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Sample records for antibody reactivity identify

  1. Cross-reactivity of anti-human cytokine monoclonal antibodies used as a tool to identify novel immunological biomarkers in domestic ruminants.

    Science.gov (United States)

    Dorneles, E M S; Araújo, M S S; Teixeira-Carvalho, A; Martins-Filho, O A; Lage, A P

    2015-01-01

    Eleven commercially available PE-labeled anti-human (IL-1-α, IL-6, IL-8, TNF-α, IL-17A, IL-5, IL-10, IL-12 and IL-13) and anti-mouse (IL-10, TNF-α) cytokine monoclonal antibodies (mAbs) were tested for cross-reactivity with cattle, goat, and sheep cytokines. Cross-reactivity was assessed by comparative analysis with the standard reactivity of the target species. Our data demonstrated that anti-human IL-1-α, IL-6, IL-8, IL-17A and IL-10 mAbs cross-react with all ruminant species tested. Anti-human IL-5 mAb showed a strong cross-reactivity with cattle and goat IL-5, while anti-human TNF-α mAb showed a selective cross-reactivity with goat TNF-α. No cross-reactivity with the ruminant cytokines was observed for anti-human IL-12 and IL-13 mAbs or for the two anti-mouse cytokine mAbs tested. The present study demonstrated the cross-reactivity of various anti-human cytokine mAbs with cattle, sheep, and goat cytokines, increasing the range of immunological biomarkers for studies in veterinary medicine. PMID:25730032

  2. Transformation-related antigens identified by monoclonal antibodies.

    OpenAIRE

    Strand, M

    1980-01-01

    Tumor-cell proteins that were antigenic in a syngeneic animal were identified by immunoprecipitation with monoclonal antibodies. Spleen cells of BALB/c mice immunized with plasma membranes of Kirsten RNA sarcoma virus-transformed BALB/3T3 cells were fused with NS-1 myeloma cells. Antibodies secreted into the culture fluid from these hybridomas were distinguished by their reactivity against proteins of different target cells. A total of 191 cultures were established; 143 produced antibodies th...

  3. [3H]Azidodantrolene photoaffinity labeling, synthetic domain peptides and monoclonal antibody reactivity identify the dantrolene binding sequence on RyR1

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    Paul-Pletzer, Kalanethee; Yamamoto, Takeshi; Bhat, Manju B.; Ma, Jianjie; Ikemoto, Noriaki; Jimenez, Leslie S.; Morimoto, Hiromi; Williams, Philip G.; Parness, Jerome

    2002-06-14

    Dantrolene is a drug that suppresses intracellular Ca2+ release from sarcoplasmic reticulum in normal skeletal muscle and is used as a therapeutic agent in individuals susceptible to malignant hyperthermia. Though its precise mechanism of action has not been elucidated, we have identified the N-terminal region (amino acids 1-1400) of the skeletal muscle isoform of the ryanodine receptor (RyR1), the primary Ca2+ release channel in sarcoplasmic reticulum, as a molecular target for dantrolene using the photoaffinity analog [3H]azidodantrolene(1). Here, we demonstrate that heterologously expressed RyR1 retains its capacity to be specifically labeled with [3H]azidodantrolene,indicating that muscle specific factors are not required for this ligand-receptor interaction. Synthetic domain peptides of RyR1, previously shown to affect RyR1 function in vitro and in vivo, were exploited as potential drug binding site mimics and used in photoaffinity labeling experiments. Only DP1 and DP1-2, peptide s containing the amino acid sequence corresponding to RyR1 residues 590-609, were specifically labeled by [3H]azidodantrolene. A monoclonal anti-RyR1 antibody which recognizes RyR1 and its 1400 amino acid N-terminal fragment, recognizes DP1 and DP1-2 in both Western blots and immunoprecipitation assays, and specifically inhibits [3H]azidodantrolene photolabeling of RyR1 and its N-terminal fragment in sarcoplasmic reticulum. Our results indicate that synthetic domain peptides can mimic a native, ligand binding conformation in vitro, and that the dantrolene binding site and the epitope for the monoclonal antibody on RyR1 are equivalent and composed of amino-acids 590-609.

  4. Reactivity of Antibodies from Syphilis Patients to a Protein Array Representing the Treponema pallidum Proteome†

    OpenAIRE

    Brinkman, Mary Beth; McKevitt, Matthew; McLoughlin, Melanie; Perez, Carla; Howell, Jerrilyn; Weinstock, George M.; Norris, Steven J; Palzkill, Timothy

    2006-01-01

    To identify antigens important in the human immune response to syphilis, the serum antibody reactivity of syphilitic patients was examined with 908 of the 1,039 proteins in the proteome of Treponema pallidum subsp. pallidum using a protein array enzyme-linked immunosorbent assay. Thirty-four proteins exhibited significant reactivity when assayed with human sera from patients in the early latent stage of syphilis. A subset of antigens identified were further scrutinized for antibody reactivity...

  5. Production of monoclonal antibodies reactive with ovine eosinophils

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    Meeusen Els NT

    2007-09-01

    Full Text Available Abstract Background There is strong evidence implicating eosinophils in host defence against parasites as well as allergic disease pathologies. However, a lack of reagents such as monoclonal antibodies (mAbs specific for eosinophils has made it difficult to confirm the functional role of eosinophils in such disease conditions. Using an established mammary model of allergic inflammation in sheep, large numbers of inflammatory cells enriched for eosinophils were collected from parasite-stimulated mammary glands and used for the generation of mAbs against ovine eosinophils. Results A panel of mAbs was raised against ovine eosinophils of which two were shown to be highly specific for eosinophils. The reactivity of mAbs 3.252 and 1.2 identified eosinophils from various cell and tissue preparations with no detectable reactivity on cells of myeloid or lymphoid lineage, tissue mast cells, dendritic cells, epithelial cells or other connective tissues. Two other mAbs generated in this study (mAbs 4.4 and 4.10 were found to have reactivity for both eosinophils and neutrophils. Conclusion This study describes the production of new reagents to identify eosinophils (as well as granulocytes in sheep that will be useful in studying the role of eosinophils in disease pathologies in parasite and allergy models.

  6. Maturation Pathways of Cross-Reactive HIV-1 Neutralizing Antibodies

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    Dimiter S. Dimitrov

    2009-11-01

    Full Text Available Several human monoclonal antibodies (hmAbs and antibody fragments, including the best characterized in terms of structure-function b12 and Fab X5, exhibit relatively potent and broad HIV-1 neutralizing activity. However, the elicitation of b12 or b12-like antibodies in vivo by vaccine immunogens based on the HIV-1 envelope glycoprotein (Env has not been successful. B12 is highly divergent from the closest corresponding germline antibody while X5 is less divergent. We have hypothesized that the relatively high degree of specific somatic hypermutations may preclude binding of the HIV-1 envelope glycoprotein (Env to closest germline antibodies, and that identifying antibodies that are intermediates in the pathways to maturation could help design novel vaccine immunogens to guide the immune system for their enhanced elicitation. In support of this hypothesis we have previously found that a germline-like b12 (monovalent and bivalent scFv as an Fc fusion protein or IgG lacks measurable binding to an Env as measured by ELISA with a sensitivity in the μM range [1]; here we present evidence confirming and expanding these findings for a panel of Envs. In contrast, a germline-like scFv X5 bound Env with high (nM affinity. To begin to explore the maturation pathways of these antibodies we identified several possible b12 intermediate antibodies and tested their neutralizing activity. These intermediate antibodies neutralized only some HIV-1 isolates and with relatively weak potency. In contrast, germline-like scFv X5 neutralized a subset of the tested HIV-1 isolates with comparable efficiencies to that of the mature X5. These results could help explain the relatively high immunogenicity of the coreceptor binding site on gp120 and the abundance of CD4-induced (CD4i antibodies in HIV-1-infected patients (X5 is a CD4i antibody as well as the maturation pathway of X5. They also can help identify antigens that can bind specifically to b12 germline and

  7. Development of a novel monoclonal antibody with reactivity to a wide range of Venezuelan equine encephalitis virus strains

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    Phelps Amanda L

    2009-11-01

    Full Text Available Abstract Background There is currently a requirement for antiviral therapies capable of protecting against infection with Venezuelan equine encephalitis virus (VEEV, as a licensed vaccine is not available for general human use. Monoclonal antibodies are increasingly being developed as therapeutics and are potential treatments for VEEV as they have been shown to be protective in the mouse model of disease. However, to be truly effective, the antibody should recognise multiple strains of VEEV and broadly reactive monoclonal antibodies are rarely and only coincidentally isolated using classical hybridoma technology. Results In this work, methods were developed to reliably derive broadly reactive murine antibodies. A phage library was created that expressed single chain variable fragments (scFv isolated from mice immunised with multiple strains of VEEV. A broadly reactive scFv was identified and incorporated into a murine IgG2a framework. This novel antibody retained the broad reactivity exhibited by the scFv but did not possess virus neutralising activity. However, the antibody was still able to protect mice against VEEV disease induced by strain TrD when administered 24 h prior to challenge. Conclusion A monoclonal antibody possessing reactivity to a wide range of VEEV strains may be of benefit as a generic antiviral therapy. However, humanisation of the murine antibody will be required before it can be tested in humans. Crown Copyright © 2009

  8. A novel antibody discovery platform identifies anti-influenza A broadly neutralizing antibodies from human memory B cells.

    Science.gov (United States)

    Xiao, Xiaodong; Chen, Yan; Varkey, Reena; Kallewaard, Nicole; Koksal, Adem C; Zhu, Qing; Wu, Herren; Chowdhury, Partha S; Dall'Acqua, William F

    2016-07-01

    Monoclonal antibody isolation directly from circulating human B cells is a powerful tool to delineate humoral responses to pathological conditions and discover antibody therapeutics. We have developed a platform aimed at improving the efficiencies of B cell selection and V gene recovery. Here, memory B cells are activated and amplified using Epstein-Barr virus infection, co-cultured with CHO-muCD40L cells, and then assessed by functional screenings. An in vitro transcription and translation (IVTT) approach was used to analyze variable (V) genes recovered from each B cell sample and identify the relevant heavy/light chain pair(s). We achieved efficient amplification and activation of memory B cells, and eliminated the need to: 1) seed B cells at clonal level (≤1 cell/well) or perform limited dilution cloning; 2) immortalize B cells; or 3) assemble V genes into an IgG expression vector to confirm the relevant heavy/light chain pairing. Cross-reactive antibodies targeting a conserved epitope on influenza A hemagglutinin were successfully isolated from a healthy donor. In-depth analysis of the isolated antibodies suggested their potential uses as anti-influenza A antibody therapeutics and uncovered a distinct affinity maturation pathway. Importantly, our results showed that cognate heavy/light chain pairings contributed to both the expression level and binding abilities of our newly isolated VH1-69 family, influenza A neutralizing antibodies, contrasting with previous observations that light chains do not significantly contribute to the function of this group of antibodies. Our results further suggest the potential use of the IVTT as a powerful antibody developability assessment tool. PMID:27049174

  9. Analysis of Cross-Reactive Neutralizing Antibodies in Human HFMD Serum with an EV71 Pseudovirus-Based Assay

    Science.gov (United States)

    Jin, Jun; Xu, Lin; Sun, Shiyang; Jiang, Liping; Li, Xiaojun; Shao, Jie; Ma, Hongxia; Huang, Xueyong; Guo, Shijie; Chen, Haiying; Cheng, Tong; Yang, Lisheng; Su, Weiheng; Kong, Wei; Liang, Zhenglun; Jiang, Chunlai

    2014-01-01

    Hand, foot and mouth disease, associated with enterovirus 71 (EV71) infections, has recently become an important public health issue throughout the world. Serum neutralizing antibodies are major indicators of EV71 infection and protective immunity. However, the potential for cross-reactivity of neutralizing antibodies for different EV71 genotypes and subgenotypes is unclear. Here we measured the cross-reactive neutralizing antibody titers against EV71 of different genotypes or subgenotypes in sera collected from EV71-infected children and vaccine-inoculated children in a phase III clinical trial (ClinicalTrials.gov Identifier: NCT01636245) using a new pseudovirus-based neutralization assay. Antibodies induced by EV71-C4a were cross-reactive for different EV71 genotypes, demonstrating that C4a is a good candidate strain for an EV71 vaccine. Our study also demonstrated that this new assay is practical for analyses of clinical samples from epidemiological and vaccine studies. PMID:24964084

  10. Analysis of cross-reactive neutralizing antibodies in human HFMD serum with an EV71 pseudovirus-based assay.

    Directory of Open Access Journals (Sweden)

    Huafei Zhang

    Full Text Available Hand, foot and mouth disease, associated with enterovirus 71 (EV71 infections, has recently become an important public health issue throughout the world. Serum neutralizing antibodies are major indicators of EV71 infection and protective immunity. However, the potential for cross-reactivity of neutralizing antibodies for different EV71 genotypes and subgenotypes is unclear. Here we measured the cross-reactive neutralizing antibody titers against EV71 of different genotypes or subgenotypes in sera collected from EV71-infected children and vaccine-inoculated children in a phase III clinical trial (ClinicalTrials.gov Identifier: NCT01636245 using a new pseudovirus-based neutralization assay. Antibodies induced by EV71-C4a were cross-reactive for different EV71 genotypes, demonstrating that C4a is a good candidate strain for an EV71 vaccine. Our study also demonstrated that this new assay is practical for analyses of clinical samples from epidemiological and vaccine studies.

  11. Dengue serotype cross-reactive, anti-E protein antibodies confound specific immune memory for one year after infection

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    Ying Xiu eToh

    2014-08-01

    Full Text Available Dengue virus has four serotypes and is endemic globally in tropical countries. Neither a specific treatment nor an approved vaccine is available, and correlates of protection are not established. The standard neutralization assay cannot differentiate between serotype-specific and serotype cross-reactive antibodies in patients early after infection, leading to an overestimation of the long-term serotype-specific protection of an antibody response. It is known that the cross-reactive response in patients is temporary but few studies have assessed kinetics and potential changes in serum antibody specificity over time. To better define the specificity of polyclonal antibodies during disease and after recovery, longitudinal samples from patients with primary or secondary DENV-2 infection were collected over a period of one year. We found that serotype cross-reactive antibodies peaked three weeks after infection and subsided within one year. Since secondary patients rapidly produced antibodies specific for the virus envelope (E protein, an E-specific ELISA was superior compared to a virus particle-specific ELISA to identify patients with secondary infections. Dengue infection triggered a massive activation and mobilization of both naïve and memory B cells possibly from lymphoid organs into the blood, providing an explanation for the surge of circulating plasmablasts and the increase in cross-reactive E protein-specific antibodies.

  12. Tick-borne encephalitis virus (TBEV) – findings on cross reactivity and longevity of TBEV antibodies in animal sera

    OpenAIRE

    Klaus, Christine; Ziegler, Ute; Kalthoff, Donata; Hoffmann, Bernd; Beer, Martin

    2014-01-01

    Background By using animal sera as sentinels, natural TBEV foci could be identified and further analyses including investigations of ticks could be initiated. However, antibody response against TBEV-related flaviviruses might adversely affect the readout of such a monitoring. Therefore, the cross-reactivity of the applied TBEV serology test systems – enzyme linked immunosorbent assay (ELISA) and virus neutralization test (VNT) – as well as the longevity of TBEV antibody titres in sheep and go...

  13. Reactivity of eleven anti-human leucocyte monoclonal antibodies with lymphocytes from several domestic animals

    DEFF Research Database (Denmark)

    Aasted, Bent; Blixenkrone-Møller, Merete; Larsen, Else Bang;

    1988-01-01

    Nine commercially available monoclonal antibodies and two monoclonal antibodies from The American Type Culture Collection, raised against various human leucocyte surface antigens, were tested on lymphocytes from cow, sheep, goat, swine, horse, cat, dog, mink, and rabbit as well as man. Four......-reactive antibody reacted with lymphocytes from mink. The anti-C3b-R antibody reacted with lymphocytes from horse, swine, dog, and cat, and the anti-HLA-DR reacted with lymphocytes from cow, goat, sheep, horse, dog, cat, and mink....... antibodies bound to lymphocytes from some of the animals. These were the antibodies against CD8 and CD4 antigen, the antibody to C3b-receptor, and the antibody to the HLA-DR antigen. The CD8 antigen-reactive antibody reacted with lymphocytes from mink, cat, dog, and sheep, while the CD4 antigen...

  14. Identifying cognitive predictors of reactive and proactive aggression

    NARCIS (Netherlands)

    Brugman, S.; Lobbestael, J.; Arntz, A.R.; Cima, M.; Schumann, T.; Dambacher, F.

    2015-01-01

    The aim of this study was to identify implicit cognitive predictors of aggressive behavior. Specifically, the predictive value of an attentional bias for aggressive stimuli and automatic association of the self and aggression was examined for reactive and proactive aggressive behavior in a non-clini

  15. Identifying cognitive predictors of reactive and proactive aggression

    NARCIS (Netherlands)

    S. Brugman; J. Lobbestael; A. Arntz; M. Cima; T. Schuhmann; F. Dambacher; A.T. Sack

    2014-01-01

    The aim of this study was to identify implicit cognitive predictors of aggressive behavior. Specifically, the predictive value of an attentional bias for aggressive stimuli and automatic association of the self and aggression was examined for reactive and proactive aggressive behavior in a non-clini

  16. Identifying reactive peptides from phage-displayed libraries

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    Eldridge, Glenn M.; Weiss, Gregory A.

    2015-01-01

    Summary Phage display enables the synthesis, selection and screening of large, polypeptide libraries (>1 × 1010). Selections from such libraries can identify binding partners to essentially any desired target (1, 2). Peptide with affinity or reactivity to small molecule probes are attractive for numerous uses including the targeted, site-specific labeling of proteins. Here, we describe selection and screening protocols for the identification of short peptides that can selectively bind to and/or react with small molecules. PMID:25616334

  17. Antibody Arrays Identify Potential Diagnostic Markers of Hepatocellular Carcinoma

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    Brian J. Peter

    2008-01-01

    Full Text Available Hepatocellular carcinoma (HCC is the third leading cause of cancer deaths worldwide. Effective treatment of HCC patients is hampered by the lack of sensitive and specific diagnostic markers of HCC. Alpha-fetoprotein (AFP, the currently used HCC marker, misses 30%–50% of HCC patients, who therefore remain undiagnosed and untreated. In order to identify novel diagnostic markers that can be used individually or in combination with AFP, we used an antibody array platform to detect the levels of candidate proteins in the plasma of HCC patients (n = 48 and patients with chronic hepatitis B or C viral infections (n = 19 (both of which are the major risk factors of HCC. We identified 7 proteins that significantly differentiate HCC patients from hepatitis patients (p < 0.05 (AFP, CTNNB, CSF1, SELL, IGFBP6, IL6R, and VCAM1.Importantly, we also identified 8 proteins that significantly differentiate HCC patients with ‘normal’ levels of AFP (<20 ng/ml from hepatitis patients (p < 0.05 (IL1RN, IFNG, CDKN1A, RETN, CXCL14, CTNNB, FGF2, and SELL. These markers are potentially important complementary markers to AFP. Using an independent immunoassay method in an independent group of 23 HCC patients and 22 hepatitis patients, we validated that plasma levels of CTNNB were significantly higher in the HCC group (p = 0.020. In conclusion, we used an antibody array platform to identify potential circulating diagnostic markers of HCC, some of which may be valuable when used in combination with AFP. The clinical utility of these newly identified HCC diagnostic markers needs to be systematically evaluated.

  18. Specificity, cross-reactivity, and function of antibodies elicited by Zika virus infection.

    Science.gov (United States)

    Stettler, Karin; Beltramello, Martina; Espinosa, Diego A; Graham, Victoria; Cassotta, Antonino; Bianchi, Siro; Vanzetta, Fabrizia; Minola, Andrea; Jaconi, Stefano; Mele, Federico; Foglierini, Mathilde; Pedotti, Mattia; Simonelli, Luca; Dowall, Stuart; Atkinson, Barry; Percivalle, Elena; Simmons, Cameron P; Varani, Luca; Blum, Johannes; Baldanti, Fausto; Cameroni, Elisabetta; Hewson, Roger; Harris, Eva; Lanzavecchia, Antonio; Sallusto, Federica; Corti, Davide

    2016-08-19

    Zika virus (ZIKV), a mosquito-borne flavivirus with homology to Dengue virus (DENV), has become a public health emergency. By characterizing memory lymphocytes from ZIKV-infected patients, we dissected ZIKV-specific and DENV-cross-reactive immune responses. Antibodies to nonstructural protein 1 (NS1) were largely ZIKV-specific and were used to develop a serological diagnostic tool. In contrast, antibodies against E protein domain I/II (EDI/II) were cross-reactive and, although poorly neutralizing, potently enhanced ZIKV and DENV infection in vitro and lethally enhanced DENV disease in mice. Memory T cells against NS1 or E proteins were poorly cross-reactive, even in donors preexposed to DENV. The most potent neutralizing antibodies were ZIKV-specific and targeted EDIII or quaternary epitopes on infectious virus. An EDIII-specific antibody protected mice from lethal ZIKV infection, illustrating the potential for antibody-based therapy. PMID:27417494

  19. Referencing cross-reactivity of detection antibodies for protein array experiments.

    Science.gov (United States)

    Lemass, Darragh; O'Kennedy, Richard; Kijanka, Gregor S

    2016-01-01

    Protein arrays are frequently used to profile antibody repertoires in humans and animals. High-throughput protein array characterisation of complex antibody repertoires requires a platform-dependent, lot-to-lot validation of secondary detection antibodies. This article details the validation of an affinity-isolated anti-chicken IgY antibody produced in rabbit and a goat anti-rabbit IgG antibody conjugated with alkaline phosphatase using protein arrays consisting of 7,390 distinct human proteins. Probing protein arrays with secondary antibodies in absence of chicken serum revealed non-specific binding to 61 distinct human proteins. The cross-reactivity of the tested secondary detection antibodies points towards the necessity of platform-specific antibody characterisation studies for all secondary immunoreagents. Secondary antibody characterisation using protein arrays enables generation of reference lists of cross-reactive proteins, which can be then excluded from analysis in follow-up experiments. Furthermore, making such cross-reactivity lists accessible to the wider research community may help to interpret data generated by the same antibodies in applications not related to protein arrays such as immunoprecipitation, Western blots or other immunoassays. PMID:27335636

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

    OpenAIRE

    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

    2013-01-01

    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 ...

  1. Neutralization of Japanese Encephalitis Virus by heme-induced broadly reactive human monoclonal antibody

    OpenAIRE

    Nimesh Gupta; Mélissanne de Wispelaere; Maxime Lecerf; Manjula Kalia; Tobias Scheel; Sudhanshu Vrati; Claudia Berek; Kaveri, Srinivas V.; Philippe Desprès; Sébastien Lacroix-Desmazes; Dimitrov, Jordan D.

    2015-01-01

    International audience Geographical expansion and re-emerging new genotypes of the Japanese encephalitis virus (JEV) require the development of novel therapeutic approaches. Here, we studied a non-conventional approach for antibody therapy and show that, upon exposure to heme, a fraction of natural human immunoglobulins acquires high-affinity reactivity with the antigenic domain-III of JEV E glycoprotein. These JEV-reactive antibodies exhibited neutralizing activity against recently domina...

  2. C-REACTIVE PROTEIN AND ANTIBODIES TO HELICOBACTER PYLORI IN THE PATIENTS WITH ISCHEMIC HEART DISEASE

    OpenAIRE

    A. Ye. Kratnov; O. N. Pavlov

    2014-01-01

    Abstract. Levels of C-reactive protein and immunoglobulin G antibody titers to H. pylori in blood at patients of ischemic heart disease were measured, dependent on clinical course of disease. It was revealed that more expressed acute-phase changes in blood (leukocytosis, increased C-reactive protein contents) in the patients with unstable angina and acute myocardial infarction, as compared with appropriate parameters in stable stenocardia, were accompanied by increased titers of IgG antibodie...

  3. Molecular Evolution of Antibody Cross-Reactivity for Two Subtypes of Type a Botulinum Neurotoxin

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    Garcia-Rodriguez, C.; Levy, R.; Arndt, J.W.; Forsyth, C.M.; Razai, A.; Lou, J.; Geren, I.; Stevens, R.C.; Marks, J.D.; /UC, San Francisco /Scripps Res. Inst.

    2007-07-09

    Broadening antibody specificity without compromising affinity should facilitate detection and neutralization of toxin and viral subtypes. We used yeast display and a co-selection strategy to increase cross-reactivity of a single chain (sc) Fv antibody to botulinum neurotoxin type A (BoNT/A). Starting with a scFv that binds the BoNT/A1 subtype with high affinity (136 pM) and the BoNT/A2 subtype with low affinity (109 nM), we increased its affinity for BoNT/A2 1,250-fold, to 87 pM, while maintaining high-affinity binding to BoNT/A1 (115 pM). To find the molecular basis for improved cross-reactivity, we determined the X-ray co-crystal structures of wild-type and cross-reactive antibodies complexed to BoNT/A1 at resolutions up to 2.6 A, and measured the thermodynamic contribution of BoNT/A1 and A2 amino acids to wild-type and cross-reactive antibody binding. The results show how an antibody can be engineered to bind two different antigens despite structural differences in the antigen-antibody interface and may provide a general strategy for tuning antibody specificity and cross-reactivity.

  4. A Spectrum of Monoclonal Antibodies Reactive with Human Mammary Tumor Cells

    Science.gov (United States)

    Colcher, D.; Horan Hand, P.; Nuti, M.; Schlom, J.

    1981-05-01

    Splenic lymphocytes of mice, immunized with membrane-enriched fractions of metastatic human mammary carcinoma tissues, were fused with the NS-1 non-immunoglobulin-secreting murine myeloma cell line. This resulted in the generation of hybridoma cultures secreting immunoglobulins reactive in solid-phase radioimmunoassays with extracts of metastatic mammary carcinoma cells from involved livers, but not with extracts of apparently normal human liver. As a result of further screening of immunoglobulin reactivities and double cloning of cultures, 11 monoclonal antibodies were chosen that demonstrated reactivities with human mammary tumor cells and not with apparently normal human tissues. These monoclonal antibodies could be placed into at least five major groups on the basis of their differential binding to the surface of various live human mammary tumor cells in culture, to extracts of mammary tumor tissues, or to tissue sections of mammary tumor cells studied by the immunoperoxidase technique. Whereas a spectrum of reactivities to mammary tumors was observed with the 11 monoclonal antibodies, no reactivity was observed to apparently normal cells of the following human tissues: breast, lymph node, lung, skin, testis, kidney, thymus, bone marrow, spleen, uterus, thyroid, intestine, liver, bladder, tonsils, stomach, prostate, and salivary gland. Several of the antibodies also demonstrated a ``pancarcinoma'' reactivity, showing binding to selected non-breast carcinomas. None of the monoclonal antibodies showed binding to purified ferritin or carcinoembryonic antigen. Monoclonal antibodies of all five major groups, however, demonstrated binding to human metastatic mammary carcinoma cells both in axillary lymph nodes and at distal sites.

  5. Pneumocystis carinii and specific fungi have a common epitope, identified by a monoclonal antibody

    DEFF Research Database (Denmark)

    Lundgren, B; Kovacs, J A; Nelson, N N; Stock, F; Martinez, Angel Ricardo; Gill, V J

    1992-01-01

    Because Pneumocystis carinii may be related to fungi, we evaluated the reactivities of monoclonal antibodies raised against P. carinii with a variety of fungi. Fifty-two fungi and six protozoa were evaluated by immunofluorescence. One of three monoclonal antibodies (MAbs) tested (MAb 7D7) reacted...... with 15 fungi but no protozoa. Saccharomyces cerevisiae showed the strongest reactivity by immunofluorescence. The reactive antigen was characterized for four fungi by the immunoblot technique. In all cases the antigen that was reactive with MAb 7D7 was larger than the P. carinii antigens that reacted...

  6. Identification of cross-reactive single-domain antibodies against serum albumin using next-generation DNA sequencing.

    Science.gov (United States)

    Henry, Kevin A; Tanha, Jamshid; Hussack, Greg

    2015-10-01

    Antibodies that cross-react with multiple isoforms or homologue of a given protein are often desirable, especially in therapeutic applications. Here, we report the identification of several unique, clonally unrelated, single-domain antibodies (sdAbs) that bind to multiple serum albumin orthologues (human, rhesus, rat and mouse) with low-to-medium nanomolar affinity from a llama immunized only with human serum albumin. Using single-round panning of a phage-displayed sdAb library against serum albumins and next-generation DNA sequencing, we were able to predict patterns of sdAb reactivity to the albumins of different species with ∼90% accuracy. We expect this strategy to be generally applicable for identifying cross-reactive sdAbs, particularly when these exist at low frequency and/or are poorly enriched by panning. Moreover, the sdAbs identified here are of potential interest for serum half-life extension of biologics. PMID:26319004

  7. Reactivity of monoclonal antibodies to species-specific antigens of Entamoeba histolytica.

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    Tachibana, H; Kobayashi, S; Nagakura, K; Kaneda, Y; Takeuchi, T

    1991-01-01

    Twenty monoclonal antibodies were produced against trophozoites of Entamoeba histolytica strains HK-9 and HM-1: IMSS. When reactivity to various enteric protozoa was examined by an indirect fluorescence antibody test, 15 of the monoclonal antibodies were strongly reactive with E. histolytica trophozoites. Species-specific antigens recognized by these monoclonal antibodies were located on the plasma membrane, nucleus, cytoplasm, and cytoskeletal structures of the trophozoites. Two of the remaining five monoclonals reacted strongly with trophozoites of the E. histolytica-like Laredo strain. The determinant antigen was located in the cytoplasm. The three remaining monoclonal antibodies were found to recognize cross-reactive antigens between E. histolytica and E. histolytica-like Laredo, E. hartmanni, E. coli, Dientamoeba fragilis, Giardia lamblia, and Trichomonas hominis. These three antibodies were also reactive with T. vaginalis and mammalian cells such as HeLa cells. Thus, the combined use of monoclonal antibodies seems capable of distinguishing E. histolytica and/or E. histolytica-like Laredo from other enteric protozoa. PMID:1724012

  8. Cross-reactivity and phospholipase A2 neutralization of anti-irradiated Bothrops jararaca venom antibodies

    International Nuclear Information System (INIS)

    The detoxified Bothrops jararaca venom, immunized rabbits with the toxoid obtained and investigated cross-reactivity of the antibodies obtained against autologous and heterelogous venoms was presented. It was also investigated the ability of the IgGs, purified by affinity chromatography, from those sera to neutralize phospholipase. A2, an ubiquous enzyme in animal venoms. Results indicate that venom irradiation leads to an attenuation of toxicity of 84%. Cross-reactivity was investigated by ELISA and Western blot and all venoms were reactive to the antibodies. On what refers to phospholipase A2 activity neutralization, the antibodies neutralized autologous venoms efficiently and, curiously, other venoms from the same genus were not neutralized, while Lachesis muta venom, a remote related specier, was neutralized by this serum. These data suggest that irradiation preserve important epitopes for induction of neutralizing antibodies and that these epitopes are not shared by all venoms assayed. (author). 8 refs, 2 figs, 3 tabs

  9. Cross-reactive polyclonal antibodies to the inner core of lipopolysaccharide from Neisseria meningitidis.

    Science.gov (United States)

    Andersen, Svein Rune; Guthrie, Terry; Guile, Geoffrey R; Kolberg, Jan; Hou, Sam; Hyland, Lisa; Wong, Simon Y C

    2002-03-01

    Sera from mice immunized with native or detergent-extracted outer membrane vesicles derived from lipopolysaccharide (LPS) mutant 44/76(Mu-4) of Neisseria meningitidis were analyzed for antibodies to LPS. The carbohydrate portion of 44/76(Mu-4) LPS consists of the complete inner core, Glc beta 1-->4[GlcNAc alpha 1-->2Hep alpha 1-->3]Hep alpha 1-->5KDO[4-->2 alpha KDO]. Immunoblot analysis revealed that some sera contained antibodies to wild-type LPS which has a fully extended carbohydrate chain of immunotype L3,7, as well as to the homologous LPS. Sera reacted only weakly to LPS from 44/76(Mu-3), which lacks the terminal glucose of the inner core. No binding to more truncated LPS was observed. Consequently, the cross-reactive epitopes are expressed mainly by the complete inner core. Dephosphorylation of wild-type LPS abolished antibody binding to LPS in all but one serum. Thus, at least two specificities of cross-reactive antibodies exist: one is dependent on phosphoethanolamine groups in LPS, and one is not. Detection of these cross-reactive antibodies strongly supports the notion that epitopes expressed by meningococcal LPS inner core are also accessible to antibodies when the carbohydrate chain is fully extended. Also, these inner core epitopes are sufficiently immunogenic to induce antibody levels detectable in polyclonal antibody responses. Meningococci can escape being killed by antibodies to LPS that bind only to a specific LPS variant, by altering the carbohydrate chain length. Cross-reactive antibodies may prevent such escape. Therefore, inner core LPS structures may be important antigens in future vaccines against meningococcal disease. PMID:11854213

  10. MONOCLONAL ANTIBODIES TO IDENTIFY TOMATO MOSAIC TOBAMOVIRUS (TOMV

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    Duarte Keila M.R.

    2001-01-01

    Full Text Available Monoclonal antibodies were obtained against Tomato mosaic tobamovirus (ToMV isolated in Brazil. One antibody (8G7G2 isotyped as IgG2b (kappa light chain showed strong specificity and very low cross reaction with the Tobacco mosaic virus (TMV. It can be used in identification of tomato mosaic virus (ToMV.

  11. Production of monoclonal antibodies anti-Taenia crassiceps cysticerci with cross-reactivity with Taenia solium antigens

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    ESPÍNDOLA Noeli M.

    2000-01-01

    Full Text Available We describe the production of the potential monoclonal antibodies (MoAbs using BALB/c mice immunized with vesicular fluid (VF-Tcra (T. crassiceps antigen. Immune sera presented anti-VF-Tcra (<20kD IgG and IgM antibodies with cross-reactivity with T. solium (Tso antigen (8-12, 14, and 18 kD. After cell fusion, we selected 33 anti-Tcra and anti-Tso reactive IgM-clones and 53 anti-Tcra specific IgG-clones, 5 of them also recognizing Tso antigens. Two clones identified the 8-14 and 18kD peptides of VF-Tcra.

  12. A conserved multi-gene family induces cross-reactive antibodies effective in defense against Plasmodium falciparum.

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    Subhash Singh

    Full Text Available BACKGROUND: Two related merozoite surface proteins, MSP3 and MSP6, have previously been identified as targets of antibody-dependent cellular inhibition (ADCI, a protective mechanism against Plasmodium falciparum malaria. Both MSP3 and MSP6 share a common characteristic small N-terminal signature amino-acid stretch (NLRNA/G, a feature similar to MSP3-like orthologs identified in other human and primate malaria parasites. METHODS/RESULTS: This signature amino-acid sequence led to the identification of eight ORFs contiguously located on P. falciparum chromosome 10. Our subsequent investigations on their expression, localization, sequence conservation, epitope sharing, immunogenicity and the functional role of antibodies in defense are reported here. Six members of P. falciparum MSP3-multigene family share similar sequence organization within their C-terminal regions, are simultaneously expressed as merozoite surface proteins and are highly conserved among parasite isolates. Each of these proteins is a target of naturally occurring antibodies effective at parasite killing in ADCI assays. Moreover, both naturally occurring antibodies and those generated by immunization display cross-reactivity with other members of the family and exhibit varied binding avidities. CONCLUSIONS/SIGNIFICANCE: The unusual characteristics of the MSP3 multi-gene family lead us to hypothesize that the simultaneous expression of targets eliciting cross-reactive antibody responses capable of controlling parasite densities could represent an immune process selected through evolution to maintain homeostasis between P. falciparum and human hosts; a process that allows the continuous transmission of the parasite without killing the host. Our observations also have practical consequences for vaccine development by suggesting MSP3 vaccine efficacy might be improved when combined with the various C-terminus regions of the MSP3 family members to generate a wider range of antibodies

  13. Limited cross-reactivity of mouse monoclonal antibodies against Dengue virus capsid protein among four serotypes

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    Noda M

    2012-11-01

    Full Text Available Megumi Noda,1 Promsin Masrinoul,1 Chaweewan Punkum,1 Chonlatip Pipattanaboon,2,3 Pongrama Ramasoota,2,4 Chayanee Setthapramote,2,3 Tadahiro Sasaki,6 Mikiko Sasayama,1 Akifumi Yamashita,1,5 Takeshi Kurosu,6 Kazuyoshi Ikuta,6 Tamaki Okabayashi11Mahidol-Osaka Center for Infectious Diseases, 2Center of Excellence for Antibody Research, 3Department of Microbiology and Immunology, 4Department of Social and Environmental Medicine, Faculty of Tropical Medicine, Mahidol University, Ratchathewi, Bangkok, Thailand; 5Graduate School of Life Science, Tohoku University, Sendai, Miyagi, 6Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, JapanBackground: Dengue illness is one of the important mosquito-borne viral diseases in tropical and subtropical regions. Four serotypes of dengue virus (DENV-1, DENV-2, DENV-3, and DENV-4 are classified in the Flavivirus genus of the family Flaviviridae. We prepared monoclonal antibodies against DENV capsid protein from mice immunized with DENV-2 and determined the cross-reactivity with each serotype of DENV and Japanese encephalitis virus.Methods and results: To clarify the relationship between the cross-reactivity of monoclonal antibodies and the diversity of these viruses, we examined the situations of flaviviruses by analyses of phylogenetic trees. Among a total of 60 prepared monoclonal antibodies specific for DENV, five monoclonal antibodies stained the nuclei of infected cells and were found to be specific to the capsid protein. Three were specific to DENV-2, while the other two were cross-reactive with DENV-2 and DENV-4. No monoclonal antibodies were cross-reactive with all four serotypes. Phylogenetic analysis of DENV amino acid sequences of the capsid protein revealed that DENV-2 and DENV-4 were clustered in the same branch, while DENV-1 and DENV-3 were clustered in the other branch. However, these classifications of the capsid protein were different from those of the

  14. C-REACTIVE PROTEIN AND ANTIBODIES TO HELICOBACTER PYLORI IN THE PATIENTS WITH ISCHEMIC HEART DISEASE

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    A. Ye. Kratnov

    2014-07-01

    Full Text Available Abstract. Levels of C-reactive protein and immunoglobulin G antibody titers to H. pylori in blood at patients of ischemic heart disease were measured, dependent on clinical course of disease. It was revealed that more expressed acute-phase changes in blood (leukocytosis, increased C-reactive protein contents in the patients with unstable angina and acute myocardial infarction, as compared with appropriate parameters in stable stenocardia, were accompanied by increased titers of IgG antibodies against H. pylori.

  15. Measurement of tumour reactive antibody and antibody conjugate by competition, quantitated by flow cytofluorimetry.

    Science.gov (United States)

    Robins, R A; Laxton, R R; Garnett, M; Price, M R; Baldwin, R W

    1986-06-24

    Binding of unlabelled monoclonal antibody preparations has been assessed by competition at saturation with fluorochrome labelled homologous antibody for binding to antigen bearing target cells. The extent of competition was measured by quantitative flow cytofluorimetry, and simple mathematical procedures have been developed to allow the interpretation of competition data in terms of antibody binding activity. In the system studied, non-specific (non-competitive) fluorescence was minimal, but an iterative method to calculate its contribution to the measured signal is given. This approach has the advantage that the antibody preparation to be tested does not need to be labelled or modified; this is particularly important when evaluating the binding activity of therapeutic antibody conjugates. Comparison with a well characterized standard antibody preparation provides a rapid, sensitive and accurate quality control procedure. This test is also simple to perform, requiring only the mixing of labelled and unlabelled antibodies with target cells, a single incubation, followed by analysis without washing of the target cells. PMID:2424997

  16. Vaccine-induced cross-genotype reactive neutralizing antibodies against hepatitis C virus

    DEFF Research Database (Denmark)

    Meunier, Jean-Christophe; Gottwein, Judith M; Houghton, Michael; Russell, Rodney S; Emerson, Suzanne U; Bukh, Jens; Purcell, Robert H

    2011-01-01

    We detected cross-reactive neutralizing antibodies (NtAb) against hepatitis C virus (HCV) in chimpanzees vaccinated with HCV-1 (genotype 1a) recombinant E1/E2 envelope glycoproteins. Five vaccinated chimpanzees, protected following HCV-1 challenge, were initially studied using the heterologous H77...

  17. Cross-reactive neutralizing antibody responses to enterovirus 71 infections in young children: implications for vaccine development.

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    Mei-Liang Huang

    Full Text Available BACKGROUND: Recently, enterovirus 71 (EV71 has caused life-threatening outbreaks involving neurological and cardiopulmonary complications in Asian children with unknown mechanism. EV71 has one single serotype but can be phylogenetically classified into 3 main genogroups (A, B and C and 11 genotypes (A, B1∼B5 and C1∼C5. In Taiwan, nationwide EV71 epidemics with different predominant genotypes occurred in 1998 (C2, 2000-2001 (B4, 2004-2005 (C4, and 2008 (B5. In this study, sera were collected to measure cross-reactive neutralizing antibody titers against different genotypes. METHODS: We collected historical sera from children who developed an EV71 infection in 1998, 2000, 2005, 2008, or 2010 and measured cross-reactive neutralizing antibody titers against all 11 EV71 genotypes. In addition, we aligned and compared the amino acid sequences of P1 proteins of the tested viruses. RESULTS: Serology data showed that children infected with genogroups B and C consistently have lower neutralizing antibody titers against genogroup A (>4-fold difference. The sequence comparisons revealed that five amino acid signatures (N143D in VP2; K18R, H116Y, D167E, and S275A in VP1 are specific for genogroup A and may be related to the observed antigenic variations. CONCLUSIONS: This study documented antigenic variations among different EV71 genogroups and identified potential immunodominant amino acid positions. Enterovirus surveillance and vaccine development should monitor these positions.

  18. Studies on Preparation and Characteristics of Monoclonal Antibodies Against Enrofloxacin and Cross-Reactivity of Related Fluoroquinolones

    Institute of Scientific and Technical Information of China (English)

    CAI Qin-ren; ZENG Zhen-ling; YANG Gui-xiang; CHEN Zhang-liu

    2004-01-01

    An ester activation method was employed to couple enrofloxacin(ENFX) to the carrier proteins BSA and OVA. The conjugates ENFX-BSA and ENFX-OVA were identified with an UV spectrophotometer and amino acid automation analysis instrument, and resulted in conjugates with 48 ENFX molecules per carrier molecule(BSA). Splenocytes from mice immunized with ENFX-BSA were fused with SP2/0 myeloma cells and hybridomas secreting antibodies against enrofloxacin were selected and cloned. Two stable monoclonal antibodies, 2C5, 5D5 of the subclass IgG2a, were isolated. Using antibody 5D5, an indirect competitive inhibition enzyme-linked immunosorbent assay (Ci-ELISA) was developed for the quantitative detection of enrofloxacin and its metabolites. The IC50 of the standard curve was 21.67 ng mL-1 and the limit of detection for enrofloxacin was 0.13 ng mL-1. This method was sensitive and had a linear range from 0.13 to 10 000 ngmL-1 (r=-0.9782). Monoclonal antibody 5D5 exhibited high relative affinity to enrofloxacin, and the cross-reactivities with ciprofloxacin,marbofloxacin, sarafloxacin and danorfloxacin were 110.8, 27.40, 71.05 and 37.41%,respectively. Three non-fluoroquinolones of cefadroxil, chloramphenicol, sulfadimethoxine were tested and there was no cross-reaction between them.

  19. Subsetting of acetylcholine receptor-reactive antibodies by preparative isoelectric focusing.

    Science.gov (United States)

    Thompson, P A; Krolick, K A

    1991-01-01

    The antibodies produced against most foreign antigens are composed of a family of immunoglobulins, a family composed of members that are of a number that often reflects the size/complexity of the molecule that stimulates their production. In other words, such responses involve the activation of a "polyclonal" B lymphocyte population. The antibody products of the B cells, although all capable of binding the original antigen, bind at various immunogenic sites (epitopes) on that antigen. Such differences in antigen-binding fine specificity is determined by amino acid residues in the antibody variable region domains found associated with the antigen combining site and tend to have a complimentary biochemistry with the molecule for which they are intended to interact. Furthermore, in addition to amino acid differences that dictate the isotypes and allotypes of antibody molecules, differences in the amino acids that compose the variable regions can produce differences in net charge of particular antibody molecules; thus, families of polyclonal antibodies, all reactive with the same antigen but with different fine specificities, can be separated and, as shown below, purified based on their isoelectric points by preparative isoelectric focusing (pIEF). PMID:1780274

  20. Human antibody responses after dengue virus infection are highly cross-reactive to Zika virus

    Science.gov (United States)

    Priyamvada, Lalita; Quicke, Kendra M.; Hudson, William H.; Onlamoon, Nattawat; Sewatanon, Jaturong; Edupuganti, Srilatha; Pattanapanyasat, Kovit; Chokephaibulkit, Kulkanya; Mulligan, Mark J.; Wilson, Patrick C.; Ahmed, Rafi; Suthar, Mehul S.; Wrammert, Jens

    2016-01-01

    Zika virus (ZIKV) is an emerging mosquito-borne flavivirus of significant public health concern. ZIKV shares a high degree of sequence and structural homology compared with other flaviviruses, including dengue virus (DENV), resulting in immunological cross-reactivity. Improving our current understanding of the extent and characteristics of this immunological cross-reactivity is important, as ZIKV is presently circulating in areas that are highly endemic for dengue. To assess the magnitude and functional quality of cross-reactive immune responses between these closely related viruses, we tested acute and convalescent sera from nine Thai patients with PCR-confirmed DENV infection against ZIKV. All of the sera tested were cross-reactive with ZIKV, both in binding and in neutralization. To deconstruct the observed serum cross-reactivity in depth, we also characterized a panel of DENV-specific plasmablast-derived monoclonal antibodies (mAbs) for activity against ZIKV. Nearly half of the 47 DENV-reactive mAbs studied bound to both whole ZIKV virion and ZIKV lysate, of which a subset also neutralized ZIKV. In addition, both sera and mAbs from the dengue-infected patients enhanced ZIKV infection of Fc gamma receptor (FcγR)-bearing cells in vitro. Taken together, these findings suggest that preexisting immunity to DENV may impact protective immune responses against ZIKV. In addition, the extensive cross-reactivity may have implications for ZIKV virulence and disease severity in DENV-experienced populations. PMID:27354515

  1. Human antibody responses after dengue virus infection are highly cross-reactive to Zika virus.

    Science.gov (United States)

    Priyamvada, Lalita; Quicke, Kendra M; Hudson, William H; Onlamoon, Nattawat; Sewatanon, Jaturong; Edupuganti, Srilatha; Pattanapanyasat, Kovit; Chokephaibulkit, Kulkanya; Mulligan, Mark J; Wilson, Patrick C; Ahmed, Rafi; Suthar, Mehul S; Wrammert, Jens

    2016-07-12

    Zika virus (ZIKV) is an emerging mosquito-borne flavivirus of significant public health concern. ZIKV shares a high degree of sequence and structural homology compared with other flaviviruses, including dengue virus (DENV), resulting in immunological cross-reactivity. Improving our current understanding of the extent and characteristics of this immunological cross-reactivity is important, as ZIKV is presently circulating in areas that are highly endemic for dengue. To assess the magnitude and functional quality of cross-reactive immune responses between these closely related viruses, we tested acute and convalescent sera from nine Thai patients with PCR-confirmed DENV infection against ZIKV. All of the sera tested were cross-reactive with ZIKV, both in binding and in neutralization. To deconstruct the observed serum cross-reactivity in depth, we also characterized a panel of DENV-specific plasmablast-derived monoclonal antibodies (mAbs) for activity against ZIKV. Nearly half of the 47 DENV-reactive mAbs studied bound to both whole ZIKV virion and ZIKV lysate, of which a subset also neutralized ZIKV. In addition, both sera and mAbs from the dengue-infected patients enhanced ZIKV infection of Fc gamma receptor (FcγR)-bearing cells in vitro. Taken together, these findings suggest that preexisting immunity to DENV may impact protective immune responses against ZIKV. In addition, the extensive cross-reactivity may have implications for ZIKV virulence and disease severity in DENV-experienced populations. PMID:27354515

  2. Antibodies reactive with Bartonella henselae and Ehrlichia canis in dogs from the communal lands of Zimbabwe

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    P.J. Kelly

    2004-06-01

    Full Text Available The prevalences of antibodies against Bartonella henselae and Ehrlichia canis were determined in sera from 228 dogs in 5 communal lands of Zimbabwe, areas where traditional subsistence agro-pastoralism is practised. The sera were collected from apparently healthy dogs during routine rabies vaccination programmes and tested with indirect fluorescent antibody assays using B. henselae (Houston-I and E. canis (Oklahoma as antigens. We found reactive antibodies (>1:80 against B. henselae in 14 % of the dogs tested. Seropositive animals were found in Bikita (41 %; 17/42, Omay (13 %; 6/48, Chinamora (5 %; 2/38 and Matusadona (15 %; 7/48. No seropositive dogs were found in Chiredzi (0 %; 0/52. Antibodies reactive with E. canis (>1:80 were found in 34%of the dogs tested, from Bikita (88 %; 37/42, Chiredzi (31 %; 16/52, Omay (17 %; 8/48, Chinamora (26 %; 10/38 and Matusadona (15 %; 7/48. Our survey shows dogs in the communal lands of Zimbabwe are frequently exposed to E. canis and B. henselae or closely related species. Further studies are indicated to determine the pathogenicity of the organisms infecting these dogs and their clinical significance.

  3. Identification of a synthetic peptide inducing cross-reactive antibodies binding to Rhipicephalus (Boophilus) decoloratus, Rhipicephalus (Boophilus) microplus, Hyalomma anatolicum anatolicum and Rhipicephalus appendiculatus BM86 homologues.

    Science.gov (United States)

    Kopp, Nadja; Diaz, Diana; Amacker, Mario; Odongo, David O; Beier, Konstantin; Nitsch, Cordula; Bishop, Richard P; Daubenberger, Claudia A

    2009-12-10

    The BM86 antigen, originally identified in Rhipicephalus (Boophilus) microplus, is the basis of the only commercialized anti-tick vaccine. The long-term goal of our study is to improve BM86 based vaccines by induction of high levels of tick gut binding antibodies that are also cross-reactive with a range of BM86 homologues expressed in other important tick species. Here we have used a BD86 derived synthetic peptide, BD86-3, to raise a series of mouse monoclonal antibodies. One of these mAbs, named 12.1, recognized BM86 homologues in immuno-histochemical analyses in four out of five tick species including R. (B.) microplus, Rhipicephalus (Boophilus) decoloratus, Hyalomma anatolicum anatolicum and Rhipicephalus appendiculatus. Our results indicate that broadly cross-reactive tick gut binding antibodies can be induced after immunization with a synthetic peptide derived from the protein BD86. PMID:19808026

  4. Referencing cross-reactivity of detection antibodies for protein array experiments [version 1; referees: 1 approved, 2 approved with reservations

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    Darragh Lemass

    2016-01-01

    Full Text Available Protein arrays are frequently used to profile antibody repertoires in humans and animals. High-throughput protein array characterisation of complex antibody repertoires requires a platform-dependent, lot-to-lot validation of secondary detection antibodies. This article details the validation of an affinity-isolated anti-chicken IgY antibody produced in rabbit and a goat anti-rabbit IgG antibody conjugated with alkaline phosphatase using protein arrays consisting of 7,390 distinct human proteins. Probing protein arrays with secondary antibodies in absence of chicken serum revealed non-specific binding to 61 distinct human proteins. The cross-reactivity of the tested secondary detection antibodies points towards the necessity of platform-specific antibody characterisation studies for all secondary immunoreagents. Secondary antibody characterisation using protein arrays enables generation of reference lists of cross-reactive proteins, which can be then excluded from analysis in follow-up experiments. Furthermore, making such cross-reactivity lists accessible to the wider research community may help to interpret data generated by the same antibodies in applications not related to protein arrays such as immunoprecipitation, Western blots or other immunoassays.

  5. Complement-dependent cytotoxicity of antibodies reactive with HIV-induced cell surface antigens in HIV-carrying haemophiliacs

    International Nuclear Information System (INIS)

    Sera obtained from HIV positive and from uninfected hemophiliacs and from healthy subjects were investigated for the presence of lymphocytotoxic antibodies. Using the 51Cr-release test, HIV-positive hemophiliacs were found to produce serum antibodies exerting a complement-dependent cytotoxic effect on HIV-infected T4 cells. The antibodies were reactive mainly when HIV-infected target cells were stimulated with concanavalin A. The results of complement-dependent antibody cytotoxicity and indirect membrane immunofluorescence tests suggest that envelope antigen(s) of HIV may be the target(s) for cytotoxic antibodies. (author)

  6. Antigenic Cross-Reactivity Anti-Birtoxin Antibody against Androctonus crassicauda Venom

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    SuhandanAdigüzel Van-Zoelen

    2015-10-01

    Full Text Available Background: Antivenom is still widely used in the treatment of envenomation as there are no vaccines or other effective agents available against animal venoms. Recently, neurotoxins named birtoxin family have been described from Parabuthus transvaalicus and Androctonus crassicauda. The aim of the present study was to test the antibirtoxinantibodies for their ability to neutralize the lethal effects of A. crassicauda scorpion venom.Methods: SDS-PAGE and Western blotting used the presence of components from A. crassicauda and P.transvaalicus scorpion venoms and to determine the degree of cross-reactivity. The Minimum Lethal Dose (MLD of venom was assessed by subcutaneously (sc injections in mice.Results: The MLD of the A. crassicauda venom was 35 μg/ 20g mouse by sc injection route. Western blotting showed the presence of components from A. crassicauda and P. transvaalicus scorpion venoms strongly cross react with the A. crassicauda antivenom. However, Western blotting of the A. crassicauda scorpion venom using the Refik Saydam Public Health Agency (RSPHA generated antibody showed that not all the venom components cross reacted with the anti-birtoxin antibody. The antibodies only cross reacted with components falling under the 19 kDa protein size of A. crassicauda venom.Conclusion: The bioassays and Western blotting of A. crassicauda venom with the anti-birtoxin antibodies produced against a synthetic peptide showed that these antibodies cross reacted but did not neutralize the venom of A. crassicauda.

  7. Antibody reactivities to glutamate-rich peptides of Plasmodium falciparum parasites in humans from areas of different malaria endemicity

    DEFF Research Database (Denmark)

    Jakobsen, P.H.; Theander, T.G.; Hvid, L; Morris-Jones, S.; Jensen, J.B.; Bayoumi, R.A.L.; Greenwood, B.M.; Bygbjerg, I.C.; Heegaard, Peter M. H.

    1996-01-01

    individuals from malaria-endemic areas of Sudan, Indonesia and The Gambia to study antibody responses to these peptides in donors living in areas of different malaria endemicity. IgG and IgM reactivities to the peptides increased with malaria endemicity, although there were no differences in reactivities to...... peptides tested were shortlived in most patients. In Gambian children with malaria, IgM reactivities but not IgG antibody reactivities against the ABRA peptide were higher in those with mild malaria than in those with severe malaria. The peptides may be useful in future epidemiological studies, especially......Synthetic P. falciparum peptides were evaluated as tools in epidemiological investigations of malaria. Plasma IgM and IgG antibody reactivities against synthetic peptides covering sequences of glutamate-rich protein (GLURP) and acidic-basic repeat antigen (ABRA) were measured by ELISA in...

  8. Rapid Screening for Potential Epitopes Reactive with a Polycolonal Antibody by Solution-Phase H/D Exchange Monitored by FT-ICR Mass Spectrometry

    Science.gov (United States)

    Zhang, Qian; Noble, Kyle A.; Mao, Yuan; Young, Nicolas L.; Sathe, Shridhar K.; Roux, Kenneth H.; Marshall, Alan G.

    2013-07-01

    The potential epitopes of a recombinant food allergen protein, cashew Ana o 2, reactive to polyclonal antibodies, were mapped by solution-phase amide backbone H/D exchange (HDX) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Ana o 2 polyclonal antibodies were purified in the serum from a goat immunized with cashew nut extract. Antibodies were incubated with recombinant Ana o 2 (rAna o 2) to form antigen:polyclonal antibody (Ag:pAb) complexes. Complexed and uncomplexed (free) rAna o 2 were then subjected to HDX-MS analysis. Four regions protected from H/D exchange upon pAb binding are identified as potential epitopes and mapped onto a homologous model.

  9. Monoclonal antibodies to human butyrylcholinesterase reactive with butyrylcholinesterase in animal plasma.

    Science.gov (United States)

    Peng, Hong; Brimijoin, Stephen; Hrabovska, Anna; Krejci, Eric; Blake, Thomas A; Johnson, Rudolph C; Masson, Patrick; Lockridge, Oksana

    2016-01-01

    Five mouse anti-human butyrylcholinesterase (BChE) monoclonal antibodies bind tightly to native human BChE with nanomolar dissociation constants. Pairing analysis in the Octet system identified the monoclonal antibodies that bind to overlapping and independent epitopes on human BChE. The nucleotide and amino acid sequences of 4 monoclonal antibodies are deposited in GenBank. Our goal was to determine which of the 5 monoclonal antibodies recognize BChE in the plasma of animals. Binding of monoclonal antibodies 11D8, B2 18-5, B2 12-1, mAb2 and 3E8 to BChE in animal plasma was measured using antibody immobilized on Pansorbin cells and on Dynabeads Protein G. A third method visualized binding by the shift of BChE activity bands on nondenaturing gels stained for BChE activity. Gels were counterstained for carboxylesterase activity. The three methods agreed that B2 18-5 and mAb2 have broad species specificity, but the other monoclonal antibodies interacted only with human BChE, the exception being 3E8, which also bound chicken BChE. B2 18-5 and mAb2 recognized BChE in human, rhesus monkey, horse, cat, and tiger plasma. A weak response was found with rabbit BChE. Monoclonal mAb2, but not B2 18-5, bound pig and bovine BChE. Gels stained for carboxylesterase activity confirmed that plasma from humans, monkey, pig, chicken, and cow does not contain carboxylesterase, but plasma from horse, cat, tiger, rabbit, guinea pig, mouse, and rat has carboxylesterase. Rabbit plasma carboxylesterase hydrolyzes butyrylthiocholine. In conclusion monoclonal antibodies B2 18-5 and mAb2 can be used to immuno extract BChE from the plasma of humans, monkey and other animals. PMID:26585590

  10. Differential reactivity of mouse monoclonal anti-HBs antibodies with recombinant mutant HBs antigens

    Institute of Scientific and Technical Information of China (English)

    Azam Roohi; Yaghoub Yazdani; Jalal Khoshnoodi; Seyed Mohammad Jazayeri; William F Carman; Mahmood Chamankhah; Manley Rashedan; Fazel Shokri

    2006-01-01

    AIM: To investigate the reactivity of a panel of 8 mouse anti-hepatitis B surface antigen (HBsAg) monoclonal antibodies (mAbs) using a collection of 9 recombinant HBsAg mutants with a variety of amino acid substitutions mostly located within the "a" region.METHODS: The entire HBs genes previously cloned into a mammalian expression vector were transiently transfected into COS7 cells. Two standard unmutated sequences of the ayw and adw subtypes served as controls. Secreted mutant proteins were collected and measured by three commercial diagnostic immunoassays to assess transfection efficiency. Reactivity of anti-HBs mAbs with mutated HBsAgs was determined by sandwich enzyme-linked immunosorbent assay (ELISA).RESULTS: Reactivity of anti-HBs mAbs with mutated HBsAgs revealed different patterns. While three mutants reacted strongly with all mAbs, two mutants reacted weakly with only two mAbs and the remaining proteins displayed variable degrees of reactivity towards different mAbs. Accordingly, four groups of mAbs with different but overlapping reactivity patterns could be envisaged. One group consisting of two mAbs (37C5-S7 and 35C6-S11) was found to recognize stable linear epitopes conserved in all mutants. Mutations outside the "a"determinant at positions 120 (P→S), 123(T→N) and 161(M→T) were found to affect reactivity of these mAbs.CONCLUSION: Our findings could have important implications for biophysical studies, vaccination strategies and immunotherapy of hepatitis B virus (HBV) mutants.

  11. C-reactive protein enhances murine antibody-mediated transfusion-related acute lung injury.

    Science.gov (United States)

    Kapur, Rick; Kim, Michael; Shanmugabhavananthan, Shanjeevan; Liu, Jonathan; Li, Yuan; Semple, John W

    2015-12-17

    Transfusion-related acute lung injury (TRALI) is a syndrome of respiratory distress triggered by blood transfusions and is the leading cause of transfusion-related mortality. TRALI has primarily been attributed to passive infusion of HLA and/or human neutrophil antigen antibodies present in transfused blood products, and predisposing factors such as inflammation are known to be important for TRALI initiation. Because the acute-phase protein C-reactive protein (CRP) is highly upregulated during infections and inflammation and can also enhance antibody-mediated responses such as in vitro phagocytosis, respiratory burst, and in vivo thrombocytopenia, we investigated whether CRP affects murine antibody-mediated TRALI induced by the anti-major histocompatibility complex antibody 34-1-2s. We found that BALB/c mice injected with 34-1-2s or CRP alone were resistant to TRALI, however mice injected with 34-1-2s together with CRP had significantly enhanced lung damage and pulmonary edema. Mechanistically, 34-1-2s injection with CRP resulted in a significant synergistic increase in plasma levels of the neutrophil chemoattractant macrophage inflammatory protein-2 (MIP-2) and pulmonary neutrophil accumulation. Importantly, murine MIP-2 is the functional homolog of human interleukin-8, a known risk factor for human TRALI. These results suggest that elevated in vivo CRP levels, like those observed during infections, may significantly predispose recipients to antibody-mediated TRALI reactions and support the notion that modulating CRP levels is an effective therapeutic strategy to reduce TRALI severity. PMID:26453659

  12. Approaches to systems biology. Four methods to study single-cell gene expression, cell motility, antibody reactivity, and respiratory metabolism

    DEFF Research Database (Denmark)

    Hagedorn, Peter

    To understand how complex systems, such as cells, function, comprehensive Measurements of their constituent parts must be made. This can be achieved by combining methods that are each optimized to measure specific parts of the system. Four such methods,each covering a different area, are presented......: Transcript profiling of one cell type extracted from a complex tissue containing several cell types; observation and recording of cell motility; measurement of antibody reactivities using microarrays; and invivo measurement of free and bound NADH in mitochondria. Detailed statistical analysis of the data...... from such measurements allows models of the system to be developed and tested. For each of the methods, such analysis and modelling approaches have beenapplied and are presented: Differentially regulated genes are identified and classified according to function; cell-specfic motility models are...

  13. An activation antigen on a subpopulation of B lymphocytes identified by the monoclonal antibody CMRF-17.

    Science.gov (United States)

    Peach, S F; Davidson, S E; McKenzie, J L; Nimmo, J C; Hart, D N

    1989-07-01

    The identification of membrane molecules expressed on subpopulations of B lymphocytes is of potential significance because these molecules may be candidates for regulating the activation, proliferation and differentiation of B cells. A new monoclonal antibody, CMRF-17, which reacts with a subpopulation of tonsil B lymphocytes has been produced. The antibody did not react with T lymphocytes in tonsil or peripheral blood nor most peripheral blood B lymphocytes but did label erythrocytes and some platelets. In tonsil, the germinal centre cells, cells in the interfollicular region and endothelial cells were positive, but mantle zone B cells were negative. Double labelling experiments showed that CMRF-17 reacted with activated tonsillar lymphocytes. The antigen recognized by CMRF-17 was heat stable, resistant to treatment with proteolytic enzymes and neuraminidase and was shown to be a carbohydrate determinant on one or more glycolipids. These characteristics of the antigen recognized by CMRF-17 and its pattern of reactivity distinguish this antibody from other monoclonal antibodies recognizing B-cell activation markers. It was notable that of the B-lymphoid malignancies tested to date, including those of probable follicular origin, few stained with CMRF-17. PMID:2474491

  14. Synthesis of HPMA-DOX-antibody conjugates. Optimization of antibody-binding reaction of a polyHPMA procursor containing thiazolidine-2-thione reactive groups

    Czech Academy of Sciences Publication Activity Database

    Šubr, Vladimír; Ulbrich, Karel

    Cardiff: Welsh School of Pharmacy , Redwood Building, Cardiff University, 2004, s. 46. [International Symposium on Polymer Therapeutics /6./. Cardiff (GB), 07.01.2004-09.01.2004] R&D Projects: GA AV ČR IBS5020101 Institutional research plan: CEZ:AV0Z4050913 Keywords : polymer-drug-antibody conjugates * thiazolidine-2-thione reactive polymers Subject RIV: CD - Macromolecular Chemistry

  15. Production and characterization of mouse monoclonal antibodies reactive to Chikungunya envelope E2 glycoprotein.

    Science.gov (United States)

    Bréhin, Anne-Claire; Rubrecht, Laetitia; Navarro-Sanchez, Martha Erika; Maréchal, Valérie; Frenkiel, Marie-Pascale; Lapalud, Priscilla; Laune, Daniel; Sall, Amadou Alpha; Desprès, Philippe

    2008-02-01

    Chikungunya fever is an arbovirosis of major impact in public health in Asia and Africa. Chikungunya (CHIK) virus is member of the genus Alphavirus and belongs to the Semliki Forest (SF) antigenic complex. We describe for the first time a panel of monoclonal antibodies (MAbs) reactive to CHIK envelope E2 glycoprotein. For the screening of E2-specific MAbs, we expressed a recombinant soluble CHIK E2 protein in Drosophila S2 cells. Analyzed by immunological methods, MAbs 3C3, 3E4, and 8A4 were selected on the basis of their reactivity. Their epitopes are located to the outer surface of CHIK virion. These MAbs have no cross reactivity with related members of SF antigenic complex with the notable exception of Igbo-Ora virus. Anti-CHIK E2 MAbs 3C3, 3E4, and 8A4 should be helpful for studying the biology of CHIK virus and pathogenesis of disease. The combination of 8A4 and 3E4 is suitable for developing a specific antigen-capture ELISA. PMID:17949772

  16. [Reactivity of antibodies to collagen types I to IV and antibodies to chondroitin sulfate in the spleen].

    Science.gov (United States)

    Galbavý, S; Ruzicková, M; Surmíková, E; Danihel, L; Porubský, J; Papincák, J; Holesa, S; Trnka, J

    1996-02-01

    Antibodies to collagen type I and III reacted negatively, antibodies to collagen type IV positively with reticulin, trabeculae and circumferent reticulum of lymphatic sheaths, poorly positively with capsula, strongly positively with subcapsular zone. Antibodies to collagen type II reacted positively with capsula, poorly with subcapsular zone, strongly with sinus wall and poorly with trabeculae. They did not react with circumferent reticulum of periarterial lymphoid sheaths. Antibodies to collagen type II and IV reacted positively with central arteries. Antibodies to chondroitinsulphate C reacted poorly and antibodies to chondroitinsulphate B strongly positively with sinus walls and oval cells spread in the white and red pulpa. Antibodies to chondroitin sulphate A reacted similarly as antibodies to chondroitinsulphate B. PMID:9560890

  17. Distinct EBV and CMV reactivation patterns following antibody-based immunosuppressive regimens in patients with severe aplastic anemia

    OpenAIRE

    Scheinberg, Phillip; Fischer, Steven H.; Li, Li; Nunez, Olga; Wu, Colin O.; Sloand, Elaine M.; Cohen, Jeffrey I.; Young, Neal S; A. John Barrett

    2007-01-01

    The natural history of EBV and CMV reactivation and the potential for serious complications following antibody-based immunosuppressive treatment for bone marrow failure syndromes in the absence of transplantation is not known. We monitored blood for EBV and CMV reactivation by polymerase chain reaction (PCR) weekly in 78 consecutive patients (total of 99 immunosuppressive courses) with aplastic anemia. Four regimens were studied: (1) HC, horse ATG/cyclosporine; (2) HCS, horse ATG/CsA/sirolimu...

  18. Cell Surface Glycoprotein of Reactive Stromal Fibroblasts as a Potential Antibody Target in Human Epithelial Cancers

    Science.gov (United States)

    Garin-Chesa, Pilar; Old, Lloyd J.; Rettig, Wolfgang J.

    1990-09-01

    The F19 antigen is a cell surface glycoprotein (M_r, 95,000) of human sarcomas and proliferating, cultured fibroblasts that is absent from resting fibroblasts in normal adult tissues. Normal and malignant epithelial cells are also F19^-. The present immunohistochemical study describes induction of F19 in the reactive mesenchyme of epithelial tumors. F19^+ fibroblasts were found in primary and metastatic carcinomas, including colorectal (18 of 18 cases studied), breast (14/14), ovarian (21/21), bladder (9/10), and lung carcinomas (13/13). In contrast, the stroma of benign colorectal adenomas, fibrocystic disease and fibroadenomas of breast, benign prostate hyperplasia, in situ bladder carcinomas, and benign ovarian tumors showed no or only moderate numbers of F19^+ fibroblasts. Analysis of dermal incision wounds revealed that F19 is strongly induced during scar formation. Comparison of F19 with the extracellular matrix protein tenascin, a putative marker of tumor mesenchyme, showed a cellular staining pattern for F19 vs. the extracellular matrix pattern for tenascin and widespread expression of tenascin in F19^- normal tissues and benign tumors. Our results suggest that the F19^+ phenotype correlates with specialized fibroblast functions in wound healing and malignant tumor growth. Because of its abundance in tumor mesenchyme, F19 may serve as a target for antibodies labeled with radioisotopes or toxic agents, or inflammatogenic antibodies, in carcinoma patients.

  19. Cross-reactivity of anti-H pylori antibodies with membrane antigens of human erythrocytes

    Institute of Scientific and Technical Information of China (English)

    Feng-Hua Guo; Fan-Ling Meng; Jian-Zhong Zhang; Xiao-Mei Yan; Chun-Xiang Fan; Fei Zhao; Yuan Hu; Di Xiao; Xun Zeng; Mao-Jun Zhang; Li-Hua He

    2007-01-01

    AIM: To investigate whether anti-H pylori antibodies have cross-reaction with antigens of erythrocyte membrane.METHODS: Blood samples were collected from 14 volunteers (8 positive and 6 negative for H pylori detected by 13C-urea breath test) of the general population. Erythrocyte membrane proteins of the subjects were examined by Western blot using antiH pylori serum. The proteins related to the positive bands were identified by mass spectrum analysis.RESULTS: Anti-H pylori antibodies had cross-reaction with the proteins of about 50 kDa of erythrocyte membranes in all samples independent of H pylori infection. One protein in the positive band was identified as Chain S, the crystal structure of the cytoplasmic domain of human erythrocyte Band-3 protein.CONCLUSION: Anti-H pylori antibodies cross-react with some antigens of human erythrocyte membrane, which may provide a clue for the relationship between H pylori infection and vascular disorders.

  20. Specific IgE antibodies to reactive dye-albumin conjugates

    International Nuclear Information System (INIS)

    Hypersensitivity to reactive dye powders has been recognised for a number of years, although the extent of sensitisation amongst dye house operatives and the immunochemistry of the dye molecules has not been investigated. The authors have developed a radioallergosorbent test (RAST) to detect specific IgE to reactive dye-human serum albumin (HSA) conjugates. From a total of 19 dye-HSA conjugates, positive RASTs were found in six workers with allergic symptoms associated with dye exposure, while six asymptomatic case-matched controls were negative. Sera with raised total IgE (up to 4300 kU/l) from unexposed workers gave negative results except for two conjugates which gave a weak positive at 4300 kU/l and one which gave weak positives at all concentrations tested (750-4300 kU/l). RAST inhibition studies demonstrated that the antibody was specific for the complete dye-HSA conjugate. Substitution of bovine serum albumin (BSA) for HSA in the conjugate markedly reduced immunoreactivity and free hapten gave lower inhibition than the complete conjugate. Comparison of the dye-HSA RAST with a RAST using dyed discs showed that the latter did not correlate well with symptoms and was influenced by the total IgE concentration. (Auth.)

