. Anti-CYP2B antibodies inhibited DCF production in EB-treated, but not corn oil groups, which is consistent with the low constitutive levels of this enzyme and its induction by EB. The data also demonstrate that CYP2B contributes to ROS production. Anti-CYP2C11 did not influence DCF production in either group. ROS formation in corn oil-treated rats as well as in ethylbenzene-treated rats was also inhibited with antibodies to anti-CYP2E1 and anti-CYP3A2. These results suggest that CYP2C11 does not appear to

  19. Human Cell Line-Derived Monoclonal IgA Antibodies for Cancer Immunotherapy

    Directory of Open Access Journals (Sweden)

    Felix Hart

    2017-05-01

    Full Text Available IgA antibodies have great potential to improve the functional diversity of current IgG antibody-based cancer immunotherapy options. However, IgA production and purification is not well established, which can at least in part be attributed to the more complex glycosylation as compared to IgG antibodies. IgA antibodies possess up to five N-glycosylation sites within their constant region of the heavy chain as compared to one site for IgG antibodies. The human GlycoExpress expression system was developed to produce biotherapeutics with optimized glycosylation and used here to generate a panel of IgA isotype antibodies directed against targets for solid (TA-mucin 1, Her2, EGFR, Thomsen–Friedenreich and hematological (CD20 cancer indications. The feasibility of good manufacturing practice was shown by the production of 11 g IgA within 35 days in a one liter perfusion bioreactor, and IgA antibodies in high purity were obtained after purification. The monoclonal IgA antibodies possessed a high sialylation degree, and no non-human glycan structures were detected. Kinetic analysis revealed increased avidity antigen binding for IgA dimers as compared to monomeric antibodies. The IgA antibodies exhibited potent Fab- and Fc-mediated functionalities against cancer cell lines, whereby especially granulocytes are recruited. Therefore, for patients who do not sufficiently benefit from therapeutic IgG antibodies, IgA antibodies may complement current regiment options and represent a promising strategy for cancer immunotherapy. In conclusion, a panel of novel biofunctional IgA antibodies with human glycosylation was successfully generated.

  20. Will a mAb-Based Immunotherapy Directed against Cancer Stem Cells Be Feasible?

    Directory of Open Access Journals (Sweden)

    Silvia Santamaria

    2017-11-01

    Full Text Available The cancer stem cell (CSC hypothesis suggests that within a tumor, there is a small subpopulation of cells with stem cell properties responsible for tumor maintenance and metastasis generation. This hypothesis also implies that new antitumor drugs, rather than targeting the bulk of the tumor mass, would be more effective if they directly targeted the CSC subpopulation. The CSCs from several types of tumors have been identified with mAbs recognizing surface antigens in these cells; however, antigens specifically or exclusively expressed in the CSC population have not yet been identified. Thus, questioning the possibility of using therapeutic antibodies directed against the CSCs. Here, we review the possibilities of using antibodies directly targeting the CSCs as therapeutic agents in the form of naked antibodies, antibodies conjugated to nanoparticles, or antibody cocktails.

  1. Lupus erythematosus cell preparation, antinuclear factor and antideoxyribonucleic acid antibody incongruity in systemic lupus erythematosus.

    Science.gov (United States)

    Chee, Y C

    1983-01-01

    'Total antinuclear antibody' (ANF) is detected by the fluorescent antinuclear antibody technique which is a screening test, positive in 99% of systemic lupus erythematosus (SLE) sera. The LE factor (positive in 75% of SLE sera), like the anti-DNA antibody, is an antinuclear antibody but directed against DNA-histone. ANF-negative SLE is a clinical entity with absence of these antibodies. A false negative ANF, in the presence of high titre anti-DNA antibody and/or LE cells, is illustrated in two cases of SLE. Postulated mechanisms for this phenomenon are interference in ANF detection by rheumatoid factor, and the prozone effect on the immunofluorescent tests.

  2. Enzymatic Inactivation of Endogenous IgG by IdeS Enhances Therapeutic Antibody Efficacy.

    Science.gov (United States)

    Järnum, Sofia; Runström, Anna; Bockermann, Robert; Winstedt, Lena; Crispin, Max; Kjellman, Christian

    2017-09-01

    Endogenous plasma IgG sets an immunologic threshold that dictates the activity of tumor-directed therapeutic antibodies. Saturation of cellular antibody receptors by endogenous antibody limits antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Here, we show how enzymatic cleavage of IgG using the bacterial enzyme IdeS can be utilized to empty both high and low affinity Fcγ-receptors and clear the entire endogenous antibody pool. Using in vitro models, tumor animal models as well as ex vivo analysis of sera collected during a previous clinical trial with IdeS, we show how clearing of competing plasma antibody levels with IdeS unblocks cellular antibody receptors. We show that therapeutic antibodies against breast cancer (trastuzumab), colon cancer (cetuximab), and lymphomas (rituximab and alemtuzumab) can be potentiated when endogenous IgG is removed. Overall, IdeS is shown to be a potent tool to reboot the human antibody repertoire and to generate a window to preferentially load therapeutic antibodies onto effector cells and thereby create an armada of dedicated tumor-seeking immune cells. Mol Cancer Ther; 16(9); 1887-97. ©2017 AACR . ©2017 American Association for Cancer Research.

  3. Initial clinical evaluation of radiolabeled MX-DTPA humanized BrE-3 antibody in patients with advanced breast cancer.

    Science.gov (United States)

    Kramer, E L; Liebes, L; Wasserheit, C; Noz, M E; Blank, E W; Zabalegui, A; Melamed, J; Furmanski, P; Peterson, J A; Ceriani, R L

    1998-07-01

    To evaluate radiometal-labeled humanized BrE-3 (huBrE-3) monoclonal antibody as a radioimmunolocalization and therapeutic agent in breast cancer patients, tumor localization, pharmacokinetics, radiation dosimetry, and immunogenicity of (111)In-labeled combined 1-p-isothiocyanatobenzyl 3-methyl- and 1-p-isothiocyanatobenzyl 4-methyldiethylenetriamine pentaacetic acid (MX-DTPA) huBrE-3 were studied. Seven women with BrE-3 antigen-positive, metastatic breast carcinoma underwent (111)In huBrE-3 infusion (5 mCi; 50 mg), followed by serial gamma camera imaging and plasma sampling. Region of interest analysis of images was used to make radiation absorbed dose estimates for (111)In huBrE-3. Data were extrapolated to 90Y huBrE-3. Human anti-human antibody (HAHA) response was measured in serum samples obtained up to 3 months after infusion. Patients tolerated infusions well. Seventy-six percent of 105 known sites of disease were identified on planar and single-photon emission computed tomography scans. For six of seven patients, a biexponential model fit the plasma time-activity curve best with an average T1/2alpha=10.6+/-8.5 (SD) h and average T1/2beta=114.2+/-39.2 h. Radiation absorbed dose estimates for (111)In huBrE-3 for whole body averaged 0.53+/-.08 rads/mCi. Dose estimates for 90Y huBrE-3 for marrow averaged 8.4+/-11.9 rads/mCi, and for tumors, 70+/-31.5 rads/mCi. Liver radioactivity uptake averaged 19.7+/-8.8% injected dose at 24 h after infusion, translating into an average radiation absorbed dose 21.1+/-12 rads/90Y mCi administered. Only one of seven patients demonstrated a low level of HAHA response. Although the plasma half-lives are longer and marrow dose higher for radiolabeled huBrE-3 compared with the murine construct, the excellent tumor localization, good tumor dosimetry, and low immunogenicity support the use of 90Y-huBrE-3 antibody for radioimmunotherapy of breast cancer.

  4. Human monoclonal antibodies: the residual challenge of antibody immunogenicity.

    Science.gov (United States)

    Waldmann, Herman

    2014-01-01

    One of the major reasons for seeking human monoclonal antibodies has been to eliminate immunogenicity seen with rodent antibodies. Thus far, there has yet been no approach which absolutely abolishes that risk for cell-binding antibodies. In this short article, I draw attention to classical work which shows that monomeric immunoglobulins are intrinsically tolerogenic if they can be prevented from creating aggregates or immune complexes. Based on these classical studies two approaches for active tolerization to therapeutic antibodies are described.

  5. Effect of bovine pellucid zone 3 monoclonal antibodies on B cell lymphoma 2 expressions of granulosa cell and mice (Mus musculus follicle diameter

    Directory of Open Access Journals (Sweden)

    Heti Ira Ayue

    2017-01-01

    Full Text Available Objective: To evaluate the effects of pellucid zone 3 monoclonal antibodies against B-cell lymphoma 2 (BCL-2 expression and mice follicle diameter at various time periods.Methods: The animal model of this study was 36 Balb/c mice (Mus musculus. A true experimental design was used with a post-test only control group approach. BCL-2 expression was observed using immunohistochemistry, while the follicle diameter was observed by haematoxylin-eosin staining. The data was analyzed using nested ANOVA to compare the results of the mean expression of BCL-2 on the 5th and 20th day of observation in the pre-antral and antral follicle between the control and treatment groups.Results: No significant differences were found in BCL-2 gene expression. There were also no significant differences in BCL-2 expression on the 10th day of pre-antral follicle analysis. Moreover, there were no significant differences between the mean follicle diameter on the 5th, 10th, and 20th day of pre-antral and antral follicle development between the control and treatment groups. The addition of bovine pellucid zone 3 (bZP3 monoclonal antibodies on the 5th and 20th day of observation did not decrease the expression of BCL-2 gene in the pre-antral and antral follicle of mice. Administering bZP3 monoclonal antibodies on the 10th day of observation did not affect BCL-2 expression in the pre-antral follicle but did decrease BCL-2 expression in the antral follicle. Supplying bZP3 monoclonal antibodies on the 5th, 10th and 20th day did not affect the diameter of pre-antral and antral follicles of the mice.Conclusion: The monoclonal antibodies bovine zona pelusida 3 has the potential to be developed as a safe immunocontraception preparation.

  6. Microbials for the production of monoclonal antibodies and antibody fragments.

    Science.gov (United States)

    Spadiut, Oliver; Capone, Simona; Krainer, Florian; Glieder, Anton; Herwig, Christoph

    2014-01-01

    Monoclonal antibodies (mAbs) and antibody fragments represent the most important biopharmaceutical products today. Because full length antibodies are glycosylated, mammalian cells, which allow human-like N-glycosylation, are currently used for their production. However, mammalian cells have several drawbacks when it comes to bioprocessing and scale-up, resulting in long processing times and elevated costs. By contrast, antibody fragments, that are not glycosylated but still exhibit antigen binding properties, can be produced in microbial organisms, which are easy to manipulate and cultivate. In this review, we summarize recent advances in the expression systems, strain engineering, and production processes for the three main microbials used in antibody and antibody fragment production, namely Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. A malaria vaccine that elicits in humans antibodies able to kill Plasmodium falciparum.

    Directory of Open Access Journals (Sweden)

    2005-11-01

    Full Text Available BACKGROUND: Plasmodium falciparum merozoite surface protein 3 is a malaria vaccine candidate that was identified, characterised, and developed based on a unique immuno-clinical approach. The vaccine construct was derived from regions fully conserved among various strains and containing B cell epitopes targeted by human antibodies (from malaria-immune adults that are able to mediate a monocyte-dependent parasite killing effect. The corresponding long synthetic peptide was administered to 36 volunteers, with either alum or Montanide ISA720 as adjuvant. METHODS AND FINDINGS: Both formulations induced cellular and humoral immune responses. With alum, the responses lasted up to 12 mo. The vaccine-induced antibodies were predominantly of cytophilic classes, i.e., able to cooperate with effector cells. In vitro, the antibodies induced an inhibition of the P. falciparum erythrocytic growth in a monocyte-dependent manner, which was in most instances as high as or greater than that induced by natural antibodies from immune African adults. In vivo transfer of the volunteers' sera into P. falciparum-infected humanized SCID mice profoundly reduced or abrogated parasitaemia. These inhibitory effects were related to the antibody reactivity with the parasite native protein, which was seen in 60% of the volunteers, and remained in samples taken 12 mo postimmunisation. CONCLUSION: This is the first malaria vaccine clinical trial to clearly demonstrate antiparasitic activity by vaccine-induced antibodies by both in vitro and in vivo methods. The results, showing the induction of long-lasting antibodies directed to a fully conserved polypeptide, also challenge current concepts about malaria vaccines, such as unavoidable polymorphism, low antigenicity, and poor induction of immune memory.

  8. Heparin binding domain of antithrombin III: Characterization using a synthetic peptide directed polyclonal antibody

    International Nuclear Information System (INIS)

    Smith, J.W.; Dey, B.; Knauer, D.J.

