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.

Construction of human antibody gene libraries and selection of antibodies by phage display.

Science.gov (United States)

Frenzel, André; Kügler, Jonas; Wilke, Sonja; Schirrmann, Thomas; Hust, Michael

2014-01-01

Antibody phage display is the most commonly used in vitro selection technology and has yielded thousands of useful antibodies for research, diagnostics, and therapy.The prerequisite for successful generation and development of human recombinant antibodies using phage display is the construction of a high-quality antibody gene library. Here, we describe the methods for the construction of human immune and naive scFv gene libraries.The success also depends on the panning strategy for the selection of binders from these libraries. In this article, we describe a panning strategy that is high-throughput compatible and allows parallel selection in microtiter plates.

Macrophages are critical effectors of antibody therapies for cancer.

Science.gov (United States)

Weiskopf, Kipp; Weissman, Irving L

2015-01-01

Macrophages are innate immune cells that derive from circulating monocytes, reside in all tissues, and participate in many states of pathology. Macrophages play a dichotomous role in cancer, where they promote tumor growth but also serve as critical immune effectors of therapeutic antibodies. Macrophages express all classes of Fcγ receptors, and they have immense potential to destroy tumors via the process of antibody-dependent phagocytosis. A number of studies have demonstrated that macrophage phagocytosis is a major mechanism of action of many antibodies approved to treat cancer. Consequently, a number of approaches to augment macrophage responses to therapeutic antibodies are under investigation, including the exploration of new targets and development of antibodies with enhanced functions. For example, the interaction of CD47 with signal-regulatory protein α (SIRPα) serves as a myeloid-specific immune checkpoint that limits the response of macrophages to antibody therapies, and CD47-blocking agents overcome this barrier to augment phagocytosis. The response of macrophages to antibody therapies can also be enhanced with engineered Fc variants, bispecific antibodies, or antibody-drug conjugates. Macrophages have demonstrated success as effectors of cancer immunotherapy, and further investigation will unlock their full potential for the benefit of patients.

A natural human monoclonal antibody targeting Staphylococcus Protein A protects against Staphylococcus aureus bacteremia

Science.gov (United States)

Varshney, Avanish K.; Sunley, Kevin M.; Bowling, Rodney A.; Kwan, Tzu-Yu; Mays, Heather R.; Rambhadran, Anu; Zhang, Yanfeng; Martin, Rebecca L.; Cavalier, Michael C.; Simard, John

2018-01-01

Staphylococcus aureus can cause devastating and life-threatening infections. With the increase in multidrug resistant strains, novel therapies are needed. Limited success with active and passive immunization strategies have been attributed to S. aureus immune evasion. Here, we report on a monoclonal antibody, 514G3, that circumvents a key S. aureus evasion mechanism by targeting the cell wall moiety Protein A (SpA). SpA tightly binds most subclasses of immunoglobulins via their Fc region, neutralizing effector function. The organism can thus shield itself with a protective coat of serum antibodies and render humoral immunity ineffective. The present antibody reactivity was derived from an individual with natural anti-SpA antibody titers. The monoclonal antibody is of an IgG3 subclass, which differs critically from other immunoglobulin subclasses since its Fc is not bound by SpA. Moreover, it targets a unique epitope on SpA that allows it to bind in the presence of serum antibodies. Consequently, the antibody opsonizes S. aureus and maintains effector function to enable natural immune mediated clearance. The data presented here provide evidence that 514G3 antibody is able to successfully rescue mice from S. aureus mediated bacteremia. PMID:29364906

New Strategies Using Antibody Combinations to Increase Cancer Treatment Effectiveness

Directory of Open Access Journals (Sweden)

Isabel Corraliza-Gorjón

2017-12-01

Full Text Available Antibodies have proven their high value in antitumor therapy over the last two decades. They are currently being used as the first-choice to treat some of the most frequent metastatic cancers, like HER2+ breast cancers or colorectal cancers, currently treated with trastuzumab (Herceptin and bevacizumab (Avastin, respectively. The impressive therapeutic success of antibodies inhibiting immune checkpoints has extended the use of therapeutic antibodies to previously unanticipated tumor types. These anti-immune checkpoint antibodies allowed the cure of patients devoid of other therapeutic options, through the recovery of the patient’s own immune response against the tumor. In this review, we describe how the antibody-based therapies will evolve, including the use of antibodies in combinations, their main characteristics, advantages, and how they could contribute to significantly increase the chances of success in cancer therapy. Indeed, novel combinations will consist of mixtures of antibodies against either different epitopes of the same molecule or different targets on the same tumor cell; bispecific or multispecific antibodies able of simultaneously binding tumor cells, immune cells or extracellular molecules; immunomodulatory antibodies; antibody-based molecules, including fusion proteins between a ligand or a receptor domain and the IgG Fab or Fc fragments; autologous or heterologous cells; and different formats of vaccines. Through complementary mechanisms of action, these combinations could contribute to elude the current limitations of a single antibody which recognizes only one particular epitope. These combinations may allow the simultaneous attack of the cancer cells by using the help of the own immune cells and exerting wider therapeutic effects, based on a more specific, fast, and robust response, trying to mimic the action of the immune system.

Antibody or Antibody Fragments : Implications for Molecular Imaging and Targeted Therapy of Solid Tumors

NARCIS (Netherlands)

Xenaki, Katerina T; Oliveira, Sabrina; van Bergen En Henegouwen, Paul M P

2017-01-01

The use of antibody-based therapeutics has proven very promising for clinical applications in cancer patients, with multiple examples of antibodies and antibody-drug conjugates successfully applied for the treatment of solid tumors and lymphomas. Given reported recurrence rates, improvements are

Antigenic modulation limits the effector cell mechanisms employed by type I anti-CD20 monoclonal antibodies.

Science.gov (United States)

Tipton, Thomas R W; Roghanian, Ali; Oldham, Robert J; Carter, Matthew J; Cox, Kerry L; Mockridge, C Ian; French, Ruth R; Dahal, Lekh N; Duriez, Patrick J; Hargreaves, Philip G; Cragg, Mark S; Beers, Stephen A

2015-03-19

Following the success of rituximab, 2 other anti-CD20 monoclonal antibodies (mAbs), ofatumumab and obinutuzumab, have entered clinical use. Ofatumumab has enhanced capacity for complement-dependent cytotoxicity, whereas obinutuzumab, a type II mAb, lacks the ability to redistribute into lipid rafts and is glycoengineered for augmented antibody-dependent cellular cytotoxicity (ADCC). We previously showed that type I mAbs such as rituximab have a propensity to undergo enhanced antigenic modulation compared with type II. Here we assessed the key effector mechanisms affected, comparing type I and II antibodies of various isotypes in ADCC and antibody-dependent cellular-phagocytosis (ADCP) assays. Rituximab and ofatumumab depleted both normal and leukemic human CD20-expressing B cells in the mouse less effectively than glycoengineered and wild-type forms of obinutuzumab, particularly when human immunoglobulin G1 (hIgG1) mAbs were compared. In contrast to mouse IgG2a, hIgG1 mAbs were ineffective in ADCC assays with murine natural killer cells as effectors, whereas ADCP was equivalent for mouse IgG2a and hIgG1. However, rituximab's ability to elicit both ADCC and ADCP was reduced by antigenic modulation, whereas type II antibodies remained unaffected. These data demonstrate that ADCP and ADCC are impaired by antigenic modulation and that ADCP is the main effector function employed in vivo. © 2015 by The American Society of Hematology.

A pharmacology guided approach for setting limits on product-related impurities for bispecific antibody manufacturing.

Science.gov (United States)

Rajan, Sharmila; Sonoda, Junichiro; Tully, Timothy; Williams, Ambrose J; Yang, Feng; Macchi, Frank; Hudson, Terry; Chen, Mark Z; Liu, Shannon; Valle, Nicole; Cowan, Kyra; Gelzleichter, Thomas

2018-04-13

bFKB1 is a humanized bispecific IgG1 antibody, created by conjoining an anti-Fibroblast Growth Factor Receptor 1 (FGFR1) half-antibody to an anti-Klothoβ (KLB) half-antibody, using the knobs-into-holes strategy. bFKB1 acts as a highly selective agonist for the FGFR1/KLB receptor complex and is intended to ameliorate obesity-associated metabolic defects by mimicking the activity of the hormone FGF21. An important aspect of the biologics product manufacturing process is to establish meaningful product specifications regarding the tolerable levels of impurities that copurify with the drug product. The aim of the current study was to determine acceptable levels of product-related impurities for bFKB1. To determine the tolerable levels of these impurities, we dosed obese mice with bFKB1 enriched with various levels of either HMW impurities or anti-FGFR1-related impurities, and measured biomarkers for KLB-independent FGFR1 signaling. Here, we show that product-related impurities of bFKB1, in particular, high molecular weight (HMW) impurities and anti-FGFR1-related impurities, when purposefully enriched, stimulate FGFR1 in a KLB-independent manner. By taking this approach, the tolerable levels of product-related impurities were successfully determined. Our study demonstrates a general pharmacology-guided approach to setting a product specification for a bispecific antibody whose homomultimer-related impurities could lead to undesired biological effects. Copyright © 2018. Published by Elsevier Inc.

Compact quantum dot-antibody conjugates for FRET immunoassays with subnanomolar detection limits

Science.gov (United States)

Mattera, Lucia; Bhuckory, Shashi; Wegner, K. David; Qiu, Xue; Agnese, Fabio; Lincheneau, Christophe; Senden, Tim; Djurado, David; Charbonnière, Loïc J.; Hildebrandt, Niko; Reiss, Peter

2016-05-01

A novel two-step approach for quantum dot (QD) functionalization and bioconjugation is presented, which yields ultra-compact, stable, and highly luminescent antibody-QD conjugates suitable for use in FRET immunoassays. Hydrophobic InPZnS/ZnSe/ZnS (emission wavelength: 530 nm), CdSe/ZnS (605 nm), and CdSeTe/ZnS (705 nm) QDs were surface functionalized with zwitterionic penicillamine, enabling aqueous phase transfer under conservation of the photoluminescence properties. Post-functionalization with a heterobifunctional crosslinker, containing a lipoic acid group and a maleimide function, enabled the subsequent coupling to sulfhydryl groups of proteins. This was demonstrated by QD conjugation with fragmented antibodies (F(ab)). The obtained F(ab)-QD conjugates range among the smallest antibody-functionalized nanoprobes ever reported, with a hydrodynamic diameter coating for FRET could be demonstrated by an 6.2 and 2.5 fold improvement of the limit of detection (LOD) for PSA compared to commercially available hydrophilic QDs emitting at 605 and 705 nm, respectively. While the commercial QDs contain identical inorganic cores responsible for their fluorescence, they are coated with a comparably thick amphiphilic polymer layer leading to much larger hydrodynamic diameters (>26 nm without biomolecules). The LODs of 0.8 and 3.7 ng mL-1 obtained in 50 μL serum samples are below the clinical cut-off level of PSA (4 ng mL-1) and demonstrate their direct applicability in clinical diagnostics.A novel two-step approach for quantum dot (QD) functionalization and bioconjugation is presented, which yields ultra-compact, stable, and highly luminescent antibody-QD conjugates suitable for use in FRET immunoassays. Hydrophobic InPZnS/ZnSe/ZnS (emission wavelength: 530 nm), CdSe/ZnS (605 nm), and CdSeTe/ZnS (705 nm) QDs were surface functionalized with zwitterionic penicillamine, enabling aqueous phase transfer under conservation of the photoluminescence properties. Post

Nuclear medicine: Monoclonal antibodies

International Nuclear Information System (INIS)

Endo, K.; Sakahara, H.; Koizumi, M.; Kawamura, Y.; Torizuka, K.; Yokoyama, A.

1986-01-01

Antitumor monoclonal antibody was successfully labeled with Tc-99m by using dithiosemicarbazone (DTS) as a bifunctional chelating agent. In the first step, DTS was coupled to antibody without loss of immunoreactivity; the compound then efficiently formed a neutral 1:1 chelate with pentavalent or tetravalent Tc-99m. Imaging with Tc-99m-labeled monoclonal antibody to human osteosarcoma (OST-7) clearly displayed a small tumor in nude mice at 6 and 24 hours after intravenous administration. The tumor-to-blood ratio of the Tc-99m-labeled monoclonal antibody was higher than that of a radioiodinated antibody and similar to that of an In-111-labeled antibody. Thus, conjugation of DTS to monoclonal antibody followed by radiometalation is a simple and efficient method of preparing Tc-99m-labeled monoclonal antibody

Immune Antibody Libraries: Manipulating The Diverse Immune Repertoire for Antibody Discovery.

Science.gov (United States)

Lim, Theam Soon; Chan, Soo Khim

2016-01-01

Antibody phage display is highly dependent on the availability of antibody libraries. There are several forms of libraries depending mainly on the origin of the source materials. There are three major classes of libraries, mainly the naïve, immune and synthetic libraries. Immune antibody libraries are designed to isolate specific and high affinity antibodies against disease antigens. The pre-exposure of the host to an infection results in the production of a skewed population of antibodies against the particular infection. This characteristic takes advantage of the in vivo editing machinery to generate bias and specific immune repertoire. The skewed but diverse repertoire of immune libraries has been adapted successfully in the generation of antibodies against a wide range of diseases. We envisage immune antibody libraries to play a greater role in the discovery of antibodies for diseases in the near future. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Avian Diagnostic and Therapeutic Antibodies

Energy Technology Data Exchange (ETDEWEB)

Bradley, David Sherman [UND SMHS

2012-12-31

A number of infectious agents have the potential of causing significant clinical symptomology and even death, but dispite this, the number of incidence remain below the level that supports producing a vaccine. Therapeutic antibodies provide a viable treatment option for many of these diseases. We proposed that antibodies derived from West Nile Virus (WNV) immunized geese would be able to treat WNV infection in mammals and potential humans. We demonstrated that WNV specific goose antibodies are indeed successful in treating WNV infection both prophylactically and therapeutically in a golden hamster model. We demonstrated that the goose derived antibodies are non-reactogenic, i.e. do not cause an inflammatory response with multiple exposures in mammals. We also developed both a specific pathogen free facility to house the geese during the antibody production phase and a patent-pending purification process to purify the antibodies to greater than 99% purity. Therefore, the success of these study will allow a cost effective rapidly producible therapeutic toward clinical testing with the necessary infrastructure and processes developed and in place.

Monoclonal antibodies: an overview of their advantages and limitations in nuclear medicine

International Nuclear Information System (INIS)

Revillard, J.P.; Cohen, J.

1982-01-01

The following topics were reviewed: antigen recognition by the immune system; development of immunoassays for antigenic components of biological fluids; monoclonal antibodies against infectious agents; monochonal antibodies against tumor and differentiation antigens; human monoclonal antibodies

A Recombinant Secondary Antibody Mimic as a Target-specific Signal Amplifier and an Antibody Immobilizer in Immunoassays.

Science.gov (United States)

Min, Junseon; Song, Eun Kyung; Kim, Hansol; Kim, Kyoung Taek; Park, Tae Joo; Kang, Sebyung

2016-04-11

We construct a novel recombinant secondary antibody mimic, GST-ABD, which can bind to the Fc regions of target-bound primary antibodies and acquire multiple HRPs simultaneously. We produce it in tenth of mg quantities with a bacterial overexpression system and simple purification procedures, significantly reducing the manufacturing cost and time without the use of animals. GST-ABD is effectively conjugated with 3 HRPs per molecule on an average and selectively bind to the Fc region of primary antibodies derived from three different species (mouse, rabbit, and rat). HRP-conjugated GST-ABD (HRP-GST-ABD) is successfully used as an alternative to secondary antibodies to amplify target-specific signals in both ELISA and immunohistochemistry regardless of the target molecules and origin of primary antibodies used. GST-ABD also successfully serves as an anchoring adaptor on the surface of GSH-coated plates for immobilizing antigen-capturing antibodies in an orientation-controlled manner for sandwich-type indirect ELISA through simple molecular recognition without any complicated chemical modification.

Radiolabeled antibody imaging

International Nuclear Information System (INIS)

Wahl, R.L.

1987-01-01

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

Purification of immunoreactive radiolabeled moniclonal antibodies with anti-iodiotypic moniclonal antibodies

International Nuclear Information System (INIS)

Temponi, M.; Pupa, S.; Ferrone, S.

1990-01-01

A method is described to purify immunoreactive moniclonal antibodies from radiolabeled monoclonal antibody preparations. The method is based on incubation of radiolabeled monoclonal antibodies with insolubilized anti-idiotypic monoclonal antibodies to idiotopes within the antigen-combining site of monoclonal antibodies to be purified an elution of bound monoclonal antibodies with a low pH buffer. The immunoreactive fraction of the purified monoclonal antibodies was at least 82%; the yeald was at least 73%. The purification procedure did not cause any detectable change in the affinity constant of the eluted monoclonal antibodies. The method is simple and rapid; the requirement for anti-idiotypic monoclonal antibodies to idiotopes within the antigen-combining site of the antibodies to be purified is not likely to represent a major limitation in the broad application of the present method, since the hybridoma technology has greatly facilitated the development of anti-idiotypic monoclonal antibodies. (author). 12 refs.; 4 figs.; 1 tab

Microangiopathic antiphospholipid antibody syndrome due to anti-phosphatidylserine/prothrombin complex IgM antibody.

Science.gov (United States)

Senda, Yumi; Ohta, Kazuhide; Yokoyama, Tadafumi; Shimizu, Masaki; Furuichi, Kengo; Wada, Takashi; Yachie, Akihiro

2017-03-01

Herein we describe a case of microangiopathic antiphospholipid syndrome (MAPS) due to anti-phosphatidylserine/prothrombin complex (aPS/PT) IgM antibody successfully treated with rituximab. A significant correlation was observed between the clinical course and the aPS/PT IgM antibody titer, which can rise earlier before the appearance of clinical symptoms. Rituximab can be safely and effectively used for MAPS. Although detection of only aPS/PT IgM antibody is rare, aPS/PT IgM antibody might be associated with the pathogenesis of MAPS and might be a useful marker of disease activity. © 2017 Japan Pediatric Society.

A case of propylthiouracil-induced antineutrophilic cytoplasmic antibody-positive vasculitis successfully treated with radioactive iodine

Directory of Open Access Journals (Sweden)

C. Bes

2013-07-01

Full Text Available Antineutrophilic cytoplasmic antibody (ANCA associated vasculitis is one of the rare complications of propylthiouracil treatment. Having a variable clinical spectrum, it may be presented with both skin limited vasculitis and life-threatening systemic vasculitis. In this study, we present a case that developed ANCA-positive vasculitis with skin and kidney involvement (hematuria and proteinuria six months after propylthiouracil treatment was initiated for toxic nodular goiter. Proteinuria recovered dramatically subsequent to radioactive iodine treatment following ceasing the drug.

A novel affinity purification method to isolate peptide specific antibodies

DEFF Research Database (Denmark)

Karlsen, Alan E; Lernmark, A; Kofod, Hans

1990-01-01

Site-specific, high affinity polyclonal antisera are effectively and successfully produced by immunizing rabbits with synthetic peptides. The use of these antisera in subsequent immune analysis is often limited because of non-specific binding. We describe a new and simple method to effectively...... affinity-purify anti-peptide antibodies. To test our system, rabbits were immunized with model peptides representing sequences of the putative rabbit growth hormone receptor and several HLA-DQ beta-chain molecules. Polystyrene plastic beads were coated with peptides. Immune serum was incubated...... with the beads and after a wash step the bound antibodies were eluted in 1 M acetic acid. The eluted material was composed predominantly of intact immunoglobulin as evidenced by the presence of heavy and light chain bands in SDS-PAGE. The eluted antibodies were peptide specific in ELISA and bound only to intact...

Development of novel monoclonal antibodies against starch and ulvan - Implications for antibody production against polysaccharides with limited immunogenicity

DEFF Research Database (Denmark)

Rydahl, Maja Gro; Kračun, Stjepan K.; Fangel, Jonatan U.

2017-01-01

Monoclonal antibodies (mAbs) are widely used and powerful research tools, but the generation of mAbs against glycan epitopes is generally more problematic than against proteins. This is especially significant for research on polysaccharide-rich land plants and algae (Viridiplantae). Most antibody...

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

Directory of Open Access Journals (Sweden)

Christina Yacoob

2016-11-01

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

Imaging of colorectal carcinoma with radiolabeled antibodies.

Science.gov (United States)

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

1989-10-01

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

Expression of recombinant Antibodies

Directory of Open Access Journals (Sweden)

André eFrenzel

2013-07-01

Full Text Available 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 transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines and transgenic plants are promising to obtain antibodies with human-like post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications.

Beyond Antibodies as Binding Partners: The Role of Antibody Mimetics in Bioanalysis.

Science.gov (United States)

Yu, Xiaowen; Yang, Yu-Ping; Dikici, Emre; Deo, Sapna K; Daunert, Sylvia

2017-06-12

The emergence of novel binding proteins or antibody mimetics capable of binding to ligand analytes in a manner analogous to that of the antigen-antibody interaction has spurred increased interest in the biotechnology and bioanalytical communities. The goal is to produce antibody mimetics designed to outperform antibodies with regard to binding affinities, cellular and tumor penetration, large-scale production, and temperature and pH stability. The generation of antibody mimetics with tailored characteristics involves the identification of a naturally occurring protein scaffold as a template that binds to a desired ligand. This scaffold is then engineered to create a superior binder by first creating a library that is then subjected to a series of selection steps. Antibody mimetics have been successfully used in the development of binding assays for the detection of analytes in biological samples, as well as in separation methods, cancer therapy, targeted drug delivery, and in vivo imaging. This review describes recent advances in the field of antibody mimetics and their applications in bioanalytical chemistry, specifically in diagnostics and other analytical methods.

Generation and selection of naïve Fab library for parasitic antigen: Anti-BmSXP antibodies for lymphatic filariasis.

Science.gov (United States)

Omar, Noorsharmimi; Hamidon, Nurul Hamizah; Yunus, Muhammad Hafiznur; Noordin, Rahmah; Choong, Yee Siew; Lim, Theam Soon

2018-05-01

Phage display has been applied successfully as a tool for the generation of monoclonal antibodies (mAbs). Naive antibody libraries are unique as they are able to overcome several limitations associated with conventional mAb generation methods like the hybridoma technology. Here, we performed an in vitro selection and generation of Fab antibodies against Brugia malayi SXP protein (BmSXP), a recombinant antigen for the detection of lymphatic filariasis. We developed a naïve multi ethnic Fab antibody library with an estimated diversity of 2.99 × 10 9 . The antibody library was used to screen for mAbs against BmSXP recombinant antigen. Soluble monoclonal Fab antibodies against BmSXP were successfully isolated from the naïve library. The Fab antibodies obtained were expressed and analyzed to show its binding capability. The diversity obtained from a pool of donors from various ethnic groups allowed for a diverse antibody library to be generated. The mAbs obtained were also functional in soluble form, which makes it useful for further downstream applications. We believe that the Fab mAbs are valuable for further studies and could also contribute to improvements in the diagnosis of filariasis. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

New perspectives on recombinant human antibodies

NARCIS (Netherlands)

J. de Kruif (John); A.-R. van der Vuurst de Vries (Anne); L. Cilenti (L.); E. Boel (E.); W. van Ewijk (Willem); T. Logtenberg (Ton)

1996-01-01

textabstractThe limited potential of murine monoclonal antibodies for human immunotherapy has driven recent progress in recombinant antibody technology. Here, de Kruif and colleagues report on advances in the development and use of phage-antibody-display libraries.

Antibody therapies for lymphoma in children

NARCIS (Netherlands)

de Zwart, Verena; Gouw, Samantha C.; Meyer-Wentrup, Friederike A. G.

2016-01-01

Lymphomas are the third most common malignancy in childhood. Cure rates are high but have reached a plateau. Therefore new treatment modalities should be developed. Antibody therapy is a successful new treatment option in adult lymphoma. However, none of the therapeutic antibodies available for

Addressing safety liabilities of TfR bispecific antibodies that cross the blood-brain barrier.

Science.gov (United States)

Couch, Jessica A; Yu, Y Joy; Zhang, Yin; Tarrant, Jacqueline M; Fuji, Reina N; Meilandt, William J; Solanoy, Hilda; Tong, Raymond K; Hoyte, Kwame; Luk, Wilman; Lu, Yanmei; Gadkar, Kapil; Prabhu, Saileta; Ordonia, Benjamin A; Nguyen, Quyen; Lin, Yuwen; Lin, Zhonghua; Balazs, Mercedesz; Scearce-Levie, Kimberly; Ernst, James A; Dennis, Mark S; Watts, Ryan J

2013-05-01

Bispecific antibodies using the transferrin receptor (TfR) have shown promise for boosting antibody uptake in brain. Nevertheless, there are limited data on the therapeutic properties including safety liabilities that will enable successful development of TfR-based therapeutics. We evaluate TfR/BACE1 bispecific antibody variants in mouse and show that reducing TfR binding affinity improves not only brain uptake but also peripheral exposure and the safety profile of these antibodies. We identify and seek to address liabilities of targeting TfR with antibodies, namely, acute clinical signs and decreased circulating reticulocytes observed after dosing. By eliminating Fc effector function, we ameliorated the acute clinical signs and partially rescued a reduction in reticulocytes. Furthermore, we show that complement mediates a residual decrease in reticulocytes observed after Fc effector function is eliminated. These data raise important safety concerns and potential mitigation strategies for the development of TfR-based therapies that are designed to cross the blood-brain barrier.