  1. ZP-binding peptides identified via phage display stimulate production of sperm antibodies in dogs.

    Science.gov (United States)

    Samoylova, Tatiana I; Cox, Nancy R; Cochran, Anna M; Samoylov, Alexandre M; Griffin, Brenda; Baker, Henry J

    2010-07-01

    Zona pellucida (ZP) glycoproteins play a central role in sperm-oocyte binding and fertilization. Sperm protein sequences that are involved in sperm-ZP recognition and have an important role in fertilization represent attractive targets for development of contraceptive vaccines, yet are currently unknown. To identify peptide sequences that recognize and bind to ZP proteins, we developed a novel selection procedure from phage display libraries that utilizes intact oocytes surrounded by ZP proteins. The major advantage of this procedure is that ZP proteins remain in their native conformation unlike a selection protocol previously published that utilized solubilized ZP on artificial solid support. Several peptides of 7 and 12 amino acids with binding specificity to canine ZP proteins were identified. Four of them (LNSFLRS, SSWYRGA, YLPIYTIPSMVY, and NNQSPILKLSIH) plus a control ZP-binding peptide (YLPVGGLRRIGG) from the literature were synthesized and tested for antigenic properties in dogs. NNQSPILKLSIH peptide stimulated production of anti-peptide antibodies. These antibodies bind to the acrosomal region of the canine sperm cell, demonstrating ability to act as sperm antibodies. The identified ZP-binding peptides (mimicking sperm cell surface antigens) may be useful in the design of immunocontraceptive agents for dogs. PMID:20434854

  2. Minimizing antibody cross-reactivity in multiplex detection of biomarkers in paper-based point-of-care assays

    Science.gov (United States)

    Dias, J. T.; Lama, L.; Gantelius, J.; Andersson-Svahn, H.

    2016-04-01

    Highly multiplexed immunoassays could allow convenient screening of hundreds or thousands of protein biomarkers simultaneously in a clinical sample such as serum or plasma, potentially allowing improved diagnostic accuracy and clinical management of many conditions such as autoimmune disorders, infections, and several cancers. Currently, antibody microarray-based tests are limited in part due to cross reactivity from detection antibody reagents. Here we present a strategy that reduces the cross-reactivity between nanoparticle-bound reporter antibodies through the application of ultrasound energy. By this concept, it was possible to achieve a sensitivity 103-fold (5 pg mL-1) lower than when no ultrasound was applied (50 ng mL-1) for the simultaneous detection of three different antigens. The detection limits and variability achieved with this technique rival those obtained with other types of multiplex sandwich assays.Highly multiplexed immunoassays could allow convenient screening of hundreds or thousands of protein biomarkers simultaneously in a clinical sample such as serum or plasma, potentially allowing improved diagnostic accuracy and clinical management of many conditions such as autoimmune disorders, infections, and several cancers. Currently, antibody microarray-based tests are limited in part due to cross reactivity from detection antibody reagents. Here we present a strategy that reduces the cross-reactivity between nanoparticle-bound reporter antibodies through the application of ultrasound energy. By this concept, it was possible to achieve a sensitivity 103-fold (5 pg mL-1) lower than when no ultrasound was applied (50 ng mL-1) for the simultaneous detection of three different antigens. The detection limits and variability achieved with this technique rival those obtained with other types of multiplex sandwich assays. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr09207h

  3. Development and evaluation of a rapid latex agglutination test using a monoclonal antibody to identify Candida dubliniensis colonies.

    Science.gov (United States)

    Marot-Leblond, Agnes; Beucher, Bertrand; David, Sandrine; Nail-Billaud, Sandrine; Robert, Raymond

    2006-01-01

    Cell components of the dimorphic pathogenic fungus Candida dubliniensis were used to prepare monoclonal antibodies (MAbs). One MAb, designated 12F7-F2, was shown by indirect immunofluorescence to be specific for a surface antigen of Candida dubliniensis yeast cells. No reactivity was observed with other fungal genera or with other Candida species, including Candida albicans, that share many phenotypic features with C. dubliniensis. The use of different chemical and physical treatments for cell component extraction suggested that the specific epitope probably resides on a protein moiety absent from C. albicans. However, we failed to identify the target protein by Western blotting, owing to its sensitivity to heat and sodium dodecyl sulfate. MAb 12F7-F2 was further used to develop a commercial latex agglutination test to identify C. dubliniensis colonies (Bichro-dubli Fumouze test; Fumouze Diagnostics). The test was validated on yeast strains previously identified by PCR and on fresh clinical isolates; these included 46 C. dubliniensis isolates, 45 C. albicans isolates, and other yeast species. The test had 100% sensitivity and specificity for C. dubliniensis isolated on Sabouraud dextrose, CHROMagar Candida, and CandiSelect media and 97.8% sensitivity for C. dubliniensis grown on Candida ID medium. The test is rapid (5 min) and easy to use and may be recommended for routine use in clinical microbiology laboratories and for epidemiological investigations. PMID:16390961

  4. Antibody

    Science.gov (United States)

    An antibody is a protein produced by the body's immune system when it detects harmful substances, called antigens. Examples ... microorganisms (bacteria, fungi, parasites, and viruses) and chemicals. Antibodies may be produced when the immune system mistakenly ...

  5. Antibody and DNA dual-labeled gold nanoparticles: Stability and reactivity

    Science.gov (United States)

    Qiao, Fei-Yan; Liu, Jun; Li, Fu-Rong; Kong, Xiao-Li; Zhang, Hao-Li; Zhou, Han-Xin

    2008-03-01

    Gold nanoparticles labeled by both antibody (IgG) and single stranded DNA (ss-DNA) have been synthesized and characterized. The stability and reactivity of the dual-labeled nanoparticles were compared with the conventional IgG or ss-DNA modified nanoparticles. It was found that the IgG adsorption significantly improved the stability of the nanoparticles in aqueous solution, which is beneficial for attaching ss-DNA. The presence of IgG also effectively prohibits the desorption of ss-DNA against dithiothreitol (DTT) displacement. The coverage on dual-labeled nanoparticles was found to be 50 ± 15 ss-DNA/nanoparticle and 10 ± 2 IgG/nanoparticle, respectively, compared to the value of 70 ± 15 ss-DNA/nanoparticle of only ss-DNA-labeled gold nanoparticles. Dot-immuno and cross-linking experiments confirmed that both the IgG and ss-DNA retained their bioactivity on the nanoparticle surface. The dual-labeled nanoparticles have potential to be used as novel bio-probes for ultrasensitive detection.

  6. [Effect of thalassemia panel reactive antibody on proliferation and apoptosis of cord blood CD34(+) cells].

    Science.gov (United States)

    Yang, Xing-Ge; Lu, Xue-Liang; Xu, Lü-Hong; Fang, Jian-Pei

    2012-02-01

    The study was purposed to explore the effect of panel reactive antibody (PRA) serum from patients with β-thalassemia on proliferation and apoptosis of the CD34(+)cells from cord blood and its mechanism. CD34(+) cells of umbilical cord blood were incubated with different sera and complement respectively. After incubation, the samples were centrifuged and the supernatants were collected for lactate dehydrogenase (LDH) detection, and the CD34(+) cells were harvested and measured for the apoptosis by flow cytometry with Annexin V/PI. The intracellular DNA synthesis were also quantified by [(3)H]TdR incorporation using liquid scintillation counter. The results showed that concentration of LDH in PRA positive groups was higher as compared with control group, and the DNA synthesis of CD34(+) cells in PRA positive groups were inhibited. There were no differences in the percentage of cell apoptosis and necrosis among different groups. It is concluded that thalassemic serum PRA impairs the cell membrane, inhibits the DNA synthesis, which can be increased by addition of the complement, but PRA had no significant effect on apoptosis of CD34(+) cells. PMID:22391181

  7. Production of Mouse Monoclonal Antibody against Morphine without Cross Reactivity with Heroin

    Directory of Open Access Journals (Sweden)

    S Kashaninan

    2014-12-01

    Conclusion: The study findings revealed that the produced antibody against morphine was comparable with other antibodies for specificity and affinity; therefore it is usable in design of diagnostic immunoassay in biologic fluids.

  8. Optimizing the Measurement of Colostrum Antibody Concentrations for Identifying BVDV Persistently Infected Calves

    Directory of Open Access Journals (Sweden)

    Caitlin J. Jenvey

    2015-03-01

    Full Text Available Colostrum contains substantially higher concentrations of immunoglobulins compared to serum, which may help to improve the utility of diagnostic tests. The aim of this study was to determine the diagnostic value of colostrum antibody concentrations in identifying Bovine Viral Diarrhoea Virus (BVDV PI (persistently infected calf carrying beef heifers following an experimental infection. Colostrum was collected within 12 hours of parturition and tested in undiluted, 1:5, 1:10, 1:100, 1:200, and 1:500 dilutions using an enzyme-linked immunosorbent assay (ELISA for BVDV antibody. Cows were determined to be carrying a PI calf based on positive quantitative Real Time-Polymerase Chain Reaction and antigen ELISA result on pre-colostral serum and ear notch samples collected from their calf. The median ELISA sample-to-positive (S/P ratio for colostrum collected from heifers that carried a PI calf were significantly higher than the median ELISA S/P ratio for colostrum collected from heifers that did not carry a PI calf at dilutions of 1:100, 1:200, and 1:500. This study provides further evidence for increased antigenic stimulation in utero by the BVDV viraemic PI calf, which can also be identified with 100% diagnostic sensitivity when using 1:500 dilution colostrum.

  9. A radiolabeled antiglobulin assay to identify human cervical mucus immunoglobulin (Ig) A and IgG antisperm antibodies

    International Nuclear Information System (INIS)

    Antisperm immunoglobulin (Ig) A and IgG antibodies in human cervical mucus (CM) were identified by a radiolabeled antiglobulin assay. Cervical mucus samples from fertile and infertile women were exposed to a 1:3,200 dilution of 2-mercaptoethanol (2-ME), and 5 micrograms of the solubilized CM protein were assayed for the presence of IgA and IgG antisperm and anti-Candida activity by the radiolabeled antiglobulin assay. Purified human secretory IgA and IgG exposed to 2-ME retained the molecular integrity and functional activity of the untreated antibody molecules. CM aliquots collected after high-performance liquid chromatography (HPLC) fractionation were assessed for antisperm antibody activity; antisperm antibody activity was retained in the appropriate IgA or IgG CM fractions. The incidence of CM antisperm antibodies was minimally affected when the radiolabeled antiglobulin assay was performed with a motile sperm population. Approximately 70% of the CM IgA antisperm antibodies were of the IgA1 subclass; CM IgG was primarily of the IgG4 subclass. When Candida antigen was substituted for sperm in the radiolabeled antiglobulin assay, the CM antisperm antibodies were found to be exclusively sperm-specific. These data indicate that the radiolabeled antiglobulin assay using 2-ME to extract CM antibodies is a specific method for the assay of antisperm antibodies in CM

  10. Novel association of APC with intermediate filaments identified using a new versatile APC antibody

    Directory of Open Access Journals (Sweden)

    Coffey Robert J

    2009-10-01

    Full Text Available Abstract Background As a key player in suppression of colon tumorigenesis, Adenomatous Polyposis Coli (APC has been widely studied to determine its cellular functions. However, inconsistencies of commercially available APC antibodies have limited the exploration of APC function. APC is implicated in spindle formation by direct interactions with tubulin and microtubule-binding protein EB1. APC also interacts with the actin cytoskeleton to regulate cell polarity. Until now, interaction of APC with the third cytoskeletal element, intermediate filaments, has remained unexamined. Results We generated an APC antibody (APC-M2 pAb raised against the 15 amino acid repeat region, and verified its reliability in applications including immunoprecipitation, immunoblotting, and immunofluorescence in cultured cells and tissue. Utilizing this APC-M2 pAb, we immunoprecipitated endogenous APC and its binding proteins from colon epithelial cells expressing wild-type APC. Using Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS, we identified 42 proteins in complex with APC, including β-catenin and intermediate filament (IF proteins lamin B1 and keratin 81. Association of lamin B1 with APC in cultured cells and human colonic tissue was verified by co-immunoprecipitation and colocalization. APC also colocalized with keratins and remained associated with IF proteins throughout a sequential extraction procedure. Conclusion We introduce a versatile APC antibody that is useful for cell/tissue immunostaining, immunoblotting and immunoprecipitation. We also present evidence for interactions between APC and IFs, independent of actin filaments and microtubules. Our results suggest that APC associates with all three major components of the cytoskeleton, thus expanding potential roles for APC in the regulation of cytoskeletal integrity.

  11. Immunoproteomic and bioinformatic approaches to identify secreted Leishmania amazonensis, L. braziliensis, and L. infantum proteins with specific reactivity using canine serum.

    Science.gov (United States)

    Lima, B S S; Fialho, L C; Pires, S F; Tafuri, W L; Andrade, H M

    2016-06-15

    Leishmania spp have a wide range of hosts, and each host can harbor several Leishmania species. Dogs, for example, are frequently infected by Leishmania infantum, where they constitute its main reservoir, but they also serve as hosts for L. braziliensis and L. amazonensis. Serological tests for antibody detection are valuable tools for diagnosis of L. infantum infection due to the high levels of antibodies induced, unlike what is observed in L. amazonensis and L. braziliensis infections. Likewise, serology-based antigen-detection can be useful as an approach to diagnose any Leishmania species infection using different corporal fluid samples. Immunogenic and secreted proteins constitute powerful targets for diagnostic methods in antigen detection. As such, we performed immunoproteomic (2-DE, western blot and mass spectrometry) and bioinformatic screening to search for reactive and secreted proteins from L. amazonensis, L. braziliensis, and L. infantum. Twenty-eight non-redundant proteins were identified, among which, six were reactive only in L. amazonensis extracts, 10 in L. braziliensis extracts, and seven in L. infantum extracts. After bioinformatic analysis, seven proteins were predicted to be secreted, two of which were reactive only in L. amazonensis extracts (52kDa PDI and the glucose-regulated protein 78), one in L. braziliensis extracts (pyruvate dehydrogenase E1 beta subunit) and three in L. infantum extracts (two conserved hypothetical proteins and elongation factor 1-beta). We propose that proteins can be suitable targets for diagnostic methods based on antigen detection. PMID:27198787

  12. Development and Evaluation of a Rapid Latex Agglutination Test Using a Monoclonal Antibody To Identify Candida dubliniensis Colonies

    OpenAIRE

    Marot-Leblond, Agnes; Beucher, Bertrand; David, Sandrine; Nail-Billaud, Sandrine; Robert, Raymond

    2006-01-01

    Cell components of the dimorphic pathogenic fungus Candida dubliniensis were used to prepare monoclonal antibodies (MAbs). One MAb, designated 12F7-F2, was shown by indirect immunofluorescence to be specific for a surface antigen of Candida dubliniensis yeast cells. No reactivity was observed with other fungal genera or with other Candida species, including Candida albicans, that share many phenotypic features with C. dubliniensis. The use of different chemical and physical treatments for cel...

  13. Large-scale analysis of B-cell epitopes on influenza virus hemagglutinin - implications for cross-reactivity of neutralizing antibodies

    Directory of Open Access Journals (Sweden)

    Jing eSun

    2014-02-01

    Full Text Available Influenza viruses continue to cause substantial morbidity and mortality worldwide. Fast gene mutation on surface proteins of influenza virus result in increasing resistance to current vaccines and available antiviral drugs. Broadly neutralizing antibodies represent targets for prophylactic and therapeutic treatments of influenza. We performed a systematic bioinformatics study of cross-reactivity of neutralizing antibodies against influenza virus surface glycoprotein hemagglutinin (HA. This study utilized the available crystal structures of HA complexed with the antibodies for the analysis of tens of thousands of HA sequences. The detailed description of B-cell epitopes, measurement of epitope area similarity among different strains, and estimation of antibody neutralizing coverage provide insights into cross-reactivity status of existing neutralizing antibodies against influenza virus. We have developed a method to assess the likely cross-reactivity potential of broadly neutralizing antibodies for influenza strains, either newly emerged or existing. Our method catalogs influenza strains by a new concept named discontinuous peptide, and then provide assessment of cross-reactivity. Potentially cross-reactive strains are those that share 100% identity with experimentally verified neutralized strains. By cataloging influenza strains and their B-cell epitopes for known broadly neutralizing antibodies, our method provides guidance for selection of representative strains for further experimental design. The knowledge of sequences, their B-cell epitopes, and differences between historical influenza strains, we enhance our preparedness and the ability to respond to the emerging pandemic threats.

  14. Functional and molecular characterization of human monoclonal antibody reactive with the immunodominant region of HIV type 1 glycoprotein 41.

    Science.gov (United States)

    Cavacini, L A; Emes, C L; Wisnewski, A V; Power, J; Lewis, G; Montefiori, D; Posner, M R

    1998-09-20

    The immunoreactivity, functional activity, and molecular features of a human monoclonal antibody (HMAb), F240, from an HIV-1-infected individual have been studied. Flow cytometric analysis demonstrated that F240 is reactive with cells infected with a broad range of laboratory isolates but not with uninfected cells. Reactivity of F240 is greatly enhanced by preincubation of infected cells with soluble CD4, and to a much lesser extent, with F105, an HMAb reactive with the CD4-binding site of gp120. This enhancement is temperature dependent, with maximum enhancement observed at 37 degrees C, and suggests that the F240 epitope may be more accessible after gp120 has bound to CD4 in vivo. Immunoblot analysis reveals antigen specificity of F240 for gp41 or its precursor gp160. F240 specificity is mapped to the immunodominant region of the gp41 ectodomain by Pepscan analysis. This epitope has been implicated in eliciting nonprotective antibodies that enhance infection in the presence of complement. Consistent with this, F240 failed to neutralize laboratory isolates and enhanced viral infection in a complement-dependent manner. The F240 VH demonstrates extensive somatic mutations compared with the product of its closest homologous germline gene VH3-3.11. Most amino acid substitutions occur in CDR2, characteristic of an antigen-driven response, and in FR3, a phenomenon observed in other anti-HIV-1 envelope HMAbs. Primary structure analysis of the F240 heavy chain revealed strong homology in the CDR domains to an HMAb (3D6) reactive with the same gp41 region, which suggests that these HMAbs could define a potential human antibody clonotype. PMID:9764911

  15. Disassembly and reassembly of human papillomavirus virus-like particles produces more virion-like antibody reactivity

    Directory of Open Access Journals (Sweden)

    Zhao Qinjian

    2012-02-01

    Full Text Available Abstract Background Human papillomavirus (HPV vaccines based on major capsid protein L1 are licensed in over 100 countries to prevent HPV infections. The yeast-derived recombinant quadrivalent HPV L1 vaccine, GARDASIL(R, has played an important role in reducing cancer and genital warts since its introduction in 2006. The L1 proteins self-assemble into virus-like particles (VLPs. Results VLPs were subjected to post-purification disassembly and reassembly (D/R treatment during bioprocessing to improve VLP immunoreactivity and stability. The post-D/R HPV16 VLPs and their complex with H16.V5 neutralizing antibody Fab fragments were visualized by cryo electron microscopy, showing VLPs densely decorated with antibody. Along with structural improvements, post-D/R VLPs showed markedly higher antigenicity to conformational and neutralizing monoclonal antibodies (mAbs H16.V5, H16.E70 and H263.A2, whereas binding to mAbs recognizing linear epitopes (H16.J4, H16.O7, and H16.H5 was greatly reduced. Strikingly, post-D/R VLPs showed no detectable binding to H16.H5, indicating that the H16.H5 epitope is not accessible in fully assembled VLPs. An atomic homology model of the entire HPV16 VLP was generated based on previously determined high-resolution structures of bovine papillomavirus and HPV16 L1 pentameric capsomeres. Conclusions D/R treatment of HPV16 L1 VLPs produces more homogeneous VLPs with more virion-like antibody reactivity. These effects can be attributed to a combination of more complete and regular assembly of the VLPs, better folding of L1, reduced non-specific disulfide-mediated aggregation and increased stability of the VLPs. Markedly different antigenicity of HPV16 VLPs was observed upon D/R treatment with a panel of monoclonal antibodies targeting neutralization sensitive epitopes. Multiple epitope-specific assays with a panel of mAbs with different properties and epitopes are required to gain a better understanding of the immunochemical

  16. Low Level of Cross-Reactive Antibodies to Pandemic Influenza (H1N1) 2009 Virus in Humans in Pre-Pandemic Period in Maharashtra, India.

    Science.gov (United States)

    Kode, Sadhana S; Pawar, Shailesh D; Tandale, Babasaheb V; Parkhi, Saurabh S; Barde, Tanaji D; Mishra, Akhilesh C

    2012-06-01

    In India, the first outbreak of pandemic influenza (H1N1) 2009 (H1N1pdm) was reported from Panchgani, Maharashtra, in June 2009. Studies from several countries have revealed different levels of pre-existing immunity to H1N1pdm 2009 in various age groups. This study was undertaken using age-stratified pre-pandemic human sera to understand baseline cross-reactivity of antibodies against H1N1pdm. Using cut off antibody titers 20 and 40, overall cross-reactivity was 2.1 and 0.9% respectively by microneutralization assay; 1.2% and 0.7% by haemagglutination inhibition assay, respectively. Results showed higher baseline antibodies and cross-reactive antibodies in the 0-19 age group whereas the elderly age group (≥60) showed no cross-reactivity to H1N1pdm. The higher baseline and cross-reactive antibodies in 0-19 years age group could be because of higher positivity to seasonal H1N1 in that age group. Overall, low level of cross-reactive antibodies to H1N1pdm virus were found in humans in pre-pandemic period in Maharashtra, India. PMID:23730000

  17. Production of IgM antibody to HHV6 in reactivation and primary infection.

    OpenAIRE

    Fox, J D; Ward, P; Briggs, M; Irving, W; Stammers, T. G.; Tedder, R S

    1990-01-01

    The cross-reaction of HHV6 antibody with that to the other herpesviruses was studied in 96 blood donors whose sera were tested for IgG antibody to human herpesvirus type 6 (HHV6), cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella zostervirus (VZV) and herpes simplex virus (HSV). No correlation was found between IgG antibody to HHV6 and that to any of the other herpesviruses in these individuals. Antibodies to HHV6 and CMV were measured in patients undergoing documented serological re...

  18. Identification of discriminant proteins through antibody profiling, methods and apparatus for identifying an individual

    Energy Technology Data Exchange (ETDEWEB)

    Apel, William A.; Thompson, Vicki S; Lacey, Jeffrey A.; Gentillon, Cynthia A.

    2016-08-09

    A method for determining a plurality of proteins for discriminating and positively identifying an individual based from a biological sample. The method may include profiling a biological sample from a plurality of individuals against a protein array including a plurality of proteins. The protein array may include proteins attached to a support in a preselected pattern such that locations of the proteins are known. The biological sample may be contacted with the protein array such that a portion of antibodies in the biological sample reacts with and binds to the proteins forming immune complexes. A statistical analysis method, such as discriminant analysis, may be performed to determine discriminating proteins for distinguishing individuals. Proteins of interest may be used to form a protein array. Such a protein array may be used, for example, to compare a forensic sample from an unknown source with a sample from a known source.

  19. Large-scale analysis of B-cell epitopes on influenza virus hemagglutinin - implications for cross-reactivity of neutralizing antibodies

    OpenAIRE

    Jing eSun; Ulrich eKudahl; Zhiwei eCao; Christian eSimon; Reinherz, Ellis L; Vladimir eBrusic

    2014-01-01

    Influenza viruses continue to cause substantial morbidity and mortality worldwide. Fast gene mutation on surface proteins of influenza virus result in increasing resistance to current vaccines and available antiviral drugs. Broadly neutralizing antibodies represent targets for prophylactic and therapeutic treatments of influenza. We performed a systematic bioinformatics study of cross-reactivity of neutralizing antibodies against influenza virus surface glycoprotein hemagglutinin (HA). This s...

  20. Restricted Isotypic Antibody Reactivity to Hepatitis C Virus Synthetic Peptides in Immunocompromised Patients

    Science.gov (United States)

    Devesa, Marisol; de Saez, Arlette; León, Graciela; Sirit, Firelei; Cosson, Clarisa; Bermúdez, Henry; Liprandi, Ferdinando; Noya, Oscar; Pujol, Flor H.

    1999-01-01

    An enzyme immunoassay based on three synthetic peptides from the core, NS4, and NS5 regions of hepatitis C virus allowed the detection of antibodies in 100% of immunocompetent infected patients and in 91% of immunocompromised patients (hemodialysis and hemophiliac patients). Immune impairment seemed to restrict the spectrum of antibody isotypes reacting to the core peptide. PMID:10066669

  1. Serological survey for antibodies reactive with Ehrlichia canis and E. chaffeensis in dogs from the Bloemfontein area, South Africa

    Directory of Open Access Journals (Sweden)

    A-M Pretorius

    1998-07-01

    Full Text Available Sera from 161 dogs in the Bloemfontein area in South Africa were tested for the presence of antibodies reactive with Ehrlichia canis and E. chaffeensis by indirect fluorescent antibody testing. Overall, 68 (42 % of the dogs had significant antibody titres (>1/64 against E. canis and 61 (38 % had significant titres (>1/64 against E. chaffeensis. Seven (11 % dogs had higher titres to E. chaffeensis than E. canis (1/2048 and 1/1024 (2 dogs; 1/1024 and 1/512 (2 dogs; 1/2048 and 1/512; 1/512 and 1/256 and 1/512 and <1/64, respectively. The remaining seropositive dogs had equal (n=26; 42 % or 2- (n=17; 25 %, 3- (n=13; 2% or 4-fold (n= 5; 7 % higher titres against E. canis. Dogs from economically depressed, high-density suburbs (60/112; 48 % had significantly higher prevalences of antibodies against E. canis than those from more affluent, low-density suburbs (8/49; 14 % (c2 19.38, p < 0.001. Higher titres to E. chaffeensis than E. canis were found in dogs from affluent, low-density suburbs (3/49 and in dogs from economically depressed, high-density suburbs (4/112.

  2. Caprine arthritis-encephalitis virus-infected goats can generate human immunodeficiency virus-gp120 cross-reactive antibodies

    International Nuclear Information System (INIS)

    Lentiviruses display surprisingly disparate clinical manifestations in their specific hosts, share complex genetic structures, and exhibit extensive diversity, particularly in their envelope genes. The envelope protein, gp135, of caprine arthritis-encephalitis virus (CAEV) has minimal primary sequence homology to gp120, the envelope protein of human immunodeficiency virus (HIV). Nevertheless, they bear certain similarities in that they both possess five variable regions, both are heavily glycosylated, and both share short sequence motifs. We establish a further relationship and demonstrate that some goats, infected with CAEV, possess gp135-specific antibodies which cross-react with gp120 from several HIV strains, provided the protein is expressed in insect cells. We show that, although the cross-reactivity of these immunoglobulins depends on the level of glycosylation, nevertheless, some antibodies recognize the protein epitopes on gp120, at least some of which are linear in character. Further characterization of this unexpected cross-reaction will define its potential therapeutic utility

  3. Reaction of rheumatoid factors with IgG3 monoclonal anti-Rh(D) antibodies: more frequent reactivity to a monoclonal antibody of the Gm allotype G3m(5) in rheumatoid patients negative for G3m(5).

    OpenAIRE

    Puttick, A H; Williamson, E.A.; Merry, A.H.; Kumpel, B M; Thompson, K. M.; Jones, V E

    1988-01-01

    Human monoclonal anti-Rh(D) antibodies of known IgG isotype and Gm allotype were bound to erythrocytes and then used as the target IgG antigens for rheumatoid factors (RFs) in a direct haemagglutination test. When serum samples from patients with rheumatoid arthritis (RA) were tested for RF specificity towards these IgG monoclonal anti-D antibodies the incidence and titre of reactivity towards an IgG3 monoclonal anti-D antibody was considerably greater than for a polyclonal anti-D antibody of...

  4. Antibody Reactivity to Omp31 from Brucella melitensis in Human and Animal Infections by Smooth and Rough Brucellae

    OpenAIRE

    Cassataro, Juliana; Pasquevich, Karina; Bruno, Laura; Wallach, Jorge C.; Fossati, Carlos A.; Baldi, Pablo C.

    2004-01-01

    Group 3 of outer membrane proteins (OMPs) of Brucella includes Omp25 and Omp31, which share 34% identity. Omp25 is highly conserved in Brucella species, and Omp31 is present in all Brucella species, except Brucella abortus. Antibodies to Brucella melitensis Omp31 have been sought only in infected sheep, and Western blotting of sera from infected sheep did not reveal anti-Omp31 reactivity. We obtained recombinant purified Omp31 (B. melitensis) and tested its recognition by sera from humans and...

  5. Assessing drivers of the IgG4 antibody reactivity to recombinant antigen Bm14 in Wuchereria bancrofti endemic populations in East Africa.

    Science.gov (United States)

    Damgaard, Johanne; Meyrowitsch, Dan W; Rwegoshora, Rwehumbiza T; Magesa, Stephen M; Mukoko, Dunstan A; Simonsen, Paul E

    2016-09-01

    A high proportion of the human population in lymphatic filariasis (LF) endemic areas is positive for filarial specific IgG4 antibodies, including many individuals without microfilariae (mf; circulating larvae in the human blood) or circulating filarial antigens (CFA; marker of adult worm infection). The antibodies are commonly regarded as markers of infection and/or exposure to filarial larvae, but a direct association between the antibodies and these indices has not been well documented. The present study assessed the role and relative effect of potential drivers of the human IgG4 antibody reactivity to the recombinant filarial antigen Bm14 in Wuchereria bancrofti endemic populations in East Africa. Sera collected during previous studies from 395 well characterized individuals with regard to age, sex, mf, CFA, household vector biting and household exposure to infective filarial larvae were tested for IgG4 antibodies to Bm14, and associations between antibody reactivity and the different variables were statistically analyzed. IgG4 reactivity to Bm14 was highly positively associated with CFA, and to a lesser extent with age. However, an expected association with household exposure to infective filarial larvae was not found. Bm14 antibody reactivity thus appeared mainly to reflect actual infection of individuals with adult filarial worms rather than ongoing exposure to transmission. The analyses moreover suggested that many of the CFA negative but Bm14 positive individuals had early or low level infections where antibodies had been induced but where CFA was not (yet?) measurable. Although the study indicated that IgG4 reactivity to Bm14 is a marker of filarial infection, assessment of this reactivity, especially in children, will still be useful for indirect monitoring of changes in transmission intensity, including break of transmission and post-elimination surveillance, in LF control. PMID:27172877

  6. Phage Display-Derived Cross-Reactive Neutralizing Antibody against Enterovirus 71 and Coxsackievirus A16.

    Science.gov (United States)

    Zhang, Xiao; Sun, Chunyun; Xiao, Xiangqian; Pang, Lin; Shen, Sisi; Zhang, Jie; Cen, Shan; Yang, Burton B; Huang, Yuming; Sheng, Wang; Zeng, Yi

    2016-01-01

    Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are members of the Picornaviridae family and are considered the main causative agents of hand, foot and mouth disease (HFMD). In recent decades large HFMD outbreaks caused by EV71 and CVA16 have become significant public health concerns in the Asia-Pacific region. Vaccines and antiviral drugs are unavailable to prevent EV71 and CVA16 infection. In the current study, a chimeric antibody targeting a highly conserved peptide in the EV71 VP4 protein was isolated by using a phage display technique. The antibody showed cross-neutralizing capability against EV71 and CVA16 in vitro. The results suggest that this phage display-derived antibody will have great potential as a broad neutralizing antibody against EV71 and CVA16 after affinity maturation and humanization. PMID:26073737

  7. Age Dependence and Isotype Specificity of Influenza Virus Hemagglutinin Stalk-Reactive Antibodies in Humans

    OpenAIRE

    Nachbagauer, Raffael; Choi, Angela; Izikson, Ruvim; Cox, Manon M; Palese, Peter; Krammer, Florian

    2016-01-01

    ABSTRACT Influenza remains a major global health burden. Seasonal vaccines offer protection but can be rendered less effective when the virus undergoes extensive antigenic drift. Antibodies that target the highly conserved hemagglutinin stalk can protect against drifted viruses, and vaccine constructs designed to induce such antibodies form the basis for a universal influenza virus vaccine approach. In this study, we analyzed baseline and postvaccination serum samples of children (6 to 59 mon...

  8. Cross-reactive Legionella antigens and the antibody response during infection

    DEFF Research Database (Denmark)

    Bangsborg, Jette Marie; Shand, G; Pearlman, E;

    1991-01-01

    In order to define cross-reactive Legionella antigens suitable for diagnostic purposes, we investigated sonicate antigens from two Legionella species, including two serogroups of L. pneumophila. The antigens were reacted with heterologous and homologous rabbit antisera in Western blot. Sera from...... seven patients with culture-verified L. pneumophila infection and nine patients with serologically confirmed L. micdadei infection were also investigated for reactivity with the corresponding antigens. Among the cross-reactive Legionella antigens defined, non-specific reactivity in patients' sera with...... the 58-kDa common antigen (CA) was noted. Specific reactions were observed with the Legionella flagellum antigen and with the macrophage infectivity potentiator (Mip) protein; with both antigens, however, the reactive sera were too few to suggest the use of a single antigen in a diagnostic test....

  9. Ankle brachial index, C-reactive protein, and central augmentation index to identify individuals with severe atherosclerosis

    DEFF Research Database (Denmark)

    Eldrup, Nikolaj; Sillesen, Henrik; Prescott, Eva;

    2006-01-01

    We examined the ability of ankle brachial index, C-reactive protein and central augmentation index to identify individuals in the general population with severe atherosclerosis, diagnosed as those with ischaemic cardiovascular disease.......We examined the ability of ankle brachial index, C-reactive protein and central augmentation index to identify individuals in the general population with severe atherosclerosis, diagnosed as those with ischaemic cardiovascular disease....

  10. Binding of Klebsiella K2 LPS to specific antibody and cross-reactivity with pneumococcal group 19 polysaccharides (PS)

    International Nuclear Information System (INIS)

    The authors have studied the chemical basis of the antigen-antibody reaction between Klebsiella K2 LPS and specific antiserum, and cross-reactivity with pneumococcal group 19 PSs. K2 LPS was isolated and purified from Klebsiella K2 strain by the methods of hot phenol-water extraction, ultracentrifugation, treatment with enzymes and gel filtration. K2 LPS was hydrolyzed with 1% acetic acid to produce 2 fractions, separated by Sephadex G-50 column. Fraction G-I was passed through Sepharose 4B column to separate into 3 peaks (4B-I, -II, and -III). Peak 4B-II showed a molecular size of Kd, 0.35 and 4B-III had Kd, 0.79. When K2 LPS was injected to mice, IgM and IgG antibody responses were induced, as determined by ELISA. In contrast, with the 19F and 19A PSs, the K2 LPS induced antibody was mostly IgM. They have studied the ability of PS fractions (4B-II, 4B-III and G-I) to inhibit the binding of K2 LPS to specific IgM and IgG antibodies. Among these fractions, 4B-II PS showed a highest inhibition to the binding activity; approximately 1000-fold greater inhibiting ability than 4B-III and G-I PS. These results indicate that 4B-II PS may serve as an immunologic determinant of K2 LPS. Pneumococcal 19F and 19A PSs induced low inhibition of the binding activity between K2 LPS and specific IgM antibody. The fraction 4B-II as immunologic determinant of K2 LPS was further characterized by 125I-K2 LPS anti-serum and Western blotting electrophoresis

  11. Human monoclonal antibodies to West Nile virus identify epitopes on the prM protein

    International Nuclear Information System (INIS)

    Hybridoma cell lines (2E8, 8G8 and 5G12) producing fully human monoclonal antibodies (hMAbs) specific for the pre-membrane (prM) protein of West Nile virus (WNV) were prepared using a human fusion partner cell line, MFP-2, and human peripheral blood lymphocytes from a blood donor diagnosed with WNV fever in 2004. Using site-directed mutagenesis of a WNV-like particle (VLP) we identified 4 amino acid residues in the prM protein unique to WNV and important in the binding of these hMAbs to the VLP. Residues V19 and L33 are important epitopes for the binding of all three hMAbs. Mutations at residue, T20 and T24 affected the binding of hMAbs, 8G8 and 5G12 only. These hMAbs did not significantly protect AG129 interferon-deficient mice or Swiss Webster outbred mice from WNV infection.

  12. An Antibody Screen of a Plasmodium vivax Antigen Library Identifies Novel Merozoite Proteins Associated with Clinical Protection.

    Directory of Open Access Journals (Sweden)

    Camila T França

    2016-05-01

    Full Text Available Elimination of Plasmodium vivax malaria would be greatly facilitated by the development of an effective vaccine. A comprehensive and systematic characterization of antibodies to P. vivax antigens in exposed populations is useful in guiding rational vaccine design.In this study, we investigated antibodies to a large library of P. vivax entire ectodomain merozoite proteins in 2 Asia-Pacific populations, analysing the relationship of antibody levels with markers of current and cumulative malaria exposure, and socioeconomic and clinical indicators. 29 antigenic targets of natural immunity were identified. Of these, 12 highly-immunogenic proteins were strongly associated with age and thus cumulative lifetime exposure in Solomon Islanders (P<0.001-0.027. A subset of 6 proteins, selected on the basis of immunogenicity and expression levels, were used to examine antibody levels in plasma samples from a population of young Papua New Guinean children with well-characterized individual differences in exposure. This analysis identified a strong association between reduced risk of clinical disease and antibody levels to P12, P41, and a novel hypothetical protein that has not previously been studied, PVX_081550 (IRR 0.46-0.74; P<0.001-0.041.These data emphasize the benefits of an unbiased screening approach in identifying novel vaccine candidate antigens. Functional studies are now required to establish whether PVX_081550 is a key component of the naturally-acquired protective immune response, a biomarker of immune status, or both.

  13. Somatic populations of PGT135-137 HIV-1-neutralizing antibodies identified by 454 pyrosequencing and bioinformatics

    Directory of Open Access Journals (Sweden)

    Jiang eZhu

    2012-09-01

    Full Text Available Select HIV-1-infected individuals develop sera capable of neutralizing diverse viral strains. The molecular basis of this neutralization is currently being deciphered by the isolation of HIV-1-neutralizing antibodies. In one infected donor, three neutralizing antibodies, PGT135-137, were identified by assessment of neutralization from individually sorted B cells and found to recognize an epitope containing an N-linked glycan at residue 332 on HIV-1 gp120. Here we use deep sequencing and bioinformatics methods to interrogate the B cell record of this donor to gain a more complete understanding of the humoral immune response. PGT135-137-gene family-specific primers were used to amplify heavy and light chain-variable domain sequences. 454 pyrosequencing produced 141,298 heavy-chain sequences of IGHV4-39 origin and 87,229 light-chain sequences of IGKV3-15 origin. A number of heavy and light chain sequences of ~90% identity to PGT137, several to PGT136, and none of high identity to PGT135 were identified. After expansion of these sequences to include close phylogenetic relatives, a total of 202 heavy-chain sequences and 72 light-chain sequences were identified. These sequences were clustered into populations of 95% identity comprising 15 for heavy chain and 10 for light chain, and a select sequence from each population was synthesized and reconstituted with a PGT137-partner chain. Reconstituted antibodies showed varied neutralization phenotypes for HIV-1 clade A and D isolates. Sequence diversity of the antibody population represented by these tested sequences was notably higher than observed with a 454 pyrosequencing-control analysis on 10 antibodies of defined sequence, suggesting that this diversity results primarily from somatic maturation. Our results thus provide an example of how pathogens like HIV-1 are opposed by a varied humoral immune response, derived from intrinsic mechanisms of antibody development, and embodied by somatic populations

  14. AbMiner: A bioinformatic resource on available monoclonal antibodies and corresponding gene identifiers for genomic, proteomic, and immunologic studies

    Directory of Open Access Journals (Sweden)

    Shankavaram Uma

    2006-04-01

    Full Text Available Abstract Background Monoclonal antibodies are used extensively throughout the biomedical sciences for detection of antigens, either in vitro or in vivo. We, for example, have used them for quantitation of proteins on "reverse-phase" protein lysate arrays. For those studies, we quality-controlled > 600 available monoclonal antibodies and also needed to develop precise information on the genes that encode their antigens. Translation among the various protein and gene identifier types proved non-trivial because of one-to-many and many-to-one relationships. To organize the antibody, protein, and gene information, we initially developed a relational database in Filemaker for our own use. When it became apparent that the information would be useful to many other researchers faced with the need to choose or characterize antibodies, we developed it further as AbMiner, a fully relational web-based database under MySQL, programmed in Java. Description AbMiner is a user-friendly, web-based relational database of information on > 600 commercially available antibodies that we validated by Western blot for protein microarray studies. It includes many types of information on the antibody, the immunogen, the vendor, the antigen, and the antigen's gene. Multiple gene and protein identifier types provide links to corresponding entries in a variety of other public databases, including resources for phosphorylation-specific antibodies. AbMiner also includes our quality-control data against a pool of 60 diverse cancer cell types (the NCI-60 and also protein expression levels for the NCI-60 cells measured using our high-density "reverse-phase" protein lysate microarrays for a selection of the listed antibodies. Some other available database resources give information on antibody specificity for one or a couple of cell types. In contrast, the data in AbMiner indicate specificity with respect to the antigens in a pool of 60 diverse cell types from nine different

  15. Dengue virus sero-cross-reactivity drives antibody-dependent enhancement of infection with zika virus.

    Science.gov (United States)

    Dejnirattisai, Wanwisa; Supasa, Piyada; Wongwiwat, Wiyada; Rouvinski, Alexander; Barba-Spaeth, Giovanna; Duangchinda, Thaneeya; Sakuntabhai, Anavaj; Cao-Lormeau, Van-Mai; Malasit, Prida; Rey, Felix A; Mongkolsapaya, Juthathip; Screaton, Gavin R

    2016-09-01

    Zika virus (ZIKV) was discovered in 1947 and was thought to lead to relatively mild disease. The recent explosive outbreak of ZIKV in South America has led to widespread concern, with reports of neurological sequelae ranging from Guillain Barré syndrome to microcephaly. ZIKV infection has occurred in areas previously exposed to dengue virus (DENV), a flavivirus closely related to ZIKV. Here we investigated the serological cross-reaction between the two viruses. Plasma immune to DENV showed substantial cross-reaction to ZIKV and was able to drive antibody-dependent enhancement (ADE) of ZIKV infection. Using a panel of human monoclonal antibodies (mAbs) to DENV, we showed that most antibodies that reacted to DENV envelope protein also reacted to ZIKV. Antibodies to linear epitopes, including the immunodominant fusion-loop epitope, were able to bind ZIKV but were unable to neutralize the virus and instead promoted ADE. Our data indicate that immunity to DENV might drive greater ZIKV replication and have clear implications for disease pathogenesis and future vaccine programs for ZIKV and DENV. PMID:27339099

  16. Development and Characterization of Mouse Monoclonal Antibodies Reactive with Chicken CD83

    Science.gov (United States)

    This study was carried out to develop and characterize mouse monoclonal antibodies (mAbs) against chicken CD83 (chCD83), a membrane-bound glycoprotein belonging to the immunoglobulin superfamily that is primarily expressed on mature dendritic cells (DCs). A recombinant chCD83/IgG4 fusion protein con...

  17. Large-scale analysis of B-cell epitopes on influenza virus hemagglutinin - implications for cross-reactivity of neutralizing antibodies

    DEFF Research Database (Denmark)

    Sun, Jing; Kudahl, Ulrich J.; Simon, Christian;

    2014-01-01

    Influenza viruses continue to cause substantial morbidity and mortality worldwide. Fast gene mutation on surface proteins of influenza virus result in increasing resistance to current vaccines and available antiviral drugs. Broadly neutralizing antibodies (bnAbs) represent targets for prophylactic...... and therapeutic treatments of influenza. We performed a systematic bioinformatics study of cross-reactivity of neutralizing antibodies (nAbs) against influenza virus surface glycoprotein hemagglutinin (HA). This study utilized the available crystal structures of HA complexed with the antibodies for...... the analysis of tens of thousands of HA sequences. The detailed description of B-cell epitopes, measurement of epitope area similarity among different strains, and estimation of antibody neutralizing coverage provide insights into cross-reactivity status of existing nAbs against influenza virus. We...

  18. Production of Nurr-1 Specific Polyclonal Antibodies Free of Cross-reactivity Against Its Close Homologs, Nor1 and Nur77.

    Science.gov (United States)

    Leblanc, Pierre; Moon, Minho; Kim, Woori; Jeong, Inhye; Kim, Chun-Hyung; Kim, Kwang-Soo

    2015-01-01

    The nuclear receptor subfamily 4 (NR4A) is composed of 3 related proteins sharing a DNA binding domain (DBD) and a ligand-binding domain (LBD). The nuclear receptor related 1 protein (Nurr1 or NR4A2) plays a key role in the maintenance of the dopaminergic system. Dopamine dysfunctions associated with the Nurr1 gene include Parkinson's disease, schizophrenia and manic depression among others. Furthermore, recent evidence indicates that Nurr1 is also expressed in other brain areas such as the hippocampus and plays critical roles for learning and memory. The other members of the family are nerve growth factor IB (Nur77 or NR4A1) and neuron-derived orphan receptor 1 (NOR1 or NR4A3). To help investigate the precise functional roles of Nurr1 in dopaminergic and other brain region-related neuronal dysfunctions antibodies devoid of cross-reactivities against Nur77 and NOR1 were needed. Since the proteins are more divergent in their LBDs than in their DNA binding domains immunization with purified LBDs should yield antibodies specific for Nurr1 with minimal reactivities against Nur77 and/or NOR1. Although anti-Nurr1 antibodies were successfully generated these showed significant immunoreactivity against the other members of the family. Affinity chromatography over immobilized Protein A followed by pre-adsorption against immobilized Nur77 and NOR1 LBDs yielded Nurr1 specific antibodies free of cross-reactivity. Here, we selectively target antibodies against a specific member of a highly conserved family of proteins by immunizing animals with their most divergent regions followed by removing cross reactive antibodies by pre-adsorption. The goal of the protocol is to increase polyclonal antibodies specificity through pre-adsorption against cross-reactive antigens. PMID:26325389

  19. Large-scale analysis of B-cell epitopes on influenza virus hemagglutinin - implications for cross-reactivity of neutralizing antibodies.

    OpenAIRE

    Sun, Jing; Kudahl, Ulrich J.; Simon, Christian; Cao, Zhiwei; Reinherz, Ellis L; Brusic, Vladimir

    2014-01-01

    Influenza viruses continue to cause substantial morbidity and mortality worldwide. Fast gene mutation on surface proteins of influenza virus result in increasing resistance to current vaccines and available antiviral drugs. Broadly neutralizing antibodies (bnAbs) represent targets for prophylactic and therapeutic treatments of influenza. We performed a systematic bioinformatics study of cross-reactivity of neutralizing antibodies (nAbs) against influenza virus surface glycoprotein hemagglutin...

  20. Large scale genome analysis shows that the epitopes for broadly cross-reactive antibodies are predominant in the pandemic 2009 influenza virus A H1N1 strain.

    Science.gov (United States)

    Lara-Ramírez, Edgar E; Segura-Cabrera, Aldo; Salazar, Ma Isabel; Rodríguez-Pérez, Mario A; Guo, Xianwu

    2013-11-01

    The past pandemic strain H1N1 (A (H1N1)pdm09) has now become a common component of current seasonal influenza viruses. It has changed the pre-existing immunity of the human population to succeeding infections. In the present study, a total of 14,210 distinct sequences downloaded from National Center for Biotechnology Information (NCBI) database were used for the analysis. The epitope compositions in A (H1N1)pdm09, classic seasonal strains, swine strains as well as highly virulent avian strain H5N1, identified with the aid of the Immune Epitope DataBase (IEDB), were compared at genomic level. The result showed that A (H1N1) pdm09 contains the 90% of B-cell epitopes for broadly cross-reactive antibodies (EBCA), which is in consonance with the recent reports on the experimental identification of new epitopes or antibodies for this virus and the binding tests with influenza virus protein HA of different subtypes. Our analysis supports that high proportional EBCA depends on the epitope pattern of A (H1N1)pdm09 virus. This study may be helpful for better understanding of A (H1N1)pdm09 and the production of new influenza vaccines. PMID:24257096

  1. Large Scale Genome Analysis Shows that the Epitopes for Broadly Cross-Reactive Antibodies Are Predominant in the Pandemic 2009 Influenza Virus A H1N1 Strain

    Directory of Open Access Journals (Sweden)

    Edgar E. Lara-Ramírez

    2013-11-01

    Full Text Available The past pandemic strain H1N1 (A (H1N1pdm09 has now become a common component of current seasonal influenza viruses. It has changed the pre-existing immunity of the human population to succeeding infections. In the present study, a total of 14,210 distinct sequences downloaded from National Center for Biotechnology Information (NCBI database were used for the analysis. The epitope compositions in A (H1N1pdm09, classic seasonal strains, swine strains as well as highly virulent avian strain H5N1, identified with the aid of the Immune Epitope DataBase (IEDB, were compared at genomic level. The result showed that A (H1N1 pdm09 contains the 90% of B-cell epitopes for broadly cross-reactive antibodies (EBCA, which is in consonance with the recent reports on the experimental identification of new epitopes or antibodies for this virus and the binding tests with influenza virus protein HA of different subtypes. Our analysis supports that high proportional EBCA depends on the epitope pattern of A (H1N1pdm09 virus. This study may be helpful for better understanding of A (H1N1pdm09 and the production of new influenza vaccines.

  2. Human Survivors of Disease Outbreaks Caused by Ebola or Marburg Virus Exhibit Cross-Reactive and Long-Lived Antibody Responses.

    Science.gov (United States)

    Natesan, Mohan; Jensen, Stig M; Keasey, Sarah L; Kamata, Teddy; Kuehne, Ana I; Stonier, Spencer W; Lutwama, Julius Julian; Lobel, Leslie; Dye, John M; Ulrich, Robert G

    2016-08-01

    A detailed understanding of serological immune responses to Ebola and Marburg virus infections will facilitate the development of effective diagnostic methods, therapeutics, and vaccines. We examined antibodies from Ebola or Marburg survivors 1 to 14 years after recovery from disease, by using a microarray that displayed recombinant nucleoprotein (NP), viral protein 40 (VP40), envelope glycoprotein (GP), and inactivated whole virions from six species of filoviruses. All three outbreak cohorts exhibited significant antibody responses to antigens from the original infecting species and a pattern of additional filoviruses that varied by outbreak. NP was the most cross-reactive antigen, while GP was the most specific. Antibodies from survivors of infections by Marburg marburgvirus (MARV) species were least cross-reactive, while those from survivors of infections by Sudan virus (SUDV) species exhibited the highest cross-reactivity. Based on results revealed by the protein microarray, persistent levels of antibodies to GP, NP, and VP40 were maintained for up to 14 years after infection, and survival of infection caused by one species imparted cross-reactive antibody responses to other filoviruses. PMID:27335383

  3. Application of a Trifunctional Reactive Linker for the Construction of Antibody-Drug Hybrid Conjugates

    OpenAIRE

    Thomas, Joshua D.; Hofer, Thomas; Rader, Christoph; Burke, Terrence R

    2008-01-01

    A flexible, trifunctional poly(ethylene glycol)-succinamide-Lysine-Lysine-maleimide (PEG-SU-Lys-Lys-mal) linker was employed to simultaneously allow biotin tagging and cell-surface targeting through an integrin α4β1-binding peptidomimetic that was regiospecifically conjugated to an IgG1-derived Fc fragment with an engineered C-terminal selenocysteine residue. The resulting antibody derivative mediates Fc receptor binding by virtue of the Fc protein and selectively targets cancer cells express...

  4. Limited cross-reactivity of mouse monoclonal antibodies against Dengue virus capsid protein among four serotypes

    OpenAIRE

    Noda M; Masrinoul P; Punkum C; Pipattanaboon C; Ramasoota P; Setthapramote C; Sasaki T; Sasayama M; Yamashita A; Kurosu T; Ikuta K; Okabayashi T

    2012-01-01

    Megumi Noda,1 Promsin Masrinoul,1 Chaweewan Punkum,1 Chonlatip Pipattanaboon,2,3 Pongrama Ramasoota,2,4 Chayanee Setthapramote,2,3 Tadahiro Sasaki,6 Mikiko Sasayama,1 Akifumi Yamashita,1,5 Takeshi Kurosu,6 Kazuyoshi Ikuta,6 Tamaki Okabayashi11Mahidol-Osaka Center for Infectious Diseases, 2Center of Excellence for Antibody Research, 3Department of Microbiology and Immunology, 4Department of Social and Environmental Medicine, Faculty of Tropical Medicine, Mahidol University, Ratchathewi, Bangko...

  5. The Effects of Anti-insulin Antibodies and Cross-reactivity with Human Recombinant Insulin Analogues in the E170 Insulin Immunometric Assay

    OpenAIRE

    Kim, Serim; Yun, Yeo Min; Hur, Mina; Moon, Hee Won; Kim, Jin Q

    2011-01-01

    Background Insulin assays are affected by varying degrees of interference from anti-insulin antibodies (IAs) and by cross-reactivity with recombinant insulin analogues. We evaluated the usefulness of the E170 insulin assay by assessing IA effects and cross-reactivity with 2 analogues. Methods Sera were obtained from 59 type 2 diabetes patients receiving continuous subcutaneous insulin infusion and 18 healthy controls. Insulin levels were determined using an E170 analyzer. To investigate the e...

  6. [Challenge to the Development of Molecular Targeted Therapy against a Novel Target Candidate Identified by Antibody Proteomics Technology].

    Science.gov (United States)

    Nagano, Kazuya

    2016-01-01

    Disease proteomics that systemically analyzes and identifies differentially expressed proteins between healthy and diseased samples is a potentially useful approach for obtaining target proteins for drug development. To date, however, very few target proteins have been identified from this field. A key issue that remains to be resolved is how to correctly identify target proteins from a number of potential candidates. To circumvent this problem, we have developed "antibody proteomics technology" in which a single chain Fv phage antibody library is utilized for proteome analysis. Here, we describe the application of this technology by primarily focusing on Eph receptor A10 (EphA10), a novel breast cancer-related protein that is a promising target for antibody drugs. To establish an effective and safe targeted cancer therapy, it is important that the target is specifically expressed in cancer tissues. Therefore, we attempted to analyze the EphA10 expression profiles. Tissue microarray analysis showed that EphA10 was expressed in all subtypes of breast cancer containing triple negative breast cancer cases. On the other hand, EphA10 was only expressed in testis tissue among 36 kinds of normal tissues. Thus, EphA10 could be a highly cancer-specific protein, making it a promising target for female breast cancer patients. Finally, we examined the anti-tumor effect by anti-EphA10 antibody, aiming for the development of a novel EphA10 targeting therapy. Administration of the antibody showed that tumor volumes were significantly inhibited. Our results suggest that targeting EphA10 in breast cancer cases might be a promising new form of therapy. PMID:26831784

  7. An Antibody Screen of a Plasmodium vivax Antigen Library Identifies Novel Merozoite Proteins Associated with Clinical Protection

    Science.gov (United States)

    França, Camila T.; Hostetler, Jessica B.; Sharma, Sumana; White, Michael T.; Lin, Enmoore; Kiniboro, Benson; Waltmann, Andreea; Darcy, Andrew W.; Li Wai Suen, Connie S. N.; Siba, Peter; King, Christopher L.; Rayner, Julian C.; Fairhurst, Rick M.; Mueller, Ivo

    2016-01-01

    Background Elimination of Plasmodium vivax malaria would be greatly facilitated by the development of an effective vaccine. A comprehensive and systematic characterization of antibodies to P. vivax antigens in exposed populations is useful in guiding rational vaccine design. Methodology/Principal Findings In this study, we investigated antibodies to a large library of P. vivax entire ectodomain merozoite proteins in 2 Asia-Pacific populations, analysing the relationship of antibody levels with markers of current and cumulative malaria exposure, and socioeconomic and clinical indicators. 29 antigenic targets of natural immunity were identified. Of these, 12 highly-immunogenic proteins were strongly associated with age and thus cumulative lifetime exposure in Solomon Islanders (P<0.001–0.027). A subset of 6 proteins, selected on the basis of immunogenicity and expression levels, were used to examine antibody levels in plasma samples from a population of young Papua New Guinean children with well-characterized individual differences in exposure. This analysis identified a strong association between reduced risk of clinical disease and antibody levels to P12, P41, and a novel hypothetical protein that has not previously been studied, PVX_081550 (IRR 0.46–0.74; P<0.001–0.041). Conclusion/Significance These data emphasize the benefits of an unbiased screening approach in identifying novel vaccine candidate antigens. Functional studies are now required to establish whether PVX_081550 is a key component of the naturally-acquired protective immune response, a biomarker of immune status, or both. PMID:27182597

  8. Development of a protein biochip to identify 6 monoclonal antibodies against subtypes of recombinant human interferons.

    Science.gov (United States)

    Xu, Zhenshan; Du, Weidong; Zhang, Ping; Wang, Xuan; Ma, Xueling; Shi, Liqin; Song, Lihua

    2010-04-01

    Recombinant human interferons (rhIFNs) are broadly used as effective therapeutic agents with antiviral, antitumor, and immune-modulating properties. Advances in protein biochip technology have benefited the medical community greatly, making true parallelism, miniaturization, and high throughput possible. In this study, 5 rhIFN proteins (IFN-alpha1b, IFN-alpha2a, IFN-alpha2b, IFN-beta, and IFN-gamma) were immobilized onto an N-hydroxysuccinimide (NHS)-modified gold-based biochip. The protein biochip was incubated with 6 specific mouse IgG antibodies (AK1, AK2, AK3, AK4, BK1, and CK1) against the human IFNs and then with Cy3-conjugated goat anti-mouse IgG antibody. The results showed that monoclonal antibody AK1 presented a unique binding characteristic to IFN-alpha1b. AK2 reacted in immunoassays equally with IFN-alpha2a and IFN-alpha2b. AK3 detected IFN-alpha1b, IFN-alpha2a, and IFN-alpha2b. AK4 had positive immunological responses directed to both IFN-alpha1b and IFN-alpha2b. Monoclonal antibodies BK1 and CK1 recognized epitope of IFN-beta and IFN-gamma, specifically. The assay specificity of the biochip was further confirmed by enzyme-linked immunosorbent assay (ELISA) and western blotting. Finally, 88 serum samples from patients treated with rhIFN-alpha2b were simultaneously tested on a single biochip. The result demonstrated that 6.8% (6 of 88 cases) presented positive reactions to anti-IFN-alpha2b antibodies, indicating that the patients under rhIFN-alpha2b therapy produced neutralized antibody against the IFN. The biochip format would offer a competitive alternative tool not only for facilitating characterization of IFN subtypes but also potentially for enabling clinical serum detection of corresponding antibodies directed against IFNs. PMID:20230300

  9. Crystal structure of the Hendra virus attachment G glycoprotein bound to a potent cross-reactive neutralizing human monoclonal antibody.

    Directory of Open Access Journals (Sweden)

    Kai Xu

    Full Text Available The henipaviruses, represented by Hendra (HeV and Nipah (NiV viruses are highly pathogenic zoonotic paramyxoviruses with uniquely broad host tropisms responsible for repeated outbreaks in Australia, Southeast Asia, India and Bangladesh. The high morbidity and mortality rates associated with infection and lack of licensed antiviral therapies make the henipaviruses a potential biological threat to humans and livestock. Henipavirus entry is initiated by the attachment of the G envelope glycoprotein to host cell membrane receptors. Previously, henipavirus-neutralizing human monoclonal antibodies (hmAb have been isolated using the HeV-G glycoprotein and a human naïve antibody library. One cross-reactive and receptor-blocking hmAb (m102.4 was recently demonstrated to be an effective post-exposure therapy in two animal models of NiV and HeV infection, has been used in several people on a compassionate use basis, and is currently in development for use in humans. Here, we report the crystal structure of the complex of HeV-G with m102.3, an m102.4 derivative, and describe NiV and HeV escape mutants. This structure provides detailed insight into the mechanism of HeV and NiV neutralization by m102.4, and serves as a blueprint for further optimization of m102.4 as a therapeutic agent and for the development of entry inhibitors and vaccines.

  10. An antibody to de-N-acetyl sialic acid containing-polysialic acid identifies an intracellular antigen and induces apoptosis in human cancer cell lines.

    Directory of Open Access Journals (Sweden)

    Lindsay M Steirer

    Full Text Available Polysialic acid (PSA, an α2,8-linked homopolymer of N-acetylneuraminic acid (Neu5Ac, is developmentally regulated and its expression is thought to be restricted to a few tissues in adults. Recently, we showed that two human pathogens expressed a derivative of PSA containing de-N-acetyl sialic acid residues (NeuPSA. Here we show that an epitope identified by the anti-NeuPSA monoclonal antibody, SEAM 3 (SEAM 3-reactive antigen or S3RA, is expressed in human melanomas, and also intracellularly in a human melanoma cell line (SK-MEL-28, a human T cell leukemia cell line (Jurkat, and two neuroblastoma cell lines (CHP-134 and SH-SY5Y. SEAM 3 binding induced apoptosis in the four cell lines tested. The unusual intracellular distribution of S3RA was similar to that described for the PSA polysialyltransferases, STX and PST, which are also expressed in the four cell lines used here. Interestingly, suppression of PST mRNA expression by transfection of SK-MEL-28 cells with PST-specific short interfering RNA (siRNA resulted in decreased SEAM 3 binding. The results suggest further studies of the utility of antibodies such as SEAM 3 as therapeutic agents for certain malignancies.

  11. Cross-Reactive Human IgM-Derived Monoclonal Antibodies that Bind to HIV-1 Envelope Glycoproteins.

    Science.gov (United States)

    Chen, Weizao; Zhu, Zhongyu; Liao, Huaxin; Quinnan, Gerald V; Broder, Christopher C; Haynes, Barton F; Dimitrov, Dimiter S

    2010-02-01

    Elicitation of antibodies with potent and broad neutralizing activity against HIV by immunization remains a challenge. Several monoclonal antibodies (mAbs) isolated from humans with HIV-1 infection exhibit such activity but vaccine immunogens based on structures containing their epitopes have not been successful for their elicitation. All known broadly neutralizing mAbs (bnmAbs) are immunoglobulin (Ig) Gs (IgGs) and highly somatically hypermutated which could impede their elicitation. Ig Ms (IgMs) are on average significantly less divergent from germline antibodies and are relevant for the development of vaccine immunogens but are underexplored compared to IgGs. Here we describe the identification and characterization of several human IgM-derived mAbs against HIV-1 which were selected from a large phage-displayed naive human antibody library constructed from blood, lymph nodes and spleens of 59 healthy donors. These antibodies bound with high affinity to recombinant envelope glycoproteins (gp140s, Envs) of HIV-1 isolates from different clades. They enhanced or did not neutralize infection by some of the HIV-1 primary isolates using CCR5 as a coreceptor but neutralized all CXCR4 isolates tested although weakly. One of these antibodies with relatively low degree of somatic hypermutation was more extensively characterized. It bound to a highly conserved region partially overlapping with the coreceptor binding site and close to but not overlapping with the CD4 binding site. These results suggest the existence of conserved structures that could direct the immune response to non-neutralizing or even enhancing antibodies which may represent a strategy used by the virus to escape neutralizing immune responses. Further studies will show whether such a strategy plays a role in HIV infection of humans, how important that role could be, and what the mechanisms of infection enhancement are. The newly identified mAbs could be used as reagents to further characterize conserved

  12. Cross-Reactive Human IgM-Derived Monoclonal Antibodies that Bind to HIV-1 Envelope Glycoproteins

    Directory of Open Access Journals (Sweden)

    Barton F. Haynes

    2010-02-01

    Full Text Available Elicitation of antibodies with potent and broad neutralizing activity against HIV by immunization remains a challenge. Several monoclonal antibodies (mAbs isolated from humans with HIV-1 infection exhibit such activity but vaccine immunogens based on structures containing their epitopes have not been successful for their elicitation. All known broadly neutralizing mAbs (bnmAbs are immunoglobulin (Ig Gs (IgGs and highly somatically hypermutated which could impede their elicitation. Ig Ms (IgMs are on average significantly less divergent from germline antibodies and are relevant for the development of vaccine immunogens but are underexplored compared to IgGs. Here we describe the identification and characterization of several human IgM-derived mAbs against HIV-1 which were selected from a large phage-displayed naive human antibody library constructed from blood, lymph nodes and spleens of 59 healthy donors. These antibodies bound with high affinity to recombinant envelope glycoproteins (gp140s, Envs of HIV-1 isolates from different clades. They enhanced or did not neutralize infection by some of the HIV-1 primary isolates using CCR5 as a coreceptor but neutralized all CXCR4 isolates tested although weakly. One of these antibodies with relatively low degree of somatic hypermutation was more extensively characterized. It bound to a highly conserved region partially overlapping with the coreceptor binding site and close to but not overlapping with the CD4 binding site. These results suggest the existence of conserved structures that could direct the immune response to non-neutralizing or even enhancing antibodies which may represent a strategy used by the virus to escape neutralizing immune responses. Further studies will show whether such a strategy plays a role in HIV infection of humans, how important that role could be, and what the mechanisms of infection enhancement are. The newly identified mAbs could be used as reagents to further

  13. Targeting natural killer cell reactivity by employing antibody to NKp46: implications for type 1 diabetes.

    Directory of Open Access Journals (Sweden)

    Rami Yossef

    Full Text Available Natural killer (NK cells belong to the innate lymphoid cells. Their cytotoxic activity is regulated by the delicate balance between activating and inhibitory signals. NKp46 is a member of the primary activating receptors of NK cells. We previously reported that the NKp46 receptor is involved in the development of type 1 diabetes (T1D. Subsequently, we hypothesized that blocking this receptor could prevent or hinder disease development. To address this goal, we developed monoclonal antibodies for murine NKp46. One mAb, named NCR1.15, recognizes the mouse homologue protein of NKp46, named Ncr1, and was able to down-regulate the surface expression of NKp46 on primary murine NK cells following antibody injection in vivo. Additionally, NCR1.15 treatments were able to down-regulate cytotoxic activity mediated by NKp46, but not by other NK receptors. To test our primary assumption, we examined T1D development in two models, non-obese diabetic mice and low-dose streptozotocin. Our results show a significantly lower incidence of diabetic mice in the NCR1.15-treated group compared to control groups. This study directly demonstrates the involvement of NKp46 in T1D development and suggests a novel treatment strategy for early insulitis.

  14. Monoclonal antibodies reactive with human breast or ovarian carcinoma: In vivo applications

    International Nuclear Information System (INIS)

    Monoclonal antibodies (MoAbs) are unique and useful bioprobes that allow in vivo targeting of membrane-associated or circulating antigens. Most of the clinical trials to date have used low dosages of radiolabeled MoAb given in a single dose. Newer studies have included antibody fragments, repeated injections, intraperitoneal (IP) administration, and other labels such as 90Y. Clinical MoAb trials are often arduous, expensive, and time-consuming to perform. Before human use, animal studies and extensive MoAb characterization are required. The production of pharmaceutical grade, radiolabeled MoAb is technically difficult and costly. Clinical trials require administrative and patient consent as well as extensive written protocols. These studies necessitate interdepartmental and intradepartmental cooperation and coordination. Furthermore, the use of in vivo radiolabeled probes impacts many levels of health care providers from janitorial, nursing, and technical staff to laboratories and physicians. Simple blood tests or disposal of body excretions may concern nursing or technical staff with the possibility of radiation exposure. The responsibility for study design, personnel involvement, and prospective use in patients without a definitive cancer diagnosis ultimately rests with the physician. While many issues have been addressed, additional clinical trials, consideration of safety issues, and standardization between institutions will be necessary before the use of radiolabeled MoAb for diagnosis, management, or therapy of human tumors becomes routine. Continued cooperation and funding should ensure its achievement. 136 references

  15. Using Soluble Reactive Phosphorus and Ammonia to Identify Point Source Discharge from Large Livestock Facilities

    Science.gov (United States)

    Borrello, M. C.; Scribner, M.; Chessin, K.

    2013-12-01

    A growing body of research draws attention to the negative environmental impacts on surface water from large livestock facilities. These impacts are mostly in the form of excessive nutrient loading resulting in significantly decreased oxygen levels. Over-application of animal waste on fields as well as direct discharge into surface water from facilities themselves has been identified as the main contributor to the development of hypoxic zones in Lake Erie, Chesapeake Bay and the Gulf of Mexico. Some regulators claim enforcement of water quality laws is problematic because of the nature and pervasiveness of non-point source impacts. Any direct discharge by a facility is a violation of permits governed by the Clean Water Act, unless the facility has special dispensation for discharge. Previous research by the principal author and others has shown runoff and underdrain transport are the main mechanisms by which nutrients enter surface water. This study utilized previous work to determine if the effects of non-point source discharge can be distinguished from direct (point-source) discharge using simple nutrient analysis and dissolved oxygen (DO) parameters. Nutrient and DO parameters were measured from three sites: 1. A stream adjacent to a field receiving manure, upstream of a large livestock facility with a history of direct discharge, 2. The same stream downstream of the facility and 3. A stream in an area relatively unimpacted by large-scale agriculture (control site). Results show that calculating a simple Pearson correlation coefficient (r) of soluble reactive phosphorus (SRP) and ammonia over time as well as temperature and DO, distinguishes non-point source from point source discharge into surface water. The r value for SRP and ammonia for the upstream site was 0.01 while the r value for the downstream site was 0.92. The control site had an r value of 0.20. Likewise, r values were calculated on temperature and DO for each site. High negative correlations

  16. Candidate vaccine antigens identified by antibodies from mice vaccinated with 15- or 50-kilorad-irradiated cercariae of Schistosoma mansoni.

    OpenAIRE

    Richter, D.; Harn, D A

    1993-01-01

    In murine schistosomiasis, the highest levels of resistance to cercarial challenge are obtained by vaccination with radiation-attenuated cercariae. To identify candidate vaccine antigens relevant to the vaccine model, we examined parasite antigens recognized by antibodies from mice vaccinated with irradiated cercariae of Schistosoma mansoni. To optimize recognition of a wide spectrum of antigens, several factors that influence the level of protection in this model were varied; specifically, w...

  17. Establishment of bovine prion peptide-based monoclonal antibodies for identifying bovine prion

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    To obtain high titer monoclonal antibodies(McAbs) which can react with mammalian prion protein(PrP),Balb/C mice were immunized with bovine(Bo) PrP peptide(BoPrP 209-228 aa) coupled to keyhole limpet hemocyanin(KLH).The hybridoma cell lines secreting monoclonal antibodies against the peptide were established by cell fusion and cloning.The obtained McAbs were applied to detect recombinant human,bovine and hamster PrP,cellular prion protein(PrPc) in normal bovine brain and pathogenic scrapie prion protein(PrPSc) accumulated in the medulla oblongata of bovine spongiform encephalopathy(BSE)specimen with Western blot and immunohistochemical detection,respectively.The current procedure might offer a simple,feasible method to raise high titer antibodies for studying biological features of PrP in mammals,as well as detection of transmissible spongiform encephalopathy(TSE) and diagnosis of BSE,in particular.

  18. Importance of polar solvation for cross-reactivity of antibody and its variants with steroids.

    Science.gov (United States)

    Kar, Parimal; Lipowsky, Reinhard; Knecht, Volker

    2011-06-16

    Understanding the factors determining the binding of ligands to receptors in detail is essential for rational drug design. Here, the free energies of binding of the steroids progesterone (PRG) and 5β-androstane-3,17-dione (5AD) to the Diels-Alderase antibody 1E9, as well as the Leu(H47)Trp/Arg(H100)Trp 1E9 double mutant (1E9dm) and the corresponding single mutants, have been estimated and decomposed using the molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) method. Also the difference in binding free energies between the PRG-1E9dm complex and the complex of PRG with the antiprogesterone antibody DB3 have been evaluated and decomposed. The steroids bind less strongly to 1E9 than to DB3, but the mutations tend to improve the steroid affinity, in quantitative agreement with experimental data. Although the complexes formed by PRG or 5AD with 1E9dm and by PRG with DB3 have similar affinity, the binding mechanisms are different. Reduced van der Waals interactions as observed for 5AD-1E9dm versus PRG-1E9dm or for PRG-1E9dm versus PRG-DB3 are energetically compensated by an increased solvation of polar groups, partly contrasting previous conclusions based on structural inspection. Our study illustrates that deducing binding mechanisms from structural models alone can be misleading. Therefore, taking into account solvation effects as in MM-PBSA calculations is essential to elucidate molecular recognition. PMID:21595427

  19. Antibodies reactive with Ehrlichia canis, Ehrlichia phagocytophila genogroup antigens and the spotted fever group rickettsial antigens, in free-ranging jackals (Canis aureus syriacus) from Israel.