    1990-01-01

    Antithrombin III (ATIII) is a plasma-borne serine protease inhibitor that apparently forms covalent complexes with thrombin. The interaction between ATIII and thrombin is enhanced several thousandfold by the glycosaminoglycan, heparin. The authors have previously proposed that the heparin binding site of ATIII residues within a region extending from amino acid residues 114-156. Computer-assisted analysis of this region revealed the presence of a 22 amino acid domain (residues 124-145), part of which shows a strong potential for the formation of an amphipathic helix: hydrophobic on one face and highly positively charged on the other. In the presence studies, polyclonal antisera were generated against a synthetic peptide corresponding to residues 124-145 in native human ATIII. Affinity-purified IgG from these antisera, as well as monovalent Fab's derived from them, specifically blocked the binding of heparin to ATIII. Additionally, occupancy of the heparin binding site by these same monovalent and bivalent IgG's at least partially substituted for heparin, accelerating linkage formation between ATIII and thrombin. These results provide the first immunological evidence that region 124-145 is directly involved in the binding of heparin to ATIII and that an antibody-induced conformational change within this region can mediate ATIII activation

  9. Monoclonal antibodies based on hybridoma technology.

    Science.gov (United States)

    Yagami, Hisanori; Kato, Hiroshi; Tsumoto, Kanta; Tomita, Masahiro

    2013-03-01

    Based on the size and scope of the present global market for medicine, monoclonal antibodies (mAbs) have a very promising future, with applications for cancers through autoimmune ailments to infectious disease. Since mAbs recognize only their target antigens and not other unrelated proteins, pinpoint medical treatment is possible. Global demand is dramatically expanding. Hybridoma technology, which allows production of mAbs directed against antigens of interest is therefore privileged. However, there are some pivotal points for further development to generate therapeutic antibodies. One is selective generation of human mAbs. Employment of transgenic mice producing human antibodies would overcome this problem. Another focus is recognition sites and conformational epitopes in antigens may be just as important as linear epitopes, especially when membrane proteins such as receptors are targeted. Recognition of intact structures is of critical importance for medical purposes. In this review, we describe patent related information for therapeutic mAbs based on hybridoma technology and also discuss new advances in hybridoma technology that facilitate selective production of stereospecific mAbs.

  10. Process Research and Development of Antibodies as Countermeasures for C. botulinum

    Science.gov (United States)

    2009-02-01

    ProteinA –Sepharose Fast Flow resin (Amersham Biosciences). Alternatively, culture supernatant was di- rectly loaded onto the rProtein A–Sepharose Fast Flow...interest; however the stationary point was a saddle point, so we move on the ridge in the direction to get the maximum response. The points on the...10 Ridge of steepest ascent of the reduced model for getting maximum antibody production with independent vari- ables X1, X2, X3. X4 and X5 are kept

  11. Improving food and agricultural production. Thailand. Application on monoclonal antibodies for progesterone measurement

    International Nuclear Information System (INIS)

    Butcher, G.W.

    1991-01-01

    The duties of the mission were to provide instructions on the maintenance of hybridoma cell lines and their culture and the harvesting of monoclonal antibodies; to assist the counterparts in Thailand to develop work plans for the use of monoclonal antibodies in radioimmunoassay measurements of progesterone; and to assess the need for and feasibility of establishing a laboratory for producing monoclonal antibodies directed against progesterone. The report contains a summary of the activities performed in fulfillment of these duties

  12. Preparation of anti-ciguatoxin monoclonal antibodies using synthetic haptens: sandwich ELISA detection of ciguatoxins.

    Science.gov (United States)

    Tsumuraya, Takeshi; Fujii, Ikuo; Hirama, Masahiro

    2014-01-01

    Ciguatera fish poisoning (CFP) is a form of food poisoning caused by the consumption of fish that have accumulated a type of sodium channel activator toxin called ciguatoxins (CTXs), which are produced by dinoflagellates of the genus Gambierdiscus through the food chain. CFP affects more than 50000 people each year. The extremely low level of CTXs in tainted fish has hampered the development of antibodies for the detection of these toxins. Monoclonal antibodies (mAbs) specific against major congeners of CTX3C, 51-hydroxyCTX3C, CTX1B, and 54-deoxyCTX1B were prepared by immunization of mice with protein conjugates of rationally designed synthetic haptens in place of the natural toxins. We found that haptenic groups possessing a surface area larger than 400 angstroms2 were required to produce mAbs that can bind strongly to CTXs. Direct sandwich ELISA utilizing two different monoclonal antibodies that bind specifically to one of the two wings of a CTX were established to detect CTXs. No cross-reactivity was observed against the other marine toxins tested, including brevetoxin A, brevetoxin B, okadaic acid, and maitotoxin.

  13. [Construction of a phage antibody library and screening of anti-epidermal growth factor receptor variant III single chain antibody].

    Science.gov (United States)

    Han, Dong-gang; Duan, Xiao-yi; Guo, You-min; Zhou, Qi; Wang, Quan-ying; Yang, Guang-xiao

    2010-01-01

    To obtain specific anti-epidermal growth factor receptor variant III (EGFRvIII) single chain antibody (ScFv) by phage antibody library display system. The total RNA was extracted from the spleen B cells of BALB/c mice immunized with pep-3-OVA protein, and the first-strand cDNA was synthesized by reverse transcription. Antibody VH and VL gene fragments were amplified and joined to a ScFv gene with the linker. The ScFv gene was ligated into the phagemid vector pCANTAB5E, which was transformed into competent E. coli TG1. The transformed cells were then infected with M13KO7 helper phage to yield the recombinant phage to construct the phage ScFv library. Pep-3-BSA protein was used to screen the phage antibody library and ELISA carried out to characterize the activity of the antibody. The VH and VL gene fragments of the antibody were about 350 bp and 320 bp in length as analyzed by agarose gel electrophoresis. The ScFv gene was 780 bp, consistent with the expected length. The recombinant phagemid with ScFv gene insert was rescued, and an immune phage ScFv library with the content of 5.0x10(6) was constructed. The recombinant ScFv phage had a titer of 3.0x10(4) cfu/ml, and the fourth phage harvest yielded 56 times as much as that of the first one. SDS-PAGE demonstrated a molecular mass of the soluble ScFv of about 28 kD. ELISA results indicated good specificity of the ScFv to bind EGFRvIII. An immune phage ScFv library is successfully constructed, and the ScFv antibody fragment is capable of specific binding to EGFRvIII.

  14. Heterologous coproduction of enterocin A and pediocin PA-1 by Lactococcus lactis : Detection by specific peptide-directed antibodies

    NARCIS (Netherlands)

    Martinez, JM; Kok, J; Sanders, JW; Hernandez, PE

    Antibodies against enterocin A were obtained by immunization of rabbits with synthetic peptides PH4 and PH5 designed, respectively, on the N- and C-terminal amino acid sequences of enterocin A and conjugated to the carrier protein KLH, Anti-PH4-KLH antibodies not only recognized enterocin A but also

  15. Differentiation of foot-and-mouth disease virus infected animals from vaccinated animals using a blocking ELISA based on baculovirus expressed FMDV 3ABC antigen and a 3ABC monoclonal antibody

    DEFF Research Database (Denmark)

    Sørensen, K.J.; de Stricker, K.; Dyrting, K.C.

    2005-01-01

    A blocking ELISA that differentiated foot-and-mouth disease virus (FMDV) infected animals from vaccinated animals was developed which uses baculovirus expressed FMDV 3ABC non-structural protein as antigen and monoclonal antibody against FMDV 3ABC non-structural protein as capture and detector...... infected with all seven serotypes of FMDV. The test detected antibodies from days 7 or 9 following experimental infection of non-vaccinated cattle and sheep, and in cattle strong positive reactions persisted for up to 395 days after infection. In vaccinated cattle that became carriers after challenge...... with homologous FMDV, positive reactions were obtained in all but one case. In some of these cattle the antibody response was detected late in comparison to the non-vaccinated infected cattle. The test gave results that compared favourably with two commercial ELISA's when used to test sera from cattle, pigs...

  16. Antigenotoxic and antimutagenic effects of Schinus terebinthifolius Raddi in Allium cepa and Swiss mice: a comparative study.

    Science.gov (United States)

    Fedel-Miyasato, L E S; Formagio, A S N; Auharek, S A; Kassuya, C A L; Navarro, S D; Cunha-Laura, A L; Monreal, A C D; Vieira, M C; Oliveira, R J

    2014-04-30

    It is estimated that 60% of anticancer drugs are derived directly or indirectly from medicinal plants. Schinus terebinthifolius Raddi (Anacardiaceae) is traditionally used in Brazilian medicine to treat inflammation, ulcers, and tumors. Because of the need to identify new antimutagenic agents and to determine their mechanism of action, this study evaluated the chemopreventive activity of the methanolic extract from leaves of S. terebinthifolius (MEST) in Allium cepa cells and in Swiss mice analyzing different protocols of MEST in association with DNA-damaging agents. The antigenotoxic and antimutagenic aspects in peripheral blood were evaluated using the comet and micronucleus assays, respectively. The percentage of damage reduction was used to compare the A. cepa and mice results. Our results showed for the first time that MEST can act as a chemopreventive compound that promotes cellular genome integrity by desmutagenic and bioantimutagenic activities in vegetal and animal models. This finding may therefore have therapeutic applications that can indirectly correlate to the prevention and/or treatment of the degenerative diseases such as cancer.

  17. Brain-Reactive Antibodies and Disease

    OpenAIRE

    Diamond, B.; Honig, G.; Mader, S.; Brimberg, L.; Volpe, B.T.

    2013-01-01

    Autoimmune diseases currently affect 5–7% of the world's population; in most diseases there are circulating autoantibodies. Brain-reactive antibodies are present in approximately 2–3% of the general population but do not usually contribute to brain pathology. These antibodies penetrate brain tissue only early in development or under pathologic conditions. This restriction on their pathogenicity and the lack of correlation between serum titers and brain pathology have, no doubt, contributed to...

  18. Dissection of antibody specificities induced by yellow fever vaccination.

    Directory of Open Access Journals (Sweden)

    Oksana Vratskikh

    Full Text Available The live attenuated yellow fever (YF vaccine has an excellent record of efficacy and one dose provides long-lasting immunity, which in many cases may last a lifetime. Vaccination stimulates strong innate and adaptive immune responses, and neutralizing antibodies are considered to be the major effectors that correlate with protection from disease. Similar to other flaviviruses, such antibodies are primarily induced by the viral envelope protein E, which consists of three distinct domains (DI, II, and III and is presented at the surface of mature flavivirions in an icosahedral arrangement. In general, the dominance and individual variation of antibodies to different domains of viral surface proteins and their impact on neutralizing activity are aspects of humoral immunity that are not well understood. To gain insight into these phenomena, we established a platform of immunoassays using recombinant proteins and protein domains that allowed us to dissect and quantify fine specificities of the polyclonal antibody response after YF vaccination in a panel of 51 vaccinees as well as determine their contribution to virus neutralization by serum depletion analyses. Our data revealed a high degree of individual variation in antibody specificities present in post-vaccination sera and differences in the contribution of different antibody subsets to virus neutralization. Irrespective of individual variation, a substantial proportion of neutralizing activity appeared to be due to antibodies directed to complex quaternary epitopes displayed on the virion surface only but not on monomeric E. On the other hand, DIII-specific antibodies (presumed to have the highest neutralizing activity as well as broadly flavivirus cross-reactive antibodies were absent or present at very low titers. These data provide new information on the fine specificity as well as variability of antibody responses after YF vaccination that are consistent with a strong influence of individual

  19. Chimeric recombinant antibody fragments in cardiac troponin I immunoassay.

    Science.gov (United States)

    Hyytiä, Heidi; Heikkilä, Taina; Brockmann, Eeva-Christine; Kekki, Henna; Hedberg, Pirjo; Puolakanaho, Tarja; Lövgren, Timo; Pettersson, Kim

    2015-03-01

    To introduce a novel nanoparticle-based immunoassay for cardiac troponin I (cTnI) utilizing chimeric antibody fragments and to demonstrate that removal of antibody Fc-part and antibody chimerization decrease matrix related interferences. A sandwich-type immunoassay for cTnI based on recombinant chimeric (mouse variable/human constant) antigen binding (cFab) antibodies and intrinsically fluorescent nanoparticles was developed. To test whether using chimeric antibody fragments helps to avoid matrix related interferences, samples (n=39) with known amounts of triglycerides, bilirubin, rheumatoid factor (RF) or human anti-mouse antibodies (HAMAs) were measured with the novel assay, along with a previously published nanoparticle-based research assay with the same antibody epitopes. The limit of detection (LoD) was 3.30ng/L. Within-laboratory precision for 29ng/L and 2819ng/L cTnI were 13.7% and 15.9%, respectively. Regression analysis with Siemens ADVIA Centaur® yielded a slope (95% confidence intervals) of 0.18 (0.17-1.19) and a y-intercept of 1.94 (-1.28-3.91) ng/L. When compared to a previously published nanoparticle-based assay, the novel assay showed substantially reduced interference in the tested interference prone samples, 15.4 vs. 51.3%. A rheumatoid factor containing sample was decreased from 241ng/L to 3.3ng/L) immunoassay for the detection of cTnI and decreased matrix related interferences, thus resulting in a lower number of falsely elevated cTnI-values. Copyright © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  20. Monoclonal antibody

    International Nuclear Information System (INIS)

    Oyamada, Hiyoshimaru

    1987-01-01

    Some aspects of monoclonal antibodies are described, centering on studies made by the author and those presented at the Second International Conference on Monoclonal Antibody Immunoconjugates for Cancer held in March this year (1987). The history of immuno-nuclear medicine and procedures for producing monoclonal antibodies are briefly outlined. Monoclonal antibodies are immunoglobulins. Here, the structure of IgG, which is used most frequently, is described. An IgG is composed of two antigen binding fragments (Fab) and one crystallizable fragment (Fc). The end portion of a Fab reacts with an antigen. One of the major applications of immuno-nuclear medicine is the diagnosis of cancer. As label nucleides, 131 I and 111 I were selected in most cases in the past while 123 I and 99m Tc are currently used more often. Advantages and disadvantages of this diagnosis method is discussed citing studies presented at the First (1986) and Second (1987) International Conference on Monoclonal Antibody Immunoconjugates for Cancer. The present status of the application of monoclonal antibodies to treatment of cancer is also described. (Nogami, K.)