Antibody Characterization Process | Office of Cancer Clinical Proteomics Research

Science.gov (United States)

The goal of the NCI's Antibody Characterization Program (ACP) is to have three monoclonal antibodies produced for each successfully expressed/purified recombinant antigen and one antibody per peptide (1 to 3 peptides per protein). To date, over 4000 clones have been screened before selecting the current 393 antibodies. They are winnowed down based on the projected end use of the antibody.

Pollination limitation to reproductive success in the Missouri evening primrose, Oenothera macrocarpa (Onagraceae).

Science.gov (United States)

Moody-Weis, J M; Heywood, J S

2001-09-01

Habitat fragmentation may result in plant populations that are less attractive to pollinators and thus susceptible to reduced reproductive output due to pollination limitation. Pollination limitation was investigated in three Missouri populations of Oenothera macrocarpa, a hawk-moth-pollinated, perennial herb. The populations represented extremes in size and habitat quality. Following supplemental pollination, mean fertilization success (proportion of ovules fertilized) across populations increased from 24.3 to 44.8% and mean seed set (proportion of ovules that matured into seed) increased from 14.7 to 27.9%. These increases were statistically significant in two of the three populations. Failure to achieve 100% fertilization and seed set following supplementation indicates that other factors, in addition to pollination, were limiting to female reproductive success. Fruit set was pollination limited in only one population. Fruits matured with as few as one seed, suggesting that fruit set was not resource limited. The degree of pollination limitation was greatest in the most disturbed population. The population located in the highest-quality habitat was not significantly pollination limited. This suggests that pollination limitation is occurring, at least in part, because of reduced pollinator activity in degraded habitats.

Frequently relapsing anti-glomerular basement membrane antibody disease with changing clinical phenotype and antibody characteristics over time

OpenAIRE

Gu, Bobby; Magil, Alex B.; Barbour, Sean J.

2016-01-01

Anti-glomerular basement membrane (GBM) antibody disease is a typically monophasic autoimmune disease with severe pulmonary and renal involvement. We report an atypical case of frequently relapsing anti-GBM antibody disease with both anti-GBM antibody?positive flares with pulmonary and renal involvement, and anti-GBM antibody?negative flares that were pulmonary limited with no histologic renal disease. This is the first report of alternating disease phenotype and anti-GBM antibody status over...

Combining Phage and Yeast Cell Surface Antibody Display to Identify Novel Cell Type-Selective Internalizing Human Monoclonal Antibodies.

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Bidlingmaier, Scott; Su, Yang; Liu, Bin

2015-01-01

Using phage antibody display, large libraries can be generated and screened to identify monoclonal antibodies with affinity for target antigens. However, while library size and diversity is an advantage of the phage display method, there is limited ability to quantitatively enrich for specific binding properties such as affinity. One way of overcoming this limitation is to combine the scale of phage display selections with the flexibility and quantitativeness of FACS-based yeast surface display selections. In this chapter we describe protocols for generating yeast surface antibody display libraries using phage antibody display selection outputs as starting material and FACS-based enrichment of target antigen-binding clones from these libraries. These methods should be widely applicable for the identification of monoclonal antibodies with specific binding properties.

Computer Aided Drug Design: Success and Limitations.

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Baig, Mohammad Hassan; Ahmad, Khurshid; Roy, Sudeep; Ashraf, Jalaluddin Mohammad; Adil, Mohd; Siddiqui, Mohammad Haris; Khan, Saif; Kamal, Mohammad Amjad; Provazník, Ivo; Choi, Inho

2016-01-01

Over the last few decades, computer-aided drug design has emerged as a powerful technique playing a crucial role in the development of new drug molecules. Structure-based drug design and ligand-based drug design are two methods commonly used in computer-aided drug design. In this article, we discuss the theory behind both methods, as well as their successful applications and limitations. To accomplish this, we reviewed structure based and ligand based virtual screening processes. Molecular dynamics simulation, which has become one of the most influential tool for prediction of the conformation of small molecules and changes in their conformation within the biological target, has also been taken into account. Finally, we discuss the principles and concepts of molecular docking, pharmacophores and other methods used in computer-aided drug design.

Successful Renal Transplantation Across HLA Barrier: Report from India.

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Aggarwal, G; Tiwari, A K; Dorwal, P; Chauhan, R; Arora, D; Dara, R C; Kher, V

2017-01-01

Organ donors are sometimes found "unsuitable" due to the presence of donor-specific anti-HLA antibodies in the recipient. In recent years, improved desensitization protocols have successfully helped to overcome HLA incompatibility hurdle. We present three cases where optimum desensitization was achieved in patients with the donor-specific anti-HLA antibody (DSA) leading to successful renal transplantation. All patient-donor pair underwent HLA typing, complement dependent cytotoxicity crossmatch (CDC-XM), flow cytometry XM (FC-XM), and panel reactive antibody. If any of the three tests was positive, single antigen bead assay was performed to determine the specificity of the anti-HLA antibody (s). Patients with DSA were offered organ-swap or anti-HLA antibody desensitization followed by transplantation. Desensitization protocol consisted of single dose rituximab and cascade plasmapheresis (CP) along with standard triple immunosuppression. The target DSA mean fluorescence index (MFI) was HLA DSA, who did not find a suitable match in organ swap program, consented to anti-HLA antibody desensitization, followed by transplantation. Mean pre-desensitization antibody MFI was 1740 (1422-2280). Mean number of CP required to achieve the target MFI was 2.3 (2-3). All the three patients are on regular follow-up and have normal renal function test at a mean follow-up of 8 months. This report underlines successful application of desensitization protocol leading to successful HLA-antibody incompatible renal transplants and their continued normal renal functions.

Successful renal transplantation across HLA barrier: Report from India

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G Aggarwal

2017-01-01

Full Text Available Organ donors are sometimes found “unsuitable” due to the presence of donor-specific anti-HLA antibodies in the recipient. In recent years, improved desensitization protocols have successfully helped to overcome HLA incompatibility hurdle. We present three cases where optimum desensitization was achieved in patients with the donor-specific anti-HLA antibody (DSA leading to successful renal transplantation. All patient–donor pair underwent HLA typing, complement dependent cytotoxicity crossmatch (CDC-XM, flow cytometry XM (FC-XM, and panel reactive antibody. If any of the three tests was positive, single antigen bead assay was performed to determine the specificity of the anti-HLA antibody (s. Patients with DSA were offered organ-swap or anti-HLA antibody desensitization followed by transplantation. Desensitization protocol consisted of single dose rituximab and cascade plasmapheresis (CP along with standard triple immunosuppression. The target DSA mean fluorescence index (MFI was <500, along with negative CDC-XM and FC-XM for both T- and B-cells. Three patients with anti-HLA DSA, who did not find a suitable match in organ swap program, consented to anti-HLA antibody desensitization, followed by transplantation. Mean pre-desensitization antibody MFI was 1740 (1422–2280. Mean number of CP required to achieve the target MFI was 2.3 (2–3. All the three patients are on regular follow-up and have normal renal function test at a mean follow-up of 8 months. This report underlines successful application of desensitization protocol leading to successful HLA-antibody incompatible renal transplants and their continued normal renal functions.

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

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

2012-06-01

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

Comparative analysis of in vivo T cell depletion with radiotherapy, combination chemotherapy, and the monoclonal antibody Campath-1G, using limiting dilution methodology

International Nuclear Information System (INIS)

Theobald, M.; Hoffmann, T.; Bunjes, D.; Heit, W.

1990-01-01

We have investigated the efficacy of standard conditioning regimens for bone marrow transplantation in depleting functional T lymphocytes in vivo and have compared it with the efficacy of the monoclonal antibody Campath-1G. Using limiting dilution techniques the frequencies of proliferating T cell precursors (PTL), cytotoxic T cell precursors (CTL-p), helper T cell precursors (HTL-p), and mature helper T cells (HTL) were determined before and after treatment. Both total body irradiation and combination chemotherapy with busulfan/cyclophosphamide were highly efficient at depleting PTL, CTL-p, and HTL-p (0-4 days) but spared HTL to a variable extent (0-99.5%). In the majority of patients treated with Campath-1G a similar degree of PTL, CTL-p, and HTL-p depletion was achieved, and, in addition, HTL were effectively removed (greater than 95.5%). These results suggest that Campath-1G could be successfully employed in depleting radio- and chemotherapy-resistant host T lymphocytes prior to T-depleted bone marrow transplantation

Baculovirus display of functional antibody Fab fragments.

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Takada, Shinya; Ogawa, Takafumi; Matsui, Kazusa; Suzuki, Tasuku; Katsuda, Tomohisa; Yamaji, Hideki

2015-08-01

The generation of a recombinant baculovirus that displays antibody Fab fragments on the surface was investigated. A recombinant baculovirus was engineered so that the heavy chain (Hc; Fd fragment) of a mouse Fab fragment was expressed as a fusion to the N-terminus of baculovirus gp64, while the light chain of the Fab fragment was simultaneously expressed as a secretory protein. Following infection of Sf9 insect cells with the recombinant baculovirus, the culture supernatant was analyzed by enzyme-linked immunosorbent assay using antigen-coated microplates and either an anti-mouse IgG or an anti-gp64 antibody. A relatively strong signal was obtained in each case, showing antigen-binding activity in the culture supernatant. In western blot analysis of the culture supernatant using the anti-gp64 antibody, specific protein bands were detected at an electrophoretic mobility that coincided with the molecular weight of the Hc-gp64 fusion protein as well as that of gp64. Flow cytometry using a fluorescein isothiocyanate-conjugated antibody specific to mouse IgG successfully detected the Fab fragments on the surface of the Sf9 cells. These results suggest that immunologically functional antibody Fab fragments can be displayed on the surface of baculovirus particles, and that a fluorescence-activated cell sorter with a fluorescence-labeled antigen can isolate baculoviruses displaying specific Fab fragments. This successful baculovirus display of antibody Fab fragments may offer a novel approach for the efficient selection of specific antibodies.

Antibodies to watch in 2014.

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Reichert, Janice M

2014-01-01

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

BRCAA1 antibody- and Her2 antibody-conjugated amphiphilic polymer engineered CdSe/ZnS quantum dots for targeted imaging of gastric cancer

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Li, Chao; Ji, Yang; Wang, Can; Liang, Shujing; Pan, Fei; Zhang, Chunlei; Chen, Feng; Fu, Hualin; Wang, Kan; Cui, Daxiang

2014-05-01

Successful development of safe and highly effective nanoprobes for targeted imaging of in vivo early gastric cancer is a great challenge. Herein, we choose the CdSe/ZnS (core-shell) quantum dots (QDs) as prototypical materials, synthesized one kind of a new amphiphilic polymer including dentate-like alkyl chains and multiple carboxyl groups, and then used the prepared amphiphilic polymer to modify QDs. The resultant amphiphilic polymer engineered QDs (PQDs) were conjugated with BRCAA1 and Her2 monoclonal antibody, and prepared BRCAA1 antibody- and Her2 antibody-conjugated QDs were used for in vitro MGC803 cell labeling and in vivo targeted imaging of gastric cancer cells. Results showed that the PQDs exhibited good water solubility, strong photoluminescence (PL) intensity, and good biocompatibility. BRCAA1 antibody- and Her2 antibody-conjugated QD nanoprobes successfully realized targeted imaging of in vivo gastric cancer MGC803 cells. In conclusion, BRCAA1 antibody- and Her2 antibody-conjugated PQDs have great potential in applications such as single cell labeling and in vivo tracking, and targeted imaging and therapeutic effects' evaluation of in vivo early gastric cancer cells in the near future.

Antibodies Targeting EMT

Science.gov (United States)

2017-10-01

these unusual antibodies can effectively be displayed on the cell surface. 5 Additionally, we successfully prepared cDNA from lymphocytes derived...from cow peripheral blood, spleen, and lymph nodes, amplified this cDNA by PCR with VH gene specific primers, and this “library” has been cloned into

RosettaAntibodyDesign (RAbD): A general framework for computational antibody design.

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Adolf-Bryfogle, Jared; Kalyuzhniy, Oleks; Kubitz, Michael; Weitzner, Brian D; Hu, Xiaozhen; Adachi, Yumiko; Schief, William R; Dunbrack, Roland L

2018-04-01

A structural-bioinformatics-based computational methodology and framework have been developed for the design of antibodies to targets of interest. RosettaAntibodyDesign (RAbD) samples the diverse sequence, structure, and binding space of an antibody to an antigen in highly customizable protocols for the design of antibodies in a broad range of applications. The program samples antibody sequences and structures by grafting structures from a widely accepted set of the canonical clusters of CDRs (North et al., J. Mol. Biol., 406:228-256, 2011). It then performs sequence design according to amino acid sequence profiles of each cluster, and samples CDR backbones using a flexible-backbone design protocol incorporating cluster-based CDR constraints. Starting from an existing experimental or computationally modeled antigen-antibody structure, RAbD can be used to redesign a single CDR or multiple CDRs with loops of different length, conformation, and sequence. We rigorously benchmarked RAbD on a set of 60 diverse antibody-antigen complexes, using two design strategies-optimizing total Rosetta energy and optimizing interface energy alone. We utilized two novel metrics for measuring success in computational protein design. The design risk ratio (DRR) is equal to the frequency of recovery of native CDR lengths and clusters divided by the frequency of sampling of those features during the Monte Carlo design procedure. Ratios greater than 1.0 indicate that the design process is picking out the native more frequently than expected from their sampled rate. We achieved DRRs for the non-H3 CDRs of between 2.4 and 4.0. The antigen risk ratio (ARR) is the ratio of frequencies of the native amino acid types, CDR lengths, and clusters in the output decoys for simulations performed in the presence and absence of the antigen. For CDRs, we achieved cluster ARRs as high as 2.5 for L1 and 1.5 for H2. For sequence design simulations without CDR grafting, the overall recovery for the native

Anti-dog IgG secondary antibody successfully detects IgG in a variety of aquatic mammals

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Roehl, Katherine; Jankowski, Mark D.; Hofmeister, Erik K.

2016-01-01

Serological tests play an important role in the detection of wildlife diseases. However, while there are many commercial assays and reagents available for domestic species, there is a need to develop efficient serological assays for wildlife. In recent years, marine mammals have represented a wildlife group with emerging infectious diseases, such as influenza, brucellosis, and leptospirosis. However, with the exception of disease-agent-specific assays or functional assays, few reports describe the use of antibody detection assays in marine mammals. In an indirect enzyme-linked immunoassay (EIA) or an immunofluorescence assay, antibody is detected using an antitarget species secondary conjugated antibody. The sensitivity of the assay depends on the avidity of the binding reaction between the bound antibody and the detection antibody. A commercial polyclonal antidog IgG conjugated antibody was tested in an EIA for its ability to sensitively detect the IgG of seven marine mammals including sea otter (Enhydra lutris), polar bear (Ursus maritimus), grey seal (Halichoerus grypus), harbor seal (Phoca vitulina), northern elephant seal (Mirounga angustirostris), California sea lion (Zalophus californianus), Pacific walrus (Odobenus rosmarus) and one freshwater mammal: Asian small-clawed otter (Aonyx cinerea). With the exception of Asian small-clawed sea otters, the detection of IgG in these marine mammals either exceeded or was nearly equal to detection of dog IgG. The use of the tested commercial antidog IgG antibody may be a valid approach to the detection of antibody response to disease in sea mammals.

Novel Antibody-Based Proteins for Cancer Immunotherapy

Energy Technology Data Exchange (ETDEWEB)

Fuenmayor, Jaheli; Montaño, Ramon F., E-mail: jfuenmay@ivic.gob.ve [Laboratorio de Patología Celular y Molecular, Centro de Medicina Experimental, Instituto Venezolano de Investigaciones Científicas. Caracas, 1020-A (Venezuela, Bolivarian Republic of)

2011-08-19

The relative success of monoclonal antibodies in cancer immunotherapy and the vast manipulation potential of recombinant antibody technology have encouraged the development of novel antibody-based antitumor proteins. Many insightful reagents have been produced, mainly guided by studies on the mechanisms of action associated with complete and durable remissions, results from experimental animal models, and our current knowledge of the human immune system. Strikingly, only a small percent of these new reagents has demonstrated clinical value. Tumor burden, immune evasion, physiological resemblance, and cell plasticity are among the challenges that cancer therapy faces, and a number of antibody-based proteins are already available to deal with many of them. Some of these novel reagents have been shown to specifically increase apoptosis/cell death of tumor cells, recruit and activate immune effectors, and reveal synergistic effects not previously envisioned. In this review, we look into different approaches that have been followed during the past few years to produce these biologics and analyze their relative success, mainly in terms of their clinical performance. The use of antibody-based antitumor proteins, in combination with standard or novel therapies, is showing significant improvements in objective responses, suggesting that these reagents will become important components of the antineoplastic protocols of the future.

Use of monoclonal antibodies against Hendra and Nipah viruses in an antigen capture ELISA

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Spiropoulou Christina F

2010-06-01

Full Text Available Abstract Background Outbreaks of Hendra (HeV and Nipah (NiV viruses have been reported starting in 1994 and 1998, respectively. Both viruses are capable of causing fatal disease in humans and effecting great economical loss in the livestock industry. Results Through screening of hybridomas derived from mice immunized with γ-irradiated Nipah virus, we identified two secreted antibodies; one reactive with the nucleocapsid (N protein and the other, the phosphoprotein (P of henipaviruses. Epitope mapping and protein sequence alignments between NiV and HeV suggest the last 14 amino acids of the carboxyl terminus of the N protein is the target of the anti-N antibody. The anti-P antibody recognizes an epitope in the amino-terminal half of P protein. These monoclonal antibodies were used to develop two antigen capture ELISAs, one for virus detection and the other for differentiation between NiV and HeV. The lower limit of detection of the capture assay with both monoclonal antibodies was 400 pfu. The anti-N antibody was used to successfully detect NiV in a lung tissue suspension from an infected pig. Conclusion The antigen capture ELISA developed is potentially affordable tool to provide rapid detection and differentiation between the henipaviruses.

Overcoming the Constraints of Anti-HIV/CD89 Bispecific Antibodies That Limit Viral Inhibition

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Xiaocong Yu

2016-01-01

Full Text Available Innovative strategies are necessary to maximize the clinical application of HIV neutralizing antibodies. To this end, bispecific constructs of human antibody F240, reactive with well-conserved gp41 epitope and antibody 14A8, reactive with the IgA receptor (CD89 on effector cells, were constructed. A F240 × 14A8 bispecific single chain variable region (scFv molecule was constructed by linking two scFvs using a conventional GGGGS linker. Despite immunoreactivity with HIV gp41 and neutrophils, this bispecific scFv failed to inhibit HIV infection. This is in sharp contrast to viral inhibition using a chemical conjugate of the Fab of these two antibodies. Therefore, we constructed two novel Fab-like bispecific antibody molecules centered on fusion of the IgG1 CH1 domain or CH1-hinge domain to the C-terminus of F240scFv and fusion of the kappa chain CL domain to the C-terminus of 14A8scFv. Both Bi-Fab antibodies showed significant ADCVI activity for multiple clade B and clade C isolates by arming the neutrophils to inhibit HIV infection. The approach presented in this study is unique for HIV immunotherapy in that the impetus of neutralization is to arm and mobilize PMN to destroy HIV and HIV infected cells.

Genetically engineered multivalent single chain antibody constructs for cancer therapy

International Nuclear Information System (INIS)

Surinder Batra

2006-01-01

Current therapeutic approaches against the advanced stages of human solid tumors are palliative rather than curative. Many modalities, including, surgery, radiation, and chemotherapy, either alone or in combination have met with only modest success for advanced metastatic cancers. Radioimmunotherapy (RIT) combines the specificity of monoclonal antibodies with cytotoxic effects of radioisotopes. It is the ''smart'' way of delivering radiation to the known and occult metastatic cancer cells and is independent of drug toxicity and/or hormone resistance. The tumor associated glycoprotein-72 (TAG-72) containing the unique disaccharide sialyl-Tn, is highly expressed in majority of adenocarcinomas, including carcinomas of the prostate, breast, ovaries, pancreas and colon (80-90%) compared to undetectable expression in normal tissues. Monoclonal antibody CC49, reactive with TAG-72, after conjugation to potent gamma- and beta-emitting radionuclides, has been useful in selective systemic radiolocalization of disease and therapy of primary and metastatic tumor sites. However, limited therapeutic responses were observed in patients. Limited success of antibody based delivery of radioisotopes can be attributed to several factors including undesirable pharmacokinetics, poor tumor uptake and high immunogenicity of intact antibodies (IgGs). The primary factors contributing towards the failure of RIT include: (1) longer serum half-lives of the intact IgG molecules resulting in the radiotoxicity, (2) generation of human antibodies against murine antibodies (HAMA) that limits the frequency of dose administration, (3) poor diffusion rates of intact IgG due to the large size and (4) high interstitial fluid pressures (IFP) encountered in solid tumors. The major goal of our multidisciplinary project was to develop specific novel radiopharmaceuticals, with desired pharmacokinetics, for the diagnosis and therapy of solid tumors. To overcome the low uptake of radioactivity by tumors and to

[International classification of various types of monoclonal antibodies].

Science.gov (United States)

Scheen, A J

2009-01-01

Significant advances in the development of monoclonal antibodies ("mabs") have been acknowledged during the last two decades. Successive developments led to the marketing of murine antibodies ("o-mab" first, followed by chimeric antibodies ("xi-mab"), humanised antibodies ("zu-mab") and, finally, human monoclonal antibodies ("u-mab"). In order to facilitate the distinction between the various monoclonal antibodies used in clinical practice, an international nomenclature has been proposed with the use of a specific suffix corresponding to the origine/source of "mabs" preceded by an infix referring to the medicine's target. The efforts in developing new types of monoclonal antibodies aimed at improving their pharmacokinetics (longer half-life), pharmacodynamics (better efficacy because of stronger affinity to human receptor), and safety profile (less antigenic and immunogenic reactions). These progresses could be obtained thanks to the remarkable development of molecular biotechnology.

Microbial platform technology for recombinant antibody fragment production: A review.

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Gupta, Sanjeev Kumar; Shukla, Pratyoosh

2017-02-01

Recombinant antibody fragments are being used for the last few years as an important therapeutic protein to cure various critical and life threatening human diseases. Several expression platforms now days employed for the production of these recombinant fragments, out of which bacterial system has emerged a promising host for higher expression. Since, a small antibody fragment unlike full antibody does not require human-like post-translational modification therefore it is potentially expressed in prokaryotic production system. Recently, small antibody fragments such as scFvs (single-chain variable fragments) and Fabs (antibody fragments) which does not require glycosylation are successfully produced in bacteria and have commercially launched for therapeutic use as these fragments shows better tissue penetration and less immunogenic to human body compared to full-size antibody. Recently developed Wacker's ESETEC secretion technology is an efficient technology for the expression and secretion of the antibody fragment (Fab) exceeded up to 4.0 g/L while scFv up to 3.5 g/L into the fermentation broth. The Pfenex system and pOP prokaryotic expression vector are another platform used for the considerably good amount of antibody fragment production successfully. In this review, we summarize the recent progress on various expression platforms and cloning approaches for the production of different forms of antibody fragments in E. coli.

Tubulin Inhibitor-Based Antibody-Drug Conjugates for Cancer Therapy

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Hao Chen

2017-08-01

Full Text Available Antibody-drug conjugates (ADCs are a class of highly potent biopharmaceutical drugs generated by conjugating cytotoxic drugs with specific monoclonal antibodies through appropriate linkers. Specific antibodies used to guide potent warheads to tumor tissues can effectively reduce undesired side effects of the cytotoxic drugs. An in-depth understanding of antibodies, linkers, conjugation strategies, cytotoxic drugs, and their molecular targets has led to the successful development of several approved ADCs. These ADCs are powerful therapeutics for cancer treatment, enabling wider therapeutic windows, improved pharmacokinetic/pharmacodynamic properties, and enhanced efficacy. Since tubulin inhibitors are one of the most successful cytotoxic drugs in the ADC armamentarium, this review focuses on the progress in tubulin inhibitor-based ADCs, as well as lessons learned from the unsuccessful ADCs containing tubulin inhibitors. This review should be helpful to facilitate future development of new generations of tubulin inhibitor-based ADCs for cancer therapy.