    Science.gov (United States)

    Waner, T; Baneth, G; Strenger, C; Keysary, A; King, R; Harrus, S

    1999-03-31

    A seroepidemiological survey was conducted to investigate the prevalence of antibodies reactive with the Ehrlichia canis and Ehrlichia phagocytophila genogroup antigens, and the spotted fever group (SFG) rickettsiae antigens in jackals in Israel (Canis aureus syriacus), to assess the possible role of the jackal in the epidemiology of these diseases. Fifty-three serum samples from jackals were assayed by the indirect immunofluorescence antibody test. Antibodies to E. canis were detected in 35.8% serum samples while 26.4% of the samples tested were positive to Ehrlichia chaffeensis. Twenty-six percent of the jackals tested were seropositive to E. phagocytophila, of which 5.7% were seropositive to E. phagocytophila alone without any seroreactivity to either E. canis or E. chaffeensis. Fifty-five percent of the jackals were seropositive to the SFG-rickettsiae antigens. The results suggest a high exposure rate of jackals in Israel to E. canis. Positive reactivity to E. chaffeensis was considered to be due to antigenic cross-reactions with E. canis. The study demonstrated for the first time the presence of E. phagocytophila antibodies in free-range jackals. The high incidence of antibodies to the SFG-rickettsiae and their relatively high antibody titers was suggestive of either recent or persistent infection. The possibility that jackals may play a role in the transmission of E. canis, E. phagocytophila and the SFG-rickettsiae for human and canine infections is discussed. PMID:10321583

  20. Generation and characterization of novel stromal specific antibodies

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Rheumatoid synovial fibroblasts were used as an immunogen to produce monoclonal antibodies selected for their reactivity with stromal cell antigens. Mice were immunised with low passage whole cell preparations and the subsequent hybridomas were screened by immunohistochemistry on rheumatoid synovium and tonsil sections. The aim was to identify those antibodies that recognised antigens that were restricted to stromal cells and were not expressed on CD45 positive leucocytes. A significant number of antibodies detected antigen that identified endothelial cells. These antibodies were further characterised to determine whether the vessels identified by these antibodies were vascular or lymphatic.From five fusions clones were identified with predominant reactivity with: 1) fibroblasts and endothelial cells; or 2)broad stromal elements (fibroblast, endothelium, epithelium, follicular dendritic cells). A fibroblast-specific antibody that did not also identify vessels was not generated. Examples of each reactivity pattern are discussed.

  1. Human Papillomavirus neutralizing and cross-reactive antibodies induced in HIV-positive subjects after vaccination with quadrivalent and bivalent HPV vaccines

    DEFF Research Database (Denmark)

    Faust, Helena; Nielsen, Lars Toft; Sehr, Peter;

    2016-01-01

    neutralizing and binding antibodies had good agreement (average Kappa for HPV types 6, 11, 16, 18, 31, 33 and 45 was 0.65). At baseline, 88% of subjects had antibodies against at least one genital HPV. Following vaccination with Cervarix™, all subjects became seropositive for HPV16 and 18. After Gardasil......™ vaccination, 96% of subjects seroconverted for HPV16 and 73% for HPV18. Levels of HPV16-specific antibodies were <1 international unit (IU) in 87% of study subjects before vaccination but >10IU in 85% of study subjects after vaccination. Antibodies against non-vaccine HPV types appeared after Gardasil......™ vaccination for >50% of vaccinated females for HPV 31, 35 and 73 and for >50% of Cervarix™-vaccinated females forHPV 31, 33, 35, 45, 56 and 58. Cross-reactivity with non-genital HPV types was also detected. In conclusion, HIV-infected subjects responded to HPV vaccination with induction of neutralizing...

  2. Referencing cross-reactivity of detection antibodies for protein array experiments [version 1; referees: 1 approved, 2 approved with reservations

    OpenAIRE

    Darragh Lemass; Richard O'Kennedy; Kijanka, Gregor S.

    2016-01-01

    Protein arrays are frequently used to profile antibody repertoires in humans and animals. High-throughput protein array characterisation of complex antibody repertoires requires a platform-dependent, lot-to-lot validation of secondary detection antibodies. This article details the validation of an affinity-isolated anti-chicken IgY antibody produced in rabbit and a goat anti-rabbit IgG antibody conjugated with alkaline phosphatase using protein arrays consisting of 7,390 distinct human protei...

  3. Characterization of antigen-expressing Plasmodium falciparum cDNA clones that are reactive with parasite inhibitory antibodies.

    Science.gov (United States)

    Horii, T; Bzik, D J; Inselburg, J

    1988-07-01

    A Plasmodium falciparum (FCR3 strain) lambda gt11 cDNA expression library was constructed from trophozoite and schizont poly(A) RNA and was screened immunologically with a pooled human immune serum from Nigeria to form a gene bank of 288 positive clones. The gene bank was subsequently screened with parasite inhibitory mouse monoclonal antibodies (mMAb) and with individual human Liberian sera. Two mMAb, 43E5 and 5H10, strongly reacted with 8 and 3 cDNA clones, respectively. Several of those clones also weakly cross-reacted with the other mMAb. Two of those weakly cross-reactive clones, cDNA#366 and cDNA#22, were shown to be located in different chromosomal regions of the parasite by Southern hybridization and so appeared to represent two different parasite genes. The genomic organization of both cDNA#366 and cDNA#22 sequences were identical in the FCR3 and the Honduras-1 strain. The nucleotide sequence of cDNA#366 and the amino acid sequence it coded for were homologous to a partial DNA and amino acid sequence previously reported for a P. falciparum (Camp strain) exoantigen designated p126. The mRNA for cDNA#366 appeared to represent an abundant message in blood stage trophozoites and schizonts. PMID:2456465

  4. The Diagnostic Value of Serum C-Reactive Protein for Identifying Pneumonia in Hospitalized Patients with Acute Respiratory Symptoms

    OpenAIRE

    Ruiz-González, Agustín; Utrillo, Laia; Bielsa, Silvia; Falguera, Miquel; PORCEL, José M.

    2016-01-01

    Background. The clinical diagnosis of pneumonia is sometimes difficult since chest radiographs are often indeterminate. In this study, we aimed to assess whether serum C-reactive protein (CRP) could assist in identifying patients with pneumonia. Methods. For one winter, all consecutive patients with acute respiratory symptoms admitted to the emergency ward of a single center were prospectively enrolled. In addition to chest radiographs, basic laboratory tests, and microbiology, serum levels o...

  5. Close topographical relationship in alpha foetoprotein (AFP) between a lens culinaris binding glycan and the epitope recognized by AFP-reactive monoclonal antibody, 18H4.

    OpenAIRE

    Suzuki, Y.; Aoyagi, Y.; Muramatsu, M; Sekine, C.; Isemura, M; Ichida, F.

    1987-01-01

    Monoclonal antibodies 18H4 and 19F12 against alpha-foetoprotein (AFP) were examined by enzyme immunoassay for binding to two forms of AFP that were separated on the basis of the reactivity with lentil lectin (LCA). LCA-binding and LCA non-binding AFP, coated on a solid phase, reacted with 18H4 but reactivity with the LCA-binding species was inhibited by 60% following pretreatment of the AFP with LCA. The lectin was a very poor inhibitor of binding of 18H4 to the AFP which did not interact wit...

  6. Presence of antibodies against a cell-surface protein, cross-reactive with DNA, in systemic lupus erythrematosus: a marker of the disease

    International Nuclear Information System (INIS)

    Antibodies against a cell-surface protein, cross-reactive with double-stranded DNA, were detected in the serum of 25 patients with active human systemic lupus erythematosus (SLE), defined on the basis of the revised American Rheumatism Association classification. Among these sera, two did not display anti-DNA antibodies, as shown by Farr assay, solid-phase radioimmunoassay, and Crithidia luciliae test. Five other SLE patients were consecutively studied in active and remission states. Antibodies against the protein were detected in the serum of the 5 SLE patients when they were in active phase but not in the serum of the same patients in inactive phase of the disease. The anti-protein antibodies were not found in the serum of 10 inactive SLE patients or in the sera of 10 normal human controls, 10 patients with rheumatoid arthritis, 5 patients with scleroderma, and 4 patients with primary sicca syndrome. Taken together, these results strongly suggest that antibodies against this cell-surface protein could provide a better diagnosis marker and activity index than anti-DNA antibodies in SLE

  7. Identifying bottlenecks in transient and stable production of recombinant monoclonal-antibody sequence variants in Chinese hamster ovary cells

    OpenAIRE

    Mason, Megan; Sweeney, Bernadette; Cain, Katharine; Stephens, Paul; Sharfstein, Susan T.

    2012-01-01

    The increasing demand for antibody-based therapeutics has emphasized the need for technologies to improve recombinant antibody titers from mammalian cell lines. Moreover, as antibody therapeutics address an increasing spectrum of indications, interest has increased in antibody engineering to improve affinity and biological activity. However, the cellular mechanisms that dictate expression and the relationships between antibody sequence and expression level remain poorly understood. Fundamenta...

  8. Interrelationship of interleukin 6, C-reactive protein and Chlamydia pneumoniae IgG antibodies in patients with acute coronary syndromes

    OpenAIRE

    Burazor Ivana; Vojdani Aristo; Burazor Mirko

    2008-01-01

    Background/Aim. Inflammation due to infection could be associated with the development of acute coronary syndromes, clinical manifestations of ongoing atherosclerosis in vessel walls. Our aim was determine whether interleukin 6, C-reactive protein and Chlamydia pneumoniae IgG antibodies are connected with the development of acute coronary syndromes, to evaluate their interrelationship and to examine whether they are predictive of new events and mortality. Methods. This prospective study inclu...

  9. Facial recognition of heroin vaccine opiates: Type 1 cross-reactivities of antibodies induced by hydrolytically stable haptenic surrogates of heroin, 6-acetylmorphine, and morphine

    OpenAIRE

    Matyas, Gary R.; Rice, Kenner C.; Cheng, Kejun; Li, Fuying; Antoline, Joshua F. G.; Iyer, Malliga R.; Jacobson, Arthur E; Mayorov, Alexander V.; Beck, Zoltan; Torres, Oscar; Alving, Carl R.

    2014-01-01

    Novel synthetic compounds similar to heroin and its major active metabolites, 6-acetylmorphine and morphine, were examined as potential surrogate haptens for the ability to interface with the immune system for a heroin vaccine. Recent studies have suggested that heroin-like haptens must degrade hydrolytically to induce independent immune responses both to heroin and to the metabolites, resulting in antisera containing mixtures of antibodies (type 2 cross-reactivity). To test this concept, two...

  10. Study of Humoral Immunity to Commensal Oral Bacteria in Human Infants Demonstrates the Presence of Secretory Immunoglobulin A Antibodies Reactive with Actinomyces naeslundii Genospecies 1 and 2 Ribotypes

    OpenAIRE

    Cole, Michael F.; Evans, Mishell K.; Kirchherr, Jennifer L.; Sheridan, Michael J.; Bowden, G. H. W.

    2004-01-01

    The mouths of three human infants were examined from birth to age 2 years to detect colonization of Actinomyces naeslundii genospecies 1 and 2. These bacteria did not colonize until after tooth eruption. The diversity of posteruption isolates was determined by ribotyping. Using immunoblotting and enzyme-linked immunosorbent assay, we determined the reactivity of secretory immunoglobulin A (SIgA) antibodies in saliva samples collected from each infant before and after colonization against cell...

  11. Mediation of cytotoxic functions by classes and subclasses of sheep antibody reactive with cell surface immunoglobulin idiotypic and constant region determinants

    International Nuclear Information System (INIS)

    Sheep antibodies, reactive with either the idiotypic or constant region antigenic determinants of the immunoglobulin light chain in guinea-pig L2C leukaemic cells, were separated into IgM and into the two subclasses of IgG, IgG1 and IgG2. Antibody of both IgG subclasses inhibited the migration of L2C cells along plastic surfaces; IgM was only weakly inhibitory. Antibody of class IgM and of subclass IgG1 mediated complement cytotoxicity against the L2C cells whereas only that of subclass IgG2 mediated K-cell cytotoxicity; the effector arms were rabbit complement and sheep peripheral leucocytes, respectively. (author)

  12. Facial recognition of heroin vaccine opiates: type 1 cross-reactivities of antibodies induced by hydrolytically stable haptenic surrogates of heroin, 6-acetylmorphine, and morphine.

    Science.gov (United States)

    Matyas, Gary R; Rice, Kenner C; Cheng, Kejun; Li, Fuying; Antoline, Joshua F G; Iyer, Malliga R; Jacobson, Arthur E; Mayorov, Alexander V; Beck, Zoltan; Torres, Oscar B; Alving, Carl R

    2014-03-14

    Novel synthetic compounds similar to heroin and its major active metabolites, 6-acetylmorphine and morphine, were examined as potential surrogate haptens for the ability to interface with the immune system for a heroin vaccine. Recent studies have suggested that heroin-like haptens must degrade hydrolytically to induce independent immune responses both to heroin and to the metabolites, resulting in antisera containing mixtures of antibodies (type 2 cross-reactivity). To test this concept, two unique hydrolytically stable haptens were created based on presumed structural facial similarities to heroin or to its active metabolites. After conjugation of a heroin-like hapten (DiAmHap) to tetanus toxoid and mixing with liposomes containing monophosphoryl lipid A, high titers of antibodies after two injections in mice had complementary binding sites that exhibited strong type 1 ("true") specific cross-reactivity with heroin and with both of its physiologically active metabolites. Mice immunized with each surrogate hapten exhibited reduced antinociceptive effects caused by injection of heroin. This approach obviates the need to create hydrolytically unstable synthetic heroin-like compounds to induce independent immune responses to heroin and its active metabolites for vaccine development. Facial recognition of hydrolytically stable surrogate haptens by antibodies together with type 1 cross-reactivities with heroin and its metabolites can help to guide synthetic chemical strategies for efficient development of a heroin vaccine. PMID:24486371

  13. Study of a Cohort of 1,886 Persons To Determine Changes in Antibody Reactivity to Borrelia burgdorferi 3 Months after a Tick Bite

    DEFF Research Database (Denmark)

    Dessau, Ram B; Fryland, Linda; Wilhelmsson, Peter;

    2015-01-01

    Lyme borreliosis is a tick-borne disease caused by the bacterium Borrelia burgdorferi. The most frequent clinical manifestation is a rash called erythema migrans. Changes in antibody reactivity to B. burgdorferi 3 months after a tick bite are measured using enzyme-linked immunosorbent assays...... (ELISAs). One assay is based on native purified flagellum antigen (IgG), and the other assay is based on a recombinant antigen called C6 (IgG or IgM). Paired samples were taken at the time of a tick bite and 3 months later from 1,886 persons in Sweden and the Åland Islands, Finland. The seroconversion or...... antigen and the C6 antigen, respectively. Graphical methods to display the antibody response and to choose thresholds for a rise in relative antibody reactivity are shown and discussed. In conclusion, 5.4% of people with tick bites showed a rise in Borrelia-specific antibodies above the 2.5% threshold in...

  14. Reactive inspection response of NRC Region III to potential technical deficiencies identified in recent Nuclear Air Cleaning Conference papers

    International Nuclear Information System (INIS)

    In order to effectively meet its responsibility to protect the public health and safety, the Nuclear Regulatory Commission (NRC) nuclear power plant licensing and inspection programs respond to potential technical deficiencies identified by conference and professional society meeting papers when deemed appropriate. The NRC staff's response mechanisms for such technical deficiencies include: generic letters, Bulletins, Information Notices, Standard Review Plan (NUREG-0800) revisions, docketed Final Safety Analysis Report (FSAR) questions, special studies, special (reactive) inspection, and inspection program revisions. This paper describes reactive inspection efforts by Region III in response to potential technical deficiencies identified in recent air cleaning conference papers, including: post-accident effluent sample line deposition losses; failure to implement good engineering practices in the design, construction, and testing of Nuclear Air Treatment Systems (NATS); filter bypass via filter housing drain lines; spinster carbon degradation; use of silicone sealants and other temporary patching material in NATS; filter housing fire protection deluge system problems; lack of charcoal batch traceability; Quality Assurance records problems involving equipment, vendor, filter, and personnel qualifications; inadequate ANSI/ASME N510 acceptance criteria and tests; and failure to adequately demonstrate control room habitability per 10 CFR 50, Appendix A, General Design Criterion-19. Region III inspections indicate that many of these deficiencies appear to be prevalent. Inspection findings and utility responses to the findings are discussed. NRC Region III and Headquarters programmatic reactions to the identified generic problem areas are also discussed

  15. Phenotypic assays to identify agents that induce reactive gliosis: a counter-screen to prioritize compounds for preclinical animal studies.

    Science.gov (United States)

    Beckerman, Samuel R; Jimenez, Joaquin E; Shi, Yan; Al-Ali, Hassan; Bixby, John L; Lemmon, Vance P

    2015-09-01

    Astrocyte phenotypes change in a process called reactive gliosis after traumatic central nervous system (CNS) injury. Astrogliosis is characterized by expansion of the glial fibrillary acidic protein (GFAP) cytoskeleton, adoption of stellate morphologies, and differential expression of some extracellular matrix molecules. The astrocytic response immediately after injury is beneficial, but in the chronic injury phase, reactive astrocytes produce inhibitory factors (i.e., chondroitin sulfate proteoglycans [CSPGs]) that limit the regrowth of injured axons. There are no drugs that promote axon regeneration or functional recovery after CNS trauma in humans. To develop novel therapeutics for the injured CNS, we screened various libraries in a phenotypic assay to identify compounds that promote neurite outgrowth. However, the effects these compounds have on astrocytes are unknown. Specifically, we were interested in whether compounds could alter astrocytes in a manner that mimics the glial reaction to injury. To test this hypothesis, we developed cell-based phenotypic bioassays to measure changes in (1) GFAP morphology/localization and (2) CSPG expression/immunoreactivity from primary astrocyte cultures. These assays were optimized for six-point dose-response experiments in 96-well plates. The GFAP morphology assay is suitable for counter-screening with a Z-factor of 0.44±0.03 (mean±standard error of the mean; N=3 biological replicates). The CSPG assay is reproducible and informative, but does not satisfy common metrics for a "screenable" assay. As proof of principle, we tested a small set of hit compounds from our neurite outgrowth bioassay and identified one that can enhance axon growth without exacerbating the deleterious characteristics of reactive gliosis. PMID:26230074

  16. JC virus antibody index in natalizumab-treated patients: correlations with John Cunningham virus DNA and C-reactive protein level

    Directory of Open Access Journals (Sweden)

    Lanzillo R

    2014-10-01

    Full Text Available Roberta Lanzillo,1 Raffaele Liuzzi,2 Luca Vallefuoco,3 Marcello Moccia,1 Luca Amato,1 Giovanni Vacca,1 Veria Vacchiano,1 Giuseppe Portella,3 Vincenzo Brescia Morra1 1Neurological Sciences Department, Federico II University, 2Institute of Biostructure and Bioimaging, National Research Council, 3Clinical Pathology Department, Federico II University, Naples, ItalyAbstract: Natalizumab-treated patients have a higher risk of developing progressive multifocal leukoencephalopathy. Exposure to John Cunningham virus (JCV is a prerequisite for PML (progressive multifocal leukoencephalopathy. To assess JCV exposure in multiple sclerosis patients, we performed a serological examination, obtained the antibody index, performed real-time polymerase chain reaction (PCR to detect JCV DNA in plasma and urine, and investigated the role of ultrasensitive C-reactive protein (usCRP as a possible biological marker of JCV reactivation. We retrospectively analyzed consecutive natalizumab-treated multiple sclerosis patients who underwent a JCV antibody test through a two-step enzyme-linked immunosorbent assay (STRATIFY test to the measure of serum usCRP levels, and to perform blood and urine JCV PCR. The studied cohort included 97 relapsing–remitting patients (60 women. Fifty-two patients (53.6% tested positive for anti-JCV antibodies. PCR showed JCV DNA in the urine of 30 out of 83 (36.1% patients and 28 out of 44 seropositive patients (63.6%, with a 6.7% false-negative rate for the STRATIFY test. Normalized optical density values were higher in urinary JCV DNA-positive patients (P<0.0001. Interestingly, the level of usCRP was higher in urinary JCV DNA-positive patients and correlated to the number of DNA copies in urine (P=0.028. As expected, patients' age correlated with JCV seropositivity and with JC viruria (P=0.02 and P=0.001, respectively. JC viruria was significantly correlated with a high JCV antibody index and high serum usCRP levels. We suggest that PCR and

  17. Humoral Immunity to Commensal Oral Bacteria in Human Infants: Salivary Antibodies Reactive with Actinomyces naeslundii Genospecies 1 and 2 during Colonization

    OpenAIRE

    Cole, Michael F.; Bryan, Stacey; Evans, Mishell K.; Pearce, Cheryl L.; Sheridan, Michael J.; Sura, Patricia A.; Wientzen, Raoul; Bowden, George H. W.

    1998-01-01

    The secretory immune response in saliva to colonization by Actinomyces naeslundii genospecies 1 and 2 was studied in 10 human infants from birth to 2 years of age. Actinomyces species were not recovered from the mouths of the infants until approximately 4 months after the eruption of teeth. However, low levels of secretory immunoglobulin A1 (SIgA1) and SIgA2 antibodies reactive with whole cells of A. naeslundii genospecies 1 and 2 were detected within the first month after birth. Although the...

  18. Serum Reactivity Against Bacterial Pyruvate Dehydrogenase: Increasing the Specificity of Anti-Mitochondrial Antibodies for the Diagnosis of Primary Biliary Cirrhosis

    Directory of Open Access Journals (Sweden)

    Carlo Selmi

    2006-01-01

    Full Text Available Antimitochondrial antibodies (AMA are the serum hallmark of primary biliary cirrhosis (PBC. However, AMA-positivity can be found in non-PBC sera when lower dilutions are used, thus raising issues about the specificity and sensitivity of the test. AMA reacts primarily with the lipoylated domains of pyruvate dehydrogenase-E2 (PDC-E2 which is highly conserved across species, including bacteria. We studied 77 serum samples, including 24 from patients with anti-PDC-E2-positive PBC and 53 controls (16 with autoimmune hepatitis (AIH, 10 with primary sclerosing cholangitis (PSC, and 27 healthy individuals for their reactivities at serial dilutions (1:10, 1:20 and 1:40 against Escherichia coli DH5 alpha lysate overexpressing human PDC-E2 using immunoblotting (IB. A murine anti-human PDC-E2 monoclonal antibody (mAB was used as control. We further studied positive sera using adsorption with a synthetic E. coli peptide sharing similarity with human PDC-E2. Finally, we verified whether a unique buffer for E. coli preparation could reduce non-specific serum reactivity. Results demonstrated that 100% of anti-PDC-E2-positive PBC and up to 38% of control sera at 1:10 dilution recognized E. coli PDC-E2 at IB while dilution tests indicated that the overall potency of PBC reactivity was 100-fold higher compared to controls. In fact, a subgroup (20-38% of non-PBC sera were positive at low titers but lost the reactivity when absorbed with the synthetic E. coli peptide. Finally, our unique buffer reduced the reactivity of non-PBC sera as measured by ELISA. In conclusion, we demonstrated that weak cross-reactivity with E. coli PDC-E2 occurs in non-PBC sera at lower dilutions and that such reactivity is not due to AMA-positivity. The use of a specific buffer might avoid the risk of false positive AMA determinations when E. coli-expressed recombinant antigens are used.

  19. Implementation of an original approach on the Mines-Douai Comparative Reactivity Method (MD-CRM) instrument to identify part of the missing OH reactivity at an urban site

    Science.gov (United States)

    Dusanter, S.; Michoud, V.; Leonardis, T.; Riffault, V.; Zhang, S.; Locoge, N.

    2015-12-01

    Due to the large number of Volatile Organic Compounds (VOCs) expected in the atmosphere (104-105) (Goldstein and Galbally, ES&T, 2007), exhaustive measurements of VOCs appear to be currently unfeasible using common analytical techniques. In this context, measurements of the total sink of OH, referred as total OH reactivity, can provide a critical test to assess the completeness of trace gas measurements during field campaigns. This can be done by comparing the measured total OH reactivity to values calculated from trace gas measurements. Indeed, large discrepancies are usually found between measured and calculated OH reactivity values revealing the presence of important unmeasured reactive species, which have yet to be identified. A Comparative Reactivity Method (CRM) instrument has been setup at Mines Douai to allow sequential measurements of VOCs and OH reactivity using the same Proton Transfer Reaction-Time of Flight Mass Spectrometer. This approach aims at identifying unmeasured reactive VOCs based on a method proposed by Kato et al. (Atmos. Environ., 2011), taking advantage of VOC oxidations occurring in the CRM sampling reactor. MD-CRM has been deployed at an urban site in Dunkirk (France) during July 2014 to test this new approach. During this campaign, a large fraction of the OH reactivity was not explained by collocated measurements of trace gases (67% on average). In this presentation, we will first describe the approach that was implemented in the CRM instrument to identify part of the observed missing OH reactivity and we will then discuss the OH reactivity budget regarding the origin of air masses reaching the measurement site.

  20. Hepatitis B reactivation in a patient with rheumatoid arthritis with antibodies to hepatitis B surface antigen treated with rituximab

    OpenAIRE

    Gigi, E; GEORGIOU T; Mougiou, D; Boura, P; Raptopoulou-Gigi, M

    2013-01-01

    Hepatitis B virus (HBV) can still be found within the hepatocytes after its clearance and the control of viral replication depends on the immune response. However during immunosuppression, seroconversion of HBsAg has been described followed by disease reactivation. Hepatitis B virus reactivation represents an emerging cause of liver disease in patients undergoing treatment with biologic agents and in particular, by the use of rituximab (anti-CD20) and alemtuzumab (anti-CD52) that cause profou...

  1. The Diagnostic Value of Serum C-Reactive Protein for Identifying Pneumonia in Hospitalized Patients with Acute Respiratory Symptoms

    Science.gov (United States)

    Utrillo, Laia; Bielsa, Silvia; Falguera, Miquel; Porcel, José M.

    2016-01-01

    Background. The clinical diagnosis of pneumonia is sometimes difficult since chest radiographs are often indeterminate. In this study, we aimed to assess whether serum C-reactive protein (CRP) could assist in identifying patients with pneumonia. Methods. For one winter, all consecutive patients with acute respiratory symptoms admitted to the emergency ward of a single center were prospectively enrolled. In addition to chest radiographs, basic laboratory tests, and microbiology, serum levels of CRP were measured at entry. Results. A total of 923 (62.3%) of 1473 patients hospitalized for acute respiratory symptoms were included. Subjects with a final diagnosis of pneumonia had higher serum CRP levels (median 187 mg/L) than those with exacerbations of chronic obstructive pulmonary disease (63 mg/L) or acute bronchitis (54 mg/L, p CRP was accurate in identifying pneumonia (area under the curve 0.84, 95% CI 0.82–0.87). The multilevel likelihood ratio (LR) for intervals of CRP provided useful information on the posttest probability of having pneumonia. CRP intervals above 200 mg/L were associated with LR+ > 5, for which pneumonia is likely, whereas CRP intervals below 75 mg/L were associated with LR CRP may be a useful addition for diagnosing pneumonia in hospitalized patients with acute respiratory symptoms. PMID:27610265

  2. The Use of Chimeric Virus-like Particles Harbouring a Segment of Hantavirus Gc Glycoprotein to Generate a Broadly-Reactive Hantavirus-Specific Monoclonal Antibody

    Directory of Open Access Journals (Sweden)

    Aurelija Zvirbliene

    2014-02-01

    Full Text Available Monoclonal antibodies (MAbs against viral glycoproteins have important diagnostic and therapeutic applications. In most cases, the MAbs specific to viral glycoproteins are raised against intact virus particles. The biosynthesis of viral glycoproteins in heterologous expression systems such as bacteria, yeast, insect or mammalian cells is often problematic due to their low expression level, improper folding and limited stability. To generate MAbs against hantavirus glycoprotein Gc, we have used initially a recombinant yeast-expressed full-length Puumala virus (PUUV Gc protein. However, this approach was unsuccessful. As an alternative recombinant antigen, chimeric virus-like particles (VLPs harboring a segment of PUUV Gc glycoprotein were generated in yeast Saccharomyces cerevisiae. A 99 amino acid (aa-long segment of Gc protein was inserted into the major capsid protein VP1 of hamster polyomavirus at previously defined positions: either site #1 (aa 80–89 or site #4 (aa 280–289. The chimeric proteins were found to self-assemble to VLPs as evidenced by electron microscopy. Chimeric VLPs induced an efficient insert-specific antibody response in immunized mice. Monoclonal antibody (clone #10B8 of IgG isotype specific to hantavirus Gc glycoprotein was generated. It recognized recombinant full-length PUUV Gc glycoprotein both in ELISA and Western blot assay and reacted specifically with hantavirus-infected cells in immunofluorescence assay. Epitope mapping studies revealed the N-terminally located epitope highly conserved among different hantavirus strains. In conclusion, our approach to use chimeric VLPs was proven useful for the generation of virus-reactive MAb against hantavirus Gc glycoprotein. The generated broadly-reactive MAb #10B8 might be useful for various diagnostic applications.

  3. A novel 95-kilodalton antigen of Wuchereria bancrofti infective larvae identified by species-specific monoclonal antibodies.

    OpenAIRE

    Burkot, T. R.; Kwan-Lim, G E; R. M. Maizels

    1996-01-01

    CBA and BALB/c mice produced polyspecific and monospecific polyclonal antibody responses, respectively, following immunization with Wuchereria bancrofti stage-3 larvae. Two monoclonal antibodies (MAbs) were produced from the immunized BALB/c mouse. These MAbs (both isotype M) recognized a previously undescribed highly expressed W. bancrofti antigen present in stage-3 larvae. The epitopes bound by the MAbs appear to be species specific for W. bancrofti since the MAbs did not bind to antigens o...

  4. Anti-NeuGcGM3 reactivity: a possible role of natural antibodies and B-1 cells in tumor immunosurveillance.

    Science.gov (United States)

    Rodriguez-Zhurbenko, Nely; Rabade-Chediak, Maura; Martinez, Darel; Griñan, Tania; Hernandez, Ana Maria

    2015-12-01

    While not naturally expressed in normal human tissues, N-glycolylated (NeuGc) gangliosides are overexpressed in several tumors and have immunosuppressive capacity, which contributes to cancer progression. Naturally occurring antibodies against NeuGcGM3 exist in healthy donors that specifically recognize and kill tumor cells expressing the antigen by complement-dependent and -independent mechanisms, the latter resembling an oncotic necrosis-type of cell death. Both the levels of anti-NeuGcGM3 antibodies in the sera of healthy donors and the percentage of donors with these natural antibodies decrease with age. Our work has shown that anti-NeuGcGM3 antibodies are not detected in the sera of non-small cell lung cancer (NSCLC) patients, compared to age- and sex-matched healthy donors, which have anti-NeuGcGM3. Interestingly, the level of serum total IgM, but not IgG, was significantly lower in cancer patients than in healthy donors. Screening of immortalized mouse splenic and peritoneal-derived hybridomas showed that peritoneal B-1 cells secrete anti-NeuGcGM3 with tumor cytotoxic capacity. Defects in the natural surveillance against tumor antigens could increase the risk of elderly donors developing cancer and affect the capacity of cancer patients to effectively fight against tumor cells. PMID:26214505

  5. Antibody Reactivity of B Cells in Lupus Patients with Increased Disease Activity and ARID3a Expression

    Directory of Open Access Journals (Sweden)

    Julie M. Ward

    2015-11-01

    Full Text Available Earlier studies showed that the DNA-binding protein, Bright/ARID3a bound to a subset of human and mouse immunoglobulin heavy chain promoters where it enhanced expression. Indeed, mice with transgenic expression of ARID3a in all B lymphocytes have expanded MZ B cells and produce anti-nuclear antibodies (ANAs. Consistent with our findings in mice, we observed that human systemic lupus erythematosus (SLE patients had expanded numbers of peripheral blood ARID3a+ B cells that were associated with increased disease activity (p = 0.0038. We hypothesized that ARID3a+ naïve B cells would eventually produce autoantibodies, explaining associations between ARID3a expression and disease activity in lupus. Unlike healthy controls, ARID3a was expressed in the naïve B cell population in SLE patients, and we hypothesized that these might represent expansions of autoreactive cells. Therefore, monoclonal antibodies were generated from single-sorted naïve B cells derived from patients with normal (ARID3aN and high (ARID3aH numbers of ARID3a+ B cells. We found that ARID3a expression did not correlate with autoantibody expression. Furthermore, measures of antigen specificities of autoreactive antibodies did not reveal skewing toward particular proteins. These data suggest that the association of increased disease activity in SLE with numbers of ARID3a+ B lymphocytes may be mediated by an antibody-independent mechanism.

  6. Molecular basis for the cross-reactivity of antibodies elicited by a natural anatoxin with alpha- and beta-toxins from the venom of Tityus serrulatus scorpion.

    Science.gov (United States)

    Chavez-Olortegui, Carlos; Molina, Franck; Granier, Claude

    2002-03-01

    A non-toxic protein (TsNTxP) isolated from the venom of the noxious scorpion Tityus serrulatus (Ts) induces polyclonal antibodies cross-reactive with several toxins from the venom, in sharp contrast to anti-toxin antibodies which are toxin specific. To try to uncover the molecular basis for these unusual properties, peptide scanning experiments were performed and indicated that the N- and C-terminal parts of TsNTxP enclose continuous epitopes (residues 1-15 and 47-61). Antibodies raised against peptides corresponding to these two regions were found to have neutralizing properties against a mixture of all toxic proteins from the T. serrulatus venom, indicating that residues 1-15 and 47-61 correspond to neutralizing epitopes. The identification of key antigenic residues within these two epitopes revealed that several of them are well conserved in the amino-acid sequences of the three main toxins (Ts II, Ts IV and Ts VII) from the venom: Glu 3, Tyr 5, Asp 8, Asp 50, Trp 55 and Lys 61. A single key-residue (Glu 58) is unique to TsNTxP. By using homology modeling, a model of the three-dimensional structure of TsNTxP was obtained. The antigenically important residues from TsNTxP were found to be surface exposed, with five of them clustered on the facet of the protein reported to enclose the active site of toxins. Residues equivalent to the seven key-residues of the anatoxin were also found to be exposed in the active toxins from T. serrulatus venom. These results show that antibodies elicited by the non-toxic protein TsNTxP recognized, within the N- and C-terminal parts of toxins of T. serrulatus, conserved and surface exposed residues which might also be involved in the toxic action of the proteins. PMID:11922945

  7. Lymphocyte Reactivity Contributes to Protection Conferred by Specific Antibody Passively Transferred to Herpes Simplex Virus-Infected Mice

    OpenAIRE

    Oakes, John E.; Davis, William B.; Taylor, John A.; Weppner, William A.

    1980-01-01

    Passively acquired immunity to herpes simplex virus (HSV) was studied in antithymocyte serum (ATS)-treated mice and athymic nude mice to determine whether immunocompetent lymphocytes contribute to the protection observed after transfer of HSV-specific antibody to infected animals. Mice were given three intraperitoneal injections of 0.1 ml of ATS at 24-h intervals. This treatment reduced concanavalin A and lipopolysaccharide stimulation of lymphocytes harvested from these animals by 90% when c...

  8. Immunoglobulin isotypes of anti-Trichomonas vaginalis antibodies in patients with vaginal trichomoniasis.

    OpenAIRE

    Wos, S M; Watt, R M

    1986-01-01

    Studies of anti-Trichomonas vaginalis antibodies in patients with vaginal trichomoniasis were undertaken in attempts to identify the predominant antibody isotype produced and to delineate clinically significant antigens. The total antibody content of serum samples from 23 patients was determined by an enzyme-linked immunosorbent assay (ELISA) that employed anti-human immunoglobulin and isotype-specific antisera. The immunochemical reactivity of these antibodies was examined by Western blot an...

  9. Sensitization prevalence, antibody cross-reactivity and immunogenic peptide profile of Api g 2, the non-specific lipid transfer protein 1 of celery.

    Directory of Open Access Journals (Sweden)

    Gabriele Gadermaier

    Full Text Available BACKGROUND: Celery (Apium graveolens represents a relevant allergen source that can elicit severe reactions in the adult population. To investigate the sensitization prevalence and cross-reactivity of Api g 2 from celery stalks in a Mediterranean population and in a mouse model. METHODOLOGY: 786 non-randomized subjects from Italy were screened for IgE reactivity to rApi g 2, rArt v 3 (mugwort pollen LTP and nPru p 3 (peach LTP using an allergen microarray. Clinical data of 32 selected patients with reactivity to LTP under investigation were evaluated. Specific IgE titers and cross-inhibitions were performed in ELISA and allergen microarray. Balb/c mice were immunized with purified LTPs; IgG titers were determined in ELISA and mediator release was examined using RBL-2H3 cells. Simulated endolysosomal digestion was performed using microsomes obtained from human DCs. RESULTS: IgE testing showed a sensitization prevalence of 25.6% to Api g 2, 18.6% to Art v 3, and 28.6% to Pru p 3 and frequent co-sensitization and correlating IgE-reactivity was observed. 10/32 patients suffering from LTP-related allergy reported symptoms upon consumption of celery stalks which mainly presented as OAS. Considerable IgE cross-reactivity was observed between Api g 2, Art v 3, and Pru p 3 with varying inhibition degrees of individual patients' sera. Simulating LTP mono-sensitization in a mouse model showed development of more congruent antibody specificities between Api g 2 and Art v 3. Notably, biologically relevant murine IgE cross-reactivity was restricted to the latter and diverse from Pru p 3 epitopes. Endolysosomal processing of LTP showed generation of similar clusters, which presumably represent T-cell peptides. CONCLUSIONS: Api g 2 represents a relevant celery stalk allergen in the LTP-sensitized population. The molecule displays common B cell epitopes and endolysosomal peptides that encompass T cell epitopes with pollen and plant-food derived LTP.

  10. Checkpoint inhibitors in cancer immunotherapy: Cross reactivity of a CTLA-4 antibody and IDO-inhibitor L-1MT in pigs

    DEFF Research Database (Denmark)

    Al-Shatrawi, Zina Adil; Frøsig, Thomas Mørch; Jungersen, Gregers

    Blockade of checkpoint inhibitors has recently shown very convincing results in the treatment of cancer. One key target is CTLA-4, which has been demonstrated to be a potent negative regulator of lymphocyte activation. The treatment with the FDA-approved fully human CTLA-4 monoclonal antibody...... a non-specific activation of porcine T cells. This will be further investigated to provide the basis for in vivo studies investigating checkpoint inhibitor blockade in combination with other cancer immunotherapies. Eventually our goal is to establish pigs as an alternative large animal model...... Ipilimumab increases anticancer T-cell reactivity and overall survival of metastatic cancer patients. Indole-amine 2,3-dioxygenase (IDO) is another checkpoint inhibitor which suppresses T-cell immunity by the depletion of tryptophan in the T-cell microenvironment, and also inhibition of IDO by L-1...

  11. Antiviral antibodies stimulate production of reactive oxygen species in cultured canine brain cells infected with canine distemper virus.

    OpenAIRE

    Bürge, T; Griot, C; Vandevelde, M; Peterhans, E

    1989-01-01

    Canine distemper is characterized mainly by respiratory, enteric, and nervous symptoms. Infection of the central nervous system results in demyelination, to which inflammation has been shown to contribute significantly. It has been proposed that macrophages play a major role as effector cells in this process. We report that cultured dog brain cells contain a population of macrophages capable of producing reactive oxygen species as measured by luminol-dependent chemiluminescence. In cultures i...

  12. Neuronal antibody biomarkers for Sydenham's chorea identify a new group of children with chronic recurrent episodic acute exacerbations of tic and obsessive compulsive symptoms following a streptococcal infection.

    Science.gov (United States)

    Singer, Harvey S; Mascaro-Blanco, Adda; Alvarez, Kathy; Morris-Berry, Christina; Kawikova, Ivana; Ben-Pazi, Hilla; Thompson, Carol B; Ali, Syed F; Kaplan, Edward L; Cunningham, Madeleine W

    2015-01-01

    Several autoantibodies (anti-dopamine 1 (D1R) and 2 (D2R) receptors, anti-tubulin, anti-lysoganglioside-GM1) and antibody-mediated activation of calcium calmodulin dependent protein kinase II (CaMKII) signaling activity are elevated in children with Sydenham's chorea (SC). Recognizing proposed clinical and autoimmune similarities between SC and PANDAS (pediatric autoimmune neuropsychiatric disorder associated with a streptococcal infection), we sought to identify serial biomarker changes in a slightly different population. Antineuronal antibodies were measured in eight children (mean 11.3 years) with chronic, dramatic, recurrent tics and obsessive-compulsive disorder (OCD) associated with a group A β-hemolytic streptococcal (GABHS) respiratory tract infection, but differing because they lacked choreiform movements. Longitudinal serum samples in most subjects included two pre-exacerbation samples, Exac), one midst Exac (abrupt recurrence of tic/OCD; temporally association with a GABHS infection in six of eight subjects), and two post-Exac. Controls included four groups of unaffected children (n = 70; mean 10.8 years) obtained at four different institutions and published controls. Clinical exacerbations were not associated with a significant rise in antineuronal antibody titers. CaMKII activation was increased at the GABHS exacerbation point in 5/6 subjects, exceeded combined and published control's 95th percentile at least once in 7/8 subjects, and median values were elevated at each time point. Anti-tubulin and anti-D2R titers did not differ from published or combined control group's 95th percentile or median values. Differences in anti-lysoganglioside-GM1 and anti-D1R titers were dependent on the selected control. Variances in antibody titers and CaMKII activation were identified among the institutional control groups. Based on comparisons to published studies, results identify two groups of PANDAS: 1) a cohort, represented by this study, which lacks choreiform

  13. Neuronal antibody biomarkers for Sydenham's chorea identify a new group of children with chronic recurrent episodic acute exacerbations of tic and obsessive compulsive symptoms following a streptococcal infection.

    Directory of Open Access Journals (Sweden)

    Harvey S Singer

    Full Text Available Several autoantibodies (anti-dopamine 1 (D1R and 2 (D2R receptors, anti-tubulin, anti-lysoganglioside-GM1 and antibody-mediated activation of calcium calmodulin dependent protein kinase II (CaMKII signaling activity are elevated in children with Sydenham's chorea (SC. Recognizing proposed clinical and autoimmune similarities between SC and PANDAS (pediatric autoimmune neuropsychiatric disorder associated with a streptococcal infection, we sought to identify serial biomarker changes in a slightly different population. Antineuronal antibodies were measured in eight children (mean 11.3 years with chronic, dramatic, recurrent tics and obsessive-compulsive disorder (OCD associated with a group A β-hemolytic streptococcal (GABHS respiratory tract infection, but differing because they lacked choreiform movements. Longitudinal serum samples in most subjects included two pre-exacerbation samples, Exac, one midst Exac (abrupt recurrence of tic/OCD; temporally association with a GABHS infection in six of eight subjects, and two post-Exac. Controls included four groups of unaffected children (n = 70; mean 10.8 years obtained at four different institutions and published controls. Clinical exacerbations were not associated with a significant rise in antineuronal antibody titers. CaMKII activation was increased at the GABHS exacerbation point in 5/6 subjects, exceeded combined and published control's 95th percentile at least once in 7/8 subjects, and median values were elevated at each time point. Anti-tubulin and anti-D2R titers did not differ from published or combined control group's 95th percentile or median values. Differences in anti-lysoganglioside-GM1 and anti-D1R titers were dependent on the selected control. Variances in antibody titers and CaMKII activation were identified among the institutional control groups. Based on comparisons to published studies, results identify two groups of PANDAS: 1 a cohort, represented by this study, which lacks

  14. Radioimmunoimaging of experimental thrombi in dogs using Tc-99m labeled monoclonal antibody fragments [MAPab-F(ab')/sub 2/] reactive with human platelets

    International Nuclear Information System (INIS)

    Radioimmunoimaging of thrombi could have great clinical value in the management of coronary artery and thromboembolic disease. In-111-oxine-labeled platelets currently used require platelet isolation, delayed imaging, background subtraction and there is also potential for damaging or contaminating platelets during labeling. Murine monoclonal antibody (IgG/sub 2/a) fragments directed against human platelet membrane components (cross-reactive with dog platelets) were labeled with Tc-99m and repurified from ''kits''. After radiolabeling, 91.5-93.3% of the Tc-99m was antibody-associated. The preparations retained immunoreactivity, as determined by the ratio of cell to plasma-associated radioactivity (ratios 54.7-63.8). Tc-99m-MAPAb-F(ab')/sub 2/ were injected i.v. into dogs with thrombi produced in peripheral and pulmonary veins and arteries. About 50% of the radioactivity was cleared from the blood in 3-6 min. and 18-24% was excreted in the urine within 3 hrs. The thrombi were consistently and easily visible within 1-3 hrs. with no need for blood pool subtraction. In some cases, intimal damage along the path of catheters was seen. No adverse side effects were observed. The advantages of this method are: short and simple preparation, no need for blood pool subtraction and early visualization of thrombi. Human studies are warranted to determine its clinical efficacy

  15. 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.

  16. Identification and characterization of a broadly cross-reactive HIV-1 human monoclonal antibody that binds to both gp120 and gp41.

    Directory of Open Access Journals (Sweden)

    Mei-Yun Zhang

    Full Text Available Identification of broadly cross-reactive HIV-1-neutralizing antibodies (bnAbs may assist vaccine immunogen design. Here we report a novel human monoclonal antibody (mAb, designated m43, which co-targets the gp120 and gp41 subunits of the HIV-1 envelope glycoprotein (Env. M43 bound to recombinant gp140 s from various primary isolates, to membrane-associated Envs on transfected cells and HIV-1 infected cells, as well as to recombinant gp120 s and gp41 fusion intermediate structures containing N-trimer structure, but did not bind to denatured recombinant gp140 s and the CD4 binding site (CD4bs mutant, gp120 D368R, suggesting that the m43 epitope is conformational and overlaps the CD4bs on gp120 and the N-trimer structure on gp41. M43 neutralized 34% of the HIV-1 primary isolates from different clades and all the SHIVs tested in assays based on infection of peripheral blood mononuclear cells (PBMCs by replication-competent virus, but was less potent in cell line-based pseudovirus assays. In contrast to CD4, m43 did not induce Env conformational changes upon binding leading to exposure of the coreceptor binding site, enhanced binding of mAbs 2F5 and 4E10 specific for the membrane proximal external region (MPER of gp41 Envs, or increased gp120 shedding. The overall modest neutralization activity of m43 is likely due to the limited binding of m43 to functional Envs which could be increased by antibody engineering if needed. M43 may represent a new class of bnAbs targeting conformational epitopes overlapping structures on both gp120 and gp41. Its novel epitope and possibly new mechanism(s of neutralization could helpdesign improved vaccine immunogens and candidate therapeutics.

  17. Cross-reactivity of antibodies with phenolic compounds in pistachios during quantification of ochratoxin A by commercial enzyme-linked immunosorbent assay kits.

    Science.gov (United States)

    Lee, Hyun Jung; Meldrum, Alexander D; Rivera, Nicholas; Ryu, Dojin

    2014-10-01

    Ochratoxin A (OTA), a nephrotoxic mycotoxin, naturally occurs in wide range of agricultural commodities. Typical screening of OTA involves various enzyme-linked immunosorbent assay (ELISA) methods. Pistachio (Pistacia vera L.) is a rich source of phenolic compounds that may result in a false positive due to structural similarities to OTA. The present study investigated the cross-reactivity profiles of phenolic compounds using two commercial ELISA test kits. High-performance liquid chromatography was used to confirm the concentration of OTA in the pistachio samples and compared with the results obtained from ELISA. When the degree of interaction and 50 % inhibitory concentration of phenolic compounds were determined, the cross-reactivity showed a pattern similar to that observed with the commercial ELSIA kits, although quantitatively different. In addition, the degree of interaction increased with the increasing concentration of phenolic compounds. The ELISA value had stronger correlations with the content of total phenolic compound, gallic acid, and catechin (R(2) = 0.757, 0.732, and 0.729, respectively) compared with epicatechin (R(2) = 0.590). These results suggest that phenolic compounds in pistachio skins may cross-react with the OTA antibody and lead to a false positive or to an overestimation of OTA concentration in ELISA-based tests. PMID:25285493

  18. A 1-year lifestyle intervention for weight loss in individuals with type 2 diabetes reduces high C-reactive protein levels and identifies metabolic predictors of change

    Science.gov (United States)

    OBJECTIVE: We examined whether a 1-year intensive lifestyle intervention (ILI) for weight loss reduced elevated high-sensitivity C-reactive protein (hs-CRP) levels in obese individuals with diabetes and identified metabolic and fitness predictors of hs-CRP change. RESEARCH DESIGN AND METHODS: Look A...

  19. Antibody-linked polymerase assay on protein blots: a novel method for identifying polymerases following SDS-polyacrylamide gel electrophoresis.

    OpenAIRE

    van der Meer, J.; Dorssers, L; Zabel, P

    1983-01-01

    We describe a method for correlating polymerase activity with a particular polypeptide band in an SDS-polyacrylamide gel which does not require renaturation of the SDS-denatured enzyme. The method involves the following steps: (i) transfer of proteins from an SDS-polyacrylamide gel onto nitrocellulose; (ii) incubation with excess antiserum raised against a partially purified polymerase preparation to link one Fab site of an antibody molecule to the denatured enzyme on the nitrocellulose; (iii...

  20. Ab-origin: an enhanced tool to identify the sourcing gene segments in germline for rearranged antibodies

    Directory of Open Access Journals (Sweden)

    Sun Jing

    2008-12-01

    Full Text Available Abstract Background In the adaptive immune system, variable regions of immunoglobulin (IG are encoded by random recombination of variable (V, diversity (D, and joining (J gene segments in the germline. Partitioning the functional antibody sequences to their sourcing germline gene segments is vital not only for understanding antibody maturation but also for promoting the potential engineering of the therapeutic antibodies. To date, several tools have been developed to perform such "trace-back" calculations. Yet, the predicting ability and processing volume of those tools vary significantly for different sets of data. Moreover, none of them give a confidence for immunoglobulin heavy diversity (IGHD identification. Developing fast, efficient and enhanced tools is always needed with the booming of immunological data. Results Here, a program named Ab-origin is presented. It is designed by batch query against germline databases based on empirical knowledge, optimized scoring scheme and appropriate parameters. Special efforts have been paid to improve the identification accuracy of the short and volatile region, IGHD. In particular, a threshold score for certain sensitivity and specificity is provided to give the confidence level of the IGHD identification. Conclusion When evaluated using different sets of both simulated data and experimental data, Ab-origin outperformed all the other five popular tools in terms of prediction accuracy. The features of batch query and confidence indication of IGHD identification would provide extra help to users. The program is freely available at http://mpsq.biosino.org/ab-origin/supplementary.html.

  1. The Diagnostic Utility of Anti-cyclic Citrullinated Peptide Antibodies, Matrix Metalloproteinase-3, Rheumatoid Factor, Erythrocyte Sedimentation Rate, and C-reactive Protein in Patients with Erosive and Non-erosive Rheumatoid Arthritis

    Directory of Open Access Journals (Sweden)

    O. Shovman

    2005-01-01

    Full Text Available Objective: To compare the diagnostic utility of laboratory variables, including matrix metalloproteinase-3 (MMP-3, anti-cyclic citrullinated peptide (CCP antibodies, rheumatoid factor (RF, erythrocyte sedimentation rate (ESR, and C-reactive protein (CRP in patients with erosive and non-erosive rheumatoid arthritis (RA.

  2. Potent and Broadly Reactive HIV-2 Neutralizing Antibodies Elicited by a Vaccinia Virus Vector Prime-C2V3C3 Polypeptide Boost Immunization Strategy▿ †

    Science.gov (United States)

    Marcelino, José Maria; Borrego, Pedro; Rocha, Cheila; Barroso, Helena; Quintas, Alexandre; Novo, Carlos; Taveira, Nuno

    2010-01-01

    Human immunodeficiency virus type 2 (HIV-2) infection affects about 1 to 2 million individuals, the majority living in West Africa, Europe, and India. As for HIV-1, new strategies for the prevention of HIV-2 infection are needed. Our aim was to produce new vaccine immunogens that elicit the production of broadly reactive HIV-2 neutralizing antibodies (NAbs). Native and truncated envelope proteins from the reference HIV-2ALI isolate were expressed in vaccinia virus or in bacteria. This source isolate was used due to its unique phenotype combining CD4 independence and CCR5 usage. NAbs were not elicited in BALB/c mice by single immunization with a truncated and fully glycosylated envelope gp125 (gp125t) or a recombinant polypeptide comprising the C2, V3, and C3 envelope regions (rpC2-C3). A strong and broad NAb response was, however, elicited in mice primed with gp125t expressed in vaccinia virus and boosted with rpC2-C3. Serum from these animals potently neutralized (median 50% neutralizing titer, 3,200) six of six highly divergent primary HIV-2 isolates. Coreceptor usage and the V3 sequence of NAb-sensitive isolates were similar to that of the vaccinating immunogen (HIV-2ALI). In contrast, NAbs were not reactive on three X4 isolates that displayed major changes in V3 loop sequence and structure. Collectively, our findings demonstrate that broadly reactive HIV-2 NAbs can be elicited by using a vaccinia virus vector-prime/rpC2-C3-boost immunization strategy and suggest a potential relationship between escape to neutralization and cell tropism. PMID:20844029

  3. An HLA-B27 Homodimer Specific Antibody Recognizes a Discontinuous Mixed-Disulfide Epitope as Identified by Affinity-Mass Spectrometry

    Science.gov (United States)

    Iuraşcu, Marius-Ionuţ; Marroquin Belaunzanar, Osiris; Cozma, Claudia; Petrausch, Ulf; Renner, Christoph; Przybylski, Michael

    2016-04-01

    HLA-B27 homodimer formation is believed to be a hallmark of HLA-B27 associated spondyloarthritides. Recently, we have generated a homodimer-specific monoclonal antibody (HD6) and have demonstrated that HLA-B27 homodimer complexes are present on monocytes of healthy HLA-B27 gene carriers at low levels, with significantly increased levels at active disease. The capability of the HD6 antibody to discriminate between correctly formed HLA-B27 heterotrimers and pathology-associated homodimers is striking and cannot be explained by the primary structure of HLA-B27. We hypothesized that HD6 accesses a unique epitope and used affinity-mass spectrometry for its identification. The HD6 antibody was immobilized on an activated sepharose affinity column, and HLA-B27 homodimer characterized for affinity. The epitope was identified by proteolytic epitope excision and MALDI mass spectrometry, and shown to comprise a discontinuous Cys-203- 257-Cys mixed-disulfide peptide structure that is not accessible in HLA-B27 heterotrimers due to protection by noncovalently linked β2-microglobulin. The epitope peptides were synthesized by solid phase peptide synthesis, and the two monomeric peptide components, HLA-B27(203-219) and HLA-B27(257-273), as well as the homo- and hetero-dimeric disulfide linked combinations prepared. The affinity binding constants KD towards the antibodies were determined using a surface acoustic wave (SAW) biosensor, and showed the highest affinity with a KD of approximately 40 nM to the HD6 antibody for the (203-219)-SS-(257-273) mixed disulfide epitope.

  4. An HLA-B27 Homodimer Specific Antibody Recognizes a Discontinuous Mixed-Disulfide Epitope as Identified by Affinity-Mass Spectrometry

    Science.gov (United States)

    Iuraşcu, Marius-Ionuţ; Marroquin Belaunzanar, Osiris; Cozma, Claudia; Petrausch, Ulf; Renner, Christoph; Przybylski, Michael

    2016-06-01

    HLA-B27 homodimer formation is believed to be a hallmark of HLA-B27 associated spondyloarthritides. Recently, we have generated a homodimer-specific monoclonal antibody (HD6) and have demonstrated that HLA-B27 homodimer complexes are present on monocytes of healthy HLA-B27 gene carriers at low levels, with significantly increased levels at active disease. The capability of the HD6 antibody to discriminate between correctly formed HLA-B27 heterotrimers and pathology-associated homodimers is striking and cannot be explained by the primary structure of HLA-B27. We hypothesized that HD6 accesses a unique epitope and used affinity-mass spectrometry for its identification. The HD6 antibody was immobilized on an activated sepharose affinity column, and HLA-B27 homodimer characterized for affinity. The epitope was identified by proteolytic epitope excision and MALDI mass spectrometry, and shown to comprise a discontinuous Cys-203- 257-Cys mixed-disulfide peptide structure that is not accessible in HLA-B27 heterotrimers due to protection by noncovalently linked β2-microglobulin. The epitope peptides were synthesized by solid phase peptide synthesis, and the two monomeric peptide components, HLA-B27(203-219) and HLA-B27(257-273), as well as the homo- and hetero-dimeric disulfide linked combinations prepared. The affinity binding constants KD towards the antibodies were determined using a surface acoustic wave (SAW) biosensor, and showed the highest affinity with a KD of approximately 40 nM to the HD6 antibody for the (203-219)-SS-(257-273) mixed disulfide epitope.

  5. The sulphation pattern in chondroitin sulphate chains investigated by chondroitinase ABC and ACII digestion and reactivity with monoclonal antibodies.

    Science.gov (United States)

    Hardingham, T E; Fosang, A J; Hey, N J; Hazell, P K; Kee, W J; Ewins, R J

    1994-03-01

    We have used progressive chondroitinase digestion of pig aggrecan in conjunction with ELISA assays and disaccharide analysis to derive information about the pattern of 4- and 6-sulphation in chondroitin sulphate chains. Digestion with chondroitinase ABC resulted in the release of mainly disaccharides from the nonreducing terminal of chondroitin sulphate chains but there was also the release of some tetra- and hexa-saccharides which were degraded to disaccharides with more extensive digestion. Chondroitinase ACII, in contrast, released only disaccharides. Analysis of the disaccharide composition of the intact and digested products at different stages of digestion showed that there was a slight increase in 6-sulphate content of the chains as they were shortened. Reaction of the partially digested proteoglycans with monoclonal antibodies 3-B-3 and 3-D-5 which recognise chains terminating in 6- or 4-sulphated disaccharides, respectively, showed major differences between chondroitinase ABC and ACII products. The results suggested that chondroitinase ABC preferentially cleaved next to 4-sulphated, rather than 6-sulphated disaccharides and this resulted in some oligosaccharides as well as disaccharide being released. Chondroitinase ACII also cleaved an additional disaccharide next to the linkage to protein of chondroitin sulphate, which was not removed by chondroitinase ABC and this disaccharide was mainly nonsulphated. PMID:7514097

  6. Outcome of asbestos exposure (lung fibrosis and antinuclear antibodies) with respect to skin reactivity: an 8-year longitudinal study

    International Nuclear Information System (INIS)

    Two hundred seventy asbestos workers were examined during an 8-year period. During this time five consecutive surveys were completed. Skin tests with streptokinase-streptodornase (SK-SD), tuberculin (PPD), and phytohemagglutinin (PHA) were performed in the middle of this period. The results of these tests were related to X-ray chest film results and the appearance of antinuclear antibodies (ANA). In all surveys, except the first, X-ray films with small irregular opacities with a profusion ≥1/1 belonged more frequently to asbestos workers who did not respond to SK-SD or PHA. Thirty-one cases of asbestosis were diagnosed at that time, 23 of them became asbestotic after the skin tests were performed. Asbestotic cases contributed more frequently to the group with energy as compared to asbestos workers lacking asbestosis. Furthermore, asbestosis was correlated with lack of response to second strength of SK-SD in males and PHA in both sexes. Lack of response to these activators was predictive of asbestosis. Asbestos workers with ANA frequently displayed a lack of response to the first strength of SK-SD, PPD, and PHA. This was partly due to the presence of asbestotic cases in the group. However, low responders to all these activators were found frequently in the group with ANA independent of the presence of asbestosis

  7. Impact of human leukocyte antigen matching and recipients' panel reactive antibodies on two-year outcome in presensitized renal allograft recipients

    Institute of Scientific and Technical Information of China (English)

    MENG Hui-lin; JIN Xun-bo; LI Xiang-tie; WANG Hong-wei; L(U) Jia-ju

    2009-01-01

    Background Renal transplantation in sensitized candidates remains a highly significant challenge worldwide. The production of panel reactive antibody (PRA) against human leukocyte antigen (HLA) is a major risk factor in presensitized recipients. The aim of this study was to evaluate the impact of HLA matching and recipients' PRA on two-year outcome in presensitized renal allograft recipients.Methods We determined the percentage of panel reactivity and specificity of anti-HLA immunoglobulin (Ig) G antibodies in 73 presensitized renal allograft recipients compared with 81 unsensitized recipients (control group). HLA genotyping of both recipients and corresponding donors was performed by PCR with sequence-specific primers (PCR-SSP). We analyzed the factors influencing the early graft outcome (two-year rejection rates and survival rates of the grafts), including HLA mismatching, class and degree of panel reactivity, and target antigen of donors.Results Presensitized recipients had a worse two-year outcome than unsensitized recipients (P=0.019 for rejection rate, P=0.01 for survival rate). The difference in number of HLA-mismatched alleles with either 6-antigen matching (Ag M) standard or amino acid residue matching (Res M) standard was not significant between the rejection and non-rejection groups of presensitized recipients or between the graft survival group and graft loss group. Compared with the control group, recipients with both PRA-Ⅰ and PRA-Ⅱ antibodies had a significantly worse two-year outcome (P=0.001 for rejection rate, P=0.002 for survival rate). The two-year outcomes of the peak PRA ≥50% group and its subgroup, at-transplant PRA ≥50% group, were significantly worse compared with the control group (P=0.025 and P=0.001 for rejection rate, P=0.043 and P=0.024 for survival rate). The rejection rates of the at-transplant target antigen positive group and its subgroup, HLA-Ⅰ target antigen positive group, were significantly higher than the control

  8. A single exposure to iron oxide nanoparticles attenuates antigen-specific antibody production and T-cell reactivity in ovalbumin-sensitized BALB/c mice

    Directory of Open Access Journals (Sweden)

    Shen CC

    2011-06-01

    Full Text Available Chien-Chang Shen1, Chia-Chi Wang1, Mei-Hsiu Liao2, Tong-Rong Jan11Department and Graduate Institute of Veterinary Medicine, School of Veterinary Medicine, National Taiwan University, Taipei, Taiwan; 2Division of Isotope Application, Institute of Energy Research, Taoyuan, TaiwanBackground: Superparamagnetic iron oxide nanoparticles have been used in clinical applications as a diagnostic contrasting agent. Previous studies showed that iron oxide nanoparticles deposited in the liver and spleen after systemic administration. The present study investigated the effect of iron oxide nanoparticles on antigen-specific immune responses in mice sensitized with the T cell-dependent antigen ovalbumin (OVA.Methods: BALB/c mice were intravenously administered with a single dose of iron oxide nanoparticles (10-60 mg Fe/kg 1 hour prior to OVA sensitization, and the serum antibody production and splenocyte reactivity were examined 7 days later.Results: The serum levels of OVA-specific IgG1 and IgG2a were significantly attenuated by treatment with iron oxide nanoparticles. The production of interferon-γ and interleukin-4 by splenocytes re-stimulated with OVA in culture was robustly suppressed in mice administered with iron oxide nanoparticles. The viability of OVA-stimulated splenocytes was also attenuated. In contrast, treatment with iron oxide nanoparticles did not affect the viability of splenocytes stimulated with concanavalin A, a T-cell mitogen.Conclusion: Collectively, these data indicate that systemic exposure to a single dose of iron oxide nanoparticles compromises subsequent antigen-specific immune reactions, including the serum production of antigen-specific antibodies, and the functionality of T cells.Keywords: iron oxide nanoparticle, antigen-specific, immune, ovalbumin

  9. Development of a 'mouse and human cross-reactive' affinity-matured exosite inhibitory human antibody specific to TACE (ADAM17) for cancer immunotherapy.

    Science.gov (United States)

    Kwok, Hang Fai; Botkjaer, Kenneth A; Tape, Christopher J; Huang, Yanchao; McCafferty, John; Murphy, Gillian

    2014-06-01

    We previously showed that a human anti-TACE antibody, D1(A12), is a potent inhibitor of TNF-α converting enzyme (TACE) ectodomain proteolysis and has pharmacokinetic properties suitable for studies of the inhibition of TACE-dependent growth factor shedding in relation to possible therapeutic applications. However, the lack of murine TACE immunoreactivity limits pre-clinical in vivo studies to human xenograft models which are poor analogies to in situ pathology and are not considered clinically predictive. Here, to overcome these limitations, we set out to develop a 'mouse and human cross-reactive' specific anti-TACE antibody. We first re-investigated the originally selected anti-TACE ectodomain phage-display clones, and isolated a lead 'mouse-human cross-reactive' anti-TACE scFv, clone A9. We reformatted scFv-A9 into an IgG2 framework for comprehensive biochemical and cellular characterization and further demonstrated that A9 is an exosite TACE inhibitor. However, surface plasmon resonance analysis and quenched-fluorescent (QF) peptide assay indicated that IgG reformatting of A9 caused low binding affinity and an 80-fold reduction in TACE ectodomain inhibition, severely limiting its efficacy. To address this, we constructed second generation phage-display randomization libraries focused on the complementarity-determining region 3, and carried out affinity selections shuffling between human and mouse TACE ectodomain as antigen in addition to an off-rate selection to increase the chance of affinity improvement. The bespoke 'three-step' selections enabled a 100-fold affinity enhancement of A9 IgG, and also improved its IC50 in a QF peptide assay to 0.2 nM. In human and mouse cancer cell assays, matured A9 IgG showed significant cell-surface TACE inhibition as a monotherapy or combination therapy with chemotherapeutic agent. Collectively, these data suggest that we successfully developed an exosite inhibitor of TACE with sub-nanomolar affinity, which possesses both

  10. Cross-Reactivity of Porcine Immunoglobulin A Antibodies with Fecal Immunoglobulins of Wild Boar (Sus scrofa) and Other Animal Species

    Science.gov (United States)

    Seo, Sang won; Yoo, Sung J.; Sunwoo, Sunyoung; Hyun, Bang hun

    2016-01-01

    Fecal samples obtained from wild boar habitats are useful for the surveillance of diseases in wild boar populations; however, it is difficult to determine the species of origin of feces collected in natural habitats. In this study, a fecal IgA ELISA was evaluated as a method for identifying the porcine species from fecal samples. Both domestic pigs (Sus scrofa domestica) and wild boars (Sus scrofa coreanus) showed significantly higher levels of fecal IgA than other animal species. Additionally, age dependent changes in the level of Ig A in wild boars and domestic pigs were identified; Titers of Ig A were highest in suckling period and lowest in weanling period. PMID:27340389

  11. Electrochemical investigations of the interaction of C-reactive protein (CRP) with a CRP antibody chemically immobilized on a gold surface

    International Nuclear Information System (INIS)

    A possibility of using a range of dc and ac electrochemical techniques to probe associative interactions of C-reactive protein (CRP) with CRP antibody (aCRP) immobilized on a gold electrode surface was investigated. It was demonstrated that the investigated electrochemical techniques can be used efficiently to probe these interactions over a wide CRP concentration range, from 1.15 x 10-5 to 1.15 mg L-1. The measured sensitivity of the techniques is in the following decreasing order: differential pulse voltammetry, charge-transfer resistance obtained from electrochemical impedance spectroscopy (EIS), cyclic voltammetry, chronoamperometry, and double-layer capacitance deduced from EIS measurements which gave the poorest sensitivity. Measurements of kinetic parameters demonstrated that the associative interactions of CRP with the immobilized aCRP reached quasi-equilibrium after 20-30 min. The kinetics of these interactions was modeled successfully using a two-step kinetic model. In this model, the first step represents reversible CRP-aCRP associative-dissociative interactions, while the second step represents the irreversible transformation of the bound CRP into a thermodynamically stable configuration. It was demonstrated that the thermodynamically stable configuration of CRP starts prevailing after 7 min of interaction of CRP with the immobilized aCRP.

  12. An amino-terminal segment of hantavirus nucleocapsid protein presented on hepatitis B virus core particles induces a strong and highly cross-reactive antibody response in mice

    International Nuclear Information System (INIS)

    Previously, we have demonstrated that hepatitis B virus (HBV) core particles tolerate the insertion of the amino-terminal 120 amino acids (aa) of the Puumala hantavirus nucleocapsid (N) protein. Here, we demonstrate that the insertion of 120 amino-terminal aa of N proteins from highly virulent Dobrava and Hantaan hantaviruses allows the formation of chimeric core particles. These particles expose the inserted foreign protein segments, at least in part, on their surface. Analysis by electron cryomicroscopy of chimeric particles harbouring the Puumala virus (PUUV) N segment revealed 90% T = 3 and 10% T = 4 shells. A map computed from T = 3 shells shows additional density splaying out from the tips of the spikes producing the effect of an extra shell of density at an outer radius compared with wild-type shells. The inserted Puumala virus N protein segment is flexibly linked to the core spikes and only partially icosahedrally ordered. Immunisation of mice of two different haplotypes (BALB/c and C57BL/6) with chimeric core particles induces a high-titered and highly cross-reactive N-specific antibody response in both mice strains

  13. Exome sequencing of extreme clopidogrel response phenotypes identifies B4GALT2 as a determinant of on-treatment platelet reactivity.

    Science.gov (United States)

    Scott, S A; Collet, J-P; Baber, U; Yang, Y; Peter, I; Linderman, M; Sload, J; Qiao, W; Kini, A S; Sharma, S K; Desnick, R J; Fuster, V; Hajjar, R J; Montalescot, G; Hulot, J-S

    2016-09-01

    Interindividual variability in platelet aggregation is common among patients treated with clopidogrel and both high on-treatment platelet reactivity (HTPR) and low on-treatment platelet reactivity (LTPR) increase risks for adverse clinical outcomes. CYP2C19 influences clopidogrel response but only accounts for ∼12% of the variability in platelet reactivity. To identify novel variants implicated in on-treatment platelet reactivity, patients with coronary artery disease (CAD) with extreme pharmacodynamic responses to clopidogrel and wild-type CYP2C19 were subjected to exome sequencing. Candidate variants that clustered in the LTPR subgroup subsequently were genotyped across the discovery cohort (n = 636). Importantly, carriers of B4GALT2 c.909C>T had lower on-treatment P2Y12 reaction units (PRUs; P = 0.0077) and residual platelet aggregation (P = 0.0008) compared with noncarriers, which remained significant after adjusting for CYP2C19 and other clinical variables in both the discovery (P = 0.0298) and replication (n = 160; PRU: P = 0.0001) cohorts. B4GALT2 is a platelet-expressed galactosyltransferase, indicating that B4GALT2 c.909C>T may influence clopidogrel sensitivity through atypical cell-surface glycoprotein processing and platelet adhesion. PMID:27213804

  14. Failed detection of Bovine viral diarrhea virus 2 subgenotype a (BVDV-2a) by direct fluorescent antibody test on tissue samples due to reduced reactivity of field isolates to raw anti-BVDV antibody.