  1. Immune Antibody Libraries: Manipulating The Diverse Immune Repertoire for Antibody Discovery.

    Science.gov (United States)

    Lim, Theam Soon; Chan, Soo Khim

    2016-01-01

    Antibody phage display is highly dependent on the availability of antibody libraries. There are several forms of libraries depending mainly on the origin of the source materials. There are three major classes of libraries, mainly the naïve, immune and synthetic libraries. Immune antibody libraries are designed to isolate specific and high affinity antibodies against disease antigens. The pre-exposure of the host to an infection results in the production of a skewed population of antibodies against the particular infection. This characteristic takes advantage of the in vivo editing machinery to generate bias and specific immune repertoire. The skewed but diverse repertoire of immune libraries has been adapted successfully in the generation of antibodies against a wide range of diseases. We envisage immune antibody libraries to play a greater role in the discovery of antibodies for diseases in the near future. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  2. Generation and Characterization of Anti-CD34 Monoclonal Antibodies that React with Hematopoietic Stem Cells

    Science.gov (United States)

    Aghebati Maleki, Leili; Majidi, Jafar; Baradaran, Behzad; Movassaghpour, Aliakbar; Abdolalizadeh, Jalal

    2014-01-01

    CD34 is a type I membrane protein with a molecular mass of approximately 110 kDa. This antigen is associated with human hematopoietic progenitor cells and is a differentiation stage-specific leukocyte antigen. In this study we have generated and characterized monoclonal antibodies (mAbs) directed against a CD34 marker. Mice were immunized with two keyhole lympet hemocyanin (KLH)-conjugated CD34 peptides. Fused cells were grown in hypoxanthine, aminopterine and thymidine (HAT) selective medium and cloned by the limiting dilution (L.D) method. Several monoclones were isolated by three rounds of limited dilutions. From these, we chose stable clones that presented sustained antibody production for subsequent characterization. Antibodies were tested for their reactivity and specificity to recognize the CD34 peptides and further screened by enzyme-linked immunosorbent assay (ELISA) and Western blotting analyses. One of the mAbs (3D5) was strongly reactive against the CD34 peptide and with native CD34 from human umbilical cord blood cells (UCB) in ELISA and Western blotting analyses. The results have shown that this antibody is highly specific and functional in biomedical applications such as ELISA and Western blot assays. This monoclonal antibodies (mAb) can be a useful tool for isolation and purification of human hematopoietic stem cells (HSCs). PMID:24611141

  3. Vaccine induced antibodies to the first variable loop of human immunodeficiency virus type 1 gp120, mediate antibody-dependent virus inhibition in macaques.

    Science.gov (United States)

    Bialuk, Izabela; Whitney, Stephen; Andresen, Vibeke; Florese, Ruth H; Nacsa, Janos; Cecchinato, Valentina; Valeri, Valerio W; Heraud, Jean-Michel; Gordon, Shari; Parks, Robyn Washington; Montefiori, David C; Venzon, David; Demberg, Thorsten; Guroff, Marjorie Robert-; Landucci, Gary; Forthal, Donald N; Franchini, Genoveffa

    2011-12-09

    The role of antibodies directed against the hyper variable envelope region V1 of human immunodeficiency virus type 1 (HIV-1), has not been thoroughly studied. We show that a vaccine able to elicit strain-specific non-neutralizing antibodies to this region of gp120 is associated with control of highly pathogenic chimeric SHIV(89.6P) replication in rhesus macaques. The vaccinated animal that had the highest titers of antibodies to the amino terminus portion of V1, prior to challenge, had secondary antibody responses that mediated cell killing by antibody-dependent cellular cytotoxicity (ADCC), as early as 2 weeks after infection and inhibited viral replication by antibody-dependent cell-mediated virus inhibition (ADCVI), by 4 weeks after infection. There was a significant inverse correlation between virus level and binding antibody titers to the envelope protein, (R=-0.83, p=0.015), and ADCVI (R=-0.84 p=0.044). Genotyping of plasma virus demonstrated in vivo selection of three SHIV(89.6P) variants with changes in potential N-linked glycosylation sites in V1. We found a significant inverse correlation between virus levels and titers of antibodies that mediated ADCVI against all the identified V1 virus variants. A significant inverse correlation was also found between neutralizing antibody titers to SHIV(89.6) and virus levels (R=-0.72 p=0.0050). However, passive inoculation of purified immunoglobulin from animal M316, the macaque that best controlled virus, to a naïve macaque, resulted in a low serum neutralizing antibodies and low ADCVI activity that failed to protect from SHIV(89.6P) challenge. Collectively, while our data suggest that anti-envelope antibodies with neutralizing and non-neutralizing Fc(R-dependent activities may be important in the control of SHIV replication, they also demonstrate that low levels of these antibodies alone are not sufficient to protect from infection. Published by Elsevier Ltd.

  4. Inactivated H7 Influenza Virus Vaccines Protect Mice despite Inducing Only Low Levels of Neutralizing Antibodies.

    Science.gov (United States)

    Kamal, Ram P; Blanchfield, Kristy; Belser, Jessica A; Music, Nedzad; Tzeng, Wen-Pin; Holiday, Crystal; Burroughs, Ashley; Sun, Xiangjie; Maines, Taronna R; Levine, Min Z; York, Ian A

    2017-10-15

    Avian influenza viruses of the H7 hemagglutinin (HA) subtype present a significant public health threat, as evidenced by the ongoing outbreak of human A(H7N9) infections in China. When evaluated by hemagglutination inhibition (HI) and microneutralization (MN) assays, H7 viruses and vaccines are found to induce lower level of neutralizing antibodies (nAb) than do their seasonal counterparts, making it difficult to develop and evaluate prepandemic vaccines. We have previously shown that purified recombinant H7 HA appear to be poorly immunogenic in that they induce low levels of HI and MN antibodies. In this study, we immunized mice with whole inactivated reverse genetics reassortant (RG) viruses expressing HA and neuraminidase (NA) from 3 different H7 viruses [A/Shanghai/2/2013(H7N9), A/Netherlands/219/2003(H7N7), and A/New York/107/2003(H7N2)] or with human A(H1N1)pdm09 (A/California/07/2009-like) or A(H3N2) (A/Perth16/2009) viruses. Mice produced equivalent titers of antibodies to all viruses as measured by enzyme-linked immunosorbent assay (ELISA). However, the antibody titers induced by H7 viruses were significantly lower when measured by HI and MN assays. Despite inducing very low levels of nAb, H7 vaccines conferred complete protection against homologous virus challenge in mice, and the serum antibodies directed against the HA head region were capable of mediating protection. The apparently low immunogenicity associated with H7 viruses and vaccines may be at least partly related to measuring antibody titers with the traditional HI and MN assays, which may not provide a true measure of protective immunity associated with H7 immunization. This study underscores the need for development of additional correlates of protection for prepandemic vaccines. IMPORTANCE H7 avian influenza viruses present a serious risk to human health. Preparedness efforts include development of prepandemic vaccines. For seasonal influenza viruses, protection is correlated with antibody

  5. A murine monoclonal anti-idiotypic antibody detects a common idiotope on human, mouse and rabbit antibodies to allergen Lol p IV.

    Science.gov (United States)

    Zhou, E M; Dzuba-Fischer, J M; Rector, E S; Sehon, A H; Kisil, F T

    1991-09-01

    A syngeneic mouse monoclonal anti-idiotypic antibody (anti-Id), designated as B1/1, was generated against a monoclonal antibody (MoAb 91) specific for Ryegrass pollen allergen Lol p IV. This anti-Id recognized an idiotope (Id) that was also present on other monoclonal antibodies with the same specificity as MoAb 91. Observations that (i) the anti-Id inhibited the binding of MoAb 91 to Lol p IV and (ii) the Id-anti-Id interaction could be inhibited by Lol p IV indicated that the Id was located within or near the antigen combining site. These properties served to characterize B1/1 as an internal image anti-Id. Evidence that an immune response in different species to Lol p IV elicits the formation of antibodies which express a common Id was provided by the observations that (i) the Id-anti-Id interactions could be inhibited by mouse, human and rabbit antisera to Lol p IV and (ii) the binding of these antisera to Lol p IV could be inhibited by the anti-Id. Interestingly, the internal image anti-Id B1/1 also recognized an Id on a monoclonal antibody which was directed to an epitope of Lol p IV, different from that recognized by MoAb 91.

  6. [Voltage-Gated Potassium Channel-Complex Antibodies Associated Encephalopathy and Related Diseases].

    Science.gov (United States)

    Watanabe, Osamu

    2016-09-01

    Voltage-gated potassium channel (VGKC) complex antibodies are auto-antibodies, initially identified in acquired neuromyotonia (aNMT; Isaacs' syndrome), which cause muscle cramps and difficulty in opening the palm of the hands. Subsequently, these antibodies were found in patients presenting with aNMT along with psychosis, insomnia, and dysautonomia, collectively termed Morvan's syndrome (MoS), and in a limbic encephalopathy (LE) patient with prominent amnesia and frequent seizures. Typical LE cases have a distinctive adult-onset, frequent, brief dystonic seizure semiology that predominantly affects the arms and ipsilateral face. It has now been termed faciobrachial dystonic seizures (FBDS). The VGKC complex is a group of proteins that are strongly associated in situ and after extraction in the mild detergent digitonin. Recent studies indicated that the VGKC complex antibodies are mainly directed toward associated proteins (for example LGI1, Caspr2) that complex with VGKCs themselves. Patients with aNMT or MoS are most likely to have Caspr2 antibodies, whereas LGI1 antibodies are found characteristically in patients with FBDS and LE. We systematically identified and quantified autoantibodies in patient sera with VGKC-complex antibody associated encephalopathy and showed the relationship between individual antibodies and patient's symptoms. Furthermore, we revealed how autoantibodies disrupt the physiological functions of target proteins. LGI1 antibodies neutralize the interaction between LGI1 and ADAM22, reducing the synaptic AMPA receptors.

  7. Selecting highly structure-specific antibodies using structured synthetic mimics of the cystine knot protein sclerostin

    NARCIS (Netherlands)

    Back, J.W.; Frisch, C.; Van Pee, K.; Boschert, V.; van Vught, R.; Puijk, W.; Mueller, T. D.; Knappik, A.; Timmerman, P.

    2012-01-01

    Antibodies directed against specific regions of a protein have traditionally been raised against full proteins, protein domains or simple unstructured peptides, containing contiguous stretches of primary sequence. We have used a new approach of selecting antibodies against restrained peptides

  8. How immunoglobulin G antibodies kill target cells: revisiting an old paradigm.

    Science.gov (United States)

    Biburger, Markus; Lux, Anja; Nimmerjahn, Falk

    2014-01-01

    The capacity of immunoglobulin G (IgG) antibodies to eliminate virtually any target cell has resulted in the widespread introduction of cytotoxic antibodies into the clinic in settings of cancer therapy, autoimmunity, and transplantation, for example. More recently, it has become apparent that also the protection from viral infection via IgG antibodies may require cytotoxic effector functions, suggesting that antibody-dependent cellular cytotoxicity (ADCC) directed against malignant or virally infected cells is one of the most essential effector mechanisms triggered by IgG antibodies to protect the host. A detailed understanding of the underlying molecular and cellular pathways is critical, therefore, to make full use of this antibody effector function. Several studies over the last years have provided novel insights into the effector pathways and innate immune effector cells responsible for ADCC reactions. One of the most notable outcomes of many of these reports is that cells of the mononuclear phagocytic system rather than natural killer cells are critical for removal of IgG opsonized target cells in vivo. © 2014 Elsevier Inc. All rights reserved.

  9. Stability of monoclonal antibodies at high-concentration

    DEFF Research Database (Denmark)

    Neergaard, Martin S; Nielsen, Anders D; Parshad, Henrik

    2014-01-01

    Few studies have so far directly compared the impact of antibody subclass on protein stability. This case study investigates two mAbs (one IgG1 and one IgG4 ) with identical variable region. Investigations of mAbs that recognize similar epitopes are necessary to identify possible differences betw...