Antibody-nanoparticle conjugates to enhance the sensitivity of ELISA-based detection methods.

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Margaret M Billingsley

Full Text Available Accurate antigen detection is imperative for clinicians to diagnose disease, assess treatment success, and predict patient prognosis. The most common technique used for the detection of disease-associated biomarkers is the enzyme linked immunosorbent assay (ELISA. In an ELISA, primary antibodies are incubated with biological samples containing the biomarker of interest. Then, detectible secondary antibodies conjugated with horseradish peroxidase (HRP bind the primary antibodies. Upon addition of a color-changing substrate, the samples provide a colorimetric signal that directly correlates to the targeted biomarker concentration. While ELISAs are effective for analyzing samples with high biomarker content, they lack the sensitivity required to analyze samples with low antigen levels. We hypothesized that the sensitivity of ELISAs could be enhanced by replacing freely delivered primary antibodies with antibody-nanoparticle conjugates that provide excess binding sites for detectible secondary antibodies, ultimately leading to increased signal. Here, we investigated the use of nanoshells (NS decorated with antibodies specific to epidermal growth factor receptor (EGFR as a model system (EGFR-NS. We incubated one healthy and two breast cancer cell lines, each expressing different levels of EGFR, with EGFR-NS, untargeted NS, or unconjugated EGFR antibodies, as well as detectable secondary antibodies. We found that EGFR-NS consistently increased signal intensity relative to unconjugated EGFR antibodies, with a substantial 13-fold enhancement from cells expressing high levels of EGFR. Additionally, 40x more unconjugated antibodies were required to detect EGFR compared to those conjugated to NS. Our results demonstrate that antibody-nanoparticle conjugates lower the detection limit of traditional ELISAs and support further investigation of this strategy with other antibodies and nanoparticles. Owing to their enhanced sensitivity, we anticipate that

Basics of Antibody Phage Display Technology.

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Ledsgaard, Line; Kilstrup, Mogens; Karatt-Vellatt, Aneesh; McCafferty, John; Laustsen, Andreas H

2018-06-09

Antibody discovery has become increasingly important in almost all areas of modern medicine. Different antibody discovery approaches exist, but one that has gained increasing interest in the field of toxinology and antivenom research is phage display technology. In this review, the lifecycle of the M13 phage and the basics of phage display technology are presented together with important factors influencing the success rates of phage display experiments. Moreover, the pros and cons of different antigen display methods and the use of naïve versus immunized phage display antibody libraries is discussed, and selected examples from the field of antivenom research are highlighted. This review thus provides in-depth knowledge on the principles and use of phage display technology with a special focus on discovery of antibodies that target animal toxins.

Generation of HER2 monoclonal antibodies using epitopes of a rabbit polyclonal antibody.

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Hu, Francis Jingxin; Uhlen, Mathias; Rockberg, Johan

2014-01-25

One of the issues in using polyclonal antibodies is the limited amount of reagent available from an immunisation, leading to batch-to-batch variation and difficulties in obtaining the same antibody performance when the same antigen is re-immunised into several separate animals. This led to the development of hybridoma technology allowing, at least theoretically, for an unlimited production of a specific binder. Nevertheless, polyclonal antibodies are widely used in research and diagnostics and there exists a need for robust methods to convert a polyclonal antibody with good binding performance into a renewable monoclonal with identical or similar binding specificity. Here we have used precise information regarding the functional recognition sequence (epitope) of a rabbit polyclonal antibody with attractive binding characteristics as the basis for generation of a renewable mouse monoclonal antibody. First, the original protein fragment antigen was used for immunisation and generation of mouse hybridoma, without obtaining binders to the same epitope region. Instead a peptide designed using the functional epitope and structural information was synthesised and used for hybridoma production. Several of the monoclonal antibodies generated were found to have similar binding characteristics to those of the original polyclonal antibody. These monoclonal antibodies detected native HER2 on cell lines and were also able to stain HER2 in immunohistochemistry using xenografted mice, as well as human normal and cancer tissues. Copyright © 2013 Elsevier B.V. All rights reserved.

Identification of anti-CD98 antibody mimotopes for inducing antibodies with antitumor activity by mimotope immunization.

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Saito, Misa; Kondo, Masahiro; Ohshima, Motohiro; Deguchi, Kazuki; Hayashi, Hideki; Inoue, Kazuyuki; Tsuji, Daiki; Masuko, Takashi; Itoh, Kunihiko

2014-04-01

A mimotope is an antibody-epitope-mimicking peptide retrieved from a phage display random peptide library. Immunization with antitumor antibody-derived mimotopes is promising for inducing antitumor immunity in hosts. In this study, we isolated linear and constrained mimotopes from HBJ127, a tumor-suppressing anti-CD98 heavy chain mAb, and determined their abilities for induction of antitumor activity equal to that of the parent antibody. We detected elevated levels of antipeptide responses, but failed to detect reactivity against native CD98-expressing HeLa cells in sera of immunized mice. Phage display panning and selection of mimotope-immunized mouse spleen-derived antibody Fab library showed that HeLa cell-reactive Fabs were successfully retrieved from the library. This finding indicates that native antigen-reactive Fab clones represented an undetectable minor population in mimotope-induced antibody repertoire. Functional and structural analysis of retrieved Fab clones revealed that they were almost identical to the parent antibody. From these results, we confirmed that mimotope immunization was promising for retrieving antitumor antibodies equivalent to the parent antibody, although the co-administration of adjuvant compounds such as T-cell epitope peptides and Toll-like receptor 4 agonist peptides is likely to be necessary for inducing stronger antitumor immunity than mimotope injection alone. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

Tumour auto-antibody screening: performance of protein microarrays using SEREX derived antigens

International Nuclear Information System (INIS)

Stempfer, René; Weinhäusel, Andreas; Syed, Parvez; Vierlinger, Klemens; Pichler, Rudolf; Meese, Eckart; Leidinger, Petra; Ludwig, Nicole; Kriegner, Albert; Nöhammer, Christa

2010-01-01

The simplicity and potential of minimal invasive testing using serum from patients make auto-antibody based biomarkers a very promising tool for use in diagnostics of cancer and auto-immune disease. Although several methods exist for elucidating candidate-protein markers, immobilizing these onto membranes and generating so called macroarrays is of limited use for marker validation. Especially when several hundred samples have to be analysed, microarrays could serve as a good alternative since processing macro membranes is cumbersome and reproducibility of results is moderate. Candidate markers identified by SEREX (serological identification of antigens by recombinant expression cloning) screenings of brain and lung tumour were used for macroarray and microarray production. For microarray production recombinant proteins were expressed in E. coli by autoinduction and purified His-tag (histidine-tagged) proteins were then used for the production of protein microarrays. Protein arrays were hybridized with the serum samples from brain and lung tumour patients. Methods for the generation of microarrays were successfully established when using antigens derived from membrane-based selection. Signal patterns obtained by microarrays analysis of brain and lung tumour patients' sera were highly reproducible (R = 0.92-0.96). This provides the technical foundation for diagnostic applications on the basis of auto-antibody patterns. In this limited test set, the assay provided high reproducibility and a broad dynamic range to classify all brain and lung samples correctly. Protein microarray is an efficient means for auto-antibody-based detection when using SEREX-derived clones expressing antigenic proteins. Protein microarrays are preferred to macroarrays due to the easier handling and the high reproducibility of auto-antibody testing. Especially when using only a few microliters of patient samples protein microarrays are ideally suited for validation of auto-antibody

Kinetics of circulating antibody response to Trichomonas vaginalis: clinical and diagnostic implications.

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Ton Nu, Phuong Anh; Rappelli, Paola; Dessì, Daniele; Nguyen, Vu Quoc Huy; Fiori, Pier Luigi

2015-12-01

Persistence of antibodies against pathogens after antimicrobial treatment is a marker of therapy failure or evolution to a chronic infection. The kinetics of antibody production decrease following antigen elimination is highly variable, and predicting the duration of soluble immunity in infectious diseases is often impossible. This hampers the development and use of immunoassays for diagnostic and seroepidemiological purposes. In the case of Trichomonas vaginalis infection, the kinetics of antibody levels decrease following therapy has never been studied. We thus investigated the clearance of circulating anti-T. vaginalis IgGs after pharmacological treatment in patients affected by trichomoniasis. 18 female patients affected by acute trichomoniasis were enrolled in this study. After metronidazole therapy administration, subjects were followed up monthly up to 5 months, and serum levels of anti-T. vaginalis IgGs were measured by ELISA. We showed that a successful therapy is characterised by a relatively fast decline of specific antibodies, until turning into negative by ELISA in 1-3 months. In a few patients we observed that the persistence of anti-T. vaginalis antibodies was associated with an evolution to chronic infection, which may be due to treatment failure or to reinfection by untreated sexual partners. Our results describe the direct correlation between the decline of a specific humoral anti-T. vaginalis response and an effective antimicrobial therapy. These findings may facilitate the follow-up approach to circumvent limitations in developing new diagnostic tools and techniques routinely used in microbiology laboratories to assess the presence of T. vaginalis in clinical samples. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Radiolabeled monoclonal antibodies: a review

International Nuclear Information System (INIS)

Toledo e Souza, I.T. de; Okada, H.

1990-05-01

Since the description by Kohler and Milstein 1975 of their technique for producing monoclonal antibodies of predefined specificity, it has become a mainstay in most laboratories that utilize immunochemical techniques to study problems in basic, applied or clinical research. Paradoxically, the very success of monoclonal antibodies has generated a literature which is now so vast and scattered that it has become difficult to obtain a perspective. This brief review represents the distillation of many publications relating to the production and use of monoclonaal antibodies as radiopharmaceuticals. Significant advances were made possible in the last few years by combined developments in the fields of tumor-associated antigens and of monoclonal antibodies. In fact monoclonal antibodies against some well defined tumor-associated antigens, has led to significantly greater practical possibilities for producing highly specific radiolabeled antibodies as radiopharmaceuticals for diagnosis and therapy of human tumors. One of the main requirements of this methodology is the availability of stable radiopharmaceutical reagents which after labeling in vivo injection retain the capacity of specific interaction with the defined antigen and their molecular integrity. Since injection into human is the objetive of this kind of study all the specifications of radiopharmaceutical have to be fulfilled e.g. sterility, apirogenicity and absence of toxicity. (author) [pt

Analysis of anti-HLA antibodies in sensitized kidney transplant candidates subjected to desensitization with intravenous immunoglobulin and rituximab.

Science.gov (United States)

Lobashevsky, Andrew L; Higgins, Nancy G; Rosner, Kevin M; Mujtaba, Muhammad A; Goggins, William C; Taber, Tim E

2013-07-27

Preexisting donor-specific antibodies against human leukocyte antigens are major risk factors for acute antibody-mediated and chronic rejection of kidney transplant grafts. Immunomodulation (desensitization) protocols may reduce antibody concentration and improve the success of transplant. We investigated the effect of desensitization with intravenous immunoglobulin and rituximab on the antibody profile in highly sensitized kidney transplant candidates. In 31 transplant candidates (calculated panel-reactive antibody [cPRA], 34%-99%), desensitization included intravenous immunoglobulin on days 0 and 30 and a single dose of rituximab on day 15. Anti-human leukocyte antigen antibodies were analyzed before and after desensitization. Reduction of cPRA from 25% to 50% was noted for anti-class I (5 patients, within 20-60 days) and anti-class II (3 patients, within 10-20 days) antibodies. After initial reduction of cPRA, the cPRA increased within 120 days. In 24 patients, decrease in mean fluorescence intensity of antibodies by more than 50% was noted at follow-up, but there was no reduction of cPRA. Rebound occurred in 65% patients for anti-class I antibodies at 350 days and anti-class II antibodies at 101 to 200 days. Probability of rebound effect was higher in patients with mean fluorescence intensity of more than 10,700 before desensitization, anti-class II antibodies, and history of previous transplant. The desensitization protocol had limited efficacy in highly sensitized kidney transplant candidate because of the short period with antibody reduction and high frequency of rebound effect.

Reshaping Human Antibodies: Grafting an Antilysozyme Activity

Science.gov (United States)

Verhoeyen, Martine; Milstein, Cesar; Winter, Greg

1988-03-01

The production of therapeutic human monoclonal antibodies by hybridoma technology has proved difficult, and this has prompted the ``humanizing'' of mouse monoclonal antibodies by recombinant DNA techniques. It was shown previously that the binding site for a small hapten could be grafted from the heavy-chain variable domain of a mouse antibody to that of a human myeloma protein by transplanting the hypervariable loops. It is now shown that a large binding site for a protein antigen (lysozyme) can also be transplanted from mouse to human heavy chain. The success of such constructions may be facilitated by an induced-fit mechanism.

Monoclonal antibodies for treating cancer

International Nuclear Information System (INIS)

Dillman, R.O.

1989-01-01

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

Successful bone marrow transplantation in sensitized recipients

International Nuclear Information System (INIS)

Levey, R.H.; Parkman, J.; Rappeport, J.; Nathan, D.G.; Rosen, F.

1979-01-01

Fourteen patients with aplastic anemia and one with the Wiskott-Aldrich syndrome who were specifically sensitized against their donors were successfully engrafted with bone marrow from those donors. Sensitivity was detected in antibody-independent and antibody-dependent cell-mediated lysis assays. In order to erase this immunity to non-MHR familial transplantation antigens, multiagent immunosuppression with cyclophosphamide, procarbazine, and whole rabbit antithymocyte serum (ATS) was used. The data suggest that ATS was largely responsible for abrogation of this sensitivity and indicate that immunity does not represent a barrier to successful transplantation

RosettaAntibodyDesign (RAbD): A general framework for computational antibody design

Science.gov (United States)

Adolf-Bryfogle, Jared; Kalyuzhniy, Oleks; Kubitz, Michael; Hu, Xiaozhen; Adachi, Yumiko; Schief, William R.

2018-01-01

A structural-bioinformatics-based computational methodology and framework have been developed for the design of antibodies to targets of interest. RosettaAntibodyDesign (RAbD) samples the diverse sequence, structure, and binding space of an antibody to an antigen in highly customizable protocols for the design of antibodies in a broad range of applications. The program samples antibody sequences and structures by grafting structures from a widely accepted set of the canonical clusters of CDRs (North et al., J. Mol. Biol., 406:228–256, 2011). It then performs sequence design according to amino acid sequence profiles of each cluster, and samples CDR backbones using a flexible-backbone design protocol incorporating cluster-based CDR constraints. Starting from an existing experimental or computationally modeled antigen-antibody structure, RAbD can be used to redesign a single CDR or multiple CDRs with loops of different length, conformation, and sequence. We rigorously benchmarked RAbD on a set of 60 diverse antibody–antigen complexes, using two design strategies—optimizing total Rosetta energy and optimizing interface energy alone. We utilized two novel metrics for measuring success in computational protein design. The design risk ratio (DRR) is equal to the frequency of recovery of native CDR lengths and clusters divided by the frequency of sampling of those features during the Monte Carlo design procedure. Ratios greater than 1.0 indicate that the design process is picking out the native more frequently than expected from their sampled rate. We achieved DRRs for the non-H3 CDRs of between 2.4 and 4.0. The antigen risk ratio (ARR) is the ratio of frequencies of the native amino acid types, CDR lengths, and clusters in the output decoys for simulations performed in the presence and absence of the antigen. For CDRs, we achieved cluster ARRs as high as 2.5 for L1 and 1.5 for H2. For sequence design simulations without CDR grafting, the overall recovery for the

Antibody-mediated rejection in kidney transplantation: a review of pathophysiology, diagnosis, and treatment options.

Science.gov (United States)

Kim, Miae; Martin, Spencer T; Townsend, Keri R; Gabardi, Steven

2014-07-01

Antibody-mediated rejection (AMR), also known as B-cell-mediated or humoral rejection, is a significant complication after kidney transplantation that carries a poor prognosis. Although fewer than 10% of kidney transplant patients experience AMR, as many as 30% of these patients experience graft loss as a consequence. Although AMR is mediated by antibodies against an allograft and results in histologic changes in allograft vasculature that differ from cellular rejection, it has not been recognized as a separate disease process until recently. With an improved understanding about the importance of the development of antibodies against allografts as well as complement activation, significant advances have occurred in the treatment of AMR. The standard of care for AMR includes plasmapheresis and intravenous immunoglobulin that remove and neutralize antibodies, respectively. Agents targeting B cells (rituximab and alemtuzumab), plasma cells (bortezomib), and the complement system (eculizumab) have also been used successfully to treat AMR in kidney transplant recipients. However, the high cost of these medications, their use for unlabeled indications, and a lack of prospective studies evaluating their efficacy and safety limit the routine use of these agents in the treatment of AMR in kidney transplant recipients. © 2014 Pharmacotherapy Publications, Inc.

The development and applications of polyclonal and monoclonal antibodies for the detection of illicit drugs in saliva samples

OpenAIRE

Fanning, Lorna M.

2002-01-01

Anti-tetrahydrocannabinol (THC), anti-cocaine and anti-morphine polyclonal antibodies were produced. These antibodies were successfully applied to an ELISA format for the detection of THC, cocaine, and morphine in saliva samples. Monoclonal antibodies against amphetamine and its derivatives were produced using two different conjugates, amphetamine-bovine serum albumin and methamphetaminebovine serum albumin. Two successful clones were produced, and the antibodies were applied to an ELISA ...

Exploration of novel strategies to enhance monoclonal antibodies targeting

International Nuclear Information System (INIS)

Khawli, L.A.; Epstein, A.L.

1997-01-01

This paper highlights the major obstacles and prospects of antibody targeting for the radio imaging and therapy of human malignant lymphomas and more challenging solid tumors. To improve the therapeutic potential of monoclonal antibodies, the authors have focused their attention on the development of new and successful methods to augment antibody uptake in the tumor. These approaches include the use of radiolabeled streptavidin to target biotinylated monoclonal antibodies already bound to tumor, pretreatment with vasoactive immunoconjugates, and the use of chemically modified antibodies. Because of the promising preclinical data obtained with these three newer approaches, plans are underway to test them in the clinic. More generally, these approaches are applicable to the use of other monoclonal antibody/tumor systems for the diagnosis and therapy of human cancers and related diseases

Insecticide-treated bed nets reduce plasma antibody levels and limit the repertoire of antibodies to Plasmodium falciparum variant surface antigens

DEFF Research Database (Denmark)

Askjaer, N; Maxwell, C; Chambo, W

2001-01-01

The use of insecticide-treated bed nets (ITN) has been documented to reduce malaria morbidity and mortality in areas with endemic malaria, but concerns have been raised that ITN usage could affect the acquisition of malaria immunity. Several lines of evidence have indicated that antibodies against...... variant surface antigens (VSA) are important in the development of naturally acquired immunity to Plasmodium falciparum malaria and may thus be good indicators of immune status. We have compared the levels of VSA antibodies in plasma from children who have used ITN for 4 years to levels in plasma from...

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

International Nuclear Information System (INIS)

Reske, S.N.; Buell, U.

1990-01-01

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

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...

Boosting antibody developability through rational sequence optimization.

Science.gov (United States)

Seeliger, Daniel; Schulz, Patrick; Litzenburger, Tobias; Spitz, Julia; Hoerer, Stefan; Blech, Michaela; Enenkel, Barbara; Studts, Joey M; Garidel, Patrick; Karow, Anne R

2015-01-01

The application of monoclonal antibodies as commercial therapeutics poses substantial demands on stability and properties of an antibody. Therapeutic molecules that exhibit favorable properties increase the success rate in development. However, it is not yet fully understood how the protein sequences of an antibody translates into favorable in vitro molecule properties. In this work, computational design strategies based on heuristic sequence analysis were used to systematically modify an antibody that exhibited a tendency to precipitation in vitro. The resulting series of closely related antibodies showed improved stability as assessed by biophysical methods and long-term stability experiments. As a notable observation, expression levels also improved in comparison with the wild-type candidate. The methods employed to optimize the protein sequences, as well as the biophysical data used to determine the effect on stability under conditions commonly used in the formulation of therapeutic proteins, are described. Together, the experimental and computational data led to consistent conclusions regarding the effect of the introduced mutations. Our approach exemplifies how computational methods can be used to guide antibody optimization for increased stability.

Purpose-Oriented Antibody Libraries Incorporating Tailored CDR3 Sequences

OpenAIRE

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

2015-01-01

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

Antiphospholipid Antibody Titers and Clinical Outcomes in Patients with Recurrent Miscarriage and Antiphospholipid Antibody Syndrome: A Prospective Study

Science.gov (United States)

Song, Yu; Wang, Hai-Yan; Qiao, Jie; Liu, Ping; Chi, Hong-Bin

2017-01-01

Background: The management of patients with recurrent miscarriage (RM) and antiphospholipid antibody syndrome (APS) includes prolonged treatment with heparin and aspirin, starting from the confirmation of pregnancy and continuing until 6 weeks after birth. This study was conducted to determine the relationship between changes in antiphospholipid antibody titers and clinical outcomes. The effect of a shortened treatment regimen was also evaluated. Methods: A prospective study of 123 patients with RM and APS between March 2012 and May 2014 was conducted. Patients were pretreated with a low dose of prednisone plus aspirin before pregnancy, and heparin was added after conception. The levels of antiphospholipid antibodies and pregnancy outcomes were evaluated. Results: All patients were positive for anti-β2-glycoprotein 1 (anti-β2-GP1) IgM. After prepregnancy treatment with low-dose prednisone plus aspirin, 99 of 123 patients became pregnant, and 87 of those pregnancies resulted in successful live births, while 12 resulted in miscarriage, showing a success rate of 87.9%. In the live birth group, levels of anti-β2-GP1 were 56.8 ± 49.0 RU/ml before the pretreatment regimen, 32.1 ± 26.0 RU/ml after 2 months of pretreatment, and 24.1 ± 23.1 RU/ml during early pregnancy (P antiphospholipid antibody titers were 52.8 ± 30.7 RU/ml before pretreatment, 38.5 ± 34.2 RU/ml after pretreatment, and 33.9 ± 24.7 RU/ml during early pregnancy; the decrease in antiphospholipid antibodies was lower in the miscarriage group than in the live birth group (P antiphospholipid antibody titers correlated with better pregnancy outcomes. The shorter treatment regimen was effective and economical. PMID:28139508

Monoclonal Antibodies for Non-Hodgkin's Lymphoma: State of the Art and Perspectives

Directory of Open Access Journals (Sweden)

Giulia Motta

2010-01-01

Full Text Available Monoclonal antibodies have been the most successful therapeutics ever brought to cancer treatment by immune technologies. The use of monoclonal antibodies in B-cell Non-Hodgkin's lymphomas (NHL represents the greatest example of these advances, as the introduction of the anti-CD20 antibody rituximab has had a dramatic impact on how we treat this group of diseases today. Despite this success, several questions about how to optimize the use of monoclonal antibodies in NHL remain open. The best administration schedules, as well as the optimal duration of rituximab treatment, have yet to be determined. A deeper knowledge of the mechanisms underlying resistance to rituximab is also necessary in order to improve the activity of this and of similar therapeutics. Finally, new antibodies and biological agents are entering the scene and their advantages over rituximab will have to be assessed. We will discuss these issues and present an overview of the most significant clinical studies with monoclonal antibodies for NHL treatment carried out to date.

Generation of monoclonal antibodies against highly conserved antigens.

Directory of Open Access Journals (Sweden)

Hongzhe Zhou

Full Text Available BACKGROUND: Therapeutic antibody development is one of the fastest growing areas of the pharmaceutical industry. Generating high-quality monoclonal antibodies against a given therapeutic target is very crucial for the success of the drug development. However, due to immune tolerance, some proteins that are highly conserved between mice and humans are not very immunogenic in mice, making it difficult to generate antibodies using a conventional approach. METHODOLOGY/PRINCIPAL FINDINGS: In this report, the impaired immune tolerance of NZB/W mice was exploited to generate monoclonal antibodies against highly conserved or self-antigens. Using two highly conserved human antigens (MIF and HMGB1 and one mouse self-antigen (TNF-alpha as examples, we demonstrate here that multiple clones of high affinity, highly specific antibodies with desired biological activities can be generated, using the NZB/W mouse as the immunization host and a T cell-specific tag fused to a recombinant antigen to stimulate the immune system. CONCLUSIONS/SIGNIFICANCE: We developed an efficient and universal method for generating surrogate or therapeutic antibodies against "difficult antigens" to facilitate the development of therapeutic antibodies.