    Science.gov (United States)

    Yan, Lifang; Pace, Lanny W; Baughman, Brittany; Wilson, Floyd D; Zhang, Shuping; Zhang, Michael Z

    2016-03-01

    Bovine viral diarrhea virus 1 (BVDV-1) is associated with mild or subclinical infections, whereas BVDV-2 is frequently implicated in outbreaks of severe thrombocytopenia and acute fatal disease. In the present study, the carcass of a beef breed cow and tissue samples of a beef calf were received for laboratory diagnosis. Both animals exhibited severe clinical signs compatible with thrombocytopenia or hemorrhagic syndrome. Direct fluorescent antibody test (DFAT) failed to detect BVDV antigen in the tissue specimens of both cases. However, immunohistochemistry (IHC) revealed the presence of BVDV antigen in oral and esophageal mucosa and Peyer patches of the beef breed cow. Real-time reverse transcription polymerase chain reaction (real-time RT-PCR) detected BVDV-2 in selected tissues of both animals. Subsequently, BVDV was isolated from both cases and subjected to genetic and serologic characterizations. Mutations in the 5'-untranslated genomic region (5'-UTR) primer and probe binding sites and the E2 gene were associated with reduced efficiency of an established real-time RT-PCR assay and amino acid alterations in the E2 glycoprotein, respectively. Both viral isolates were classified by real-time RT-PCR and phylogenetic analysis as BVDV-2 subgenotype a. Unlike BVDV reference strains Singer and 125c, the isolates cross-reacted with anti-BVDV-1 and anti-BVDV-2 reference sera, indicating antigenic variations in field isolates. The isolates also showed reduced reactivity to porcine anti-BVDV antiserum (the raw serum used to produce BVDV DFA conjugate). In summary, data from the present investigation indicated that genetic and antigenic variations affected the performance of detection assays, especially DFAT, highlighting the need for regular evaluation and modification of BVDV tests. PMID:26965235

  15. Radioimmunotargeting of malignant glioma by monoclonal antibody D2C7 reactive against both wild-type and variant III mutant epidermal growth factor receptors

    International Nuclear Information System (INIS)

    Introduction: Malignant glioma remains a significant therapeutic challenge, and immunotherapeutics might be a beneficial approach for these patients. A monoclonal antibody (MAb) specific for multiple molecular targets could expand the treatable patient population and the fraction of tumor cells targeted, with potentially increased efficacy. This motivated the generation of MAb D2C7, which recognizes both wild-type epidermal growth factor receptor (EGFRwt) and a tumor-specific mutant, EGFRvIII. Methods: D2C7 binding affinity was determined by surface plasmon resonance and its specificity characterized through comparison to EGFRwt-specific EGFR.1 and EGFRvIII-specific L8A4 MAbs by flow cytometry and immunohistochemical analysis. The three MAbs were labeled with 125I or 131I using Iodogen, and paired-label internalization assays and biodistribution experiments in athymic mice with human tumor xenografts were performed. Results: The affinity of D2C7 for EGFRwt and EGFRvIII was 5.2×109 M−1 and 3.6×109 M−1, and cell-surface reactivity with both receptors was documented by flow cytometry. Immunohistochemical analyses revealed D2C7 reactivity with malignant glioma tissue from 90 of 101 patients. Internalization assays performed on EGFRwt-expressing WTT cells and EGFRvIII-expressing NR6M cells indicated a threefold lower degradation of 125I-labeled D2C7 compared with 131I-labeled EGFR.1. Uptake of 125I-labeled D2C7 in NR6M xenografts (52.45±13.97 %ID g−1 on Day 3) was more than twice that of 131I-labeled L8A4; a threefold to fivefold tumor delivery advantage was seen when compared to 131I-labeled EGFR.1 in mice with WTT xenografts. Conclusions: These results suggest that D2C7 warrants further evaluation for the development of MAb-based therapeutics against cancers expressing EGFRwt and EGFRvIII.

  16. Antibodies to envelope glycoprotein of dengue virus during the natural course of infection are predominantly cross-reactive and recognize epitopes containing highly conserved residues at the fusion loop of domain II.

    Science.gov (United States)

    Lai, Chih-Yun; Tsai, Wen-Yang; Lin, Su-Ru; Kao, Chuan-Liang; Hu, Hsien-Ping; King, Chwan-Chuen; Wu, Han-Chung; Chang, Gwong-Jen; Wang, Wei-Kung

    2008-07-01

    The antibody response to the envelope (E) glycoprotein of dengue virus (DENV) is known to play a critical role in both protection from and enhancement of disease, especially after primary infection. However, the relative amounts of homologous and heterologous anti-E antibodies and their epitopes remain unclear. In this study, we examined the antibody responses to E protein as well as to precursor membrane (PrM), capsid, and nonstructural protein 1 (NS1) of four serotypes of DENV by Western blot analysis of DENV serotype 2-infected patients with different disease severity and immune status during an outbreak in southern Taiwan in 2002. Based on the early-convalescent-phase sera tested, the rates of antibody responses to PrM and NS1 proteins were significantly higher in patients with secondary infection than in those with primary infection. A blocking experiment and neutralization assay showed that more than 90% of anti-E antibodies after primary infection were cross-reactive and nonneutralizing against heterologous serotypes and that only a minor proportion were type specific, which may account for the type-specific neutralization activity. Moreover, the E-binding activity in sera of 10 patients with primary infection was greatly reduced by amino acid replacements of three fusion loop residues, tryptophan at position 101, leucine at position 107, and phenylalanine at position 108, but not by replacements of those outside the fusion loop of domain II, suggesting that the predominantly cross-reactive anti-E antibodies recognized epitopes involving the highly conserved residues at the fusion loop of domain II. These findings have implications for our understanding of the pathogenesis of dengue and for the future design of subunit vaccine against DENV as well. PMID:18448542

  17. Isolation of Potent CGRP Neutralizing Antibodies Using Four Simple Assays.

    Science.gov (United States)

    Neal, Frances; Arnold, Joanne; Rossant, Christine J; Podichetty, Sadhana; Lowne, David; Dobson, Claire; Wilkinson, Trevor; Colley, Caroline; Howes, Rob; Vaughan, Tristan J

    2016-01-01

    Calcitonin gene-related peptide (CGRP) is a small neuropeptide and a potent vasodilator that is widely associated with chronic pain and migraine. An antibody that inhibits CGRP function would be a potential therapeutic for treatment of these disorders. Here we describe the isolation of highly potent antibodies to CGRP from phage and ribosome display libraries and characterization of their epitope, species cross-reactivity, kinetics, and functional activity. Homogenous time-resolved fluorescence (HTRF) binding assays identified antibodies with the desired species cross-reactivity from naïve libraries, and HTRF epitope competition assays were used to characterize and group scFv by epitope. The functional inhibition of CGRP and species cross-reactivity of purified scFv and antibodies were subsequently confirmed using cAMP assays. We show that epitope competition assays could be used as a surrogate for functional cell-based assays during affinity maturation, in combination with scFv off-rate ranking by biolayer interferometry (BLI). This is the first time it has been shown that off-rate ranking can be predictive of functional activity for anti-CGRP antibodies. Here we demonstrate how, by using just four simple assays, diverse panels of antibodies to CGRP can be identified. These assay formats have potential utility in the identification of antibodies to other therapeutic targets. PMID:26450103

  18. Poly(3,4-ethylenedioxythiophene) Bearing Phosphorylcholine Groups for Metal-Free, Antibody-Free, and Low-Impedance Biosensors Specific for C-Reactive Protein.

    Science.gov (United States)

    Goda, Tatsuro; Toya, Masahiro; Matsumoto, Akira; Miyahara, Yuji

    2015-12-16

    Conducting polymers possessing biorecognition elements are essential for developing electrical biosensors sensitive and specific to clinically relevant biomolecules. We developed a new 3,4-ethylenedioxythiophene (EDOT) derivative bearing a zwitterionic phosphorylcholine group via a facile synthesis through the Michael-type addition thiol-ene "click" reaction for the detection of an acute-phase biomarker human C-reactive protein (CRP). The phosphorylcholine group, a major headgroup in phospholipid, which is the main constituent of plasma membrane, was also expected to resist nonspecific adsorption of other proteins at the electrode/solution interface. The biomimetic EDOT derivative was randomly copolymerized with EDOT, via an electropolymerization technique with a dopant sodium perchlorate, onto a glassy carbon electrode to make the synthesized polymer film both conductive and target-responsive. The conducting copolymer films were characterized by cyclic voltammetry, scanning electron microscopy, attenuated total reflection Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and electrochemical impedance spectroscopy. The specific interaction of CRP with phosphorylcholine in a calcium-containing buffer solution was determined by differential pulse voltammetry, which measures the altered redox reaction between the indicators ferricyanide/ferrocyanide as a result of the binding event. The conducting polymer-based protein sensor achieved a limit of detection of 37 nM with a dynamic range of 10-160 nM, covering the dynamically changing CRP levels in circulation during the acute phase. The results will enable the development of metal-free, antibody-free, and low-impedance electrochemical biosensors for the screening of nonspecific biomarkers of inflammation and infection. PMID:26588324

  19. Current trend of induction and maintenance treatment in positive panel-reactive antibody patients: a report on OPTN/UNOS kidney transplant registry data

    Institute of Scientific and Technical Information of China (English)

    Junchao Cai; Paul I. Terasaki

    2011-01-01

    Background The status of sensitization in kidney transplant recipients in the last 10 years and the trend of induction and maintenance therapy in patients of different panel-reactive antibody (PRA) levels have not been analyzed. The aim of this study was to investigate the current status of pre-transplant sensitization and its association with graft outcome.Methods A total of 155 570 kidney transplants reported to United Network for Organ Sharing (UNOS) during 2000-2009 were included in this study. We investigated the current status of pre-transplant sensitization and its association with graft outcome, and also compared the usage trend of 16 induction agents and 7 maintenance immunosuppressants in patients at different PRA levels. The difference of distributions of categorical variables between groups was investigated using the chi-square test. Unpaired t test or one-way analysis of variance (ANOVA) were used for numerical variables. The survival rates of transplant recipients were estimated using Kaplan-Meier methods and significance was determined by Log-rank test. Two-side P value <0.05 was considered statistically significant. All statistical analyses were performed using STATA 10 with all available updates as of March 2010 (StataCorp LP, College Station, Texas 77845, USA).Results Despite the fact of the decreased percentages of kidney transplant recipients with presensitization history, the mean PRA levels of all kidney recipients has been increasing in the last 7 years, which was possibly due to the introduction of more sensitive antibody testing techniques. The percentage of patients with treated rejection episodes within one year post-transplant were significantly higher in sensitized patients (PRA=50%-100%:14.3% and PRA=1%-49%:13.9%) than in non-sensitized patients (12.4%). Both 1- and 5-year graft survival rates improved in the last 10 years; this was more significant in high PRA patients. Thymoglobulin was the most commonly used induction agent in

  20. The evolution of food immune reactivity testing: why immunoglobulin G or immunoglobulin A antibody for food may not be reproducible from one lab to another.

    Science.gov (United States)

    Vojdani, Aristo

    2015-01-01

    The gold standard for identifying food reactions is the elimination-provocation diet. Embarking on this long, tedious journey takes an expert practitioner and a completely dedicated patient, with a whole lot of patience from both. In the contemporary fast lane, microwave, "give me a pill" popping, I-want-satisfaction-now society, many clinicians have turned to laboratory assessments for quick answers to food reactivity. From the introduction of cytotoxic testing for food allergies in 1947 until today, food reactivity testing has evolved and branched; it has been both pseudoimproved and scientifically improved. With multiple available options for methodology, specimen types, and clinical lab, how is a clinician expected to find the one that fits the requirements of a particular practice? How, indeed, when one self-promoting paper supports a particular methodology, while another criticizes it? In this article, with the benefit of his years of training and experience as a research scientist and test development expert, the author, who is trained in both microbiology and immunology, discusses the history of food testing, analyzes the criticisms of it, reviews the scientific literature, and tours the methodologies. PMID:25599182

  1. A novel stem cell associated marker identified by monoclonal antibody HESC5:3 differentiates between neoplastic lesions in follicular thyroid neoplasms.

    Science.gov (United States)

    Heikkilä, Annukka; Fermér, Christian; Hagström, Jaana; Louhimo, Johanna; Mäenpää, Hanna; Siironen, Päivi; Heiskanen, Ilkka; Nilsson, Olle; Arola, Johanna; Haglund, Caj

    2015-07-01

    Follicular thyroid lesions are the bane of cytopathology. Differentiation between adenoma and carcinoma is impossible, and often these neoplasms are indistinguishable even from uninodular goitre. In other cancers as well, a theory of stem cells as the origin of cancer has been discussed in thyroid carcinogenesis. We aimed to examine a novel stem cell associated marker identified by monoclonal antibody HESC5:3 in follicular lesions in an attempt to find a marker for differential diagnosis in thyroid cytopathology. HESC5:3 was raised against and is specific for undifferentiated human embryonic stem cells. The epitope of this novel antibody is to be defined. Immunohistochemical expression of HESC5:3 was examined in clinical material comprised of follicular neoplasms (83 adenomas, 43 carcinomas) and non-neoplastic lesions (41 goitrous, 22 hyperplastic, 23 normal tissue specimens). Staining differed significantly between neoplastic and non-neoplastic lesions. Nuclear staining was increased in non-neoplastic cells, whereas in neoplastic cells expression was mainly cytoplasmic. There was no difference between benign and malignant lesions, suggesting a role in early tumourigenesis. In conclusion, the HESC5:3 epitope may be of benefit as a neoplasia marker in distinguishing between uninodular goitre and neoplasia. Characterization of the epitope would increase the interest in this promising new stem cell associated marker. PMID:25960045

  2. Reactive Oxygen Species (ROS)-Activated ATM-Dependent Phosphorylation of Cytoplasmic Substrates Identified by Large-Scale Phosphoproteomics Screen.

    Science.gov (United States)

    Kozlov, Sergei V; Waardenberg, Ashley J; Engholm-Keller, Kasper; Arthur, Jonathan W; Graham, Mark E; Lavin, Martin

    2016-03-01

    Ataxia-telangiectasia, mutated (ATM) protein plays a central role in phosphorylating a network of proteins in response to DNA damage. These proteins function in signaling pathways designed to maintain the stability of the genome and minimize the risk of disease by controlling cell cycle checkpoints, initiating DNA repair, and regulating gene expression. ATM kinase can be activated by a variety of stimuli, including oxidative stress. Here, we confirmed activation of cytoplasmic ATM by autophosphorylation at multiple sites. Then we employed a global quantitative phosphoproteomics approach to identify cytoplasmic proteins altered in their phosphorylation state in control and ataxia-telangiectasia (A-T) cells in response to oxidative damage. We demonstrated that ATM was activated by oxidative damage in the cytoplasm as well as in the nucleus and identified a total of 9,833 phosphorylation sites, including 6,686 high-confidence sites mapping to 2,536 unique proteins. A total of 62 differentially phosphorylated peptides were identified; of these, 43 were phosphorylated in control but not in A-T cells, and 19 varied in their level of phosphorylation. Motif enrichment analysis of phosphopeptides revealed that consensus ATM serine glutamine sites were overrepresented. When considering phosphorylation events, only observed in control cells (not observed in A-T cells), with predicted ATM sites phosphoSerine/phosphoThreonine glutamine, we narrowed this list to 11 candidate ATM-dependent cytoplasmic proteins. Two of these 11 were previously described as ATM substrates (HMGA1 and UIMCI/RAP80), another five were identified in a whole cell extract phosphoproteomic screens, and the remaining four proteins had not been identified previously in DNA damage response screens. We validated the phosphorylation of three of these proteins (oxidative stress responsive 1 (OSR1), HDGF, and ccdc82) as ATM dependent after H2O2 exposure, and another protein (S100A11) demonstrated ATM

  3. Analysis of Platelet-Reactive Antibodies in Patients Who Were Refractory to Platelet Transfusions in Foshan Area%佛山地区血小板输注无效患者的血小板抗体分析

    Institute of Scientific and Technical Information of China (English)

    伍伟健; 王文敬; 卢瑾; 周健欣; 朱业华

    2014-01-01

    目的 探讨佛山地区血小板输注无效(PTR)患者血小板(PLT)抗体特异性、抗体阳性率及其相关因素,为制定PLT输注策略提供参考依据.方法 选择2012年1月至2013年8月,佛山地区5所医院收治的55例PTR患者作为研究对象(本研究遵循的程序符合各病例收集医院人体试验委员会制定的伦理学标准,得到该委员会批准,并征得受试对象的知情同意,并与之签订临床研究知情同意书).采用固相凝集法检测PTR患者血清中的PLT抗体.计算PLT同种抗体检出率和人类白细胞抗原(HLA)、人类血小板抗原(HPA)抗体阳性率,并分析年龄和输血次数对PLT抗体阳性率的影响.结果 PTR患者PLT同种抗体检出率为58.2% (32/55),其中同种HPA抗体阳性率为21.8% (12/55),同种HLA抗体阳性率为50.9%(28/55),抗-HLA占所有免疫性抗体的87.5% (28/32);在检出HPA抗体的患者中有66.7%(8/12)的患者同时检出HLA抗体.PLT自身抗体阳性率为7.3%(4/55).女性患者中PLT抗体阳性率为60.61%(20/33),略高于男性的54.5%(12/22),但两者比较差异无统计学意义(x2=0.01,P>0.05).PLT抗体阳性率随输血次数增加而呈增高趋势(x2趋势=13.14,P<0.05),以输血次数为4~6次时增幅最大.结论 佛山地区PTR患者中PLT同种抗体以HLA抗体多见,其次为HPA抗体,少数患者还存在PLT自身抗体;PLT同种抗体阳性率随输血次数增加呈增高趋势,而与患者性别无关.%Objective To detect and determine the correlation factors,specificity and positive rate of platelet (PLT)-reactive antibodies in patients who were refractory to platelet transfusions (RPT) in Foshan area,and to provide a reference basis for formulating marketing strategy of PLT transfusions.Methods From January 2012 to August 2013,55 RPT patients who were inspected by five different hospitals in Foshan area were included in this study.The study protocol was approved by the Ethical Review Board of

  4. The NTS-DBL2X region of VAR2CSA Induces cross-reactive antibodies that inhibit adhesion of several Plasmodium falciparum isolates to chondroitin sulfate A

    DEFF Research Database (Denmark)

    Bigey, Pascal; Gnidehou, Sédami; Doritchamou, Justin;

    2011-01-01

    -adapted parasite lines and field isolates expressing VAR2CSA. Competition enzyme-linked immunosorbent assay (ELISA) was employed to analyze functional resemblance between antibodies induced in animals and those naturally acquired by immune multigravidae. Results. Antibodies targeting the N-terminal sequence (NTS......) up to DBL2X (NTS-DBL2X) efficiently blocked parasite adhesion to chondroitin sulfate A in a manner similar to that of antibodies raised against the entire VAR2CSA extracellular domain. Interestingly, naturally acquired antibodies and those induced by vaccination against NTS-DBL2X target overlapping...

  5. Identification of anticitrullinated protein antibody reactivities in a subset of anti-CCP-negative rheumatoid arthritis: association with cigarette smoking and HLA-DRB1 ‘shared epitope’ alleles

    Science.gov (United States)

    Wagner, Catriona A; Sokolove, Jeremy; Lahey, Lauren J; Bengtsson, Camilla; Saevarsdottir, Saedis; Alfredsson, Lars; Delanoy, Michelle; Lindstrom, Tamsin M; Walker, Roger P; Bromberg, Reuven; Chandra, Piyanka E; Binder, Steven R; Klareskog, Lars; Robinson, William H

    2015-01-01

    Introduction A hallmark of rheumatoid arthritis (RA) is the development of autoantibodies targeting proteins that contain citrulline. Anticitrullinated protein antibodies (ACPAs) are currently detected by the commercial cyclic citrullinated peptide (CCP) assay, which uses a mix of cyclised citrullinated peptides as an artificial mimic of the true antigen(s). To increase the sensitivity of ACPA detection and dissect ACPA specificities, we developed a multiplex assay that profiles ACPAs by measuring their reactivity to the citrullinated peptides and proteins derived from RA joint tissue. Methods We created a bead-based, citrullinated antigen array to profile ACPAs. This custom array contains 16 citrullinated peptides and proteins detected in RA synovial tissues. We used the array to profile ACPAs in sera from a cohort of patients with RA and other non-inflammatory arthritides, as well as sera from an independent cohort of RA patients for whom data were available on carriage of HLA-DRB1 ‘shared epitope’ (SE) alleles and history of cigarette smoking. Results Our multiplex assay showed that at least 10% of RA patients who tested negative in the commercial CCP assay possessed ACPAs. Carriage of HLA-DRB1 SE alleles and a history of cigarette smoking were associated with an increase in ACPA reactivity—in anti-CCP+ RA and in a subset of anti-CCP− RA. Conclusions Our multiplex assay can identify ACPA-positive RA patients missed by the commercial CCP assay, thus enabling greater diagnostic sensitivity. Further, our findings suggest that cigarette smoking and possession of HLA-DRB1 SE alleles contribute to the development of ACPAs in anti-CCP− RA. PMID:24297382

  6. Human Papillomavirus neutralizing and cross-reactive antibodies induced in HIV-positive subjects after vaccination with quadrivalent and bivalent HPV vaccines

    DEFF Research Database (Denmark)

    Faust, Helena; Nielsen, Lars Toft; Sehr, Peter;

    2016-01-01

    Ninety-one HIV-infected individuals (61 men and 30 women) were randomized to vaccination either with quadrivalent (Gardasil™) or bivalent (Cervarix™) HPV vaccine. Neutralizing and specific HPV-binding serum antibodies were measured at baseline and 12 months after the first vaccine dose. Presence...... of neutralizing and binding antibodies had good agreement (average Kappa for HPV types 6, 11, 16, 18, 31, 33 and 45 was 0.65). At baseline, 88% of subjects had antibodies against at least one genital HPV. Following vaccination with Cervarix™, all subjects became seropositive for HPV16 and 18. After Gardasil......™ vaccination, 96% of subjects seroconverted for HPV16 and 73% for HPV18. Levels of HPV16-specific antibodies were 10IU in 85% of study subjects after vaccination. Antibodies against non-vaccine HPV types appeared after Gardasil...

  7. Antibody reactivity to conserved linear epitopes of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1)

    DEFF Research Database (Denmark)

    Staalsø, T; Khalil, E A; Elhassan, I M;

    1998-01-01

    The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family of protein antigens are involved in adhesion of P. falciparum infected erythrocytes to the capillary endothelium of the host. Antibodies to variable regions of these proteins, measured by agglutination, correlates with...... synthetic peptides derived from conserved regions of PfEMP1. The antibody responses to these peptides increased with age and were higher in individuals with asymptomatic P. falciparum infection compared to individuals presenting with fever attributable to falciparum malaria. This indicates that antibodies...... recognising the conserved regions of PfEMP1 arise upon exposure to the parasite, and that these may be involved in the development of protection against malaria. Antibodies to the Pfalhesin peptide of the human aniontransporter, band3, were measured by the same method. The magnitude of this antibody response...

  8. TCR-like antibodies distinguish conformational and functional differences in two vs. four-domain auto-reactive MHC II-peptide complexes

    Science.gov (United States)

    Dahan, Rony; Tabul, Moran; Chou, Yuan K.; Meza-Romero, Roberto; Andrew, Shayne; Ferro, Adolph J.; Burrows, Gregory G.; Offner, Halina; Vandenbark, Arthur A.; Reiter, Yoram

    2011-01-01

    SUMMARY Antigen presenting cell-associated four-domain MHC class-II molecules play a central role in activating autoreactive CD4+ T-cells involved in Multiple Sclerosis (MS) and Type 1 Diabetes (T1D). In contrast, two-domain MHC-II structures with the same covalently-attached self peptide (Recombinant T-cell receptor Ligands=RTLs) can regulate pathogenic CD4+ T-cells and reverse clinical signs of experimental autoimmune diseases. RTL1000, comprised of the β1α1 domains of HLA-DR2 linked to the encephalitogenic human MOG-35-55 peptide, was recently shown to be safe and well-tolerated in a Phase I clinical trial in MS. To evaluate the opposing biological effects of four- vs. two-domain class-II structures, we screened phage Fab antibodies (Abs) for neutralizing activity of RTL1000. . Five different TCR-like Abs were identified that could distinguish between the two- vs. four-domain MHC peptide complexes, while the cognate TCR was unable to make such a distinction. Moreover, Fab detection of native two-domain HLA-DR structures in human plasma implies that there are naturally-occurring regulatory MHC-peptide complexes. These results demonstrate for the first time distinct conformational determinants characteristic of activating vs. tolerogenic MHC-peptide complexes involved in human autoimmunity. PMID:21469129

  9. TCR-like antibodies distinguish conformational and functional differences in two- versus four-domain auto reactive MHC class II-peptide complexes.

    Science.gov (United States)

    Dahan, Rony; Tabul, Moran; Chou, Yuan K; Meza-Romero, Roberto; Andrew, Shayne; Ferro, Adolph J; Burrows, Gregory G; Offner, Halina; Vandenbark, Arthur A; Reiter, Yoram

    2011-05-01

    Antigen-presenting cell-associated four-domain MHC class II (MHC-II) molecules play a central role in activating autoreactive CD4(+) T cells involved in multiple sclerosis (MS) and type 1 diabetes (T1D). In contrast, two-domain MHC-II structures with the same covalently attached self-peptide (recombinant T-cell receptor ligands (RTLs)) can regulate pathogenic CD4(+) T cells and reverse clinical signs of experimental autoimmune diseases. RTL1000, which is composed of the β1α1 domains of human leukocyte antigen (HLA)-DR2 linked to the encephalitogenic human myelin oligodendrocyte glycoprotein (MOG)-35-55 peptide, was recently shown to be safe and well tolerated in a phase I clinical trial in MS. To evaluate the opposing biological effects of four- versus two-domain MHC-II structures, we screened phage Fab antibodies (Abs) for the neutralizing activity of RTL1000. Five different TCR-like Abs were identified that could distinguish between the two- versus four-domain MHC-peptide complexes while the cognate TCR was unable to make such a distinction. Moreover, Fab detection of native two-domain HLA-DR structures in human plasma implies that there are naturally occurring regulatory MHC-peptide complexes. These results demonstrate for the first time distinct conformational determinants characteristic of activating versus tolerogenic MHC-peptide complexes involved in human autoimmunity. PMID:21469129

  10. Serum strain-specific or cross-reactive neuraminidase inhibiting antibodies against pandemic А/California/07/2009(H1N1) influenza in healthy volunteers

    OpenAIRE

    Desheva, Yulia A; Smolonogina, Tatiana A; Donina, Svetlana A; Rudenko, Larisa G

    2015-01-01

    Background Pre-existing antibodies to influenza virus neuraminidase may provide protection against infection influenza viruses containing novel hemagglutinin (HA). The aim of our study was to evaluate serum neuraminidase-inhibiting (NI) antibodies against А/California/07/2009(H1N1) [H1N1/2009pdm] and А/New Caledonia/20/1999(H1N1) [H1N1/1999] influenza viruses in relation with the age of participants and hemagglutination-inhibition (HI) antibody levels. Anti-H1N1/2009pdm neuraminidase and anti...

  11. Mapping the binding site of a cross-reactive Plasmodium falciparum PfEMP1 monoclonal antibody inhibitory of ICAM-1 binding

    DEFF Research Database (Denmark)

    Lennartz, Frank; Bengtsson, Anja; Olsen, Rebecca W;

    2015-01-01

    The virulence of Plasmodium falciparum is linked to the ability of infected erythrocytes (IE) to adhere to the vascular endothelium, mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1). In this article, we report the functional characterization of an mAb that recognizes a panel of PfEMP......1s and inhibits ICAM-1 binding. The 24E9 mouse mAb was raised against PFD1235w DBLβ3_D4, a domain from the group A PfEMP1s associated with severe malaria. 24E9 recognizes native PfEMP1 expressed on the IE surface and shows cross-reactivity with and cross-inhibition of the ICAM-1 binding capacity of...... domain cassette 4 PfEMP1s. 24E9 Fab fragments bind DBLβ3_D4 with nanomolar affinity and inhibit ICAM-1 binding of domain cassette 4-expressing IE. The antigenic regions targeted by 24E9 Fab were identified by hydrogen/deuterium exchange mass spectrometry and revealed three discrete peptides that are...

  12. Development of a monoclonal antibody-based broad-specificity ELISA for fluroquinolone antibiotics in foods and molecular modeling studies of cross-reactive compounds

    Science.gov (United States)

    Development of a competitive indirect enzyme-linked immunosorbent assay (ciELISA) with monoclonal antibodies (Mabs) having broad specificity for fluoroquinolone (FQ) antibiotics is described. Four FQs, ciprofloxacin (CIP), norfloxacin (NOR), enrofloxacin (ENR) and ofloxacin (OFL) were conjugated to...

  13. Prevalence and chemotherapy-induced reactivation of occult hepatitis B virus among hepatitis B surface antigen negative patients with diffuse large B-cell lymphoma: Significance of hepatitis B core antibodies screening

    International Nuclear Information System (INIS)

    Background: Occult hepatitis B infection (OBI) is characterized by negative hepatitis B surface antigen (HBsAg) and detectable hepatitis B virus (HBV)-DNA in the liver and/or serum, with or without hepatitis B core antibody (anti-HBc). Anti-HBc is the most sensitive marker of previous HBV. HBV reactivation in patients under immunosuppressive treatment is life-threatening, occurring in both overt and occult HBV especially in hematological malignancies. Aim of the work: To evaluate the prevalence and chemotherapy-induced reactivation of OBI among hepatitis B surface antigen negative patients with diffuse large B-cell lymphoma (DLBCL) patients and to determine the significance of anti-HBc screening among this group of patients before receiving chemotherapy. Patients and methods: This cross-sectional study included 72 DLBCL patients negative for HBsAg, HBsAb and hepatitis C virus antibodies (anti-HCV). Patients were subjected to investigations including anti-HBc. All patients underwent alanine transaminase (ALT) monitoring before each cycle of chemotherapy and monthly for 12 months after the end of chemotherapy. Patients with suspected OBI were tested for HBV-DNA using real-time polymerase chain reaction (PCR). Results: Anti-HBc was detected in 10 of 72 HBsAg negative sera (13.89%) (95% confidence interval 6.9-22.2%). Five of the 10 anti-HBc positive patients in this study had OBI reactivation. Conclusion: The study concluded that anti-HBc screening is mandatory before chemotherapy. HBsAg-negative/anti-HBc-positive patients should be closely observed for signs of HBV reactivation through the regular monitoring of ALT. Prophylaxis lamivudine is recommended for anti-HBc positive patients before chemotherapy.

  14. Development of antibody against sulfamethazine

    International Nuclear Information System (INIS)

    Sulfamethazine (SMT) is widely used to treat bacterial and protozoan infections in food animals. So its residue has been detected in various food products, and in Europe, the tolerance level for sulfonamides in meat and milk is 100 ng/g. To ensure that residues in animal food products do not exceed this limit, a simple, sensitive, and rapid method to determinate their residues in animal tissues is needed. In this paper the development of polyclonal or monoclonal antibodies against sulfamethazine (SMT) and a simplified method to identify residual sulfamethazine by radio immunoassay (RIA) is presented. Polyclonal antibodies (PcAbs) against sulfamethazine (SMT) were obtained by immunizing rabbits with SMT-conjugated bovine serum albumin (BSA). The association constants (Ka) of the PcAbs were higher than 108 and the cross-reactivities with Sulfadiazine(SD), Sulfaquinoxaline(SQX) which were structurally related compounds were lower than 0.05%(RIA). Simultaneous, six strains of hybridoma cell were prepared which can secrete monoclonal antibodies (McAbs) against SMT . The Ka of the McAbs against SMT were higher than 107 and the cross-reactivities with SD, SQX were lower than 0.1%(RIA). (authors)

  15. Generation of a Novel Bacteriophage Library Displaying scFv Antibody Fragments from the Natural Buffalo Host to Identify Antigens from Adult Schistosoma japonicum for Diagnostic Development.

    Directory of Open Access Journals (Sweden)

    Christopher G Hosking

    2015-12-01

    Full Text Available The development of effective diagnostic tools will be essential in the continuing fight to reduce schistosome infection; however, the diagnostic tests available to date are generally laborious and difficult to implement in current parasite control strategies. We generated a series of single-chain antibody Fv domain (scFv phage display libraries from the portal lymph node of field exposed water buffaloes, Bubalus bubalis, 11-12 days post challenge with Schistosoma japonicum cercariae. The selected scFv-phages showed clear enrichment towards adult schistosomes and excretory-secretory (ES proteins by immunofluorescence, ELISA and western blot analysis. The enriched libraries were used to probe a schistosome specific protein microarray resulting in the recognition of a number of proteins, five of which were specific to schistosomes, with RNA expression predominantly in the adult life-stage based on interrogation of schistosome expressed sequence tags (EST. As the libraries were enriched by panning against ES products, these antigens may be excreted or secreted into the host vasculature and hence may make good targets for a diagnostic assay. Further selection of the scFv library against infected mouse sera identified five soluble scFv clones that could selectively recognise soluble whole adult preparations (SWAP relative to an irrelevant protein control (ovalbumin. Furthermore, two of the identified scFv clones also selectively recognised SWAP proteins when spiked into naïve mouse sera. These host B-cell derived scFvs that specifically bind to schistosome protein preparations will be valuable reagents for further development of a cost effective point-of-care diagnostic test.

  16. Generation of a Novel Bacteriophage Library Displaying scFv Antibody Fragments from the Natural Buffalo Host to Identify Antigens from Adult Schistosoma japonicum for Diagnostic Development.

    Science.gov (United States)

    Hosking, Christopher G; McWilliam, Hamish E G; Driguez, Patrick; Piedrafita, David; Li, Yuesheng; McManus, Donald P; Ilag, Leodevico L; Meeusen, Els N T; Veer, Michael J de

    2015-12-01

    The development of effective diagnostic tools will be essential in the continuing fight to reduce schistosome infection; however, the diagnostic tests available to date are generally laborious and difficult to implement in current parasite control strategies. We generated a series of single-chain antibody Fv domain (scFv) phage display libraries from the portal lymph node of field exposed water buffaloes, Bubalus bubalis, 11-12 days post challenge with Schistosoma japonicum cercariae. The selected scFv-phages showed clear enrichment towards adult schistosomes and excretory-secretory (ES) proteins by immunofluorescence, ELISA and western blot analysis. The enriched libraries were used to probe a schistosome specific protein microarray resulting in the recognition of a number of proteins, five of which were specific to schistosomes, with RNA expression predominantly in the adult life-stage based on interrogation of schistosome expressed sequence tags (EST). As the libraries were enriched by panning against ES products, these antigens may be excreted or secreted into the host vasculature and hence may make good targets for a diagnostic assay. Further selection of the scFv library against infected mouse sera identified five soluble scFv clones that could selectively recognise soluble whole adult preparations (SWAP) relative to an irrelevant protein control (ovalbumin). Furthermore, two of the identified scFv clones also selectively recognised SWAP proteins when spiked into naïve mouse sera. These host B-cell derived scFvs that specifically bind to schistosome protein preparations will be valuable reagents for further development of a cost effective point-of-care diagnostic test. PMID:26684756

  17. Detection of Campylobacter species using monoclonal antibodies

    Science.gov (United States)

    Young, Colin R.; Lee, Alice; Stanker, Larry H.

    1999-01-01

    A panel of species specific monoclonal antibodies were raised to Campylobacter coli, Campylobacter jejuni and Campylobacter lari. The isotypes, and cross-reactivity profiles of each monoclonal antibody against an extensive panel of micro- organisms, were determined.

  18. Fusion loop peptide of the West Nile virus envelope protein is essential for pathogenesis and recognized by a therapeutic cross-reactive human monoclonal antibody

    OpenAIRE

    Sultana, Hameeda; Foellmer, Harald G.; Neelakanta, Girish; Oliphant, Theodore; Engle, Michael; Ledizet, Michel; Krishnan, Manoj; Bonafe, Nathalie; Anthony, Karen G.; Marasco, Wayne A.; Kaplan, Paul; Montgomery, Ruth R.; Diamond, Michael S.; Koski, Raymond A.; Fikrig, Erol

    2009-01-01

    West Nile virus is an emerging pathogen that can cause fatal neurological disease. A recombinant human monoclonal antibody, mAb11, has been described as a candidate for the prevention and treatment of West Nile disease. Using a yeast surface display epitope mapping assay and neutralization escape mutant, we show that mAb11 recognizes the fusion loop, at the distal end of domain II of the West Nile virus envelope protein. Antibody mAb11 cross-reacts with all four dengue viruses and provides pr...

  19. Thyroid Antibodies

    Science.gov (United States)

    ... be limited. Home Visit Global Sites Search Help? Thyroid Antibodies Share this page: Was this page helpful? Also known as: Thyroid Autoantibodies; Antithyroid Antibodies; Antimicrosomal Antibody; Thyroid Microsomal Antibody; ...

  20. Sensitization Prevalence, Antibody Cross-Reactivity and Immunogenic Peptide Profile of Api g 2, the Non-Specific Lipid Transfer Protein 1 of Celery

    OpenAIRE

    Gadermaier, G.; Hauser, M.; Egger, M.; Ferrara, R; Briza, P.; Santos, K.S.; Zennaro, D.; Girbl, T.; Zuidmeer-Jongejan, L.; Mari, A.; F. Ferreira

    2011-01-01

    Background: Celery (Apium graveolens) represents a relevant allergen source that can elicit severe reactions in the adult population. To investigate the sensitization prevalence and cross-reactivity of Api g 2 from celery stalks in a Mediterranean population and in a mouse model. Methodology: 786 non-randomized subjects from Italy were screened for IgE reactivity to rApi g 2, rArt v 3 (mugwort pollen LTP) and nPru p 3 (peach LTP) using an allergen microarray. Clinical data of 32 selected pati...

  1. SERUM HIGH SENSITIVE C-REACTIVE PROTEIN AND ANTICARDIOLIPIN ANTIBODY LEVEL IN CAD PATIENTS WITH AND WITHOUT TYPE 2 DIABETES MELLITUS.

    Directory of Open Access Journals (Sweden)

    Sachu Philip

    2012-08-01

    Full Text Available The increased incidence of coronary artery disease (CAD in Type 2 Diabetes mellitus is not fully explained by the conventional risk factors. Our aim was to determine the association of biomarkers, high sensitive CRP and anticardiolipin antibody (acL with severity of coronary artery disease in patients with and without type 2 DM. In our study, hsCRP level was significantly high in CAD with DM and found to be positively correlated with severity (p<0.01 while anticardiolipin antibody does not show any significant change among the two groups. Our study concluded that increased risk of CAD in type 2 DM patients is not only because of dyslipidemia but inflammatory events also play a major role. hsCRP was found to be a valuable predictor for CAD in type 2 DM.

  2. The Use of Chimeric Virus-like Particles Harbouring a Segment of Hantavirus Gc Glycoprotein to Generate a Broadly-Reactive Hantavirus-Specific Monoclonal Antibody

    OpenAIRE

    Aurelija Zvirbliene; Indre Kucinskaite-Kodze; Ausra Razanskiene; Rasa Petraityte-Burneikiene; Boris Klempa; Ulrich, Rainer G.; Alma Gedvilaite

    2014-01-01

    Monoclonal antibodies (MAbs) against viral glycoproteins have important diagnostic and therapeutic applications. In most cases, the MAbs specific to viral glycoproteins are raised against intact virus particles. The biosynthesis of viral glycoproteins in heterologous expression systems such as bacteria, yeast, insect or mammalian cells is often problematic due to their low expression level, improper folding and limited stability. To generate MAbs against hantavirus glycoprotein Gc, we have us...

  3. [Antibody therapy for Alzheimer's disease].

    Science.gov (United States)

    Tabira, Takeshi; Matsumoto, Shin-Ei; Jin, Haifeng

    2011-11-01

    In order to avoid Abeta-induced autoimmune encephalitis, several monoclonal and polyclonal antibodies are in clinical trials. These are bapineuzumab, solanezumab, ponezumab, gantenerumab, BAN2401, gammaguard and octagam. Since each antibody has a different antigen epitope of Abeta, anti-amyloid activities are different. It is unknown which antibody is effective for Alzheimer disease, and we must wait for the result of clinical trials. Some patients who developed tissue amyloid plaque immuno-reactive (TAPIR) antibody showed slower decline after AN-1792 vaccination. We developed TAPIR-like monoclonal antibody, which was found to react with Abeta oligomers preferentially. PMID:22277519

  4. Conformational epitopes of myelin oligodendrocyte glycoprotein are targets of potentially pathogenic antibody responses in multiple sclerosis

    OpenAIRE

    Menge Til; Lalive Patrice H; von Büdingen H -Christian; Genain Claude P

    2011-01-01

    Abstract Background Myelin/oligodendrocyte glycoprotein (MOG) is a putative autoantigen in multiple sclerosis (MS). Establishing the pathological relevance and validity of anti-MOG antibodies as biomarkers has yielded conflicting reports mainly due to different MOG isoforms used in different studies. Because epitope specificity may be a key factor determining anti-MOG reactivity we aimed at identifying a priori immunodominant MOG epitopes by monoclonal antibodies (mAbs) and at assessing clini...

  5. Use of flow cytometry to identify monoclonal antibodies that recognize conserved epitopes on orthologous leukocyte differentiation antigens in goats, llamas, and rabbits

    Czech Academy of Sciences Publication Activity Database

    Davis, W. C.; Drbal, Karel; El-Aziz, A.; Mosaad, A.E.; Elbagory, A.R.M.; TIbary, A.; Barrington, G.M.; Park, Y.H.; Hamilton, M.J.

    2007-01-01

    Roč. 119, 1-2 (2007), s. 123-130. ISSN 0165-2427 Institutional research plan: CEZ:AV0Z50520514 Keywords : flow cytometry * monoclonal antibodies * leukocytes Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.957, year: 2007

  6. Neuronal Antibody Biomarkers for Sydenham’s Chorea Identify a New Group of Children with Chronic Recurrent Episodic Acute Exacerbations of Tic and Obsessive Compulsive Symptoms Following a Streptococcal Infection

    Science.gov (United States)

    Singer, Harvey S.; Mascaro-Blanco, Adda; Alvarez, Kathy; Morris-Berry, Christina; Kawikova, Ivana; Ben-Pazi, Hilla; Thompson, Carol B.; Ali, Syed F.; Kaplan, Edward L.; Cunningham, Madeleine W.

    2015-01-01

    Several autoantibodies (anti-dopamine 1 (D1R) and 2 (D2R) receptors, anti-tubulin, anti-lysoganglioside-GM1) and antibody-mediated activation of calcium calmodulin dependent protein kinase II (CaMKII) signaling activity are elevated in children with Sydenham’s chorea (SC). Recognizing proposed clinical and autoimmune similarities between SC and PANDAS (pediatric autoimmune neuropsychiatric disorder associated with a streptococcal infection), we sought to identify serial biomarker changes in a slightly different population. Antineuronal antibodies were measured in eight children (mean 11.3 years) with chronic, dramatic, recurrent tics and obsessive-compulsive disorder (OCD) associated with a group A β-hemolytic streptococcal (GABHS) respiratory tract infection, but differing because they lacked choreiform movements. Longitudinal serum samples in most subjects included two pre-exacerbation samples, Exac), one midst Exac (abrupt recurrence of tic/OCD; temporally association with a GABHS infection in six of eight subjects), and two post-Exac. Controls included four groups of unaffected children (n = 70; mean 10.8 years) obtained at four different institutions and published controls. Clinical exacerbations were not associated with a significant rise in antineuronal antibody titers. CaMKII activation was increased at the GABHS exacerbation point in 5/6 subjects, exceeded combined and published control’s 95th percentile at least once in 7/8 subjects, and median values were elevated at each time point. Anti-tubulin and anti-D2R titers did not differ from published or combined control group’s 95th percentile or median values. Differences in anti-lysoganglioside-GM1 and anti-D1R titers were dependent on the selected control. Variances in antibody titers and CaMKII activation were identified among the institutional control groups. Based on comparisons to published studies, results identify two groups of PANDAS: 1) a cohort, represented by this study, which lacks

  7. Identification and in vitro reactivity of celiac immunoactive peptides in an apparent gluten-free beer.

    Directory of Open Access Journals (Sweden)

    Ana Real

    Full Text Available Gluten content from barley, rye, wheat and in certain oat varieties, must be avoid in individuals with celiac disease. In most of the Western countries, the level of gluten content in food to be considered as gluten-free products is below 20 parts per million measured by ELISA based on specific anti-gluten peptide antibody. However, in beverages or food suffering complex hydrolytic processes as beers, the relative proportion of reactive peptides for celiac patients and the analytical techniques may differ, because of the diversity of the resulting peptide populations after fermentations. A beer below 20 parts per million of gluten but yet detectable levels of gluten peptides by anti-gliadin 33-mer antibodies (G12 and A1 was analyzed. We identified and characterized the relevant peptides for either antibody recognition or immunoactivity in celiac patients. The beer was fractionated by HPLC. The relative reactivity of the different HPLC fractions to the G12/A1 antibodies correlated to the reactivity of peripheral blood mononuclear cells isolated from 14 celiac individuals. Peptides from representative fractions classified according to the relative reactivity to G12/A1 antibodies were identified by mass spectrometry. The beer peptides containing sequences with similarity to those of previously described G12 and A1 epitopes were synthesized and confirmed significant reactivity for the antibodies. The most reactive peptides for G12/A1 also confirmed the highest immunogenicity by peripheral blood mononuclear cell activation and interferon γ production from celiac patients. We concluded that preparative HPLC combined with anti-gliadin 33-mer G12/A1 antibodies were very sensitive and specific methods to analyze the relevant immunogenic peptides in hydrolyzed gluten.

  8. Most Anti-BrdU Antibodies React with 2′-Deoxy-5-Ethynyluridine — The Method for the Effective Suppression of This Cross-Reactivity

    Czech Academy of Sciences Publication Activity Database

    Liboska, Radek; Ligasová, Anna; Strunin, Dmytro; Rosenberg, Ivan; Koberna, Karel

    2012-01-01

    Roč. 7, č. 12 (2012), e51679/1-e51679/10. E-ISSN 1932-6203 R&D Projects: GA AV ČR KAN200520801; GA ČR GBP302/12/G157 Grant ostatní: GA ČR(CZ) GA204/09/0973 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50520514; CEZ:AV0Z50040702 Keywords : 2´-deoxy-5-ethynyluridine * 5-bromo-2´-deoxyuridine * DNA replication * anti-BrdU antibodies * immunocytochemistry Subject RIV: CC - Organic Chemistry Impact factor: 3.730, year: 2012

  9. Immunoglobulin G antibody reactivity to a group A Plasmodium falciparum erythrocyte membrane protein 1 and protection from P. falciparum malaria

    DEFF Research Database (Denmark)

    Magistrado, Pamela A; Lusingu, John; Vestergaard, Lasse S;

    2007-01-01

    Variant surface antigens (VSA) on the surface of Plasmodium falciparum-infected red blood cells play a major role in the pathogenesis of malaria and are key targets for acquired immunity. The best-characterized VSA belong to the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family. In areas...... where P. falciparum is endemic, parasites causing severe malaria and malaria in young children with limited immunity tend to express semiconserved PfEMP1 molecules encoded by group A var genes. Here we investigated antibody responses of Tanzanians who were 0 to 19 years old to PF11_0008, a group A PfEMP...

  10. Engineered Antibodies for Monitoring of Polynuclear Aromatic Hydrocarbons

    Energy Technology Data Exchange (ETDEWEB)

    Alexander E. Karu Ph.D; Victoria A. Roberts Ph.D.; Qing X. Li, Ph.D.

    2002-01-17

    This project was undertaken to fill needs in ODE's human and ecosystem health effects research, site remediation, rapid emergency response, and regulatory compliance monitoring programs. Doe has greatly stimulated development and validation of antibody-based, rapid, field-portable detection systems for small hazardous compounds. These range from simple dipsticks, microplate enzyme-linked immunosorbent assays (ELISAs), and hand-held colorimeters, to ultrasensitive microfluidic reactors, fiber-optic sensors and microarrays that can identify multiple analytes from patterns of cross-reactivity. Unfortunately, the technology to produce antibodies with the most desirable properties did not keep pace. Lack of antibodies remains a limiting factor in production and practical use of such devices. The goals of our project were to determine the chemical and structural bases for the antibody-analyte binding interactions using advanced computational chemistry, and to use this information to create useful new binding properties through in vitro genetic engineering and combinatorial library methods.

  11. Reactive Transport Modeling of Chemical and Isotope Data to Identify Degradation Processes of Chlorinated Ethenes in a Diffusion-Dominated Media

    Science.gov (United States)

    Chambon, J. C.; Damgaard, I.; Jeannottat, S.; Hunkeler, D.; Broholm, M. M.; Binning, P. J.; Bjerg, P. L.

    2012-12-01

    Chlorinated ethenes are among the most widespread contaminants in the subsurface and a major threat to groundwater quality at numerous contaminated sites. Many of these contaminated sites are found in low-permeability media, such as clay tills, where contaminant transport is controlled by diffusion. Degradation and transport processes of chlorinated ethenes are not well understood in such geological settings, therefore risk assessment and remediation at these sites are particularly challenging. In this work, a combined approach of chemical and isotope analysis on core samples, and reactive transport modeling has been used to identify the degradation processes occurring at the core scale. The field data was from a site located at Vadsby, Denmark, where chlorinated solvents were spilled during the 1960-70's, resulting in contamination of the clay till and the underlying sandy layer (15 meters below surface). The clay till is heavily contaminated between 4 and 15 mbs, both with the mother compounds PCE/TCE and TCA and the daughter products (DCE, VC, ethene, DCA), indicating the occurrence of natural dechlorination of both PCE/TCE and TCA. Intact core samples of length 0.5m were collected from the source zone (between 6 and 12 mbs). Concentrations and stable isotope ratios of the mother compounds and their daughter products, as well as redox parameters, fatty acids and microbial data, were analyzed with discrete sub-sampling along the cores. More samples (each 5 mm) were collected around the observed higher permeability zones such as sand lenses, sand stringers and fractures, where a higher degradation activity was expected. This study made use of a reactive transport model to investigate the appropriateness of several conceptual models. The conceptual models considered the location of dechlorination and degradation pathways (biotic reductive dechlorination or abiotic β-elimination with iron minerals) in three core profiles. The model includes diffusion in the matrix

  12. Multiple antibody targets on herpes B glycoproteins B and D identified by screening sera of infected rhesus macaques with peptide microarrays.

    Directory of Open Access Journals (Sweden)

    Sven-Kevin Hotop

    Full Text Available Herpes B virus (or Herpesvirus simiae or Macacine herpesvirus 1 is endemic in many populations of macaques, both in the wild and in captivity. The virus elicits only mild clinical symptoms (if any in monkeys, but can be transmitted by various routes, most commonly via bites, to humans where it causes viral encephalitis with a high mortality rate. Hence, herpes B constitutes a considerable occupational hazard for animal caretakers, veterinarians and laboratory personnel. Efforts are therefore being made to reduce the risk of zoonotic infection and to improve prognosis after accidental exposure. Among the measures envisaged are serological surveillance of monkey colonies and specific diagnosis of herpes B zoonosis against a background of antibodies recognizing the closely related human herpes simplex virus (HSV. 422 pentadecapeptides covering, in an overlapping fashion, the entire amino acid sequences of herpes B proteins gB and gD were synthesized and immobilized on glass slides. Antibodies present in monkey sera that bind to subsets of the peptide collection were detected by microserological techniques. With 42 different rhesus macaque sera, 114 individual responses to 18 different antibody target regions (ATRs were recorded, 17 of which had not been described earlier. This finding may pave the way for a peptide-based, herpes B specific serological diagnostic test.

  13. In vivo biotinylation of the vasculature in B-cell lymphoma identifies BST-2 as a target for antibody-based therapy.

    Science.gov (United States)

    Schliemann, Christoph; Roesli, Christoph; Kamada, Haruhiko; Borgia, Beatrice; Fugmann, Tim; Klapper, Wolfram; Neri, Dario

    2010-01-21

    The discovery of accessible markers of lymphoma may facilitate the development of antibody-based therapeutic strategies. Here, we describe the results of a chemical proteomic study, based on the in vivo biotinylation of vascular proteins in lymphoma-bearing mice followed by mass spectrometric and bioinformatic analysis, to discover proteins expressed at the tissue-blood border of disseminated B-cell lymphoma. From a list of 58 proteins, which were more than 10-fold up-regulated in nodal and extranodal lymphoma lesions compared with their levels in the corresponding normal host organs, we validated BST-2 as a novel vascular marker of B-cell lymphoma, using immunochemical techniques and in vivo biodistribution studies. Furthermore, targeting BST-2 with 2 independent monoclonal antibodies delayed lymphoma growth in a syngeneic mouse model of the disease. The results of this study delineate a strategy for the treatment of systemic B-cell lymphoma in humans and suggest that anti-BST-2 antibodies may facilitate pharmacodelivery approaches that target the tumor-stroma interface. PMID:19903902

  14. Production and characterization of monoclonal antibodies against mink leukocytes

    DEFF Research Database (Denmark)

    Chen, W.S.; Pedersen, Mikael; Gram-Nielsen, S.;

    1997-01-01

    Three monoclonal antibodies (mAbs) were generated against mink leukocytes. One antibody reacted with all T lymphocytes, one with all monocytes and one had platelet reactivity. Under reducing conditions, the T lymphocyte reactive antibody immunoprecipitated 18 kDa, 23 kDa, 25 kDa and 32-40 kDa pol...

  15. Rapid Purification of a New Humanized Single-chain Fv Antibody/Human Interleukin-2 Fusion Protein Reactive against HER2 Receptor

    Institute of Scientific and Technical Information of China (English)

    Wei-Yun ZHANG; Tak-Chun YIP; Cheuk-Sang KWOK

    2004-01-01

    Human embryonic kidney 293 cells were transfected with plasmid pcDNA-H520C9scFv-rhIL2 containing a chimeric cDNA encoding the humanized 520C9 scFv/recombinant human IL-2 fusion protein (H520C9scFv-rhIL-2). The transfected cells in plateau growing phase were cultured in serum-free medium for three days. The supernatant was collected, concentrated and purified using an affinity column packed with CNBr-activated Sepharose 4B coupled with anti-rhIL-2 mouse monoclonal antibody. The purified fusion protein was analyzed by ELISA, SDS-PAGE and Western blot. The fusion protein showed only one band in both silver stained electrophoresis gel and Western blot developed by ECL chemiluminescence system.Its molecular weight was confirmed to be about 45 kD. This fusion protein possessed binding specificity against p 185 positive SKOV3 and B 16/neu cells, and it might stimulate IL-2-dependent CTLL-2 cell proliferation as well.

  16. Determination of MBT-waste reactivity - An infrared spectroscopic and multivariate statistical approach to identify and avoid failures of biological tests.

    Science.gov (United States)

    Böhm, K; Smidt, E; Binner, E; Schwanninger, M; Tintner, J; Lechner, P

    2010-04-01

    The Austrian Landfill Ordinance provides limit values regarding the reactivity for the disposal of mechanically biologically treated (MBT) waste before landfilling. The potential reactivity determined by biological tests according to the Austrian Standards (OENORM S 2027 1-2) can be underestimated if the microbial community is affected by environmental conditions. New analytical tools have been developed as an alternative to error-prone and time-consuming biological tests. Fourier Transform Infrared (FT-IR) spectroscopy in association with Partial Least Squares Regression (PLS-R) was used to predict the reactivity parameters respiration activity (RA(4)) and gas generation sum (GS(21)) as well as to detect errors resulting from inhibiting effects on biological tests. For this purpose 250 MBT-waste samples from different Austrian MBT-plants were investigated using FT-IR spectroscopy in the mid (MIR) and near infrared (NIR) area and biological tests. Spectroscopic results were compared with those from biological tests. Arising problems caused by interferences of RA(4) and GS(21) are discussed. It is shown that FT-IR spectroscopy predicts RA(4) and GS(21) reliably to assess stability of MBT-waste materials and to detect errors. PMID:19854633

  17. A plant-derived quadrivalent virus like particle influenza vaccine induces cross-reactive antibody and T cell response in healthy adults.

    Science.gov (United States)

    Pillet, Stéphane; Aubin, Éric; Trépanier, Sonia; Bussière, Diane; Dargis, Michèle; Poulin, Jean-François; Yassine-Diab, Bader; Ward, Brian J; Landry, Nathalie

    2016-07-01

    Recent issues regarding efficacy of influenza vaccines have re-emphasized the need of new approaches to face this major public health issue. In a phase 1-2 clinical trial, healthy adults received one intramuscular dose of a seasonal influenza plant-based quadrivalent virus-like particle (QVLP) vaccine or placebo. The hemagglutination inhibition (HI) titers met all the European licensure criteria for the type A influenza strains at the 3μg/strain dose and for all four strains at the higher dosages 21days after immunization. High HI titers were maintained for most of the strains 6months after vaccination. QVLP vaccine induced a substantial and sustained increase of hemagglutinin-specific polyfunctional CD4 T cells, mainly transitional memory and TEMRA effector IFN-γ(+) CD4 T cells. A T cells cross-reactive response was also observed against A/Hong-Kong/1/1968 H3N2 and B/Massachusetts/2/2012. Plant-based QVLP offers an attractive alternative manufacturing method for producing effective and HA-strain matching seasonal influenza vaccines. PMID:26987887

  18. Identification of anti-HPA-1a allo-antibodies using IgG platelet antibody detection and crossmatch system assay with Galileo Echo.

    Science.gov (United States)

    Di Cristofaro, Julie; Frassati, Coralie; Montagnie, Rolande; Basire, Agnes; Merieux, Yves; Picard, Christophe

    2015-01-01

    Fetal/neonatal allo-immune thrombocytopenia is the most frequent and the most dangerous clinical condition involving anti-human platelet antigens (HPA)-1a allo-antibodies. Anti-HPA-1a allo-immunization requires rapid and accurate diagnosis to determine appropriate treatment. The Capture-P Ready-Screen assay (C-PRS) is a new qualitative immunoassay to detect IgG anti-human leukocyte antigen (HLA) and anti-HPA allo-antibodies. The aim of this study is to assess the identification of anti-HPA-1a allo-antibodies using the C-PRS assay, associated with HLA class I stripping reagents, on the automated benchtop analyzer Galileo Echo. Forty-nine sera were analyzed: without anti-HLA class I or anti-HPA allo-antibodies, with anti-HLA class I allo-antibodies, with anti-HPA-1a allo-antibodies, among which with anti-HLA class I allo-antibodies. None of the samples without allo-antibodies were reactive. Only anti-HLA antibodies, detected by cytotoxicity-dependent complement and not by Luminex, remained positive before and after stripping reagents. Of the 13 samples, anti-HPA-1a allo-antibodies that were correctly identified before and after incubation with HLA assassin reagent were 70% and 85%, respectively. Anti-glycoprotein auto-antibodies and anti-HLA allo-antibodies do not interfere with the detection of anti-HPA-1a antibodies. This preliminary study indicates that further improvement of the test will be helpful in developing a clinically useful assay in the future. PMID:25101933

  19. 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 to an increased risk ...

  20. Memory B cell antibodies to HIV-1 gp140 cloned from individuals infected with clade A and B viruses.

    Directory of Open Access Journals (Sweden)

    Hugo Mouquet

    Full Text Available Understanding the antibody response to HIV-1 in humans that show broad neutralizing serologic activity is a crucial step in trying to reproduce such responses by vaccination. Investigating antibodies with cross clade reactivity is particularly important as these antibodies may target conserved epitopes on the HIV envelope gp160 protein. To this end we have used a clade B YU-2 gp140 trimeric antigen and single-cell antibody cloning methods to obtain 189 new anti-gp140 antibodies representing 51 independent B cell clones from the IgG memory B cells of 3 patients infected with HIV-1 clade A or B viruses and exhibiting broad neutralizing serologic activity. Our results support previous findings showing a diverse antibody response to HIV gp140 envelope protein, characterized by differentially expanded B-cell clones producing highly hypermutated antibodies with heterogenous gp140-specificity and neutralizing activity. In addition to their high-affinity binding to the HIV spike, the vast majority of the new anti-gp140 antibodies are also polyreactive. Although none of the new antibodies are as broad or potent as VRC01 or PG9, two clonally-related antibodies isolated from a clade A HIV-1 infected donor, directed against the gp120 variable loop 3, rank in the top 5% of the neutralizers identified in our large collection of 185 unique gp140-specific antibodies in terms of breadth and potency.

  1. Anticorpos antipromastigotas vivas de Leishmania (Viannia braziliensis, detectados pela citometria de fluxo, para identificação da infecção ativa na leishmaniose tegumentar americana Anti-live Leishmania (Viannia braziliensis promastigote antibodies, detected by flow cytometry, to identify active infection in american cutaneous leishmaniasis

    Directory of Open Access Journals (Sweden)

    Roberta Dias Rodrigues Rocha

    2002-12-01

    Full Text Available Neste estudo, descrevemos etapas iniciais de padronização de uma nova metodologia para detecção de anticorpos antipromastigotas vivas de Leishmania (Viannia braziliensis, pela citometria de fluxo e a análise de sua aplicabilidade para estudos clínicos. Foram avaliados 39 indivíduos com sorologia convencional (RIFI positiva para leishmaniose, classificados quanto à ausência/presença de lesão (L- e L+. Os resultados foram expressos sob a forma de percentual de parasitas fluorescentes positivos (PPFP. A análise dos dados, na diluição 1:1.024, permitiu distinguir 95% dos pacientes L+ como um grupo de alta reatividade (PPFP>50% e 72% dos indivíduos L- como um grupo de baixa reatividade (PPFPIn the current study we described initial standardization steps of a new methodology to detect anti-live Leishmania (Viannia braziliensis promastigote antibodies by flow cytometry, followed by analysis of its applicability to clinical studies. We have studied 39 individuals with positive conventional serology to leishmaniasis, classified according to the absence/presence of cutaneous lesions (L- and L+. The results were expressed as percentage of positive fluorescent parasites (PPFP. Data analysis at dilution of 1:1,024, allowed the distinction of 95% of L+ patients as a group of high reactivity (PPFP>50% and 72% of L- individuals as a group of low reactivity (PPFP<50%. The analysis of immunofluorescence assay titers did not show any relationship with the absence/presence of lesion. Together, our data support the applicability of flow cytometry to identify cases of active infection, which has not been possible through conventional serological reactions.

  2. Interaction of monoclonal antibodies directed against bromodeoxyuridine with pyrimidine bases, nucleosides, and DNA

    International Nuclear Information System (INIS)

    Although antibodies directed against bromodeoxyuridine (BrdU) are being used in both clinical and basic research laboratories as tools to study and monitor DNA synthesis, little is known about the epitopes with which they react. Four monoclonal antibodies directed against BrdU were produced and were characterized to learn more about the epitopes on BrdU which are important for antibody recognition, to identify compounds other than BrdU which react with the antibodies and which might interfere with immunologic assays for BrdU, and to characterize the reaction of these antibodies with BrdU-containing DNA. By radioimmunoassays, the antibodies generally reacted well with 5-iododeoxyuridine, 5-fluorodeoxyuridine, and 5-nitrouracil. However, none of the antibodies reacted well with uridine - indicating that a substituent on uridine C5 was essential for antibody reactivity - or with 5-bromo or iodo-cytosine, indicating that the region around pyrimidine C4 is important for antibody recognition. Although the antibodies reacted with 5-halogen-substituted uracil bases, the antibodies reacted much better with the corresponding halogenated nucleosides, indicating that the sugar moiety was important for recognition. The presence of a triphosphate group of C'5 of BrdU (i.e., BrdUTP) did not detectably alter antibody recognition. S1 nuclease treatment of purified DNA suggested that all four monoclonal antibodies reacted exclusively with single-stranded regions of BrdU-containing DNA. Comparison of detecting DNA synthesis by [3H]TdR incorporation followed by autoradiography with that by BrdU incorporation followed by indirect immunofluorescence indicated that the latter technique was both an accurate and a sensitive measure of DNA synthesis

  3. Interfacial metal and antibody recognition

    OpenAIRE

    Zhou, Tongqing; Hamer, Dean H.; Hendrickson, Wayne A.; Sattentau, Quentin J.; Kwong, Peter D.

    2005-01-01

    The unique ligation properties of metal ions are widely exploited by proteins, with approximately one-third of all proteins estimated to be metalloproteins. Although antibodies use various mechanisms for recognition, to our knowledge, none has ever been characterized that uses an interfacial metal. We previously described a family of CD4-reactive antibodies, the archetype being Q425. CD4:Q425 engagement does not interfere with CD4:HIV-1 gp120 envelope glycoprotein binding, but it blocks subse...

  4. Selection of apoptotic cell specific human antibodies from adult bone marrow.

    Directory of Open Access Journals (Sweden)

    Caroline Grönwall

    Full Text Available Autoreactive antibodies that recognize neo-determinants on apoptotic cells in mice have been proposed to have protective, homeostatic and immunoregulatory properties, although our knowledge about the equivalent antibodies in humans has been much more limited. In the current study, human monoclonal antibodies with binding specificity for apoptotic cells were isolated from the bone marrow of healthy adults using phage display technology. These antibodies were shown to recognize phosphorylcholine (PC-associated neo-determinants. Interestingly, three of the four identified apoptotic cell-specific antibody clones were encoded by VH3 region rearrangements with germline or nearly germline configuration without evidence of somatic hypermutation. Importantly, the different identified antibody clones had diverse heavy chain CDR3 and deduced binding surfaces as suggested by structure modeling. This may suggest a potentially great heterogeneity in human antibodies recognizing PC-related epitopes on apoptotic cells. To re-construct the postulated structural format of the parental anti-PC antibody, the dominant clone was also expressed as a recombinant human polymeric IgM, which revealed a substantially increased binding reactivity, with dose-dependent and antigen-inhibitable binding of apoptotic cells. Our findings may have implication for improved prognostic testing and therapeutic interventions in human inflammatory disease.

  5. Antibodies to age-β2 glycoprotein I in patients with anti-phospholipid antibody syndrome.

    Science.gov (United States)

    Sorice, M; Buttari, B; Capozzi, A; Profumo, E; Facchiano, F; Truglia, S; Recalchi, S; Alessandri, C; Conti, F; Misasi, R; Valesini, G; Riganò, R

    2016-05-01

    Anti-phospholipid antibody syndrome (APS) is a systemic autoimmune disease characterized clinically by arterial and/or venous thromboses, recurrent abortions or fetal loss and serologically by the presence of 'anti-phospholipid antibodies' (aPL). The main target antigen of the antibodies is β2 glycoprotein I (β2 GPI). Post-translational oxidative modifications of the protein have been widely described. In this study we aimed to analyse sera reactivity to glucose-modified β2 GPI (G-β2 GPI). Sera collected from 43 patients with APS [15 primary APS (PAPS) and 28 APS associated with systemic lupus erythematosus (SLE) (SAPS)], 30 with SLE, 30 with rheumatoid arthritis (RA) and 40 healthy subjects were analysed by an enzyme-linked immunosorbent assay (ELISA) using a G-β2 GPI. Nine of 15 consecutive PAPS out-patients (60%) and 16 of 28 SAPS (57.1%) showed serum antibodies [immunoglobulin (Ig)G class] against G-β2 GPI (anti-G-β2 GPI) by ELISA. The occurrence of anti-G-β2 GPI was significantly higher in APS patients compared to patients suffering from SLE. No RA patients or control healthy subjects resulted positive for anti-G-β2 GPI. Of note, aG-β2 GPI prompted to identify some APS patients (four PAPS and seven SAPS), who were negative in the classical anti-β2 GPI test. Moreover, in APS patients, anti-G-β2 GPI titre was associated significantly with venous thrombosis and seizure in APS patients. This study demonstrates that G-β2 GPI is a target antigen of humoral immune response in patients with APS, suggesting that β2 GPI glycation products may contain additional epitopes for anti-β2 GPI reactivity. Searching for these antibodies may be useful for evaluating the risk of clinical manifestations. PMID:26702877

  6. EpViX: A cloud-based tool for epitope reactivity analysis and epitope virtual crossmatching to identify low immunologic risk donors for sensitized recipients.

    Science.gov (United States)

    Anunciação, Fernando Antonio Costa; Sousa, Luiz Claudio Demes da Mata; da Silva, Adalberto Socorro; Marroquim, Mário Sérgio Coelho; Coelho, Antônio Gilberto Borges; Willcox, Glauco Henrique; de Andrade, João Marcelo Medeiros; Corrêa, Bruno de Melo; Guimarães, Elisabeth Lima; do Monte, Semiramis Jamil Hadad

    2015-11-01

    One of the challenges facing solid organ transplantation programs globally is the identification of low immunological risk donors for sensitized recipients by HLA allele genotype. Because recognition of donor HLA alleles by host antibodies is at the core of organ rejection, the objective of this work was to develop a new version of the EpHLA software, named EpViX, which uses an HLAMatchmaker algorithm and performs automated epitope virtual crossmatching at the initiation of the organ donation process. EpViX is a free, web-based application developed for use over the internet on a tablet, smartphone or computer. This program was developed using the Ruby programming language and the Ruby-on-Rails framework. To improve the user experience, the EpViX software interface was developed based on the best human–computer interface practices. To simplify epitope analysis and virtual crossmatching, the program was integrated with important available web-based resources, such as OPTN, IMGT/HLA and the International HLA Epitope Registry. We successfully developed a program that allows people to work collaboratively and effectively during the donation process by accurately predicting negative crossmatches, saving time and other resources. PMID:26531328

  7. Vaccination of cattle with TickGARD induces cross-reactive antibodies binding to conserved linear peptides of Bm86 homologues in Boophilus decoloratus.

    Science.gov (United States)

    Odongo, David; Kamau, Lucy; Skilton, Robert; Mwaura, Stephen; Nitsch, Cordula; Musoke, Anthony; Taracha, Evans; Daubenberger, Claudia; Bishop, Richard

    2007-01-26

    Vaccines based on recombinant Bm86 gut antigen from Boophilus microplus are a useful component of integrated control strategies against B. microplus infestations of cattle. The capacity of such vaccines to control heterologous infestations by two African tick species was investigated. The mean weight of engorged female ticks and mean egg mass per tick were significantly reduced in B. decoloratus infestations, but there was no effect of the vaccine against adult Rhipicephalus appendiculatus. We cloned, sequenced and expressed two Bm86 homologues (Bd86) from B. decoloratus. Amino acid sequence identity between Bd86 homologues (Bd86-1 and Bd86-2) and Bm86 was 86% and 85%, respectively, compared to 93% identity between the variants. Native Bd86 protein in B. decoloratus tick mid-gut sections and recombinant Bd86-1 reacted strongly with sera from TickGARD vaccinated cattle. TickGARD can therefore protect against a heterologous tick species with multiple antigen sequences. Epitope mapping using sera from TickGARD-vaccinated cattle identified two linear peptides conserved between the Bd86 homologues and Bm86. These epitopes represent candidate synthetic peptide vaccines for control of Boophilus spp. and the pathogens transmitted by these tick vectors. PMID:17070625

  8. Fully automated high-throughput chromatin immunoprecipitation for ChIP-seq: Identifying ChIP-quality p300 monoclonal antibodies

    OpenAIRE

    Gasper, William C.; Marinov, Georgi K.; Pauli-Behn, Florencia; Scott, Max T.; Newberry, Kimberly; DeSalvo, Gilberto; Ou, Susan; Myers, Richard M.; Vielmetter, Jost; Wold, Barbara J

    2014-01-01

    Chromatin immunoprecipitation coupled with DNA sequencing (ChIP-seq) is the major contemporary method for mapping in vivo protein-DNA interactions in the genome. It identifies sites of transcription factor, cofactor and RNA polymerase occupancy, as well as the distribution of histone marks. Consortia such as the ENCyclopedia Of DNA Elements (ENCODE) have produced large datasets using manual protocols. However, future measurements of hundreds of additional factors in many cell types and physio...

  9. C-reactive protein in patients with advanced metastatic renal cell carcinoma: Usefulness in identifying patients most likely to benefit from initial nephrectomy

    International Nuclear Information System (INIS)

    C-reactive protein (CRP) is considered a useful serum marker for patients with RCC. However, its clinical utility in advanced metastatic renal cell carcinoma (AM-RCC), particularly in deciding whether to perform nephrectomy at the onset, is not well studied. We retrospectively evaluated 181 patients with AM-RCC, including 18 patients underwent potentially curative surgery, 111 underwent cytoreductive nephrectomy, and 52 received medical treatment only. CRP cutoff points were determined by receiver operating characteristic (ROC) curve analysis. Kaplan-Meier and Cox regression analyses were used for survival tests. ROC analysis suggested that grouping patients according to 3 CRP ranges was a rational model. Patients with highly elevated CRP (≥67.0 mg/L) presented remarkably poor prognosis despite treatment (nephrectomy or medical treatment only). Cox regression models demonstrated that risk factors of overall survival for patients who underwent nephrectomy were the CRP ranges defined in this study (≤18.0 mg/L, >18.0 and <67.0 mg/L, and ≥67.0 mg/L), ECOG PS (0, 1, and ≥2), and number of metastatic organ sites (0–1 and ≥2). The retrospective design is a limitation of this study. Our study demonstrated that the serum CRP level is a statistically significant prognostic parameter for patients with AM-RCC. The data also indicated that pretreatment serum CRP level provides useful prognostic information that helps in deciding whether to perform initial nephrectomy for patients with AM-RCC

  10. Monoclonal antibodies to Pneumocystis carinii

    DEFF Research Database (Denmark)

    Kovacs, J A; Halpern, J L; Lundgren, B; Swan, J C; Parrillo, J E; Masur, H

    1989-01-01

    To increase understanding of the antigenic structure of Pneumocystis carinii, we developed monoclonal antibodies to rat and human P. carinii. The specificity of the antibodies was demonstrated by immunofluorescence and immunoblot studies. Only one of five monoclonal antibodies to rat P. carinii...... reacted with human P. carinii, and none of four monoclonal antibodies to human P. carinii reacted with rat P. carinii. Two antibodies to human P. carinii reacted by immunofluorescence with only one human P. carinii isolate. Immunoblot studies identified major antigens of rat P. carinii with molecular...