  10. Structure and function of broadly reactive antibody PG16 reveal an H3 subdomain that mediates potent neutralization of HIV-1

    Energy Technology Data Exchange (ETDEWEB)

    Pejchal, Robert; Walker, Laura M.; Stanfield, Robyn L.; Phogat, Sanjay K.; Koff, Wayne C.; Poignard, Pascal; Burton, Dennis R.; Wilson, Ian A. (Scripps); (IAVI)

    2010-11-15

    Development of an effective vaccine against HIV-1 will likely require elicitation of broad and potent neutralizing antibodies against the trimeric surface envelope glycoprotein (Env). Monoclonal antibodies (mAbs) PG9 and PG16 neutralize {approx}80% of HIV-1 isolates across all clades with extraordinary potency and target novel epitopes preferentially expressed on Env trimers. As these neutralization properties are ideal for a vaccine-elicited antibody response to HIV-1, their structural basis was investigated. The crystal structure of the antigen-binding fragment (Fab) of PG16 at 2.5 {angstrom} resolution revealed its unusually long, 28-residue, complementarity determining region (CDR) H3 forms a unique, stable subdomain that towers above the antibody surface. A 7-residue 'specificity loop' on the 'hammerhead' subdomain was identified that, when transplanted from PG16 to PG9 and vice versa, accounted for differences in the fine specificity and neutralization of these two mAbs. The PG16 electron density maps also revealed that a CDR H3 tyrosine was sulfated, which was confirmed for both PG9 (doubly) and PG16 (singly) by mass spectral analysis. We further showed that tyrosine sulfation plays a role in binding and neutralization. An N-linked glycan modification is observed in the variable light chain, but not required for antigen recognition. Further, the crystal structure of the PG9 light chain at 3.0 {angstrom} facilitated homology modeling to support the presence of these unusual features in PG9. Thus, PG9 and PG16 use unique structural features to mediate potent neutralization of HIV-1 that may be of utility in antibody engineering and for high-affinity recognition of a variety of therapeutic targets.

  11. The effect of treatment on the age-antibody relationship in children infected with Schistosoma mansoni and Schistosoma haematobium

    Directory of Open Access Journals (Sweden)

    Mutapi Francisca

    2002-01-01

    Full Text Available The effect of praziquantel treatment on the age-antibody relationship was studied in 174 children aged between 6 and 17 years from a schistosome endemic area in Zimbabwe. The children were co-infected with Schistosoma mansoni and S. haematobium with infection prevalences of 74% and 53% respectively. Antibody levels for the isotypes IgA, IgE, IgM, IgG1, IgG2, IgG3 and IgG4, directed against soluble egg antigen were measured using an indirect ELISA assay. Treatment resulted in a significant increase in levels of IgG2 and IgG3 while levels of IgA decreased significantly. In untreated children there were significant decreases in levels of IgG4. Treatment also resulted in significant alteration in the age-antibody profiles for the isotypes IgE, IgM, IgG1 and IgG2 in treated children but not in untreated children. The results are discussed in the context of factors believed to give rise to the age-antibody relationship; i.e. age-related exposure patterns, age-related development of acquired immunity, age-related hormonal changes and age-related changes in innate susceptibility to infection.

  12. In silico peptide prediction for antibody generation to recognize 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in genetically modified organisms.

    Science.gov (United States)

    Marani, Mariela M; Costa, Joana; Mafra, Isabel; Oliveira, Maria Beatriz P P; Camperi, Silvia A; Leite, José Roberto de Souza Almeida

    2015-03-01

    For the prospective immunorecognition of 5-enolpyruvylshikimate-3-phosphate synthase (CP4-EPSPS) as a biomarker protein expressed by transgenic soybean, an extensive in silico evaluation of the referred protein was performed. The main objective of this study was the selection of a set of peptides that could function as potential immunogens for the production of novel antibodies against CP4-EPSPS protein. For this purpose, the protein was in silico cleaved with trypsin/chymotrypsin and the resultant peptides were extensively analyzed for further selection of the best candidates for antibody production. The analysis enabled the successful proposal of four peptides with potential immunogenicity for their future use as screening biomarkers of genetically modified organisms. To our knowledge, this is the first attempt to select and define potential linear epitopes for the immunization of animals and, subsequently, to generate adequate antibodies for CP4-EPSPS recognition. The present work will be followed by the synthesis of the candidate peptides to be incubated in animals for antibody generation and potential applicability for the development of an immunosensor for CP4-EPSPS detection. © 2015 Wiley Periodicals, Inc.

  13. Characterization of an extracellular epitope antibody to the neuronal K-Cl cotransporter, KCC2.

    Science.gov (United States)

    Gagnon, Kenneth Be; Fyffe, Robert Ew; Adragna, Norma C; Lauf, Peter K

    2007-07-01

    1. Ion gradients across the cell membrane are important for proper cellular communication and homeostasis. With the exception of erythrocytes, chloride (Cl), one of the most important free anions in animal cells, is not distributed at thermodynamic equilibrium across the plasma membrane. The K-Cl cotransporter (COT), consisting of at least four isoforms, utilizes the larger outwardly directed chemical driving force of K to expel Cl from the cell against its inwardly directed chemical gradient and has been implicated recently as one of the main Cl extruders in developing neurons. 2. Previous in situ hybridization studies have indicated widespread mRNA distribution of the neuronal-specific K-Cl COT isoform (KCC2) throughout the rat central nervous system (CNS). However, immunohistochemical studies have been limited owing to the availability of a more selective antibody to KCC2. The goal of the present study was to develop a new molecular tool for the immunohistochemical identification and neuronal distribution of KCC2. 3. Herein, we present evidence of immunohistochemical corroboration of the widespread KCC2 mRNA expression using a novel extracellular anti-peptide antibody directed against the second extracellular loop (ECL2) of KCC2. Immunoperoxidase and immunofluorescent labelling revealed widespread post-synaptic somatic and dendritic localization of KCC2 in multiple neuronal populations in the cerebral cortex, hippocampus, brainstem, lumbar spinal cord and cerebellum. We also demonstrate that binding of the antibody to an extracellular epitope within ECL2 does not alter cotransporter function. In essence, the present study reports on a new molecular tool for structural and functional studies of KCC2.

  14. ISOLATION OF VIRUS IN PRODUCTS OF CONCEPTION IN SPONTANEOUS ABORTION

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    KAMYAB S.D. AZARI S.D. NATEGH R

    1980-08-01

    Full Text Available Two hundred pa t ient s o f t he l ow socioe conomic g roup wi th an aborti on o f the f i r s t t r i mest e r were stud 1ed . The s ter i l e produc ts o f concept ion were c ul t ured on Gr e e n Monke y Kidney ti s sue cul t ure f or v i r a l de t e c - tion. Four po s i t i ve Herpe s virus, two positive Rubella"nv~ru s and t hree pos i tive unidentifie d Entero v irus l ike Vlrus were obtained The sera of 192 of these patients were titrated for Rubella antibody. The sera of a control group of 150 normal pregnant women were titrated for Rubella antibody and 8.8 per cent of these patients had a negative titre and 21.3 per cent , 1 had a tltre over 40

  15. Primary biliary cirrhosis is characterized by IgG3 antibodies cross-reactive with the major mitochondrial autoepitope and its Lactobacillus mimic.

    Science.gov (United States)

    Bogdanos, Dimitrios-Petrou; Baum, Harold; Okamoto, Manabu; Montalto, Paolo; Sharma, Umesh C; Rigopoulou, Eirini I; Vlachogiannakos, John; Ma, Yun; Burroughs, Andrew K; Vergani, Diego

    2005-08-01

    The serological hallmark of primary biliary cirrhosis (PBC) is the presence of pyruvate dehydrogenase complex E2 subunit (PDC-E2) antimitochondrial antibodies (AMAs). Anti-PDC-E2 antibodies cross-react specifically with mycobacterial hsp65, and we have demonstrated that the motif SxGDL[ILV]AE shared by PDC-E2(212-226) and hsp's is a cross-reactive target. Having found that this same motif is present only in beta-galactosidase of Lactobacillus delbrueckii (BGAL LACDE), we hypothesized that this homology would also lead to cross-reactivity. The mimics were tested via ELISA for reactivity and competitive cross-reactivity using sera from 100 AMA-positive and 23 AMA-negative PBC patients and 190 controls. An Escherichia coli (ECOLI) PDC-E2 mimic that has been pathogenetically linked to PBC but lacks this motif has been also tested. Anti-BGAL(266-280) LACDE antibodies were restricted to AMA-positive patients (54 of 95, 57%) and belonged to immunoglobulin (Ig) G3. Of the 190 controls, 22 (12%; P ECOLI PDC-E2 reactivity was virtually absent. BGAL(266-280)/PDC-E2(212-226) reactivity of the IgG3 isotype was found in 52 (52%) AMA-positive PBC patients but in only 1 of the controls (P ECOLI PDC-E2 mimics. In conclusion, IgG3 antibodies to BGAL LACDE cross-react with the major mitochondrial autoepitope and are characteristic of PBC.

  16. Radioimmunoscintigraphy with technetium-99m labelled monoclonal antibody, 1A3, in colorectal cancer

    International Nuclear Information System (INIS)

    Granowska, M.; Britton, K.E.; Mather, S.J.; Morris, G.; Ellison, D.; Soobramoney, S.; Talbot, I.C.; Northover, J.M.A.

    1993-01-01

    This study of radioimmunoscintigraphy (RIS) was primarily undertaken to determine how the imaging results related to surgical findings. Technetium-99m radiolabelled 1A3, a monoclonal antibody against a columnar cell surface antigen, was used. No adverse effects or thyroid uptake was observed in 127 studies. The 85 primary colorectal cancers were all image positive. In the assessment of recurrent tumour in the abdomen or pelvis, the accuracy was 33/35 (94%), including true-positive findings in some whose serum carcinoembryonic antigen was normal. There was a positive predictive value for abdominal or pelvic recurrence of 92% and a negative predictive value of 100%, at a prevalence of 66%. In those patients whose liver was able to be evaluated, the accuracy was 72/79 (91%). There was a positive predictive value for liver metastases of 88% and a negative predictive value of 93%, at a prelavence of 32%. The simple procedure for labelling antibody with 99m Tc and its ready availability allow a completed report to be given within 24 h of the request. (orig.)

  17. Characterization and biodistribution of recombinant and recombinant/chimeric constructs of monoclonal antibody B72.3

    International Nuclear Information System (INIS)

    Colcher, D.; Milenic, D.; Roselli, M.

    1989-01-01

    Radiolabeled B72.3 has been administered both i.v. and i.p. in patients with colorectal and ovarian cancer as well as other carcinomas and has been shown to selectively bind to approximately 70-80% of metastatic lesions. Greater than 50% of the patients that have been treated with B72.3 have developed an immunological response to murine IgG after a single injection. In an attempt to minimize the immune response of these patients to the administered murine monoclonal antibody, we developed a recombinant form of the murine B72.3 as well as a recombinant/chimeric antibody, using the variable regions of the murine B72.3 and human heavy chain (gamma 4) and light chain (kappa) constant regions. We report here that both the recombinant B72.3 [rB72.3] and the recombinant/chimeric B72.3 [cB72.3(gamma 4)] IgGs maintain the tissue binding and idiotypic specificity of the native murine IgG. The native B72.3, rB72.3, and cB72.3(gamma 4) IgGs were radiolabeled and the biodistribution of these IgGs was studied in athymic mice bearing human colon carcinoma xenografts (LS-174T). Differences were observed between the cB72.3(gamma 4) and the native B72.3 in the percentage of injected dose/g that localized in the tumor. The somewhat lower absolute amounts of the cB72.3(gamma 4) in the tumor are mostly likely due to the observed more rapid clearance from the blood and body of the mouse as compared to the native B72.3 and rB72.3. All three forms [native B72.3, rB72.3, and cB72.3(gamma 4)] of the IgG, however, were able to localize the colon tumor with similar radiolocalization indices [percentage of injected dose/g in tumor divided by the percentage of injected dose/g in normal tissue

  18. Comparative efficacy of antigen and antibody detection tests for human trichinellosis

    International Nuclear Information System (INIS)

    Ivanoska, D.; Cuperlovic, K.; Gamble, H.R.; Murrell, K.D.