Neutralizing and non-neutralizing monoclonal antibodies against dengue virus E protein derived from a naturally infected patient

Directory of Open Access Journals (Sweden)

Isern Sharon

2010-02-01

Full Text Available Abstract Background Antibodies produced in response to infection with any of the four serotypes of dengue virus generally provide homotypic immunity. However, prior infection or circulating maternal antibodies can also mediate a non-protective antibody response that can enhance the course of disease in a subsequent heterotypic infection. Naturally occurring human monoclonal antibodies can help us understand the protective and pathogenic roles of the humoral immune system in dengue virus infection. Results Epstein-Barr Virus (EBV transformation of B cells isolated from the peripheral blood of a human subject with previous dengue infection was performed. B cell cultures were screened by ELISA for antibodies to dengue (DENV envelope (E protein. ELISA positive cultures were cloned by limiting dilution. Three IgG1 human monoclonal antibodies (HMAbs were purified and their binding specificity to E protein was verified by ELISA and biolayer interferometry. Neutralization and enhancement assays were conducted in epithelial and macrophage-like cell lines, respectively. All three HMAbs bound to E from at least two of the four DENV serotypes, one of the HMAbs was neutralizing, and all were able to enhance DENV infection. Conclusions HMAbs against DENV can be successfully generated by EBV transformation of B cells from patients at least two years after naturally acquired DENV infections. These antibodies show different patterns of cross-reactivity, neutralizing, and enhancement activity.

Novel anti-Sialyl-Tn monoclonal antibodies and antibody-drug conjugates demonstrate tumor specificity and anti-tumor activity.

Science.gov (United States)

Prendergast, Jillian M; Galvao da Silva, Ana Paula; Eavarone, David A; Ghaderi, Darius; Zhang, Mai; Brady, Dane; Wicks, Joan; DeSander, Julie; Behrens, Jeff; Rueda, Bo R

Targeted therapeutics that can differentiate between normal and malignant tumor cells represent the ideal standard for the development of a successful anti-cancer strategy. The Sialyl-Thomsen-nouveau antigen (STn or Sialyl-Tn, also known as CD175s) is rarely seen in normal adult tissues, but it is abundantly expressed in many types of human epithelial cancers. We have identified novel antibodies that specifically target with high affinity the STn glycan independent of its carrier protein, affording the potential to recognize a wider array of cancer-specific sialylated proteins. A panel of murine monoclonal anti-STn therapeutic antibodies were generated and their binding specificity and efficacy were characterized in vitro and in in vivo murine cancer models. A subset of these antibodies were conjugated to monomethyl auristatin E (MMAE) to generate antibody-drug conjugates (ADCs). These ADCs demonstrated in vitro efficacy in STn-expressing cell lines and significant tumor growth inhibition in STn-expressing tumor xenograft cancer models with no evidence of overt toxicity.

Antibody-radioisotope conjugates for tumor localization and treatment

International Nuclear Information System (INIS)

Larson, S.M.; Carrasquillo, J.A.

1985-01-01

In principle, anti-tumor antibodies can be used to carry radioactivity to tumors for in-vivo diagnosis and treatment of cancer. First, for diagnostic purposes, an antibody that targets a specific antigen (for example, the p97 antigen of human melanoma tumor), is labeled with a tracer amount of radioactivity. When this antibody-radioisotope conjugate is injected into the blood stream, the antibody carries the radioactivity throughout the body and in time, percolates through all the tissues of the body. Because the tumor has specific antigens to which the antibody can bind, the antibody conjugate progressively accumulates in the tumor. Using conventional nuclear medicine imaging equipment, the body of the patient is scanned for radioactivity content, and a map of the distribution of the radioactivity is displayed on photographic film. The tumor shows up as a dense area of radio-activity. These same antibody-radioisotope conjugates may be used for therapy of tumors, except that in this case large amounts of radioactivity are loaded on the antibody. After localization of the conjugate there is sufficient radiation deposited in the tumor of radiotherapy. The success of this approach in the clinic is determined in large measure by the concentration gradient that can be achieved between tissue antibody conjugate in tumor versus normal tissue

Successful treatment of acute autoimmune limbic encephalitis with negative VGKC and NMDAR antibodies.

Science.gov (United States)

Modoni, Anna; Masciullo, Marcella; Spinelli, Pietro; Marra, Camillo; Tartaglione, Tommaso; Andreetta, Francesca; Tonali, Pietro; Silvestri, Gabriella

2009-03-01

To describe a case of acute nonherpetic limbic encephalitis (LE) with negative testing for antibodies directed against onconeuronal and cell membrane antigens, including voltage-gated potassium channels and N-methyl-D-aspartate receptor, that showed a dramatic response to immune therapy. A 30-year-old woman manifested generalized seizures, altered consciousness, and memory impairment shortly after a prodromal viral illness. Few days later the patient developed a drug-resistant epileptic status. Electroencephalograph showed bitemporal slowing and paroxysmal slow wave bursts. Brain magnetic resonance imaging showed bilateral swelling in the medial temporal lobes. Cerebrospinal fluid analysis ruled out viral etiologies. A diagnostic search for cancer, including serum testing for known onconeuronal antibodies proved negative. Screening for cell membrane antigen antibodies, including voltage-gated potassium channels and N-methyl-D-aspartate receptor, was also negative. Suspecting an autoimmune etiology, we started an immunomodulatory treatment with intravenous immunoglobulin followed by a short course of oral prednisone, obtaining a full clinical recovery. Our report confirms previous observations of "seronegative" autoimmune LE, suggesting the presence of other, still unknown central nervous system antigens representing a target of a postinfectious, autoimmune response in these patients. Moreover, it emphasizes the importance of early recognition and treatment of acute autoimmune LE, to reduce the risk of intensive care unit-related complications and the occurrence of permanent cognitive or behavioral defects.

Effect of monoclonal antibodies on limited proteolysis of native glycoprotein gD of herpes simplex virus type 1

International Nuclear Information System (INIS)

Eisenberg, R.J.; Long, D.; Pereira, L.; Hampar, B.; Zweig, M.; Cohen, G.H.

1982-01-01

We examined the properties of 17 monoclonal antibodies to glycoprotein gD of herpes simplex type 1 (HSV-1) (gD-1) and HSV-2 (gD-2). The antibodies recognized eight separate determinants of gD, based on differences in radioimmuno-precipitation and neutralization assays. The determinants were distributed as follows: three were gD-1 specific, one was gD-2 specific, and four were type common. Several type-specific and type-common determinants appeared to be involved in neutralization. We developed a procedure for examining the effect that binding of monoclonal antibody has on proteolysis of native gD-1 by Staphylococcus aureus protease V8. We showed that several different patterns of protease V8 cleavage were obtained, depending on the monoclonal antibody used. The proteolysis patterns were generally consistent with the immunological groupings. With four groups of antibodies, we found that fragments of gD-1 remained bound to antibody after V8 treatment. A 38,000-dalton fragment remained bound to antibodies in three different groups of monoclonal antibodies. This fragment appeared to contain one type-common and two type-specific determinants. A 12,000-dalton fragment remained bound to antibodies belonging to one type-common group of monoclonal antibodies. Tryptic peptide analysis revealed that the 12,000-dalton fragment represented a portion of the 38,000-dalton fragment and was enriched in a type-common arginine tryptic peptide

Microsphere-liposome complexes protect adenoviral vectors from neutralising antibody without losses in transfection efficiency, in-vitro.

Science.gov (United States)

Steel, Jason C; Cavanagh, Heather M A; Burton, Mark A; Kalle, Wouter H J

2004-11-01

Adenoviral vectors have been commonly used in gene therapy protocols but the success of their use is often limited by the induction of host immunity to the vector. Following exposure to the adenoviral vector, adenoviral-specific neutralising antibodies are produced, which limits further administration. This study examines the effectiveness of a novel combination of microspheres and liposomes for the shielding of adenovirus from neutralising antibodies in an in-vitro setting. We show that liposomes are effective in the protection of adenovirus from neutralising antibody and that the conjugation of these complexes to microspheres augments the level of protection. This study further reveals that previously neutralised adenovirus may still be transported into the cell via liposome-cell interactions and is still capable of expressing its genes, making this vector an effective tool for circumvention of the humoral immune response. We also looked at possible side effects of using the complexes, namely increases in cytotoxicity and reductions in transfection efficiency. Our results showed that varying the liposome:adenovirus ratio can reduce the cytotoxicity of the vector as well as increase the transfection efficiency. In addition, in cell lines that are adenoviral competent, transfection efficiencies on par with uncomplexed adenoviral vectors were achievable with the combination vector.

Current status of cancer immunodetection with radiolabeled human monoclonal antibodies.

Science.gov (United States)

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

1993-04-01

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

Serum Antibody Biomarkers for ASD

Science.gov (United States)

2015-10-01

typically developing control. US, unaffected sibling control. 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a...typically developing (TD) children (e.g., Warren et al., 1990; Singh, 2009). The goal of this study is to identify a serum antibody biomarker for ASD using...50% less IgG1 antibody in ASD boys vs . TD boys (p=0.0096). The level of ASD1 binding to the AM group was the same as to the ASD boys. These data

Modification of liposomal concentration in liposome/adenoviral complexes allows significant protection of adenoviral vectors from neutralising antibody, in vitro.

Science.gov (United States)

Steel, Jason C; Cavanagh, Heather M A; Burton, Mark A; Dingwall, Daniel J; Kalle, Wouter H J

2005-06-01

Adenoviral vectors have been commonly used in gene therapy protocols, however the success of their use is often limited by the induction of host immunity to the vector. Following exposure to the adenoviral vector, adenoviral-specific neutralising antibodies are produced which limits further administration. This study examines the efficacy of complexing liposomes to adenovirus for the protection of the adenovirus from neutralising antibodies in an in vitro setting. Dimethyldioctadecylammonium bromide (DDAB)-dioleoyl-l-phosphatidylethanolamine (DOPE) liposomes were bound at varying concentrations to adenovirus to form AL complexes and tested these complexes' ability to prevent adenoviral neutralisation. It is shown that by increasing the concentration of liposomes in the adenoviral-liposome (AL) complexes we can increase the level of immuno-shielding afforded the adenovirus. It is also shown that the increase in liposomal concentration may lead to drawbacks such as increased cytotoxicity and reductions in expression levels.

Antibody guided diagnosis and therapy of brain gliomas using radiolabeled monoclonal antibodies against epidermal growth factor receptor and placental alkaline phosphatase

International Nuclear Information System (INIS)

Kalofonos, H.P.; Pawlikowska, T.R.; Hemingway, A.

1989-01-01

Twenty-seven patients with brain glioma were scanned using 123 I-labeled monoclonal antibodies against epidermal growth factor receptor (EGFR1) or placental alkaline phosphatase (H17E2). Successful localization was achieved in 18 out of 27 patients. Eleven out of 27 patients were also studied using a nonspecific control antibody (11.4.1) of the same immunoglobulin subclass and observable tumor localization was also achieved in five patients. The specificity of targeting was assessed by comparing images obtained with specific and nonspecific antibodies and by examining tumor and normal tissue biopsies after dual antibody administration. Ten patients with recurrent grade III or IV glioma who showed good localization of radiolabeled antibody were treated with 40-140 mCi of 131 I-labeled antibody delivered to the tumor area intravenously (n = 5) or by infusion into the internal carotid artery (n = 5). Six patients showed clinical improvement lasting from 6 mo to 3 yr. One patient continues in remission (3 yr after therapy), but the other five who responded initially relapsed 6-9 mo after therapy and died. No major toxicity was attributable to antibody-guided irradiation. Targeted irradiation by monoclonal antibody may be clinically useful and should be explored further in the treatment of brain gliomas resistant to conventional forms of treatment

Aggregates in monoclonal antibody manufacturing processes.

Science.gov (United States)

Vázquez-Rey, María; Lang, Dietmar A

2011-07-01

Monoclonal antibodies have proved to be a highly successful class of therapeutic products. Large-scale manufacturing of pharmaceutical antibodies is a complex activity that requires considerable effort in both process and analytical development. If a therapeutic protein cannot be stabilized adequately, it will lose partially or totally its therapeutic properties or even cause immunogenic reactions thus potentially further endangering the patients' health. The phenomenon of protein aggregation is a common issue that compromises the quality, safety, and efficacy of antibodies and can happen at different steps of the manufacturing process, including fermentation, purification, final formulation, and storage. Aggregate levels in drug substance and final drug product are a key factor when assessing quality attributes of the molecule, since aggregation might impact biological activity of the biopharmaceutical. In this review it is analyzed how aggregates are formed during monoclonal antibody industrial production, why they have to be removed and the manufacturing process steps that are designed to either minimize or remove aggregates in the final product. Copyright © 2011 Wiley Periodicals, Inc.

Radioimmunotherapy of Non-Hodgkin's Lymphoma. The interaction of radiation and antibody with lymphoma cells

International Nuclear Information System (INIS)

Illidge, T.M.

1999-06-01

Whilst many patients with indolent Non-Hodgkin's Lymphoma (NHL) can achieve clinical remissions to first-line chemotherapy and/or radiotherapy, most will relapse. Current treatment options for relapsing patients are limited since most patients become resistant to repeated chemotherapy. Death usually occurs within 10 years of diagnosis. Overall, these disappointing results have not changed significantly in a quarter of a century and clearly advocate the urgent priority to research into potential new therapeutic approaches into this diverse and increasingly prevalent group of human tumours. Radioimmunotherapy (RIT) is currently under investigation as a new approach for the treatment of this disease. In this form of treatment, radionuclide-labeled monoclonal antibodies are able to deliver selective systemic irradiation by recognising tumour-associated antigens. The use of RIT with radiolabeled anti-CD20 antibodies in patients with recurrent B-cell lymphoma has resulted in extremely high rates of durable complete remissions. The optimal approach and mechanisms of action of successful RIT remain however largely unknown. The work described in this thesis has focused on clarifying some of the important determinants and mechanisms of effective RIT of syngeneic B-cell lymphoma, both in vivo and in vitro. A successful animal model of RIT in B cell lymphomas was established by initially generating a panel of antibodies against mouse B cell antigens. The in vitro characteristics of these antibodies have been compared with their subsequent performance, in biodistribution studies and RIT in vivo. For the first time in an in vivo model the relative contributions of antibody and irradiation are described. Some antibodies including anti-MHC Class II were shown to be effective delivery vehicles of low doses of Iodine-131. These antibodies, which appear to be inactive delivery vehicles can cure animals with low burdens of tumour. However antibodies such as anti-idiotype and anti-CD40

High-resolution mapping of linear antibody epitopes using ultrahigh-density peptide microarrays

DEFF Research Database (Denmark)

Buus, Søren; Rockberg, Johan; Forsström, Björn

2012-01-01

Antibodies empower numerous important scientific, clinical, diagnostic, and industrial applications. Ideally, the epitope(s) targeted by an antibody should be identified and characterized, thereby establishing antibody reactivity, highlighting possible cross-reactivities, and perhaps even warning...... against unwanted (e.g. autoimmune) reactivities. Antibodies target proteins as either conformational or linear epitopes. The latter are typically probed with peptides, but the cost of peptide screening programs tends to prohibit comprehensive specificity analysis. To perform high-throughput, high......-resolution mapping of linear antibody epitopes, we have used ultrahigh-density peptide microarrays generating several hundred thousand different peptides per array. Using exhaustive length and substitution analysis, we have successfully examined the specificity of a panel of polyclonal antibodies raised against...

Clinical use of antibodies

International Nuclear Information System (INIS)

Baum, R.P.; Hoer, Gustav; Cox, P.H.; Buraggi, G.L.

1991-01-01

Use of monoclonal antibodies as tumour specific carrier molecules for therapeutic agents or as in vivo diagnostic reagents when labelled with radionuclides or NMR signal enhancers is attracting more and more attention. The potential is enormous but the technical problems are also considerable requiring the concerted action of many different scientific disciplines. This volume is based upon a symposium organised in Frankfurt in 1990 under the auspices of the European Association of Nuclear Medicines' Specialist Task Groups on Cardiology and the Utility of Labelled Antibodies. It gives a multidisciplinary review of the state of the art and of problems to be solved as well as recording the not inconsiderable successes which have been booked to date. The book will be of value as a reference to both clinicians and research scientists. refs.; figs.; tabs

Antithyroglobulin antibody

Science.gov (United States)

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

Immunotherapy for the treatment of colorectal tumors: focus on approved and in-clinical-trial monoclonal antibodies

Directory of Open Access Journals (Sweden)

Françoso A

2017-01-01

Full Text Available Alex Françoso,1 Patricia Ucelli Simioni1–3 1Department of Biomedical Science, Faculty of Americana, Americana, 2Department of Genetics, Evolution and Bioagents, Institute of Biology, University of Campinas, Campinas, 3Department of Biochemistry and Microbiology, Institute of Biosciences, Universidade Estadual Paulista, Rio Claro, São Paulo, Brazil Abstract: Colorectal cancer is considered a disease of the elderly population. Since the number of geriatric patients continues to rise, monoclonal antibody therapy is the most promising therapy in the recent research. Presently, the monoclonal antibodies most frequently used in the treatment of colorectal tumors are bevacizumab, cetuximab, panitumumab, and ramucirumab. Bevacizumab is a monoclonal antibody that acts on VEGF. Cetuximab and panitumumab act on EGFR. Ramucirumab binds directly to the ligand-binding pocket of VEGFR-2 to block the binding of VEGF-A, VEGF-C, and VEGF-D. These monoclonal antibodies, alone or in association with radiotherapy or chemotherapy, are presenting good results and are increasing patient survival, despite the side effects. Due to the limited number of molecules available, several studies are trying to develop new monoclonal antibodies for the treatment of colorectal tumors. Among those being studied, some recent molecules are in phase I and/or II trials and are yielding advantageous results, such as anti-DR5, anti-Fn14, anti-IGF-1R, anti-EGFR, anti-NRP1, and anti-A33 antibodies. This has been successful in reducing side effects and in treating nonresponsive patients. Keywords: monoclonal antibodies, colorectal tumor, bevacizumab, cetuximab, panitumumab, ramucirumab

Successful management of aortic thrombi resulting in spinal cord infarction in a patient with antiphospholipid antibody syndrome and acute cholecystitis

Directory of Open Access Journals (Sweden)

Izumi M

2011-12-01

Full Text Available Manabu Izumi, Shoko Teraoka, Keisuke Yamashita, Kenji Matsumoto, Tomohiro Muronoi, Yoshimitsu Izawa, Chikara Yonekawa, Masaki Ano, Masayuki SuzukawaDepartment of Emergency and Critical Care Medicine, Jichi Medical University, Tochigi, JapanAbstract: A 74-year-old man with coronary artery disease was suffering from acute nonobstructive cholecystitis and was admitted to a nearby hospital. Dual antiplatelet (aspirin and ticlopidine therapy was discontinued before preparation for surgical resection of the gall bladder. During his time in hospital he was aware of lumbar pain and weakness in both legs. He was transferred to our hospital for further evaluation and therapy. Diffuse intra-aortic thrombi were revealed by computed tomography with contrast media, and magnetic resonance imaging showed spinal cord infarction. However, computed tomography scans of the descending aorta obtained 4 months before admission exhibited no signs of atherosclerotic plaques or intra-aortic thrombi. Laboratory data suggest that antiphospholipid antibody syndrome might have caused these acute multiple intra-arterial thrombi. By restarting dual antiplatelet therapy and increasing the dose of heparin (from 10,000 IU/day to 15,000 IU/day we successfully managed the patient's clinical condition and symptoms. It is important to understand that stopping antiplatelet therapy may rapidly grow thrombi in patients with a hypercoagulative state.Keywords: intra-aortic thrombus, antiphospholipid antibody syndrome, spinal cord infarction

Growth control of genetically modified cells using an antibody/c-Kit chimera.

Science.gov (United States)

Kaneko, Etsuji; Kawahara, Masahiro; Ueda, Hiroshi; Nagamune, Teruyuki

2012-05-01

Gene therapy has been regarded as an innovative potential treatment against serious congenital diseases. However, applications of gene therapy remain limited, partly because its clinical success depends on therapeutic gene-transduced cells acquiring a proliferative advantage. To address this problem, we have developed the antigen-mediated genetically modified cell amplification (AMEGA) system, which uses chimeric receptors to enable the selective proliferation of gene-transduced cells. In this report, we describe mimicry of c-Kit signaling and its application to the AMEGA system. We created an antibody/c-Kit chimera in which the extracellular domain of c-Kit is replaced with an anti-fluorescein single-chain Fv antibody fragment and the extracellular D2 domain of the erythropoietin receptor. A genetically modified mouse pro-B cell line carrying this chimera showed selective expansion in the presence of fluorescein-conjugated BSA (BSA-FL) as a growth inducer. By further engineering the transmembrane domain of the chimera to reduce interchain interaction we attained stricter ligand-dependency. Since c-Kit is an important molecule in the expansion of hematopoietic stem cells (HSCs), this antibody/c-Kit chimera could be a promising tool for gene therapy targeting HSCs. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Antibodies to AB blood group antigens mimic anti-salivary duct autoantibodies in patients with limited sicca symptoms.

Science.gov (United States)

Goldblatt, F; Beroukas, D; Gillis, D; Cavill, D; Bradwell, A; Rischmueller, M; Gordon, T P

2000-10-01

We evaluated the clinical relevance and pathogenic significance of anti-salivary duct autoantibodies (ASDA) in Sjögren's syndrome (SS) and rheumatoid arthritis (RA) by examining (1) their frequency in healthy controls, patients with sicca symptoms, and patients with various autoimmune and infective disorders; (2) their localization by confocal microscopy; and (3) their tissue distribution and cross reactivity with blood group antigens. Indirect immunofluorescence (IF) was performed on commercial cryostat sections of monkey parotid salivary gland. Sections were examined by fluorescence and confocal laser scanning microscopy. Sera giving positive staining on the ducts were tested by IF on a range of monkey tissues and salivary glands from several mammalian species. Blocking experiments were performed with human erythrocytes of different ABO blood groups and AB antigens. We identified 2 distinct ductal staining patterns. The first resembled ASDA described in earlier studies and showed patchy bright staining of the apical (luminal) surfaces of the ducts and staining of apical cytoplasmic vesicles. The other was only observed with anti-mitochondrial antibody positive sera and stained the mitochondrial-rich ductal epithelium in a distinctive punctate pattern. Antibodies staining the apical surface of ducts were detected rarely in patients with antiRo/La autoantibody-positive primary SS (1/76) and RA (1/36) and were found in only 1115 with RA and secondary SS. ASDA were detected in sera from 13/51 (25.5%) of patients referred to our clinic with limited sicca symptoms who were anti-Ro/La antibody-negative and had no typical clinical or laboratory features of classical primary SS. The apical ductal staining pattern was not observed with sera from 63 healthy controls without sicca symptoms or in patients with autoimmune and infective disorders. Twelve of the 13 patients whose sera gave ASDA-like staining were blood group O and one group A. Ductal staining was abolished in

Postbooster Antibodies from Humans as Source of Diphtheria Antitoxin.

Science.gov (United States)

Bermejo-Martin, Jesús F; Avila-Alonso, Ana; González-Rivera, Milagros; Tamayo, Eduardo; Eiros, Jose María; Almansa, Raquel

2016-07-01

Diphtheria antitoxin for therapeutic use is in limited supply. A potential source might be affinity-purified antibodies originally derived from plasma of adults who received a booster dose of a vaccine containing diphtheria toxoid. These antibodies might be useful for treating even severe cases of diphtheria.

Labeling an anti-CD20 monoclonal antibody with 90Y

International Nuclear Information System (INIS)

Perera Pintado, Alejandro; Leyva Montaña, René; Prats Capote, Anaís; Góngora Bravo, Magdiel; Alberti Ramírez, Alejandro; León, Mariela; Hernández González, Ignacio; Dorvignit, Denise

2016-01-01

Lymphomas are among the 10 leading causes of death, both in Cuba and in the world, with an increasing incidence in recent years. Follicular lymphoma low-grade (indolent) is one of the most common in the Western world, representing 1/3 of all non-Hodgkin lymphomas (NHL). More than 90% of patients present with disseminated disease at diagnosis and generally have a slow evolution and good response to conventional treatment; but radically changed its forecast to relapse, resistance to therapeutic and histologic transformation can occur. The monoclonal antibody therapy has been a promising therapeutic. In this respect CD20 antigen it has been considered one of the most attractive targets in the therapy of follicular B cell lymphoma This is expressed in more than 90% of cases, while not present in stem cells and lines progenitors. Despite the success of immunotherapy, the relapse rate is still considerable. In order to increase the cytotoxic potential of immunotherapy, marked with beta emitting radionuclides alpha particles or monoclonal antibodies are used today. Despite encouraging results in patients with non-Hodgkin lymphomas refractory to other treatments, the extremely high costs of these commercial radiopharmaceuticals have greatly limited its application, even in the first world. A sustainable alternative is the marking of other anti-CD20 monoclonal antibodies, so researchers from several countries have concentrated their efforts on rituximaby other similar antibodies labeled with therapeutic radionuclides, as a possible cost-effectively to more problem. Today in Cuba it has an electrolytic generator 90 Sr- 90 Y Isotope Center, which ensures the availability of the radionuclide. In addition, the chimeric MAb rituximab is applied as part of the therapy of NHL in its health system and, recently, the Center for Molecular Immunology has obtained a chimeric monoclonal anti-CD20 antibody biosimilar rituximab, which is in phase clinical trial; which opens prospects for

Anti sperm antibodies detection in infertile patients by radioimmunometry

International Nuclear Information System (INIS)

ELnabarawy, F.; Megahed, Y.M.; Tadrous, G.A.; Hamada, T.; Elbadry, A.