  11. Human serum antibodies against EBV latent membrane protein 1 cross-react with α-synuclein

    Science.gov (United States)

    Gray, Madison T.; Ganesh, Munisha S.; Middeldorp, Jaap M.

    2016-01-01

    Objectives: To identify the epitope on α-synuclein (α-syn) to which antibodies against the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) bind and to determine whether antibodies targeting this mimicry domain are present in human sera. Methods: Reactivity of the α-syn-cross-reacting anti-LMP1 monoclonal antibody CS1-4 to a synthetic peptide containing the putative mimicry domain was compared to those in which this domain was mutated and to murine and rat α-syn (which differ from human α-syn at this site) in Western blots. Using ELISA, sera from EBV+ (n = 4) and EBV− (n = 12) donors as well as those with infectious mononucleosis (IM; n = 120), and Hodgkin disease (HD; n = 33) were interrogated for antibody reactivity to synthetic peptides corresponding to regions of α-syn and LMP1 containing the mimicry domain. Results: CS1-4 showed strong reactivity to wild-type human α-syn, but not to the mutant peptides or rodent α-syn. Control EBV− and EBV+ sera showed no reactivity to α-syn or LMP1 peptides. However, a significant proportion of IM and HD sera contained immunoglobulin M (IgM) (59% and 70%, in IM and HD, respectively), immunoglobulin G (IgG) (40% and 48%), and immunoglobulin A (IgA) (28% and 36%) antibodies to both peptides, as well as a significant correlation in the titers of IgM (ρ = 0.606 and 0.664, for IM and HD, respectively), IgG (0.526 and 0.836), and IgA (0.569 and 0.728) antibodies targeting LMP1 and α-syn peptides. Conclusions: Anti-EBV-LMP1 antibodies cross-reacting with a defined epitope in α-syn are present in human patients. These findings may have implications for the pathogenesis of synucleinopathies. PMID:27218119

  12. 氨苄青霉素单克隆抗体及其与青霉素类抗生素交叉反应性%Anti-ampicillin Monoclonal Antibodies and Their Cross-reactivity to Penicillin Antibiotics

    Institute of Scientific and Technical Information of China (English)

    李青梅; 刘庆堂; 王寅彪; 柴书军; 王自良; 郭军庆; 职爱民; 孟红丽; 张改平

    2011-01-01

    Ampicillin ( Amp) was coupled to carrier proteins of bovine serum albumin ( BSA ) and ovalbumin (OVA) to synthetize immunogen BSA-Amp and detection antigen OVA-Amp. Four hybridoma cell lines secreting monoclonal antibodies (mAbs) specific for Amp were established by immunizing BALB/c mice with BSA-Amp u-sing hybridoma technology. The antibody titers of mAb ascites (1G7, 2A11, 2D10 and 3F6) were determined as 1:2.56×107, 1:1.28×107, 1 :5.12 × 106 and 1:5.12 × 106 in ELISA, and their half maximal inhibitory concentration (IC50) of Amp were 10.22, 3.58, 4.03,4.95 ng/mL in the inhibitive ELISA, respectively. The Amp mAbs showed significant cross-reactivity to the penicillin antibiotics of carbenicillin (1G7, 2A11, 2D10 and 3F6 ) , penicillin G (1G7 and 3F6) , and amoxicillin (1G7) , whereas no obvious cross-reactivity to dicloxacillin or clox-acillin was found.%以戊二醛法将氨苄青霉素(Ampicillin,Amp)偶联于牛血清白蛋白(BSA)和卵清白蛋白(OVA),合成免疫抗原BSA-Amp和检测抗原0VA-Amp,并进行了鉴定;用BSA-Amp免疫BALB/c小鼠,应用杂交瘤技术建立了4株分泌抗Amp单克隆抗体的杂交瘤细胞株.ELISA和竞争ELISA结果显示,单克隆抗体1G7、2A11、2D10和3F6的腹水抗体效价分别为1:2.56×107、1:1.28×107、1:5.12 ×106和1:5.12×106,Amp的半数抑制浓度(IC50)分别为10.22,3.58,4.03,4.95 ng/mL.Amp单克隆抗体与羧苄青霉素(1G7、2A11、2D10和3F6)、青霉素G(1G7和3F6)、阿莫西林(1G7)等青霉素类抗生素存在显著的交叉反应,而与双氯霉素和邻氯霉素未见明显交叉反应.

  13. C595--a monoclonal antibody against the protein core of human urinary epithelial mucin commonly expressed in breast carcinomas.

    OpenAIRE

    Price, M. R.; Pugh, J. A.; Hudecz, F.; Griffiths, W.; Jacobs, E.; Symonds, I. M.; Clarke, A J; Chan, W. C.; Baldwin, R. W.

    1990-01-01

    Urinary mucins which express determinants for the anti-breast carcinoma monoclonal antibody, NCRC-11 (IgM), closely resemble the mammary mucins found in milk fat globules and carcinomas. An IgG3 monoclonal antibody, C595, was prepared against urinary mucins isolated on a NCRC-11 antibody affinity column, and this 'second generation' antibody was shown to have a very similar pattern of reactivity to the original NCRC-11 antibody. By immunohistology, the profile of reactivity of both antibodies...

  14. Reactive transport modeling of chemical and isotope data to identify degradation processes of chlorinated ethenes in a diffusion-dominated media

    DEFF Research Database (Denmark)

    Chambon, Julie Claire Claudia; Damgaard, Ida; Jeannottat, Simon;

    Chlorinated ethenes are among the most widespread contaminants in the subsurface and a major threat to groundwater quality at numerous contaminated sites. Many of these contaminated sites are found in low-permeability media, such as clay tills, where contaminant transport is controlled by diffusi...... important finding, that is further supported by microbial and chemical data. Improved understanding of degradation processes in clay tills is useful for improving the reliability of risk assessment and the design of remediation schemes for chlorinated solvents....... modeling has been used to identify the degradation processes occurring at the core scale. The field data was from a site located at Vadsby, Denmark, where chlorinated solvents were spilled during the 1960-70’s, resulting in contamination of the clay till and the underlying sandy layer (15 meters below...... source zone (between 6 and 12 mbs). Concentrations and stable isotope ratios of the mother compounds and their daughter products, as well as redox parameters, fatty acids and microbial data, were analyzed with discrete sub-sampling along the cores. More samples (each 5 mm) were collected around the...

  15. Anti-G antibody in alloimmunized pregnant women: Report of two cases.

    Science.gov (United States)

    Makroo, Raj Nath; Kaul, Anita; Bhatia, Aakanksha; Agrawal, Soma; Singh, Chanchal; Karna, Prashant

    2015-01-01

    Anti-G has been reported as a possible cause of hemolytic disease of the fetus and newborn (HDFN), either independently or in association with anti-D, anti-C or both. The antibody mimics the pattern of anti-C and anti-D reactivity in the identification panel and is often present along with either or both of these antibodies. The differentiation of anti-D, -C and-G in routine pretransfusion workup is particularly essential in antenatal cases. We report two antenatal cases where anti-G was identified on advanced immunohematological workup, in addition to other alloantibodies. PMID:26420948

  16. Anti-G antibody in alloimmunized pregnant women: Report of two cases

    Directory of Open Access Journals (Sweden)

    Raj Nath Makroo

    2015-01-01

    Full Text Available Anti-G has been reported as a possible cause of hemolytic disease of the fetus and newborn (HDFN, either independently or in association with anti-D, anti-C or both. The antibody mimics the pattern of anti-C and anti-D reactivity in the identification panel and is often present along with either or both of these antibodies. The differentiation of anti-D, -C and-G in routine pretransfusion workup is particularly essential in antenatal cases. We report two antenatal cases where anti-G was identified on advanced immunohematological workup, in addition to other alloantibodies.

  17. A simple set of validation steps identifies and removes false results in a sandwich enzyme-linked immunosorbent assay caused by anti-animal IgG antibodies in plasma from arthritis patients

    OpenAIRE

    Kragstrup, Tue W; Vorup-Jensen, Thomas; Deleuran, Bent; Hvid, Malene

    2013-01-01

    Rheumatoid arthritis (RA) and spondyloarthritis (SpA) are chronic diseases characterized by activation of the immune system and production of antibodies. Thus, rheumatoid factor, anti-animal IgG antibodies and heterophilic antibodies in plasma samples from arthritis patients can interfere with immunoassays such as sandwich enzyme-linked immunosorbent assay (ELISA) systems often used in arthritis research. However, standard methodologies on how to test for false results caused by these antibod...

  18. Monoclonal antibodies for treating cancer

    International Nuclear Information System (INIS)

    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

  19. Antibody informatics for drug discovery

    DEFF Research Database (Denmark)

    Shirai, Hiroki; Prades, Catherine; Vita, Randi;

    2014-01-01

    infrastructure for these large data sets has become necessary. In this article, we first identify and discuss the typical obstacles faced during the antibody drug discovery process. We then summarize the current status of three sub-fields of antibody informatics as follows: (i) recent progress in technologies...... (iii) antibody numbering and IMGT. Here, we review “antibody informatics,” which may integrate the above three fields so that bridging the gaps between industrial needs and academic solutions can be accelerated. This article is part of a Special Issue entitled: Recent advances in molecular engineering...

  20. Antigenic analysis of classical swine fever virus E2 glycoprotein using pig antibodies identifies residues contributing to antigenic variation of the vaccine C-strain and group 2 strains circulating in China

    Directory of Open Access Journals (Sweden)

    Peng Jinrong

    2010-12-01

    Full Text Available Abstract Background Glycoprotein E2, the immunodominant protein of classical swine fever virus (CSFV, can induce neutralizing antibodies and confer protective immunity in pigs. Our previous phylogenetic analysis showed that subgroup 2.1 viruses branched away from subgroup 1.1, the vaccine C-strain lineage, and became dominant in China. The E2 glycoproteins of CSFV C-strain and recent subgroup 2.1 field isolates are genetically different. However, it has not been clearly demonstrated how this diversity affects antigenicity of the protein. Results Antigenic variation of glycoprotein E2 was observed not only between CSFV vaccine C-strain and subgroup 2.1 strains, but also among strains of the same subgroup 2.1 as determined by ELISA-based binding assay using pig antisera to the C-strain and a representative subgroup 2.1 strain QZ-07 currently circulating in China. Antigenic incompatibility of E2 proteins markedly reduced neutralization efficiency against heterologous strains. Single amino acid substitutions of D705N, L709P, G713E, N723S, and S779A on C-strain recombinant E2 (rE2 proteins significantly increased heterologous binding to anti-QZ-07 serum, suggesting that these residues may be responsible for the antigenic variation between the C-strain and subgroup 2.1 strains. Notably, a G713E substitution caused the most dramatic enhancement of binding of the variant C-strain rE2 protein to anti-QZ-07 serum. Multiple sequence alignment revealed that the glutamic acid residue at this position is conserved within group 2 strains, while the glycine residue is invariant among the vaccine strains, highlighting the role of the residue at this position as a major determinant of antigenic variation of E2. A variant Simpson's index analysis showed that both codons and amino acids of the residues contributing to antigenic variation have undergone similar diversification. Conclusions These results demonstrate that CSFV vaccine C-strain and group 2 strains

  1. Seroprevalence of unexpected red blood cell antibodies among pregnant women in Uganda.

    Science.gov (United States)

    Eipl, K; Nakabiito, C; Bwogi, K; Motevalli, M; Roots, A; Blagg, L; Jackson, J B

    2012-01-01

    We conducted a population-based, cross-sectional study among pregnant women in Kampala, Uganda, to determine ABO and D blood types and to determine the percentage who have unexpected red blood cell (RBC) antibodies and their specificities. De-identified blood samples from routine testing of 1001 pregnant women at the Mulago Hospital antenatal clinics in Kampala were typed for ABO and D and screened for the presence of unexpected RBC antibodies with confirmation and subsequent antibody identification. Of the 1001 blood samples tested, 48.9 percent, 26.4 percent, 21.0 percent, and 3.8 percent tested positive for blood groups 0, A, B, and AB, respectively. Of these samples, 23 (2.3%)were negative forD, and 55 (5.5%) showed initial reactivity with at least one screening RBC. The RBC antibody screen was repeated on these 55 samples, and antibody identification was performed at the Johns Hopkins Hospital Blood Bank in Baltimore, Maryland. Twenty-one of the 55 samples were confirmed to have evidence of agglutination. Nine of the 21 samples demonstrated the presence of clinically significant RBC antibodies with anti-S being the most common, 8 samples demonstrated the presence of benign or naturally occurring antibodies, and 4 had only inconclusive reactivity. This study revealed a relatively high frequency of D and a low frequency of demonstrable clinically significant alloantibodies that may cause hemolytic disease of the newborn or hemolytic transfusion reactions among pregnant women in Kampala, with anti-S being the most frequent antibody specificity. PMID:23421539

  2. A simple set of validation steps identifies and removes false results in a sandwich enzyme-linked immunosorbent assay caused by anti-animal IgG antibodies in plasma from arthritis patients.

    Science.gov (United States)

    Kragstrup, Tue W; Vorup-Jensen, Thomas; Deleuran, Bent; Hvid, Malene

    2013-12-01

    Rheumatoid arthritis (RA) and spondyloarthritis (SpA) are chronic diseases characterized by activation of the immune system and production of antibodies. Thus, rheumatoid factor, anti-animal IgG antibodies and heterophilic antibodies in plasma samples from arthritis patients can interfere with immunoassays such as sandwich enzyme-linked immunosorbent assay (ELISA) systems often used in arthritis research. However, standard methodologies on how to test for false results caused by these antibodies are lacking. The objective of this study was to design a simple set of steps to validate a sandwich ELISA before using it for measuring analytes in plasma from arthritis patients. An interleukin-24 (IL-24) sandwich ELISA system was prepared with a monoclonal mouse capture antibody and a polyclonal goat detection antibody and tested for interference by rheumatoid factor, anti-animal IgG antibodies and heterophilic antibodies. Plasma samples from 23 patients with RA and SpA were used. No differences were found between plasma samples measured in wells coated with anti-IL-24 specific antibody and in wells coated with isotype control antibody (false positive results), and recombinant human IL-24 was not recovered in spiked samples (false negative results). This interference was removed after preincubating the plasma samples from patients with arthritis with goat or bovine IgG, suggesting that anti-animal IgG antibodies found in the plasma of the arthritis patients caused the false results. Additional testing showed that the signal-to-noise ratio could be increased by titration of the capture and detection antibodies and by using the ELAST amplification system. Finally, the calculated concentration of IL-24 was increased in ethylenediaminetetraacetic acid (EDTA) plasma compared to heparin plasma and serum and decreased with repetitive freeze/thaw cycles of the samples illustrating how sample handling could additionally contribute to the variations reported by different

  3. Bispecific antibodies.

    Science.gov (United States)

    Kontermann, Roland E; Brinkmann, Ulrich

    2015-07-01

    Bispecific antibodies (bsAbs) combine specificities of two antibodies and simultaneously address different antigens or epitopes. BsAbs with 'two-target' functionality can interfere with multiple surface receptors or ligands associated, for example with cancer, proliferation or inflammatory processes. BsAbs can also place targets into close proximity, either to support protein complex formation on one cell, or to trigger contacts between cells. Examples of 'forced-connection' functionalities are bsAbs that support protein complexation in the clotting cascade, or tumor-targeted immune cell recruiters and/or activators. Following years of research and development (R&D), the first bsAb was approved in 2009. Another bsAb entered the market in December 2014 and several more are in clinical trials. Here, we describe the potentials of bsAbs to become the next wave of antibody-based therapies, focusing on molecules in clinical development. PMID:25728220

  4. Pharmacological selection of antibodies for immunoscintigraphy

    International Nuclear Information System (INIS)

    The recent development of hybridoma technology has resulted in the production of monoclonal antibodies that recognize a variety of tumor antigens. Many antibodies have been developed and some of them are used with different success in clinical practice. A list of criteria is proposed for the selection of antibodies suitable for imaging studies illustrated with the example of two monoclonal antibodies anti-CEA and 19.9 used in colorectal carcinoma imaging. Monoclonal antibodies obtained today are not truly tumor-specific, they are tumor-associated; this suggests that some cross-reactions with normal tissues exist. For immunoscintigraphical use it is important to select antibodies which procedure high tumor cell staining with limited reactivity against normal tissues. Antibodies can be separated into F(ab')2 and Fab fragments which diffuse more easily into the tumor with a rapid clearance from the circulation giving higher tumor to normal tissues ratio at an early time. Antibodies with both high affinity and avidity towards tumor cell receptors produce better imaging results. Antibodies can be labelled directly with iodine or technetium and with indium using chelating agents. In vivo kinetics of radiolabelled antibodies are very different considering the nuclide and the labelling method used. Pharmacokinetics on nude mice grated with human tumors are very useful for selecting the most appropriate nuclide antibody fragment and the most efficient labelling technique for a given application. (author)

  5. Antigenic specificity of serum antibodies in mice fed soy protein

    DEFF Research Database (Denmark)

    Christensen, Hanne Risager; Bruun, S.W.; Frøkiær, Hanne

    2003-01-01

    Background: Soybean protein is used in a number of food products but unfortunately is also a common cause of food allergy. Upon ingestion of soy protein, healthy mice like other animals and humans generate a soy-specific antibody response in the absence of signs of illness. Not much is known about...... the relationship between the immunogenic proteins involved in this nondeleterious antibody response and the pathological response associated with food allergy. The objective of the present study was to characterize the antigenic specificity of the soy protein-specific antibody response generated in....... Moreover, antibody reactivity was found towards the native quaternary structure of glycinin. Conclusions: Mice ingesting soy protein generate an antibody response with reactivity towards glycinin and beta-conglycinin. Antibody reactivity found towards the native quaternary structure of glycinin indicates...

  6. Tumor imaging with monoclonal antibodies

    International Nuclear Information System (INIS)

    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 123I is the best candidate for radioimmunodetection purposes. The labeling of antibodies through chelates makes it possible to use metal radioisotopes like 111In, 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

  7. Verification of the Cross Immunoreactivity of A60, a Mouse Monoclonal Antibody against Neuronal Nuclear Protein

    Science.gov (United States)

    Mao, Shanping; Xiong, Guoxiang; Zhang, Lei; Dong, Huimin; Liu, Baohui; Cohen, Noam A.; Cohen, Akiva S.

    2016-01-01

    A60, the mouse monoclonal antibody against the neuronal nuclear protein (NeuN), is the most widely used neuronal marker in neuroscience research and neuropathological assays. Previous studies identified fragments of A60-immunoprecipitated protein as Synapsin I (Syn I), suggesting the antibody will demonstrate cross immunoreactivity. However, the likelihood of cross reactivity has never been verified by immunohistochemical techniques. Using our established tissue processing and immunofluorescent staining protocols, we found that A60 consistently labeled mossy fiber terminals in hippocampal area CA3. These A60-positive mossy fiber terminals could also be labeled by Syn I antibody. After treating brain slices with saponin in order to better preserve various membrane and/or vesicular proteins for immunostaining, we observed that A60 could also label additional synapses in various brain areas. Therefore, we used A60 together with a rabbit monoclonal NeuN antibody to confirm the existence of this cross reactivity. We showed that the putative band positive for A60 and Syn I could not be detected by the rabbit anti-NeuN in Western blotting. As efficient as Millipore A60 to recognize neuronal nuclei, the rabbit NeuN antibody demonstrated no labeling of synaptic structures in immunofluorescent staining. The present study successfully verified the cross reactivity present in immunohistochemistry, cautioning that A60 may not be the ideal biomarker to verify neuronal identity due to its cross immunoreactivity. In contrast, the rabbit monoclonal NeuN antibody used in this study may be a better candidate to substitute for A60. PMID:27242450

  8. Synthetic B-Cell Epitopes Eliciting Cross-Neutralizing Antibodies: Strategies for Future Dengue Vaccine.

    Science.gov (United States)

    Ramanathan, Babu; Poh, Chit Laa; Kirk, Kristin; McBride, William John Hannan; Aaskov, John; Grollo, Lara

    2016-01-01

    Dengue virus (DENV) is a major public health threat worldwide. A key element in protection from dengue fever is the neutralising antibody response. Anti-dengue IgG purified from DENV-2 infected human sera showed reactivity against several peptides when evaluated by ELISA and epitope extraction techniques. A multi-step computational approach predicted six antigenic regions within the E protein of DENV-2 that concur with the 6 epitopes identified by the combined ELISA and epitope extraction approach. The selected peptides representing B-cell epitopes were attached to a known dengue T-helper epitope and evaluated for their vaccine potency. Immunization of mice revealed two novel synthetic vaccine constructs that elicited good humoral immune responses and produced cross-reactive neutralising antibodies against DENV-1, 2 and 3. The findings indicate new directions for epitope mapping and contribute towards the future development of multi-epitope based synthetic peptide vaccine. PMID:27223692

  9. Synthetic B-Cell Epitopes Eliciting Cross-Neutralizing Antibodies: Strategies for Future Dengue Vaccine.

    Directory of Open Access Journals (Sweden)

    Babu Ramanathan

    Full Text Available Dengue virus (DENV is a major public health threat worldwide. A key element in protection from dengue fever is the neutralising antibody response. Anti-dengue IgG purified from DENV-2 infected human sera showed reactivity against several peptides when evaluated by ELISA and epitope extraction techniques. A multi-step computational approach predicted six antigenic regions within the E protein of DENV-2 that concur with the 6 epitopes identified by the combined ELISA and epitope extraction approach. The selected peptides representing B-cell epitopes were attached to a known dengue T-helper epitope and evaluated for their vaccine potency. Immunization of mice revealed two novel synthetic vaccine constructs that elicited good humoral immune responses and produced cross-reactive neutralising antibodies against DENV-1, 2 and 3. The findings indicate new directions for epitope mapping and contribute towards the future development of multi-epitope based synthetic peptide vaccine.

  10. TISSUE DISTRIBUTION OF THE C3D/EBV-RECEPTOR - CD21 MONOCLONAL-ANTIBODIES REACTIVE WITH A VARIETY OF EPITHELIAL-CELLS, MEDULLARY THYMOCYTES, AND PERIPHERAL T-CELLS

    NARCIS (Netherlands)

    TIMENS, W; BOES, A; VOS, H; POPPEMA, S

    1991-01-01

    The CD21 antigen has been described to represent CR2, the receptor for the complement fragment C3d and also the receptor for the Epstein-Barr virus (EBV). Monoclonal antibodies B2, HB5, and B-ly4 belong to the CD21 cluster, recognizing different epitopes of the CD21-molecule. Immunohistology of lymp

  11. Reactive Arthritis

    Directory of Open Access Journals (Sweden)

    Eren Erken

    2013-06-01

    Full Text Available Reactive arthritis is an acute, sterile, non-suppurative and inflammatory arthropaty which has occured as a result of an infectious processes, mostly after gastrointestinal and genitourinary tract infections. Reiter syndrome is a frequent type of reactive arthritis. Both reactive arthritis and Reiter syndrome belong to the group of seronegative spondyloarthropathies, associated with HLA-B27 positivity and characterized by ongoing inflammation after an infectious episode. The classical triad of Reiter syndrome is defined as arthritis, conjuctivitis and urethritis and is seen only in one third of patients with Reiter syndrome. Recently, seronegative asymmetric arthritis and typical extraarticular involvement are thought to be adequate for the diagnosis. However, there is no established criteria for the diagnosis of reactive arthritis and the number of randomized and controlled studies about the therapy is not enough. [Archives Medical Review Journal 2013; 22(3.000: 283-299

  12. Reactive Safety

    OpenAIRE

    Rüdiger Ehlers; Bernd Finkbeiner

    2011-01-01

    The distinction between safety and liveness properties is a fundamental classification with immediate implications on the feasibility and complexity of various monitoring, model checking, and synthesis problems. In this paper, we revisit the notion of safety for reactive systems, i.e., for systems whose behavior is characterized by the interplay of uncontrolled environment inputs and controlled system outputs. We show that reactive safety is a strictly larger class of properties than standard...

  13. Reactivity measurements

    International Nuclear Information System (INIS)

    Digital reactivity meter, realized as an off-line Fortran program, the input to which is a record of 500 consecutive values of n(tsub(i)) obtained by on-line program on CDC 1700 from the linear power channel of the TRIGA reactor, has been tested at low powers at which the reactor fuel temperature feedback reactivity is negligible. Calibration of the meter by the regulating rod, the reactivity of which has been determined by the assymptotic reactor period, shows that the absolute error is below 1,6% for reactivities up to 1 $. The accuracy of the reactivity meter is proportional to the square of the product of the sampling interval and the period at which the neutron density changes. So the relative error of the reactivity remains at all operational states below 0.2% at 1 second sampling intervals and even at 3 seconds sampling it does not rises above 2.0%. The meter is useful for measurements of control rod drops into the reactor at sampling intervals of 0.1 sec. The meter sensitivity is 0.5 c/s at 1 sec sampling

  14. Laboratory and epidemiologic evaluation of an enzyme immunoassay for antibodies to HTLV-III

    International Nuclear Information System (INIS)

    The enzyme immunoassays (EIAs) for antibody to human T-cell lymphotropic virus type III (HTLV-III) were rapidly adopted for screening donated blood and plasma. To evaluate the significance of a positive EIA reaction, test performance was examined in a blood bank screening program. Specimens were tested by EIA, Western blot assay, and HTLV-III/lymphadenopathy-associated virus (LAV) culture. The EIA was positive in 0.25% of 67 190 blood donations. Specimens were categorized and 57.3% had low (weak) reactivity, 12.7% had moderate reactivity, and 30.0% had high reactivity. Highly reactive specimens were strongly associated with a positive Western blot or culture (86.7%) in contrast to moderately and weekly reactive specimens (1.9%). Twenty-five of 29 donors interviewed with a highly reactive EIA had risk factors for HTLV-III/LAV infection. Risk factors were not identified for 74 of 75 interviewed donors with specimens of lower reactivity. The minimum calculated specificity was 99.82%. The use of the HTLV-III EIA has virtually eliminated the use of blood and plasma for HTLV-III/LAV infected donors

  15. Comprehensive mapping of common immunodominant epitopes in the West Nile virus nonstructural protein 1 recognized by avian antibody responses.

    Science.gov (United States)

    Sun, Encheng; Zhao, Jing; Liu, Nihong; Yang, Tao; Xu, Qingyuan; Qin, Yongli; Bu, Zhigao; Yang, Yinhui; Lunt, Ross A; Wang, Linfa; Wu, Donglai

    2012-01-01

    West Nile virus (WNV) is a mosquito-borne flavivirus that primarily infects birds but occasionally infects humans and horses. Certain species of birds, including crows, house sparrows, geese, blue jays and ravens, are considered highly susceptible hosts to WNV. The nonstructural protein 1 (NS1) of WNV can elicit protective immune responses, including NS1-reactive antibodies, during infection of animals. The antigenicity of NS1 suggests that NS1-reactive antibodies could provide a basis for serological diagnostic reagents. To further define serological reagents for diagnostic use, the antigenic sites in NS1 that are targeted by host immune responses need to be identified and the potential diagnostic value of individual antigenic sites also needs to be defined. The present study describes comprehensive mapping of common immunodominant linear B-cell epitopes in the WNV NS1 using avian WNV NS1 antisera. We screened antisera from chickens, ducks and geese immunized with purified NS1 for reactivity against 35 partially overlapping peptides covering the entire WNV NS1. This study identified twelve, nine and six peptide epitopes recognized by chicken, duck and goose antibody responses, respectively. Three epitopes (NS1-3, 14 and 24) were recognized by antibodies elicited by immunization in all three avian species tested. We also found that NS1-3 and 24 were WNV-specific epitopes, whereas the NS1-14 epitope was conserved among the Japanese encephalitis virus (JEV) serocomplex viruses based on the reactivity of avian WNV NS1 antisera against polypeptides derived from the NS1 sequences of viruses of the JEV serocomplex. Further analysis showed that the three common polypeptide epitopes were not recognized by antibodies in Avian Influenza Virus (AIV), Newcastle Disease Virus (NDV), Duck Plague Virus (DPV) and Goose Parvovirus (GPV) antisera. The knowledge and reagents generated in this study have potential applications in differential diagnostic approaches and subunit vaccines

  16. Comprehensive mapping of common immunodominant epitopes in the West Nile virus nonstructural protein 1 recognized by avian antibody responses.

    Directory of Open Access Journals (Sweden)

    Encheng Sun

    Full Text Available West Nile virus (WNV is a mosquito-borne flavivirus that primarily infects birds but occasionally infects humans and horses. Certain species of birds, including crows, house sparrows, geese, blue jays and ravens, are considered highly susceptible hosts to WNV. The nonstructural protein 1 (NS1 of WNV can elicit protective immune responses, including NS1-reactive antibodies, during infection of animals. The antigenicity of NS1 suggests that NS1-reactive antibodies could provide a basis for serological diagnostic reagents. To further define serological reagents for diagnostic use, the antigenic sites in NS1 that are targeted by host immune responses need to be identified and the potential diagnostic value of individual antigenic sites also needs to be defined. The present study describes comprehensive mapping of common immunodominant linear B-cell epitopes in the WNV NS1 using avian WNV NS1 antisera. We screened antisera from chickens, ducks and geese immunized with purified NS1 for reactivity against 35 partially overlapping peptides covering the entire WNV NS1. This study identified twelve, nine and six peptide epitopes recognized by chicken, duck and goose antibody responses, respectively. Three epitopes (NS1-3, 14 and 24 were recognized by antibodies elicited by immunization in all three avian species tested. We also found that NS1-3 and 24 were WNV-specific epitopes, whereas the NS1-14 epitope was conserved among the Japanese encephalitis virus (JEV serocomplex viruses based on the reactivity of avian WNV NS1 antisera against polypeptides derived from the NS1 sequences of viruses of the JEV serocomplex. Further analysis showed that the three common polypeptide epitopes were not recognized by antibodies in Avian Influenza Virus (AIV, Newcastle Disease Virus (NDV, Duck Plague Virus (DPV and Goose Parvovirus (GPV antisera. The knowledge and reagents generated in this study have potential applications in differential diagnostic approaches and

  17. Detection of antitrophoblast antibodies in the sera of patients with anticardiolipin antibodies and fetal loss.

    Science.gov (United States)

    McCrae, K R; DeMichele, A M; Pandhi, P; Balsai, M J; Samuels, P; Graham, C; Lala, P K; Cines, D B

    1993-11-01

    Women with anticardiolipin antibodies (ACLA) are at increased risk for fetal loss. One potential explanation for this outcome is that sera from these individuals contain antibodies reactive with trophoblast cells, which are involved in the establishment of the uteroplacental vasculature and maintenance of placental blood fluidity. To examine this hypothesis, we compared the incidence of trophoblast-reactive antibodies in 27 patients with ACLA and a history of fetal loss with that in 29 normal pregnant women. Sera from 20 patients, but only one control, contained trophoblast-reactive antibodies (P trophoblast cells. In most cases, sera from which ACLA were adsorbed by cardiolipin-containing liposomes maintained reactivity against cells. In addition, patient Ig fractions immunoprecipitated an approximately 62-kD protein from the trophoblast cell surface, stimulated the release of arachidonic acid and thromboxane A2 by trophoblasts, and inhibited the binding of prourokinase to trophoblast urokinase receptors. These observations show that sera from women with ACLA and a history of fetal loss contain antitrophoblast antibodies. These antibodies may be serologically distinct from ACLA, and may contribute to the pathogenesis of fetal demise. PMID:7693045

  18. Reactive arthritis.

    Science.gov (United States)

    Keat, A

    1999-01-01

    Reactive arthritis is one of the spondyloarthropathy family of clinical syndromes. The clinical features are those shared by other members of the spondyloarthritis family, though it is distinguished by a clear relationship with a precipitating infection. Susceptibility to reactive arthritis is closely linked with the class 1 HLA allele B27; it is likely that all sub-types pre-dispose to this condition. The link between HLA B27 and infection is mirrored by the development of arthritis in HLA B27-transgenic rats. In this model, arthritis does not develop in animals maintained in a germ-free environment. Infections of the gastrointestinal, genitourinary and respiratory tract appear to provoke reactive arthritis and a wide range of pathogens has now been implicated. Although mechanistic parallels may exist, reactive arthritis is distinguished from Lyme disease, rheumatic fever and Whipple's disease by virtue of the distinct clinical features and the link with HLA B27. As in these conditions both antigens and DNA of several micro-organisms have been detected in joint material from patients with reactive arthritis. The role of such disseminated microbial elements in the provocation or maintenance of arthritis remains unclear. HLA B27-restricted T-cell responses to microbial antigens have been demonstrated and these may be important in disease pathogenesis. The importance of dissemination of bacteria from sites of mucosal infection and their deposition in joints has yet to be fully understood. The role of antibiotic therapy in the treatment of reactive arthritis is being explored; in some circumstances, both the anti-inflammatory and anti-microbial effects of certain antibiotics appear to be valuable. The term reactive arthritis should be seen as a transitory one, reflecting a concept which may itself be on the verge of replacement, as our understanding of the condition develops. Nevertheless it appropriately describes arthritis that is associated with demonstrable

  19. Antibody-Directed Phototherapy (ADP

    Directory of Open Access Journals (Sweden)

    M. Adil Butt

    2013-04-01

    Full Text Available Photodynamic therapy (PDT is a clinically-approved but rather under-exploited treatment modality for cancer and pre-cancerous superficial lesions. It utilises a cold laser or LED to activate a photochemical reaction between a light activated drug (photosensitiser-drug and oxygen to generate cytotoxic oxygen species. These free radical species damage cellular components leading to cell death. Despite its benefits, the complexity, limited potency and side effects of PDT have led to poor general usage. However, the research area is very active with an increasing understanding of PDT-related cell biology, photophysics and significant progress in molecular targeting of disease. Monoclonal antibody therapy is maturing and the next wave of antibody therapies includes antibody-drug conjugates (ADCs, which promise to be more potent and curable. These developments could lift antibody-directed phototherapy (ADP to success. ADP promises to increase specificity and potency and improve drug pharmacokinetics, thus delivering better PDT drugs whilst retaining its other benefits. Whole antibody conjugates with first generation ADP-drugs displayed problems with aggregation, poor pharmacokinetics and loss of immuno-reactivity. However, these early ADP-drugs still showed improved selectivity and potency. Improved PS-drug chemistry and a variety of conjugation strategies have led to improved ADP-drugs with retained antibody and PS-drug function. More recently, recombinant antibody fragments have been used to deliver ADP-drugs with superior drug loading, more favourable pharmacokinetics, enhanced potency and target cell selectivity. These improvements offer a promise of better quality PDT drugs.

  20. Antibody-mediated neutralization of virus is abrogated by mycoplasma.

    OpenAIRE

    Dickson, C; Elkington, J; Hales, A.; Weiss, R.

    1980-01-01

    The ability of a mouse mammary tumor cell line to abrogate antibody neutralization of vesicular stomatitis virus was shown to be due to the presence of mycoplasma. The mycoplasma was isolated from the cell line and typed as Mycoplasma orale. Colonies of this mycoplasma were used to deliberately infect cell cultures which then gained the capacity to reactivate antibody-neutralized virus. The extent of the reactivation depended on the source of neutralizing antiserum. Other species of mycoplasm...

  1. Vaccine-Induced Antibodies that Neutralize Group 1 and Group 2 Influenza A Viruses.

    Science.gov (United States)

    Joyce, M Gordon; Wheatley, Adam K; Thomas, Paul V; Chuang, Gwo-Yu; Soto, Cinque; Bailer, Robert T; Druz, Aliaksandr; Georgiev, Ivelin S; Gillespie, Rebecca A; Kanekiyo, Masaru; Kong, Wing-Pui; Leung, Kwanyee; Narpala, Sandeep N; Prabhakaran, Madhu S; Yang, Eun Sung; Zhang, Baoshan; Zhang, Yi; Asokan, Mangaiarkarasi; Boyington, Jeffrey C; Bylund, Tatsiana; Darko, Sam; Lees, Christopher R; Ransier, Amy; Shen, Chen-Hsiang; Wang, Lingshu; Whittle, James R; Wu, Xueling; Yassine, Hadi M; Santos, Celia; Matsuoka, Yumiko; Tsybovsky, Yaroslav; Baxa, Ulrich; Mullikin, James C; Subbarao, Kanta; Douek, Daniel C; Graham, Barney S; Koup, Richard A; Ledgerwood, Julie E; Roederer, Mario; Shapiro, Lawrence; Kwong, Peter D; Mascola, John R; McDermott, Adrian B

    2016-07-28

    Antibodies capable of neutralizing divergent influenza A viruses could form the basis of a universal vaccine. Here, from subjects enrolled in an H5N1 DNA/MIV-prime-boost influenza vaccine trial, we sorted hemagglutinin cross-reactive memory B cells and identified three antibody classes, each capable of neutralizing diverse subtypes of group 1 and group 2 influenza A viruses. Co-crystal structures with hemagglutinin revealed that each class utilized characteristic germline genes and convergent sequence motifs to recognize overlapping epitopes in the hemagglutinin stem. All six analyzed subjects had sequences from at least one multidonor class, and-in half the subjects-multidonor-class sequences were recovered from >40% of cross-reactive B cells. By contrast, these multidonor-class sequences were rare in published antibody datasets. Vaccination with a divergent hemagglutinin can thus increase the frequency of B cells encoding broad influenza A-neutralizing antibodies. We propose the sequence signature-quantified prevalence of these B cells as a metric to guide universal influenza A immunization strategies. PMID:27453470

  2. A Novel Domain Cassette Identifies Plasmodium falciparum PfEMP1 Proteins Binding ICAM-1 and Is a Target of Cross-Reactive, Adhesion-Inhibitory Antibodies

    DEFF Research Database (Denmark)

    Bengtsson, Anja; Jørgensen, Louise; Rask, Thomas Salhøj;

    2013-01-01

    Cerebral Plasmodium falciparum malaria is characterized by adhesion of infected erythrocytes (IEs) to the cerebral microvasculature. This has been linked to parasites expressing the structurally related group A subset of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family of IE adhes...

  3. Monoclonal antibodies

    International Nuclear Information System (INIS)

    Monoclonal antibodies (MAbs) are antibodies having single specificity for a given antigen site (epitope). The development of hybridoma technology and the relative ease by which MAbs can be prepared has revolutionized many aspects of serological applications in diagnosis and differentiation of disease producing agents. The property of monospecificity offers advantages in diagnostic applications over polyclonal sera in that tests can be defined exactly with regard to the antigen detected and the affinity of reaction between the given antigenic site and the monoclonal reagent. In addition, MAbs offer better possibilities for test standardization, because the same reagent can be used in different laboratories. Such an MAb can be supplied by a central laboratory or 'grown' from hybridoma cells, ensuring that the resultant product is identical from laboratory to laboratory and that the part of the test involving the MAb reaction is the same. The methodologies for inoculation regimes, mice, cloning methods, selection of fusion partners, etc., have been validated extensively in developed country laboratories. The decision to establish a MAb production facility must be examined on a strict cost-benefit basis, since it is still expensive to produce a product. There are many MAbs available that should be sought to allow exploitation in developing tests. If a production facility is envisaged, it should produce reagents for national needs, i.e. there should be a clear problem oriented approach whereby exact needs are defined. In the field of veterinary applications, MAbs are the central reagent in many immunoassays based on the enzyme linked immunosorbent assay (ELISA). The development of specific tests for diagnosing diseases is dominated by MAbs and has been fuelled by a strong research base, mainly in developed countries allied to developing countries through the study of related diseases. Thus, there are very many assays dependent on MAbs, some of which form the basis of

  4. Antibody-mediated immune suppression is improved when blends of anti-RBC monoclonal antibodies are used in mice.

    Science.gov (United States)

    Bernardo, Lidice; Amash, Alaa; Marjoram, Danielle; Lazarus, Alan H

    2016-08-25

    Although the prevention of hemolytic disease of the fetus and newborn is highly effective using polyclonal anti-D, a recombinant alternative is long overdue. Unfortunately, anti-D monoclonal antibodies have been, at best, disappointing. To determine the primary attribute defining an optimal antibody, we assessed suppression of murine red blood cell (RBC) immunization by single-monoclonal antibodies vs defined blends of subtype-matched antibodies. Allogeneic RBCs expressing the HOD antigen (hen egg lysozyme [HEL]-ovalbumin-human transmembrane Duffy(b)) were transfused into naïve mice alone or together with selected combinations of HEL-specific antibodies, and the resulting suppressive effect was assessed by evaluating the antibody response. Polyclonal HEL antibodies dramatically inhibited the antibody response to the HOD antigen, whereas single-monoclonal HEL antibodies were less effective despite the use of saturating doses. A blend of monoclonal HEL-specific antibodies reactive with different HEL epitopes significantly increased the suppressive effect, whereas a blend of monoclonal antibodies that block each other's binding to the HEL protein did not increase suppression. In conclusion, these data show that polyclonal antibodies are superior to monoclonal antibodies at suppressing the immune response to the HOD cells, a feature that can be completely recapitulated using monoclonal antibodies to different epitopes. PMID:27330002

  5. Accumulation of reactivity to MBP sensitizes TRAIL mediated oligodendrocyte apoptosis in adult sub cortical white matter in a model for human multiple sclerosis.

    Science.gov (United States)

    Mir, Sajad; Ali, Farrah; Chauhan, Deepika; Arora, Rajesh; Khan, Haider A

    2016-04-01

    Reactivity to myelin associated proteins is the hallmark of human multiple sclerosis (M.S) and its experimental counterparts. However, the nature of such reactivity has not been described fully. Herein, we report that myelin basic protein (MBP) reactivity accumulates in a rat model for M.S. over a period of time and sensitizes TRAIL mediated progressive oligodendrocyte apoptosis. We used active immunization by Myelin Oligodendrocyte Glycoprotein (MOG, 50 μg) to study chronic remitting relapsing encephalomyelitis in rats. A time point analysis of the progressive disease revealed cumulative accumulation of anti myelin basic protein antibodies during the disease progression with minimal change in the anti-MOG antibodies. Increased reactivity to MBP was studied to sensitize TNF related apoptosis-inducing ligand (TRAIL) and other proinflammatory cytokines in a cumulative fashion leading to the Caspase dependent apoptosis of oligodendrocytes and myelin loss. In a rescue experiment, we could limit the demyelination and prevent disease progression by neutralizing the effector, TRAIL in an early stage of the disease. This is the first study to identify the accumulation of MBP antibodies in MOG induced EAE which possibly leads to TRAIL sensitized oligodendrocyte apoptosis in the white mater of EAE rats. This finding stresses on the need to study MBP antibody titers in M.S. patients and therefore might serve as an alternate marker for progressive demyelination. PMID:26477945

  6. Radioimmunoguided surgery using monoclonal antibody

    International Nuclear Information System (INIS)

    The potential proficiency of radioimmunoguided surgery in the intraoperative detection of tumors was assessed using labeled monoclonal antibody B72.3 in 66 patients with tissue-proved tumor. Monoclonal antibody B72.3 was injected 5 to 42 days preoperatively, and the hand-held gamma-detecting probe was used intraoperatively to detect the presence of tumor. Intraoperative probe counts of less than 20 every 2 seconds, or tumor-to-adjacent normal tissue ratios less than 2:1 were considered negative (system failure). Positive probe counts were detected in 5 of 6 patients with primary colon cancer (83 percent), in 31 of 39 patients with recurrent colon cancer (79 percent), in 4 of 5 patients with gastric cancer (80 percent), in 3 of 8 patients with breast cancer (37.5 percent), and in 4 of 8 patients with ovarian cancer (50 percent) undergoing second-look procedures. Additional patients in each group were scored as borderline positive. Overall, radioimmunoguided surgery using B72.3 identified tumors in 47 patients (71.2 percent), bordered on positive in 6 patients (9.1 percent), and failed to identify tumor in 13 patients (19.7 percent). Improved selection of patients for antigen-positive tumors, the use of higher affinity second-generation antibodies, alternate routes of antibody administration, alternate radionuclides, and more sophisticatedly bioengineered antibodies and antibody combinations should all lead to improvements in radioimmunoguided surgery

  7. ANTIBODIES DEFINING RAT ENDOTHELIAL-CELLS - RECA-1, A PAN-ENDOTHELIAL CELL-SPECIFIC MONOCLONAL-ANTIBODY

    NARCIS (Netherlands)

    DUIJVESTIJN, AM; VANGOOR, H; KLATTER, F; MAJOOR, GD; VANBUSSEL, E; VRIESMAN, PJCV

    1992-01-01

    We have been searching for antibodies reactive with rat endothelial cells. Two monoclonal antibodies (mAb), named RECA-1 and RECA-2 were produced and tested in immunoperoxidase staining on frozen sections of various rat tissues. Staining patterns were compared to those obtained with the mAbs OX-2, O

  8. Monoclonal antibodies.

    Science.gov (United States)

    2009-01-01

    The ability to produce and exploit monoclonal antibodies (mAbs) has revolutionized many areas of biological sciences. The unique property of an mAb is that it is a single species of immunoglobulin (IG) molecule. This means that the specificity of the interaction of the paratopes on the IG, with the epitopes on an antigenic target, is the same on every molecule. This property can be used to great benefit in immunoassays to provide tests of defined specificity and sensitivity, which improve the possibilities of standardization. The performance of assays can often be determined relating the actual weight of antibody (hence the number of molecules) to the activity. Often the production of an mAb against a specific epitope is the only way that biological entities can be differentiated. This chapter outlines the areas involving the development of assays based on mAbs. The problems involved address include the physical aspects of mAbs and how they may affect assay design and also the implications of results based on monospecific reagents. Often these are not fully understood, leading to assays that are less than satisfactory, which does not justify the relatively high cost of preparing and screening of mAbs. There are many textbooks and reviews dealing with the preparation of mAbs, the principles involved, and various purification and manipulative methods for the preparation of fragments and conjugation. There has been little general information attempting to summarize the best approaches to assay design using mAbs. Much time can be wasted through bad planning, and this is particularly relevant to mAbs. A proper understanding of some basic principles is essential. It is beyond the scope of this chapter to discuss all aspects, but major areas are highlighted. PMID:19219589

  9. Characterization of antibodies against ferret immunoglobulins, cytokines and CD markers

    DEFF Research Database (Denmark)

    Martel, Cyril Jean-Marie; Aasted, Bent

    immunoglobulins, we identified and characterized polyclonal antibodies towards ferret IgG, IgM and IgA. We also identified 22 monoclonal antibodies (mAbs) raised mostly against human CD markers which cross-reacted with ferret leukocytes. These antibodies were originally specific against human CD8, CD9, CD14, CD18...

  10. A study of cross-reactivity in serum samples from dogs positive for Leishmania sp., Babesia canis and Ehrlichia canis in enzyme-linked immunosorbent assay and indirect fluorescent antibody test.

    Science.gov (United States)

    Oliveira, Trícia Maria F de Sousa; Furuta, Patrícia I; de Carvalho, Débora; Machado, Rosangela Z

    2008-01-01

    To verify the presence of cross-reaction among leishmaniosis, ehrlichiosis and babesiosis in serological diagnostics used in human visceral leishmaniasis control programs, serum samples from leishmaniasis endemic and non-endemic areas were collected and tested by Indirect Fluorescent Antibody (IFAT) and Enzyme-linked immunosorbent assay (ELISA). All serum samples from endemic areas were positive for Leishmania sp., by ELISA and IFAT, 51% positive for Babesia canis and 43% for Ehrlichia canis by IFAT. None of the serum samples from non-endemic areas were positive for Leishmania sp., by IFAT, but 67% were positive for B. canis and 78% for E. canis using the same test. When tested by ELISA for Leishmania sp., four samples from non-endemic area were positive. These dogs were then located and no clinical signs, parasites or antibody was detected in new tests for a six month period. Only one of these 4 samples was positive for B. canis by IFAT and ELISA and three for E. canis by IFAT. The results of the work suggest a co-infection in the endemic area and no serological cross-reaction among these parasites by IFAT and ELISA. PMID:18554433

  11. Phage displayed peptides and anti-idiotype antibodies recognised by a monoclonal antibody directed against a diagnostic antigen of Mycoplasma capricolum subsp. capripneumoniae.

    Science.gov (United States)

    Bengurić, D R; Dungu, B; Thiaucourt, F; du Plessis, D H

    2001-07-26

    A monoclonal antibody (Mab 4.52) raised against Mycoplasma capricolum subsp. capripneumoniae (Mccp) cell lysate was used as a template to obtain substitute antigens recognised by its paratope. Two approaches were investigated: a 17-mer random peptide library displayed on the surface of a filamentous phage was screened by panning on the immobilised Mab 4.52 and anti-idiotype antibodies were generated by immunising a chicken with the F(ab')(2) fragments of the antibody. Analysis of the peptide sequences displayed by the isolated phages identified two peptides. Both contained two cysteine residues and had identical or similar amino acids in positions 5 (P), 8 (I/L) and 13 (L). The fusion phages were also recognised by Mab 4.52 in enzyme-linked immunosorbent assay (ELISA) and binding was shown by surface plasmon resonance. One of the peptides was a markedly better inhibitor (67%) of the binding of Mab 4.52 to its original antigen than the other (20%) at 1mg/ml. After absorption, to remove isotypic and allotypic reactivities, the anti-idiotype IgY was specifically recognised by Mab 4.52 in ELISA and was able to inhibit its binding to the original antigen, whereas anti-idiotype antibodies raised against a bluetongue virus-specific antibody had no effect. In spite of unequivocal binding of the anti-idiotype antibodies and the fusion phages to the paratope of Mab 4.52, goat antisera appeared not to react with either of the surrogate antigens. In contrast, the test sera bound to the original antigen suggesting that Mab 4.52 does not recognise exactly the same antigenic site as antibodies in the goat antisera. PMID:11376960

  12. Reactive Systems

    DEFF Research Database (Denmark)

    Aceto, Luca; Ingolfsdottir, Anna; Larsen, Kim Guldstrand;

    A reactive system comprises networks of computing components, achieving their goals through interaction among themselves and their environment. Thus even relatively small systems may exhibit unexpectedly complex behaviours. As moreover reactive systems are often used in safety critical systems......, the need for mathematically based formal methodology is increasingly important. There are many books that look at particular methodologies for such systems. This book offers a more balanced introduction for graduate students and describes the various approaches, their strengths and weaknesses, and when...... they are best used. Milner's CCS and its operational semantics are introduced, together with the notions of behavioural equivalences based on bisimulation techniques and with recursive extensions of Hennessy-Milner logic. In the second part of the book, the presented theories are extended to take timing issues...

  13. Hepatitis B virus reactivation in hepatitis B virus surface antigen negative patients receiving immunosuppression: A hidden threat

    OpenAIRE

    2013-01-01

    AIM: To present the characteristics and the course of a series of anti-hepatitis B virus core antibody (HBc) antibody positive patients, who experienced hepatitis B virus (HBV) reactivation after immunosuppression.

  14. Antibody-Mediated Lung Transplant Rejection

    OpenAIRE

    Hachem, Ramsey

    2012-01-01

    Antibody-mediated rejection after lung transplantation remains enigmatic. However, emerging evidence over the past several years suggests that humoral immunity plays an important role in allograft rejection. Indeed, the development of donor-specific antibodies after transplantation has been identified as an independent risk factor for acute cellular rejection and bronchiolitis obliterans syndrome. Furthermore, cases of acute antibody-mediated rejection resulting in severe allograft dysfunctio...

  15. Antibody therapy for Ebola

    Science.gov (United States)

    Qiu, Xiangguo; Kobinger, Gary P

    2014-01-01

    Ebola viruses can cause severe hemorrhagic fever in humans and nonhuman primates with fatality rates up to 90%, and are identified as biosafety level 4 pathogens and CDC Category A Agents of Bioterrorism. To date, there are no approved therapies and vaccines available to treat these infections. Antibody therapy was estimated to be an effective and powerful treatment strategy against infectious pathogens in the late 19th, early 20th centuries but has fallen short to meet expectations to widely combat infectious diseases. Passive immunization for Ebola virus was successful in 2012, after over 15 years of failed attempts leading to skepticism that the approach would ever be of potential benefit. Currently, monoclonal antibody (mAbs)-based therapies are the most efficient at reversing the progression of a lethal Ebola virus infection in nonhuman primates, which recapitulate the human disease with the highest similarity. Novel combinations of mAbs can even fully cure lethally infected animals after clinical symptoms and circulating virus have been detected, days into the infection. These new developments have reopened the door for using antibody-based therapies for filovirus infections. Furthermore, they are reigniting hope that these strategies will contribute to better control the spread of other infectious agents and provide new tools against infectious diseases. PMID:24503566

  16. Characterization of periplasmic protein BP26 epitopes of Brucella melitensis reacting with murine monoclonal and sheep antibodies.

    Directory of Open Access Journals (Sweden)

    Jinlang Qiu

    Full Text Available More than 35,000 new cases of human brucellosis were reported in 2010 by the Chinese Center for Disease Control and Prevention. An attenuated B. melitensis vaccine M5-90 is currently used for vaccination of sheep and goats in China. In the study, a periplasmic protein BP26 from M5-90 was characterized for its epitope reactivity with mouse monoclonal and sheep antibodies. A total of 29 monoclonal antibodies (mAbs against recombinant BP26 (rBP26 were produced, which were tested for reactivity with a panel of BP26 peptides, three truncated rBP26 and native BP26 containing membrane protein extracts (NMP of B. melitensis M5-90 in ELISA and Western-Blot. The linear, semi-conformational and conformational epitopes from native BP26 were identified. Two linear epitopes recognized by mAbs were revealed by 28 of 16mer overlapping peptides, which were accurately mapped as the core motif of amino acid residues ⁹³DRDLQTGGI¹⁰¹ (position 93 to 101 or residues ¹⁰⁴QPIYVYPD¹¹¹, respectively. The reactivity of linear epitope peptides, rBP26 and NMP was tested with 137 sheep sera by ELISAs, of which the two linear epitopes had 65-70% reactivity and NMP 90% consistent with the results of a combination of two standard serological tests. The results were helpful for evaluating the reactivity of BP26 antigen in M5-90.

  17. The Mutation Glu151Asp in the B-Component of the Bacillus cereus Non-Hemolytic Enterotoxin (Nhe) Leads to a Diverging Reactivity in Antibody-Based Detection Systems

    Science.gov (United States)

    Didier, Andrea; Jeßberger, Nadja; Krey, Victoria; Dietrich, Richard; Scherer, Siegfried; Märtlbauer, Erwin

    2015-01-01

    The ability of Bacillus cereus to cause foodborne toxicoinfections leads to increasing concerns regarding consumer protection. For the diarrhea-associated enterotoxins, the assessment of the non-hemolytic enterotoxin B (NheB) titer determined by a sandwich enzyme immunoassay (EIA) correlates best with in vitro cytotoxicity. In general, the regulation of enterotoxin expression of B. cereus is a coordinately-regulated process influenced by environmental, and probably also by host factors. As long as these factors are not completely understood, the currently-applied diagnostic procedures are based on indirect approaches to assess the potential virulence of an isolate. To date, sandwich EIA results serve as a surrogate marker to categorize isolates as either potentially low or highly toxic. Here, we report on a single amino acid exchange in the NheB sequence leading to an underestimation of the cytotoxic potential in a limited number of strains. During the screening of a large panel of B. cereus isolates, six showed uncommon features with low sandwich EIA titers despite high cytotoxicity. Sequence analysis revealed the point-mutation Glu151Asp in the potential binding region of the capture antibody. Application of this antibody also results in low titers in an indirect EIA format and shows variable detection intensities in Western-immunoblots. A commercially-available assay based on a lateral flow device detects all strains correctly as NheB producers in a qualitative manner. In conclusion, isolates showing low NheB titers should additionally be assayed in an indirect EIA or for their in vitro cytotoxicity to ensure a correct classification as either low or highly toxic. PMID:26569304

  18. The Mutation Glu151Asp in the B-Component of the Bacillus cereus Non-Hemolytic Enterotoxin (Nhe Leads to a Diverging Reactivity in Antibody-Based Detection Systems

    Directory of Open Access Journals (Sweden)

    Andrea Didier

    2015-11-01

    Full Text Available The ability of Bacillus cereus to cause foodborne toxicoinfections leads to increasing concerns regarding consumer protection. For the diarrhea-associated enterotoxins, the assessment of the non-hemolytic enterotoxin B (NheB titer determined by a sandwich enzyme immunoassay (EIA correlates best with in vitro cytotoxicity. In general, the regulation of enterotoxin expression of B. cereus is a coordinately-regulated process influenced by environmental, and probably also by host factors. As long as these factors are not completely understood, the currently-applied diagnostic procedures are based on indirect approaches to assess the potential virulence of an isolate. To date, sandwich EIA results serve as a surrogate marker to categorize isolates as either potentially low or highly toxic. Here, we report on a single amino acid exchange in the NheB sequence leading to an underestimation of the cytotoxic potential in a limited number of strains. During the screening of a large panel of B. cereus isolates, six showed uncommon features with low sandwich EIA titers despite high cytotoxicity. Sequence analysis revealed the point-mutation Glu151Asp in the potential binding region of the capture antibody. Application of this antibody also results in low titers in an indirect EIA format and shows variable detection intensities in Western-immunoblots. A commercially-available assay based on a lateral flow device detects all strains correctly as NheB producers in a qualitative manner. In conclusion, isolates showing low NheB titers should additionally be assayed in an indirect EIA or for their in vitro cytotoxicity to ensure a correct classification as either low or highly toxic.

  19. The mutation Glu151Asp in the B-component of the Bacillus cereus non-hemolytic enterotoxin (Nhe) leads to a diverging reactivity in antibody-based detection systems.

    Science.gov (United States)

    Didier, Andrea; Jeßberger, Nadja; Krey, Victoria; Dietrich, Richard; Scherer, Siegfried; Märtlbauer, Erwin

    2015-11-01

    The ability of Bacillus cereus to cause foodborne toxicoinfections leads to increasing concerns regarding consumer protection. For the diarrhea-associated enterotoxins, the assessment of the non-hemolytic enterotoxin B (NheB) titer determined by a sandwich enzyme immunoassay (EIA) correlates best with in vitro cytotoxicity. In general, the regulation of enterotoxin expression of B. cereus is a coordinately-regulated process influenced by environmental, and probably also by host factors. As long as these factors are not completely understood, the currently-applied diagnostic procedures are based on indirect approaches to assess the potential virulence of an isolate. To date, sandwich EIA results serve as a surrogate marker to categorize isolates as either potentially low or highly toxic. Here, we report on a single amino acid exchange in the NheB sequence leading to an underestimation of the cytotoxic potential in a limited number of strains. During the screening of a large panel of B. cereus isolates, six showed uncommon features with low sandwich EIA titers despite high cytotoxicity. Sequence analysis revealed the point-mutation (Glu)151(Asp) in the potential binding region of the capture antibody. Application of this antibody also results in low titers in an indirect EIA format and shows variable detection intensities in Western-immunoblots. A commercially-available assay based on a lateral flow device detects all strains correctly as NheB producers in a qualitative manner. In conclusion, isolates showing low NheB titers should additionally be assayed in an indirect EIA or for their in vitro cytotoxicity to ensure a correct classification as either low or highly toxic. PMID:26569304

  20. Differentiation of Naegleria fowleri from Acanthamoeba species by using monoclonal antibodies and flow cytometry.

    OpenAIRE

    Flores, B M; Garcia, C A; Stamm, W E; Torian, B E

    1990-01-01

    Monoclonal antibodies to Naegleria fowleri and Acanthamoeba polyphaga were analyzed by enzyme-linked immunosorbent assay, indirect immunofluorescence microscopy, and fluorescence flow cytometry to assess specificity and cross-reactivity with axenically cultured N. fowleri and Acanthamoeba spp. Four monoclonal antibodies to N. fowleri were specific for N. fowleri and had no reactivity to A. polyphaga. Similarly, four monoclonal antibodies to A. polyphaga did not react with N. fowleri. Two of t...

  1. What Is Reactive Arthritis?

    Science.gov (United States)

    ... Arthritis PDF Version Size: 69 KB November 2014 What is Reactive Arthritis? Fast Facts: An Easy-to- ... Information About Reactive Arthritis and Other Related Conditions What Causes Reactive Arthritis? Sometimes, reactive arthritis is set ...

  2. High throughput ranking of recombinant avian scFv antibody fragments from crude lysates using the Biacore A100.

    Science.gov (United States)

    Leonard, Paul; Säfsten, Pär; Hearty, Stephen; McDonnell, Barry; Finlay, William; O'Kennedy, Richard

    2007-06-30

    Advances in molecular evolution strategies have made it possible to identify antibodies with exquisite specificities and also to fine-tune their biophysical properties for practically any specified application. Depending on the desired function, antibody/antigen interactions can be long-lived or short-lived and, therefore, particular attention is needed when seeking to identify antibodies with specific reaction-rate and affinity properties. Surface plasmon resonance (SPR) biosensors routinely generate sensitive and reliable kinetic data from antibody/antigen interactions for both therapeutic and diagnostic applications. However, many kinetic-based screening assays require rigorous sample preparation and purification prior to analysis. To ameliorate this problem, we developed a rapid and reliable assay for characterising recombinant scFv antibody fragments, directly from crude bacterial lysates. Ninety-six scFv antibodies derived from chickens immunised with C-reactive protein (CRP) were selected by phage display and evaluated using the Biacore A100 protein interaction array system. Antibodies were captured from crude bacterial extracts on the sensor chip surface and ranked based on the percentage of the complex left (% left) after dissociation in buffer. Kinetic rate constants (k(a) and k(d)) and affinity (K(D)) data were obtained for six clones that bound monomeric CRP across a broad affinity range (2.54 x 10(-8) to 3.53 x 10(-10) M). Using this assay format the A100 biosensor yielded high quality kinetic data, permitting the screening of nearly 400 antibody clones per day. PMID:17532001

  3. The reactivity meter and core reactivity

    International Nuclear Information System (INIS)

    The point kinetic equations and the characteristics of the point kinetic reactivity meter are discussed, particularly for large negative reactivities. From a given input signal representing the neutron flux seen by a detector, the meter computes a value of reactivity in dollars (ρ/β), based on inverse point kinetics. The prompt jump point of view is emphasised. A simple point model of the reactor and a local flux distortion factor are used to generate input signals into a simulated reactivity meter. The obtained results show how the reading of the reactivity meter will approach the reactivity of the core model, if the reactivity is lower than -1 dollars. However, for reactivity values higher than -1 dollars, the influence of the flux distortion on the reading of the reactivity meter persists. Reactivity meter measurements taken during typical rod drop experiments in VVER-440 reactors do not produce accurate indications of the (static) core reactivity. (author)

  4. Antibody Treatment of Ebola and Sudan Virus Infection via a Uniquely Exposed Epitope within the Glycoprotein Receptor-Binding Site.

    Science.gov (United States)

    Howell, Katie A; Qiu, Xiangguo; Brannan, Jennifer M; Bryan, Christopher; Davidson, Edgar; Holtsberg, Frederick W; Wec, Anna Z; Shulenin, Sergey; Biggins, Julia E; Douglas, Robin; Enterlein, Sven G; Turner, Hannah L; Pallesen, Jesper; Murin, Charles D; He, Shihua; Kroeker, Andrea; Vu, Hong; Herbert, Andrew S; Fusco, Marnie L; Nyakatura, Elisabeth K; Lai, Jonathan R; Keck, Zhen-Yong; Foung, Steven K H; Saphire, Erica Ollmann; Zeitlin, Larry; Ward, Andrew B; Chandran, Kartik; Doranz, Benjamin J; Kobinger, Gary P; Dye, John M; Aman, M Javad

    2016-05-17

    Previous efforts to identify cross-neutralizing antibodies to the receptor-binding site (RBS) of ebolavirus glycoproteins have been unsuccessful, largely because the RBS is occluded on the viral surface. We report a monoclonal antibody (FVM04) that targets a uniquely exposed epitope within the RBS; cross-neutralizes Ebola (EBOV), Sudan (SUDV), and, to a lesser extent, Bundibugyo viruses; and shows protection against EBOV and SUDV in mice and guinea pigs. The antibody cocktail ZMapp™ is remarkably effective against EBOV (Zaire) but does not cross-neutralize other ebolaviruses. By replacing one of the ZMapp™ components with FVM04, we retained the anti-EBOV efficacy while extending the breadth of protection to SUDV, thereby generating a cross-protective antibody cocktail. In addition, we report several mutations at the base of the ebolavirus glycoprotein that enhance the binding of FVM04 and other cross-reactive antibodies. These findings have important implications for pan-ebolavirus vaccine development and defining broadly protective antibody cocktails. PMID:27160900

  5. 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.

  6. Factors associated with anti-human leukocyte antigen antibodies in patients supported with continuous-flow devices and effect on probability of transplant and post-transplant outcomes

    DEFF Research Database (Denmark)

    Alba, Ana C; Tinckam, Kathryn; Foroutan, Farid;

    2015-01-01

    outcomes. METHODS: We included 143 consecutive heart failure patients who received a CF-VAD as a bridge-to-transplant at 3 institutions. Factors associated with post-VAD peak panel reactive antibodies (PRA) among several measurements were identified using multivariable linear regression. A parametric......BACKGROUND: One major disadvantage of ventricular assist device (VAD) therapy is the development of human-leukocyte antigen (HLA) antibodies. We aimed to identify factors associated with HLA antibodies during continuous flow (CF)-VAD support and assess the effect on transplant probability and...... survival model was used to assess transplant waiting time and probability, risk of rejection, and a composite outcome of rejection, graft failure, and death. RESULTS: Thirty-six patients (25%) were female; mean age was 47 ± 13 years. Eighty-one patients (57%) had a pre-VAD PRA of 0%, and 16 were highly...

  7. Multiplex giant magnetoresistive biosensor microarrays identify interferon-associated autoantibodies in systemic lupus erythematosus

    Science.gov (United States)

    Lee, Jung-Rok; Haddon, D. James; Wand, Hannah E.; Price, Jordan V.; Diep, Vivian K.; Hall, Drew A.; Petri, Michelle; Baechler, Emily C.; Balboni, Imelda M.; Utz, Paul J.; Wang, Shan X.

    2016-06-01

    High titer, class-switched autoantibodies are a hallmark of systemic lupus erythematosus (SLE). Dysregulation of the interferon (IFN) pathway is observed in individuals with active SLE, although the association of specific autoantibodies with chemokine score, a combined measurement of three IFN-regulated chemokines, is not known. To identify autoantibodies associated with chemokine score, we developed giant magnetoresistive (GMR) biosensor microarrays, which allow the parallel measurement of multiple serum antibodies to autoantigens and peptides. We used the microarrays to analyze serum samples from SLE patients and found individuals with high chemokine scores had significantly greater reactivity to 13 autoantigens than individuals with low chemokine scores. Our findings demonstrate that multiple autoantibodies, including antibodies to U1-70K and modified histone H2B tails, are associated with IFN dysregulation in SLE. Further, they show the microarrays are capable of identifying autoantibodies associated with relevant clinical manifestations of SLE, with potential for use as biomarkers in clinical practice.

  8. Isolation of Balamuthia mandrillaris-specific antibody fragments from a bacteriophage antibody display library.

    Science.gov (United States)

    Siddiqui, Ruqaiyyah; Kulsoom, Huma; Lalani, Salima; Khan, Naveed Ahmed

    2016-07-01

    Balamuthia mandrillaris is a protist pathogen that can cause encephalitis with a mortality rate of more than 95%. Early diagnosis followed by aggressive treatment is a pre-requisite for successful prognosis. Current methods for identifying this organism rely on culture and microscopy, antibody-based methods using animals, or involve the use of molecular tools that are expensive. Here, we describe the isolation of antibody fragments that can be used for the unequivocal identification of B. mandrillaris. B. mandrillaris-specific antibody fragments were isolated from a bacteriophage antibody display library. Individual clones were studied by enzyme-linked immunosorbent assay, and immunofluorescence. Four antibody clones showed specific binding to B. mandrillaris. The usefulness of phage antibody display technology as a diagnostic tool for isolating antibody fragments against B. mandrillaris antigens and studying their biological role(s) is discussed further. PMID:27055361

  9. Characterization and evaluation of monoclonal antibodies developed for typing influenza A and influenza B viruses.

    OpenAIRE

    Walls, H H; Harmon, M W; Slagle, J J; Stocksdale, C; Kendal, A P

    1986-01-01

    Monoclonal antibodies that are broadly reactive with influenza A or influenza B viruses were produced as stable reagents for typing influenza viruses. Monoclonal antibodies to influenza A were specific for either matrix protein or nucleoprotein. The antibodies to influenza B were specific for nucleoprotein or hemagglutinin protein. In an enzyme immunoassay procedure, influenza A antibodies detected H1N1, H2N2, and H3N2 influenza A virus strains collected between 1934 and 1984. Each of the inf...

  10. Anti-Taenia solium monoclonal antibodies for the detection of parasite antigens in body fluids from patients with neurocysticercosis.

    Science.gov (United States)

    Paredes, Adriana; Sáenz, Patricia; Marzal, Miguel W; Orrego, Miguel A; Castillo, Yesenia; Rivera, Andrea; Mahanty, Siddhartha; Guerra-Giraldez, Cristina; García, Hector H; Nash, Theodore E

    2016-07-01

    Neurocysticercosis (NCC), an infection of the brain by Taenia solium (Ts) cysts, is the most common cause of adult-onset epilepsy in developing countries. Serological testing consists primarily of varying methods to detect antibodies in body fluids and more recently antigen (Ag) detection assays to identify individuals or animals with viable parasites. Antigen assays currently in use employ monoclonal antibodies (mAbs) raised against T. saginata, which have known cross reactivity to animal cestodes but are highly specific in human samples. We produced, characterized and tested 21 mAbs raised against T. solium whole cyst antigens, vesicular fluid or excretory secretory products. Reactivity of the TsmAbs against specific cyst structures was determined using immunofluorescence and immunohistochemistry on histological sections of Ts muscle cysts. Four TsmAbs reacted to vesicular space alone, 9 to the neck and cyst wall, one to the neck and vesicular space and 7 to the neck, cyst wall and vesicular space. An in-house ELISA assay to detect circulating Ts antigen, using the TsmAbs as capture antibodies and a rabbit polyclonal anti-Ts whole cyst antibody as a detector antibody demonstrated that eight of the 21 TsmAbs detected antigens in known NCC-positive human sera and three of these also in urine samples. Reactivity was expressed as normalized ratios of optical densities (OD positive control/OD negative control). Three TsmAbs had ratios >10 and five between 2 and 10. The TsmAbs have potential utility for the diagnosis and post-treatment monitoring of patients with viable NCC infections. PMID:27018063

  11. Antibodies and Selection of Monoclonal Antibodies.

    Science.gov (United States)

    Hanack, Katja; Messerschmidt, Katrin; Listek, Martin

    2016-01-01

    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. PMID:27236550

  12. Specific immunoradiometric assay of insulin-like growth factor I with use of monoclonal antibodies

    International Nuclear Information System (INIS)

    We identified two monoclonal antibodies that bind spatially distinct epitopes on insulin-like growth factor I (IGF-I). Using these two antibodies, we developed a simultaneous, two-site immunoradiometric assay (IRMA) specific for IGF-I. This IRMA has no detectable cross reactivity with insulin, proinsulin, prolactin, or somatotropin, and less than 2% crossreactivity with IGF-II. The assay response varies linearly with IGF-I concentrations of 0-800 micrograms/L in serum; the detection limit is about 10 micrograms/L. A comparison of 26 IGF-I serum values from the IRMA and from a previously reported IGF-I specific RIA gave a correlation coefficient of 0.96 with no substantial bias (slope = 1.10). IGF-I values for serum, as an aid in assessing growth abnormalities, are easily (only three pipetting steps) obtained in less than 4 h

  13. Specific immunoradiometric assay of insulin-like growth factor I with use of monoclonal antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Scott, M.G.; Cuca, G.C.; Petersen, J.R.; Lyle, L.R.; Burleigh, B.D.; Daughaday, W.H.