    1989-01-01

    Sera collected from patients with suspected or confirmed exposure to Trichinella spiralis were tested for circulating parasite antigens and antiparasite antibodies. Using an immunoradiometric assay, excretory--secretory antigens from muscle-stage larvae of T. spiralis were detected in the sera of 47% of 62 patients with clinical trichinellosis and 13% of 39 patients without clinical signs but suspected of exposure to infected meat. In comparison, antibodies were detected using an indirect immunofluorescent test in the circulation of 100% of the 62 patients with clinical trichinellosis and 46% of the 39 patients with suspected exposure. The presence of antibodies specific to excretory-secretory products of T. spiralis muscle larvae was confirmed in the majority of the samples tested by a monoclonal antibody-based competitive inhibition assay. These results indicate that antibody detection is a more sensitive diagnostic method for human trichinellosis, but that antigen detection might be a useful confirmatory test because it is a direct demonstration of parasite products in the circulation

  19. DARPA Antibody Technology Program. Standardized Test Bed for Antibody Characterization: Characterization of an MS2 ScFv Antibody Produced by Illumina

    Science.gov (United States)

    2016-08-01

    ECBC-TR-1395 DARPA ANTIBODY TECHNOLOGY PROGRAM STANDARDIZED TEST BED FOR... ANTIBODY CHARACTERIZATION: CHARACTERIZATION OF AN MS2 SCFV ANTIBODY PRODUCED BY ILLUMINA Patricia E. Buckley Alena M. Calm Heather Welsh Roy...4. TITLE AND SUBTITLE DARPA Antibody Technology Program Standardized Test Bed for Antibody Characterization: Characterization of an MS2 ScFv

  20. RosettaAntibodyDesign (RAbD): A general framework for computational antibody design.

    Science.gov (United States)

    Adolf-Bryfogle, Jared; Kalyuzhniy, Oleks; Kubitz, Michael; Weitzner, Brian D; Hu, Xiaozhen; Adachi, Yumiko; Schief, William R; Dunbrack, Roland L

    2018-04-01

    A structural-bioinformatics-based computational methodology and framework have been developed for the design of antibodies to targets of interest. RosettaAntibodyDesign (RAbD) samples the diverse sequence, structure, and binding space of an antibody to an antigen in highly customizable protocols for the design of antibodies in a broad range of applications. The program samples antibody sequences and structures by grafting structures from a widely accepted set of the canonical clusters of CDRs (North et al., J. Mol. Biol., 406:228-256, 2011). It then performs sequence design according to amino acid sequence profiles of each cluster, and samples CDR backbones using a flexible-backbone design protocol incorporating cluster-based CDR constraints. Starting from an existing experimental or computationally modeled antigen-antibody structure, RAbD can be used to redesign a single CDR or multiple CDRs with loops of different length, conformation, and sequence. We rigorously benchmarked RAbD on a set of 60 diverse antibody-antigen complexes, using two design strategies-optimizing total Rosetta energy and optimizing interface energy alone. We utilized two novel metrics for measuring success in computational protein design. The design risk ratio (DRR) is equal to the frequency of recovery of native CDR lengths and clusters divided by the frequency of sampling of those features during the Monte Carlo design procedure. Ratios greater than 1.0 indicate that the design process is picking out the native more frequently than expected from their sampled rate. We achieved DRRs for the non-H3 CDRs of between 2.4 and 4.0. The antigen risk ratio (ARR) is the ratio of frequencies of the native amino acid types, CDR lengths, and clusters in the output decoys for simulations performed in the presence and absence of the antigen. For CDRs, we achieved cluster ARRs as high as 2.5 for L1 and 1.5 for H2. For sequence design simulations without CDR grafting, the overall recovery for the native

  1. Diagnostic of tumours of epithelial origin with the monoclonal antibody IOR EGF/R3 murino

    International Nuclear Information System (INIS)

    Ramos, M.

    1997-01-01

    Despite of the advantages on anti tumoral therapy, the cancer of epithelial origin constitutes one of the first causes of death worldwide. That kind of tumors have a 10-30-fold overexpression of the epidermal growth factor receptor (EGFr). Monoclonal antibody ior egf/r3 is a lgG2a, which recognizes the epidermal growth factor receptor. The aim of the present work was the evaluate the diagnostic efficacy of the 99m Tc-labelled ior egf/r3 for the detection of epithelial derived tumors, its metastasis and its recurrences

  2. Maternofetal transplacental transport of recombinant IgG antibodies lacking effector functions

    DEFF Research Database (Denmark)

    Mathiesen, Line; Nielsen, Leif K; Andersen, Jan Terje

    2013-01-01

    alloimmunity, which may be lethal. A novel strategy to control pathogenic antibodies would be administration of a non-destructive IgG antibody blocking antigen binding while retaining binding to FcRn. We report on two human IgG3 antibodies with a hinge deletion and a C131S point mutation (IgG3ΔHinge...

  3. The prevalence of ANA antibodies, anticentromere antibodies, and anti-cyclic citrullinated peptide antibodies in patients with primary Sjögren’s syndrome compared to patients with dryness symptoms without primary Sjögren’s syndrome confirmation

    Directory of Open Access Journals (Sweden)

    Maria Maślińska

    2017-07-01

    Full Text Available Objectives : Our study analyses the prevalence of ANA, anti-SS-A, anti-SS-B, and ACA and ACPA antibodies in patients with pSS and with dryness symptoms without pSS confirmation, and the association of ACPA and ACA antibodies with specific clinical symptoms. Materials and methods : 113 patients were divided into two groups: I – with diagnosed pSS (N = 75; and II – with dryness without pSS evidence (N = 38. Diagnostics: indirect immunofluorescence (IF; Hep-2 cell line of antinuclear antibodies (ANA, anti-SS-A anti-SS-B antibodies determined with semi-quantitative method, autoantibody profile (14 antigens, ANA Profil 3 EUROLINE; basic laboratory, ophthalmic examination tests, minor salivary gland biopsy with focus score (FS, joint and lung evaluation, and ESSDAI questionnaire (pSS activity. Results : 88% of group I had ANA antibodies (1 : 320 titre, 5.3% at 1 : 160. Anti-SS-A antibodies were present in 88% of group I, including all ANA 1 : 160. Anti-SS-A antibodies positively correlated with greater and moderate activity of ESSDAI 5 (p = 0.046 and FS. The presence of SS-B antibodies significantly affected disease activity. ACPA present: group I – 13% (associated with higher arthritis incidence; p = 0.003; group II – 8%. ACA antibodies present in 4% of group I, but not in group II. No ACA association with interstitial lung changes (small ACA + group excludes full conclusions. Conclusions : ANA antibodies should also be considered in a titre of less than 1 : 320, but the presence of anti-SS-A antibodies is still the most important immunological marker for pSS. Anti-SS-A antibodies correlate with higher disease activity (ESSDAI ≥ 5 and higher FS. The presence of the anti-SS-B antibody was significantly affected by higher activity of the disease. The incidence of arthritis was higher in patients with ACPA+ pSS compared to ACPA– (p = 0.003. There was no relationship between ACPA and arthritis in patients with dry-type syndrome without

  4. IgE antibodies in toxoplasmosis.

    Science.gov (United States)

    Matowicka-Karna, Joanna; Kemona, Halina

    2014-05-15

    Toxoplasmosis is a worldwide infection caused by the intracellular parasite Toxoplasma gondii. At least a third of the world human population is infected with the parasite, making it one of the most successful parasitic infections. Primary maternal infection may cause health-threatening sequelae for the fetus, or even cause death of the uterus. Reactivation of a latent infection in immune deficiency conditions such as AIDS and organ transplantation can cause fatal toxoplasmic encephalitis. Toxoplasmosis is a major cause of chorioretinitis, especially in individuals with impaired immune systems. In the acute phase, directly after invading the body, T. gondii begins to multiply rapidly. In the majority of cases acquired toxoplasmosis is asymptomatic. In the second week of infection, specific IgM antibodies are present in the blood. IgE antibodies appear at the same time, slightly preceding specific IgA antibodies. The concentration of IgE can be one of the parameters used for diagnosing an infection with T. gondii. Laboratory diagnosis, i.e. IgE and serologic assays, plays the main role in the diagnosis of congenital infection and assists in the confirmatory diagnosis of toxoplasmic encephalitis and ocular toxoplasmosis. This article is a review of IgE in toxoplasmosis.

  5. Preliminary studies of monoclonal antibody lymphoscintigraphy in malignant melanoma

    International Nuclear Information System (INIS)

    Nelp, W.B.; Eary, J.F.; Jones, R.F.; Hellstrom, K.E.; Hellstrom, I.; Beaumier, P.L.; Krohn, K.A.

    1987-01-01

    Lymphoscintigraphy was performed at 3 and 20 hr following subcutaneous injection of 131 I anti-melanoma antibody (Fab) in 11 patients who had surgical resection of lymph nodes (neck, axilla, groin) at 24 hr for suspected metastatic melanoma. Comparable amounts of 125 I nonspecific control antibody (Fab) were co-administered. Six patients had nodal metastases and three showed positive images at both time periods. Five patients had no metastases though one was image positive. Four other nondiseased inguinal node groups were image negative. A total of 28 tumored nodes and 110 normal nodes were removed, counted and histologically examined. All metastatic tumors expressed antigen against which the specific Fab was directed. The concentrations of both specific and nonspecific Fab were similar in tumored nodes and both were significantly greater than in normal nodes showed essentially identical intranodal spatial distribution of the specific and control Fab in areas containing tumor. These preliminary results suggest the increased concentration of murine immunoglobulin (Fab) retained in diseased nodes was a nonspecific phenomenon

  6. Isolation of monoclonal antibodies with predetermined conformational epitope specificity.

    Directory of Open Access Journals (Sweden)

    Anton M Sholukh

    Full Text Available Existing technologies allow isolating antigen-specific monoclonal antibodies (mAbs from B cells. We devised a direct approach to isolate mAbs with predetermined conformational epitope specificity, using epitope mimetics (mimotopes that reflect the three-dimensional structure of given antigen subdomains. We performed differential biopanning using bacteriophages encoding random peptide libraries and polyclonal antibodies (Abs that had been affinity-purified with either native or denatured antigen. This strategy yielded conformational mimotopes. We then generated mimotope-fluorescent protein fusions, which were used as baits to isolate single memory B cells from rhesus monkeys (RMs. To amplify RM immunoglobulin variable regions, we developed RM-specific PCR primers and generated chimeric simian-human mAbs with predicted epitope specificity. We established proof-of-concept of our strategy by isolating mAbs targeting the conformational V3 loop crown of HIV Env; the new mAbs cross-neutralized viruses of different clades. The novel technology allows isolating mAbs from RMs or other hosts given experimental immunogens or infectious agents.

  7. Seroprevalence rates of antibodies againstLeishmania infantum and other protozoan and rickettsial parasites in dogs

    Directory of Open Access Journals (Sweden)

    Silvana de Cássia Paulan

    Full Text Available Canine visceral leishmaniasis (CVL is caused by the protozoan Leishmania infantum, which infects dogs and humans in many regions of Brazil. The present study involved an indirect fluorescent antibody test (IFAT to analyze L. infantum,Ehrlichia spp., Babesia canis,Toxoplasma gondii and Neospora caninuminfection rates in serum samples from 93 dogs in a rural settlement in Ilha Solteira, SP, Brazil. The seroprevalence rates of anti-L. infantum, anti-Ehrlichia, anti-B. canis, anti-T. gondii and anti-N. caninum antibodies were 37.6%, 75.3%, 72%, 47.3% and 6.4%, respectively. In addition to IFAT, direct microscopic examination of popliteal lymph node aspirates revealed 26.9% of CVL positive dogs. Serological tests revealed that 17.2% of the dogs were seropositive for a single parasite, 29% for two parasites, 33% for three, 16.1% for four, and 1.1% for five parasites, while 3.2% were seronegative for five parasites. The presence of antibodies against these parasites in serum samples from dogs confirmed their exposure to these parasites in this rural area. Because of the potential zoonotic risk of these diseases, mainly leishmaniasis, ehrlichiosis and toxoplasmosis, special attention should focus on programs for the improvement of diagnostic assays and control measures against these parasites.

  8. Antiparietal cell antibody test

    Science.gov (United States)

    APCA; Anti-gastric parietal cell antibody; Atrophic gastritis - anti-gastric parietal cell antibody; Gastric ulcer - anti-gastric parietal cell antibody; Pernicious anemia - anti-gastric parietal cell antibody; ...

  9. Antibody responses of ponies to initial and challenge infections of Strongylus vulgaris.

    Science.gov (United States)

    Klei, T R; Chapman, M R; Torbert, B J; McClure, J R

    1983-05-01

    An indirect fluorescent antibody assay (IFA) was developed using Strongylus vulgaris third stage larvae (L3) as antigens. Observations using the IFA indicate that a species-specific antibody response to S. vulgaris L3 develops in S. vulgaris-infected ponies and that some surface L3 antigens are shared by adult worms. Sequential antibody levels against S. vulgaris were measured in strongyle-naive and in immune ponies following initial and challenge infections using the IFA and an indirect hemagglutination assay (IHA). Antibody levels measured by IFA increased faster following initial infections than did levels measured by IHA. Antibody levels appear to increase following challenge infections of immune ponies when measured with the IFA, but not with the IHA. Significant differences in antibody titers were not seen between ponies which developed colic following challenge infections and those that did not develop colic. Antibodies were not detectable in ponies unexposed to larval migrations, but which received surgical implantation of S. vulgaris adults into the cecum.

  10. Targeting of indium 111-labeled bivalent hapten to human melanoma mediated by bispecific monoclonal antibody conjugates: Imaging of tumors hosted in nude mice

    International Nuclear Information System (INIS)

    Le Doussal, J.M.; Gruaz-Guyon, A.; Martin, M.; Gautherot, E.; Delaage, M.; Barbet, J.