1992-01-01

Three different methods of testing for anti sperm antibodies were compared: complement cytotoxicity, sperm agglutination, and radiolabelled anti globulin antibody technique, for detection of anti sperm antibodies in serum and secretions (seminal plasma and cervical mucus). Sample from 120 patients with infertility were investigated by the previous three methods. The results of unexplained infertile patients revealed wide variations in figures, concerning the positivity of anti sperm antibody whether in their serum or secretions, by using the cytotoxicity or sperm agglutination tests. Using a specific radiolabelled anti globulin test, a subset of patients (44.9% in the serum of men and 50% in seminal plasma) with IgG anti sperm antibody was identified, and this antibody was present in 65.4% and 78,6% of infertile wives sera and cervical mucus, respectively. Therefore, this test has been used to identify and quantitate antibodies directed toward other human cell surfaces. It was concluded that this radiolabelled method is a clinically useful and a potentially versatile procedure that can be successfully applied to the diagnosis and management of patients with suspected immunologic infertility. 1 fig., 5 tab

Rapid isolation of antibody from a synthetic human antibody library by repeated fluorescence-activated cell sorting (FACS.

Directory of Open Access Journals (Sweden)

Sung Sun Yim

Full Text Available Antibodies and their derivatives are the most important agents in therapeutics and diagnostics. Even after the significant progress in the technology for antibody screening from huge libraries, it takes a long time to isolate an antibody, which prevents a prompt action against the spread of a disease. Here, we report a new strategy for isolating desired antibodies from a combinatorial library in one day by repeated fluorescence-activated cell sorting (FACS. First, we constructed a library of synthetic human antibody in which single-chain variable fragment (scFv was expressed in the periplasm of Escherichia coli. After labeling the cells with fluorescent antigen probes, the highly fluorescent cells were sorted by using a high-speed cell sorter, and these cells were reused without regeneration in the next round of sorting. After repeating this sorting, the positive clones were completely enriched in several hours. Thus, we screened the library against three viral antigens, including the H1N1 influenza virus, Hepatitis B virus, and Foot-and-mouth disease virus. Finally, the potential antibody candidates, which show K(D values between 10 and 100 nM against the target antigens, could be successfully isolated even though the library was relatively small (∼ 10(6. These results show that repeated FACS screening without regeneration of the sorted cells can be a powerful method when a rapid response to a spreading disease is required.

[Prokaryotic expression of Nanog gene and preparation of anti-Nanog antibody].

Science.gov (United States)

Li, Jun; Wang, Xiao-min; Dou, Zhong-ying; Li, Yong

2012-07-01

To express Nanog fusion protein in Escherichia coli ( E.coli), and to prepare rabbit anti-mouse polyclonal antibodies to the Nanog fusion protein. Mouse Nanog gene was amplified from the pNA992 recombinant plasmid and inserted into pET-32a vector to construct a recombinant expression vector pET-32a-Nanog. The recombinant vector was transfected into E.coli BL21 and induced by IPTG to express in them. The acquired Nanog fusion protein was purified with HisTrap affinity column and injected as an antigen into rabbits for preparing polyclonal antibodies. At last, the titer and specificity of the polyclonal antibodies were analyzed with indirect ELISA, Western blotting and immunocytochemical staining, respectively. The recombinant expression vector pET-32a-Nanog was successfully prepared, transfected and induced to obtain the high expression of the Nanog fusion protein in a form of inclusion bodies in E.coli. After purification, its purity was up to 97%. The titer of anti-Nanog antibodies was 1:32 000 in the immunized rabbit serum, and exhibited a high specificity to Nanog protein. The rabbit anti-mouse polyclonal antibodies have been prepared successfully with a high titer and specificity to the Nanog fusion protein.

Refractory disease in antineutrophil cytoplasmic antibodies associated vasculitis

NARCIS (Netherlands)

Rutgers, Abraham; Kallenberg, Cornelis

Purpose of review Induction treatment of antineutrophil cytoplasmic antibodies (ANCA) associated vasculitis (AAV) is not always successful and nonresponding patients are considered refractory. Recent findings Refractory disease should be subdefined to the treatment that was received.

Antiphospholipid Antibody Induced by Nivolumab

Directory of Open Access Journals (Sweden)

Ahmed Aburahma

2018-01-01

Full Text Available Nivolumab is a monoclonal antibody against the programmed death protein 1 and is used for patients with advanced melanoma. It is associated with potentially immune-related adverse events, including disorders of the skin, GI tract, and the thyroid; these disorders were successfully treated with prednisone and infliximab. Other immunotherapeutic agents were observed to induce the formation of antiphospholipid antibody (APA including α-interferon and interleukin-2. We present a case of APA development after the third dose of nivolumab in a 71-year-old male with advanced melanoma. The APA was detected after finding a prolonged aPTT; the lupus anticoagulant assay tested positive. The patient was treated with prednisone but, unfortunately, he expired a few days later.

Next Generation Antibody Therapeutics Using Bispecific Antibody Technology.

Science.gov (United States)

Igawa, Tomoyuki

2017-01-01

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

Radioimmunoassay with heterologous antibody (hetero-antibody RIA)

International Nuclear Information System (INIS)

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

1991-01-01

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

Development and characterization of a pre-treatment procedure to eliminate human monoclonal antibody therapeutic drug and matrix interference in cell-based functional neutralizing antibody assays.

Science.gov (United States)

Xu, Weifeng; Jiang, Hao; Titsch, Craig; Haulenbeek, Jonathan R; Pillutla, Renuka C; Aubry, Anne-Françoise; DeSilva, Binodh S; Arnold, Mark E; Zeng, Jianing; Dodge, Robert W

2015-01-01

Biological therapeutics can induce an undesirable immune response resulting in the formation of anti-drug antibodies (ADA), including neutralizing antibodies (NAbs). Functional (usually cell-based) NAb assays are preferred to determine NAb presence in patient serum, but are often subject to interferences from numerous serum factors, such as growth factors and disease-related cytokines. Many functional cell-based NAb assays are essentially drug concentration assays that imply the presence of NAbs by the detection of small changes in functional drug concentration. Any drug contained in the test sample will increase the total amount of drug in the assay, thus reducing the sensitivity of NAb detection. Biotin-drug Extraction with Acid Dissociation (BEAD) has been successfully applied to extract ADA, thereby removing drug and other interfering factors from human serum samples. However, to date there has been no report to estimate the residual drug level after BEAD treatment when the drug itself is a human monoclonal antibody; mainly due to the limitation of traditional ligand-binding assays. Here we describe a universal BEAD optimization procedure for human monoclonal antibody (mAb) drugs by using a LC-MS/MS method to simultaneously measure drug (a mutant human IgG4), NAb positive control (a mouse IgG), and endogenous human IgGs as an indicator of nonspecific carry-over in the BEAD eluate. This is the first report demonstrating that residual human mAb drug level in clinical sample can be measured after BEAD pre-treatment, which is critical for further BEAD procedure optimization and downstream immunogenicity testing. Copyright © 2014 Elsevier B.V. All rights reserved.

Developability studies before initiation of process development: improving manufacturability of monoclonal antibodies.

Science.gov (United States)

Yang, Xiaoyu; Xu, Wei; Dukleska, Svetlana; Benchaar, Sabrina; Mengisen, Selina; Antochshuk, Valentyn; Cheung, Jason; Mann, Leslie; Babadjanova, Zulfia; Rowand, Jason; Gunawan, Rico; McCampbell, Alexander; Beaumont, Maribel; Meininger, David; Richardson, Daisy; Ambrogelly, Alexandre

2013-01-01

Monoclonal antibodies constitute a robust class of therapeutic proteins. Their stability, resistance to stress conditions and high solubility have allowed the successful development and commercialization of over 40 antibody-based drugs. Although mAbs enjoy a relatively high probability of success compared with other therapeutic proteins, examples of projects that are suspended due to the instability of the molecule are not uncommon. Developability assessment studies have therefore been devised to identify early during process development problems associated with stability, solubility that is insufficient to meet expected dosing or sensitivity to stress. This set of experiments includes short-term stability studies at 2-8 þC, 25 þC and 40 þC, freeze-thaw studies, limited forced degradation studies and determination of the viscosity of high concentration samples. We present here three case studies reflecting three typical outcomes: (1) no major or unexpected degradation is found and the study results are used to inform early identification of degradation pathways and potential critical quality attributes within the Quality by Design framework defined by US Food and Drug Administration guidance documents; (2) identification of specific degradation pathway(s) that do not affect potency of the molecule, with subsequent definition of proper process control and formulation strategies; and (3) identification of degradation that affects potency, resulting in program termination and reallocation of resources.

Utility of HLA Antibody Testing in Kidney Transplantation

Science.gov (United States)

Konvalinka, Ana

2015-01-01

HLA antigens are polymorphic proteins expressed on donor kidney allograft endothelium and are critical targets for recipient immune recognition. HLA antibodies are risk factors for acute and chronic rejection and allograft loss. Solid-phase immunoassays for HLA antibody detection represent a major advance in sensitivity and specificity over cell-based methods and are widely used in organ allocation and pretransplant risk assessment. Post-transplant, development of de novo donor–specific HLA antibodies and/or increase in donor-specific antibodies from pretransplant levels are associated with adverse outcomes. Although single antigen bead assays have allowed sensitive detection of recipient HLA antibodies and their specificities, a number of interpretive considerations must be appreciated to understand test results in clinical and research contexts. This review, which is especially relevant for clinicians caring for transplant patients, discusses the technical aspects of single antigen bead assays, emphasizes their quantitative limitations, and explores the utility of HLA antibody testing in identifying and managing important pre- and post-transplant clinical outcomes. PMID:25804279

New tools for immunochemistry: internally labelled monoclonal antibodies

International Nuclear Information System (INIS)

Galfre, G.; Cuello, A.C.

1981-01-01

Labelled antibodies are routinely used in a wide variety of immunochemical methods. Over the years several labelling techniques have been developed and the discussion of some of them forms a substantial part of this course. Common to all the procedures is the need to purify the antibodies. The labelling itself consists of coupling the antibodies to a ''label'' molecule by means of a chemical reaction. Preparation in vitro of monoclonal antibodies offers the unique possibility to internally label them. Although this is restricted to radiolabelling, and the specific activity achieved is limited, the procedure is extremely simple, does not require purification prior to labelling and chemical manipulation is not necessary as the antibodies themselves are synthesized from radioactive amino acids. Moreover, different labels can be used ( 14 C, 35 S, 3 H) which have a much longer half-life than 125 I. The choice of labelled amino acid precurors and labelling procedure is discussed. The uses of internally-labelled monoclonal antibodies are indicated. (Auth.)

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

International Nuclear Information System (INIS)

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

1993-01-01

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

Separation of hemagglutination-inhibiting immunoglobulin M antibody to rubella virus in human serum by high-performance liquid chromatography.

OpenAIRE

Kobayashi, N; Suzuki, M; Nakagawa, T; Matumoto, M

1986-01-01

High-performance liquid chromatography was successfully used to separate hemagglutination-inhibiting immunoglobulin M (IgM) rubella virus antibody from IgG rubella virus antibody in human serum. The fractionation by high-performance liquid chromatography was as effective as sucrose density gradient centrifugation in separating IgM antibody from IgG antibody.

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

International Nuclear Information System (INIS)

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

1983-01-01

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

rapid assessment of polio virus antibodies prevalence amongst

African Journals Online (AJOL)

ISSN 1597-6343. Polio Virus Antibodies Prevalence Amongst Children In Kano State ... poliovirus serotypes (types 1, 2 or 3) which cause poliomyelitis. They are spread by ..... all those that have contributed to the overall success of this work.

Severe antiphospholipid antibody syndrome - response to plasmapheresis and rituximab.

Science.gov (United States)

Gkogkolou, Paraskevi; Ehrchen, Jan; Goerge, Tobias

2017-09-01

Antiphospholipid antibody syndrome (APS) is a systemic autoimmune disease characterized by arterial and/or venous thrombosis, recurrent abortions and detection of antiphospholipid antibodies. In fulminant cases, involvement of multiple organs can lead to significant morbidity and even fatal outcomes, so that a rapid, interdisciplinary treatment is needed. Here, we describe the case of a 39-year-old woman with a severe hard-to-treat APS with arterial occlusion and progressive skin necrosis, who was successfully treated with a combination therapy with plasmapheresis and rituximab. The treatment led to complete remission of the skin lesions for over a year. Clinical response correlated with a long-lasting reduction of antiphospholipid antibodies and B-cell depletion. This case demonstrates the use of antiphospholipid antibodies for monitoring APS-activity and shows that this severe vascular disease requires rigorous therapeutic approaches.

Tetrafluorophenolate of HBED-CC: a versatile conjugation agent for 68Ga-labeled small recombinant antibodies

International Nuclear Information System (INIS)

Eder, Matthias; Waengler, Bjoern; Eisenhut, Michael; Knackmuss, Stefan; LeGall, Fabrice; Little, Melvyn; Haberkorn, Uwe; Mier, Walter

2008-01-01

The success of 68 Ga-labeled peptides for positron emission tomography of neuroendocrine tumors is mainly depending on the complex chemistry of this radioisotope. 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), the chelator of choice has however limitations if its application is expanded to heat-sensitive proteins. Recombinant antibodies like single chain Fv or diabodies belong to this class of proteins. They are suited to provide imaging contrast despite the short-lived 68 Ga because of their rapid blood clearances and nanomolar affinities. The heterobifunctional agent N,N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid (HBED-CC) was chosen as an alternative ligand because this agent is complexing [ 68 Ga]Ga 3+ much faster than DOTA at ambient temperatures. A versatile technology for HBED-CC conjugation of proteins and 68 Ga-labeling has been developed. This included HBED-CC-tetrafluorophenol (TFP) ester synthesis, coupling to the antibody at various pH and complexation reactions performed in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer under different conditions. The synthesis of the monoreactive 2,3,5,6-tetrafluorophenolate of HBED-CC at a carboxyl group not participating in complex formation used [Fe(HBED-CC)] - for ester formation. The removal of Fe 3+ from purified (HBED-CC)TFP ester was achieved with RP 18 cartridge technology. The conjugation chemistry was performed with mAb425 which binds to the epidermal growth factor receptor (EGFR). This protein was used for optimizing purposes only. The influence of complexation parameters like temperature, pH, reaction time, and HBED-CC/antibody ratio on the biological activity of this model antibody was investigated. Furthermore, the outcome of this labeling procedure on the biological activity of a recombinant diabody (50 kDa) was studied. It is known that small HBED-CC/antibody ratios are prerequisites for minimal interference of labels with antigen

Addressing the Immunogenicity of the Cargo and of the Targeting Antibodies with a Focus on Deimmunized Bacterial Toxins and on Antibody-Targeted Human Effector Proteins

Science.gov (United States)

Grinberg, Yehudit; Benhar, Itai

2017-01-01

Third-generation immunotoxins are composed of a human, or humanized, targeting moiety, usually a monoclonal antibody or an antibody fragment, and a non-human effector molecule. Due to the non-human origin of the cytotoxic domain, these molecules stimulate potent anti-drug immune responses, which limit treatment options. Efforts are made to deimmunize such immunotoxins or to combine treatment with immunosuppression. An alternative approach is using the so-called “human cytotoxic fusion proteins”, in which antibodies are used to target human effector proteins. Here, we present three relevant approaches for reducing the immunogenicity of antibody-targeted protein therapeutics: (1) reducing the immunogenicity of the bacterial toxin, (2) fusing human cytokines to antibodies to generate immunocytokines and (3) addressing the immunogenicity of the targeting antibodies. PMID:28574434

Radioimmunotherapy of Non-Hodgkin's Lymphoma. The interaction of radiation and antibody with lymphoma cells

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Illidge, T.M

1999-06-01

Whilst many patients with indolent Non-Hodgkin's Lymphoma (NHL) can achieve clinical remissions to first-line chemotherapy and/or radiotherapy, most will relapse. Current treatment options for relapsing patients are limited since most patients become resistant to repeated chemotherapy. Death usually occurs within 10 years of diagnosis. Overall, these disappointing results have not changed significantly in a quarter of a century and clearly advocate the urgent priority to research into potential new therapeutic approaches into this diverse and increasingly prevalent group of human tumours. Radioimmunotherapy (RIT) is currently under investigation as a new approach for the treatment of this disease. In this form of treatment, radionuclide-labeled monoclonal antibodies are able to deliver selective systemic irradiation by recognising tumour-associated antigens. The use of RIT with radiolabeled anti-CD20 antibodies in patients with recurrent B-cell lymphoma has resulted in extremely high rates of durable complete remissions. The optimal approach and mechanisms of action of successful RIT remain however largely unknown. The work described in this thesis has focused on clarifying some of the important determinants and mechanisms of effective RIT of syngeneic B-cell lymphoma, both in vivo and in vitro. A successful animal model of RIT in B cell lymphomas was established by initially generating a panel of antibodies against mouse B cell antigens. The in vitro characteristics of these antibodies have been compared with their subsequent performance, in biodistribution studies and RIT in vivo. For the first time in an in vivo model the relative contributions of antibody and irradiation are described. Some antibodies including anti-MHC Class II were shown to be effective delivery vehicles of low doses of Iodine-131. These antibodies, which appear to be inactive delivery vehicles can cure animals with low burdens of tumour. However antibodies such as anti-idiotype and anti

Aligning physics and physiology: Engineering antibodies for radionuclide delivery.

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Tsai, Wen-Ting K; Wu, Anna M

2018-03-14

The exquisite specificity of antibodies and antibody fragments renders them excellent agents for targeted delivery of radionuclides. Radiolabeled antibodies and fragments have been successfully used for molecular imaging and radioimmunotherapy (RIT) of cell surface targets in oncology and immunology. Protein engineering has been used for antibody humanization essential for clinical applications, as well as optimization of important characteristics including pharmacokinetics, biodistribution, and clearance. Although intact antibodies have high potential as imaging and therapeutic agents, challenges include long circulation time in blood, which leads to later imaging time points post-injection and higher blood absorbed dose that may be disadvantageous for RIT. Using engineered fragments may address these challenges, as size reduction and removal of Fc function decreases serum half-life. Radiolabeled fragments and pretargeting strategies can result in high contrast images within hours to days, and a reduction of RIT toxicity in normal tissues. Additionally, fragments can be engineered to direct hepatic or renal clearance, which may be chosen based on the application and disease setting. This review discusses aligning the physical properties of radionuclides (positron, gamma, beta, alpha, and Auger emitters) with antibodies and fragments and highlights recent advances of engineered antibodies and fragments in preclinical and clinical development for imaging and therapy. Copyright © 2018 John Wiley & Sons, Ltd.

Antithyroglobulin Antibodies and Antimicrosomal Antibodies in Various Thyroid Diseases

International Nuclear Information System (INIS)

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

1979-01-01

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

Quantitative relationship between antibody affinity and antibody avidity

International Nuclear Information System (INIS)

Griswold, W.R.

1987-01-01

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

Binding-site analysis of opioid receptors using monoclonal anti-idiotypic antibodies

International Nuclear Information System (INIS)

Conroy, W.G.

1988-01-01

Structural relatedness between the variable region of anti-ligand antibodies and opioid binding sites allowed the generation of anti-idiotypic antibodies which recognized opioid receptors. The IgG 3 k antibodies which bound to opioid receptors were obtained when an anti-morphine antiserum was the idiotype. Both antibodies bound to opioid receptors, but only one of these blocked the binding of [ 3 H]naloxone. The antibody which did not inhibit the binding of [ 3 H]naloxone was itself displaced from the receptor by opioid ligands. The unique binding properties displayed by this antibody indicated that anti-idiotypic antibodies are not always a perfect image of the original ligand, and therefore may be more useful than typical ligands as probes for the receptor. An auto-anti-idiotypic technique was successfully used to obtain anti-opioid receptor antibodies. Another IgG 3 k antibody that blocked the binding of [ 3 H]naloxone to rat brain opioid receptors was obtained when a mouse was immunized with naloxone conjugated to bovine serum albumin. These data confirmed that an idiotype-anti-idiotype network which can generate an anti-receptor antibody normally functions when an opioid ligand is introduced into an animal in an immunogenic form

Success and limitations in the prevention of violence: a case study of nine Brazilian experiences

Directory of Open Access Journals (Sweden)

Romeu Gomes

Full Text Available The present study analyzes the success of Brazilian experiences engaged in prevention of violence as well as their limitations, and seeks to show ways for a solution of the problem. The investigation underlying this article consisted in a case study of nine experiences, using triangulation of methods. For this purpose, we used questionnaires and interviewed individuals and groups. Successful results were mainly: informed citizenship, work preparation, involvement of children and adolescents in recreating and educative activities, and increased self-esteem of the children and teens involved in the experiences. The greatest difficulties found were the lack of funds and of articulation of proposals with other groups and institutions. These experiences are not the only successful ones carried out in the country, but they point to a new horizon by innovating and creating a different practical approach to violence.

Anti-VEGF antibody conjugated CdHgTe quantum dots as a fluorescent probe for imaging in living mouse

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Pang, Lili; Cui, Hongjing [Department of Analytical Chemistry, China Pharmaceutical University, Nanjing (China); Liu, Yu [Department of Biochemistry, School of Life Science and Technology, China Pharmaceutical University, Nanjing (China); Zhong, Wenying, E-mail: wyzhong@cpu.edu.cn [Department of Analytical Chemistry, China Pharmaceutical University, Nanjing (China); Key laboratory of Biomedical Functional Materials, China Pharmaceutical University, Nanjing (China)

2016-05-15

The dual-function anti-VEGF antibody conjugated CdHgTe quantum dots with good targeting property was successfully prepared. In this system, anti-VEGF antibody is not only a target agent but also a therapeutic drug. The anti-VEGF antibody conjugated near-infrared quantum dots can achieve the purposes of detection and treatment at the same time. As-prepared dual-function fluorescent probe in this work has been successfully applied for in vivo and in vitro imaging, which is promising in rapid tumor detection.

Antibody-functionalized porous silicon nanoparticles for vectorization of hydrophobic drugs.

Science.gov (United States)

Secret, Emilie; Smith, Kevin; Dubljevic, Valentina; Moore, Eli; Macardle, Peter; Delalat, Bahman; Rogers, Mary-Louise; Johns, Terrance G; Durand, Jean-Olivier; Cunin, Frédérique; Voelcker, Nicolas H

2013-05-01

We describe the preparation of biodegradable porous silicon nanoparticles (pSiNP) functionalized with cancer cell targeting antibodies and loaded with the hydrophobic anti-cancer drug camptothecin. Orientated immobilization of the antibody on the pSiNP is achieved using novel semicarbazide based bioconjugate chemistry. To demonstrate the generality of this targeting approach, the three antibodies MLR2, mAb528 and Rituximab are used, which target neuroblastoma, glioblastoma and B lymphoma cells, respectively. Successful targeting is demonstrated by means of flow cytometry and immunocytochemistry both with cell lines and primary cells. Cell viability assays after incubation with pSiNPs show selective killing of cells expressing the receptor corresponding to the antibody attached on the pSiNP. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Antithyroglobulin Antibodies and Antimicrosomal Antibodies in Various Thyroid Diseases

Energy Technology Data Exchange (ETDEWEB)

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

1979-03-15

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

78 FR 18999 - Prospective Grant of Start-Up Exclusive License: Photosensitizing Antibody-Fluorophore Conjugates...

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2013-03-28

...-Up Exclusive License: Photosensitizing Antibody-Fluorophore Conjugates for Photoimmunotherapy AGENCY...-205-2010/0-US-01), and entitled ``Photosensitizing Antibody- Fluorophore Conjugates,'' to Aspyrian... invention. The field of use may be limited to ``use of photosensitizing antibody-fluorophore conjugate by...

Integration of Antibody Array Technology into Drug Discovery and Development.

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Huang, Wei; Whittaker, Kelly; Zhang, Huihua; Wu, Jian; Zhu, Si-Wei; Huang, Ruo-Pan

Antibody arrays represent a high-throughput technique that enables the parallel detection of multiple proteins with minimal sample volume requirements. In recent years, antibody arrays have been widely used to identify new biomarkers for disease diagnosis or prognosis. Moreover, many academic research laboratories and commercial biotechnology companies are starting to apply antibody arrays in the field of drug discovery. In this review, some technical aspects of antibody array development and the various platforms currently available will be addressed; however, the main focus will be on the discussion of antibody array technologies and their applications in drug discovery. Aspects of the drug discovery process, including target identification, mechanisms of drug resistance, molecular mechanisms of drug action, drug side effects, and the application in clinical trials and in managing patient care, which have been investigated using antibody arrays in recent literature will be examined and the relevance of this technology in progressing this process will be discussed. Protein profiling with antibody array technology, in addition to other applications, has emerged as a successful, novel approach for drug discovery because of the well-known importance of proteins in cell events and disease development.