    1987-11-01

    We identified two monoclonal antibodies that bind spatially distinct epitopes on insulin-like growth factor I (IGF-I). Using these two antibodies, we developed a simultaneous, two-site immunoradiometric assay (IRMA) specific for IGF-I. This IRMA has no detectable cross reactivity with insulin, proinsulin, prolactin, or somatotropin, and less than 2% crossreactivity with IGF-II. The assay response varies linearly with IGF-I concentrations of 0-800 micrograms/L in serum; the detection limit is about 10 micrograms/L. A comparison of 26 IGF-I serum values from the IRMA and from a previously reported IGF-I specific RIA gave a correlation coefficient of 0.96 with no substantial bias (slope = 1.10). IGF-I values for serum, as an aid in assessing growth abnormalities, are easily (only three pipetting steps) obtained in less than 4 h.

  14. 42 CFR 493.865 - Standard; Antibody identification.

    Science.gov (United States)

    2010-10-01

    ... 42 Public Health 5 2010-10-01 2010-10-01 false Standard; Antibody identification. 493.865 Section..., Or Any Combination of These Tests § 493.865 Standard; Antibody identification. (a) Failure to attain... proficiency testing event. (e) Failure to identify the same antibody in two consecutive or two out of...

  15. Quantitative Assessment of Antibody Internalization with Novel Monoclonal Antibodies against Alexa Fluorophores

    OpenAIRE

    Liao-Chan, Sindy; Daine-Matsuoka, Barbara; Heald, Nathan; Wong, Tiffany; Lin, Tracey; Cai, Allen G.; Lai, Michelle; D’Alessio, Joseph A.; Theunissen, Jan-Willem

    2015-01-01

    Antibodies against cell surface antigens may be internalized through their specific interactions with these proteins and in some cases may induce or perturb antigen internalization. The anti-cancer efficacy of antibody-drug conjugates is thought to rely on their uptake by cancer cells expressing the surface antigen. Numerous techniques, including microscopy and flow cytometry, have been used to identify antibodies with desired cellular uptake rates. To enable quantitative measurements of inte...

  16. Development of monoclonal antibodies suitable for rabies virus antibody and antigen detection.

    Science.gov (United States)

    Chander, Vishal; Singh, R P; Verma, P C

    2012-12-01

    The control of an infectious viral disease as rabies is made easier by rapid and accurate diagnosis. Successful rabies prophylaxis is dependent upon the active immunization with vaccine along with passive administration of rabies virus neutralizing antibodies which together clear the virus before widespread infection of central nervous system occurs. The present study aimed at the development of monoclonal antibodies (MAbs) suitable for rabies virus antibody and antigen detection. For the production of rabies specific MAbs, immunization of Swiss albino mice with a commercially available vaccine was done and Polyethylene glycol mediated fusion of spleenocytes with myeloma cells was performed. The positive clones were selected on the basis of distinct reactivity by cell Enzyme linked immunosorbent assay and fluorescence in Indirect Fluorescent antibody test. The positive clones obtained were subjected to single cell cloning by limiting dilution method. The reactive clones were further titrated and employed for virus titration and virus neutralization. The neutralizing activity was evaluated using Fluorescence Activated Cell Sorter technique. Three MAb clones showed a distinct percent inhibition in the presence of positive serum. One of the MAb clone No. 5C3 was relatively more specific in detecting rabies antibodies and also found suitable for competitive ELISA to assess the antibody level in vaccinated subjects. PMID:24293819

  17. Simultaneous identification of Trypanosoma cruzi surface and internal antigens reactive to different immunoglobulin classes (radio-immunoblotting)

    International Nuclear Information System (INIS)

    A radioactive Western blotting technique was developed by which the reactivity of Immunoglobulins (IGs) from different classes to both membrane radiolabelled and internal parasite antigens is simultaneously identified. The method includes radioiodination of parasites, polypeptide fractionation by SDS-PAGE, Western-blot transfer and autoradiography of the immunoblots developed with anti-Igs conjugates labelled with enzymes. The analysis is then performed by the comparison of common bands on the autoradiograms and the respective substrate stained nitrocellulose blots. This technique was used to analyse. T.cruzi trypomastigote surface labelled antigens reactive to IgM, IgA and IgC specific antibodies. A different pattern of reactivity with acute Chagas disease patients sera was thus obtained. (author)

  18. Anti-HmuY antibodies specifically recognize Porphyromonas gingivalis HmuY protein but not homologous proteins in other periodontopathogens.

    Directory of Open Access Journals (Sweden)

    Michał Śmiga

    Full Text Available Given the emerging evidence of an association between periodontal infections and systemic conditions, the search for specific methods to detect the presence of P. gingivalis, a principal etiologic agent in chronic periodontitis, is of high importance. The aim of this study was to characterize antibodies raised against purified P. gingivalis HmuY protein and selected epitopes of the HmuY molecule. Since other periodontopathogens produce homologs of HmuY, we also aimed to characterize responses of antibodies raised against the HmuY protein or its epitopes to the closest homologous proteins from Prevotella intermedia and Tannerella forsythia. Rabbits were immunized with purified HmuY protein or three synthetic, KLH-conjugated peptides, derived from the P. gingivalis HmuY protein. The reactivity of anti-HmuY antibodies with purified proteins or bacteria was determined using Western blotting and ELISA assay. First, we found homologs of P. gingivalis HmuY in P. intermedia (PinO and PinA proteins and T. forsythia (Tfo protein and identified corrected nucleotide and amino acid sequences of Tfo. All proteins were overexpressed in E. coli and purified using ion-exchange chromatography, hydrophobic chromatography and gel filtration. We demonstrated that antibodies raised against P. gingivalis HmuY are highly specific to purified HmuY protein and HmuY attached to P. gingivalis cells. No reactivity between P. intermedia and T. forsythia or between purified HmuY homologs from these bacteria and anti-HmuY antibodies was detected. The results obtained in this study demonstrate that P. gingivalis HmuY protein may serve as an antigen for specific determination of serum antibodies raised against this bacterium.

  19. Anti-HmuY antibodies specifically recognize Porphyromonas gingivalis HmuY protein but not homologous proteins in other periodontopathogens.

    Science.gov (United States)

    Śmiga, Michał; Bielecki, Marcin; Olczak, Mariusz; Smalley, John W; Olczak, Teresa

    2015-01-01

    Given the emerging evidence of an association between periodontal infections and systemic conditions, the search for specific methods to detect the presence of P. gingivalis, a principal etiologic agent in chronic periodontitis, is of high importance. The aim of this study was to characterize antibodies raised against purified P. gingivalis HmuY protein and selected epitopes of the HmuY molecule. Since other periodontopathogens produce homologs of HmuY, we also aimed to characterize responses of antibodies raised against the HmuY protein or its epitopes to the closest homologous proteins from Prevotella intermedia and Tannerella forsythia. Rabbits were immunized with purified HmuY protein or three synthetic, KLH-conjugated peptides, derived from the P. gingivalis HmuY protein. The reactivity of anti-HmuY antibodies with purified proteins or bacteria was determined using Western blotting and ELISA assay. First, we found homologs of P. gingivalis HmuY in P. intermedia (PinO and PinA proteins) and T. forsythia (Tfo protein) and identified corrected nucleotide and amino acid sequences of Tfo. All proteins were overexpressed in E. coli and purified using ion-exchange chromatography, hydrophobic chromatography and gel filtration. We demonstrated that antibodies raised against P. gingivalis HmuY are highly specific to purified HmuY protein and HmuY attached to P. gingivalis cells. No reactivity between P. intermedia and T. forsythia or between purified HmuY homologs from these bacteria and anti-HmuY antibodies was detected. The results obtained in this study demonstrate that P. gingivalis HmuY protein may serve as an antigen for specific determination of serum antibodies raised against this bacterium. PMID:25658942

  20. Antibody profiling sensitivity through increased reporter antibody layering

    Energy Technology Data Exchange (ETDEWEB)

    Apel, William A; Thompson, Vicki S

    2013-02-26

    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  1. Antibody profiling sensitivity through increased reporter antibody layering

    Energy Technology Data Exchange (ETDEWEB)

    Apel, William A.; Thompson, Vicki S.

    2013-02-26

    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  2. Antibody profiling sensitivity through increased reporter antibody layering

    Energy Technology Data Exchange (ETDEWEB)

    Apel, William A.; Thompson, Vicki S

    2010-04-13

    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  3. Antibody profiling sensitivity through increased reporter antibody layering

    International Nuclear Information System (INIS)

    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  4. "Unconventional" Neutralizing Activity of Antibodies Against HIV

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Neutralizing antibodies are recognized to be one of the essential elements of the adaptive immune response that must be induced by an effective vaccine against HIV. However, only a limited number of antibodies have been identified to neutralize a broad range of primary isolates of HIV-1 and attempts to induce such antibodies by immunization were unsuccessful. The difficulties to generate such antibodies are mainly due to intrinsic properties of HIV-1 envelope spikes, such as high sequence diversity, heavy glycosylation, and inducible and transient nature of certain epitopes. In vitro neutralizing antibodies are identified using "conventional" neutralization assay which uses phytohemagglutinin (PHA)-stimulated human PBMCs as target cells. Thus, in essence the assay evaluates HIV-1 replication in CD4+ T cells. Recently, several laboratories including us demonstrated that some monoclonal antibodies and HIV-1-specific polyclonal IgG purified from patient sera, although they do not have neutralizing activity when tested by the "conventional" neutralization assay, do exhibit potent and broad neutralizing activity in "unconventional" ways. The neutralizing activity of these antibodies and IgG fractions is acquired through post-translational modifications, through opsonization of virus particles into macrophages and immature dendritic cells (iDCs), or through expression of antibodies on the surface of HIV-1-susceptible cells. This review will focus on recent findings of this area and point out their potential applications in the development of preventive strategies against HIV.

  5. Diagnostic potential of recombinant scFv antibodies generated against hemagglutinin protein of influenza A virus

    Directory of Open Access Journals (Sweden)

    Roopali eRajput

    2015-09-01

    Full Text Available Human influenza A viruses have been the cause of enormous socio-economic losses worldwide. In order to combat such a notorious pathogen, hemagglutinin protein (HA has been a preferred target for generation of neutralizing-antibodies, as potent therapeutic/ diagnostic agents. In the present study, recombinant anti-HA single chain variable fragment (scFv antibodies were constructed using the phage display technology to aid in diagnosis and treatment of human influenza A virus infections. Spleen cells of mice hyper-immunized with A/New Caledonia/20/99 (H1N1 virus were used as the source for recombinant antibody (rAb production. The antigen-binding phages were quantified after 6 rounds of bio-panning against A/New Caledonia/20/99 (H1N1, A/California/07/2009 (H1N1-like, or A/Udorn/307/72(H3N2 viruses. The phage yield was maximum for the A/New Caledonia/20/99 (H1N1, however, considerable cross-reactivity was observed for the other virus strains as well. The HA-specific polyclonal rAb preparation was subjected to selection of single clones for identification of high reactive relatively conserved epitopes. The high affinity rAbs were tested against certain known conserved HA epitopes by peptide ELISA. Three recombinant mAbs showed reactivity with both the H1N1 strains and one (C5 showed binding with all the three viral strains. The C5 antibody was thus used for development of an ELISA test for diagnosis of influenza virus infection. Based on the sample size in the current analysis, the ELISA test demonstrated 83.9% sensitivity and 100% specificity. Thus, the ELISA, developed in our study, may prove as a cheaper alternative to the presently used real time RT-PCR test for detection of human influenza A viruses in clinical specimens, which will be beneficial, especially in the developing countries. Since, the two antibodies identified in this study are reactive to conserved HA epitopes; these may prove as potential therapeutic agents as well.

  6. Antiphospholipid Antibody Syndrome

    Science.gov (United States)

    ... page from the NHLBI on Twitter. What Is Antiphospholipid Antibody Syndrome? Antiphospholipid (AN-te-fos-fo-LIP-id) antibody ... weeks or months. This condition is called catastrophic antiphospholipid syndrome (CAPS). People who have APS also are at ...

  7. Quality control of antibodies for assay development.

    Science.gov (United States)

    Schumacher, Sarah; Seitz, Harald

    2016-09-25

    Antibodies are used as powerful tools in basic research, for example, in biomarker identification, and in various forms for diagnostics, for example, identification of allergies or autoimmune diseases. Due to their robustness and ease of handling, immunoassays are favourite methods for investigation of various biological or medical questions. Nevertheless in many cases, additional analyses such as mass spectrometry are used to validate or confirm the results of immunoassays. To minimize the workload and to increase confidence in immunoassays, there are urgent needs for antibodies which are both highly specific and well validated. Unfortunately many commercially available antibodies are neither well characterized nor fully tested for cross-reactivities. Adequate quality control and validation of an antibody is time-consuming and can be frustrating. Such validation needs to be performed for every assay/application. However, where an antibody validation is successful, a highly specific and stable reagent will be on hand. This article describes the validation processes of antibodies, including some often neglected factors, as well as unspecific binding to other sample compounds in a multiparameter diagnostic assay. The validation consists of different immunological methods, with important assay controls, and is performed in relation to the development of a diagnostic test. PMID:26873787

  8. Pathological conformations involving the amino terminus of tau occur early in Alzheimer's disease and are differentially detected by monoclonal antibodies.

    Science.gov (United States)

    Combs, Benjamin; Hamel, Chelsey; Kanaan, Nicholas M

    2016-10-01

    Conformational changes involving the amino terminus of the tau protein are among the earliest alterations associated with tau pathology in Alzheimer's disease and other tauopathies. This region of tau contains a phosphatase-activating domain (PAD) that is aberrantly exposed in pathological forms of the protein, an event that is associated with disruptions in anterograde fast axonal transport. We utilized four antibodies that recognize the amino terminus of tau, TNT1, TNT2 (a novel antibody), Tau12, and Tau13, to further study this important region. Using scanning alanine mutations in recombinant tau proteins, we refined the epitopes of each antibody. We examined the antibodies' relative abilities to specifically label pathological tau in non-denaturing and denaturing assays to gain insight into some of the mechanistic details of PAD exposure. We then determined the pattern of tau pathology labeled by each antibody in human hippocampal sections at various disease stages in order to characterize PAD exposure in the context of disease progression. The characteristics of reactivity for the antibodies fell into two groups. TNT1 and TNT2 recognized epitopes within amino acids 7-12 and specifically identified recombinant tau aggregates and pathological tau from Alzheimer's disease brains in a conformation-dependent manner. These antibodies labeled early pre-tangle pathology from neurons in early Braak stages and colocalized with thiazine red, a marker of fibrillar pathology, in classic neurofibrillary tangles. However, late tangles were negative for TNT1 and TNT2 indicating a loss of the epitope in later stages of tangle evolution. In contrast, Tau12 and Tau13 both identified discontinuous epitopes in the amino terminus and were unable to differentiate between normal and pathological tau in biochemical and tissue immunohistological assays. Despite the close proximity of these epitopes, the antibodies demonstrated remarkably different abilities to identify pathological

  9. The antibody mining toolbox

    OpenAIRE

    D'Angelo, Sara; Glanville, Jacob; Ferrara, Fortunato; Naranjo, Leslie; Gleasner, Cheryl D.; Shen, Xiaohong; Bradbury, Andrew RM; Kiss, Csaba

    2013-01-01

    In vitro selection has been an essential tool in the development of recombinant antibodies against various antigen targets. Deep sequencing has recently been gaining ground as an alternative and valuable method to analyze such antibody selections. The analysis provides a novel and extremely detailed view of selected antibody populations, and allows the identification of specific antibodies using only sequencing data, potentially eliminating the need for expensive and laborious low-throughput ...

  10. 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...

  11. Hepatitis A virus antibody

    International Nuclear Information System (INIS)

    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 125I-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)

  12. Assembly and antigenicity of the Neisseria gonorrhoeae pilus mapped with antibodies.

    Science.gov (United States)

    Forest, K T; Bernstein, S L; Getzoff, E D; So, M; Tribbick, G; Geysen, H M; Deal, C D; Tainer, J A

    1996-02-01

    The relationship between the sequence of Neisseria gonorrhoeae pilin and its quaternary assembly into pilus fibers was studied with a set of site-directed antibody probes and by mapping the specificities of antipilus antisera with peptides. Buried and exposed peptides in assembled pili were identified by competitive immunoassays and immunoelectron microscopy with polyclonal antibodies raised against 11 peptides spanning the pilin sequence. Pili did not compete significantly with pilin subunits for binding to antibodies against residues 13 to 31 (13-31) and 18-36. Pilus fibers competed well with pilin protein subunits for binding to antibodies raised against peptides 37-56, 58-78, 110-120, 115-127, 122-139, and 140-159 and competed weakly for antibodies against residues 79-93 and 94-108. Antibodies to sequence-conserved residues 37-56 and to semiconserved residues 94-108 preferentially bound pilus ends as shown by immunoelectron microscopy. The exposure of pilus regions to the immune system was tested by peptide mapping of antiserum specificities against sets of overlapping peptides representing all possible hexameric or octameric peptides from the N. gonorrhoeae MS11 pilin sequence. The immunogenicity of exposed peptides incorporating semiconserved residues 49-56 and 121-126 was revealed by strong, consistent antigenic reactivity to these regions measured in antipilus sera from rabbits, mice, and human and in sera from human volunteers with gonorrhea. The conservation and variation of antigenic responses among these three species clarify the relevance of immunological studies of other species to the human immune response against pathogens. Overall, our results explain the extreme conservation of the entire N-terminal one-third of the pilin protein by its dominant role in pilus assembly: hydrophobic residues 1-36 are implicated in buried lateral contacts, and polar residues 37-56 are implicated in longitudinal contacts within the pilus fiber. PMID:8550220

  13. Generation and characterization of function-blocking anti-ectodysplasin A (EDA) monoclonal antibodies that induce ectodermal dysplasia.

    Science.gov (United States)

    Kowalczyk-Quintas, Christine; Willen, Laure; Dang, Anh Thu; Sarrasin, Heidi; Tardivel, Aubry; Hermes, Katharina; Schneider, Holm; Gaide, Olivier; Donzé, Olivier; Kirby, Neil; Headon, Denis J; Schneider, Pascal

    2014-02-14

    Development of ectodermal appendages, such as hair, teeth, sweat glands, sebaceous glands, and mammary glands, requires the action of the TNF family ligand ectodysplasin A (EDA). Mutations of the X-linked EDA gene cause reduction or absence of many ectodermal appendages and have been identified as a cause of ectodermal dysplasia in humans, mice, dogs, and cattle. We have generated blocking antibodies, raised in Eda-deficient mice, against the conserved, receptor-binding domain of EDA. These antibodies recognize epitopes overlapping the receptor-binding site and prevent EDA from binding and activating EDAR at close to stoichiometric ratios in in vitro binding and activity assays. The antibodies block EDA1 and EDA2 of both mammalian and avian origin and, in vivo, suppress the ability of recombinant Fc-EDA1 to rescue ectodermal dysplasia in Eda-deficient Tabby mice. Moreover, administration of EDA blocking antibodies to pregnant wild type mice induced in developing wild type fetuses a marked and permanent ectodermal dysplasia. These function-blocking anti-EDA antibodies with wide cross-species reactivity will enable study of the developmental and postdevelopmental roles of EDA in a variety of organisms and open the route to therapeutic intervention in conditions in which EDA may be implicated. PMID:24391090

  14. Generation and Characterization of Function-blocking Anti-ectodysplasin A (EDA) Monoclonal Antibodies That Induce Ectodermal Dysplasia*

    Science.gov (United States)

    Kowalczyk-Quintas, Christine; Willen, Laure; Dang, Anh Thu; Sarrasin, Heidi; Tardivel, Aubry; Hermes, Katharina; Schneider, Holm; Gaide, Olivier; Donzé, Olivier; Kirby, Neil; Headon, Denis J.; Schneider, Pascal

    2014-01-01

    Development of ectodermal appendages, such as hair, teeth, sweat glands, sebaceous glands, and mammary glands, requires the action of the TNF family ligand ectodysplasin A (EDA). Mutations of the X-linked EDA gene cause reduction or absence of many ectodermal appendages and have been identified as a cause of ectodermal dysplasia in humans, mice, dogs, and cattle. We have generated blocking antibodies, raised in Eda-deficient mice, against the conserved, receptor-binding domain of EDA. These antibodies recognize epitopes overlapping the receptor-binding site and prevent EDA from binding and activating EDAR at close to stoichiometric ratios in in vitro binding and activity assays. The antibodies block EDA1 and EDA2 of both mammalian and avian origin and, in vivo, suppress the ability of recombinant Fc-EDA1 to rescue ectodermal dysplasia in Eda-deficient Tabby mice. Moreover, administration of EDA blocking antibodies to pregnant wild type mice induced in developing wild type fetuses a marked and permanent ectodermal dysplasia. These function-blocking anti-EDA antibodies with wide cross-species reactivity will enable study of the developmental and postdevelopmental roles of EDA in a variety of organisms and open the route to therapeutic intervention in conditions in which EDA may be implicated. PMID:24391090

  15. Antibody-Mediated Lung Transplant Rejection

    Science.gov (United States)

    Hachem, Ramsey

    2012-01-01

    Antibody-mediated rejection after lung transplantation remains enigmatic. However, emerging evidence over the past several years suggests that humoral immunity plays an important role in allograft rejection. Indeed, the development of donor-specific antibodies after transplantation has been identified as an independent risk factor for acute cellular rejection and bronchiolitis obliterans syndrome. Furthermore, cases of acute antibody-mediated rejection resulting in severe allograft dysfunction have been reported, and these demonstrate that antibodies can directly injure the allograft. However, the incidence and toll of antibody-mediated rejection are unknown because there is no widely accepted definition and some cases may be unrecognized. Clearly, humoral immunity has become an important area for research and clinical investigation. PMID:23002428

  16. Monoclonal antibodies and cancer

    International Nuclear Information System (INIS)

    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 111In, 67Ga and 131I. 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

  17. Chain specificity assignment of monoclonal antibodies to human laminins by using recombinant laminin beta1 and gamma1 chains.

    Science.gov (United States)

    Geberhiwot, T; Wondimu, Z; Salo, S; Pikkarainen, T; Kortesmaa, J; Tryggvason, K; Virtanen, I; Patarroyo, M

    2000-05-01

    In the present study, the chain specificity of 16 commonly used monoclonal antibodies to human laminin(s) was analysed by using recombinant laminin beta1 and gamma1 chains. By ELISA, all antibodies reacted with purified placenta laminin, and most antibodies recognised either recombinant beta1 or gamma1 chains. Reactivity and chain specificity was confirmed against the recombinant chains in Western blotting under non-reducing conditions, and only a few antibodies were reactive under reducing conditions. Most antibodies were able to immunoprecipitate associated laminin beta1/gamma1 chains from platelet lysates. Based on these results and data from the literature, a tentative epitope map is presented. PMID:10842099

  18. Influenza-Specific Antibody-Dependent Phagocytosis

    Science.gov (United States)

    Ana-Sosa-Batiz, Fernanda; Vanderven, Hillary; Jegaskanda, Sinthujan; Johnston, Angus; Rockman, Steven; Laurie, Karen; Barr, Ian; Reading, Patrick; Lichtfuss, Marit; Kent, Stephen J.

    2016-01-01

    Background Immunity to human influenza A virus (IAV) infection is only partially understood. Broadly non-neutralizing antibodies may assist in reducing disease but have not been well characterized. Methods We measured internalization of opsonized, influenza protein-coated fluorescent beads and live IAV into a monocytic cell line to study antibody-dependent phagocytosis (ADP) against multiple influenza hemagglutinin (HA) subtypes. We analyzed influenza HA-specific ADP in healthy human donors, in preparations of intravenous immunoglobulin (IVIG), and following IAV infection of humans and macaques. Results We found that both sera from healthy adults and IVIG preparations had broad ADP to multiple seasonal HA proteins and weak cross-reactive ADP to non-circulating HA proteins. The ADP in experimentally influenza-infected macaque plasma and naturally influenza-infected human sera mediated phagocytosis of both homologous and heterologous IAVs. Further, the IAV phagocytosed in an antibody-mediated manner had reduced infectivity in vitro. Conclusion We conclude that IAV infections in humans and macaques leads to the development of influenza-specific ADP that can clear IAV infection in vitro. Repeated exposure of humans to multiple IAV infections likely leads to the development of ADP that is cross-reactive to strains not previously encountered. Further analyses of the protective capacity of broadly reactive influenza-specific ADP is warranted. PMID:27124730

  19. Effect of anti-mosquito hemolymph antibodies on fecundity and on the infectivity of malarial parasite Plasmodium vivax to Anopheles stephensi (Diptera:Insecta).

    Science.gov (United States)

    Gulia, Monika; Suneja, Amita; Gakhar, Surendra K

    2002-06-01

    Rabbit antibodies to hemolymph antigens (102.5, 101, 100, 96, 88, 80, 64, 55, 43, 29, and 23 kDa) of Anopheles stephensi reduced fecundity as well as viability in An. stephensi. However, ingestion of these antibodies was not associated with a marked effect on the engorgement of mosquitoes but egg laying was significantly delayed. Antisera raised against hemolymph proteins were also used to identify cross reactive antigens/epitopes present in other tissues by Western blotting, as well as by in vivo ELISA. In addition, a significant reduction in oocyst development was also observed in An. stephensi mosquitoes that ingested anti-hemolymph antibodies along with Plasmodium vivax. The results confirmed the feasibility of targeting mosquito antigens as a novel anti-mosquito strategy, as well as confirmed the usefulness of such antigens for the development of a transmission-blocking vaccine. PMID:12195047

  20. Mechanisms of Mitochondrial Damage in Keratinocytes by Pemphigus Vulgaris Antibodies*

    OpenAIRE

    Kalantari-Dehaghi, Mina; Chen, Yumay; Deng, Wu; Chernyavsky, Alex; Marchenko, Steve; Wang, Ping H.; Grando, Sergei A.

    2013-01-01

    Background: Previous studies suggested that mitochondrial antibodies contribute to pemphigus vulgaris (PV).Results: PV sera elicited mitochondrial damage, and mitochondria-protecting drugs exhibited protective effect in cell culture and mouse skin.Conclusion: PV antibodies altered O2 respiration, disrupted electron transfer chain, and increased reactive oxygen species.Significance: Results provide the mechanism of therapeutic action and justify the use of mitochondria-protecting drugs in PV.T...

  1. Comprehensive Mapping Antigenic Epitopes of NS1 Protein of Japanese Encephalitis Virus with Monoclonal Antibodies.

    Science.gov (United States)

    Hua, Rong-Hong; Liu, Li-Ke; Chen, Zhen-Shi; Li, Ye-Nan; Bu, Zhi-Gao

    2013-01-01

    Japanese encephalitis virus (JEV) non-structural protein 1 (NS1) contributes to virus replication and elicits protective immune responses during infection. JEV NS1-specific antibody responses could be a target in the differential diagnosis of different flavivirus infections. However, the epitopes on JEV NS1 are poorly characterized. The present study describes the full mapping of linear B-cell epitopes in JEV NS1. We generated eleven NS1-specific monoclonal antibodies from mice immunized with recombinant NS1. For epitope mapping of monoclonal antibodies, a set of 51 partially-overlapping peptides covering the entire NS1 protein were expressed with a GST-tag and then screened using monoclonal antibodies. Through enzyme-linked immunosorbent assay (ELISA), five linear epitope-containing peptides were identified. By sequentially removing amino acid residues from the carboxy and amino terminal of peptides, the minimal units of the five linear epitopes were identified and confirmed using monoclonal antibodies. Five linear epitopes are located in amino acids residues (5)AIDITRK(11), (72)RDELNVL(78), (251)KSKHNRREGY(260), (269)DENGIVLD(276), and (341)DETTLVRS(348). Furthermore, it was found that the epitopes are highly conserved among JEV strains through sequence alignment. Notably, none of the homologous regions on NS1 proteins from other flaviviruses reacted with the MAbs when they were tested for cross-reactivity, and all five epitope peptides were not recognized by sera against West Nile virus or Dengue virus. These novel virus-specific linear B-cell epitopes of JEV NS1 would benefit the development of new vaccines and diagnostic assays. PMID:23825668

  2. Comprehensive Mapping Antigenic Epitopes of NS1 Protein of Japanese Encephalitis Virus with Monoclonal Antibodies.

    Directory of Open Access Journals (Sweden)

    Rong-Hong Hua

    Full Text Available Japanese encephalitis virus (JEV non-structural protein 1 (NS1 contributes to virus replication and elicits protective immune responses during infection. JEV NS1-specific antibody responses could be a target in the differential diagnosis of different flavivirus infections. However, the epitopes on JEV NS1 are poorly characterized. The present study describes the full mapping of linear B-cell epitopes in JEV NS1. We generated eleven NS1-specific monoclonal antibodies from mice immunized with recombinant NS1. For epitope mapping of monoclonal antibodies, a set of 51 partially-overlapping peptides covering the entire NS1 protein were expressed with a GST-tag and then screened using monoclonal antibodies. Through enzyme-linked immunosorbent assay (ELISA, five linear epitope-containing peptides were identified. By sequentially removing amino acid residues from the carboxy and amino terminal of peptides, the minimal units of the five linear epitopes were identified and confirmed using monoclonal antibodies. Five linear epitopes are located in amino acids residues (5AIDITRK(11, (72RDELNVL(78, (251KSKHNRREGY(260, (269DENGIVLD(276, and (341DETTLVRS(348. Furthermore, it was found that the epitopes are highly conserved among JEV strains through sequence alignment. Notably, none of the homologous regions on NS1 proteins from other flaviviruses reacted with the MAbs when they were tested for cross-reactivity, and all five epitope peptides were not recognized by sera against West Nile virus or Dengue virus. These novel virus-specific linear B-cell epitopes of JEV NS1 would benefit the development of new vaccines and diagnostic assays.

  3. Reatividade do anticorpo IgM anti-Treponema pallidum na soroconversão e na resposta sorológica ao tratamento de sífilis IgM antibody reactivity to Treponema pallidum in seroconversion and in the serologic response to syphilis treatment

    Directory of Open Access Journals (Sweden)

    Neuza Satomi Sato

    2012-12-01

    Full Text Available INTRODUÇÃO: A utilidade da detecção de anticorpos da imunoglobulina da classe M (IgM no diagnóstico da sífilis tem sido discutida há tempos. OBJETIVO: No presente estudo foi analisada a ocorrência de anticorpo IgM anti-T. pallidum (Tp-IgMAc nas amostras de pacientes com sífilis recente, na fase de soroconversão e no monitoramento da resposta sorológica pós-tratamento. MÉTODOS: Amostras séricas de 11 indivíduos. RESULTADOS: Na soroconversão, o Tp-IgMAc foi detectado nas amostras de 10 indivíduos, e em um paciente a reatividade IgM ocorreu anteriormente ao Venereal Disease Research Laboratory (VDRL. A sororreversão foi evidenciada nas amostras de três pacientes com sífilis secundária tratada, e em um indivíduo com reinfecção. CONCLUSÃO: A detecção de Tp-IgMAc mostrou ser um potencial marcador diagnóstico de sífilis ativa e o desempenho do ensaio imunoenzimático de captura de IgM (ELISA-IgM para o monitoramento pós-tratamento foi similar ao da VDRL.INTRODUCTION: The appropriateness of IgM antibody detection in the diagnosis of syphilis has been extensively discussed. OBJECTIVE: This study aimed at assessing the detection of anti-T. pallidum IgM antibody (TP-IgMAb in serum samples from patients with recent syphilis in seroconversion and in the monitoring of post-treatment serological response. METHODS: Serum samples from 11 individuals. RESULTS: At seroconversion, positive Tp-IgMAb was detected in 10 samples and IgM reactivity previous to Venereal Disease Research Laboratory (VDRL was detected in one sample. Seroreversion was found in samples from three treated patients with secondary syphilis and in one individual with reinfection. CONCLUSION: Tp-IgMAb detection proved to be a potential diagnostic marker for active syphilis, and IgM capture enzyme linked immunosorbent assay (ELISA-IgM performance was similar to VDRL in post-treatment monitoring.

  4. The reactive extrusion of thermoplastic polyurethane

    OpenAIRE

    Verhoeven, Vincent Wilhelmus Andreas

    2006-01-01

    The objective of this thesis was to increase the understanding of the reactive extrusion of thermoplastic polyurethane. Overall, several issues were identified: • Using a relative simple extrusion model, the reactive extrusion process can be described. This model can be used to further investigate and optimize the reactive extrusion of thermoplastic polyurethane. • Premixing has a small beneficiary effect on the efficiency of the extrusion process and the quality of the product formed. • The ...

  5. Monoclonal antibody imaging of human melanoma. Radioimmunodetection by subcutaneous or systemic injection

    International Nuclear Information System (INIS)

    Fab fragments of monoclonal antibodies (MoAb) to melanoma, radiolabeled with 131I, were evaluated as diagnostic reagents to determine their ability to localize systemic--MoAb injected intravenously (IV)--or nodal metastatic disease--injected subcutaneously (SQ) at a site proximal to draining lymph nodes. Sixty-one scans were performed (40 IV, 21 SQ) in 59 patients who had injections of 0.2-50 mg of 131I coupled (0.2-12 mCi) antibody. These included 48.7, which identifies a high molecular weight antigen (HMW), or 96.5, which identifies a transferrin like molecule, p97. 125I coupled nonspecific Fab 1.4, reacting with murine leukemia virus, or the whole antibody BL3, reactive with a human B cell idiotypic determinant, was generally used in tandem with the patients injected SQ as a nonspecific control. All patients had immunohistochemical studies performed and demonstrated binding to the antibodies injected. Of the IV patients, 22/38 (58%) had (+) scans, 13 at SQ or nodal sites, four at visceral sites, and five at visceral and SQ sites. Patients with clinical stage II disease had SQ injection of MoAb, including 11 additional patients injected with the whole antibody 9.2.27 (anti-HMW) labeled with 111In (6 patients) or 131I (5 patients). Nodal dissection was performed 2-4 days later. All 111In coupled antibodies demonstrated excellent nodal delineation without specific identification of tumor deposits. Of the 21 patients injected SQ with MoAb, 17 had confirmed tumor in nodes. Of patients injected with Fab fragments, 50% had specific uptake of MoAb, although only two were successfully imaged. Increased uptake of antimelanoma antibodies was observed in some patients in lymph nodes not containing tumor. Clearance of labeled antibody from the injection site occurred with a half life of 16-50 hours. Toxicity was limited to local discomfort at the site of SQ injection

  6. Multifunctional reactive nanocomposite materials

    Science.gov (United States)

    Stamatis, Demitrios

    Many multifunctional nanocomposite materials have been developed for use in propellants, explosives, pyrotechnics, and reactive structures. These materials exhibit high reaction rates due to their developed reaction interfacial area. Two applications addressed in this work include nanocomposite powders prepared by arrested reactive milling (ARM) for burn rate modifiers and reactive structures. In burn rate modifiers, addition of reactive nanocomposite powders to aluminized propellants increases the burn rate of aluminum and thus the overall reaction rate of an energetic formulation. Replacing only a small fraction of aluminum by 8Al·MoO3 and 2B·Ti nanocomposite powders enhances the reaction rate with little change to the thermodynamic performance of the formulation; both the rate of pressure rise and maximum pressure measured in the constant volume explosion test increase. For reactive structures, nanocomposite powders with bulk compositions of 8Al·MoO3, 12Al·MoO3, and 8Al·3CuO were prepared by ARM and consolidated using a uniaxial die. Consolidated samples had densities greater than 90% of theoretical maximum density while maintaining their high reactivity. Pellets prepared using 8Al·MoO3 powders were ignited by a CO2 laser. Ignition delays increased at lower laser powers and greater pellet densities. A simplified numerical model describing heating and thermal initiation of the reactive pellets predicted adequately the observed effects of both laser power and pellet density on the measured ignition delays. To investigate the reaction mechanisms in nanocomposite thermites, two types of nanocomposite reactive materials with the same bulk compositions 8Al·MoO3 were prepared by different methods. One of the materials was manufactured by ARM and the other, so called metastable interstitial composite (MIC), by mixing of nano-scaled individual powders. Clear differences in the low-temperature redox reactions, welldetectable by differential scanning calorimetry

  7. Persistent strong anti-HLA antibody at high titer is complement binding and associated with increased risk of antibody-mediated rejection in heart transplant recipients

    Science.gov (United States)

    Zeevi, Adriana; Lunz, John; Feingold, Brian; Shullo, Michael; Bermudez, Christian; Teuteberg, Jeffery; Webber, Steven

    2013-01-01

    BACKGROUND Sensitized heart transplant candidates are evaluated for donor-specific anti-HLA IgG antibody (DSA) by Luminex single-antigen bead (SAB) testing (SAB-IgG) to determine donor suitability and help predict a positive complement-dependent cytotoxicity crossmatch (CDC-XM) by virtual crossmatching (VXM). However, SAB testing used for VXM does not correlate perfectly with CDC-XM results and individual transplant programs have center-specific permissible thresholds to predict crossmatch positivity. A novel Luminex SAB-based assay detecting C1q-binding HLA antibodies (SAB-C1q) contributes functional information to SAB testing, but the relationship between SAB strength and complement-binding ability is unclear. METHODS In this retrospective study, we identified 15 pediatric and adult heart allograft candidates with calculated panel-reactive antibody (cPRA) >50% by SAB-IgG and compared conventional SAB-IgG results with SAB-C1q testing. RESULTS Pre- and post-transplant DSA by SAB-C1q correlated with DSA by SAB-IgG and also with CDC-XM results and early post-transplant endomyocardial biopsy findings. Individual HLA antibodies by SAB-IgG in undiluted sera correlated poorly with SAB-C1q; however, when sera were diluted 1:16, SAB-IgG results were well correlated with SAB-C1q. In some sera, HLA antibodies with low mean fluorescent intensity (MFI) by SAB-IgG exhibited high SAB-C1q MFIs for the same HLA antigens. Diluting or heat-treating these sera increased SAB-IgG MFI, consistent with SAB-C1q results. In 13 recipients, SAB-C1q–positive DSA was associated with positive CDC-XM and with early clinical post-transplant antibody-mediated rejection (cAMR). CONCLUSIONS Risk assessment for positive CDC-XM and early cAMR in sensitized heart allograft recipients are correlated with SAB-C1q reactivity. PMID:23142561

  8. Increased specificity in human cardiac-myosin radioimmunoassay utilizing two monoclonal antibodies in a double sandwich assay

    International Nuclear Information System (INIS)

    An immunoradiometric assay that simultaneously measured two different epitopes on the same molecule was devised to differential between cardiac- and skeletal-myosin light chains. Three monoclonal antibodies were examined that were 100% (lC5), 25% (2B9) and 17% (4F10) cross reactive, respectively, between the two antigens. One antibody of the pair to be studied was immobilized to cyanogen bromide-activated Sepharose 4B while the other was iodinated with 125I using the lactoperoxidase method. The antigen was mixed with the immobilized antibody, the labeled antibody was added and the precipitate then washed and counted in a gamma counter. When both antibodies of the pair to be studied (immobilized and labeled) were the same (2B9), no radioactivity above background was bound to the precipitate, indicating that the second antibody could not bind to an already occupied epitope. When two different antibodies were employed, the specificity of the assay increased over that of a single antibody. The cross reactivity of a pair approximated the product of the cross reactivities of the individual antibodies. Thus, lC5 and 2B9 were 25% cross reactive together, lC5 and 4F10 17% cross reactive, and 2B9 and 4F10 4.3% cross reactive. (author)

  9. Sequence homology: A poor predictive value for profilins cross-reactivity

    Directory of Open Access Journals (Sweden)

    Pazouki Nazanin

    2005-09-01

    Full Text Available Summary Background Profilins are highly cross-reactive allergens which bind IgE antibodies of almost 20% of plant-allergic patients. This study is aimed at investigating cross-reactivity of melon profilin with other plant profilins and the role of the linear and conformational epitopes in human IgE cross-reactivity. Methods Seventeen patients with melon allergy were selected based on clinical history and a positive skin prick test to melon extract. Melon profilin has been cloned and expressed in E. coli. The IgE binding and cross-reactivity of the recombinant profilin were measured by ELISA and inhibition ELISA. The amino acid sequence of melon profilin was compared with other profilin sequences. A combination of chemical cleavage and immunoblotting techniques were used to define the role of conformational and linear epitopes in IgE binding. Comparative modeling was used to construct three-dimensional models of profilins and to assess theoretical impact of amino acid differences on conformational structure. Results Profilin was identified as a major IgE-binding component of melon. Alignment of amino acid sequences of melon profilin with other profilins showed the most identity with watermelon profilin. This melon profilin showed substantial cross-reactivity with the tomato, peach, grape and Cynodon dactylon (Bermuda grass pollen profilins. Cantaloupe, watermelon, banana and Poa pratensis (Kentucky blue grass displayed no notable inhibition. Our experiments also indicated human IgE only react with complete melon profilin. Immunoblotting analysis with rabbit polyclonal antibody shows the reaction of the antibody to the fragmented and complete melon profilin. Although, the well-known linear epitope of profilins were identical in melon and watermelon, comparison of three-dimensional models of watermelon and melon profilins indicated amino acid differences influence the electric potential and accessibility of the solvent-accessible surface of

  10. Serum Levels of Anticyclic Citrullinated Peptide Antibodies, Interleukin-6, Tumor Necrosis Factor-α, and C-Reactive Protein Are Associated with Increased Carotid Intima-Media Thickness: A Cross-Sectional Analysis of a Cohort of Rheumatoid Arthritis Patients without Cardiovascular Risk Factors

    Directory of Open Access Journals (Sweden)

    Mónica Vázquez-Del Mercado

    2015-01-01

    Full Text Available The main cause of death in rheumatoid arthritis (RA is cardiovascular events. We evaluated the relationship of anticyclic citrullinated peptide (anti-CCP antibody levels with increased carotid intima-media thickness (cIMT in RA patients. Methods. Forty-five anti-CCP positive and 37 anti-CCP negative RA patients, and 62 healthy controls (HC were studied. All groups were assessed for atherogenic index of plasma (AIP and cIMT. Anti-CCP, C-reactive protein (CRP, and levels of tumor necrosis factor alpha (TNFα and interleukin-6 (IL-6 were measured by enzyme-linked immunosorbent assay (ELISA. Results. The anti-CCP positive RA patients showed increased cIMT compared to HC and anti-CCP negative (P<0.001. Anti-CCP positive versus anti-CCP negative RA patients, had increased AIP, TNFα and IL-6 (P<0.01, and lower levels of high density lipoprotein cholesterol (HDL-c (P=0.02. The cIMT correlated with levels of anti-CCP (r=0.513, P=0.001, CRP (r=0.799, P<0.001, TNFα (r=0.642, P=0.001, and IL-6 (r=0.751, P<0.001. In multiple regression analysis, cIMT was associated with CRP (P<0.001 and anti-CCP levels (P=0.03. Conclusions. Levels of anti-CCP and CRP are associated with increased cIMT and cardiovascular risk supporting a clinical role of the measurement of cIMT in RA in predicting and preventing cardiovascular events.

  11. Serum Levels of Anticyclic Citrullinated Peptide Antibodies, Interleukin-6, Tumor Necrosis Factor-α, and C-Reactive Protein Are Associated with Increased Carotid Intima-Media Thickness: A Cross-Sectional Analysis of a Cohort of Rheumatoid Arthritis Patients without Cardiovascular Risk Factors

    Science.gov (United States)

    Vázquez-Del Mercado, Mónica; Nuñez-Atahualpa, Lourdes; Figueroa-Sánchez, Mauricio; Gómez-Bañuelos, Eduardo; Rocha-Muñoz, Alberto Daniel; Martín-Márquez, Beatriz Teresita; Martínez-García, Erika Aurora; Macias-Reyes, Héctor; Gamez-Nava, Jorge Ivan; Navarro-Hernandez, Rosa Elena; Nuñez-Atahualpa, María Alejandra; Andrade-Garduño, Javier

    2015-01-01

    The main cause of death in rheumatoid arthritis (RA) is cardiovascular events. We evaluated the relationship of anticyclic citrullinated peptide (anti-CCP) antibody levels with increased carotid intima-media thickness (cIMT) in RA patients. Methods. Forty-five anti-CCP positive and 37 anti-CCP negative RA patients, and 62 healthy controls (HC) were studied. All groups were assessed for atherogenic index of plasma (AIP) and cIMT. Anti-CCP, C-reactive protein (CRP), and levels of tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6) were measured by enzyme-linked immunosorbent assay (ELISA). Results. The anti-CCP positive RA patients showed increased cIMT compared to HC and anti-CCP negative (P < 0.001). Anti-CCP positive versus anti-CCP negative RA patients, had increased AIP, TNFα and IL-6 (P < 0.01), and lower levels of high density lipoprotein cholesterol (HDL-c) (P = 0.02). The cIMT correlated with levels of anti-CCP (r = 0.513, P = 0.001), CRP (r = 0.799, P < 0.001), TNFα (r = 0.642, P = 0.001), and IL-6 (r = 0.751, P < 0.001). In multiple regression analysis, cIMT was associated with CRP (P < 0.001) and anti-CCP levels (P = 0.03). Conclusions. Levels of anti-CCP and CRP are associated with increased cIMT and cardiovascular risk supporting a clinical role of the measurement of cIMT in RA in predicting and preventing cardiovascular events. PMID:25821796

  12. Immunohistochemical diagnosis of systemic bovine zygomycosis by murine monoclonal antibodies

    DEFF Research Database (Denmark)

    Jensen, H.E.; Aalbaek, B.; Lind, Peter; Krogh, H.V.

    1996-01-01

    97%]) the Mab and the polyclonal antibodies reacted in a similar pattern, i.e., positively for zygomycosis in 89 lesions, negatively in 41 aspergillosis lesions, and negatively in 10 undiagnosed lesions. Hyphae within two of four lesions in lymph nodes, which were not stained by the polyclonal...... antibodies, reacted with the specific Mab. However, in another three lesions of lymph nodes stained by the polyclonal antibodies no reactivity was seen with the Mab-WSSA-RA-1. The immunoreactivity of the Mabs (Mab-WSSA-RA-1 through Mab-WSSA-RA-4) raised against WSSA of R. arrhizus justify their application...... for the in situ diagnosis of systemic bovine zygomycosis....

  13. Antigenic specificity of serum antibodies in mice fed soy protein

    DEFF Research Database (Denmark)

    Christensen, Hanne Risager; Bruun, S.W.; Frøkiær, Hanne

    2003-01-01

    the relationship between the immunogenic proteins involved in this nondeleterious antibody response and the pathological response associated with food allergy. The objective of the present study was to characterize the antigenic specificity of the soy protein-specific antibody response generated in...... healthy mice ingesting soy protein. Methods: Blood from mice fed a soy-containing diet was analyzed using ELISA and immunoblot for antibody reactivity towards various soy protein fractions and pure soy proteins/subunits. Mice bred on a soy-free diet were used as controls. Results: The detectable antigenic...

  14. Serum peptide reactivities may distinguish neuromyelitis optica subgroups and multiple sclerosis

    Science.gov (United States)

    Metz, Imke; Beißbarth, Tim; Ellenberger, David; Pache, Florence; Stork, Lidia; Ringelstein, Marius; Aktas, Orhan; Jarius, Sven; Wildemann, Brigitte; Dihazi, Hassan; Friede, Tim; Ruprecht, Klemens; Paul, Friedemann

    2016-01-01

    Objective: To assess in an observational study whether serum peptide antibody reactivities may distinguish aquaporin-4 (AQP4) antibody (Ab)–positive and -negative neuromyelitis optica spectrum disorders (NMOSD) and relapsing-remitting multiple sclerosis (RRMS). Methods: We screened 8,700 peptides that included human and viral antigens of potential relevance for inflammatory demyelinating diseases and random peptides with pooled sera from different patient groups and healthy controls to set up a customized microarray with 700 peptides. With this microarray, we tested sera from 66 patients with AQP4-Ab-positive (n = 16) and AQP4-Ab-negative (n = 19) NMOSD, RRMS (n = 11), and healthy controls (n = 20). Results: Differential peptide reactivities distinguished NMOSD subgroups from RRMS in 80% of patients. However, the 2 NMOSD subgroups were not well-discriminated, although those patients are clearly separated by their antibody reactivities against AQP4 in cell-based assays. Elevated reactivities to myelin and Epstein-Barr virus peptides were present in RRMS and to AQP4 and AQP1 peptides in AQP4-Ab-positive NMOSD. Conclusions: While AQP4-Ab-positive and -negative NMOSD subgroups are not well-discriminated by peptide antibody reactivities, our findings suggest that peptide antibody reactivities may have the potential to distinguish between both NMOSD subgroups and MS. Future studies should thus concentrate on evaluating peptide antibody reactivities for the differentiation of AQP4-Ab-negative NMOSD and MS. PMID:26894206

  15. Recognition of Candida albicans Als3 by the Germ Tube-Specific Monoclonal Antibody 3D9.3

    OpenAIRE

    Beucher, Bertrand; Marot-Leblond, Agnès; Billaud-Nail, Sandrine; OH, SOON-HWAN; Hoyer, Lois L.; Robert, Raymond

    2009-01-01

    Monoclonal antibody 3D9.3 (MAb 3D9.3) reacts with the surface of Candida albicans germ tubes and recognizes a protein epitope. We used a two-step chromatography procedure to purify and identify the antigen (3D9) from C. albicans strain 66396 germ tubes. MAb 3D9.3 recognized two intense protein bands at 140 and 180 kDa. A comparative analysis between theoretical and experimental mass spectrum peaks showed that both bands corresponded to Als3. This conclusion was supported by lack of reactivity...

  16. Immunoproteomics analysis of the murine antibody response to vaccination with an improved Francisella tularensis live vaccine strain (LVS.

    Directory of Open Access Journals (Sweden)

    Susan M Twine

    Full Text Available BACKGROUND: Francisella tularensis subspecies tularensis is the causative agent of a spectrum of diseases collectively known as tularemia. An attenuated live vaccine strain (LVS has been shown to be efficacious in humans, but safety concerns have prevented its licensure by the FDA. Recently, F. tularensis LVS has been produced under Current Good Manufacturing Practice (CGMP guidelines. Little is known about the immunogenicity of this new vaccine preparation in comparison with extensive studies conducted with laboratory passaged strains of LVS. Thus, the aim of the current work was to evaluate the repertoire of antibodies produced in mouse strains vaccinated with the new LVS vaccine preparation. METHODOLOGY/PRINCIPAL FINDINGS: In the current study, we used an immunoproteomics approach to examine the repertoire of antibodies induced following successful immunization of BALB/c versus unsuccessful vaccination of C57BL/6 mice with the new preparation of F. tularensis LVS. Successful vaccination of BALB/c mice elicited antibodies to nine identified proteins that were not recognized by antisera from vaccinated but unprotected C57BL/6 mice. In addition, the CGMP formulation of LVS stimulated a greater repertoire of antibodies following vaccination compared to vaccination with laboratory passaged ATCC LVS strain. A total of 15 immunoreactive proteins were identified in both studies, however, 16 immunoreactive proteins were uniquely reactive with sera from the new formulation of LVS. CONCLUSIONS/SIGNIFICANCE: This is the first report characterising the antibody based immune response of the new formulation of LVS in the widely used murine model of tularemia. Using two mouse strains, we show that successfully vaccinated mice can be distinguished from unsuccessfully vaccinated mice based upon the repertoire of antibodies generated. This opens the door towards downselection of antigens for incorporation into tularemia subunit vaccines. In addition, this work

  17. Choriocarcinoma: blocking factor and monoclonal antibody iodine 131 imaging

    Energy Technology Data Exchange (ETDEWEB)

    Pattillo, R.A.; Khazaeli, M.B.; Ruckert, A.C.; Hussa, R.O.; Collier, B.D.; Beierwaltes, W.; Mattingly, R.F.

    1984-04-01

    Postoperative iodine 131 monoclonal antibody localization in metastatic choriocarcinoma was accomplished in this study. The monoclonal antibody was prepared to male choriocarcinoma which cross reacted with gestational choriocarcinoma. The antibody was raised against whole choriocarcinoma cells and human chorionic gonadotropin (hCG) cross reactivity was excluded. The purified antibody was iodinated with /sup 131/I and successfully imaged BeWo choriocarcinoma transplanted in nude mice; however, imaging of choriocarcinoma in a patient was verified only after resection. It is our belief that failure to sufficiently concentrate the antibody in the tumor before operation was due to blocking factor in the serum of the patient. Blocking factor and hCG dropped postoperatively. Blocking factor activity in 15 patients with metastatic trophoblastic disease was monitored and, like hCG, was found to be a sensitive indicator of the presence of disease. Its efficacy may be in the small number of patients without hCG but with persistent disease.

  18. Engineering antibody therapeutics.

    Science.gov (United States)

    Chiu, Mark L; Gilliland, Gary L

    2016-06-01

    The successful introduction of antibody-based protein therapeutics into the arsenal of treatments for patients has within a few decades fostered intense innovation in the production and engineering of antibodies. Reviewed here are the methods currently used to produce antibodies along with how our knowledge of the structural and functional characterization of immunoglobulins has resulted in the engineering of antibodies to produce protein therapeutics with unique properties, both biological and biophysical, that are leading to novel therapeutic approaches. Antibody engineering includes the introduction of the antibody combining site (variable regions) into a host of architectures including bi and multi-specific formats that further impact the therapeutic properties leading to further advantages and successes in patient treatment. PMID:27525816

  19. The Fingerprint of Anti-Bromodeoxyuridine Antibodies and Its Use for the Assessment of Their Affinity to 5-Bromo-2'-Deoxyuridine in Cellular DNA under Various Conditions

    Science.gov (United States)

    Ligasová, Anna; Liboska, Radek; Rosenberg, Ivan; Koberna, Karel

    2015-01-01

    We have developed a simple system for the analysis of the affinity of anti-bromodeoxyuridine antibodies. The system is based on the anchored oligonucleotides containing 5-bromo-2'-deoxyuridine (BrdU) at three different positions. It allows a reliable estimation of the reactivity of particular clones of monoclonal anti-bromodeoxyuridine antibodies with BrdU in fixed and permeabilized cells. Using oligonucleotide probes and four different protocols for the detection of BrdU incorporated in cellular DNA, we identified two antibody clones that evinced sufficient reactivity to BrdU in all the tested protocols. One of these clones exhibited higher reactivity to 5-iodo-2'-deoxyuridine (IdU) than to BrdU. It allowed us to increase the sensitivity of the used protocols without a negative effect on the cell physiology as the cytotoxicity of IdU was comparable with BrdU and negligible when compared to 5-ethynyl-2'-deoxyuridine. The combination of IdU and the improved protocol for oxidative degradation of DNA provided a sensitive and reliable approach for the situations when the low degradation of DNA and high BrdU signal is a priority. PMID:26161977

  20. THE OCCURRENCE OF ADENOVIRUS PRECIPITATING ANTIBODIES IN HUMAN SERA IN IRAN

    Directory of Open Access Journals (Sweden)

    A. AFSHAR

    1969-01-01

    Full Text Available By irnmunocdiffusion tests 312 serum samples from human were examined for the presence of antibodies to the group.reactive precipitatng antigen of adenoviruses. of the total sera from female, 19.6% and from male, 15.8% had antibody to the group reactive antigen prepared from bovine adenovirus type 3. No significant difference was demo., nstrated on account of sex or age. The results suggest that adenovirus infection is widespread among the human population in Iran.

  1. Transfusion-Related Acute Lung Injury: The role of donor antibodies

    OpenAIRE

    Mathijssen-van Stein, Danielle

    2015-01-01

    markdownabstractAbstract Transfusion-related acute lung injury (TRALI) is a serious complication of blood transfusion, which causes serious morbidity and is the leading cause of transfusion-associated mortality according to the FDA. The majority of TRALI cases (up to 89%) are thought to be antibody-mediated TRALI, caused by the passive infusion of white blood cell (WBC)- reactive antibodies, present in plasma-containing blood products. This thesis focuses on the role of donor WBC-reactive ant...

  2. Monoclonal Antibody Analysis of Keratin Expression in the Central Nervous System

    Science.gov (United States)

    Franko, Maryellen C.; Gibbs, Clarence J.; Rhoades, Dorothy A.; Carleton Gajdusek, D.

    1987-05-01

    A monoclonal antibody directed against a 65-kDa brain protein demonstrates an epitope found in keratin from human epidermis. By indirect immunofluorescence, the antibody decorates intracytoplasmic filaments in a subclass of astrocytes and Purkinje cells of adult hamster brain. Double-label immunofluorescence study using antibody to glial fibrillary acidic protein and this antibody reveals the 65-kDa protein to be closely associated with glial filaments in astrocytes of fetal mouse brain cultures. Immunoblot analysis of purified human epidermal keratin and hamster brain homogenate confirms the reactivity of this antibody to epidermal keratin polypeptides. All the major epidermal keratins were recognized by this antibody. It did not bind to the remaining major intermediate filament proteins. These findings suggest that monoclonal antibody 34C9 recognizes a cytoskeletal structure connected with intermediate filaments. In addition, the monoclonal antibody demonstrates that epidermal keratins share an epitope not only among themselves but also with a ``neural keratin.''

  3. Differences in PfEMP1s recognized by antibodies from patients with uncomplicated or severe malaria

    DEFF Research Database (Denmark)

    Duffy, Michael F; Noviyanti, Rintis; Tsuboi, Takafumi;

    2016-01-01

    BACKGROUND: Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) variants are encoded by var genes and mediate pathogenic cytoadhesion and antigenic variation in malaria. PfEMP1s can be broadly divided into three principal groups (A, B and C) and they contain conserved arrangements of...... functional domains called domain cassettes. Despite their tremendous diversity there is compelling evidence that a restricted subset of PfEMP1s is expressed in severe disease. In this study antibodies from patients with severe and uncomplicated malaria were compared for differences in reactivity with a range...... of PfEMP1s to determine whether antibodies to particular PfEMP1 domains were associated with severe or uncomplicated malaria. METHODS: Parts of expressed var genes in a severe malaria patient were identified by RNAseq and several of these partial PfEMP1 domains were expressed together with others...

  4. Reactive Kripke semantics

    CERN Document Server

    Gabbay, Dov M

    2013-01-01

    This text offers an extension to the traditional Kripke semantics for non-classical logics by adding the notion of reactivity. Reactive Kripke models change their accessibility relation as we progress in the evaluation process of formulas in the model. This feature makes the reactive Kripke semantics strictly stronger and more applicable than the traditional one. Here we investigate the properties and axiomatisations of this new and most effective semantics, and we offer a wide landscape of applications of the idea of reactivity. Applied topics include reactive automata, reactive grammars, rea

  5. Probing the antibody-catalyzed water-oxidation pathway at atomic resolution

    OpenAIRE

    Zhu, Xueyong; Wentworth, Paul; Wentworth, Anita D.; Eschenmoser, Albert; Lerner, Richard A.; Wilson, Ian A.

    2004-01-01

    Antibodies can catalyze the generation of hydrogen peroxide (H2O2) from singlet dioxygen (1O*2) and water via the postulated intermediacy of dihydrogen trioxide (H2O3) and other trioxygen species. Nine different crystal structures were determined to elucidate the chemical consequences to the antibody molecule itself of exposure to such reactive intermediates and to provide insights into the location on the antibody where these species could be generated. Herein, we report structural evidence ...

  6. Analysis of T-cell-dependent and -independent antigens of Rickettsia conorii with monoclonal antibodies.

    OpenAIRE

    Feng, H M; Walker, D H; Wang, J. G.

    1987-01-01

    Four monoclonal antibodies from euthymic mice and two monoclonal antibodies from athymic mice were directed against antigens of Rickettsia conorii, as shown by both indirect immunofluorescence and an enzyme immunoassay. There was extensive cross-reactivity with other spotted fever group rickettsiae. Euthymic monoclonal antibodies 3-2 and 9-2 (immunoglobulin G2a [IgG2a]) and 27-10 (IgG1) distinctly outlined the acetone-fixed rickettsial surface, as determined by indirect immunofluorescence; on...

  7. The production of recombinant single chain antibody fragments for the detection of illicit drug residues

    OpenAIRE

    Brennan, Joanne

    2005-01-01

    Recombinant antibodies represent a more sensitive and specific detection tool for immunoanalysis. The research carried out for this thesis describes the production of genetically-derived single chain antibody fragments to detect illicit drugs. A variety of novel recombinant antibody fragments against morphine-3-glucuronide, a metabolite of heroin has been produced. A monomeric, dimeric and enzyme-labelled scFv were characterised with respect to their binding abilities and cross reactiviti...

  8. Schistosoma mansoni. Anti-egg monoclonal antibodies protect against cercarial challenge in vivo

    OpenAIRE

    1984-01-01

    Monoclonal antibodies that bind to surface membranes of developing schistosomula and/or cercarial tails were generated from mice immunized with living schistosome eggs or soluble egg antigen. These monoclonal antibodies detected at least three different surface epitopes. One surface antigen detected by anti-egg monoclonal antibody EG1C4B1 (E.1) persisted on the surface of developing schistosomula for 96 h posttransformation . The same or a cross-reactive antigen was also detected on the surfa...

  9. Prospective isolation of mesenchymal stem cells from human bone marrow using novel antibodies directed against Sushi domain containing 2.

    Science.gov (United States)

    Sivasubramaniyan, Kavitha; Harichandan, Abhishek; Schumann, Susanne; Sobiesiak, Malgorzata; Lengerke, Claudia; Maurer, Andreas; Kalbacher, Hubert; Bühring, Hans-Jörg

    2013-07-01

    Several strategies have been developed to facilitate the prospective isolation of bone marrow-derived mesenchymal stem/stromal cells (BM-MSCs) based on the selective expression or absence of surface markers. Recently, we described the monoclonal antibodies W3D5 and W5C5, which selectively react with BM-MSCs, but not with hematopoietic cells. Both antibodies showed an identical reactivity pattern, indicating that they may recognize the same molecule. To identify the cognate antigen, cultured MSCs were sorted for cells expressing either very high levels of W5C5/W3D5 antigen or for cells which were negative for this antigen. Further processing of these cells for microarray analysis revealed a 20-fold enrichment of the type 1 integral membrane protein Sushi domain containing 2 (SUSD2) in the in W5C5(+) subset. To confirm the identity of the W5C5/W3D5 antigen to SUSD2, HEK293 cells were transfected with the full-length coding sequence of human SUSD2 followed by reactivity analysis of W5C5 and W3D5 antibodies with the transfected line. Flow cytometric analysis showed that both antibodies selectively recognized HEK293/huSUSD2 cells, but not the parental cell line. In line with this, SUSD2 siRNA treatment of SUSD2(+) WERI-RB-1 retinoblastoma cells reduced the expression levels of W3D5 and W5C5 antigens to ~39% and 37%, respectively. Finally, FACSorting and colony assays revealed that only SUSD2(+), but not SUSD2(-) BM cells give rise to colony-forming units-fibroblasts and are able to differentiate into osteoblasts, adipocytes, and chondrocytes. In conclusion, we identified SUSD2 as a novel and specific marker for the prospective isolation of BM-MSCs. PMID:23406305

  10. Neurobehavioral foundation of environmental reactivity.

    Science.gov (United States)

    Moore, Sarah R; Depue, Richard A

    2016-02-01

    Sensitivity to environmental context has been of interest for many years, but the nature of individual differences in environmental sensitivity has become of particular focus over the past 2 decades. What is particularly uncertain are the neural variables and processes that mediate the effects of environment on developmental outcomes. Accordingly, we provide a neurobehavioral foundation of reactivity to the environment in several steps. First, the different patterns of environmental sensitivity are defined to identify the significant factors involved in the manifestation of these patterns. Second, we focus on neurobiological reactivity as the construct underlying variation in sensitivity to the environment by (a) providing an organizing threshold model of elicitation of neurobiology by environmental context; and (b) integrating the literature on 2 sets of neuromodulators in terms of each modulator's (a) contribution to neural and behavioral reactivity to stimulation, and (b) relation to emotional-motivational systems (dopamine, opiates and oxytocin, corticotropin-releasing hormone) or the general modulation of those systems (serotonin, norepinephrine, and GABA). Discussion concludes with (a) a comprehensive neurobehavioral framework of environmental reactivity based on a combinatorial model of a supertrait, (b) methodological implications of the model, and (c) a developmental perspective on environmental reactivity. PMID:26479069

  11. Affinity purification of antibodies

    Science.gov (United States)

    Antibodies are provided in a variety of formats that includes antiserum, hybridoma culture supernatant or ascites. They can all be used successfully in crude form for the detection of target antigens by immunoassay. However, it is advantageous to use purified antibody in defined quantity to facil...

  12. Therapeutic Recombinant Monoclonal Antibodies

    Science.gov (United States)

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  13. Production Of Human Antibodies

    Science.gov (United States)

    Sammons, David W.; Neil, Garry A.

    1993-01-01

    Process for making human monoclonal antibodies based on combination of techniques. Antibodies made active against specific antigen. Process involves in vivo immunization of human B lymphocyte cells in mice. B cells of interest enriched in vitro before fusion. Method potentially applicable to any antigen. Does not rely on use of Epstein-Barr virus at any step. Human lymphocytes taken from any source.

  14. RBC Antibody Screen

    Science.gov (United States)

    ... the baby is Rh-positive and the mother's antibody status is negative for anti-D, the mother is given additional RhIG. This test also may be used to help diagnose autoimmune-related hemolytic anemia ... when a person produces antibodies against his or her own RBC antigens. This ...

  15. Antibody affinity maturation

    DEFF Research Database (Denmark)

    Skjødt, Mette Louise

    Yeast surface display is an effective tool for antibody affinity maturation because yeast can be used as an all-in-one workhorse to assemble, display and screen diversified antibody libraries. By employing the natural ability of yeast Saccharomyces cerevisiae to efficiently recombine multiple DNA...

  16. Reactive perforating collagenosis

    Directory of Open Access Journals (Sweden)

    Yadav Mukesh

    2009-01-01

    Full Text Available Reactive perforating collagenosis is a rare cutaneous disorder of unknown etiology. We hereby describe a case of acquired reactive perforating collagenosis in a patient of diabetes and chronic renal failure.

  17. Reactive perforating collagenosis

    OpenAIRE

    Yadav Mukesh; Sangal B; Bhargav Puneet; Jai P; Goyal Mukul

    2009-01-01

    Reactive perforating collagenosis is a rare cutaneous disorder of unknown etiology. We hereby describe a case of acquired reactive perforating collagenosis in a patient of diabetes and chronic renal failure.

  18. Characterization of a panel of anti-phosphoprotein monoclonal antibodies generated against the raccoon strain of rabies virus.

    Science.gov (United States)

    Nadin-Davis, Susan A; Fehlner-Gardiner, Christine; Sheen, Mary; Wandeler, Alexander I

    2010-09-01

    The generation of a new panel of 32 monoclonal antibodies (MAbs) reactive with the P protein of the raccoon strain of rabies virus is described. Through a series of analyses employing competitive ELISA and immunoblotting, these MAbs were classified into eight groups, each defining an antigenic site, thereby increasing the number of sites now recognized along the length of the P protein. Studies on MAb reactivity with a collection of diverse lyssaviruses identified sites that were highly conserved, moderately conserved and highly variable. Several groups of MAbs were highly specific for the raccoon rabies virus (RRV) strain and may be useful for inclusion into panels used for antigenic typing of rabies viruses. The utility of these MAbs to detect truncated versions of the P product may facilitate more fundamental studies on the function of this rabies virus protein. PMID:20600390

  19. Selection of Recombinant Human Antibodies.

    Science.gov (United States)

    Tomszak, Florian; Weber, Susanne; Zantow, Jonas; Schirrmann, Thomas; Hust, Michael; Frenzel, André

    2016-01-01

    Since the development of therapeutic antibodies the demand of recombinant human antibodies is steadily increasing. Traditionally, therapeutic antibodies were generated by immunization of rat or mice, the generation of hybridoma clones, cloning of the antibody genes and subsequent humanization and engineering of the lead candidates. In the last few years, techniques were developed that use transgenic animals with a human antibody gene repertoire. Here, modern recombinant DNA technologies can be combined with well established immunization and hybridoma technologies to generate already affinity maturated human antibodies. An alternative are in vitro technologies which enabled the generation of fully human antibodies from antibody gene libraries that even exceed the human antibody repertoire. Specific antibodies can be isolated from these libraries in a very short time and therefore reduce the development time of an antibody drug at a very early stage.In this review, we describe different technologies that are currently used for the in vitro and in vivo generation of human antibodies. PMID:27236551

  20. The antibody mining toolbox: an open source tool for the rapid analysis of antibody repertoires.

    Science.gov (United States)

    D'Angelo, Sara; Glanville, Jacob; Ferrara, Fortunato; Naranjo, Leslie; Gleasner, Cheryl D; Shen, Xiaohong; Bradbury, Andrew R M; Kiss, Csaba

    2014-01-01

    In vitro selection has been an essential tool in the development of recombinant antibodies against various antigen targets. Deep sequencing has recently been gaining ground as an alternative and valuable method to analyze such antibody selections. The analysis provides a novel and extremely detailed view of selected antibody populations, and allows the identification of specific antibodies using only sequencing data, potentially eliminating the need for expensive and laborious low-throughput screening methods such as enzyme-linked immunosorbant assay. The high cost and the need for bioinformatics experts and powerful computer clusters, however, have limited the general use of deep sequencing in antibody selections. Here, we describe the AbMining ToolBox, an open source software package for the straightforward analysis of antibody libraries sequenced by the three main next generation sequencing platforms (454, Ion Torrent, MiSeq). The ToolBox is able to identify heavy chain CDR3s as effectively as more computationally intense software, and can be easily adapted to analyze other portions of antibody variable genes, as well as the selection outputs of libraries based on different scaffolds. The software runs on all common operating systems (Microsoft Windows, Mac OS X, Linux), on standard personal computers, and sequence analysis of 1-2 million reads can be accomplished in 10-15 min, a fraction of the time of competing software. Use of the ToolBox will allow the average researcher to incorporate deep sequence analysis into routine selections from antibody display libraries. PMID:24423623

  1. The humoral immune response to Chlamydia trachomatis in patients with acute reactive arthritis

    DEFF Research Database (Denmark)

    Larsen, B; Birkelund, Svend; Mordhorst, CH;

    1994-01-01

    . trachomatis cysteine rich outer membrane protein 2 (Omp2) and lipopolysaccharide (LPS) were detected in 10 patients. Thus 40% of the patients presented antibodies specific for C. trachomatis. There was no correlation between acute reactive arthritis and antibodies to heat-shock proteins GroEL, GroES and DnaK....

  2. Transfusion management of patients with red blood cell antibodies

    Directory of Open Access Journals (Sweden)

    Bujandrić Nevenka B.

    2013-01-01

    Full Text Available Introduction. Red blood cell antibodies may cause a positive result of pre-transfusion blood compatibility testing (crossmatch test. It can be a problem to provide suitable blood units for patients with clinically significant antibodies to high-frequency antigens as well as for those with multiple alloantibody specificities. This study was aimed at identifying transfused patients in the population of South-Backa who had developed clinically significant red blood cell alloantibodies. Material and methods. We analyzed the records of crossmatch results and antibody screening performed at the Blood Transfusion Institute of Vojvodina during 2012. Results. Antibodies were found in 103 patients: A 63 patients with single antibodies: 1 16 with antibodies of unknown specificity (3 autoantibodies, 13 alloantibodies; 2 39 with clinically significant antibodies (23 from Rh system (2 anti-C, 2 anti-D, 12 anti-E, 7 anti-c, 4 anti-K, 3 anti-Fya, 7 anti-Jka, 2 anti-S; 3 8 with usually not significant antibodies (6 anti-M, 1 anti-A1, 1 anti- Cw; B 40 patients developed multiple antibodies: 1 all patients had at least one clinically significant antibody from various blood group system (44 Rh, 13 Kell, 7 Kidd, 7 MNSs (S, s; 2 3 patients had usually not significant antibodies (1 Lewis, 2 Lutheran; 3 3 patients occasionally had clinically significant antibody (3 anti- Yta; 4 3 patients had antibodies of unknown specificity (2 autoantibodies, 1alloantibody. Antibodies detected in the majority of patients (65-63.1% had a specificity of Rh and/or the Kell system. Conclusions. The main goal of pre-transfusion blood compatibility testing is to detect clinically significant antibodies. The provision of antigen negative blood units for those patients is a special challenge for blood establishments. Database with a sufficient number of typed blood donors can help to resolve this problem.