    1990-01-01

    Antibody conjugates were prepared by coupling F(ab')2 or Fab' fragments of an antibody specific for the human high molecular weight-melanoma associated antigen to Fab' fragments of an antibody specific for indium-diethylenetriaminepentaacetate complexes. Monovalent and bivalent haptens were synthesized by reacting the dipeptide tyrosyl-lysine with diethylenetriaminepentaacetic cyclic anhydride. In vitro, the antibody conjugate mediated binding of the 111In-labeled haptens to melanoma cells. In vivo, it allowed specific localization of the haptens in A375 tumors. The bivalent hapten exhibited much higher efficiency at targeting 111In onto cells, both in vitro and in vivo. Antibody conjugate and hapten doses (2 micrograms and 1 pmol, respectively) and the delay between antibody conjugate and tracer injections (24 h) were adjusted to maximize tumor uptake (4% injected dose/g) and tumor to normal tissue contrast (greater than 3) obtained 3 h after injection of the 111In-labeled bivalent hapten. This two-step technique, when compared to direct targeting of 111In-labeled F(ab')2 fragments, provided lower localization of injected activity into the tumor (x 0.25), but higher tumor/tissue ratios, especially with respect to liver (x 7), spleen (x 8), and kidneys (x 10). In addition, high contrast images were obtained within 3 hours, instead of days. Thus, antibody conjugate-mediated targeting of small bivalent haptens, labeled with short half-life isotopes, is proposed as a general method for improving tumor radioimmunolocalization

  11. Demonstration of two distinct antigenic determinants on hepatitis B e antigen by monoclonal antibodies

    International Nuclear Information System (INIS)

    Imai, M.; Nomura, M.; Gotanda, T.; Sano, T.; Tachibana, K.; Miyamoto, H.; Takahashi, K.; Toyama, S.; Miyakawa, Y.; Mayumi, M.

    1982-01-01

    Mice were immunized against hepatitis B e antigen (HBeAg) isolated from sera of asymptomatic carriers of hepatitis B virus. Their spleen cells were fused with mouse myeloma (NS-1) cells, and 5 clones of hybridoma cells secreting antibody against HBeAg (anti-HBe) were isolated. For the production of anti-HBe in large scale, cells were cultivated both in vitro and in the peritoneal cavity of ascitic mice. Although monoclonal antibodies produced by these clones showed a strong reactivity of anti-HBe in hemagglutination tests, individual monoclonal anti-HBe did not reveal any precipitin line in immunodiffusion. When 2 of the 5 monoclonal antibodies were mixed together, however, some combinations showed a precipitin line against HBeAg, whereas others did not. Utilizing solid-phase radioimmunoassay involving a number of combinations of monoclonal antibodies used for solid-phase and radiolabeling, the 5 antibodies were classified into 2 groups. Three of the anti-HBe antibodies were found to be directed to 1 determinant of HBeAg (determinant a); the remaining 2 to the other determinant (determinant b). Determinants a and b were detected on HBeAg in the serum, as well as on the polypeptide of 19,000 daltons (P19) derived from the nucleocapsid of hepatitis B virus. Monoclonal anti-HBe antibodies with different specificities may provide useful tools in delineating the antigenic structure of HBeAg and also in evaluating immune responses of the host directed to its subdeterminants

  12. Highly multiplexed and quantitative cell-surface protein profiling using genetically barcoded antibodies.

    Science.gov (United States)

    Pollock, Samuel B; Hu, Amy; Mou, Yun; Martinko, Alexander J; Julien, Olivier; Hornsby, Michael; Ploder, Lynda; Adams, Jarrett J; Geng, Huimin; Müschen, Markus; Sidhu, Sachdev S; Moffat, Jason; Wells, James A

    2018-03-13

    Human cells express thousands of different surface proteins that can be used for cell classification, or to distinguish healthy and disease conditions. A method capable of profiling a substantial fraction of the surface proteome simultaneously and inexpensively would enable more accurate and complete classification of cell states. We present a highly multiplexed and quantitative surface proteomic method using genetically barcoded antibodies called phage-antibody next-generation sequencing (PhaNGS). Using 144 preselected antibodies displayed on filamentous phage (Fab-phage) against 44 receptor targets, we assess changes in B cell surface proteins after the development of drug resistance in a patient with acute lymphoblastic leukemia (ALL) and in adaptation to oncogene expression in a Myc-inducible Burkitt lymphoma model. We further show PhaNGS can be applied at the single-cell level. Our results reveal that a common set of proteins including FLT3, NCR3LG1, and ROR1 dominate the response to similar oncogenic perturbations in B cells. Linking high-affinity, selective, genetically encoded binders to NGS enables direct and highly multiplexed protein detection, comparable to RNA-sequencing for mRNA. PhaNGS has the potential to profile a substantial fraction of the surface proteome simultaneously and inexpensively to enable more accurate and complete classification of cell states. Copyright © 2018 the Author(s). Published by PNAS.

  13. Polyclonal antibodies for the detection of Trypanosoma cruzi circulating antigens.

    Directory of Open Access Journals (Sweden)

    Edith S Málaga-Machaca

    2017-11-01

    Full Text Available Detection of Trypanosoma cruzi antigens in clinical samples is considered an important diagnostic tool for Chagas disease. The production and use of polyclonal antibodies may contribute to an increase in the sensitivity of immunodiagnosis of Chagas disease.Polyclonal antibodies were raised in alpacas, rabbits, and hens immunized with trypomastigote excreted-secreted antigen, membrane proteins, trypomastigote lysate antigen and recombinant 1F8 to produce polyclonal antibodies. Western blot analysis was performed to determine specificity of the developed antibodies. An antigen capture ELISA of circulating antigens in serum, plasma and urine samples was developed using IgY polyclonal antibodies against T. cruzi membrane antigens (capture antibody and IgG from alpaca raised against TESA. A total of 33 serum, 23 plasma and 9 urine samples were analyzed using the developed test. Among serum samples, compared to serology, the antigen capture ELISA tested positive in 55% of samples. All plasma samples from serology positive subjects were positive in the antigen capture ELISA. All urine positive samples had corresponding plasma samples that were also positive when tested by the antigen capture ELISA.Polyclonal antibodies are useful for detection of circulating antigens in both the plasma and urine of infected individuals. Detection of antigens is direct evidence of the presence of the parasite, and could be a better surrogate of current infection status.

  14. The HIV-1 V3 domain on field isolates: participation in generation of escape virus in vivo and accessibility to neutralizing antibodies

    DEFF Research Database (Denmark)

    Arendrup, M; Akerblom, L; Heegaard, P M

    1995-01-01

    The V3 domain is highly variable and induces HIV neutralizing antibodies (NA). Here we addressed the issues of 1) the participation of mutations in V3 in generation of neutralization resistant escape virus in vivo and 2) the applicability of synthetic V3 peptides corresponding to field isolates...... patterns against V3 peptides corresponding to sequential primary and escape field isolates, with the strongest reactivity against late isolated escape virus. These observations suggest that the neutralization epitope was influenced by the appearance of mutations. When used as immunogen in rabbits, V3...... to induce neutralizing immune sera. Seven peptides corresponding to the V3 region of primary and escape virus from 3 HIV-1 infected patients were synthesized and used for antibody (Abs) studies and immunizations. The anti-V3 Abs titre in patient serum was generally low against peptides corresponding...

  15. Hemolytic disease of the fetus and newborn due to anti-Ge3: combined antibody-dependent hemolysis and erythroid precursor cell growth inhibition.

    Science.gov (United States)

    Blackall, Douglas P; Pesek, Gina D; Montgomery, Matthew M; Oza, Krishna K; Arndt, Patricia A; Garratty, George; Shahcheraghi, Ali; Denomme, Gregory A

    2008-10-01

    The Gerbich (Ge) antigens are a collection of high-incidence antigens carried on the red blood cell membrane glycoproteins, glycophorins C and D. Antibodies against these antigens are uncommon, and there have been only rare case reports of hemolytic disease of the fetus and newborn due to anti-Ge. In this case report, we present a neonate with severe anemia and hyperbilirubinemia due to anti-Ge3. Routine and special laboratory studies undertaken in this case suggested two mechanisms for the patient's hemolysis and persistent anemia. Antibody-dependent hemolysis was associated with early-onset hyperbilirubinemia, anemia, and a mild reticulocytosis, and inhibition of erythroid progenitor cell growth was associated with late anemia and normal bilirubin and reticulocyte values. Though rare, anti-Ge3 can be a dangerous antibody in pregnancy. Affected neonates may require intensive initial therapy and close follow-up for at least several weeks after delivery.

  16. Impact of pretransplant anti-HLA antibodies on outcomes in lung transplant candidates.

    Science.gov (United States)

    Kim, Miae; Townsend, Keri R; Wood, Isabelle G; Boukedes, Steve; Guleria, Indira; Gabardi, Steven; El-Chemaly, Souheil; Camp, Phillip C; Chandraker, Anil K; Milford, Edgar L; Goldberg, Hilary J

    2014-05-15

    The prevalence of anti-HLA antibodies in lung transplant candidates and their impact on waitlist and transplant outcomes is not known. We examined the prevalence of pretransplant anti-HLA antibodies at varying thresholds and evaluated their impact on outcomes before and after lung transplantation. We performed a single-center retrospective cohort study including all patients listed for lung transplantation between January 2008 and August 2012. Per protocol, transplant candidates were assessed by solid phase LABscreen mixed Class I and II and LABscreen Single Antigen assays. Among 224 patients, 34% had anti-HLA antibodies at mean fluorescent intensity (MFI) greater than or equal to 3,000 (group III), and 24% had antibodies at MFI 1,000 to 3,000 (group II). Ninety percent of the patients with pretransplant anti-HLA antibodies had class I antibodies, whereas only seven patients developed class II alone. Patients in group III were less likely to receive transplants than patients without any anti-HLA antibodies (group I) (45.5 vs. 67.7%, P = 0.005). Wait time to transplant was longer in group III than group I, although this difference did not meet statistical significance, and waitlist mortality was similar. Among transplant recipients, antibody-mediated rejection (AMR) was more frequent in group III than in group II (20% vs. 0%, P = 0.01) or group I (6.3%, P = 0.05). The presence of anti-HLA antibodies at the high MFI threshold (>3,000) was associated with lower transplant rate and higher rates of AMR. Screening for anti-HLA antibodies using the 3,000 MFI threshold may be important in managing transplant candidates and recipients.

  17. Screening for single-chain variable fragment antibodies against multiple Cry1 toxins from an immunized mouse phage display antibody library.

    Science.gov (United States)

    Dong, Sa; Bo, Zongyi; Zhang, Cunzheng; Feng, Jianguo; Liu, Xianjin

    2018-04-01

    Single-chain variable fragment (scFv) is a kind of antibody that possess only one chain of the complete antibody while maintaining the antigen-specific binding abilities and can be expressed in prokaryotic system. In this study, scFvs against Cry1 toxins were screened out from an immunized mouse phage displayed antibody library, which was successfully constructed with capacity of 6.25 × 10 7  CFU/mL. Using the mixed and alternative antigen coating strategy and after four rounds of affinity screening, seven positive phage-scFvs against Cry1 toxins were selected and characterized. Among them, clone scFv-3H9 (MG214869) showing relative stable and high binding abilities to six Cry1 toxins was selected for expression and purification. SDS-PAGE indicated that the scFv-3H9 fragments approximately 27 kDa were successfully expressed in Escherichia coli HB2151 strain. The purified scFv-3H9 was used to establish the double antibody sandwich enzyme-linked immunosorbent assay method (DAS-ELISA) for detecting six Cry1 toxins, of which the lowest detectable limits (LOD) and the lowest quantitative limits (LOQ) were 3.14-11.07 and 8.22-39.44 ng mL -1 , respectively, with the correlation coefficient higher than 0.997. The average recoveries of Cry1 toxins from spiked rice leaf samples were ranged from 84 to 95%, with coefficient of variation (CV) less than 8.2%, showing good accuracy for the multi-residue determination of six Cry1 toxins in agricultural samples. This research suggested that the constructed phage display antibody library based on the animal which was immunized with the mixture of several antigens under the same category can be used for the quick and effective screening of generic antibodies.

  18. DEVELOPMENT OF MONOCLONAL ANTIBODIES AGAINST FATHEAD MINNOW (PIMEPHALES PROMELAS) VITELLOGENIN

    Science.gov (United States)

    We have obtained a panel of monoclonal antibodies directed against fathead minnow vitellogenin (Vtg) for use in sensitive ELISAs to quantify the response of exposure in vivo to estrogen or estrogen mimics.

  19. Generation, Characterization and Application of Antibodies Directed against HERV-H Gag Protein in Colorectal Samples.