Radiometric immunosorbent assay for the detection of anti-hormone-binding protein antibodies

International Nuclear Information System (INIS)

Pierce, E.A.; Dame, M.C.; DeLuca, H.F.

1986-01-01

A radiometric immunosorbent assay (RISA) for the detection of monoclonal antibodies to hormone-binding proteins has been developed. The assay involves incubating hybridoma supernatants in microtiter wells that have been coated with goat anti-mouse IgG antibodies. Any mouse IgG in the test supernatant is thus specifically retained in the wells. Radioactive ligand-binding protein complexes are then incubated in the wells. The presence of anti-binding protein antibodies in the supernatant is indicated by specific retention of radioactive ligand-binding protein complexes in the wells. Crude antigen preparations, such as tissue homogenates, can be used to detect antibodies. The assay is capable of detecting antibody at concentrations 20 ng/ml (approx. 100 pM IgG). The RISA has been used successfully to screen for monoclonal antibodies to the intracellular receptor for 1,25-dihydroxyvitamin D 3 and should be useful for the detection of antibodies to ligand-binding proteins in general

Affinity chromatography: A versatile technique for antibody purification.

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Arora, Sushrut; Saxena, Vikas; Ayyar, B Vijayalakshmi

2017-03-01

Antibodies continue to be extremely utilized entities in myriad applications including basic research, imaging, targeted delivery, chromatography, diagnostics, and therapeutics. At production stage, antibodies are generally present in complex matrices and most of their intended applications necessitate purification. Antibody purification has always been a major bottleneck in downstream processing of antibodies, due to the need of high quality products and associated high costs. Over the years, extensive research has focused on finding better purification methodologies to overcome this holdup. Among a plethora of different techniques, affinity chromatography is one of the most selective, rapid and easy method for antibody purification. This review aims to provide a detailed overview on affinity chromatography and the components involved in purification. An array of support matrices along with various classes of affinity ligands detailing their underlying working principles, together with the advantages and limitations of each system in purifying different types of antibodies, accompanying recent developments and important practical methodological considerations to optimize purification procedure are discussed. Copyright © 2016 Elsevier Inc. All rights reserved.

Nanoparticles for the delivery of therapeutic antibodies: Dogma or promising strategy?

Science.gov (United States)

Sousa, Flávia; Castro, Pedro; Fonte, Pedro; Kennedy, Patrick J; Neves-Petersen, Maria Teresa; Sarmento, Bruno

2017-10-01

Over the past two decades, therapeutic antibodies have demonstrated promising results in the treatment of a wide array of diseases. However, the application of antibody-based therapy implies multiple administrations and a high cost of antibody production, resulting in costly therapy. Another disadvantage inherent to antibody-based therapy is the limited stability of antibodies and the low level of tissue penetration. The use of nanoparticles as delivery systems for antibodies allows for a reduction in antibody dosing and may represent a suitable alternative to increase antibody stability Areas covered: We discuss different nanocarriers intended for the delivery of antibodies as well as the corresponding encapsulation methods. Recent developments in antibody nanoencapsulation, particularly the possible toxicity issues that may arise from entrapment of antibodies into nanocarriers, are also assessed. In addition, this review will discuss the alterations in antibody structure and bioactivity that occur with nanoencapsulation. Expert opinion: Nanocarriers can protect antibodies from degradation, ensuring superior bioavailability. Encapsulation of therapeutic antibodies may offer some advantages, including potential targeting, reduced immunogenicity and controlled release. Furthermore, antibody nanoencapsulation may aid in the incorporation of the antibodies into the cells, if intracellular components (e.g. intracellular enzymes, oncogenic proteins, transcription factors) are to be targeted.

Desensitization protocol enabling pediatric crossmatch-positive renal transplantation: successful HLA-antibody-incompatible renal transplantation of two highly sensitized children.

Science.gov (United States)

Adamusiak, Anna M; Stojanovic, Jelena; Shaw, Olivia; Vaughan, Robert; Sebire, Neil J; Drage, Martin; Kessaris, Nicos; Marks, Stephen D; Mamode, Nizam

2017-02-01

Renal transplantation improves quality of life (QoL) and survival in children requiring renal replacement therapy (RRT). Sensitization with development of a broad-spectrum of anti-HLA antibodies as a result of previous transplantation or after receiving blood products is an increasing problem. There are no published reports of desensitization protocols in children allowing renal transplantation from HLA-antibody-incompatible living donors. We adopted our well-established adult desensitization protocol for this purpose and undertook HLA antibody-incompatible living donor renal transplants in two children: a 14-year-old girl and a 13-year-old boy. After 2 and 1.5 years of follow-up, respectively, both patients have stable renal allograft function despite a rise in donor-specific antibodies in one case. HLA-incompatible transplantation should be considered in selected cases for sensitized children.

Recent progress of diagnostic and therapeutic approach to cancers using polyclonal or monoclonal antibodies

International Nuclear Information System (INIS)

Koji, Toshihiko

1982-01-01

Among the major topics of interest in cancer immunology, immunodiagnosis and immunotherapy with the antibodies are summarized historically and prospectively. The concept of injecting anti-tumor cell antibodies to localize tumors was first introduced in experimental systems by Pressman (1957). Since then, various trials have been achieved with human tumors using specific or nonspecific tumor-localizing antibodies diagnostically or therapeutically. In 1970's, successes in immunodiagnosis with the antibodies to oncofetal proteins also have been reported. Recently, there are numerous papers dealed with a series of external scanning or serotherapeutic trials by the use of monoclonal antibodies that bind selectively to tumor cells. Various relevant problems with them are discussed. (author)

Bone marrow dosimetry for monoclonal antibody therapy

International Nuclear Information System (INIS)

Bigler, R.E.; Zanzonico, P.B.; Leonard, R.

1986-01-01

Immunoglobulins must permeate through the basement membrane of capillaries in order to enter the extracellular space (ECS) of tissue. Since the process is quite slow, the blood plasma activity in various organs contributes considerably to the radiation dose of the dose-limiting tissues. In bone marrow the basement membrane is absent and the blood circulation is functionally open. Therefore, blood plasma and marrow ECS maintain equal concentrations of labeled immunoglobulins. A combination of factors including intravenous administration, slow absorption into most tissues, slow breakdown and elimination of labeled immunoglobulin, and rapid entry into bone marrow ECS as well as known radiosensitivity of marrow led the authors to expect this tissue would prove to be the primary tissue at risk for systemic monoclonal antibody therapy. They have developed and applied in a Phase I clinical study of 131 I labeled CEA antibody a procedure for estimation of radiation dose to red bone marrow. Serieal measurements of blood plasma and total body retention are carried out. Binding of labeled antibody to the cellular components of blood is verified to be very low. They have observed bone marrow depression at doses greater than 400 rad. If no special procedures are used to reconstitute marrow after radiation treatment, this level represents a much greater than generally recognized limitation to radiolabeled monoclonal antibody therapy. 25 references, 4 tables

Effects of genetic engineering on the pharmacokinetics of antibodies

International Nuclear Information System (INIS)

Colcher, D.; Goel, A.; Pavlinkova, G.; Beresford, G.; Booth, B.; Batra, S.K.

1999-01-01

Monoclonal antibodies (MAbs) may be considered 'magic bullets' due to their ability to recognize and eradicate malignant cells. MAbs, however, have practical limitations for their rapid application in the clinics. The structure of the antibody molecules can be engineered to modify functional domains such as antigen-binding sites and/or effectors functions. Advanced in genetic engineering have provided rapid progress the development of new immunoglobulin constructs of MAbs with defined research and therapeutic application. Recombinant antibody constructs are being engineered, such as human mouse chimeric, domain-dispositioned, domain-deleted, humanized and single-chain Fv fragments. Genetically-engineered antibodies differ in size and rate of catabolism. Pharmacokinetics studies show that the intact IgG (150 kD), enzymatically derived fragments Fab' (50 kD) and single chain Fv (28 kD) have different clearance rates. These antibody forms clear 50% from the blood pool in 2.1 days, 30 minutes and 10 minutes, respectively. Genetically-engineered antibodies make a new class of immunotherapeutic tracers for cancer treatment

Antibody-Based Agents in the Management of Antibiotic-Resistant Staphylococcus aureus Diseases

Science.gov (United States)

Speziale, Pietro; Rindi, Simonetta

2018-01-01

Staphylococcus aureus is a human pathogen that can cause a wide spectrum of diseases, including sepsis, pneumonia, arthritis, and endocarditis. Ineffective treatment of a number of staphylococcal infections with antibiotics is due to the development and spread of antibiotic-resistant strains following decades of antibiotic usage. This has generated renewed interest within the scientific community in alternative therapeutic agents, such as anti-S. aureus antibodies. Although the role of antibodies in the management of S. aureus diseases is controversial, the success of this pathogen in neutralizing humoral immunity clearly indicates that antibodies offer the host extensive protection. In this review, we report an update on efforts to develop antibody-based agents, particularly monoclonal antibodies, and their therapeutic potential in the passive immunization approach to the treatment and prevention of S. aureus infections. PMID:29533985

Site-specific antibody-drug conjugates: the nexus of bioorthogonal chemistry, protein engineering, and drug development.

Science.gov (United States)

Agarwal, Paresh; Bertozzi, Carolyn R

2015-02-18

Antibody-drug conjugates (ADCs) combine the specificity of antibodies with the potency of small molecules to create targeted drugs. Despite the simplicity of this concept, generation of clinically successful ADCs has been very difficult. Over the past several decades, scientists have learned a great deal about the constraints on antibodies, linkers, and drugs as they relate to successful construction of ADCs. Once these components are in hand, most ADCs are prepared by nonspecific modification of antibody lysine or cysteine residues with drug-linker reagents, which results in heterogeneous product mixtures that cannot be further purified. With advances in the fields of bioorthogonal chemistry and protein engineering, there is growing interest in producing ADCs by site-specific conjugation to the antibody, yielding more homogeneous products that have demonstrated benefits over their heterogeneous counterparts in vivo. Here, we chronicle the development of a multitude of site-specific conjugation strategies for assembly of ADCs and provide a comprehensive account of key advances and their roots in the fields of bioorthogonal chemistry and protein engineering.

Purification of antibody against Ara h 2 by a homemade immunoaffinity chromatography column.

Science.gov (United States)

Wu, Zhihua; Li, Kun; Zhan, Shaode; Tong, Ping; Li, Xin; Yang, Anshu; Chen, Hongbing

2017-09-14

Antibodies are used extensively in numerous applications both in vivo and in vitro. To purify anti-Ara h 2 polyclonal antibody, a homemade immunoaffinity chromatography (IAC) column method was established. The properties of homemade column were compared with those of the mAb affinity protein G (MPG) agarose high flow, a commercially available column successfully used in capturing polyclonal antibodies. During antibody purification from rabbits' antiserum against Ara h 2, the column capacity, recovery, and purification factor were characterized for IAC and MPG. The homemade IAC could separate the corresponding antibody with higher specificity and lower cost but with lower recovery and column capacity than those of MPG. Thus, the homemade IAC is a specific, inexpensive, and suitable method that can be used for various laboratory purifications.

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

Science.gov (United States)

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

1992-02-01

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

Human Cell Line-Derived Monoclonal IgA Antibodies for Cancer Immunotherapy

Directory of Open Access Journals (Sweden)

Felix Hart

2017-05-01

Full Text Available IgA antibodies have great potential to improve the functional diversity of current IgG antibody-based cancer immunotherapy options. However, IgA production and purification is not well established, which can at least in part be attributed to the more complex glycosylation as compared to IgG antibodies. IgA antibodies possess up to five N-glycosylation sites within their constant region of the heavy chain as compared to one site for IgG antibodies. The human GlycoExpress expression system was developed to produce biotherapeutics with optimized glycosylation and used here to generate a panel of IgA isotype antibodies directed against targets for solid (TA-mucin 1, Her2, EGFR, Thomsen–Friedenreich and hematological (CD20 cancer indications. The feasibility of good manufacturing practice was shown by the production of 11 g IgA within 35 days in a one liter perfusion bioreactor, and IgA antibodies in high purity were obtained after purification. The monoclonal IgA antibodies possessed a high sialylation degree, and no non-human glycan structures were detected. Kinetic analysis revealed increased avidity antigen binding for IgA dimers as compared to monomeric antibodies. The IgA antibodies exhibited potent Fab- and Fc-mediated functionalities against cancer cell lines, whereby especially granulocytes are recruited. Therefore, for patients who do not sufficiently benefit from therapeutic IgG antibodies, IgA antibodies may complement current regiment options and represent a promising strategy for cancer immunotherapy. In conclusion, a panel of novel biofunctional IgA antibodies with human glycosylation was successfully generated.

Small founding number and low genetic diversity in an introduced species exhibiting limited invasion success (speckled dace, Rhinichthys ocsulus)

Science.gov (United States)

Andrew Kinziger; Rodney Nakamoto; Eric Anderson; Bret Harvey

2011-01-01

Molecular evaluations of successful invaders are common, however studies of introduced species that have had limited invasion success, or have died out completely, are rare. We studied an introduced population of speckled dace (Rhinichthys osculus) from northern California, USA that has rapidly increased in abundance but remained restricted to a 25-km stretch of river...

Conjugation of E. coli O157:H7 Antibody to CdSe/ZnS Quantum Dots

Directory of Open Access Journals (Sweden)

N. T. Vo

2015-01-01

Full Text Available The conjugation of antibody to semiconductor quantum dots plays a very important role in many applications such as bioimaging, biomarking, and biosensing. In this research, we present some results of highly luminescent core/shell structure CdSe/ZnS on which the E. coli antibody was conjugated. The CdSe core was synthesized successfully with chemical “green” method. For biological applications, the capping surfactant, trioctylphosphine oxide, was substituted by a new one, mercaptopropionic acid (MPA, before the antibody attachment step. Finally, the E. coli antibody was attached to quantum dots CdSe/ZnS. Morphology, structure, and optical properties were investigated with PL, UV-Vis, TEM, and XRD methods. The successful ligand substitution and antibody attachment were confirmed by zeta potential measurement, FTIR spectroscopy, and TEM. The results showed quantum dots size of 2.3 nm, uniform distribution, and high luminescence. CdSe/ZnS core/shell structure had better stability and enhanced the luminescence efficiency up to threefold compared with the core CdSe. MPA ligand shifted the initial hydrophobic quantum dots to hydrophilic ones, which helped to dissolve them in organic solvents and attach the antibody.

Protective antibodies against placental malaria and poor outcomes during pregnancy, Benin

DEFF Research Database (Denmark)

Ndam, Nicaise Tuikue; Denoeud-Ndam, Lise; Doritchamou, Justin

2015-01-01

Placental malaria is caused by Plasmodium falciparum-infected erythrocytes that bind to placental tissue. Binding is mediated by VAR2CSA, a parasite antigen coded by the var gene, which interacts with chondroitin sulfate A (CSA). Consequences include maternal anemia and fetal growth retardation....... Antibody-mediated immunity to placental malaria is acquired during successive pregnancies, but the target of VAR2CSA-specific protective antibodies is unclear. We assessed VAR2CSA-specific antibodies in pregnant women and analyzed their relationships with protection against placental infection, preterm...... birth, and low birthweight. Antibody responses to the N-terminal region of VAR2CSA during early pregnancy were associated with reduced risks for infections and low birthweight. Among women infected during pregnancy, an increase in CSA binding inhibition was associated with reduced risks for placental...

[Progress of research on genetic engineering antibody and its application in prevention and control of parasitic diseases].

Science.gov (United States)

Yao, Yuan; Yu, Chuan-xin

2013-08-01

Antibody has extensive application prospects in the biomedical field. The inherent disadvantages of traditional polyclonal antibody and monoclonal antibody limit their application values. The humanized and fragmented antibody remodeling has given a rise to a series of genetic engineered antibody variant. This paper reviews the progress of research on genetic engineering antibody and its application in prevention and control of parasitic diseases.

Homogeneous plate based antibody internalization assay using pH sensor fluorescent dye.

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Nath, Nidhi; Godat, Becky; Zimprich, Chad; Dwight, Stephen J; Corona, Cesear; McDougall, Mark; Urh, Marjeta

2016-04-01

Receptor-mediated antibody internalization is a key mechanism underlying several anti-cancer antibody therapeutics. Delivering highly toxic drugs to cancer cells, as in the case of antibody drug conjugates (ADCs), efficient removal of surface receptors from cancer cells and changing the pharmacokinetics profile of the antibody drugs are some of key ways that internalization impacts the therapeutic efficacy of the antibodies. Over the years, several techniques have been used to study antibody internalization including radiolabels, fluorescent microscopy, flow cytometry and cellular toxicity assays. While these methods allow analysis of internalization, they have limitations including a multistep process and limited throughput and are generally endpoint assays. Here, we present a new homogeneous method that enables time and concentration dependent measurements of antibody internalization. The method uses a new hydrophilic and bright pH sensor dye (pHAb dye), which is not fluorescent at neutral pH but becomes highly fluorescent at acidic pH. For receptor mediated antibody internalization studies, antibodies against receptors are conjugated with the pHAb dye and incubated with the cells expressing the receptors. Upon binding to the receptor, the dyes conjugated to the antibody are not fluorescent because of the neutral pH of the media, but upon internalization and trafficking into endosomal and lysosomal vesicles the pH drops and dyes become fluorescent. The enabling attributes of the pHAb dyes are the hydrophilic nature to minimize antibody aggregation and bright fluorescence at acidic pH which allows development of simple plate based assays using a fluorescent reader. Using two different therapeutic antibodies--Trastuzumab (anti-HER2) and Cetuximab (anti-EGFR)--we show labeling with pHAb dye using amine and thiol chemistries and impact of chemistry and dye to antibody ration on internalization. We finally present two new approaches using the pHAb dye, which will be

Antithyroid microsomal antibody

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Thyroid antimicrosomal antibody; Antimicrosomal antibody; Microsomal antibody; Thyroid peroxidase antibody; TPOAb ... Granulomatous thyroiditis Hashimoto thyroiditis High levels of these antibodies have also been linked with an increased risk ...

Advances in recombinant antibody manufacturing.

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Kunert, Renate; Reinhart, David

2016-04-01

Since the first use of Chinese hamster ovary (CHO) cells for recombinant protein expression, production processes have steadily improved through numerous advances. In this review, we have highlighted several key milestones that have contributed to the success of CHO cells from the beginning of their use for monoclonal antibody (mAb) expression until today. The main factors influencing the yield of a production process are the time to accumulate a desired amount of biomass, the process duration, and the specific productivity. By comparing maximum cell densities and specific growth rates of various expression systems, we have emphasized the limiting parameters of different cellular systems and comprehensively described scientific approaches and techniques to improve host cell lines. Besides the quantitative evaluation of current systems, the quality-determining properties of a host cell line, namely post-translational modifications, were analyzed and compared to naturally occurring polyclonal immunoglobulin fractions from human plasma. In summary, numerous different expression systems for mAbs are available and also under scientific investigation. However, CHO cells are the most frequently investigated cell lines and remain the workhorse for mAb production until today.

Intracellular trafficking of new anticancer therapeutics: antibody-drug conjugates.

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Kalim, Muhammad; Chen, Jie; Wang, Shenghao; Lin, Caiyao; Ullah, Saif; Liang, Keying; Ding, Qian; Chen, Shuqing; Zhan, Jinbiao

2017-01-01

Antibody-drug conjugate (ADC) is a milestone in targeted cancer therapy that comprises of monoclonal antibodies chemically linked to cytotoxic drugs. Internalization of ADC takes place via clathrin-mediated endocytosis, caveolae-mediated endocytosis, and pinocytosis. Conjugation strategies, endocytosis and intracellular trafficking optimization, linkers, and drugs chemistry present a great challenge for researchers to eradicate tumor cells successfully. This inventiveness of endocytosis and intracellular trafficking has given considerable momentum recently to develop specific antibodies and ADCs to treat cancer cells. It is significantly advantageous to emphasize the endocytosis and intracellular trafficking pathways efficiently and to design potent engineered conjugates and biological entities to boost efficient therapies enormously for cancer treatment. Current studies illustrate endocytosis and intracellular trafficking of ADC, protein, and linker strategies in unloading and also concisely evaluate practically applicable ADCs.

Development of Antibody-Based Vaccines Targeting the Tumor Vasculature.

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Zhuang, Xiaodong; Bicknell, Roy

2016-01-01

A functional vasculature is essential for tumor progression and malignant cell metastasis. Endothelial cells lining blood vessels in the tumor are exposed to a unique microenvironment, which in turn induces expression of specific proteins designated as tumor endothelial markers (TEMs). TEMs either localized at the plasma membrane or secreted into the extracellular matrix are accessible for antibody targeting, which can be either infused or generated de novo via vaccination. Recent studies have demonstrated vaccines against several TEMs can induce a strong antibody response accompanied by a potent antitumor effect in animal models. These findings present an exciting field for novel anticancer therapy development. As most of the TEMs are self-antigens, breaking tolerance is necessary for a successful vaccine. This chapter describes approaches to efficiently induce a robust antibody response against the tumor vasculature.

A cocktail of humanized anti-pertussis toxin antibodies limits disease in murine and baboon models of whooping cough.

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Nguyen, Annalee W; Wagner, Ellen K; Laber, Joshua R; Goodfield, Laura L; Smallridge, William E; Harvill, Eric T; Papin, James F; Wolf, Roman F; Padlan, Eduardo A; Bristol, Andy; Kaleko, Michael; Maynard, Jennifer A

2015-12-02

Despite widespread vaccination, pertussis rates are rising in industrialized countries and remain high worldwide. With no specific therapeutics to treat disease, pertussis continues to cause considerable infant morbidity and mortality. The pertussis toxin is a major contributor to disease, responsible for local and systemic effects including leukocytosis and immunosuppression. We humanized two murine monoclonal antibodies that neutralize pertussis toxin and expressed them as human immunoglobulin G1 molecules with no loss of affinity or in vitro neutralization activity. When administered prophylactically to mice as a binary cocktail, antibody treatment completely mitigated the Bordetella pertussis-induced rise in white blood cell counts and decreased bacterial colonization. When administered therapeutically to baboons, antibody-treated, but not untreated control animals, experienced a blunted rise in white blood cell counts and accelerated bacterial clearance rates. These preliminary findings support further investigation into the use of these antibodies to treat human neonatal pertussis in conjunction with antibiotics and supportive care. Copyright © 2015, American Association for the Advancement of Science.

Ranitidine improves postoperative suppression of antibody response to preoperative vaccination

DEFF Research Database (Denmark)

Nielsen, Hans Jørgen; Hammer, J H; Moesgaard, F

1992-01-01

The effect of the histamine-2 receptor antagonist ranitidine (100 mg intravenously every 12 hours for 72 hours) on postoperative serum antibody responses to preoperative immunization with six limit of flocculation tetanus toxoid and six limit of flocculation diphtheria toxoid was assessed...... in a double-blind, placebo-controlled randomized study in 26 patients undergoing major abdominal surgery. The preoperative antitetanus antibody level was less than 0.1 IU/ml in all patients, and they were inoculated with both antigens 48 hours before surgery. Serum samples for analysis of antitetanus toxoid...... and antidiphtheria toxoid were drawn before skin incision and on postoperative days 1, 3, 5, 7, 10, 14, 21, and 28. Ranitidine significantly increased the postoperative antibody response to tetanus toxoid, (p less than 0.01) and insignificantly increased that to diphtheria toxoid vaccination (p less than 0...

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

International Nuclear Information System (INIS)

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

1996-01-01

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

Antiprothrombin Antibodies

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Polona Žigon

2015-05-01

Full Text Available In patients with the antiphospholipid syndrome (APS, the presence of a group of pathogenic autoantibodies called antiphospholipid antibodies causes thrombosis and pregnancy complications. The most frequent antigenic target of antiphospholipid antibodies are phospholipid bound β2-glycoprotein 1 (β2GPI and prothrombin. The international classification criteria for APS connect the occurrence of thrombosis and/or obstetric complications together with the persistence of lupus anticoagulant, anti-cardiolipin antibodies (aCL and antibodies against β2GPI (anti-β2GPI into APS. Current trends for the diagnostic evaluation of APS patients propose determination of multiple antiphospholipid antibodies, among them also anti-prothrombin antibodies, to gain a common score which estimates the risk for thrombosis in APS patients. Antiprothrombin antibodies are common in APS patients and are sometimes the only antiphospholipid antibodies being elevated. Methods for their determination differ and have not yet been standardized. Many novel studies confirmed method using phosphatidylserine/prothrombin (aPS/PT ELISA as an antigen on solid phase encompass higher diagnostic accuracy compared to method using prothrombin alone (aPT ELISA. Our research group developed an in-house aPS/PT ELISA with increased analytical sensitivity which enables the determination of all clinically relevant antiprothrombin antibodies. aPS/PT exhibited the highest percentage of lupus anticoagulant activity compared to aCL and anti-β2GPI. aPS/PT antibodies measured with the in-house method associated with venous thrombosis and presented the strongest independent risk factor for the presence of obstetric complications among all tested antiphospholipid antibodies. 