  3. Reactivity and neutron dynamics

    International Nuclear Information System (INIS)

    Basing on the results of experiments mad with the full-scale WWER-1000 reactor core the problems of simulating neutron distributed transients, reactivity role in their description and uncertainties connected with reactivity value determination are discussed. Adiabatic approximation application and reactivity insertions lead to multiplicative representation of the solution kinetics equation including amplitude multiplier and form function

  4. Fracture Reactivation in Chemically Reactive Rock Systems

    Science.gov (United States)

    Eichhubl, P.; Hooker, J. N.

    2013-12-01

    Reactivation of existing fractures is a fundamental process of brittle failure that controls the nucleation of earthquake ruptures, propagation and linkage of hydraulic fractures in oil and gas production, and the evolution of fault and fracture networks and thus of fluid and heat transport in the upper crust. At depths below 2-3 km, and frequently shallower, brittle processes of fracture growth, linkage, and reactivation compete with chemical processes of fracture sealing by mineral precipitation, with precipitation rates similar to fracture opening rates. We recently found rates of fracture opening in tectonically quiescent settings of 10-20 μm/m.y., rates similar to euhedral quartz precipitation under these conditions. The tendency of existing partially or completely cemented fractures to reactivate will vary depending on strain rate, mineral precipitation kinetics, strength contrast between host rock and fracture cement, stress conditions, degree of fracture infill, and fracture network geometry. Natural fractures in quartzite of the Cambrian Eriboll Formation, NW Scotland, exhibit a complex history of fracture formation and reactivation, with reactivation involving both repeated crack-seal opening-mode failure and shear failure of fractures that formed in opening mode. Fractures are partially to completely sealed with crack-seal or euhedral quartz cement or quartz cement fragmented by shear reactivation. Degree of cementation controls the tendency of fractures for later shear reactivation, to interact elastically with adjacent open fractures, and their intersection behavior. Using kinematic, dynamic, and diagenetic criteria, we determine the sequence of opening-mode fracture formation and later shear reactivation. We find that sheared fracture systems of similar orientation display spatially varying sense of slip We attribute these inconsistent directions of shear reactivation to 1) a heterogeneous stress field in this highly fractured rock unit and 2

  5. A radioimmunoassay for determination of anti-actin antibodies

    International Nuclear Information System (INIS)

    The reaction of spontaneously occurring human anti-actin antibodies and experimentally produced rabbit anti-actin antibodies was investigated in a solid-phase radioimmunoassay (RIA). Three structurally different in vitro forms of actin, monomeric G-actin, filamentous F-actin and aggregated denatured actin were used as antigens. Human anti-actin antibodies reacted with F- and G-actin but not with aggregated actin, while rabbit anti-actin antibodies gave a strong reaction with all 3 forms of actin indicating differences in antibody specificities. The results of the anti-actin RIA were compared with those obtained by indirect immunofluorescence (IFL) on cryostat sections of rat stomach. The anti-actin RIA discriminated between patients' sera and control sera in most cases, although the indirect IFL test gave more conclusive results. The seemingly low sensitivity of the anti-actin RIA compared with that of indirect IFL test for detection of human anti-actin antibodies is probably due to favourable antigen distribution in tissue, not available in the solid phase. The anti-actin RIA was able to detect anti-actin antibodies in 8 out of 8 immunized rabbits although only two produced antibodies detectable by indirect IFL. The differences in reactivity between the two methods may depend on the presence of aggregated denatured actin in the antigen preparation used for immunization and exposure of the corresponding antigenic determinants of actin on the solid phase. (Auth.)

  6. A monoclonal antibody (OPT1 to T cells which is available for paraffin-embedded materials.

    Directory of Open Access Journals (Sweden)

    Mukuzono,Hiroshi

    1991-06-01

    Full Text Available A monoclonal antibody (MAb, OPT1, reactive with T cells in formalin-fixed, paraffin-embedded tissue sections, has been identified through immunization with activated T cells from peripheral blood lymphocytes (PBL. The antibody is an IgG1 antibody as demonstrated by the Ouchterlony technique. By cytofluorometric analysis, almost all CD3+ lymphocytes and only a few CD20+ lymphocytes of peripheral blood expressed the OPT1 antigen. Nonhematolymphoid cell lines were negative for OPT1 by the immunoperoxidase staining using acetone-fixed cell lines. On the contrary, peripheral T cells, cells of two T cell lines out of four and a part of the cells of one B cell line out of two were positive for OPT1. The immunoperoxidase staining of paraffin-embedded tissue sections revealed that most of lymphocytes in T cell areas of lymph nodes expressed OPT1 antigen. Some lymphocytes in both cortex and medulla of the thymus and erythroid precursors of the bone marrow were OPT1+. In the malignant lymphoma series, approximately 90% of T cell lymphomas and 6% of B cell lymphomas reacted with OPT1. None of the Reed-Sternberg cells nor Hodgkin cells in Hodgkin's disease were positive. Consequently, OPT1 may be useful for the diagnosis and study of malignant lymphomas and other related lesions.

  7. Enhancing antibody: a novel component of the immune response.

    OpenAIRE

    Nemazee, D A; Sato, V L

    1982-01-01

    Current descriptions of the immune response identify two classes of antigenic stimuli that result in the production of specific antibody: (i) exogenous antigens and (ii) endogenous variable-region determinants of the immune system. We expand this scheme to include a third class of antigenic stimulus--new determinants created by the binding of antibody to antigen. This paper describes a set of monoclonal antibodies which arose after repeated immunization with antigen alone but which bound anti...

  8. HLA Genotyping and Antibody Characterization Using the Luminex™ Multiplex Technology

    OpenAIRE

    Heinemann, Falko Markus

    2009-01-01

    The Luminex-based human leukocyte antigen (HLA) antibody screening technology is widespread used in laboratories affiliated to kidney transplantation programs and enables both screening (i.e. the definition of positive or negative antibody status) and antibody identification with high sensitivity and specificity. HLA typing at different levels of resolution with Luminex technology uses sequence-specific oligonucleotide probes bound to color-coded microbeads in order to identify HLA alleles en...

  9. Development of antibodies to human embryonic stem cell antigens

    OpenAIRE

    Stanley Marisa; Rao Mahendra S; Olson Judith M; Cai Jingli; Taylor Eva; Ni Hsiao-Tzu

    2005-01-01

    Abstract Background Using antibodies to specific protein antigens is the method of choice to assign and identify cell lineage through simultaneous analysis of surface molecules and intracellular markers. Embryonic stem cell research can be benefited from using antibodies specific to transcriptional factors/markers that contribute to the "stemness" phenotype or critical for cell lineage. Results In this report, we have developed and validated antibodies (either monoclonal or polyclonal) specif...

  10. Preparation and Preliminary Application of MAdCAM-1 Polyclonal Antibody in Dairy Cows with Subclinical Mastitis.

    Science.gov (United States)

    Xu, Chuang; Chen, Yuanyuan; Chang, Qiaocheng; Xia, Cheng; Yang, Wei; Zhang, Hongyou

    2015-08-01

    MAdCAM-1 plays an important role in mediating immune response and inflammation. This study aimed to express and purify a fusion protein of MAdCAM-1 in prokaryotic cells and to prepare rat anti-bovine MAdCAM-1 polyclonal antibodies. Prokaryotic expression vector pGEX-4T-1-MAdCAM-1 and pET-28a-MAdCAM-1 were constructed, respectively. The above plasmids were transformed into BL21 Escherichia coli strain. These recombinant strains were induced by IPTG and identified by Western blot analysis and SDS-PAGE. Wistar rats were immunized with recombinant protein (pET-28a-MAdCAM-1) emulsified with Freund's adjuvant, and antibody titers were measured by indirect ELISA. Antibody titers reached the highest value (1:128,000) after the third immunization. Western blot showed that rat anti-bovine MAdCAM-1 polyclonal antibody can not only recognize recombinant MAdCAM-1 protein expressed in E. coli but also recognizes natural MAdCAM-1 protein extracted from bovine tissues. However, commercial anti-mouse MAdCAM-1 monoclonal antibodies did not recognize the recombinant MAdCAM-1 protein or natural protein, which indicated no cross-reactivity between bovine MAdCAM-1 and mouse MAdCAM-1. Real-time fluorescence quantitative polymerase chain reaction and Western blot analysis showed that MAdCAM-1 expression was limited in mammary lymphoid nodes of subclinical mastitis in dairy cows. We speculate that MAdCAM-1 expression is inconsistent in different periods of the dairy cows. The successful preparation of rat anti-bovine MAdCAM-1 polyclonal antibody and its preliminary application in dairy cows provide the foundation for further study of the mechanism of anti-inflammation of MAdCAM-1 in dairy cows with subclinical mastitis. PMID:26301930

  11. Antibody discovery: sourcing of monoclonal antibody variable domains.

    Science.gov (United States)

    Strohl, William R

    2014-03-01

    Historically, antibody variable domains for therapeutic antibodies have been sourced primarily from the mouse IgG repertoire, and typically either chimerized or humanized. More recently, human antibodies from transgenic mice producing human IgG, phage display libraries, and directly from human B lymphocytes have been used more broadly as sources of antibody variable domains for therapeutic antibodies. Of the total 36 antibodies approved by major maket regulatory agencies, the variable domain sequences of 26 originate from the mouse. Of these, four are marketed as murine antibodies (of which one is a mouse-rat hybrid IgG antibody), six are mouse-human chimeric antibodies, and 16 are humanized. Ten marketed antibodies have originated from human antibody genes, three isolated from phage libraries of human antibody genes and seven from transgenic mice producing human antibodies. Five antibodies currently in clinical trials have been sourced from camelids, as well as two from non-human primates, one from rat, and one from rabbit. Additional sources of antibody variable domains that may soon find their way into the clinic are potential antibodies from sharks and chickens. Finally, the various methods for retrieval of antibodies from humans, mouse and other sources, including various display technologies and amplification directly from B cells, are described. PMID:24168292

  12. 基于有功与无功相对大小的变压器励磁涌流鉴别新方法%A strategy based on the relative size of active and reactive power to identify transformer inrush current

    Institute of Scientific and Technical Information of China (English)

    周霏霏; 徐岩

    2011-01-01

    This paper presents a new strategy based on comparison of the relative size of active and reactive power to identify transformer inrush current. This paper analyzes the different change trend of active and reactive power in the conditions of magnetizing inrush and internal fault. When the transformer switches on a no-load transformer in a state of internal fault, due to the presence of fault slip, active power is always larger than the reactive power for a period of time; while when the inrush current occurs, even if the active power is larger than the reactive power, the duration time is very short (non-saturation time is 3~5 ms). There are significant differences between the two cases. By using this point, this strategy not only is able to quickly determine whether the transformer internal fault occurs, but also is easy to implement and has less calculation. The results of dynamic analog testing confirm that the strategy is not affected by inrush current and is able to quickly and reliably reflect the transformer internal fault, and the performance is better than that of the strategy based on harmonic restraint.%提出了一种基于有功功率和无功功率相对大小特征的变压器励磁涌流鉴别新方法.分析了励磁涌流和内部故障情况下,有功和无功功率不同的变化趋势.当变压器空投于故障时,由于故障支路的存在,有功功率在一段时间内始终大于无功功率,而发生励磁涌流时,即使有功大于无功,持续时间也很短(非饱和阶段的时间3~5 ms),这两种情况区别非常明显.利用这点不同构成判据,判断是否发生故障.该方法易于实现,计算量小,动作可靠、迅速.利用变压器各种运行状态的动模实验数据进行验证,结果表明该方法不受励磁涌流的影响且能够迅速、可靠地反映变压器内部故障,在性能上明显优于谐波制动判据.

  13. Radiation safety issues related to radiolabeled antibodies

    International Nuclear Information System (INIS)

    Techniques related to the use of radiolabeled antibodies in humans are reviewed and evaluated in this report. It is intended as an informational resource for the US Nuclear Regulatory Commission (NRC) and NRC licensees. Descriptions of techniques and health and safety issues are provided. Principal methods for labeling antibodies are summarized to help identify related radiation safety problems in the preparation of dosages for administration to patients. The descriptions are derived from an extensive literature review and consultations with experts in the field. A glossary of terms and acronyms is also included. An assessment was made of the extent of the involvement of organizations (other than the NRC) with safety issues related to radiolabeled antibodies, in order to identify regulatory issues which require attention. Federal regulations and guides were also reviewed for their relevance. A few (but significant) differences between the use of common radiopharmaceuticals and radiolabeled antibodies were observed. The clearance rate of whole, radiolabeled immunoglobulin is somewhat slower than common radiopharmaceuticals, and new methods of administration are being used. New nuclides are being used or considered (e.g., Re-186 and At-211) for labeling antibodies. Some of these nuclides present new dosimetry, instrument calibration, and patient management problems. Subjects related to radiation safety that require additional research are identified. 149 refs., 3 figs., 20 tabs

  14. Detection of novel diagnostic antibodies in ankylosing spondylitis: An overview.

    Science.gov (United States)

    Quaden, Dana H F; De Winter, Liesbeth M; Somers, Veerle

    2016-08-01

    Ankylosing spondylitis (AS) is a debilitating, chronic, rheumatic disease characterized by inflammation and new bone formation resulting in fusion of the spine and sacroiliac joints. Since early treatment is impeded by a delayed diagnosis, it is highly important to find new biomarkers that improve early diagnosis and may also contribute to a better assessment of disease activity, prognosis and therapy response in AS. Because of the absence of rheumatoid factor, AS was long assumed to have a seronegative character and antibodies are thus not considered a hallmark of the disease. However, emerging evidence suggests plasma cells and autoantibodies to be involved in the disease course. In this review, the role of B cells and antibodies in AS is discussed. Furthermore, an overview is provided of antibodies identified in AS up till now, and their diagnostic potential. Many of these antibody responses were based on small study populations and further validation is lacking. Moreover, most were identified by a hypothesis-driven approach and thus limited to antibodies against targets that are already known to be involved in AS pathogenesis. Hence, we propose an unbiased approach to identify novel diagnostic antibodies. The already successfully applied techniques cDNA phage display and serological antigen selection will be used to identify antibodies against both known and new antigen targets in AS plasma. These newly identified antibodies will enhance early diagnosis of AS and provide more insight into the underlying disease pathology, resulting in a more effective treatment strategy and eventually an improved disease outcome. PMID:27288842

  15. Antikörpernachweis bei der Notfalltransfusion [Antibody detection in emergency transfusions. A comparison of 3 different methods

    OpenAIRE

    Heim, M. U.; Alraun, K.; Hansen, Ernil; Pachmann, U; Böck, M.; Mempel, W.

    1988-01-01

    We compared the manual Polybrene technique to three standard methods (albumin/Coombs, LISS/Coombs, and enzyme/papain) on 113 red cell antibodies. Polybrene identified 8 antibodies of the Rhesus system missed by standard methods, whereas 3 antibodies could only be detected by the standard techniques. Four antibodies were identified only in saline solution at room temperature; 6 were found by use of Polybrene exclusively in the additional Coombs phase. In addition, 61 antibodies were tested by ...

  16. Antibody remodeling: a general solution to the design of a metal-coordination site in an antibody binding pocket.

    OpenAIRE

    Roberts, V A; Iverson, B L; Iverson, S A; Benkovic, S J; Lerner, R A; Getzoff, E D; Tainer, J A

    1990-01-01

    To develop a general approach to designing cofactor-binding sites for catalytic antibodies, we characterized structural patterns in the binding sites of antibodies and zinc enzymes. Superposition of eight sets of antibody light- and heavy-chain variable domains identified structurally conserved sites within the sequence-variable complementarity determining regions. The pattern for catalytic zinc sites included two ligands close in sequence, a sequence-distant ligand, and a main-chain hydrogen...

  17. Radiolabelled antibodies in imaging

    International Nuclear Information System (INIS)

    Recent technological advances make it possible to produce pure (monoclonal) antibodies in unlimited quantities without the need for continuous immunization of animals and to label these antibodies with a variety of radionuclides which can be traced by single-photon computed tomography. An outline review of the state of the art is presented, with particular reference to the imaging of myocardial infarcts and to tumour imaging studies using labelled monoclonal antibodies (sup(99m)Tc and 125I). Lengthy bibliography. (U.K.)

  18. Rapid discovery and optimization of therapeutic antibodies against emerging infectious diseases

    OpenAIRE

    Rogers, J.; Schoepp, R J; Schröder, O; Clements, T.L.; Holland, T.F.; Li, J. Q.; Li, J.; Lewis, L.M.; Dirmeier, R.P.; Frey, G.J.; Tan, X; Wong, K.; Woodnutt, G; M. Keller; Reed, D.S.

    2008-01-01

    Using a comprehensive set of discovery and optimization tools, antibodies were produced with the ability to neutralize SARS coronavirus (SARS-CoV) infection in Vero E6 cells and in animal models. These anti-SARS antibodies were discovered using a novel DNA display method, which can identify new antibodies within days. Once neutralizing antibodies were identified, a comprehensive and effective means of converting the mouse sequences to human frameworks was accomplished using HuFR™ (human frame...

  19. Wildtype p53-specific Antibody and T-Cell Responses in Cancer Patients

    DEFF Research Database (Denmark)

    Pedersen, Anders Elm; Stryhn, Anette; Justesen, Sune;

    2011-01-01

    Mutation in the p53 gene based on single amino acid substitutions is a frequent event in human cancer. Accumulated mutant p53 protein is released to antigen presenting cells of the immune system and anti-p53 immune responses even against wt p53 is induced and observed in a number of human cancer...... patients. Detection of antibodies against wt p53 protein has been used as a diagnostic and prognostic marker and discovery of new T-cell epitopes has enabled design of cancer vaccination protocols with promising results. Here, we identified wt p53-specific antibodies in various cancer patients and...... identified a broad range of responses against wt p53 protein and 15-mer peptides using a novel print array technology. Likewise, using bioinformatic tools in silico, we identified CD8 T-cell specificity or reactivity against HLA-A*02:01 binding peptides wt p53(65-73), wt p53(187-197), and wt p53(264-272) in...

  20. Oilseed rape and bronchial reactivity.

    OpenAIRE

    Soutar, A; Harker, C; Seaton, A.; Packe, G

    1995-01-01

    OBJECTIVES--To investigate atopy and changes in symptoms, peak flow rate, and bronchial reactivity in people complaining of symptoms during the oilseed rape flowering season. METHODS--37 people who had given positive answers to questions about the presence of symptoms in relation to the flowering season of oilseed rape and 24 controls with no such symptoms were studied, although not all took part in all parts of the study. All had been previously identified in a cross sectional survey of a ra...

  1. Method for preparation of single chain antibodies

    Science.gov (United States)

    Cheung, Nai-Kong V.; Guo, Hong-fen

    2012-04-03

    This invention provides a method for identifying cells expressing a target single chain antibody (scFv) directed against a target antigen from a collection of cells that includes cells that do not express the target scFv, comprising the step of combining the collection of cells with an anti-idiotype directed to an antibody specific for the target antigen and detecting interaction, if any, of the anti-idiotype with the cells, wherein the occurrence of an interaction identifies the cell as one which expresses the target scFv. This invention also provides a method for making a single chain antibody (scFv) directed against an antigen, wherein the selection of clones is made based upon interaction of those clones with an appropriate anti-idiotype, and heretofore inaccessible scFv so made. This invention provides the above methods or any combination thereof. Finally, this invention provides various uses of these methods.

  2. Production and characterization of monoclonal antibodies to IgM of Pacific herring (Clupea pallasii)

    Science.gov (United States)

    Purcell, Maureen K.; Bromage, Erin S.; Silva, Jessica; Hansen, John D.; Badil, Samantha M.; Woodson, James C.; Hershberger, Paul K.

    2012-01-01

    Pacific herring (Clupea pallasii) have a central role in the North Pacific ecosystem as a forage fish species and are natural reservoirs of several important finfish pathogens, including Viral hemorrhagic septicemia virus (VHSV). Here, we report the identification of the gene encoding the immunoglobulin mu (IgM) heavy chain, as well as the development and characterization of monoclonal antibodies (MAbs) that specifically react with Pacific herring IgM. Pacific herring immunoglobulin was purified and consisted of heavy and light chains of approximately 80 and 25 kDa. Three hybridoma clones were initially identified by ELISA as reactive with purified immunoglobulin but only one clone was able to detect an 80 kDa protein in Pacific and Atlantic herring (Clupea harengus) whole plasma by denaturing western blot. However, all three MAbs were able to precipitate an 80 kDa protein from Pacific herring and LCMS sequencing of peptide fragments derived from this protein matched the predicted amino acid sequence of the cloned, heavy chain gene. In addition, two of the MAbs stained cells within the putative lymphocyte gates for the spleen, anterior kidney and posterior kidney but were not reactive for myeloid/granulocyte gates, which is consistent with these MAbs reacting with surface IgM+ B-cells. To our knowledge, this is the first report of IgM-related gene sequences and anti-IgM monoclonal antibodies from any member of the family Clupeidae. The antibodies produced in this study are critical for achieving our long-term goal of conducting serological surveillance to assess pathogen exposure in natural populations of Pacific herring.

  3. Anti-sulfotyrosine antibodies

    Science.gov (United States)

    Bertozzi, Carolyn R.; Kehoe, John; Bradbury, Andrew M.

    2009-09-15

    The invention provides anti-sulfotyrosine specific antibodies capable of detecting and isolating polypeptides that are tyrosine-sulfated. The sulfotyrosine antibodies and antibody fragments of the invention may be used to discriminate between the non-sulfated and sulfated forms of such proteins, using any number of immunological assays, such ELISAs, immunoblots, Western Blots, immunoprecipitations, and the like. Using a phage-display system, single chain antibodies (scFvs) were generated and screened against tyrosine-sulfated synthetic peptide antigens, resulting in the isolation of scFvs that specifically recognize sulfotyrosine-containing peptides and/or demonstrate sulfotyrosine-specific binding in tyrosine sulfated proteins. The VH and VL genes from one such sulfotyrosine-specific scFv were employed to generate a full length, sulfotyrosine-specific immunoglobulin.

  4. HIV Antibody Test

    Science.gov (United States)

    ... be limited. Home Visit Global Sites Search Help? HIV Antibody and HIV Antigen (p24) Share this page: Was this page helpful? Also known as: HIV Screening Tests; AIDS Test; AIDS Screen; HIV Serology; ...

  5. Antinuclear antibody panel

    Science.gov (United States)

    ... blood may be due to: Chronic liver disease Collagen vascular disease Drug-induced lupus erythematosus Myositis (inflammatory muscle disease) ... Saunders; 2011:chap 51. Read More Antibody Arthritis Collagen vascular disease Drug-induced lupus erythematosus Liver disease Scleroderma Systemic ...

  6. PRODUCTION OF MONOCLONAL ANTIBODIES

    Directory of Open Access Journals (Sweden)

    TOLKOVA E.S.

    2015-01-01

    Full Text Available The article considers the use of monoclonal antibodies in immunotherapy and immunodiagnostics of oncological diseases and their production using hybridoma technolody with flow diagram and technological scheme of manufacturing process

  7. PRODUCTION OF MONOCLONAL ANTIBODIES

    OpenAIRE

    TOLKOVA E.S.

    2015-01-01

    The article considers the use of monoclonal antibodies in immunotherapy and immunodiagnostics of oncological diseases and their production using hybridoma technolody with flow diagram and technological scheme of manufacturing process

  8. The INNs and outs of antibody nonproprietary names.

    Science.gov (United States)

    Jones, Tim D; Carter, Paul J; Plückthun, Andreas; Vásquez, Max; Holgate, Robert G E; Hötzel, Isidro; Popplewell, Andrew G; Parren, Paul W H I; Enzelberger, Markus; Rademaker, Hendrik J; Clark, Michael R; Lowe, David C; Dahiyat, Bassil I; Smith, Victoria; Lambert, John M; Wu, Herren; Reilly, Mary; Haurum, John S; Dübel, Stefan; Huston, James S; Schirrmann, Thomas; Janssen, Richard A J; Steegmaier, Martin; Gross, Jane A; Bradbury, Andrew R M; Burton, Dennis R; Dimitrov, Dimiter S; Chester, Kerry A; Glennie, Martin J; Davies, Julian; Walker, Adam; Martin, Steve; McCafferty, John; Baker, Matthew P

    2016-01-01

    An important step in drug development is the assignment of an International Nonproprietary Name (INN) by the World Health Organization (WHO) that provides healthcare professionals with a unique and universally available designated name to identify each pharmaceutical substance. Monoclonal antibody INNs comprise a -mab suffix preceded by a substem indicating the antibody type, e.g., chimeric (-xi-), humanized (-zu-), or human (-u-). The WHO publishes INN definitions that specify how new monoclonal antibody therapeutics are categorized and adapts the definitions to new technologies. However, rapid progress in antibody technologies has blurred the boundaries between existing antibody categories and created a burgeoning array of new antibody formats. Thus, revising the INN system for antibodies is akin to aiming for a rapidly moving target. The WHO recently revised INN definitions for antibodies now to be based on amino acid sequence identity. These new definitions, however, are critically flawed as they are ambiguous and go against decades of scientific literature. A key concern is the imposition of an arbitrary threshold for identity against human germline antibody variable region sequences. This leads to inconsistent classification of somatically mutated human antibodies, humanized antibodies as well as antibodies derived from semi-synthetic/synthetic libraries and transgenic animals. Such sequence-based classification implies clear functional distinction between categories (e.g., immunogenicity). However, there is no scientific evidence to support this. Dialog between the WHO INN Expert Group and key stakeholders is needed to develop a new INN system for antibodies and to avoid confusion and miscommunication between researchers and clinicians prescribing antibodies. PMID:26716992

  9. Association of Immunofluorescence pattern of Antinuclear Antibody with Specific Autoantibodies in the Bangladeshi Population.

    Science.gov (United States)

    Sharmin, S; Ahmed, S; Abu Saleh, A; Rahman, F; Choudhury, M R; Hassan, M M

    2014-08-01

    Antinuclear antibody (ANA) is useful in the diagnosis of connective tissue disorder (CTD). Association of specific autoantibodies with the immunofluorescence pattern of ANA in CTD, noted in western literature has been considered as reference in all over the world. However, in Bangladesh no such research work or data correlating the autoantibodies and their ANA patterns is found. Objective of the study was to identify an association between immunofluorescence patterns of antinuclear antibody on HEp-2 cell and more specific antinuclear reactivities (e.g. anti-dsDNA and anti-extractable nuclear antigen) in the serum samples of CTD patients. Serum samples of 152 CTD patients (Systemic lupus erythematosus, Rhumatoid arthritis, Sjogren's syndrome, Systemic sclerosis, Polymyositis, Mixed connective tissue disease) were diagnosed clinically, attending at Bangabandhu Sheikh Mujib Medical University (BSMMU) during the study period of January, 2010 to December, 2010. Samples were subjected for ANA testing by Indirect Immunofluorescence (IIF) on HEp-2 cell (ALPHADIA) in dilution of 1:40, anti-dsDNA by ELISA and anti- extractable nuclear antigen (anti-ENA) by Dot Immunoblot. Dot blot strips were tested for anti-Sm, anti-RNP, anti-SSA/Ro, anti-SSB/La, anti-Scl-70 and anti-Jo-1. Out of 152 patients 110 (72.3%) cases were ANA positive by IIF on HEp-2 cell. ANA positive sera exhibited four fluorescence patterns such as speckled (50.8%), peripheral (21.6%) , homogenous (18.1%) and nucleolar pattern (9%). Peripheral pattern and homogenous pattern was predominantly associated with anti-dsDNA (p RNP (25.7%) then anti-Scl-70 (20%), anti-SSA (14.2%) and anti-SSB (5.7%). Multiple anti-ENA reactivities were identified in 34.28% cases. Peripheral and homogenous pattern is strongly associated with anti-dsDNA and speckled pattern may predict anti-ENA (specially ribonucleoprotiens). As a definite correlation between the ANA patterns and the group of antibodies was detected by dot immunoblot

  10. Expression of Recombinant Antibodies

    OpenAIRE

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transg...

  11. A region of the insulin receptor important for ligand binding (residues 450-601) is recognized by patients' autoimmune antibodies and inhibitory monoclonal antibodies.

    OpenAIRE

    Zhang, B; Roth, R A

    1991-01-01

    Chimeric receptors containing different portions of the homologous human insulin receptor, insulin-like growth factor I receptor, and insulin receptor-related receptor were utilized to identify the epitopes recognized by various anti-insulin receptor antibodies. The antibodies studied included 12 monoclonal antibodies to the extracellular domain of the human insulin receptor as well as 15 patients' sera with autoimmune anti-insulin receptor antibodies. All of the patients' sera and all 8 mono...

  12. Purification of Murine Monoclonal IgM Antibody

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    This paper presents the purification of a monoclonal IgM antibody against human tumor associated antigen Lewis-Y by ion exchange chromatography and gel filtration.Enzyme-linked immunosorbent assay (ELISA) and SDS-polyacrylamide gel electrophoresis (PAGE) were used to identify purified IgM antibody.In flow cytometry analysis, the purified IgM antibody recognizes human breast tumor cell line MCF-7 which expresses Lewis-Y antigen.This work presents a new way for the purification of murine monoclonal IgM antibody.

  13. H7N9 influenza virus neutralizing antibodies that possess few somatic mutations.

    Science.gov (United States)

    Thornburg, Natalie J; Zhang, Heng; Bangaru, Sandhya; Sapparapu, Gopal; Kose, Nurgun; Lampley, Rebecca M; Bombardi, Robin G; Yu, Yingchun; Graham, Stephen; Branchizio, Andre; Yoder, Sandra M; Rock, Michael T; Creech, C Buddy; Edwards, Kathryn M; Lee, David; Li, Sheng; Wilson, Ian A; García-Sastre, Adolfo; Albrecht, Randy A; Crowe, James E

    2016-04-01

    Avian H7N9 influenza viruses are group 2 influenza A viruses that have been identified as the etiologic agent for a current major outbreak that began in China in 2013 and may pose a pandemic threat. Here, we examined the human H7-reactive antibody response in 75 recipients of a monovalent inactivated A/Shanghai/02/2013 H7N9 vaccine. After 2 doses of vaccine, the majority of donors had memory B cells that secreted IgGs specific for H7 HA, with dominant responses against single HA subtypes, although frequencies of H7-reactive B cells ranged widely between donors. We isolated 12 naturally occurring mAbs with low half-maximal effective concentrations for binding, 5 of which possessed neutralizing and HA-inhibiting activities. The 5 neutralizing mAbs exhibited narrow breadth of reactivity with influenza H7 strains. Epitope-mapping studies using neutralization escape mutant analysis, deuterium exchange mass spectrometry, and x-ray crystallography revealed that these neutralizing mAbs bind near the receptor-binding pocket on HA. All 5 neutralizing mAbs possessed low numbers of somatic mutations, suggesting the clones arose from naive B cells. The most potent mAb, H7.167, was tested as a prophylactic treatment in a mouse intranasal virus challenge study, and systemic administration of the mAb markedly reduced viral lung titers. PMID:26950424

  14. Monoclonal antibody identification of subpopulations of cerebral cortical neurons affected in Alzheimer's disease

    International Nuclear Information System (INIS)

    Neuronal degeneration is one of the hallmarks of Alzheimer's disease (AD). Given the paucity of molecular markers available for the identification of neuronal subtypes, the specificity of neuronal loss within the cerebral cortex has been difficult to evaluate. With a panel of four monoclonal antibodies (mAbs) applied to central nervous system tissues from AD patients, the authors have immunocytochemically identified a population of vulnerable cortical neurons; a subpopulation of pyramidal neurons is recognized by mAbs 3F12 and 44.1 in the hippocampus and neocortex, and clusters of multipolar neurons in the entorhinal cortex reactive with mAb 44.1 show selective degeneration. Closely adjacent stellate-like neurons in these regions, identified by mAb 6A2, show striking preservation in AD. The neurons recognized by mAbs 3F12 and 44.1 do not comprise a single known neurotransmitter system. mAb 3A4 identifies a phosphorylated antigen that is undetectable in normal brain but accumulates early in the course of AD in somas of vulnerable neurons. Antigen 3A4 is distinct from material reactive with thioflavin S or antibody generated against paired helical filaments. Initially, antigen 3A4 is localized to neurons in the entorhinal cortex and subiculum, later in the association neocortex, and, ultimately in cases of long duration, in primary sensory cortical regions. mAb 3F12 recognizes multiple bands of immunoblots of homogenates of normal and AD cortical tissues, whereas mAb 3A4 does not bind to immunoblots containing neurofilament proteins or brain homogenates from AD patients. Ultrastructurally, antigen 3A4 is localized to paired-helical filaments. Using these mAbs, further molecular characterization of the affected cortical neurons is now possible

  15. Three monoclonal antibodies against the serpin protease nexin-1 prevent protease translocation

    DEFF Research Database (Denmark)

    Kousted, Tina M; Skjødt, Karsten; Petersen, Steen V;

    2014-01-01

    , conversion to an inactive conformation or induction of serpin substrate behaviour. Until now, no inhibitory antibodies against PN-1 have been thoroughly characterised. Here we report the development of three monoclonal antibodies binding specifically and with high affinity to human PN-1. The antibodies all...... serpin presenting its so-called reactive centre loop as a substrate to its target protease, resulting in a covalent complex with the inactivated enzyme. Previously, three mechanisms have been proposed for the inactivation of serpins by monoclonal antibodies: steric blockage of protease recognition...... abolish the protease inhibitory activity of PN-1. In the presence of the antibodies, PN-1 does not form a complex with its target proteases, but is recovered in a reactive centre cleaved form. Using site-directed mutagenesis, we mapped the three overlapping epitopes to an area spanning the gap between the...

  16. Characterization of Antibodies for Grain-Specific Gluten Detection.

    Science.gov (United States)

    Sharma, Girdhari M; Rallabhandi, Prasad; Williams, Kristina M; Pahlavan, Autusa

    2016-03-01

    Gluten ingestion causes immunoglobulin E (IgE)-mediated allergy or celiac disease in sensitive individuals, and a strict gluten-free diet greatly limits food choices. Immunoassays such as enzyme-linked immunosorbent assay (ELISA) are used to quantify gluten to ensure labeling compliance of gluten-free foods. Anti-gluten antibodies may not exhibit equal affinity to gluten from wheat, rye, and barley. Moreover, because wheat gluten is commonly used as a calibrator in ELISA, accurate gluten quantitation from rye and barley contaminated foods may be compromised. Immunoassays utilizing grain-specific antibodies and calibrators may help improve gluten quantitation. In this study, polyclonal antibodies raised against gluten-containing grain-specific peptides were characterized for their immunoreactivity to gluten from different grain sources. Strong immunoreactivity to multiple gluten polypeptides from wheat, rye, and barley was observed in the range 34 to 43 kDa with anti-gliadin, 11 to 15 and 72 to 95 kDa with anti-secalin, and 30 to 43 kDa with anti-hordein peptide antibodies, respectively. Minimal or no cross-reactivity with gluten from other grains was observed among these antibodies. The anti-consensus peptide antibody raised against a repetitive amino acid sequence of proline and glutamine exhibited immunoreactivity to gluten from wheat, rye, barley, and oat. The antibodies exhibited similar immunoreactivity with most of the corresponding grain cultivars by ELISA. The high specificity and minimal cross-reactivity of grain-specific antibodies suggest their potential use in immunoassays for accurate gluten quantitation. PMID:26878584

  17. Use of immunomagnetic reduction for C-reactive protein assay in clinical samples

    Directory of Open Access Journals (Sweden)

    Chang CH

    2012-08-01

    Full Text Available Chien-Hsi Chang,1 Zhi-Xian Lai,1 Hsiu-Li Lin,2 Che-Chuan Yang,3 Hsin-Hsien Chen,3,4 Shieh-Yueh Yang,5 Herng-Er Horng,3 Chin-Yih Hong,6 Hong-Chang Yang,4 Hsiu-Chen Lin1,71Department of Laboratory Medicine, Taipei Medical University Hospital, 2Department of Neurology, General Cathay Hospital, Sijhih Branch, 3Institute of Electro-optical Science and Technology, National Taiwan Normal University, 4Department of Physics, National Taiwan University, 5MagQu Co, Ltd, Sindian District, New Taipei City, 6Department of Mechanical Engineering, Nan-Kai University of Technology, Nan-tau County, 7Department of Pediatrics, School of Medicine, College of Medicine, Taipei Medical University, Taipei, TaiwanBackground: Magnetic nanoparticles biofunctionalized with antibodies are able to recognize and bind to the corresponding antigens. In this work, anti-C-reactive protein (CRP antibody was covalently conjugated onto the surface of magnetic nanoparticles to label CRP specifically in serum.Methods: The level of serum CRP was detected by immunomagnetic reduction (IMR assay, which identifies the changes in the magnetic signal representing the level of interaction between antibody-conjugated magnetic nanoparticles and CRP proteins. To investigate the feasibility of IMR for clinical application, pure CRP solutions and 40 human serum samples were tested for IMR detection of CRP to characterize sensitivity, specificity, and interference.Results: In comparison with the immunoturbidimetry assay, the results of the IMR assay indicated higher sensitivity and had a high correlation with those of the current immunoturbidimetry assay.Conclusion: We have developed a novel and promising way to assay CRP in human serum using immunomagnetic reduction in clinical diagnosis.Keywords: magnetic nanoparticles, immunomagnetic reduction, C-reactive protein

  18. Capture ELISA for IgM antibodies against Plasmodium falciparum glutamate rich protein

    DEFF Research Database (Denmark)

    Dziegiel, M; Borre, Mette; Petersen, E; Högh, B; Jepsen, S; Vuust, J; Axelsen, N

    1992-01-01

    -galactosidase fusion protein and used as antigen after purification and biotinylation. Specific IgM antibodies were found in 51% (39/77) of sera from adult Liberians immune to malaria. The binding of IgM antibodies was specific for the malaria portion of the fusion protein and no cross-reactivity was found in sera...... from patients with IgM antibodies due to other diseases. Inhibition studies with a fusion protein containing amino acid residues 816-1134 (GLURP816-1134) representing the carboxy-terminal repeat region suggested a different use of epitopes for IgM antibodies in different individuals....

  19. Antibody-Dependent NK Cell Activation Is Associated with Late Kidney Allograft Dysfunction and the Complement-Independent Alloreactive Potential of Donor-Specific Antibodies.

    Science.gov (United States)

    Legris, Tristan; Picard, Christophe; Todorova, Dilyana; Lyonnet, Luc; Laporte, Cathy; Dumoulin, Chloé; Nicolino-Brunet, Corinne; Daniel, Laurent; Loundou, Anderson; Morange, Sophie; Bataille, Stanislas; Vacher-Coponat, Henri; Moal, Valérie; Berland, Yvon; Dignat-George, Francoise; Burtey, Stéphane; Paul, Pascale

    2016-01-01

    Although kidney transplantation remains the best treatment for end-stage renal failure, it is limited by chronic humoral aggression of the graft vasculature by donor-specific antibodies (DSAs). The complement-independent mechanisms that lead to the antibody-mediated rejection (ABMR) of kidney allografts remain poorly understood. Increasing lines of evidence have revealed the relevance of natural killer (NK) cells as innate immune effectors of antibody-dependent cellular cytotoxicity (ADCC), but few studies have investigated their alloreactive potential in the context of solid organ transplantation. Our study aimed to investigate the potential contribution of the antibody-dependent alloreactive function of NK cells to kidney graft dysfunction. We first conducted an observational study to investigate whether the cytotoxic function of NK cells is associated with chronic allograft dysfunction. The NK-Cellular Humoral Activation Test (NK-CHAT) was designed to evaluate the recipient and antibody-dependent reactivity of NK cells against allogeneic target cells. The release of CD107a/Lamp1(+) cytotoxic granules, resulting from the recognition of rituximab-coated B cells by NK cells, was analyzed in 148 kidney transplant recipients (KTRs, mean graft duration: 6.2 years). Enhanced ADCC responsiveness was associated with reduced graft function and identified as an independent risk factor predicting a decline in the estimated glomerular filtration rate over a 1-year period (hazard ratio: 2.83). In a second approach, we used the NK-CHAT to reveal the cytotoxic potential of circulating alloantibodies in vitro. The level of CD16 engagement resulting from the in vitro recognition of serum-coated allogeneic B cells or splenic cells was further identified as a specific marker of DSA-induced ADCC. The NK-CHAT scoring of sera obtained from 40 patients at the time of transplant biopsy was associated with ABMR diagnosis. Our findings indicate that despite the administration of

  20. PRODUCTION OF MONOCLONAL ANTIBODY AGAINST HUMAN IMMUNOGLOBULIN

    Directory of Open Access Journals (Sweden)

    J. Majidi

    2000-04-01

    Full Text Available Immunoglobulin E is one of the five classes of immonoglobulins that plays an important role in allergic diseases. Production of monoclonal antibodies by a single clonotype against different epitopes of immunoglobulin E has high priority in development of diagnostic kits.In this study, an attempt was made to produce monoclonal antibodies against human immunoglobulin E. Balb/c mice were immunized with semipurified immunoglobulin E and spleen cells fused with SP2.0 mouse myeloma eel! line in the presence of polyethylene glycol. Supernatant of hybridoma cells was screened for detection of antibody by enzyme linked immonosorbent assay method. Cloning of selective high absorbance wells were done with limiting dilution method. The suitable clone (monoclone was selected by enzyme linked immunosorbent assay and confirmed by immunoblot. The subclass of the chosen monoclonal antibodies was determined and the clones freezed and kept in liquid nitrogen.During this study three successful fusions were carried out, which resulted in development of 156 clones with high production of anti-IgE. Fourteen clones with the highest titres were selected for cloning. After limiting dilution more than 100 monoclonal antibodies were produced and the suitable (me (GJ0F7, i.e.; the clone which displayed the high absorbance in reaction with purified immunoglobulin E and the lowest cross-reactivity with immunoglobulin M, immunoglobulin G and immoglobulin A was chosen. In immunoblotting, presence of high density band in reaction with immunoglobulin E was confirmed. The suitable mab was shown to be IgG 1 subclass with kappa light chain. It seems that, this mab could be successfully used in diagnostic kits.

  1. Interim Guidance for Interpretation of Zika Virus Antibody Test Results.

    Science.gov (United States)

    Rabe, Ingrid B; Staples, J Erin; Villanueva, Julie; Hummel, Kimberly B; Johnson, Jeffrey A; Rose, Laura; Hills, Susan; Wasley, Annemarie; Fischer, Marc; Powers, Ann M

    2016-01-01

    Zika virus is a single-stranded RNA virus in the genus Flavivirus and is closely related to dengue, West Nile, Japanese encephalitis, and yellow fever viruses (1,2). Among flaviviruses, Zika and dengue virus share similar symptoms of infection, transmission cycles, and geographic distribution. Diagnostic testing for Zika virus infection can be accomplished using both molecular and serologic methods. For persons with suspected Zika virus disease, a positive real-time reverse transcription-polymerase chain reaction (rRT-PCR) result confirms Zika virus infection, but a negative rRT-PCR result does not exclude infection (3-7). In these cases, immunoglobulin (Ig) M and neutralizing antibody testing can identify additional recent Zika virus infections (6,7). However, Zika virus antibody test results can be difficult to interpret because of cross-reactivity with other flaviviruses, which can preclude identification of the specific infecting virus, especially when the person previously was infected with or vaccinated against a related flavivirus (8). This is important because the results of Zika and dengue virus testing will guide clinical management. Pregnant women with laboratory evidence of Zika virus infection should be evaluated and managed for possible adverse pregnancy outcomes and be reported to the U.S. Zika Pregnancy Registry or the Puerto Rico Zika Active Pregnancy Surveillance System for clinical follow-up (9,10). All patients with clinically suspected dengue should have proper management to reduce the risk for hemorrhage and shock (11). If serologic testing indicates recent flavivirus infection that could be caused by either Zika or dengue virus, patients should be clinically managed for both infections because they might have been infected with either virus. PMID:27254248

  2. Differential recognition of calmodulin-enzyme complexes by a conformation-specific anti-calmodulin monoclonal antibody

    International Nuclear Information System (INIS)

    An anti-calmodulin monoclonal antibody having an absolute requirement for Ca2+ has been produced from mice immunized with a mixture of calmodulin and calmodulin-binding proteins. Radioimmune assays were developed for the determination of its specificity. The epitope for this antibody resides on the COOH-terminal half of the mammalian protein. Plant calmodulin or toponin C had little reactivity. The apparent affinity of the antibody for calmodulin was increased approximately 60-fold in the presence of heart calmodulin-dependent phosphodiesterase. The presence of heart phosphodiesterase in the radioimmune assay greatly enhanced the sensitivity for calmodulin. The intrinsic calmodulin subunit of phosphorylase kinase and calmodulin which was bound to brain phosphodiesterases was also recognized with high affinity by the antibody. In direct binding experiments, most of the calmodulin-binding proteins studied were unreactive with the antibody. This selectivity allowed purification of heart and two brain calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes on immobilized antibody affinity columns. The data suggest that the binding of ligands to Ca2+/calmodulin induce conformation changes in calmodulin which alter reactivity with the anti-calmodulin monoclonal antibody. The differential antibody reactivity toward calmodulin-enzyme complexes indicates that target proteins either induce very different conformations in calmodulin and/or interact with different geometries relative to the antibody binding site. The anti-calmodulin monoclonal antibody should be useful for the purification of other calmodulin-dependent phosphodiesterases as well as isozymes of phosphorylase kinase

  3. Computational screening of the human TF-glycome provides a structural definition for the specificity of anti-tumor antibody JAA-F11.

    Directory of Open Access Journals (Sweden)

    Matthew B Tessier

    Full Text Available Recombinant antibodies are of profound clinical significance; yet, anti-carbohydrate antibodies are prone to undesirable cross-reactivity with structurally related-glycans. Here we introduce a new technology called Computational Carbohydrate Grafting (CCG, which enables a virtual library of glycans to be assessed for protein binding specificity, and employ it to define the scope and structural origin of the binding specificity of antibody JAA-F11 for glycans containing the Thomsen-Friedenreich (TF human tumor antigen. A virtual library of the entire human glycome (GLibrary-3D was constructed, from which 1,182 TF-containing human glycans were identified and assessed for their ability to fit into the antibody combining site. The glycans were categorized into putative binders, or non-binders, on the basis of steric clashes with the antibody surface. The analysis employed a structure of the immune complex, generated by docking the TF-disaccharide (Galβ1-3GalNAcα into a crystal structure of the JAA-F11 antigen binding fragment, which was shown to be consistent with saturation transfer difference (STD NMR data. The specificities predicted by CCG were fully consistent with data from experimental glycan array screening, and confirmed that the antibody is selective for the TF-antigen and certain extended core-2 type mucins. Additionally, the CCG analysis identified a limited number of related putative binding motifs, and provided a structural basis for interpreting the specificity. CCG can be utilized to facilitate clinical applications through the determination of the three-dimensional interaction of glycans with proteins, thus augmenting drug and vaccine development techniques that seek to optimize the specificity and affinity of neutralizing proteins, which target glycans associated with diseases including cancer and HIV.

  4. Phage Display Approaches for the Isolation of Monoclonal Antibodies Against Dengue Virus Envelope Domain III from Human and Mouse Derived Libraries

    Directory of Open Access Journals (Sweden)

    Subhash G. Vasudevan

    2012-02-01

    Full Text Available Domain III of the dengue virus envelope protein (EDIII, aa295-395 has an immunoglobulin fold and is the proposed receptor-binding domain of the virus. Previous studies have shown that monoclonal antibodies against EDIII can be neutralizing and have therapeutic potential. Here, cloned Fab-phage libraries of human and mouse origin were screened for DENV specific antibodies. Firstly, bacterially expressed EDIII or whole virus particles were used as bait in biopanning against a large naïve human Fab-phage library ( > 10 billion independent clones. Multiple panning strategies were employed, and in excess of 1000 clones were screened, but all of the antibodies identified bound the envelope in regions outside EDIII suggesting EDIII antibodies are virtually absent from the naïve human repertoire. Next, a chimeric Fab-phage library was constructed from a panel of EDIII specific mouse hybridomas by pooling the VH and VL chain sequences from the hybridomas and cloning these into the pComb3X phagemid vector with human CH and CL encoding sequences. Biopanning against EDIII identified a unique antibody (C9 that cross-reacts with EDIII from DENV1-3 and, in the IgG format, binds and neutralizes DENV2 in cell-based assays. Sequence analysis and saturation mutagenesis of complementary determining regions (CDR in the C9 light chain suggest an antigen recognition model in which the LCDR3 is a key determinant of EDIII specificity, while modifications in LCDR1 and LCDR2 affect DENV serotype cross-reactivity. Overall, this study supports the current prevailing opinion that neutralizing anti-EDIII monoclonal antibodies can be readily generated in murine systems, but in humans the anti-DENV immune response is directed away from domain III.

  5. Monoclonal antibody against human ovarian tumor-associated antigens

    International Nuclear Information System (INIS)

    Mouse monoclonal antibodies (OV-TL 3) were raised against human ovarian tumor-associated antigens for diagnostic purposes. A cloned hybridoma cell line was obtained by fusion of murine myeloma cells with spleen lymphocytes from BALB/c mice immunized with a tumor cell suspension prepared from an ovarian endometrioid carcinoma. The antibodies were initially screened for their ability to bind on frozen sections of human ovarian carcinoma tissue and a negative reaction on gastric carcinoma tissue by indirect immunofluorescence. The reactivity of the selected OV-TL 3 clone (IgG1 subclass) was studied on normal and neoplastic tissues as well as on a cell line derived from the original tumor cell suspension used for immunization. OV-TL 3 antibodies stained frozen sections of human ovarian carcinomas of the following histological types: serous, mucinous, endometrioid, and clear cell. No reaction was found with breast cancers or other nongynecological tumors. No differences in staining pattern were observed between primary and metastatic ovarian carcinomas. OV-TL 3 antibodies brightly stained ovarian carcinoma cell clusters in ascitic fluids and left unstained mesothelial cells and peripheral blood cells. The OV-TL 3-defined antigen also remained strongly expressed on a cell line derived from the endometrioid ovarian carcinoma originally used for generation of OV-TL 3 clone. Reactivity was weak and irregular in a few ovarian cysts, while traces of fluorescence were sometimes detected in epithelial cells lining the female genital tract. In only 3 specimens of 15 endometrium carcinomas was weak focal reactivity with OV-TL 3 antibodies observed. The results of the immunofluorescence study were confirmed by the more sensitive avidin-biotin method and by 125I-labeled OV-TL 3 antibodies

  6. Pulse labeling of small nuclear ribonucleoproteins in vivo reveals distinct patterns of antigen recognition by human autoimmune antibodies.

    OpenAIRE

    Fisher, D E; Reeves, W H; Conner, G E; Blobel, G; Kunkel, H. G.

    1984-01-01

    Antibodies directed against small nuclear ribonucleoprotein ( snRNP ) particles are found in the Sm and RNP autoimmune sera from numerous patients with systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD). These two reactivities differ in disease distribution as well as antigen specificity. Although sera from both of these autoimmune syndromes contain snRNP reactive antibodies, distinction in antigen binding specificity have been difficult to define because of the par...

  7. Comprehensive Mapping of Common Immunodominant Epitopes in the West Nile Virus Nonstructural Protein 1 Recognized by Avian Antibody Responses

    OpenAIRE

    Sun, EnCheng; Jing ZHAO; Liu, Nihong; Yang, Tao; Xu, Qingyuan; Qin, Yongli; Bu, Zhigao; Yang, Yinhui; Lunt, Ross A.; Wang, Linfa; Wu, Donglai

    2012-01-01

    West Nile virus (WNV) is a mosquito-borne flavivirus that primarily infects birds but occasionally infects humans and horses. Certain species of birds, including crows, house sparrows, geese, blue jays and ravens, are considered highly susceptible hosts to WNV. The nonstructural protein 1 (NS1) of WNV can elicit protective immune responses, including NS1-reactive antibodies, during infection of animals. The antigenicity of NS1 suggests that NS1-reactive antibodies could provide a basis for se...

  8. A monoclonal antibody that tracks endospore formation in the microsporidium Nosema bombycis.

    Directory of Open Access Journals (Sweden)

    Yanhong Li

    Full Text Available Nosema bombycis, the first identified microsporidium, is a destructive pathogen of the silkworm Bombyx mori and causes severe worldwide economic losses in sericulture. Major microsporidian structural proteins, such as the spore wall protein (SWP, are known to be involved in host invasion. In this study, the reactivity of the monoclonal antibody 2B10 was tested against an endospore protein of N. bombycis with a molecular weight size at 50-kDa, using Western blotting. The antigen was purified after immunoprecipitation and was further identified as EOB13320 according to MALDI-TOF MS assay. We found that EOB13320 locates to the surface of the different developmental stages of the parasite, mostly the sporoblast stage and the mature spore after immunoelectron microscopy examination. EOB13320 was also widely distributed in the developing endospore, especially at the sporoblast stage. This endospore protein also accumulated in the cytoplasm of both the merogony and sporoblast stages. These results imply that EOB13320 detected by monoclonal antibody 2B10 is expressed throughout the life cycle of the parasite, notably during the stage when the endospore is formed, and that this protein is important for spore-coat formation and parasite maintenance. Our study could be instrumental in the understanding of spore wall formation and will help to gain greater insight into the biology of this parasite.

  9. Antibody recognition of porcine circovirus type 2 capsid protein epitopes after vaccination, infection, and disease.

    Science.gov (United States)

    Trible, Benjamin R; Kerrigan, Maureen; Crossland, Nicholas; Potter, Megan; Faaberg, Kay; Hesse, Richard; Rowland, Raymond R R

    2011-05-01

    Open reading frame 2 (ORF2) of porcine circovirus type 2 (PCV2) codes for the 233-amino-acid capsid protein (CP). Baculovirus-based vaccines that express only ORF2 are protective against clinical disease following experimental challenge or natural infection. The goal of this study was to identify regions in CP preferentially recognized by sera from experimentally infected and vaccinated pigs and to compare these responses to those of pigs diagnosed with porcine circovirus-associated disease (PCVAD), including porcine multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS). The approach was to react porcine sera with CP polypeptide fragments followed by finer mapping studies using overlapping oligopeptides that covered amino acids 141 to 200. The results showed that vaccinated pigs preferentially recognized only the largest polypeptide fragment, CP(43-233). A subset of experimentally infected pigs and pigs with PDNS showed strong reactivity against a CP oligopeptide, 169-STIDYFQPNNKR-180. Alanine scanning identified Y-173, F-174, Q-175, and K-179 as important for antibody recognition. The results from this study support the notion of PCV2 modulation of immunity, including antibody responses that may represent a precursor for disease. The recognition of CP(169-180) and other polypeptides provides opportunities to devise diagnostic tests for monitoring the immunological effectiveness of vaccination. PMID:21430122

  10. Reactions of peripheral blood mononuclear cells (PBMC) of camels with monoclonal antibodies against ruminant leukocytes.

    Science.gov (United States)

    Ungar-Waron, H; Yagil, R; Brenner, J; Paz, R; Partosh, N; Van Creveld, C; Lubashevsky, E; Trainin, Z

    2003-03-01

    The particular immune system of the camel has been but little investigated. In this work circulating camel peripheral blood mononuclear cells (PBMC) were studied by flow cytometry. Monoclonal antibodies (mAbs) raised against ruminant leukocytes were used for the detection of cell surface antigens. Monoclonals to T-cell markers, CD4 (CACT138A) and CD8 (CACT80C), exhibited no reactivity towards camel PBMC in contrast to their reactivity to PBMC of other ruminant species and those of cattle in particular. A relatively high percentage (29.1+/-8.9%) of camel PBMC reacted with a non-immunoglobulin cell surface marker, B-B2, comparable to the reactivity of bovine PBMC. The B-B7 cell marker revealed 22.4+/-10.0% of reactive camel PBMC while the CD45 leukocyte common antigen was identified only on 19.4+/-3.1% of camel PBMC as compared to 74.7+/-4.9% for bovine PBMC. IgM (PIg45A) was detected on 9.1+/-1.4% of camel PBMC and on 46.6+/-19.5% of the bovine PBMC. Double fluorescent labeling with two B-cell markers and an anti-ruminant lambda light-chain mAb revealed 7-9% of cells bearing both B and lambda L-chain markers. Light chain reactivity was also assessed using an anti-goat F(ab')(2) antiserum. The values obtained, 14.3+/-5.8% for the camel and 47.8+/-2.7% for the cattle, are close to the values observed for surface IgM. These data suggest that camels, like other ruminants, possess L-chain bearing cells of the B-cell lineage. However, in the camel, Igs are different in that in addition to regular four chain Igs, about 65% of them possess two heavy chain Igs devoid of light chains. Because different sets of V(H) gene segments are used by four and two chain Igs, it is possible that there might be two lineages of B-cells each secreting a different form of antibodies. PMID:12493494

  11. Computer based data acquisition for on-line reactivity computation and reactivity balance for FBTR [Paper No.:E1

    International Nuclear Information System (INIS)

    Reactivity calculations are important for fast reactors. With the availability of low cost industrial personal computers, on-line reactivity computation and reactivity balance can be effectively carried out by computer based systems. In Fast Breeder Test Reactor (FBTR), on-line reactivity computation is carried out by industrial PC based system. It derives reactivity by solving point kinetic equations. Software can be tuned very easily to incorporate the changes in the values of decay constants and delay neutron fraction, when the core configuration changes. It has features like on-line color graphic display of reactivity, history storage for post mortem analysis and hard copy. On-line calculation of reactivity balance is very useful to identify any anomalous reactivity. The software calculates negative reactivity contribution due to rise in coolant temperature, coolant flow, reactor power and burn up. It calculates the positive reactivity contribution due to withdrawal of control rods. Any anomalous reactivity can be identified from reactivity balance table and corrective action can be taken. (author). 3 refs.,1 tab

  12. Epitope mapping of 7S cashew antigen in complex with antibody by solution-phase H/D exchange monitored by FT-ICR mass spectrometry.

    Science.gov (United States)

    Guan, Xiaoyan; Noble, Kyle A; Tao, Yeqing; Roux, Kenneth H; Sathe, Shridhar K; Young, Nicolas L; Marshall, Alan G

    2015-06-01

    The potential epitope of a recombinant food allergen protein, cashew Ana o 1, reactive to monoclonal antibody, mAb 2G4, has been mapped by solution-phase amide backbone H/D exchange (HDX) monitored by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Purified mAb 2G4 was incubated with recombinant Ana o 1 (rAna o 1) to form antigen:monoclonal antibody (Ag:mAb) complexes. Complexed and uncomplexed (free) rAna o 1 were then subjected to HDX-MS analysis. Five regions protected from H/D exchange upon mAb binding are identified as potential conformational epitope-contributing segments. PMID:26169135

  13. Yeast killer toxin-like anti-idiotypic antibodies.

    OpenAIRE

    POLONELLI, L.; Morace, G

    1988-01-01

    Anti-idiotypic antibodies (anti-Ids) were raised in a rabbit against a murine monoclonal antibody (MAb) neutralizing the yeast killer toxin produced by a strain of Pichia (Hansenula) anomala. In an immunodiffusion test, the anti-Ids produced in the rabbit recognized the antigen-binding site of the MAb used as the immunogen (KT4) but not that of another heterologous MAb. The absence of any significant cross-reactivity among the anti-Ids raised in a rabbit for a heterologous MAb suggested that ...

  14. Flows of Reactive Fluids

    CERN Document Server

    Prud'homme, Roger

    2010-01-01

    The modeling of reactive flows has progressed mainly with advances in aerospace, which gave birth to a new science called aerothermochemistry, as well as through developments in chemical and process engineering. The methods employed, the phenomena investigated, and the aims of modeling differ for each field; however, in all cases, the results obtained have considerably enriched the working knowledge of reactive flows. This work examines basic concepts and methods necessary to study reactive flows and transfer phenomena in areas such as fluid mechanics, thermodynamics, and chemistry. Specific topics covered include: * Equations of state * Transfer phenomena and chemical kinetics * Balance equations of reactive flows * Dimensionless numbers and similarity * Chemical reactors * Coupled phenomena * Turbulent flow concepts * Boundary layers and fluid layers * Reactive and nonreactive waves * Interface phenomena * Multiphase flow concepts The book presents tools of interest to graduate students, researchers in math...

  15. Latent Herpes Viral Reactivation in Astronauts

    Science.gov (United States)

    Pierson, D. L.; Mehta, S. K.; Stowe, R.

    2008-01-01

    Latent viruses are ubiquitous and reactivate during stressful periods with and without symptoms. Latent herpes virus reactivation is used as a tool to predict changes in the immune status in astronauts and to evaluate associated health risks. Methods: Viral DNA was detected by real time polymerase chain reaction in saliva and urine from astronauts before, during and after short and long-duration space flights. Results and Discussion: EpsteinBarr virus (EBV), cytomegalovirus (CMV), and varicella zoster virus (VZV) reactivated, and viral DNA was shed in saliva (EBV and VZV) or urine (CMV). EBV levels in saliva during flight were 10fold higher than baseline levels. Elevations in EBV specific CD8+ T-cells, viral antibody titers, and specific cytokines were consistent with viral reactivation. Intracellular levels of cytokines were reduced in EBVspecific Tcells. CMV, rarely present in urine of healthy individuals, was shed in urine of 27% of astronauts during all phases of spaceflight. VZV, not found in saliva of asymptomatic individuals, was found in saliva of 50% of astronauts during spaceflight and 35 days after flight. VZV recovered from astronaut saliva was found to be live, infectious virus. DNA sequencing demonstrated that the VZV recovered from astronauts was from the common European strain of VZV. Elevation of stress hormones accompanied viral reactivation indicating involvement of the hypothalmic-pituitary-adrenal and sympathetic adrenal-medullary axes in the mechanism of viral reactivation in astronauts. A study of 53 shingles patients found that all shingles patients shed VZV DNA in their saliva and the VZV levels correlated with the severity of the disease. Lower VZV levels in shingles patients were similar to those observed in astronauts. We proposed a rapid, simple, and cost-effective assay to detect VZV in saliva of patients with suspected shingles. Early detection of VZV infection allows early medical intervention.

  16. Natural hidden antibodies reacting with DNA or cardiolipin bind to thymocytes and evoke their death.

    Science.gov (United States)

    Zamulaeva, I A; Lekakh, I V; Kiseleva, V I; Gabai, V L; Saenko, A S; Shevchenko, A S; Poverenny, A M

    1997-08-18

    Both free and hidden natural antibodies to DNA or cardiolipin were obtained from immunoglobulins of a normal donor. The free antibodies reacting with DNA or cardiolipin were isolated by means of affinity chromatography. Antibodies occurring in an hidden state were disengaged from the depleted immunoglobulins by ion-exchange chromatography and were then affinity-isolated on DNA or cardiolipin sorbents. We used flow cytometry to study the ability of free and hidden antibodies to bind to rat thymocytes. Simultaneously, plasma membrane integrity was tested by propidium iodide (PI) exclusion. The hidden antibodies reacted with 65.2 +/- 10.9% of the thymocytes and caused a fast plasma membrane disruption. Cells (28.7 +/- 7.1%) were stained with PI after incubation with the hidden antibodies for 1 h. The free antibodies bound to a very small fraction of the thymocytes and did not evoke death as compared to control without antibodies. The possible reason for the observed effects is difference in reactivity of the free and hidden antibodies to phospholipids. While free antibodies reacted preferentially with phosphotidylcholine, hidden antibodies reacted with cardiolipin and phosphotidylserine. PMID:9280287

  17. Induction of neutralizing antibodies against Tityus serrulatus scorpion toxins by immunization with a mixture of defined synthetic epitopes.

    Science.gov (United States)

    Alvarenga, L M; Diniz, C R; Granier, C; Chávez-Olórtegui, C

    2002-01-01

    We have used the Spot method of multiple peptide synthesis to prepare sets of immobilized overlapping peptides of uniform size (15 mer), covering the complete amino acid sequences of TsNTxP a non-toxic and immunogenic protein and TsIV, an alpha-type toxin that is the major lethal component of the venom of scorpion Tityus serrulatus. Anti-TsNTxP antibodies binding to peptides, revealed three antigenic regions, one in the N-terminal, the second in the central part and the other in the C-terminal part of TsNTxP. One peptide epitope in the C-terminal part of TsIV was identified with anti-TsIV neutralizing rabbit antibodies. Anti-peptide antibodies were raised against these four peptides all together covalently coupled to keyhole limpet hemocyanin (KLH) and found to neutralize in vitro the toxic effects of the T. serrulatus venom. Quantities of venom equivalent to 13.5 LD(50) were effectively neutralized by 1ml of the anti-peptide serum. The antigenic specificities of the anti-peptides were compared by an indirect enzyme-linked immunosorbent assay (ELISA) using synthetic peptides and crude venoms from T. serrulatus, T. bahiensis, T. cambridgei, T. stigmurus, Androctonus autralis Hector and Centruroides sculpturatus to coat the microtitration plates. The anti-peptide antibodies had a comparable high reactivity with the crude venom of T. serrulatus, moderate binding to T. bahiensis, T. cambridgei, T. stigmurus and Centruroides sculpturatus venoms but were unable to recognize the venom of Androctonus autralis Hector. These results show that by using peptides derived from the sequence of scorpion toxins, the generation of anti-peptide antibodies able to neutralize the cognate venom appears to be an alternative strategy for the easy preparation of antivenoms. PMID:11602284

  18. Comprehensive mapping of common immunodominant epitopes in the eastern equine encephalitis virus E2 protein recognized by avian antibody responses.

    Directory of Open Access Journals (Sweden)

    Encheng Sun

    Full Text Available Eastern equine encephalitis virus (EEEV is a mosquito-borne virus that can cause both human and equine encephalitis with high case fatality rates. EEEV can also be widespread among birds, including pheasants, ostriches, emu, turkeys, whooping cranes and chickens. The E2 protein of EEEV and other Alphaviruses is an important immunogenic protein that elicits antibodies of diagnostic value. While many therapeutic and diagnostic applications of E2 protein-specific antibodies have been reported, the specific epitopes on E2 protein recognized by the antibody responses of different susceptible hosts, including avian species, remain poorly defined. In the present study, the avian E2-reactive polyclonal antibody (PAb response was mapped to linear peptide epitopes using PAbs elicited in chickens and ducks following immunization with recombinant EEEV E2 protein and a series of 42 partially overlapping peptides covering the entire EEEV E2 protein. We identified 12 and 13 peptides recognized by the chicken and duck PAb response, respectively. Six of these linear peptides were commonly recognized by PAbs elicited in both avian species. Among them five epitopes recognized by both avian, the epitopes located at amino acids 211-226 and 331-352 were conserved among the EEEV antigenic complex, but not other associated alphaviruses, whereas the epitopes at amino acids 11-26, 30-45 and 151-166 were specific to EEEV subtype I. The five common peptide epitopes were not recognized by avian PAbs against Avian Influenza Virus (AIV and Duck Plague Virus (DPV. The identification and characterization of EEEV E2 antibody epitopes may be aid the development of diagnostic tools and facilitate the design of epitope-based vaccines for EEEV. These results also offer information with which to study the structure of EEEV E2 protein.

  19. The Influence of Sub-Unit Composition and Expression System on the Functional Antibody Response in the Development of a VAR2CSA Based Plasmodium falciparum Placental Malaria Vaccine.

    Science.gov (United States)

    Nielsen, Morten A; Resende, Mafalda; de Jongh, Willem A; Ditlev, Sisse B; Mordmüller, Benjamin; Houard, Sophie; Ndam, Nicaise Tuikue; Agerbæk, Mette Ø; Hamborg, Mette; Massougbodji, Achille; Issifou, Saddou; Strøbæk, Anette; Poulsen, Lars; Leroy, Odile; Kremsner, Peter G; Chippaux, Jean-Philippe; Luty, Adrian J F; Deloron, Philippe; Theander, Thor G; Dyring, Charlotte; Salanti, Ali

    2015-01-01

    The disease caused by Plasmodium falciparum (Pf) involves different clinical manifestations that, cumulatively, kill hundreds of thousands every year. Placental malaria (PM) is one such manifestation in which Pf infected erythrocytes (IE) bind to chondroitin sulphate A (CSA) through expression of VAR2CSA, a parasite-derived antigen. Protection against PM is mediated by antibodies that inhibit binding of IE in the placental intervillous space. VAR2CSA is a large antigen incompatible with large scale recombinant protein expression. Vaccines based on sub-units encompassing the functionally constrained receptor-binding domains may, theoretically, circumvent polymorphisms, reduce the risk of escape-mutants and induce cross-reactive antibodies. However, the sub-unit composition and small differences in the borders, may lead to exposure of novel immuno-dominant antibody epitopes that lead to non-functional antibodies, and furthermore influence the folding, stability and yield of expression. Candidate antigens from the pre-clinical development expressed in High-Five insect cells using the baculovirus expression vector system were transitioned into the Drosophila Schneider-2 cell (S2) expression-system compliant with clinical development. The functional capacity of antibodies against antigens expressed in High-Five cells or in S2 cells was equivalent. This enabled an extensive down-selection of S2 insect cell-expressed antigens primarily encompassing the minimal CSA-binding region of VAR2CSA. In general, we found differential potency of inhibitory antibodies against antigens with the same borders but of different var2csa sequences. Likewise, we found that subtle size differences in antigens of the same sequence gave varying levels of inhibitory antibodies. The study shows that induction of a functional response against recombinant subunits of the VAR2CSA antigen is unpredictable, demonstrating the need for large-scale screening in order to identify antigens that induce a

  20. The Influence of Sub-Unit Composition and Expression System on the Functional Antibody Response in the Development of a VAR2CSA Based Plasmodium falciparum Placental Malaria Vaccine.

    Directory of Open Access Journals (Sweden)

    Morten A Nielsen

    Full Text Available The disease caused by Plasmodium falciparum (Pf involves different clinical manifestations that, cumulatively, kill hundreds of thousands every year. Placental malaria (PM is one such manifestation in which Pf infected erythrocytes (IE bind to chondroitin sulphate A (CSA through expression of VAR2CSA, a parasite-derived antigen. Protection against PM is mediated by antibodies that inhibit binding of IE in the placental intervillous space. VAR2CSA is a large antigen incompatible with large scale recombinant protein expression. Vaccines based on sub-units encompassing the functionally constrained receptor-binding domains may, theoretically, circumvent polymorphisms, reduce the risk of escape-mutants and induce cross-reactive antibodies. However, the sub-unit composition and small differences in the borders, may lead to exposure of novel immuno-dominant antibody epitopes that lead to non-functional antibodies, and furthermore influence the folding, stability and yield of expression. Candidate antigens from the pre-clinical development expressed in High-Five insect cells using the baculovirus expression vector system were transitioned into the Drosophila Schneider-2 cell (S2 expression-system compliant with clinical development. The functional capacity of antibodies against antigens expressed in High-Five cells or in S2 cells was equivalent. This enabled an extensive down-selection of S2 insect cell-expressed antigens primarily encompassing the minimal CSA-binding region of VAR2CSA. In general, we found differential potency of inhibitory antibodies against antigens with the same borders but of different var2csa sequences. Likewise, we found that subtle size differences in antigens of the same sequence gave varying levels of inhibitory antibodies. The study shows that induction of a functional response against recombinant subunits of the VAR2CSA antigen is unpredictable, demonstrating the need for large-scale screening in order to identify antigens

  1. Adaptive responses to antibody based therapy.

    Science.gov (United States)

    Rodems, Tamara S; Iida, Mari; Brand, Toni M; Pearson, Hannah E; Orbuch, Rachel A; Flanigan, Bailey G; Wheeler, Deric L

    2016-02-01

    Receptor tyrosine kinases (RTKs) represent a large class of protein kinases that span the cellular membrane. There are 58 human RTKs identified which are grouped into 20 distinct families based upon their ligand binding, sequence homology and structure. They are controlled by ligand binding which activates intrinsic tyrosine-kinase activity. This activity leads to the phosphorylation of distinct tyrosines on the cytoplasmic tail, leading to the activation of cell signaling cascades. These signaling cascades ultimately regulate cellular proliferation, apoptosis, migration, survival and homeostasis of the cell. The vast majority of RTKs have been directly tied to the etiology and progression of cancer. Thus, using antibodies to target RTKs as a cancer therapeutic strategy has been intensely pursued. Although antibodies against the epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) have shown promise in the clinical arena, the development of both intrinsic and acquired resistance to antibody-based therapies is now well appreciated. In this review we provide an overview of the RTK family, the biology of EGFR and HER2, as well as an in-depth review of the adaptive responses undertaken by cells in response to antibody based therapies directed against these receptors. A greater understanding of these mechanisms and their relevance in human models will lead to molecular insights in overcoming and circumventing resistance to antibody based therapy. PMID:26808665

  2. Monoclonal antibodies technology. Protocols

    International Nuclear Information System (INIS)

    reached at least 3/4 confluency, 2 ampules/cell culture. Save the supernatants for further studies. Regarding to separate the interested cells of irrelevant cells, you must been do a limited diluted procedures. Ascitis production. To produce ascitis the number of cells depend of the every particular clone. Usually we inoculated ip between 1 to 2 x106 cells/ml/mice in PBS buffer. The animals must be inoculated intraperitonealy with Pristan (Sigma) or mineral oil using 0.5 ml per mice. The inoculation could be occurred 7 to 10 days before to inoculated the clone. The weight of the animals must be controlled in order to obtain better yield. The bioreactors are an alternative method to produce AcMs without it have not used animals models. IgG purification process on protein A columns. Mouse antibodies of the IgG1 subclass do not have a high affinity for protein A. Every subclass could be eluted at different peaks using a gradient pH. Usually we use 3M NaCl/1.5 M Gly pH 8.9 as a coupling buffer. For eluting IgGs we use 0.1M Citric Acid in a gradient pH (6 to IgG1, 5 to IgG2a or IgG2b, 4 IgG3). 1. Adjust a Sepharose Protein A (Pharmacia) beads using NaCl/1.5 M Gly pH 8.9 (coupling buffer). 2. Diluted the ascitis 1:10 in coupling buffer using a filter paper for remove the aggregations. 3. Pass the solution trough a Protein A bead column at a standarized flow according your equipment. The capacity of the Protein A beads for IgGl is approximately 5 mg/ml of wet beads. Ascitis contain between 1-10 mg/ml. 4. Wash the colums with 10 volumes of coupling buffer. 5. Elute the column with 0.1 M Citric Acid in a gradient pH starting at 6 to 4. Collect the eluate in appropriate tube, and identify the immunoglobulin-containing fractions by absorbance a 280 nm (1 DO = 0.8 mg/ml)

  3. The effects of variations in the specificities of the antibody components on a two-site immunoradiometric assay for ferritin

    International Nuclear Information System (INIS)

    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.)