    Science.gov (United States)

    Mullins, Christina S; Hühns, Maja; Krohn, Mathias; Peters, Sven; Cheynet, Valérie; Oriol, Guy; Guillotte, Michèle; Ducrot, Sandrine; Mallet, François; Linnebacher, Michael

    2016-01-01

    A substantial part of the human genome originates from transposable elements, remnants of ancient retroviral infections. Roughly 8% of the human genome consists of about 400,000 LTR elements including human endogenous retrovirus (HERV) sequences. Mainly, the interplay between epigenetic and post-transcriptional mechanisms is thought to silence HERV expression in most physiological contexts. Interestingly, aberrant reactivation of several HERV-H loci appears specific to colorectal carcinoma (CRC). The expression of HERV-H Gag proteins (Gag-H) was assessed using novel monoclonal mouse anti Gag-H antibodies. In a flow cytometry screen four antibody clones were tested on a panel of primary CRC cell lines and the most well performing ones were subsequently validated in western blot analysis. Finally, Gag-H protein expression was analyzed by immune histology on cell line cytospins and on clinical samples. There, we found a heterogeneous staining pattern with no background staining of endothelial, stromal and infiltrating immune cells but diffuse staining of the cytoplasm for positive tumor and normal crypt cells of the colonic epithelium. Taken together, the Gag-H antibody clone(s) present a valuable tool for staining of cells with colonic origin and thus form the basis for future more detailed investigations. The observed Gag-H protein staining in colonic epithelium crypt cells demands profound analyses of a potential role for Gag-H in the normal physiology of the human gut.

  20. Protective measures and human antibody response during an avian influenza H7N3 outbreak in poultry in British Columbia, Canada.

    Science.gov (United States)

    Skowronski, Danuta M; Li, Yan; Tweed, S Aleina; Tam, Theresa W S; Petric, Martin; David, Samara T; Marra, Fawziah; Bastien, Nathalie; Lee, Sandra W; Krajden, Mel; Brunham, Robert C

    2007-01-02

    In 2004 an outbreak of avian influenza of the H7N3 subtype occurred among poultry in British Columbia, Canada. We report compliance with recommended protective measures and associated human infections during this outbreak. We sought voluntary participation by anyone (cullers, farmers and their families) involved in efforts to control the poultry outbreak. Recruitment was by advertisements at the worker deployment site, in local media and through newsletters sent directly to farmers. Sera were tested for antibody to H7N3 by microneutralization assay. A subset of 16 sera (including convalescent sera from 2 unprotected workers with conjunctivitis from whom virus had been isolated) was further tested by Western blot and routine and modified hemagglutination inhibition assays. A total of 167 people (20% to 25% of all workers) participated between May 7 and July 26, 2004. Of these, 19 had experienced influenza-like illness and 21 had experienced red or watery eyes. There was no significant association between illness reports and exposure to infected birds. Among 65 people who entered barns with infected birds, 55 (85%) had received influenza vaccine, 48 (74%) had received oseltamivir, and 55 (85%), 54 (83%) and 36 (55%) reported always wearing gloves, mask or goggles, respectively. Antibody to the H7 subtype was not detected in any sera. During the BC outbreak, compliance with recommended protective measures, especially goggles, was incomplete. Multiple back-up precautions, including oseltamivir prophylaxis, may prevent human infections and should be readily accessible and consistently used by those involved in the control of future outbreaks of avian influenza in poultry. Localized human avian influenza infections may not result in serologic response despite confirmed viral detection and culture.

  1. Anti-insulin antibody test

    Science.gov (United States)

    Insulin antibodies - serum; Insulin Ab test; Insulin resistance - insulin antibodies; Diabetes - insulin antibodies ... Normally, there are no antibodies against insulin in your blood. ... different laboratories. Some labs use different measurements or ...

  2. A review of human anti-globulin antibody (HAGA, HAMA, HACA, HAHA) responses to monoclonal antibodies. Not four letter words.

    Science.gov (United States)

    Mirick, G R; Bradt, B M; Denardo, S J; Denardo, G L

    2004-12-01

    The United States Food and Drug Administration (FDA) has approved unconjugated monoclonal antibodies (MAbs) for immunotherapy (IT) of B-cell lymphoma, breast cancer and acute myeloid leukemia. More recently, approval has been given for conjugated ZevalinTM ((90)yttrium ibritumomab tiuxetan, IDEC-Y2B8, Biogen Idec, Cambridge, MA) and BexxarTM ((131)I-tositumomab, Corixa, Corp., Seattle, WA and GlaxoSmithKline, Philadelphia, PA) anti-CD20 MAbs for use in radioimmunotherapy (RIT) of non-Hodgkin's lymphoma (NHL), thus redefining the standard care of cancer patients. Because of, and despite a lack of basis for concern about allergic reactions due to human antibody responses to these foreign proteins, assays were developed to determine HAGA (human anti-globulin antibody) levels that developed in patient sera following treatment with MAbs. Strategies were also devised to ''humanize'' MAbs and to temporarily block patient immune function with drugs in order to decrease the seroconversion rates, with considerable success. On the other hand, a survival advantage has been observed in some patients who developed a HAGA following treatment. This correlates with development of an anti-idiotype antibody cascade directed toward the MAbs used to treat these patients. What follows is a selective review of HAGA and its effect on cancer treatment over the past 2 decades.

  3. A review of human anti-globulin antibody (HAGA, HAMA, HACA, HAHA) responses to monoclonal antibodies. Not four letter words

    International Nuclear Information System (INIS)

    Mirick, G. R.; Bradt, B. M.; Denardo, S. J.; Denardo, G. L.

    2004-01-01

    The United States Food and Drugs Administration (FDA) has approved unconjugated monoclonal antibodies (MAbs) for immunotherapy (IT) of B-cell lymphoma, breast cancer and acute myeloid leukemia. More recently, approval has been given for conjugated ZevalinTM ( 9 0yttrium ibritumomab tiuxetan, IDEC-Y2B8, Biogen Idec, Cambridge, MA) and BexxarTM ( 1 31I-tositumomab, Corixa, Corp., Seattle, WA and GlaxoSmithKline, Philadelphia, PA) antiCD20 MAns for use in radioimmunotherapy (RIT) of non-Hodgikin's lymphoma (NHL), thus redefining the standard care of cancer patients. Because of, and despite a lack of basis for concern about allergic reactions due to human antibody responses to these foreign proteins, essays were developed to determine HAGE (human anti-globulin antibody) levels that developed in patient sera following treatment with MAbs. Strategies were also devised to humanize MAbs and to temporarily block patient immune function with drugs in order to decrease the seroconversion rates, with considerable success. On the other hand, a survival advantage has been observed in some patients who developed a HAGA following treatment. This correlates with development of an anti-idiotype antibody cascade directed toward the MAbs used to treat these patients. What follows is a selective review of HAGA and its effect on cancer treatment over the past 2 decades

  4. Antibody responses to a major Pneumocystis carinii antigen in human immunodeficiency virus-infected patients with and without P. carinii pneumonia

    DEFF Research Database (Denmark)

    Lundgren, Bettina; Lundgren, Jens Dilling; Nielsen, T

    1992-01-01

    of pulmonary symptoms. Significantly more patients with P. carinii pneumonia (PCP) had detectable antibodies compared with HIV-infected patients without PCP and with HIV-negative controls (50 [66%] of 76 vs. 18 [34%] of 53 and 7 [35%] of 20, respectively; P less than .001), and the level of antibody response......Antibody responses to a major purified human Pneumocystis carinii surface antigen (gp95) were determined by ELISA in human immunodeficiency virus (HIV)-infected patients. Serum IgG directed against gp95 was measured in 129 consecutive HIV-infected patients who underwent bronchoscopy for evaluation...... response, compared with only 1 (3%) of 31 patients without PCP (P less than .001). This patient had PCP on the basis of clinical criteria, including response to therapy. Thus, despite severe immunosuppression, a proportion of HIV-infected patients with PCP can mount a specific IgG-mediated antibody...

  5. Imaging thrombus with radiolabelled monoclonal antibody to platelets

    Energy Technology Data Exchange (ETDEWEB)

    Peters, A.M.; Lavender, J.P.; Needham, S.G.; Loutfi, I.; Snook, D.; Epenetos, A.A.; Lumley, P.; Keery, R.J.; Hogg, N.

    1986-12-13

    A study was conducted evaluating a method of imaging thrombus with platelets radiolabelled with a /sup 111/In labelled monoclonal antibody, P256, directed to the platelet surface glycoprotein complex IIb/IIIa. when the number of receptors occupied by P256 was less than 3% of the total available on the platelet surface, platelet function was undisturbed. P256 was radiolabelled with /sup 111/In using diethylenetriaminepenta-acetic acid, which achieved a specific activity of 185 MBq (5 mCi)/mg. No impairment of immunoreactivity was detected at this specific activity. Platelets were labelled with radiolabelled monoclonal antibody in vitro in two patients at a receptor occupancy of 6% and in vivo in six patients at a receptor occupancy of 1%. In vivo recovery and biodistribution kinetics suggested that after in vitro labelling platelets were minimally activated. The /sup 111/In kinetics recorded after intravenous P256 suggested rapid and efficient radiolabelling of platelets and gave no indication of platelet activation. Of the six patients who received intravenous P256, three had documented thrombus, two of whom gave positive results on P256 platelet scintigraphy. The third had chronic deep venous thrombosis and was scintigraphically negative.

  6. Hepatitis A virus antibody

    International Nuclear Information System (INIS)

    Novak, J.; Kselikova, M.; Urbankova, J.

    1980-01-01

    A description is presented of a radioimmunoassay designed to prove the presence of the antibody against the hepatitis A virus (HA Ab, anti-Ha) using an Abbott HAVAB set. This proof as well as the proof of the antibody against the nucleus of the hepatitis B virus is based on competition between a normal antibody against hepatitis A virus and a 125 I-labelled antibody for the binding sites of a specific antigen spread all over the surface of a tiny ball; this is then indirect proof of the antibody under investigation. The method is described of reading the results from the number of impulses per 60 seconds: the higher the titre of the antibody against the hepatitis A virus in the serum examined, the lower the activity of the specimen concerned. The rate is reported of incidence of the antibody against the hepatitis A virus in a total of 68 convalescents after hepatitis A; the antibody was found in 94.1%. The immunoglobulin made from the convalescents' plasma showed the presence of antibodies in dilutions as high as 1:250 000 while the comparable ratio for normal immunoglobulin Norga was only 1:2500. Differences are discussed in the time incidence of the antibodies against the hepatitis A virus, the antibodies against the surface antigen of hepatitis B, and the antibody against the nucleus of the hepatitis V virus. (author)

  7. Anti-Ma2 antibody related paraneoplastic limbic/brain stem encephalitis associated with breast cancer expressing Ma1, Ma2, and Ma3 mRNAs.

    Science.gov (United States)

    Sahashi, K; Sakai, K; Mano, K; Hirose, G

    2003-09-01

    A 69 year old woman presented with cognitive impairment and supranuclear gaze palsy caused by paraneoplastic limbic/brain stem encephalitis associated with atypical medullary breast carcinoma. The cerebrospinal fluid from the patient harboured an anti-neuronal cell antibody against Ma2 antigen, but not against Ma1 or Ma3 antigen. Despite the antibody being restricted to the Ma2 antigen, the patient's cancer tissue expressed Ma1, Ma2, and Ma3 mRNAs. These results, and the expression of Ma2 mRNA in an atypical medullar breast carcinoma in another patient without paraneoplastic encephalitis, indicate that the induction of anti-Ma2 antibody depends on host immunoreponsiveness and not on the presence of the antigen itself in the cancer.

  8. Replacement of Antibodies in Pseudo-ELISAs: Molecularly Imprinted Nanoparticles for Vancomycin Detection.

    Science.gov (United States)

    Canfarotta, Francesco; Smolinska-Kempisty, Katarzyna; Piletsky, Sergey

    2017-01-01

    The enzyme-linked immunosorbent assay (ELISA) is a widely employed analytical test used to quantify a given molecule. It relies on the use of specific antibodies, linked to an enzyme, to target the desired molecule. The reaction between the enzyme and its substrate gives rise to the analytical signal that can be quantified. Thanks to their robustness and low cost, molecularly imprinted polymer nanoparticles (nanoMIPs) are a viable alternative to antibodies. Herein, we describe the synthesis of nanoMIPs imprinted for vancomycin and their subsequent application in an ELISA-like format for direct replacement of antibodies.

  9. A monoclonal antibody against PDGF B-chain inhibits PDGF-induced DNA synthesis in C3H fibroblasts and prevents binding of PDGF to its receptor.

    Science.gov (United States)

    Vassbotn, F S; Langeland, N; Hagen, I; Holmsen, H

    1990-09-01

    A monoclonal antibody (MAb 6D11) against platelet-derived growth factor (PDGF) was studied. We found that the MAb 6D11 in concentrations equimolar to PDGF blocked the [3H]thymidine incorporation in C3H/10T1/2 C18 fibroblasts stimulated by PDGF B-B and PDGF A-B. This inhibition was overcome by high doses of PDGF. The [3H]thymidine incorporation stimulated by other growth factors (aFGF, bFGF and bombesin) was not inhibited by the antibody. The MAb 6D11 blocked receptor binding of PDGF B-B, but not PDGF A-A. These findings suggest that the MAb 6D11 abolishes PDGF-induced DNA synthesis by blocking PDGF receptor binding. In this communication we demonstrate an isoform-specific monoclonal antibody against PDGF.

  10. High-throughput immunoturbidimetric assays for in-process determination of polyclonal antibody concentration and functionality in crude samples

    DEFF Research Database (Denmark)

    Bak, Hanne; Kyhse-Andersen, J.; Thomas, O.R.T.