[Construction of a phage antibody library and screening of anti-epidermal growth factor receptor variant III single chain antibody].

Science.gov (United States)

Han, Dong-gang; Duan, Xiao-yi; Guo, You-min; Zhou, Qi; Wang, Quan-ying; Yang, Guang-xiao

2010-01-01

To obtain specific anti-epidermal growth factor receptor variant III (EGFRvIII) single chain antibody (ScFv) by phage antibody library display system. The total RNA was extracted from the spleen B cells of BALB/c mice immunized with pep-3-OVA protein, and the first-strand cDNA was synthesized by reverse transcription. Antibody VH and VL gene fragments were amplified and joined to a ScFv gene with the linker. The ScFv gene was ligated into the phagemid vector pCANTAB5E, which was transformed into competent E. coli TG1. The transformed cells were then infected with M13KO7 helper phage to yield the recombinant phage to construct the phage ScFv library. Pep-3-BSA protein was used to screen the phage antibody library and ELISA carried out to characterize the activity of the antibody. The VH and VL gene fragments of the antibody were about 350 bp and 320 bp in length as analyzed by agarose gel electrophoresis. The ScFv gene was 780 bp, consistent with the expected length. The recombinant phagemid with ScFv gene insert was rescued, and an immune phage ScFv library with the content of 5.0x10(6) was constructed. The recombinant ScFv phage had a titer of 3.0x10(4) cfu/ml, and the fourth phage harvest yielded 56 times as much as that of the first one. SDS-PAGE demonstrated a molecular mass of the soluble ScFv of about 28 kD. ELISA results indicated good specificity of the ScFv to bind EGFRvIII. An immune phage ScFv library is successfully constructed, and the ScFv antibody fragment is capable of specific binding to EGFRvIII.

Nanobodies and Nanobody-Based Human Heavy Chain Antibodies As Antitumor Therapeutics

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Peter Bannas

2017-11-01

Full Text Available Monoclonal antibodies have revolutionized cancer therapy. However, delivery to tumor cells in vivo is hampered by the large size (150 kDa of conventional antibodies. The minimal target recognition module of a conventional antibody is composed of two non-covalently associated variable domains (VH and VL. The proper orientation of these domains is mediated by their hydrophobic interface and is stabilized by their linkage to disulfide-linked constant domains (CH1 and CL. VH and VL domains can be fused via a genetic linker into a single-chain variable fragment (scFv. scFv modules in turn can be fused to one another, e.g., to generate a bispecific T-cell engager, or they can be fused in various orientations to antibody hinge and Fc domains to generate bi- and multispecific antibodies. However, the inherent hydrophobic interaction of VH and VL domains limits the stability and solubility of engineered antibodies, often causing aggregation and/or mispairing of V-domains. Nanobodies (15 kDa and nanobody-based human heavy chain antibodies (75 kDa can overcome these limitations. Camelids naturally produce antibodies composed only of heavy chains in which the target recognition module is composed of a single variable domain (VHH or Nb. Advantageous features of nanobodies include their small size, high solubility, high stability, and excellent tissue penetration in vivo. Nanobodies can readily be linked genetically to Fc-domains, other nanobodies, peptide tags, or toxins and can be conjugated chemically at a specific site to drugs, radionuclides, photosensitizers, and nanoparticles. These properties make them particularly suited for specific and efficient targeting of tumors in vivo. Chimeric nanobody-heavy chain antibodies combine advantageous features of nanobodies and human Fc domains in about half the size of a conventional antibody. In this review, we discuss recent developments and perspectives for applications of nanobodies and nanobody

Nanobodies and Nanobody-Based Human Heavy Chain Antibodies As Antitumor Therapeutics.

Science.gov (United States)

Bannas, Peter; Hambach, Julia; Koch-Nolte, Friedrich

2017-01-01

Monoclonal antibodies have revolutionized cancer therapy. However, delivery to tumor cells in vivo is hampered by the large size (150 kDa) of conventional antibodies. The minimal target recognition module of a conventional antibody is composed of two non-covalently associated variable domains (VH and VL). The proper orientation of these domains is mediated by their hydrophobic interface and is stabilized by their linkage to disulfide-linked constant domains (CH1 and CL). VH and VL domains can be fused via a genetic linker into a single-chain variable fragment (scFv). scFv modules in turn can be fused to one another, e.g., to generate a bispecific T-cell engager, or they can be fused in various orientations to antibody hinge and Fc domains to generate bi- and multispecific antibodies. However, the inherent hydrophobic interaction of VH and VL domains limits the stability and solubility of engineered antibodies, often causing aggregation and/or mispairing of V-domains. Nanobodies (15 kDa) and nanobody-based human heavy chain antibodies (75 kDa) can overcome these limitations. Camelids naturally produce antibodies composed only of heavy chains in which the target recognition module is composed of a single variable domain (VHH or Nb). Advantageous features of nanobodies include their small size, high solubility, high stability, and excellent tissue penetration in vivo . Nanobodies can readily be linked genetically to Fc-domains, other nanobodies, peptide tags, or toxins and can be conjugated chemically at a specific site to drugs, radionuclides, photosensitizers, and nanoparticles. These properties make them particularly suited for specific and efficient targeting of tumors in vivo . Chimeric nanobody-heavy chain antibodies combine advantageous features of nanobodies and human Fc domains in about half the size of a conventional antibody. In this review, we discuss recent developments and perspectives for applications of nanobodies and nanobody-based human heavy

IgE antibodies in toxoplasmosis.

Science.gov (United States)

Matowicka-Karna, Joanna; Kemona, Halina

2014-05-15

Toxoplasmosis is a worldwide infection caused by the intracellular parasite Toxoplasma gondii. At least a third of the world human population is infected with the parasite, making it one of the most successful parasitic infections. Primary maternal infection may cause health-threatening sequelae for the fetus, or even cause death of the uterus. Reactivation of a latent infection in immune deficiency conditions such as AIDS and organ transplantation can cause fatal toxoplasmic encephalitis. Toxoplasmosis is a major cause of chorioretinitis, especially in individuals with impaired immune systems. In the acute phase, directly after invading the body, T. gondii begins to multiply rapidly. In the majority of cases acquired toxoplasmosis is asymptomatic. In the second week of infection, specific IgM antibodies are present in the blood. IgE antibodies appear at the same time, slightly preceding specific IgA antibodies. The concentration of IgE can be one of the parameters used for diagnosing an infection with T. gondii. Laboratory diagnosis, i.e. IgE and serologic assays, plays the main role in the diagnosis of congenital infection and assists in the confirmatory diagnosis of toxoplasmic encephalitis and ocular toxoplasmosis. This article is a review of IgE in toxoplasmosis.

Human antibody technology and the development of antibodies against cytomegalovirus.

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Ohlin, Mats; Söderberg-Nauclér, Cecilia

2015-10-01

Cytomegalovirus (CMV) is a virus that causes chronic infections in a large set of the population. It may cause severe disease in immunocompromised individuals, is linked to immunosenescence and implied to play an important role in the pathogenesis of cardiovascular diseases and cancer. Modulation of the immune system's abilities to manage the virus represent a highly viable therapeutic option and passive immunotherapy with polyclonal antibody preparations is already in clinical use. Defined monoclonal antibodies offer many advantages over polyclonal antibodies purified from serum. Human CMV-specific monoclonal antibodies have consequently been thoroughly investigated with respect to their potential in the treatment of diseases caused by CMV. Recent advances in human antibody technology have substantially expanded the breadth of antibodies for such applications. This review summarizes the fundamental basis for treating CMV disease by use of antibodies, the basic technologies to be used to develop such antibodies, and relevant human antibody specificities available to target this virus. Copyright © 2015 Elsevier Ltd. All rights reserved.

Formation of infectious dengue virus-antibody immune complex in vivo in marmosets (Callithrix jacchus) after passive transfer of anti-dengue virus monoclonal antibodies and infection with dengue virus.

Science.gov (United States)

Moi, Meng Ling; Ami, Yasushi; Shirai, Kenji; Lim, Chang-Kweng; Suzaki, Yuriko; Saito, Yuka; Kitaura, Kazutaka; Saijo, Masayuki; Suzuki, Ryuji; Kurane, Ichiro; Takasaki, Tomohiko

2015-02-01

Infection with a dengue virus (DENV) serotype induces cross-reactive, weakly neutralizing antibodies to different dengue serotypes. It has been postulated that cross-reactive antibodies form a virus-antibody immune complex and enhance DENV infection of Fc gamma receptor (FcγR)-bearing cells. We determined whether infectious DENV-antibody immune complex is formed in vivo in marmosets after passive transfer of DENV-specific monoclonal antibody (mAb) and DENV inoculation and whether infectious DENV-antibody immune complex is detectable using FcγR-expressing cells. Marmosets showed that DENV-antibody immune complex was exclusively infectious to FcγR-expressing cells on days 2, 4, and 7 after passive transfer of each of the mAbs (mAb 4G2 and mAb 6B6C) and DENV inoculation. Although DENV-antibody immune complex was detected, contribution of the passively transferred antibody to overall viremia levels was limited in this study. The results indicate that DENV cross-reactive antibodies form DENV-antibody immune complex in vivo, which is infectious to FcγR-bearing cells but not FcγR-negative cells. © The American Society of Tropical Medicine and Hygiene.

Antimitochondrial antibody

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... page: //medlineplus.gov/ency/article/003529.htm Antimitochondrial antibody To use the sharing features on this page, please enable JavaScript. Antimitochondrial antibodies (AMA) are substances ( antibodies ) that form against mitochondria. ...

NMR Detection of Semi-Specific Antibody Interactions in Serum Environments

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Saeko Yanaka

2017-09-01

Full Text Available Although antibody functions are executed in heterogeneous blood streams characterized by molecular crowding and promiscuous intermolecular interaction, detailed structural characterizations of antibody interactions have thus far been performed under homogeneous in vitro conditions. NMR spectroscopy potentially has the ability to study protein structures in heterogeneous environments, assuming that the target protein can be labeled with NMR-active isotopes. Based on our successful development of isotope labeling of antibody glycoproteins, here we apply NMR spectroscopy to characterize antibody interactions in heterogeneous extracellular environments using mouse IgG-Fc as a test molecule. In human serum, many of the HSQC peaks originating from the Fc backbone exhibited attenuation in intensity of various magnitudes. Similar spectral changes were induced by the Fab fragment of polyclonal IgG isolated from the serum, but not by serum albumin, indicating that a subset of antibodies reactive with mouse IgG-Fc exists in human serum without preimmunization. The metaepitopes recognized by serum polyclonal IgG cover the entire molecular surface of Fc, including the binding sites to Fc receptors and C1q. In-serum NMR observation will offer useful tools for the detailed characterization of biopharamaceuticals, including therapeutic antibodies in physiologically relevant heterogeneous environments, also giving deeper insight into molecular recognition by polyclonal antibodies in the immune system.

A review of human anti-globulin antibody (HAGA, HAMA, HACA, HAHA) responses to monoclonal antibodies. Not four letter words.

Science.gov (United States)

Mirick, G R; Bradt, B M; Denardo, S J; Denardo, G L

2004-12-01

The United States Food and Drug Administration (FDA) has approved unconjugated monoclonal antibodies (MAbs) for immunotherapy (IT) of B-cell lymphoma, breast cancer and acute myeloid leukemia. More recently, approval has been given for conjugated ZevalinTM ((90)yttrium ibritumomab tiuxetan, IDEC-Y2B8, Biogen Idec, Cambridge, MA) and BexxarTM ((131)I-tositumomab, Corixa, Corp., Seattle, WA and GlaxoSmithKline, Philadelphia, PA) anti-CD20 MAbs for use in radioimmunotherapy (RIT) of non-Hodgkin's lymphoma (NHL), thus redefining the standard care of cancer patients. Because of, and despite a lack of basis for concern about allergic reactions due to human antibody responses to these foreign proteins, assays were developed to determine HAGA (human anti-globulin antibody) levels that developed in patient sera following treatment with MAbs. Strategies were also devised to ''humanize'' MAbs and to temporarily block patient immune function with drugs in order to decrease the seroconversion rates, with considerable success. On the other hand, a survival advantage has been observed in some patients who developed a HAGA following treatment. This correlates with development of an anti-idiotype antibody cascade directed toward the MAbs used to treat these patients. What follows is a selective review of HAGA and its effect on cancer treatment over the past 2 decades.

A review of human anti-globulin antibody (HAGA, HAMA, HACA, HAHA) responses to monoclonal antibodies. Not four letter words

International Nuclear Information System (INIS)

Mirick, G. R.; Bradt, B. M.; Denardo, S. J.; Denardo, G. L.

2004-01-01

The United States Food and Drugs Administration (FDA) has approved unconjugated monoclonal antibodies (MAbs) for immunotherapy (IT) of B-cell lymphoma, breast cancer and acute myeloid leukemia. More recently, approval has been given for conjugated ZevalinTM ( 9 0yttrium ibritumomab tiuxetan, IDEC-Y2B8, Biogen Idec, Cambridge, MA) and BexxarTM ( 1 31I-tositumomab, Corixa, Corp., Seattle, WA and GlaxoSmithKline, Philadelphia, PA) antiCD20 MAns for use in radioimmunotherapy (RIT) of non-Hodgikin's lymphoma (NHL), thus redefining the standard care of cancer patients. Because of, and despite a lack of basis for concern about allergic reactions due to human antibody responses to these foreign proteins, essays were developed to determine HAGE (human anti-globulin antibody) levels that developed in patient sera following treatment with MAbs. Strategies were also devised to humanize MAbs and to temporarily block patient immune function with drugs in order to decrease the seroconversion rates, with considerable success. On the other hand, a survival advantage has been observed in some patients who developed a HAGA following treatment. This correlates with development of an anti-idiotype antibody cascade directed toward the MAbs used to treat these patients. What follows is a selective review of HAGA and its effect on cancer treatment over the past 2 decades

Immobilization and functional reconstitution of antibody Fab fragment by solid-phase refolding.

Science.gov (United States)

Kumada, Yoichi; Hamasaki, Kyoto; Nakagawa, Aya; Sasaki, Eiju; Shirai, Tatsunori; Okumura, Masahiro; Inoue, Manami; Kishimoto, Michimasa

2013-12-31

In this study, we demonstrated the successful preparation of a Fab antibody-immobilized hydrophilic polystyrene (phi-PS) plate via one- and two-step solid-phase refolding methods. Both polystyrene-binding peptide (PS-tag)-fused Fd fragment of heavy chain (Fab H-PS) and full-length of light-chain (Fab L-PS) were individually produced in insoluble fractions of Escherichia coli cells, and they were highly purified in the presence of 8M of urea. Antigen-binding activities of Fab antibody immobilized were correctly recovered by the one-step solid-phase refolding method that a mixture of Fab H-PS and Fab L-PS was immobilized in the presence of 0.5-2M urea, followed by surface washing of the phi-PS plate with PBST. These results indicate that by genetic fusion of a PS-tag, a complex between Fab H and Fab L was efficiently immobilized on the surface of a phi-PS plate even in the presence of a low concentration of urea, and was then correctly refolded to retain its high antigen-binding activity via removal of the urea. A two-step solid-phase refolding method whereby Fab H-PS and Fab L-PS were successively refolded on the surface of a phi-PS plate also resulted in Fab antibody formation on the plate. Furthermore, both the binding affinity and the specificity of the Fab antibody produced by the two-step method were highly maintained, according to the results of sandwich ELISA and competitive ELISA using Fab antibody-immobilized plate via two-step solid-phase refolding. Thus, the solid-phase refolding method demonstrated in this study should be quite useful for the preparation of a Fab antibody-immobilized PS surface with high efficiency from individually produced Fab H-PS and Fab L-PS. This method will be applicable to the preparation of a large Fab antibody library on the surface of a PS plate for use in antibody screening. © 2013. Published by Elsevier B.V. All rights reserved.

Unexplained infertility: identification of anti sperm antibodies using radiometric immunoassay

International Nuclear Information System (INIS)

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

1992-01-01

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

The development of methods for obtaining monoclonal antibody-producing cells

Directory of Open Access Journals (Sweden)

Michał Skowicki

2016-04-01

Full Text Available Monoclonal antibodies (mAbs are biomolecules of great scientific and practical significance. In contrast to polyclonal antibodies from immune sera, they are homogeneous and monospecific, since they are produced by hybridoma cells representing a clone arising from a single cell. The successful technology was described for the first time in 1975; the inventors were later awarded the Nobel Prize. Currently, mAbs are broadly used as a research tool, in diagnostics and medicine in particular for the treatment of cancer or in transplantology. About 47 therapeutics based on monoclonal antibodies are now available in the US and Europe, and the number is still growing. Production of monoclonal antibodies is a multistage, time-consuming and costly process. Growing demand for these molecules creates space for research focused on improvements in hybridoma technology. Lower costs, human labor, and time are important goals of these attempts. In this article, a brief review of current methods and their advances is given.

Cancer imaging with CEA antibodies: historical and current perspectives.

Science.gov (United States)

Goldenberg, D M

1992-01-01

This article reviews the history and status of cancer imaging with radiolabeled antibodies against carcinoembryonic antigen (CEA). Although CEA and many other cancer-associated antigens are not distinct for neoplasia, the quantitative increase of these markers in malignant tissues provides a sufficient differential for selective antibody targeting. Animal studies with xenografted human tumors provided the first evidence of the prospects of this technology, followed by initial clinical success with purified goat whole IgG antibodies to CEA, labeled with 131I and with the use of dual-isotope subtraction methods. Subsequently, improved and earlier imaging could be accomplished with monoclonal antibody fragments, which then would permit the use of shorter-lived radionuclides, such as 111In, 123I, and 99mTc. The preferred use of a monoclonal anti-CEA IgG Fab' fragment, labeled with 99mTc by a recently developed, simple and rapid kit, has enabled the detection of small lesions, including those in the liver, within 4 h of injection. By means of SPECT imaging, a high sensitivity and specificity for RAID could be achieved.

Selection of phage-displayed accessible recombinant targeted antibodies (SPARTA): methodology and applications.

Science.gov (United States)

D'Angelo, Sara; Staquicini, Fernanda I; Ferrara, Fortunato; Staquicini, Daniela I; Sharma, Geetanjali; Tarleton, Christy A; Nguyen, Huynh; Naranjo, Leslie A; Sidman, Richard L; Arap, Wadih; Bradbury, Andrew Rm; Pasqualini, Renata

2018-05-03

We developed a potentially novel and robust antibody discovery methodology, termed selection of phage-displayed accessible recombinant targeted antibodies (SPARTA). This combines an in vitro screening step of a naive human antibody library against known tumor targets, with in vivo selections based on tumor-homing capabilities of a preenriched antibody pool. This unique approach overcomes several rate-limiting challenges to generate human antibodies amenable to rapid translation into medical applications. As a proof of concept, we evaluated SPARTA on 2 well-established tumor cell surface targets, EphA5 and GRP78. We evaluated antibodies that showed tumor-targeting selectivity as a representative panel of antibody-drug conjugates (ADCs) and were highly efficacious. Our results validate a discovery platform to identify and validate monoclonal antibodies with favorable tumor-targeting attributes. This approach may also extend to other diseases with known cell surface targets and affected tissues easily isolated for in vivo selection.

Radioimmunoassay of IgM, IgG, and IgA brucella antibodies

International Nuclear Information System (INIS)

Parrett, D.; Nielson, K.H.; White, R.G.; Payne, D.J.H.

1977-01-01

A radioimmunoassay (R.I.A.) has been devised to measure the serum antibody against Brucella abortus in each of the immunoglobulin classes IgM, IgG, and IgA. This test was applied to 46 sera from individuals with various clinical types of brucellosis, and the results were compared with the results of conventional direct and indirect agglutination and complement-fixation tests. The R.I.A. provided a highly sensitive primary-type assay which avoided the difficulties with blocking or non-agglutinating antibody, and thus has many advantages in the diagnosis of acute and chronic stages of brucella infection in man. The R.I.A. was successful in detection of antibody in many instances in which conventional serological tests were negative, and such antibody could (if IgM) be associated with acute or (if IgG or IgA) with chronic cases of brucellosis. One case in which B.abortus was isolated by blood culture but which failed to yield antibody by conventional tests, nevertheless showed substantial levels of IgM and IgG antibody by R.I.A. In other cases the R.I.A. test helped to eliminate the diagnosis of brucellosis by revealing absent or low antibody levels. (author)

Antibody neutralization of retargeted measles viruses

Science.gov (United States)

Lech, Patrycja J.; Pappoe, Roland; Nakamura, Takafumi; Tobin, Gregory J.; Nara, Peter L.; Russell, Stephen J.

2014-01-01

The measles virus (MV) vaccine lineage is a promising oncolytic but prior exposure to the measles vaccine or wild-type MV strains limits treatment utility due to the presence of anti-measles antibodies. MV entry can be redirected by displaying a polypeptide ligand on the Hemagglutinin (H) C-terminus. We hypothesized that retargeted MV would escape neutralization by monoclonal antibodies (mAbs) recognizing the H receptor-binding surface and be less susceptible to neutralization by human antisera. Using chimeric H proteins, with and without mutations that ablate MV receptor binding, we show that retargeted MVs escape mAbs that target the H receptor-binding surface by virtue of mutations that ablate infection via SLAM and CD46. However, C-terminally displayed domains do not mediate virus entry in the presence of human antibodies that bind to the underlying H domain. In conclusion, utility of retargeted oncolytic measles viruses does not extend to evasion of human serum neutralization. PMID:24725950

ANTI·RED CELL ACTIVITY OF LYMPHOCYTOTOXIC ANTIBODIES ...

African Journals Online (AJOL)

1971-05-08

May 8, 1971 ... it can be concluded that the Iymphocytes also share anti- gens with thymocytes .... antibodies, yet seem to lack the power to prolong graft survival, indicates ..... This limitation affects our knowledge of its in vivo activity when ...

Single-Domain Antibodies As Versatile Affinity Reagents for Analytical and Diagnostic Applications

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Gualberto Gonzalez-Sapienza

2017-08-01

Full Text Available With just three CDRs in their variable domains, the antigen-binding site of camelid heavy-chain-only antibodies (HcAbs has a more limited structural diversity than that of conventional antibodies. Even so, this does not seem to limit their specificity and high affinity as HcAbs against a broad range of structurally diverse antigens have been reported. The recombinant form of their variable domain [nanobody (Nb] has outstanding properties that make Nbs, not just an alternative option to conventional antibodies, but in many cases, these properties allow them to reach analytical or diagnostic performances that cannot be accomplished with conventional antibodies. These attributes include comprehensive representation of the immune specificity in display libraries, easy adaptation to high-throughput screening, exceptional stability, minimal size, and versatility as affinity building block. Here, we critically reviewed each of these properties and highlight their relevance with regard to recent developments in different fields of immunosensing applications.

Comparative tumour localization properties of radiolabelled monoclonal antibody preparations of defined immunoreactivities

International Nuclear Information System (INIS)

Pimm, M.V.; Baldwin, R.W.

1987-01-01

The immunoreactive fraction of an anti-CEA monoclonal antibody preparation has been progressively decreased by the addition of increasing proportions of impurity in the form of immunologically inert mouse immunoglobulin. Following radioiodination, the immunoreactive fractions of the preparations were determined and their localization in a human tumour xenograft in nude mice was assessed. There was a progressive decline in tumour localization, from tumour to blood ratios of 2:1 with unadulterated antibody to 0.6:1 with preparations only 15% with respect to the initial antibody. These findings demonstrate that the immunoreactive fraction of monoclonal antibody preparations is a major limiting factor in tumour localization and this has implications for experimental and clinical applications of monoclonal antibodies. (orig.)

Challenges for bovine viral diarrhoea virus antibody detection in bulk milk by antibody enzyme-linked immunosorbent assays due to changes in milk production levels

DEFF Research Database (Denmark)

Foddai, Alessandro; Enøe, Claes; Stockmarr, Anders

2015-01-01

Background: Bovine viral diarrhoea (BVD) is considered eradicated from Denmark. Currently, very few (if any) Danish cattle herds could be infected with BVD virus (BVDV). The Danish antibody blocking enzyme-linked immunosorbent assay (ELISA) has been successfully used during the Danish BVD eradica...

Immobilization of antibodies and enzyme-labeled antibodies by radiation polymerization

International Nuclear Information System (INIS)

Kumakura, M.; Kaetsu, I.; Suzuki, M.; Adachi, S.