  4. The effects of variations in the specificities of the antibody components on a two-site immunoradiometric assay for ferritin

    International Nuclear Information System (INIS)

    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 radio-labelled 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 before 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 than antibodies prepared on liver ferritin immunoadsorbant. (author)

  5. Pre-existing Antibody: Biotherapeutic Modality-Based Review.

    Science.gov (United States)

    Gorovits, Boris; Clements-Egan, Adrienne; Birchler, Mary; Liang, Meina; Myler, Heather; Peng, Kun; Purushothama, Shobha; Rajadhyaksha, Manoj; Salazar-Fontana, Laura; Sung, Crystal; Xue, Li

    2016-03-01

    Pre-existing antibodies to biotherapeutic drugs have been detected in drug-naïve subjects for a variety of biotherapeutic modalities. Pre-existing antibodies are immunoglobulins that are either specific or cross-reacting with a protein or glycan epitopes on a biotherapeutic compound. Although the exact cause for pre-existing antibodies is often unknown, environmental exposures to non-human proteins, glycans, and structurally similar products are frequently proposed as factors. Clinical consequences of the pre-existing antibodies vary from an adverse effect on patient safety to no impact at all and remain highly dependent on the biotherapeutic drug modality and therapeutic indication. As such, pre-existing antibodies are viewed as an immunogenicity risk factor requiring a careful evaluation. Herein, the relationships between biotherapeutic modalities to the nature, prevalence, and clinical consequences of pre-existing antibodies are reviewed. Initial evidence for pre-existing antibody is often identified during anti-drug antibody (ADA) assay development. Other interfering factors known to cause false ADA positive signal, including circulating multimeric drug target, rheumatoid factors, and heterophilic antibodies, are discussed. PMID:26821802

  6. Production and characterization of a hybridoma-derived antibody to Blastomyces dermatitidis.

    OpenAIRE

    Young, K D; Larsh, H W

    1982-01-01

    A hybridoma cell line was isolated which produced monoclonal antibody to one protein component of a yeast-phase cytoplasmic antigenic complex of Blastomyces dermatitidis. The immunoglobulin M antibody product was characterized by immunodiffusion, autoradiography of polyacrylamide gels, and cellulose acetate electrophoresis. By attaching the antibody to an affinity gel, one major protein band was identified by polyacrylamide gel electrophoresis as the antigen for which the antibody was specific.

  7. The second century of the antibody. Molecular perspectives in regulation, pathophysiology, and therapeutic applications.

    OpenAIRE

    Braun, J; Saxon, A; Wall, R; Morrison, S. L.

    1992-01-01

    The modern age of immunology began in 1890 with the discovery of antibodies as a major component of protective immunity. The 2nd century of the antibody begins with a focus on the molecular physiology and pathophysiology of immunoglobulin production. Numerous human variable-region antibody genes have been identified through advances in molecular cloning and anti-variable-region monoclonal antibodies. Some of these variable-region genes are now known to be involved in specific stages of B-lymp...

  8. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants

    OpenAIRE

    Hehle, Verena K; Paul, Matthew J.; Roberts, Victoria A.; van Dolleweerd, Craig J.; Ma, Julian K.-C.

    2015-01-01

    This study examined the degradation pattern of a murine IgG1κ monoclonal antibody expressed in and extracted from transformed Nicotiana tabacum. Gel electrophoresis of leaf extracts revealed a consistent pattern of recombinant immunoglobulin bands, including intact and full-length antibody, as well as smaller antibody fragments. N-terminal sequencing revealed these smaller fragments to be proteolytic cleavage products and identified a limited number of protease-sensitive sites in the antibody...

  9. Prediction of antibody persistency from antibody titres to natalizumab

    DEFF Research Database (Denmark)

    Jensen, Poul Erik H; Koch-Henriksen, Nils; Sellebjerg, Finn; Sørensen, Per S

    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....

  10. Immunosuppression associated with novel chemotherapy agents and monoclonal antibodies.

    Science.gov (United States)

    Morrison, Vicki A

    2014-11-15

    The introduction of novel agents to the therapeutic armamentarium for oncologic, rheumatologic, and neurologic disorders has resulted in major clinical advances. These agents impact immune function, resulting in a discrete spectrum of infectious complications. Purine analogues and alemtuzumab alter cell-mediated immunity, resulting in opportunistic viral/fungal infections. Herpes zoster incidence increases with bortezomib. Hepatitis B reactivation may occur with rituximab. Cases of progressive multifocal leukoencephalopathy have occurred following monoclonal antibody therapy. Tumor necrosis factor-α inhibitor therapy is complicated by tuberculosis reactivation and fungal infections. We summarize the impact of these therapies on pathogenesis and spectrum of infection complicating their usage. PMID:25352632

  11. Monoclonal Antibodies for Lipid Management.

    Science.gov (United States)

    Feinstein, Matthew J; Lloyd-Jones, Donald M

    2016-07-01

    In recent years, biochemical and genetic studies have identified proprotein convertase subtilisin/kexin type 9 (PCSK9) as a major mediator of low-density lipoprotein cholesterol (LDL-c) levels and thereby a potential novel target for reducing risk of coronary heart disease (CHD). These observations led to the development of PCSK9 inhibitors, which lower LDL-c levels more than any other non-invasive lipid-lowering therapy presently available. The PCSK9 inhibitors furthest along in clinical trials are subcutaneously injected monoclonal antibodies. These PCSK9 inhibitors have demonstrated LDL-c-lowering efficacy with acceptable safety in phase III clinical trials and may offer a useful therapy in addition to maximally tolerated HMG-CoA reductase inhibitors (statins) in certain patient groups. Longer-term data are required to ensure sustained efficacy and safety of this new class of medications. This review provides an overview of the biology, genetics, development, and clinical trials of monoclonal antibodies designed to inhibit PCSK9. PMID:27221501

  12. Prediction of Antibody Epitopes

    DEFF Research Database (Denmark)

    Nielsen, Morten; Marcatili, Paolo

    2015-01-01

    self-proteins. Given the sequence or the structure of a protein of interest, several methods exploit such features to predict the residues that are more likely to be recognized by an immunoglobulin.Here, we present two methods (BepiPred and DiscoTope) to predict linear and discontinuous antibody...

  13. Monoclonal antibodies in myeloma

    DEFF Research Database (Denmark)

    Sondergeld, P.; van de Donk, N. W. C. J.; Richardson, P. G.;

    2015-01-01

    The development of monoclonal antibodies (mAbs) for the treatment of disease goes back to the vision of Paul Ehrlich in the late 19th century; however, the first successful treatment with a mAb was not until 1982, in a lymphoma patient. In multiple myeloma, mAbs are a very recent and exciting add...

  14. Red Blood Cell Antibody Identification

    Science.gov (United States)

    ... limited. Home Visit Global Sites Search Help? RBC Antibody Identification Share this page: Was this page helpful? Also known as: Alloantibody Identification; Antibody ID, RBC; RBC Ab ID Formal name: Red ...

  15. Anti-insulin antibody test

    Science.gov (United States)

    Insulin antibodies - serum; Insulin Ab test ... Normally, there are no antibodies against insulin in your blood. Normal value ranges may vary slightly among different laboratories. Some labs use different measurements or ...

  16. The Art of Making Antibodies.

    Science.gov (United States)

    Headon, Denis R.

    1986-01-01

    Provides background information for teachers on the nature and production of antibodies. Points out that the production of monoclonal antibodies blends the malignant with the beneficial to create a medical tool of exciting potential. (JN)

  17. Lupus anticoagulants and antiphospholipid antibodies

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/000547.htm Lupus anticoagulants and antiphospholipid antibodies To use the sharing features on this page, please enable JavaScript. Lupus anticoagulants are antibodies against substances in the lining ...

  18. Preparation and identification of monoclonal antibodies against Ractopamine

    International Nuclear Information System (INIS)

    In order to prepare the monoclonal antibody against Ractopamine (RCT), RCT-conjugated antigen was produced by methods of mixed acid anhydride. The spleen cells of BALB/c mice immunized by RCT-BSA were fused with SP2/0 plasmacytoma cells using PEG4000. A hybridoma cell line of 3E1-C9-E10 was screened for specificity to RCT and cloned by limited dilution method, which secreted stable monoclonal antibodies against RCT with indirect ELISA titers of 1 x 105 in supernatant, 1 x 107 in ascites. The McAb of 3E1-C9-E10 generally had 24% cross-reactivity to Dobutamine, and showed little or no cross-reactivity to Salbutamol and Clenbuterol. (authors)

  19. Recombinant antibodies and tumor targeting

    OpenAIRE

    Sheikholvaezin, Ali

    2006-01-01

    Different antibody derived constructs are rapidly advancing as putative tools for treatment of malignant diseases. Antibody engineering has added significant new technologies to modify size, affinities, solubility, stability and biodistribution properties for immunoconjugates. In the present thesis, the aim was to increase our knowledge on how new recombinant antibodies could be tailored to optimize localization to experimental tumors in mice. One hybridoma, producing the monoclonal antibody ...

  20. Antibody Engineering and Therapeutics Conference

    OpenAIRE

    Larrick, James W; Parren, Paul WHI; Huston, James S; Plückthun, Andreas; Bradbury, Andrew; Tomlinson, Ian M; Chester, Kerry A.; Burton, Dennis R.; Adams, Gregory P; Weiner, Louis M.; Scott, Jamie K; Alfenito, Mark R; Veldman, Trudi; Reichert, Janice M

    2013-01-01

    The Antibody Engineering and Therapeutics conference, which serves as the annual meeting of The Antibody Society, will be held in Huntington Beach, CA from Sunday December 8 through Thursday December 12, 2013. The scientific program will cover the full spectrum of challenges in antibody research and development, and provide updates on recent progress in areas from basic science through approval of antibody therapeutics. Keynote presentations will be given by Leroy Hood (Institute of System Bi...

  1. Successful kidney transplantation after desensitization in a patient with positive flow crossmatching and donor-specific anti-HLA-DP antibody

    Science.gov (United States)

    Song, Seung Hwan; Park, Borae G.; Lee, Juhan; Kim, Myoung Soo; Kim, Yu Seun; Kim, Hyon-Suk

    2016-01-01

    Abstract Background: Traditionally, the presence of antibodies against human leukocyte antigen (HLA)-C and DP was considered to be associated with only a low risk of antibody-mediated rejection (ABMR) in kidney transplantation (KT), because the antigenicities of these proteins are weak. However, the clinical effects of HLA-C and -DP donor-specific HLA antibodies (DSHAs) have recently been reevaluated. Methods: Here, we report the case of a retransplant patient with positive flow cytometry crossmatch (FCXM) and high level of HLA-DP DSHA who was desensitized using rituximab, plasmapheresis, and intravenous immunoglobulin. Results: The epitope-based antibody reactivity was identified that the positive B-cell FCXM in our patient was attributable to the specific epitope. The patient underwent a successful retransplantation and has continued to do well for 10 month after KT. Conclusion: If an HLA-DP DSHA is present, it is important to detect any mismatched HLA-DP epitope pretransplantation and to monitor HLA-DP levels carefully. According to previous reports, anti-HLA-DP DSHA can induce ABMR soon after transplantation, but such ABMR can be prevented by pretransplantation desensitization and careful monitoring of DSHA levels. PMID:27512872

  2. A retrospective analysis of cross-reacting cetuximab IgE antibody and its association with severe infusion reactions

    International Nuclear Information System (INIS)

    Severe infusion reactions (SIRs) at rates of 5% or less are known side effects of biological agents, including mAbs such as cetuximab. There are currently no prospectively validated risk factors to aid physicians in identifying patients who may be at risk of experiencing an SIR following administration of any of these drugs. A retrospective analysis of 545 banked serum or plasma samples from cancer patients participating in clinical trials of cetuximab was designed to evaluate whether the presence of pretreatment IgE antibodies against cetuximab, as determined by a commercially available assay system, is associated with SIRs during the initial cetuximab infusion. Patients with a positive test indicating the presence of pretreatment antibodies had a higher risk of experiencing an SIR; however, at the prespecified cutoff utilized in this analysis, the test has a relatively low-positive predictive value (0.577 [0.369–0.766]) and a negative predictive value of 0.961 (0.912–0.987) in an unselected patient population. Data collected in this large retrospective validation study support prior observations of an association between the presence of pretreatment IgE antibodies cross-reactive with cetuximab and SIRs. Further analysis of the test's ability to predict patients at risk of an SIR would be required before this assay could be used reliably in this patient population

  3. Immunoproteomic Analysis of Antibody Responses to Extracellular Proteins of Candida albicans Revealing the Importance of Glycosylation for Antigen Recognition.

    Science.gov (United States)

    Luo, Ting; Krüger, Thomas; Knüpfer, Uwe; Kasper, Lydia; Wielsch, Natalie; Hube, Bernhard; Kortgen, Andreas; Bauer, Michael; Giamarellos-Bourboulis, Evangelos J; Dimopoulos, George; Brakhage, Axel A; Kniemeyer, Olaf

    2016-08-01

    During infection, the human pathogenic fungus Candida albicans undergoes a yeast-to-hypha transition, secretes numerous proteins for invasion of host tissues, and modulates the host's immune response. Little is known about the interplay of C. albicans secreted proteins and the host adaptive immune system. Here, we applied a combined 2D gel- and LC-MS/MS-based approach for the characterization of C. albicans extracellular proteins during the yeast-to-hypha transition, which led to a comprehensive C. albicans secretome map. The serological responses to C. albicans extracellular proteins were investigated by a 2D-immunoblotting approach combined with MS for protein identification. On the basis of the screening of sera from candidemia and three groups of noncandidemia patients, a core set of 19 immunodominant antibodies against secreted proteins of C. albicans was identified, seven of which represent potential diagnostic markers for candidemia (Xog1, Lip4, Asc1, Met6, Tsa1, Tpi1, and Prx1). Intriguingly, some secreted, strongly glycosylated protein antigens showed high cross-reactivity with sera from noncandidemia control groups. Enzymatic deglycosylation of proteins secreted from hyphae significantly impaired sera antibody recognition. Furthermore, deglycosylation of the recombinantly produced, secreted aspartyl protease Sap6 confirmed a significant contribution of glycan epitopes to the recognition of Sap6 by antibodies in patient's sera. PMID:27386892

  4. Nucleic Acid, Antibody, and Virus Culture Methods to Detect Xenotropic MLV-Related Virus in Human Blood Samples

    Directory of Open Access Journals (Sweden)

    M. F. Kearney

    2011-01-01

    Full Text Available The MLV-related retrovirus, XMRV, was recently identified and reported to be associated with both prostate cancer and chronic fatigue syndrome. At the National Cancer Institute-Frederick, MD (NCI-Frederick, we developed highly sensitive methods to detect XMRV nucleic acids, antibodies, and replication competent virus. Analysis of XMRV-spiked samples and/or specimens from two pigtail macaques experimentally inoculated with 22Rv1 cell-derived XMRV confirmed the ability of the assays used to detect XMRV RNA and DNA, and culture isolatable virus when present, along with XMRV reactive antibody responses. Using these assays, we did not detect evidence of XMRV in blood samples ( or prostate specimens ( from two independent cohorts of patients with prostate cancer. Previous studies detected XMRV in prostate tissues. In the present study, we primarily investigated the levels of XMRV in blood plasma samples collected from patients with prostate cancer. These results demonstrate that while XMRV-related assays developed at the NCI-Frederick can readily measure XMRV nucleic acids, antibodies, and replication competent virus, no evidence of XMRV was found in the blood of patients with prostate cancer.

  5. Radiolabeled antibodies as imaging agents

    International Nuclear Information System (INIS)

    The author gives a survey of the progress made on radioimmunodetection. Antibodies may now be more readily used in scintigraphy as a result of the development of labeling methods that apply more suitable radionuclides without significant loss of the antigen-binding activity. Antibodies to tumor-specific or tumor-associated antigens can now be produced in large quantities by monoclonal antibody technology

  6. When is arthritis reactive?

    Science.gov (United States)

    Hamdulay, S S; Glynne, S J; Keat, A

    2006-07-01

    Reactive arthritis is an important cause of lower limb oligoarthritis, mainly in young adults. It is one of the spondyloarthropathy family; it is distinguishable from other forms of inflammatory arthritis by virtue of the distribution of affected sites and the high prevalence of characteristic extra-articular lesions. Many terms have been used to refer to this and related forms of arthritis leading to some confusion. Reactive arthritis is precipitated by an infection at a distant site and genetic susceptibility is marked by possession of the HLA-B27 gene, although the mechanism remains uncertain. Diagnosis is a two stage process and requires demonstration of a temporal link with a recognised "trigger" infection. The identification and management of "sexually acquired" and "enteric" forms of reactive arthritis are considered. Putative links with HIV infection are also discussed. The clinical features, approach to investigation, diagnosis, and management of reactive arthritis are reviewed. PMID:16822921

  7. Targeting a cross-reactive Gly m 5 soy peptide as responsible for hypersensitivity reactions in a milk allergy mouse model.

    Directory of Open Access Journals (Sweden)

    Renata Curciarello

    Full Text Available BACKGROUND: Cross-reactivity between soybean allergens and bovine caseins has been previously reported. In this study we aimed to map epitopes of the major soybean allergen Gly m 5 that are co-recognized by casein specific antibodies, and to identify a peptide responsible for the cross-reactivity. METHODS: Cow's milk protein (CMP-specific antibodies were used in different immunoassays (immunoblotting, ELISA, ELISA inhibition test to evaluate the in vitro recognition of soybean proteins (SP. Recombinant Gly m 5 (α, a truncated fragment containing the C-terminal domain (α-T and peptides of α-T were obtained and epitope mapping was performed with an overlapping peptide assay. Bioinformatics tools were used for epitope prediction by sequence alignment, and for modelling the cross-recognized soy proteins and peptides. The binding of SP to a monoclonal antibody was studied by surface Plasmon resonance (SPR. Finally, the in vivo cross-recognition of SP was assessed in a mouse model of milk allergy. RESULTS: Both α and α-T reacted with the different CMP-specific antibodies. α-T contains IgG and IgE epitopes in several peptides, particularly in the peptide named PA. Besides, we found similar values of association and dissociation constants between the α-casein specific mAb and the different milk and soy components. The food allergy mouse model showed that SP and PA contain the cross-reactive B and T epitopes, which triggered hypersensitivity reactions and a Th2-mediated response on CMP-sensitized mice. CONCLUSIONS: Gly m 5 is a cross-reactive soy allergen and the α-T portion of the molecule contains IgG and IgE immunodominant epitopes, confined to PA, a region with enough conformation to be bound by antibodies. These findings contribute to explain the intolerance to SP observed in IgE-mediated CMA patients, primarily not sensitised to SP, as well as it sets the basis to propose a mucosal immunotherapy for milk allergy using this soy peptide.

  8. RNAi-based validation of antibodies for reverse phase protein arrays

    Directory of Open Access Journals (Sweden)

    Sahin Özgür

    2010-12-01

    Full Text Available Abstract Background Reverse phase protein arrays (RPPA have been demonstrated to be a useful experimental platform for quantitative protein profiling in a high-throughput format. Target protein detection relies on the readout obtained from a single detection antibody. For this reason, antibody specificity is a key factor for RPPA. RNAi allows the specific knockdown of a target protein in complex samples and was therefore examined for its utility to assess antibody performance for RPPA applications. Results To proof the feasibility of our strategy, two different anti-EGFR antibodies were compared by RPPA. Both detected the knockdown of EGFR but at a different rate. Western blot data were used to identify the most reliable antibody. The RNAi approach was also used to characterize commercial anti-STAT3 antibodies. Out of ten tested anti-STAT3 antibodies, four antibodies detected the STAT3-knockdown at 80-85%, and the most sensitive anti-STAT3 antibody was identified by comparing detection limits. Thus, the use of RNAi for RPPA antibody validation was demonstrated to be a stringent approach to identify highly specific and highly sensitive antibodies. Furthermore, the RNAi/RPPA strategy is also useful for the validation of isoform-specific antibodies as shown for the identification of AKT1/AKT2 and CCND1/CCND3-specific antibodies. Conclusions RNAi is a valuable tool for the identification of very specific and highly sensitive antibodies, and is therefore especially useful for the validation of RPPA-suitable detection antibodies. On the other hand, when a set of well-characterized RPPA-antibodies is available, large-scale RNAi experiments analyzed by RPPA might deliver useful information for network reconstruction.

  9. Detection of newly antibody-defined epitopes on HLA class I alleles reacting with antibodies induced during pregnancy.

    Science.gov (United States)

    Duquesnoy, R J; Hönger, G; Hösli, I; Marrari, M; Schaub, S

    2016-08-01

    The determination of HLA mismatch acceptability at the epitope level can be best performed with epitopes that have been verified experimentally with informative antibodies. The website-based International Registry of HLA Epitopes (http://www.epregistry.com.br) has a list of 81 antibody-verified HLA-ABC epitopes but more epitopes need to be added. Pregnancy offers an attractive model to study antibody responses to mismatched HLA epitopes which can be readily determined from the HLA types of child and mother. This report describes a HLAMatchmaker-based analysis of 16 postpregnancy sera tested in single HLA-ABC allele binding assays. Most sera reacted with alleles carrying epitopes that have been antibody-verified, and this study focused on the reactivity of additional alleles that share other epitopes corresponding to eplets and other amino acid residue configurations. This analysis led in the identification of 16 newly antibody-defined epitopes, seven are equivalent to eplets and nine correspond to combinations of eplets in combination with other nearby residue configurations. These epitopes will be added to the repertoire of antibody-verified epitopes in the HLA Epitope Registry. PMID:27312793

  10. Species difference in reactivity to lignin-like enzymatically polymerized polyphenols on interferon-γ synthesis and involvement of interleukin-2 production in mice.

    Science.gov (United States)

    Yamanaka, Daisuke; Ishibashi, Ken-Ichi; Adachi, Yoshiyuki; Ohno, Naohito

    2016-09-01

    Recent studies have revealed that lignin-like polymerized polyphenols can activate innate immune systems. In this study, we aimed to evaluate whether these polymerized polyphenols could activate leukocytes from different murine strains. Splenocytes from 12 mouse strains were investigated. Our results revealed species differences in reactivity to phenolic polymers on interferon-γ (IFN-γ) release. Mice that possessed the H2(a) or H2(k) haplotype antigens were the highly responsive strains. To clarify these different points in soluble factors, multiplex cytokine profiling analysis was carried out and we identified interleukin (IL)-2 as a key molecule for IFN-γ induction by polymerized polyphenols. Furthermore, inhibition of IL-2 and IL-2Rα by neutralizing antibodies significantly decreased cytokine production in the highly responsive mice strains. Our results indicate that species difference in reactivity to phenolic polymers is mediated by adequate release of IL-2 and its receptor, IL-2Rα. PMID:27376855

  11. Production and characterization of polyclonal antibody against a synthetic peptide from β-actin protein

    Directory of Open Access Journals (Sweden)

    Nazila Amini

    2014-06-01

    Full Text Available Objective(s:Antibodies against actin, as one of the most widely studied structural and multifunctional housekeeping proteins in eukaryotic cells, are used as internal loading controls in western blot analyses. The aim of this study was to produce polyclonal antibody against a synthetic peptide derived from N-terminal region of β-actin protein to be used as a protein loading control in western blot and other assay systems. Materials and Methods: A synthetic peptide derived from β-actin protein was designed and conjugated to Keyhole limpet hemocyanin (KLH (and used to immunize a white New Zealand rabbit. The antibody was purified from serum by affinity chromatography column. The purity of the antibody was determined by SDS-PAGE and its ability to recognize the immunizing peptide was measured by ELISA. The reactivity of the antibody with β-actin protein in a panel of different cell lysates was then evaluated by western blot. In addition, the reactivity of the antibody with the corresponding protein was also evaluated by Immunocytochemistry and Immunohistochemistry in different samples. Results: The antibody could recognize the immunizing peptide in ELISA. It could also recognize            β-actin protein in western blot as well as in immunocytochemistry and immunohistochemistry. Conclusion: Our data suggest that this antibody may be used as an internal control in western blot analyses as well as in other immunological applications such as ELISA,immunocytochemistry and immunohistochemistry.

  12. 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; Houen, Gunnar; Trier, Nicole Hartwig

    2014-01-01

    ), where the amino acids Ser, Ile and Asp were found to be essential for antibody reactivity. These amino acids were found to contribute to the antibody-antigen interface through side-chain interactions, possibly in combination with a positively charged amino acid in position 77. Moreover, the amino acids...... in the N-terminal end (Pro and His) were found to contribute to the interface through backbone contributions. No notable reactivity was found with RA-positive patient sera, thus screening of RA33 antibodies does not seem to be a supplementary for the diagnosis of RA....

  13. Reactive sputter deposition

    CERN Document Server

    Mahieu, Stijn

    2008-01-01

    In this valuable work, all aspects of the reactive magnetron sputtering process, from the discharge up to the resulting thin film growth, are described in detail, allowing the reader to understand the complete process. Hence, this book gives necessary information for those who want to start with reactive magnetron sputtering, understand and investigate the technique, control their sputtering process and tune their existing process, obtaining the desired thin films.

  14. Reactive plasma spraying

    OpenAIRE

    Al-Sabouni, Omar

    1999-01-01

    Reactive Plasma Spraying (RPS) with a hydrocarbon gas has been studied as a method to improve the mechanical properties of a commercially available 80: 20 nickel-chromium alloy, and subsequently as a method to reduce the oxygen content of sprayed MCrAlY coatings. A conventional d. c. plasma torch has been modified by attaching a conical graphite tube (reactor) onto the end of the gun. The powder is then sprayed through the reactor with injected reactive hydrocarbon gas. The ...

  15. When is arthritis reactive?

    OpenAIRE

    Hamdulay, S. S.; Glynne, S J; Keat, A

    2006-01-01

    Reactive arthritis is an important cause of lower limb oligoarthritis, mainly in young adults. It is one of the spondyloarthropathy family; it is distinguishable from other forms of inflammatory arthritis by virtue of the distribution of affected sites and the high prevalence of characteristic extra‐articular lesions. Many terms have been used to refer to this and related forms of arthritis leading to some confusion. Reactive arthritis is precipitated by an infection at a distant site and gen...

  16. Interactive Chemical Reactivity Exploration

    OpenAIRE

    Haag, Moritz P.; Vaucher, Alain C.; Bosson, Mael; Redon, Stephane; Reiher, Markus

    2014-01-01

    Elucidating chemical reactivity in complex molecular assemblies of a few hundred atoms is, despite the remarkable progress in quantum chemistry, still a major challenge. Black-box search methods to find intermediates and transition-state structures might fail in such situations because of the high-dimensionality of the potential energy surface. Here, we propose the concept of interactive chemical reactivity exploration to effectively introduce the chemist's intuition into the search process. ...

  17. 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. PMID:25264572

  18. Protein A immunoadsorption combined with rituximab in renal transplant recipients positive for panel reactive antibody(Report of 7 cases)%蛋白A免疫吸附联合利妥昔单抗在PRA阳性肾移植受者中的应用七例报告

    Institute of Scientific and Technical Information of China (English)

    胡小鹏; 尹航; 刘航; 李晓北; 王玮; 任亮; 杨晓勇; 万昊; 张小东

    2009-01-01

    transplant recipients positive for panel reactive antibody(PRA).Methods Five cases received second transplantation,and 2 cases had the history of multiple pregnancies.All the cases received living-related kidney transplantation and all grafts came from male donors.Recipients and donors had the same blood type,and complement dependent cytotoxic (CDC)was negative.HLA mismatch number was 2-5.Three out of 7 cases had high PRA of both class Ⅰ and 11,2 cases had high class Ⅰ,and the rest 2 had high class Ⅱ.Mean immunoglobulin IgG,IgA,and IgM levels were 13.4,1.78,and 1.47g/L respectively.All patients were pretreated with immunoabsorption for 2-10 times(mean 5 times).Immunoglobulin and PRA changes were monitored before and after absorption.Transplantation was done after PRA or immunoglobulin levels were reduced to normal or below normal values.Before the transplantations single dose of RTX(375 mg/m2)was infused together with polyclonal antibody induction.All patients received immunosuppressive agents such as tacrolimus or cyclosporin A,mycophenolate mofetil and prednisone after transplantation.Creatinine(Cr),and creatinine clearance rate(Ccr)were monitored after transplantatioa Biopsy would be performed when rejection occurred.The percentage of peripheral blood B lymphocytes CD19 and CD20 was determined in 3 patients by using flow cytometry before transplantation and one week posttransplantation.Results There was no adverse reactions during the combined use of protein A immunoadsorption and RTX.The PRA of class Ⅰ and Ⅱ was descended after administration of protein A immunoadsorption.The average levels of IgG,IgA and IgM were also descended.Seven cases of kidney transplantation had a good recovery,with no delayed graft function (DGF).Three cases had acute rejection 8,10 and 14 days after transplantation respectively.Successful reversion was achieved after treatment.CD 19 before transplantation was 1.98 %,4.64 % and 4.45 % respectively,and 0.39 %,0.98 % and 0

  19. Nano antibody therapy for cancer

    International Nuclear Information System (INIS)

    Nanomedicine, an offshoot of nanotechnology, refers to highly specific medical intervention at the molecular scale for curing disease or repairing damaged tissues, such as bone, muscle, or nerve. Nanotechnology can have an early, paradigm-changing impact on how clinicians will detect cancer in its earliest stages. Exquisitely sensitive devices constructed of nanoscale components-such as nanocantilevers, nanowires and nanochannels-offer the potential for detecting even the rarest molecular signals associated with malignancy. One of the most pressing needs in clinical oncology is for imaging agents that can identify tumors that are far smaller than is possible with today's technology, at a scale of 100,000 cells rather than 1,000,000,000 cells. A new approach in nanotechnology for treating cancer incorporates nano iron particles and attaches them to an antibody that has targets only cancer cells and not healthy cells. The treatment works in two steps. This treatment is an ingenious way to make localized tumor ablation a systemic treatment. The advantages are incredible. There are absolutely no side effects from this treatment. It is not painful or even uncomfortable. The iron particles get flushed harmlessly from the body. It is not a drug and so the cancer cannot build up a resistance to the treatment. It is a systematic treatment; even cancer cells and tumors that are not known about get heated up and ablated. This treatment can even be used to enhance imaging of the cancer because once the cancer cells are coated with the iron particles, they are easy to identify. Everything depends on how reliably the antibodies target cancer cells and not healthy cells. When used in conjunction with other systemic treatments, such as vaccine treatments, we could be looking at a time when even advanced cancers can be brought under control. (author)

  20. Antibodies recognizing both IgM isotypes in Atlantic salmon

    DEFF Research Database (Denmark)

    Hedfors, Ida Aagård; Bakke, Hege; Skjødt, Karsten; Grimholt, Unni

    2012-01-01

    defined, mostly due to the lack of appropriate working tools like antibodies and functional assays. Membrane bound molecules like immunoglobulins (Ig) serve as cell surface markers for specific cell subsets and the identification of cells relies upon the production of specific antibodies towards these...... molecules. The present study aimed at identifying tools to separate IgM positive (IgM(+)) B cells from IgM negative (IgM(-)) non-B cell populations using flow cytometry. Several monoclonal antibodies (mAbs), and one polyclonal antibody (pAb) to both rainbow trout (Oncorhynchus mykiss) and Atlantic salmon...... IgM(+) cells in the respective tissues in salmon. To our surprise, this seemingly simple task did not reveal similar staining patterns for all antibodies as expected, but rather large differences in the number of positively stained cells were discovered. In short, positively stained cells by each...

  1. Chronic Renal Allograft Dysfunction Antibody-Mediated: An Update

    Directory of Open Access Journals (Sweden)

    Maurizio Salvadori,

    2014-07-01

    Full Text Available This paper reviews the most important studies on chronic antibody-mediated rejection (cABMR, which is an important cause of late graft dysfunction after renal transplantation. Several antibodies seem to be responsible for chronic rejection; new techniques have allowed us to identify these antibodies in circulation. The pathogenetic role of the antibodies generally includes the complement pathway, but may also be complement-independent. This paper also examines the pathogenesis of chronic endothelial lesions, as well as the histopathological aspects. Antibodies responsible for chronic rejection may preexist before transplantation or may develop after transplantation. The possible therapeutic approaches are poor and principally based on early identification and desensitisation techniques. New B cell targeting drugs are aimed at an improved control of the relevant condition.

  2. Monoclonal antibodies against human placental glutathione transferase (class pi).

    Science.gov (United States)

    Massoud, R; Lo Bello, M; De Stefano, E; Molino, A; Zelaschi, D; Federici, G

    1991-02-01

    Five monoclonal antibodies (MAbs) were produced in a mouse hybridoma system against human placental glutathione transferase (GST pi). Four of these monoclonal antibodies, named 461 to 464, were of immunoglobulin G class, whereas the monoclonal antibody 465 was of IgA class. All these MAbs specifically recognized the glutathione transferase from human placenta (class pi) showing no cross reactivity against the basic and the neutral forms of GST from human liver. When each MAb was incubated with the GST pi, no inhibition of enzymatic activity towards 1-chloro-2,4-dinitrobenzene was observed except for MAb 465 which showed a slight inhibition to a serial dilution of 1:128. PMID:1709614

  3. Structural Repertoire of HIV-1-Neutralizing Antibodies Targeting the CD4 Supersite in 14 Donors.

    Science.gov (United States)

    Zhou, Tongqing; Lynch, Rebecca M; Chen, Lei; Acharya, Priyamvada; Wu, Xueling; Doria-Rose, Nicole A; Joyce, M Gordon; Lingwood, Daniel; Soto, Cinque; Bailer, Robert T; Ernandes, Michael J; Kong, Rui; Longo, Nancy S; Louder, Mark K; McKee, Krisha; O'Dell, Sijy; Schmidt, Stephen D; Tran, Lillian; Yang, Zhongjia; Druz, Aliaksandr; Luongo, Timothy S; Moquin, Stephanie; Srivatsan, Sanjay; Yang, Yongping; Zhang, Baoshan; Zheng, Anqi; Pancera, Marie; Kirys, Tatsiana; Georgiev, Ivelin S; Gindin, Tatyana; Peng, Hung-Pin; Yang, An-Suei; Mullikin, James C; Gray, Matthew D; Stamatatos, Leonidas; Burton, Dennis R; Koff, Wayne C; Cohen, Myron S; Haynes, Barton F; Casazza, Joseph P; Connors, Mark; Corti, Davide; Lanzavecchia, Antonio; Sattentau, Quentin J; Weiss, Robin A; West, Anthony P; Bjorkman, Pamela J; Scheid, Johannes F; Nussenzweig, Michel C; Shapiro, Lawrence; Mascola, John R; Kwong, Peter D

    2015-06-01

    The site on the HIV-1 gp120 glycoprotein that binds the CD4 receptor is recognized by broadly reactive antibodies, several of which neutralize over 90% of HIV-1 strains. To understand how antibodies achieve such neutralization, we isolated CD4-binding-site (CD4bs) antibodies and analyzed 16 co-crystal structures -8 determined here- of CD4bs antibodies from 14 donors. The 16 antibodies segregated by recognition mode and developmental ontogeny into two types: CDR H3-dominated and VH-gene-restricted. Both could achieve greater than 80% neutralization breadth, and both could develop in the same donor. Although paratope chemistries differed, all 16 gp120-CD4bs antibody complexes showed geometric similarity, with antibody-neutralization breadth correlating with antibody-angle of approach relative to the most effective antibody of each type. The repertoire for effective recognition of the CD4 supersite thus comprises antibodies with distinct paratopes arrayed about two optimal geometric orientations, one achieved by CDR H3 ontogenies and the other achieved by VH-gene-restricted ontogenies. PMID:26004070

  4. Tabhu: tools for antibody humanization

    DEFF Research Database (Denmark)

    Olimpieri, Pier Paolo; Marcatili, Paolo; Tramontano, Anna

    2015-01-01

    and time-consuming experiments. Here we present tools for antibody humanization (Tabhu) a web server for antibody humanization. Tabhu includes tools for human template selection, grafting, back-mutation evaluation, antibody modelling and structural analysis, helping the user in all the critical steps......Antibodies are rapidly becoming essential tools in the clinical practice, given their ability to recognize their cognate antigens with high specificity and affinity, and a high yield at reasonable costs in model animals. Unfortunately, when administered to human patients, xenogeneic antibodies can...

  5. Monoclonal Antibody Production against Human Spermatozoal Surface Antigens

    Directory of Open Access Journals (Sweden)

    M Jedi-Tehrani

    2005-10-01

    Full Text Available Introduction: As monoclonal antibodies are potential tools for characterization of soluble or cellular surface antigens, use of these proteins has always been considered in infertility and reproduction research. Therefore, in this study, monoclonal antibodies against human sperm surface antigens were produced. Material and Methods: To produce specific clones against human sperm surface antigens, proteins were extracted using solubilization methods. Balb/c mice were immunized intraperitoneally with the proteins using complete Freund’s adjuvant in the first injection and incomplete Adjuvant in the following booster injections. Hybridoma cells producing ASA were cloned by limiting dilution. Results: Five stable ASA producing hybridoma clones were achieved and their antibody isotypes were determined by ELISA. All the isotypes were of IgG class. Their cross reactivity with rat and mice spermatozoa was examined but they did not have any cross reactivity. Conclusion: The produced antibodies can be used in further studies to characterize and evaluate each of the antigens present on human sperm surface and determining their role in fertilization.

  6. Use of monoclonal antibodies to distinguish pathogenic Naegleria fowleri (cysts, trophozoites, or flagellate forms) from other Naegleria species.

    OpenAIRE

    Sparagano, O.; Drouet, E.; Brebant, R; Manet, E; Denoyel, G A; Pernin, P.

    1993-01-01

    Monoclonal antibodies (MAbs) reactive to the pathogenic amoeba Naegleria fowleri were analyzed by enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay, Western blotting (immunoblotting), and radioimmunoprecipitation assay (RIPA). Two MAbs (3A4 and 5D12) showed reactivity by ELISA with all N. fowleri strains tested and no reactivity with the five other Naegleria species, N. lovaniensis, N. gruberi, N. australiensis, N. jadini, and N. andersoni. These MAbs reacted with t...

  7. Monoclonal antibody as radiopharmaceutical

    International Nuclear Information System (INIS)

    The purification of anti-CEA monoclonal antibody 4C11 belonging to IgG sub(2a) subclass from mouse ascitis, donated by Ludwig Institute, Brazil was developed. The fragmentation of purified IgG sub(2a) by pepsin digestion and analytical studies by polyacrilamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) were done as preliminary assessment for their specific application in immunoscintigraphy. (author)

  8. Anticardiolipin antibodies in leptospirosis.

    OpenAIRE

    Rugman, F P; Pinn, G.; Palmer, M. F.; Waite, M.; Hay, C. R.

    1991-01-01

    The clinical course and serology of 16 cases of leptospirosis in an area with an unusually high endemic infection rate were studied to gain further insight into the pathology of the secondary immune phase that is typical of the disease. IgG anticardiolipin antibody concentrations were measured by immunoassay and found to be increased in eight serologically confirmed cases with severe complicated disease, compared with eight patients with relatively uncomplicated leptospirosis who had IgG anti...

  9. A monoclonal antibody against leptin.

    Science.gov (United States)

    Mahmoudian, Jafar; Jeddi-Tehrani, Mahmood; Bayat, Ali Ahmad; Mahmoudi, Ahmad Reza; Vojgani, Yasaman; Tavangar, Banafsheh; Hadavi, Reza; Zarei, Saeed

    2012-10-01

    Leptin is an important protein that regulates energy storage and homeostasis in humans and animals. Leptin deficiency results in various abnormalities such as diabetes, obesity, and infertility. Producing a high affinity monoclonal antibody against human leptin provides an important tool to monitor and trace leptin function in different biological fluids. In this study, recombinant human leptin was conjugated to KLH and injected into mice. After immunization, mouse myeloma SP2/0 cells were fused with murine splenocytes followed by selection of antibody-producing hybridoma cells. After screening of different hybridoma colonies by ELISA, a high affinity antibody was selected and purified by affinity chromatography. The affinity constant of the antibody was measured by ELISA. Western blot, immunocytochemistry, and flow cytometry experiments were used to characterize the antibody. The anti-leptin antibody had a high affinity (around 1.13 × 10(-9) M) for its antigen. The saturation of the antibody with leptin (20 moles leptin per 1 mole antibody) in Western blot analysis proved that the antibody had specific binding to its antigen. Immunocytochemistry and flow cytometry on JEG-3 (human placental choriocarcinoma cell) cells revealed that the anti-leptin antibody recognized intracellular leptin. In conclusion, we report here the production and characterization of a murine anti-leptin antibody with high affinity for human leptin. PMID:23098305

  10. Radioimmunoimaging of 131I-anti human colon carcinoma monoclonal antibodies in nude mice with tumor xenografts

    International Nuclear Information System (INIS)

    The paired labeled antibodies, 131-labeled anti-human colon carcinoma monoclonal antibody 2C10 and 125I-labeled mice IgG, were used in the radioimmunolocalization study in nude mice with human colon carcinoma xenograft. The antibodies were radioiodinated with Iodogen method, and the incroporation efficiency and immune activity of the labeled antibodies were satisfactory. 96 hours after injection of the antibodies, good tumor localization was observed. Tumor/L. intestinal ratio was 7.08, tumor/S. intestinal 6.02, tumor/muscle 7.53, tumor/blood 1.10. Tumor imaging with camera was clear. The entrance of the antibodies into tumor tissues is a slow passive process and the accumulation of the antibodies in tumor is a combined result of specific immune reactivity and nonspecific deposition

  11. Targeting Reactive Carbonyl Species with Natural Sequestering Agents

    OpenAIRE

    Sung Won Hwang; Yoon-Mi Lee; Giancarlo Aldini; Kyung-Jin Yeum

    2016-01-01

    Reactive carbonyl species generated by the oxidation of polyunsaturated fatty acids and sugars are highly reactive due to their electrophilic nature, and are able to easily react with the nucleophilic sites of proteins as well as DNA causing cellular dysfunction. Levels of reactive carbonyl species and their reaction products have been reported to be elevated in various chronic diseases, including metabolic disorders and neurodegenerative diseases. In an effort to identify sequestering agents...

  12. The use of monoclonal antibodies to detect Bacteroides gingivalis in biological samples.

    OpenAIRE

    Chen, P; Bochacki, V; Reynolds, H S; Beanan, J; Tatakis, D.N.; Zambon, J J; Genco, R J

    1986-01-01

    Hybridomas were established which produce monoclonal antibodies specific for Bacteroides gingivalis, a pathogen associated with human periodontal disease. Spleen cells from BALB/c mice immunized with formalinized B. gingivalis were fused with Sp2/0-Ag14 myeloma cells. Of 1,050 wells with positive growth, 60 contained antibody reactive with the immunizing strain of B. gingivalis by enzyme-linked immunosorbent assay. Expansion of these cultures and cloning by limited dilution resulted in 28 clo...

  13. Production of Antibodies against Multipass Membrane Proteins Expressed in Human Tumor Cells Using Dendritic Cell Immunization

    OpenAIRE

    Takahiko Tamura; Joe Chiba

    2009-01-01

    Antibody mediated therapeutic strategies against human malignant tumors have been widely authorized and clinically applied to cancer patients. In order to develop methods to generate antibodies reactive to the extracellular domains of multipass plasma membrane proteins specifically expressed in malignant tumors, we examined the use of dendritic cells (DCs) for immunization. DCs were transduced with genes encoding the human six transmembrane epithelial antigen of prostate 1 (STEAP1), STEAP4, a...

  14. Effective Binding of a Phosphatidylserine-Targeting Antibody to Ebola Virus Infected Cells and Purified Virions

    OpenAIRE

    Dowall, S. D.; Graham, V A; Corbin-Lickfett, K; C. Empig; Schlunegger, K.; Bruce, C B; Easterbrook, L.; Hewson, R.

    2015-01-01

    Ebola virus is responsible for causing severe hemorrhagic fevers, with case fatality rates of up to 90%. Currently, no antiviral or vaccine is licensed against Ebola virus. A phosphatidylserine-targeting antibody (PGN401, bavituximab) has previously been shown to have broad-spectrum antiviral activity. Here, we demonstrate that PGN401 specifically binds to Ebola virus and recognizes infected cells. Our study provides the first evidence of phosphatidylserine-targeting antibody reactivity again...

  15. Patterned Immobilization of Antibodies within Roll-to-Roll Hot Embossed Polymeric Microfluidic Channels

    OpenAIRE

    Feyssa, Belachew; Liedert, Christina; Kivimaki, Liisa; Johansson, Leena-Sisko; Jantunen, Heli; Hakalahti, Leena

    2013-01-01

    This paper describes a method for the patterned immobilization of capture antibodies into a microfluidic platform fabricated by roll-to-roll (R2R) hot embossing on poly (methyl methacrylate) (PMMA). Covalent attachment of antibodies was achieved by two sequential inkjet printing steps. First, a polyethyleneimine (PEI) layer was deposited onto oxygen plasma activated PMMA foil and further cross-linked with glutaraldehyde (GA) to provide an amine-reactive aldehyde surface (PEI-GA). This step wa...

  16. Solid phase measurements of antibody and lectin binding to xenogenic carbohydrate antigens

    DEFF Research Database (Denmark)

    Kirkeby, Svend; André, Sabine; Gabius, Hans-Joachim

    2004-01-01

    naturally occurring subfraction from human serum, to Galalpha containing neoglycoproteins and mouse laminin that were immobilized on microtiter plates. RESULTS: Galalpha reactive antibodies with similar monosaccharide specificity have distinct structural preference for sugar ligands. Laminin and......-Galalpha1 antibodies that both have been raised against glycans on rabbit red blood cells may recognize Galalpha-antigens with varying specificities. Binding results obtained after digestion with alpha-galactosidase indicate that some xenoreactive Galalpha groups are not directly accessible for removal by...

  17. Radioimmunoassay of digoxin in serum using monoclonal antibodies and assessment of interference by digoxin-like immunoreactive substances

    International Nuclear Information System (INIS)

    We used 7 monoclonal antibodies (MoAbs) and one polyclonal antibody to develop radioimmunoassays (RIAs) for digoxin in serum or plasma. These RIAs were tested for measuring apparent digoxin concentrations in serum from patients receiving the drug, from normal individuals, and in cord blood plasma. We found that two MoAbs cross-reacted significantly with substances in cord blood. The magnitude of cross-reactivity was dependent on the incubation time and temperature. Under equilibrium conditions, one antibody gave apparent digoxin values in cord blood plasma averaging 2.15 ng/ml. We suggest that this cross-reactivity is partially due to progesterone and 17-hydroxyprogesterone in cord blood plasma. The antibody that shows high cross-reactivity with digoxin-like immunoreactive substances may prove a useful tool for studies dealing with characterization of the cross-reacting compounds

  18. A multiple antigen ELISA to detect Neospora-specific antibodies in bovine sera, bovine foetal fluids, ovine and caprine sera.

    Science.gov (United States)

    Osawa, T; Wastling, J; Maley, S; Buxton, D; Innes, E A

    1998-09-01

    Neospora caninum is a cyst-forming coccidian parasite recently identified as a cause of abortion in cattle. The epidemiology of neosporosis is poorly understood, partly because accurate diagnosis of infection is difficult. In this paper we describe the development of a multiple antigen-based enzyme-linked immunosorbent assay (ELISA) to detect antibodies to N. caninum in sera from cattle, sheep and goats as well as from bovine foetal fluids. A water-soluble fraction (wsf) of sonicated NC-1 strain tachyzoites was used as the antigen in the ELISA. Minimum optical density (OD) values that were considered to be Neospora antibody-positive, that is, the cut-off OD values were determined separately for bovine maternal sera, bovine foetal fluids, ovine sera and caprine sera; they were 0.40, 0.17, 0.23 and 0.41 OD, respectively. The ELISA gave a high signal/noise ratio, giving good sensitivity and specificity, correlating well with the indirect fluorescent antibody test (IFAT) currently used to diagnose Neospora infection in cattle, sheep and goats. In both the ELISA and immunoblot analysis using the same antigen, there was no significant cross-reactivity with sera from cattle, sheep or goats that had been infected with Toxoplasma gondii. The ELISA also showed no cross-reactivity in sera from cattle infected with Sarcocystis cruzi, Babesia divergens, B. bovis and B. bigemina. The wsf fraction of sonicated N. caninum tachyzoites used in this ELISA can be easily prepared and may be more sensitive than a single antigen ELISA, whilst still retaining good specificity. PMID:9777723

  19. Antiphospholipid Antibody and Antiphospholipid Syndrome

    Institute of Scientific and Technical Information of China (English)

    吴竞生

    2008-01-01

    @@ Antiphospholipid antibodies (APA) APA is a big category for all kinds of negative charge phospholipid or lecithin - a protein complex autoantibodies or the same antibody, through its recognition of antigen (target protein) different, and phospholipids or lecithin - protein complex combination of various rely on the interference Phospholipid clotting and anti-coagulation factor, and promote endothelial cells, platelets, complement activation and play a role. APA including lupus anticoagulant(LA) and anticardiolipin antibody (ACA), In addition, there are anti-β2 glycoprotein-I (β2-GPI) antibody, anti-prothrombin (a- PT) antibody, anti-lysophosphatidic acid antibody and anti-phosphatidylserine antibody, and so on. APA as the main target of phospholipid-binding protein, including β2-GPI, prothrombin, annexin, protein C (PC) and protein S (PS), plasminogen, and so on.

  20. Structural Comparison of Different Antibodies Interacting with Parvovirus Capsids

    Energy Technology Data Exchange (ETDEWEB)

    Hafenstein, Susan; Bowman, Valorie D.; Sun, Tao; Nelson, Christian D.S.; Palermo, Laura M.; Chipman, Paul R.; Battisti, Anthony J.; Parrish, Colin R.; Rossmann, Michael G.; Cornell; Purdue

    2009-05-13

    The structures of canine parvovirus (CPV) and feline parvovirus (FPV) complexed with antibody fragments from eight different neutralizing monoclonal antibodies were determined by cryo-electron microscopy (cryoEM) reconstruction to resolutions varying from 8.5 to 18 {angstrom}. The crystal structure of one of the Fab molecules and the sequence of the variable domain for each of the Fab molecules have been determined. The structures of Fab fragments not determined crystallographically were predicted by homology modeling according to the amino acid sequence. Fitting of the Fab and virus structures into the cryoEM densities identified the footprints of each antibody on the viral surface. As anticipated from earlier analyses, the Fab binding sites are directed to two epitopes, A and B. The A site is on an exposed part of the surface near an icosahedral threefold axis, whereas the B site is about equidistant from the surrounding five-, three-, and twofold axes. One antibody directed to the A site binds CPV but not FPV. Two of the antibodies directed to the B site neutralize the virus as Fab fragments. The differences in antibody properties have been linked to the amino acids within the antibody footprints, the position of the binding site relative to the icosahedral symmetry elements, and the orientation of the Fab structure relative to the surface of the virus. Most of the exposed surface area was antigenic, although each of the antibodies had a common area of overlap that coincided with the positions of the previously mapped escape mutations.

  1. [Guideline for interpretation and report of the antibody to hepatitis C virus. Grupo de Desarrollo de la Guía ].

    Science.gov (United States)

    Contreras, Ana M; Ochoa-Jiménez, Rodolfo J; Kershenobich, David; Granados-García, Víctor; Conde-González, Carlos J; Celis, Alfredo; Pérez-Gómez, Héctor R; Ruelas-Hernández, Sara; Romero-Flores, Patricia; Alcántar-Luna, Ernesto; Sierra-García de Quevedo, Julio; Ancona-Pisté, Oscar

    2012-01-01

    Patients with hepatitis C virus (HCV) infection are detected by testing for the presence of antibodies to HCV (Anti-HCV). A positive Anti-HCV test represents a true positive result only in a variable proportion of subjects (35 to 95%). The qualitative interpretation as positive or negative Anti-HCV report is associated with a general lack of understanding regarding the interpretation of results, when more specific testing should be performed, and which tests should be considered for this purpose. Therefore, a substantial variation in supplemental testing practices exists among laboratories and physicians. This guideline was developed on the basis of the best available evidence to classify positive antibody in two (low and high) or three levels (very low, low and high) according to the signal to cutoff (S/CO) ratio: the very low level of the Anti-HCV identifies false-positive results and further diagnostic testing is not necessary. The low antibody level is frequently related with false-positive results and testing with Immunoblot is recommended; only Immunoblot-positive subjects require HCV RNA testing because of a low possibility of being viremic. The high Anti-HCV level is an accurate serological marker for predicting viremia and denotes the need of routine HCV RNA testing in order to efficiently confirm hepatitis C. Cost-effectiveness analysis, based on the Anti-HCV level, recommends the use of the two or three-levels to choose the confirmatory test of positive antibody. This approach can be implemented without increasing test costs because the S/CO ratio is automatically generated in most laboratory analyzers and would provide health care professionals with useful information for counseling and evaluating patients, to eliminate unwarranted notifications in cases of false antibody reactivity, and correctly identifying those Anti-HCV-positive patients who are infected and need antiviral treatment. The written report should include the antibody level (S/CO ratio

  2. Isolation and characterisation of Ebolavirus-specific recombinant antibody fragments from murine and shark immune libraries.

    Science.gov (United States)

    Goodchild, Sarah A; Dooley, Helen; Schoepp, Randal J; Flajnik, Martin; Lonsdale, Stephen G

    2011-09-01

    Members of the genus Ebolavirus cause fulminating outbreaks of disease in human and non-human primate populations with a mortality rate up to 90%. To facilitate rapid detection of these pathogens in clinical and environmental samples, robust reagents capable of providing sensitive and specific detection are required. In this work recombinant antibody libraries were generated from murine (single chain variable domain fragment; scFv) and nurse shark, Ginglymostoma cirratum (IgNAR V) hosts immunised with Zaire ebolavirus. This provides the first recorded IgNAR V response against a particulate antigen in the nurse shark. Both murine scFv and shark IgNAR V libraries were panned by phage display technology to identify useful antibodies for the generation of immunological detection reagents. Two murine scFv were shown to have specificity to the Zaire ebolavirus viral matrix protein VP40. Two isolated IgNAR V were shown to bind to the viral nucleoprotein (NP) and to capture viable Zaire ebolavirus with a high degree of sensitivity. Assays developed with IgNAR V cross-reacted to Reston ebolavirus, Sudan ebolavirus and Bundibugyo ebolavirus. Despite this broad reactivity, neither of IgNAR V showed reactivity to Côte d'Ivoire ebolavirus. IgNAR V was substantially more resistant to irreversible thermal denaturation than murine scFv and monoclonal IgG in a comparative test. The demonstrable robustness of the IgNAR V domains may offer enhanced utility as immunological detection reagents in fieldable biosensor applications for use in tropical or subtropical countries where outbreaks of Ebolavirus haemorrhagic fever occur. PMID:21752470

  3. Detection of enterovirus 70 with monoclonal antibodies.

    OpenAIRE

    Anderson, L J; Hatch, M. H.; Flemister, M R; Marchetti, G E

    1984-01-01

    To improve the ability to identify enterovirus-70 (EV-70) from patients with acute hemorrhagic conjunctivitis, we developed four monoclonal antibodies (MAbs) to EV-70. We reacted the four MAbs against nine previously characterized strains of EV-70 and heterologous viruses by virus neutralization, indirect immunofluorescence, and enzyme-linked immunosorbent assay (ELISA). Two of the MAbs neutralized all nine strains of EV-70 and none of the other enterovirus types tested. Two of the MAbs gave ...

  4. Antibody-Catalyzed Degradation of Cocaine

    Science.gov (United States)

    Landry, Donald W.; Zhao, Kang; Yang, Ginger X.-Q.; Glickman, Michael; Georgiadis, Taxiarchis M.

    1993-03-01

    Immunization with a phosphonate monoester transition-state analog of cocaine provided monoclonal antibodies capable of catalyzing the hydrolysis of the cocaine benzoyl ester group. An assay for the degradation of radiolabeled cocaine identified active enzymes. Benzoyl esterolysis yields ecgonine methyl ester and benzoic acid, fragments devoid of cocaine's stimulant activity. Passive immunization with such an artificial enzyme could provide a treatment for dependence by blunting reinforcement.

  5. Radioimmunoassay of bovine leukosis virus antibodies

    International Nuclear Information System (INIS)

    A RIA method was developed for identifying the presence of serum antibodies to the bovine leukosis virus. The chosen procedure uses the ability of the virus antigen to bind to the solid phase of a polystyrene carrier. The method was compared with the ELISA method and with the pseudoneutralization and immunodiffusion tests. A high level of agreement was achieved between the RIA and the ELISA methods (95%). By its accuracy the RIA method proves superior to the immunodiffusion test. (author)

  6. Antibody induction therapy for lung transplant recipients

    DEFF Research Database (Denmark)

    Penninga, Luit; Møller, Christian H; Penninga, Ida Elisabeth Irene;

    2013-01-01

    Lung transplantation has become a valuable and well-accepted treatment option for most end-stage lung diseases. Lung transplant recipients are at risk of transplanted organ rejection, and life-long immunosuppression is necessary. Clear evidence is essential to identify an optimal, safe and effect...... effective immunosuppressive treatment strategy for lung transplant recipients. Consensus has not yet been achieved concerning use of immunosuppressive antibodies against T-cells for induction following lung transplantation....

  7. The Reactivity and Allergenic Potential of Hazelnut Peptides

    OpenAIRE

    Lavinia Florina Calinoiu; Dan Cristian Vodnar; Carmen Socaciu

    2013-01-01

    The aim of this paper was to focus on proteins present in some food products, like hazelnuts and to investigate their allergenic potential. Several techniques were used to characterize these extracted proteins, with respect to their composition, degradability by digestive proteolytic enzymes and their reactivity with specific antibodies. It was important to analyse which proteins were present in the hazelnuts, to see if there were proteins present to trigger an allergic reaction and if the di...

  8. Atherosclerosis-related functions of C-reactive protein

    OpenAIRE

    Agrawal, Alok; Hammond, David J.; Singh, Sanjay K.

    2010-01-01

    C-reactive protein (CRP) is secreted by hepatocytes as a pentameric molecule made up of identical monomers, circulates in the plasma as pentamers, and localizes in atherosclerotic lesions. In some cases, localized CRP was detected by using monoclonal antibodies that did not react with native pentameric CRP but were specific for isolated monomeric CRP. It has been reported that, once CRP is bound to certain ligands, the pentameric structure of CRP is altered so that it can dissociate into mono...

  9. 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

  10. Heterosubtypic antiviral activity of hemagglutinin-specific antibodies induced by intranasal immunization with inactivated influenza viruses in mice.

    Directory of Open Access Journals (Sweden)

    Mieko Muramatsu

    Full Text Available Influenza A virus subtypes are classified on the basis of the antigenicity of their envelope glycoproteins, hemagglutinin (HA; H1-H17 and neuraminidase. Since HA-specific neutralizing antibodies are predominantly specific for a single HA subtype, the contribution of antibodies to the heterosubtypic immunity is not fully understood. In this study, mice were immunized intranasally or subcutaneously with viruses having the H1, H3, H5, H7, H9, or H13 HA subtype, and cross-reactivities of induced IgG and IgA antibodies to recombinant HAs of the H1-H16 subtypes were analyzed. We found that both subcutaneous and intranasal immunizations induced antibody responses to multiple HAs of different subtypes, whereas IgA was not detected remarkably in mice immunized subcutaneously. Using serum, nasal wash, and trachea-lung wash samples of H9 virus-immunized mice, neutralizing activities of cross-reactive antibodies were then evaluated by plaque-reduction assays. As expected, no heterosubtypic neutralizing activity was detected by a standard neutralization test in which viruses were mixed with antibodies prior to inoculation into cultured cells. Interestingly, however, a remarkable reduction of plaque formation and extracellular release of the H12 virus, which was bound by the H9-induced cross-reactive antibodies, was observed when infected cells were subsequently cultured with the samples containing HA-specific cross-reactive IgA. This heterosubtypic plaque reduction was interfered when the samples were pretreated with anti-mouse IgA polyclonal serum. These results suggest that the majority of HA-specific cross-reactive IgG and IgA antibodies produced by immunization do not block cellular entry of viruses, but cross-reactive IgA may have the potential to inhibit viral egress from infected cells and thus to play a role in heterosubtypic immunity against influenza A viruses.

  11. Digital reactivity meter

    International Nuclear Information System (INIS)

    Digital reactivity meters (DRM) are mostly used as measuring instruments, e.g. for calibration of control rods, and there are only a few cases of their incorporation into the control systems of the reactors. To move in this direction there is more development work needed. First of all, fast algorithms are needed for inverse kinetics equations to relieve the computer for more important tasks of reactor model solving in real time. The next problem, currently under investigation, is the incorporation of the reactor thermal-hydraulic model into the DRM so that it can be used in the power range. Such an extension of DHM allows presentation not only of the instantaneous reactivity of the system, but also the inserted reactivity can be estimated from the temperature reactivity feed-backs. One of the applications of this concept is the anomalous digital reactivity monitor (ADRN) as part of the reactor protection system. As a solution of the first problem, a fast algorithm for solving the inverse kinetics equations has been implemented in the off-line program RODCAL on CDC 1700 computer and tested for its accuracy by performing different control rod calibrations on the reactor TRIGA

  12. Antibody response to hidden epitope of influenza a hemagglutinin elicited by anti-idiotypic antibodies

    International Nuclear Information System (INIS)

    Monoclonal antibody (MoAb) IIF4 defines an epitope on the HA2 part of influenza hemagglutinin (HA)It was also found this epitope becomes fully accessible after pH 5 treatment of the antigen and is shared by strains of H3 subtype. In this study we found binding of MoAb IIF4 also to some strains belonging to H2, H4, H7, and H10 subtypes. We prepared rabbit polyclonal anti-IIF4 anti-idiotype (anti-Id) antibody. In competitive assays, the inhibition potential of anti-Id was considerably higher than that of native HA. Anti-Id was used for the preparation of mouse Ab3 (anti-anti-IIF4) serum. Reactivity pattern of Ab3 with influenza virus strains differed from Ab1 in in (i) appearance of binding to some strains of H2 and H7 subtype and (ii) decreased dependency of Ab3 binding on the pH forms of antigen. The reactivity of Ab1 and Ab3 with two amantadine-resistant virus mutants indicates that IIF4 epitope (and its related region recognized by Ab3) becomes accessible in consequence of destabilization of trimeric arrangement of HA and it it also correlates with expulsion of N-terminus of HA2. (author)

  13. Immunodot blot assay to detect Helicobacter pylori using monoclonal antibodies against the 26 kDa protein.

    Science.gov (United States)

    Amini Najafabadi, Hossein; Paknejad, Maliheh; Farshad, Shohreh; Mohammadian, Taher; Seyyed Ebrahimi, Shadi Sadat; Amini Najafabadi, Azadeh

    2012-12-01

    Development of a specific immunoassay to detect Helicobacter pylori infection in stool samples requires monoclonal antibody against the specific antigen. The aims of this study were to establish monoclonal antibodies against the 26 kDa protein of H. pylori and develop an immunodot blot for their application to recognize H. pylori infection using stool samples. Mice were immunized intraperitoneally with homogenized gel containing the 26 kDa band of cell surface proteins of H. pylori in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The monoclonal antibodies were produced using the hybridoma technique. Reactivity of monoclonal antibodies was tested with the purified 26 kDa antigen and cell surface proteins from cultured H. pylori by ELISA. Furthermore reactivity of monoclonal antibodies was tested on negative and positive stool samples for H. pylori and suspensions of several major bacteria in stool by immunodot blot assay. Five stable hybridoma monoclones were obtained. The concordant reactivity of the monoclonal antibodies with H. pylori present in the stool samples, which had been tested previously using an ACON ELISA kit for H. pylori stool antigen testing, and unreactivity with several different major fecal bacteria in immunodot blotting indicates high specificity of the immunodot blot based on the reaction of produced monoclonal antibodies with the H. pylori antigen in stools. The findings indicate that the novel immunodot blot developed based on new monoclonal antibodies for stool antigens would be useful as a noninvasive method of diagnosing H. pylori infection. PMID:23244318

  14. Risk of hepatitis B virus reactivation in rheumatoid arthritis patients undergoing biologic treatment: Extending perspective from old to newer drugs

    OpenAIRE

    Nard, Francesca De; Todoerti, Monica; Grosso, Vittorio; Monti, Sara; Breda, Silvia; Rossi, Silvia; Montecucco, Carlomaurizio; Caporali, Roberto

    2015-01-01

    Hepatitis B virus (HBV) reactivation in rheumatoid arthritis (RA) patients undergoing biological therapy is not infrequent. This condition can occur in patients with chronic hepatitis B as well as in patients with resolved HBV infection. Current recommendations are mainly focused on prevention and management strategies of viral reactivation under tumor necrosis factor-α inhibitors or chimeric monoclonal antibody rituximab. In recent years, growing data concerning HBV reactivation in RA patien...

  15. Production of monoclonal antibodies against canine leukocytes.

    Science.gov (United States)

    Aguiar, Paulo Henrique Palis; Borges dos Santos, Roberto Robson; Lima, Carla Andrade; Rios de Sousa Gomes, Hilton; Larangeira, Daniela Farias; Santos, Patrícia Meira; Barrouin-Melo, Stella Maria; Conrado dos-Santos, Washington Luis; Pontes-de-Carvalho, Lain

    2004-04-01

    A panel of anti-canine leukocyte monoclonal antibodies (MAbs) was produced by immunizing BALB/c mice with canine peripheral blood mononuclear cells (PBMC), either resting or stimulated with concanavalin A (ConA). Three out of 28 clones-IH1, AB6, and HG6-screened by ELISA and producing antibody with the highest specificity for canine cell immunostaining, were subjected to three subsequent subcloning steps by limiting dilution, and selected for further characterization. These MAbs belonged to IgG1 (HG6 and IH1) and IgG2a (AB6) isotypes. The distribution of cell populations expressing the antigen recognized by the antibodies was identified by indirect immunoflorescence on canine PBMC and on tissue sections of lymph node, spleen, liver and skin. The possible crossreactivity with human PBMC was also examined in immunocytochemistry. One of the antibodies specifically recognized macrophages. The MAbs presented here can be foreseen as possible valuable diagnostic and research tools to study immune functions in dogs. PMID:15165486

  16. Developing recombinant antibodies for biomarker detection

    Energy Technology Data Exchange (ETDEWEB)

    Baird, Cheryl L.; Fischer, Christopher J.; Pefaur, Noah B.; Miller, Keith D.; Kagen, Jacob; Srivastava, Sudhir; Rodland, Karin D.

    2010-10-01

    Monoclonal antibodies (mAbs) have an essential role in biomarker validation and diagnostic assays. A barrier to pursuing these applications is the reliance on immunization and hybridomas to produce mAbs, which is time-consuming and may not yield the desired mAb. We recommend a process flow for affinity reagent production that utilizes combinatorial protein display systems (eg, yeast surface display or phage display) rather than hybridomas. These systems link a selectable phenotype-binding conferred by an antibody fragment-with a means for recovering the encoding gene. Recombinant libraries obtained from immunizations can produce high-affinity antibodies (<10 nM) more quickly than other methods. Non-immune libraries provide an alternate route when immunizations are not possible, or when suitable mAbs are not recovered from an immune library. Directed molecular evolution (DME) is an integral part of optimizing mAbs obtained from combinatorial protein display, but can also be used on hybridoma-derived mAbs. Variants can easily be obtained and screened to increase the affinity of the parent mAb (affinity maturation). We discuss examples where DME has been used to tailor affinity reagents to specific applications. Combinatorial protein display also provides an accessible method for identifying antibody pairs, which are necessary for sandwich-type diagnostic assays.

  17. Dual-Quantum-Dots-Labeled Lateral Flow Strip Rapidly Quantifies Procalcitonin and C-reactive Protein

    OpenAIRE

    Qi, Xiaoping; Huang, YunYe; Lin, ZhongShi; Xu, Liang; Yu, Hao

    2016-01-01

    In the article, a dual-quantum-dots-labeled (dual-QDs-labeled) lateral flow strip (LFS) method was developed for the simultaneous and rapid quantitative detection of procalcitonin (PCT) and C-reactive protein (CRP) in the blood. Two QD-antibody conjugates with different fluorescence emission spectra were produced and sprayed on the LFS to capture PCT and CRP in the blood. Furthermore, a double antibody sandwich method for PCT and, meanwhile, a competitive inhibition method for CRP were employ...

  18. Interactive Chemical Reactivity Exploration

    CERN Document Server

    Haag, Moritz P; Bosson, Mael; Redon, Stephane; Reiher, Markus

    2014-01-01

    Elucidating chemical reactivity in complex molecular assemblies of a few hundred atoms is, despite the remarkable progress in quantum chemistry, still a major challenge. Black-box search methods to find intermediates and transition-state structures might fail in such situations because of the high-dimensionality of the potential energy surface. Here, we propose the concept of interactive chemical reactivity exploration to effectively introduce the chemist's intuition into the search process. We employ a haptic pointer device with force-feedback to allow the operator the direct manipulation of structures in three dimensions along with simultaneous perception of the quantum mechanical response upon structure modification as forces. We elaborate on the details of how such an interactive exploration should proceed and which technical difficulties need to be overcome. All reactivity-exploration concepts developed for this purpose have been implemented in the Samson programming environment.

  19. 40 CFR 721.9717 - Azo monochloro triazine reactive dye.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Azo monochloro triazine reactive dye... Substances § 721.9717 Azo monochloro triazine reactive dye. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as an azo monochloro...

  20. Second antibody clearance of radiolabeled antibody in cancer radioimmunodetection.

    OpenAIRE

    Sharkey, R M; Primus, F J; Goldenberg, D. M.

    1984-01-01

    The imaging of tumors using radiolabeled antibodies previously has required the implementation of computer-assisted subtraction techniques to reduce background radioactivity. A decrease in radioactivity in the blood of hamsters bearing human colonic tumor xenografts has been achieved by administering a second antibody directed against a radiolabeled primary antibody to carcinoembryonic antigen (CEA). This method was found to reduce the level of blood radioactivity by a factor of 4 within 2 hr...