    2007-01-01

    We present fast, simple immunoturbidimetric assays suitable for direct determination of antibody 'concentration' and 'functionality' in crude samples, such as in-process samples taken at various stages during antibody purification. Both assays display excellent linearity and analytical recovery. ...... antibodies, require only basic laboratory equipment, are robust, fast, cheap, easy to perform, and readily adapted to automation....

  11. Antibody induction versus placebo, no induction, or another type of antibody induction for liver transplant recipients

    DEFF Research Database (Denmark)

    Penninga, Luit; Wettergren, André; Wilson, Colin H

    2014-01-01

    . All 19 trials were with high risk of bias. Of the 19 trials, 16 trials were two-arm trials, and three trials were three-arm trials. Hence, we found 25 trial comparisons with antibody induction agents: interleukin-2 receptor antagonist (IL-2 RA) versus no induction (10 trials with 1454 participants....... Furthermore, serum creatinine was statistically significantly higher when T-cell specific antibody induction was compared with no induction (MD 3.77 μmol/L, 95% CI 0.33 to 7.21; low-quality evidence), as well as when polyclonal T-cell specific antibody induction was compared with no induction, but this small...... T-cell specific antibody induction, drug-related adverse events were less common among participants treated with interleukin-2 receptor antagonists (RR 0.23, 95% CI 0.09 to 0.63; low-quality evidence), but this was caused by the results from one trial, and trial sequential analysis could not exclude...

  12. Radioimmunoassay of IgM, IgG, and IgA brucella antibodies

    International Nuclear Information System (INIS)

    Parrett, D.; Nielson, K.H.; White, R.G.; Payne, D.J.H.

    1977-01-01

    A radioimmunoassay (R.I.A.) has been devised to measure the serum antibody against Brucella abortus in each of the immunoglobulin classes IgM, IgG, and IgA. This test was applied to 46 sera from individuals with various clinical types of brucellosis, and the results were compared with the results of conventional direct and indirect agglutination and complement-fixation tests. The R.I.A. provided a highly sensitive primary-type assay which avoided the difficulties with blocking or non-agglutinating antibody, and thus has many advantages in the diagnosis of acute and chronic stages of brucella infection in man. The R.I.A. was successful in detection of antibody in many instances in which conventional serological tests were negative, and such antibody could (if IgM) be associated with acute or (if IgG or IgA) with chronic cases of brucellosis. One case in which B.abortus was isolated by blood culture but which failed to yield antibody by conventional tests, nevertheless showed substantial levels of IgM and IgG antibody by R.I.A. In other cases the R.I.A. test helped to eliminate the diagnosis of brucellosis by revealing absent or low antibody levels. (author)

  13. Nuclear medicine: Monoclonal antibodies

    International Nuclear Information System (INIS)

    Endo, K.; Sakahara, H.; Koizumi, M.; Kawamura, Y.; Torizuka, K.; Yokoyama, A.

    1986-01-01

    Antitumor monoclonal antibody was successfully labeled with Tc-99m by using dithiosemicarbazone (DTS) as a bifunctional chelating agent. In the first step, DTS was coupled to antibody without loss of immunoreactivity; the compound then efficiently formed a neutral 1:1 chelate with pentavalent or tetravalent Tc-99m. Imaging with Tc-99m-labeled monoclonal antibody to human osteosarcoma (OST-7) clearly displayed a small tumor in nude mice at 6 and 24 hours after intravenous administration. The tumor-to-blood ratio of the Tc-99m-labeled monoclonal antibody was higher than that of a radioiodinated antibody and similar to that of an In-111-labeled antibody. Thus, conjugation of DTS to monoclonal antibody followed by radiometalation is a simple and efficient method of preparing Tc-99m-labeled monoclonal antibody

  14. Antibodies to UV irradiated DNA: the monitoring of DNA damage by ELISA and indirect immunofluorescence

    Energy Technology Data Exchange (ETDEWEB)

    Wani, A A; Gibson-D' Ambrosio, R E; D' Ambrosio, S M [Ohio State Univ., Columbus (USA). Dept. of Radiology

    1984-10-01

    The enzyme-linked immunosorbant assay (ELISA) was modified to (1) characterize antibodies raised in rabbits against UV-irradiated single-stranded DNA (UVssDNA) complexed with methylated BSA and (2) directly detect pyrimidine dimers in irradiated DNA. The antisera specifically bound to UVssDNA, UVpoly(dT) and to a limited extent to UVdsDNA and UVpoly(dC). Fifty per cent of the maximum antibody binding was observed at a 1-5000 dilution against UVssDNA. Binding to ssDNA and poly(dT) was observed only at much higher concentrations of antibody, whereas no binding to double stranded DNA (dsDNA) was observed. The extent of binding of the antibody was dependent on the UV dose to DNA and the concentration of antigen immobilized on the plate. The ability of various irradiated molecules, DNA, homopolymers and linkers to act as inhibitors of antibody binding establishes that the antigenic determinants are mainly thymine homodimers with lower affinity for cytosine dimers. Potential usefulness of the antibodies to directly quantitate pyrimidine dimers in cells exposed to UV radiation was determined by indirect immunofluorescence. Flow cytometric analysis of immunostained human lymphocytes irradiated with 254 nm radiation indicated that greater than 50% of the population had significantly higher fluorescent intensity than unirradiated cells.

  15. Pichia pastoris-Expressed Bivalent Virus-Like Particulate Vaccine Induces Domain III-Focused Bivalent Neutralizing Antibodies without Antibody-Dependent Enhancement in Vivo

    OpenAIRE

    Rahul Shukla; Ravi K. Rajpoot; Upasana Arora; Ankur Poddar; Sathyamangalam Swaminathan; Navin Khanna; Navin Khanna; Navin Khanna

    2018-01-01

    Dengue, a significant public health problem in several countries around the world, is caused by four different serotypes of mosquito-borne dengue viruses (DENV-1, -2, -3, and -4). Antibodies to any one DENV serotype which can protect against homotypic re-infection, do not offer heterotypic cross-protection. In fact, cross-reactive antibodies may augment heterotypic DENV infection through antibody-dependent enhancement (ADE). A recently launched live attenuated vaccine (LAV) for dengue, which ...

  16. Making Recombinant Monoclonal Antibody And Radiolabelling For Medical Purpose

    International Nuclear Information System (INIS)

    Nguyen Thi Thu; Duong Van Dong; Vo Thi Cam Hoa; Bui Van Cuong; Chu Van Khoa; Vu Bich Huong; Le Quang Huan

    2008-01-01

    Recombinant monoclonal antibody labeling with 131 I specific to tumor cell has been studied and prepared for treatment of Hodgkin lymphoma. In this study, a recombinant monoclonal antibody with two specific properties is a hybrid molecule created by coupling an antibody variable fragments with peptide melittin. The gene coding the antibody fragment has been obtained from human synthetic Fv libraries using for panning and screening on populations of lymphocytes fragmented from human blood cells with Hodgkin diseases. The gene encoding peptit melittin has been cloned from honeybee Apis cerana DNA. The gene coding recombinant monoclonal antibody has been expressed in E.coli BL21 (DE3) at 37 o C and was induced with 0.6 mM IPTG. The recombinant compound has been purified by affinity chromatography with HiTrap affinity column. The obtained recombinant monoclonal antibody has showed cytolytic activities when added to cell culture medium for LU cancer cell line with the amount of 100 - 200 mg/ml. This monoclonal antibody is labeled with 131 I using chloramine T procedure. ChT mass for the oxidation of 50 μg monoclonal antibody in 76 MBq was 10 μg. Sodium metabisulfite was used as a reducing agent. Reaction time was above 3 mins. The radiochemical purity was determined using electrophoresis and TLC methods. Radiochemical yield was > 97%. Radiochemical purity after purification was > 99%. Nuclear purity was > 99%. Stability of the label antibody was 12 days. This is the product promise potential used in the diagnostic and therapeutic of Hodgkin lymphoma. (author)

  17. The effects of variations in the specificities of the antibody components on a two-site immunoradiometric assay for ferritin

    International Nuclear Information System (INIS)

    Cowan, S.I.; Stagg, B.H.; Niemann, E.

    1977-01-01

    Variations in the sub-unit antigenic structure of ferritins derived from various human tissues are reflected in the differing specificities of antisera raised against these ferritin preparations. In this study it was shown that antibody specificity played an important role in determining the sensitivity and overall binding of labelled antibody in a two-site immunoradiometric assay for ferritin. Homologous assay systems, in which solid phase and radiolabelled antibodies were of similar specificities, were generally less sensitive and showed lower binding than heterologous assay systems, in which solid phase and labelled antibodies were of different specificities. The source of the ferritin which was used as assay standard also played an important part in determining the sensitivity and overall binding in homologous antibody systems, spleen ferritin standards yielding assays superior to those obtained with placenta or liver ferritin standards. However, these differences between standards were not seen in a heterologous system employing solid phase antibodies directed against liver ferritin and labelled antibodies directed against placenta ferritin. The nature of the ferritin used to prepare immunoadsorbant for the purification of antibodies prior to radioiodination also affected the assay characteristics; antibodies prepared on spleen ferritin immunoadsorbant being more reactive than antibodies prepared on placenta ferritin immunoadsorbant, which in turn were more reactive then antibodies prepared on liver ferritin immunoadsorbant. (orig.) [de

  18. Evaluation of capture ELISA for detection of antineutrophil cytoplasmic antibodies directed against proteinase 3 in Wegener's granulomatosis : first results from a multicentre study

    NARCIS (Netherlands)

    Csernok, E; Holle, J; Hellmich, B; Willem, J; Tervaert, C; Kallenberg, CGM; Limburg, PC; Niles, J; Pan, GL; Specks, U; Westman, K; Wieslander, J; Gross, WL

    Objective: To evaluate the performance characteristics of direct and capture ELISA for the detection of PR3-ANCA in Wegener's granulomatosis (WG) in international ANCA reference laboratories. Methods: Serum samples were derived from patients with histological and clinical diagnosis of WG (n = 60),

  19. Heterologous Coproduction of Enterocin A and Pediocin PA-1 by Lactococcus lactis: Detection by Specific Peptide-Directed Antibodies

    Science.gov (United States)

    Martínez, José M.; Kok, Jan; Sanders, Jan W.; Hernández, Pablo E.

    2000-01-01

    Antibodies against enterocin A were obtained by immunization of rabbits with synthetic peptides PH4 and PH5 designed, respectively, on the N- and C-terminal amino acid sequences of enterocin A and conjugated to the carrier protein KLH. Anti-PH4-KLH antibodies not only recognized enterocin A but also pediocin PA-1, enterocin P, and sakacin A, three bacteriocins which share the N-terminal class IIa consensus motif (YGNGVXC) that is contained in the sequence of the peptide PH4. In contrast, anti-PH5-KLH antibodies only reacted with enterocin A because the amino acid sequences of the C-terminal parts of class IIa bacteriocins are highly variable. Enterocin A and/or pediocin PA-1 structural and immunity genes were introduced in Lactococcus lactis IL1403 to achieve (co)production of the bacteriocins. The level of production of the two bacteriocins was significantly lower than that obtained by the wild-type producers, a fact that suggests a low efficiency of transport and/or maturation of these bacteriocins by the chromosomally encoded bacteriocin translocation machinery of IL1403. Despite the low production levels, both bacteriocins could be specifically detected and quantified with the anti-PH5-KLH (anti-enterocin A) antibodies isolated in this study and the anti-PH2-KLH (anti-pediocin PA-1) antibodies previously generated (J. M. Martínez, M. I. Martínez, A. M. Suárez, C. Herranz, P. Casaus, L. M. Cintas, J. M. Rodríguez, and P. E. Hernández, Appl. Environ. Microbiol. 64:4536–4545, 1998). In this work, the availability of antibodies for the specific detection and quantification of enterocin A and pediocin PA-1 was crucial to demonstrate coproduction of both bacteriocins by L. lactis IL1403(pJM04), because indicator strains that are selectively inhibited by each bacteriocin are not available. PMID:10919819

  20. Monoclonal antibodies in oncology. Review article

    Energy Technology Data Exchange (ETDEWEB)

    Chan, S Y.T.; Sikora, K

    1986-05-01

    Monoclonal antibodies (MCAs) can be used to differentiate between normal and neoplastic cells and thus exploited for diagnostic and, ultimately, therapeutic gain. The evidence for the existence of human tumour antigens is reviewed. Several areas of diagnosis are already benefiting from the application of the monoclonal technology. Immunohistology can help the pathologist with difficult diagnostic problems. New classifications of lymphoma and leukaemia can be based on specific surface molecules. Similarly, the detection of shed tumour antigens is already established as part of the routine assessment of many patients with common solid tumours. Isotopically labeled monoclonal antibodies have been used to localise primary and metastatic tumours. The use of antibodies in this way is not only a promising diagnostic tool but also the first step in studying the possibility of arming antibodies to provide therapeutic agents. Such trials are currently in progress. 69 refs.; 7 figs.; 3 tabs.