1983-01-01

Immobilization of antibodies and enzyme-labeled antibodies by radiation polymerization at low temperatures was studied. The antibody activity of antibody was not affected by irradiation at an irradiation dose of below 8 MR and low temperatures. Immobilization of peroxidase-labeled anti-rabbit IgG goat IgG, anti-peroxidase, peroxidase, and anti-alpha-fetoprotein was carried out with hydrophilic and hydrophobic monomers. The activity of the immobilized enzyme-labeled antibody membranes varied with the thickness of the membranes and increased with decreasing membrane thickness. The activity of the immobilized antibody particles was varied by particle size. Immobilized anti-alpha-fetoprotein particles and membranes can be used for the assay of alpha-fetoprotein by the antigen-antibody reaction, such as a solid-phase sandwich method with high sensitivity

[VGKC-complex antibodies].

Science.gov (United States)

Watanabe, Osamu

2013-04-01

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

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.

Targeted in vivo inhibition of specific protein-protein interactions using recombinant antibodies.

Directory of Open Access Journals (Sweden)

Matej Zábrady

Full Text Available With the growing availability of genomic sequence information, there is an increasing need for gene function analysis. Antibody-mediated "silencing" represents an intriguing alternative for the precise inhibition of a particular function of biomolecules. Here, we describe a method for selecting recombinant antibodies with a specific purpose in mind, which is to inhibit intrinsic protein-protein interactions in the cytosol of plant cells. Experimental procedures were designed for conveniently evaluating desired properties of recombinant antibodies in consecutive steps. Our selection method was successfully used to develop a recombinant antibody inhibiting the interaction of ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 3 with such of its upstream interaction partners as the receiver domain of CYTOKININ INDEPENDENT HISTIDINE KINASE 1. The specific down-regulation of the cytokinin signaling pathway in vivo demonstrates the validity of our approach. This selection method can serve as a prototype for developing unique recombinant antibodies able to interfere with virtually any biomolecule in the living cell.

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

Energy Technology Data Exchange (ETDEWEB)

Jin, Hongjun; Zangar, Richard C.

2017-01-17

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

Antibody Engineering and Therapeutics

Science.gov (United States)

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

2014-01-01

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

Prediction of antibody persistency from antibody titres to natalizumab

DEFF Research Database (Denmark)

Jensen, Poul Erik H; Koch-Henriksen, Nils; Sellebjerg, Finn

2012-01-01

In a subgroup of patients with multiple sclerosis natalizumab therapy causes generation of anti-natalizumab antibodies that may be transient or persistent. It is recommended to discontinue natalizumab therapy in persistently antibody-positive patients.......In a subgroup of patients with multiple sclerosis natalizumab therapy causes generation of anti-natalizumab antibodies that may be transient or persistent. It is recommended to discontinue natalizumab therapy in persistently antibody-positive patients....

Successful combination immunotherapy of anti-gamma aminobutyric acid (GABA)A receptor antibody-positive encephalitis with extensive multifocal brain lesions.

Science.gov (United States)

Fukami, Yuki; Okada, Hiroaki; Yoshida, Mari; Yamaguchi, Keiji

2017-08-31

A 78-year old woman who presented with akinetic mutism was admitted to our hospital. Brain MRI showed multifocal increased T 2 /FLAIR signal with extensive cortical-subcortical involvement. We suspected autoimmune encephalitis and the patient received methylprednisolone pulse. Her conscious level gradually recovered, but later relapsed again and presented with refractory status epilepticus. We treated her with intravenous immunoglobulin, plasma exchange and pulsed cyclophosphamide, with satisfactory response. A brain biopsy showed perivascular lymphocytic infiltrates and reactive gliosis. Anti-gamma aminobutyric acid (GABA) A receptor antibodies test came back to be positive after her recovery, and the diagnosis of anti-GABA A receptor antibody-positive encephalitis was made. This is a very rare case where brain biopsies were performed in a patient with anti-GABA A receptor antibody-positive encephalitis.

Noninvasive diagnosis of axillary node metastases with monoclonal antibody lymphoscintigraphy

International Nuclear Information System (INIS)

Fig, L.M.; Von Moll, L.; Brown, R.; Harness, J.; Appleman, H.; Stevens, R.; Johnson, J.W.; Mudgett, E.; Colcher, D.; Schlom, J.; Lichter, A.; Wicha, M.; Wahl, R.L.

1989-01-01

This study was undertaken to determine whether 131-I labeled B72.3 monoclonal antibody, when injected subcutaneously in patients with known breast cancer, successfully detects lymph node metastases. Eleven women with biopsy-proven B72.3 antibody-reactive breast cancer (determined by immunoperoxidase staining) received subcutaneous injections of 500 μ Ci 131-I B72.3 in ipsilateral finger web spaces (or, in three cases, intralesional injections into the site of the breast tumor). The antibody is a IgGlk reactive with a high molecular weight antigen found on most breast carcinomas. Images of the axilla were obtained immediately after injection and serially to 72 hours. Nodal uptake was scored on a 0-3+ scale in a blinded fashion and correlated with pathologic findings from lymph node dissection

A mechanistic compartmental model for total antibody uptake in tumors.

Science.gov (United States)

Thurber, Greg M; Dane Wittrup, K

2012-12-07

Antibodies are under development to treat a variety of cancers, such as lymphomas, colon, and breast cancer. A major limitation to greater efficacy for this class of drugs is poor distribution in vivo. Localization of antibodies occurs slowly, often in insufficient therapeutic amounts, and distributes heterogeneously throughout the tumor. While the microdistribution around individual vessels is important for many therapies, the total amount of antibody localized in the tumor is paramount for many applications such as imaging, determining the therapeutic index with antibody drug conjugates, and dosing in radioimmunotherapy. With imaging and pretargeted therapeutic strategies, the time course of uptake is critical in determining when to take an image or deliver a secondary reagent. We present here a simple mechanistic model of antibody uptake and retention that captures the major rates that determine the time course of antibody concentration within a tumor including dose, affinity, plasma clearance, target expression, internalization, permeability, and vascularization. Since many of the parameters are known or can be estimated in vitro, this model can approximate the time course of antibody concentration in tumors to aid in experimental design, data interpretation, and strategies to improve localization. Copyright © 2012 Elsevier Ltd. All rights reserved.

Development and evaluation of single domain antibodies for vaccinia and the L1 antigen.

Directory of Open Access Journals (Sweden)

Scott A Walper

Full Text Available There is ongoing interest to develop high affinity, thermal stable recognition elements to replace conventional antibodies in biothreat detection assays. As part of this effort, single domain antibodies that target vaccinia virus were developed. Two llamas were immunized with killed viral particles followed by boosts with the recombinant membrane protein, L1, to stimulate the immune response for envelope and membrane proteins of the virus. The variable domains of the induced heavy chain antibodies were selected from M13 phage display libraries developed from isolated RNA. Selection via biopanning on the L1 antigen produced single domain antibodies that were specific and had affinities ranging from 4×10(-9 M to 7.0×10(-10 M, as determined by surface plasmon resonance. Several showed good ability to refold after heat denaturation. These L1-binding single domain antibodies, however, failed to recognize the killed vaccinia antigen. Useful vaccinia binding single domain antibodies were isolated by a second selection using the killed virus as the target. The virus binding single domain antibodies were incorporated in sandwich assays as both capture and tracer using the MAGPIX system yielding limits of detection down to 4×10(5 pfu/ml, a four-fold improvement over the limit obtained using conventional antibodies. This work demonstrates the development of anti-vaccinia single domain antibodies and their incorporation into sandwich assays for viral detection. It also highlights the properties of high affinity and thermal stability that are hallmarks of single domain antibodies.

Anti-Glycoprotein G Antibodies of Herpes Simplex Virus 2 Contribute to Complete Protection after Vaccination in Mice and Induce Antibody-Dependent Cellular Cytotoxicity and Complement-Mediated Cytolysis

Directory of Open Access Journals (Sweden)

Staffan Görander

2014-11-01

Full Text Available We investigated the role of antibodies against the mature portion of glycoprotein G (mgG-2 of herpes simplex virus 2 (HSV-2 in protective immunity after vaccination. Mice were immunized intramuscularly with mgG-2 and oligodeoxynucleotides containing two CpG motifs plus alum as adjuvant. All C57BL/6 mice survived and presented no genital or systemic disease. High levels of immunoglobulin G subclass 1 (IgG1 and IgG2 antibodies were detected and re-stimulated splenic CD4+ T cells proliferated and produced IFN-γ. None of the sera from immunized mice exhibited neutralization, while all sera exerted antibody-dependent cellular cytotoxicity (ADCC and complement-mediated cytolysis (ACMC activity. Passive transfer of anti-mgG-2 monoclonal antibodies, or immune serum, to naive C57BL/6 mice did not limit disease progression. Immunized B‑cell KO mice presented lower survival rate and higher vaginal viral titers, as compared with vaccinated B-cell KO mice after passive transfer of immune serum and vaccinated C57BL/6 mice. Sera from mice that were vaccinated subcutaneously and intranasally with mgG-2 presented significantly lower titers of IgG antibodies and lower ADCC and ACMC activity. We conclude that anti-mgG-2 antibodies were of importance to limit genital HSV‑2 infection. ADCC and ACMC activity are potentially important mechanisms in protective immunity, and could tentatively be evaluated in future animal vaccine studies and in clinical trials.

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

Post-infection immunodeficiency virus control by neutralizing antibodies.

Directory of Open Access Journals (Sweden)

Hiroyuki Yamamoto

Full Text Available BACKGROUND: Unlike most acute viral infections controlled with the appearance of virus-specific neutralizing antibodies (NAbs, primary HIV infections are not met with such potent and early antibody responses. This brings into question if or how the presence of potent antibodies can contribute to primary HIV control, but protective efficacies of antiviral antibodies in primary HIV infections have remained elusive; and, it has been speculated that even NAb induction could have only a limited suppressive effect on primary HIV replication once infection is established. Here, in an attempt to answer this question, we examined the effect of passive NAb immunization post-infection on primary viral replication in a macaque AIDS model. METHODS AND FINDINGS: The inoculums for passive immunization with simian immunodeficiency virus mac239 (SIVmac239-specific neutralizing activity were prepared by purifying polyclonal immunoglobulin G from pooled plasma of six SIVmac239-infected rhesus macaques with NAb induction in the chronic phase. Passive immunization of rhesus macaques with the NAbs at day 7 after SIVmac239 challenge resulted in significant reduction of set-point plasma viral loads and preservation of central memory CD4 T lymphocyte counts, despite the limited detection period of the administered NAb responses. Peripheral lymph node dendritic cell (DC-associated viral RNA loads showed a remarkable peak with the NAb administration, and DCs stimulated in vitro with NAb-preincubated SIV activated virus-specific CD4 T lymphocytes in an Fc-dependent manner, implying antibody-mediated virion uptake by DCs and enhanced T cell priming. CONCLUSIONS: Our results present evidence indicating that potent antibody induction post-infection can result in primary immunodeficiency virus control and suggest direct and indirect contribution of its absence to initial control failure in HIV infections. Although difficulty in achieving requisite neutralizing titers for

DARPA Antibody Technology Program Standardized Test Bed for Antibody Characterization: Characterization of an MS2 Human IgG Antibody Produced by AnaptysBio, Inc.

Science.gov (United States)

2016-02-01

Enzyme- linked immunosorbent assay (ELISA) Quality Testing MS2 coat protein (MS2CP) 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF...primarily relied on the performance of an antibody in an enzyme- linked immunosorbent assay (ELISA), with little regard for quantifying the full spectrum...Protection, and Multi-Year Stabilization, in High Concentration Protein Solutions, Using Ionic Liquids. Chem. Commun. (Camb) 2007, 26, 2714–2716. 3. Bio

Single-Domain Antibodies As Therapeutics against Human Viral Diseases

Directory of Open Access Journals (Sweden)

Yanling Wu

2017-12-01

Full Text Available In full-size formats, monoclonal antibodies have been highly successful as therapeutics against cancer and immune diseases. However, their large size leads to inaccessibility of some epitopes and relatively high production costs. As an alternative, single-domain antibodies (sdAbs offer special advantages compared to full-size antibodies, including smaller size, larger number of accessible epitopes, relatively low production costs and improved robustness. Currently, sdAbs are being developed against a number of viruses, including human immunodeficiency virus-1 (HIV-1, influenza viruses, hepatitis C virus (HCV, respiratory syncytial virus (RSV, and enteric viruses. Although sdAbs are very potent inhibitors of viral infections, no sdAbs have been approved for clinical use against virial infection or any other diseases. In this review, we discuss the current state of research on sdAbs against viruses and their potential as therapeutics against human viral diseases.

Thyroid Antibodies

Science.gov (United States)

... PF4 Antibody Hepatitis A Testing Hepatitis B Testing Hepatitis C Testing HER2/neu Herpes Testing High-sensitivity C-reactive Protein (hs-CRP) Histamine Histone Antibody HIV Antibody and HIV Antigen (p24) HIV Antiretroviral Drug Resistance Testing, Genotypic HIV Viral Load HLA Testing HLA- ...

SUCCESSFUL APPLICATION OF PERIPHERAL VENO-ARTERIAL EXTRACORPOREAL MEMBRANE OXYGENATION FOR CARDIAC ALLOGRAFT ANTIBODY-MEDIATED REJECTION WITH SEVERE HEMODYNAMIC COMPROMISE

Directory of Open Access Journals (Sweden)

V. N. Poptsov

2015-01-01

Full Text Available Introduction. Acute antibody-mediated rejection (AMR is one of the severe complications of early and late period after heart transplantation (HT. Only few case reports and studies presented of mechanical circulatory support (MCS application for refractory acute rejection causing hemodynamic compromise. Aim. We report the case of a woman with cardiogenic shock caused by severe AMR that was successfully treatment by peripheral venoarterial extracorporeal membrane oxygenation (VA ECMO. Material and methods. In december 2014, a 60-year-old woman with dilated cardiomyopathy was operated for HT. The patient had a good initial cardiac allograft function and no and was discharged from ICU on the 4th day after HT. 1st endomyocardial biopsy (EMB (the 7th day after HT showed absence of acute cellular and antibody-mediated rejection. On the 11th day after HT patient aggravated and presented clinical signs of life-threatening acute cardiac allograft dysfunction: arterial blood pressure 78/49/38 mm Hg, HR 111 in min, CVP 20 mm Hg, PAP 47/34/25 mm Hg, PCWP 25 mm Hg, CI 1.5 l/min/m2, adrenalin 110 ng/kg/min, dopamine 15 mcg/kg/min. ECG showed impairment of systolic left (LVEF 25% and right (RVEF 15% ventricle function, left and right ventricle diffuse hypokinesis, thickness of IVS, LV and RV wall 1.7, 1.4 and 0.8 cm, tricuspid and mitral valve regurgitation 2–3 degrees. EMB presented AMR. In conscience peripheral VA ECMO was installed. We used peripheral transcutaneous cannulation technique via femoral vessels – arterial cannula 15 F, venous cannula – 23 F, vascular catheter 14 G for anterograde leg’s perfusion. ACT 130–150 sec. AMR therapy included: methylprednisolon pulse-therapy (10 mg/kg for 5 day, IgG, plasmapheresis (No 7, rituximab. Results. Under MCS by VA ECMO we noted quick improvement of hemodynamic, metabolic homeostasis and organ functions. On the 6th day of VA ECMO (blood flow 1.8 l/min: arterial blood pressure 133/81/54 mm Hg, CVP 5 mm

Validation of the rapid fluorescent focus inhibition test for rabies virus-neutralizing antibodies in clinical samples

NARCIS (Netherlands)

Kostense, Stefan; Moore, Susan; Companjen, Arjen; Bakker, Alexander B. H.; Marissen, Wilfred E.; von Eyben, Rie; Weverling, Gerrit Jan; Hanlon, Cathleen; Goudsmit, Jaap

2012-01-01

Monoclonal antibodies are successful biologics in treating a variety of diseases, including the prevention or treatment of viral infections. CL184 is a 1:1 combination of two human monoclonal IgG1 antibodies (CR57 and CR4098) against rabies virus, produced in the PER.C6 human cell line. The two

Development of antibody against sulfamethazine

International Nuclear Information System (INIS)

Li Ziying; Xi Wenge; Liu Yibing; Zhang Liling; Guo Weizheng; Han Shiquan

2004-01-01

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)

Human monoclonal antibodies: the residual challenge of antibody immunogenicity.

Science.gov (United States)

Waldmann, Herman

2014-01-01

One of the major reasons for seeking human monoclonal antibodies has been to eliminate immunogenicity seen with rodent antibodies. Thus far, there has yet been no approach which absolutely abolishes that risk for cell-binding antibodies. In this short article, I draw attention to classical work which shows that monomeric immunoglobulins are intrinsically tolerogenic if they can be prevented from creating aggregates or immune complexes. Based on these classical studies two approaches for active tolerization to therapeutic antibodies are described.

A review of human anti-globulin antibody (HAGA, HAMA, HACA, HAHA) responses to monoclonal antibodies. Not four letter words

Energy Technology Data Exchange (ETDEWEB)

Mirick, G. R.; Bradt, B. M.; Denardo, S. J.; Denardo, G. L. [Calfornia Univ., Sacramento (United States). Davis Medical Center

2004-12-01

The United States Food and Drugs Administration (FDA) has approved unconjugated monoclonal antibodies (MAbs) for immunotherapy (IT) of B-cell lymphoma, breast cancer and acute myeloid leukemia. More recently, approval has been given for conjugated ZevalinTM ({sup 9}0yttrium ibritumomab tiuxetan, IDEC-Y2B8, Biogen Idec, Cambridge, MA) and BexxarTM ({sup 1}31I-tositumomab, Corixa, Corp., Seattle, WA and GlaxoSmithKline, Philadelphia, PA) antiCD20 MAns for use in radioimmunotherapy (RIT) of non-Hodgikin's lymphoma (NHL), thus redefining the standard care of cancer patients. Because of, and despite a lack of basis for concern about allergic reactions due to human antibody responses to these foreign proteins, essays were developed to determine HAGE (human anti-globulin antibody) levels that developed in patient sera following treatment with MAbs. Strategies were also devised to humanize MAbs and to temporarily block patient immune function with drugs in order to decrease the seroconversion rates, with considerable success. On the other hand, a survival advantage has been observed in some patients who developed a HAGA following treatment. This correlates with development of an anti-idiotype antibody cascade directed toward the MAbs used to treat these patients. What follows is a selective review of HAGA and its effect on cancer treatment over the past 2 decades.

Microbials for the production of monoclonal antibodies and antibody fragments.

Science.gov (United States)

Spadiut, Oliver; Capone, Simona; Krainer, Florian; Glieder, Anton; Herwig, Christoph

2014-01-01

Monoclonal antibodies (mAbs) and antibody fragments represent the most important biopharmaceutical products today. Because full length antibodies are glycosylated, mammalian cells, which allow human-like N-glycosylation, are currently used for their production. However, mammalian cells have several drawbacks when it comes to bioprocessing and scale-up, resulting in long processing times and elevated costs. By contrast, antibody fragments, that are not glycosylated but still exhibit antigen binding properties, can be produced in microbial organisms, which are easy to manipulate and cultivate. In this review, we summarize recent advances in the expression systems, strain engineering, and production processes for the three main microbials used in antibody and antibody fragment production, namely Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli. Copyright © 2013 Elsevier Ltd. All rights reserved.

Evaluation of pre-existing antibody presence as a risk factor for posttreatment anti-drug antibody induction: analysis of human clinical study data for multiple biotherapeutics.

Science.gov (United States)

Xue, Li; Rup, Bonita

2013-07-01

Biotherapeutic-reactive antibodies in treatment-naïve subjects (i.e., pre-existing antibodies) have been commonly detected during clinical immunogenicity assessments; however information on pre-existing antibody prevalence, physiological effects, and impact on posttreatment anti-drug antibody (ADA) induction remains limited. In this analysis, pre-existing antibody prevalence and impact on posttreatment ADA induction were determined using ADA data from 12 biotherapeutics analyzed in 32 clinical studies. Approximately half (58%) of the biotherapeutics were associated with some level of pre-existing antibodies and 67% of those were associated with posttreatment ADA induction. Across all studies, 5.6% of study subjects demonstrated presence of pre-existing antibodies, among which, 17% of the individual subjects had posttreatment increases in their ADA titers while 16% had decreased titers and 67% had no change in titers. However, in studies conducted in the rheumatoid arthritis (RA) population, 14.8% of RA patients were associated with pre-existing antibodies and 30% of those had posttreatment titer increases. The results suggest that in most study subjects, pre-existing antibodies pose a low risk for posttreatment ADA induction. That said, the high risk of induction implicated for RA patients, primarily observed in treatments evaluating novel antibody-based constructs, indicates that further understanding of the contribution of product and disease-specific factors is needed. Cross-industry efforts to collect and analyze a larger data set would enhance understanding of the prevalence, nature, and physiological consequences of pre-existing antibodies, better inform the immunogenicity risk profiles of products associated with these antibodies and lead to better fit-for-purpose immunogenicity management and mitigation strategies.

Enzymatic Inactivation of Endogenous IgG by IdeS Enhances Therapeutic Antibody Efficacy.

Science.gov (United States)

Järnum, Sofia; Runström, Anna; Bockermann, Robert; Winstedt, Lena; Crispin, Max; Kjellman, Christian

2017-09-01

Endogenous plasma IgG sets an immunologic threshold that dictates the activity of tumor-directed therapeutic antibodies. Saturation of cellular antibody receptors by endogenous antibody limits antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Here, we show how enzymatic cleavage of IgG using the bacterial enzyme IdeS can be utilized to empty both high and low affinity Fcγ-receptors and clear the entire endogenous antibody pool. Using in vitro models, tumor animal models as well as ex vivo analysis of sera collected during a previous clinical trial with IdeS, we show how clearing of competing plasma antibody levels with IdeS unblocks cellular antibody receptors. We show that therapeutic antibodies against breast cancer (trastuzumab), colon cancer (cetuximab), and lymphomas (rituximab and alemtuzumab) can be potentiated when endogenous IgG is removed. Overall, IdeS is shown to be a potent tool to reboot the human antibody repertoire and to generate a window to preferentially load therapeutic antibodies onto effector cells and thereby create an armada of dedicated tumor-seeking immune cells. Mol Cancer Ther; 16(9); 1887-97. ©2017 AACR . ©2017 American Association for Cancer Research.

Rapid analysis of charge variants of monoclonal antibodies using non-linear salt gradient in cation-exchange high performance liquid chromatography.

Science.gov (United States)

Joshi, Varsha; Kumar, Vijesh; Rathore, Anurag S

2015-08-07

A method is proposed for rapid development of a short, analytical cation exchange high performance liquid chromatography method for analysis of charge heterogeneity in monoclonal antibody products. The parameters investigated and optimized include pH, shape of elution gradient and length of the column. It is found that the most important parameter for development of a shorter method is the choice of the shape of elution gradient. In this paper, we propose a step by step approach to develop a non-linear sigmoidal shape gradient for analysis of charge heterogeneity for two different monoclonal antibody products. The use of this gradient not only decreases the run time of the method to 4min against the conventional method that takes more than 40min but also the resolution is retained. Superiority of the phosphate gradient over sodium chloride gradient for elution of mAbs is also observed. The method has been successfully evaluated for specificity, sensitivity, linearity, limit of detection, and limit of quantification. Application of this method as a potential at-line process analytical technology tool has been suggested. Copyright © 2015 Elsevier B.V. All rights reserved.

Antibodies: From novel repertoires to defining and refining the structure of biologically important targets.

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Conroy, Paul J; Law, Ruby H P; Caradoc-Davies, Tom T; Whisstock, James C

2017-03-01

Antibodies represent a highly successful class of molecules that bind a wide-range of targets in therapeutic-, diagnostic- and research-based applications. The antibody repertoire is composed of the building blocks required to develop an effective adaptive immune response against foreign insults. A number of species have developed novel genetic and structural mechanisms from which they derive these antibody repertoires, however, traditionally antibodies are isolated from human, and rodent sources. Due to their high-value therapeutic, diagnostic, biotechnological and research applications, much innovation has resulted in techniques and approaches to isolate novel antibodies. These approaches are bolstered by advances in our understanding of species immune repertoires, next generation sequencing capacity, combinatorial antibody discovery and high-throughput screening. Structural determination of antibodies and antibody-antigen complexes has proven to be pivotal to our current understanding of the immune repertoire for a range of species leading to advances in man-made libraries and fine tuning approaches to develop antibodies from immune-repertoires. Furthermore, the isolation of antibodies directed against antigens of importance in health, disease and developmental processes, has yielded a plethora of structural and functional insights. This review highlights the significant contribution of antibody-based crystallography to our understanding of adaptive immunity and its application to providing critical information on a range of human-health related indications. Copyright © 2017 Elsevier Inc. All rights reserved.

Study on the preparation of antibody coated tubes for radioimmunoassay kit production

International Nuclear Information System (INIS)

Nguyen Thi Thu; Le Van So; Vo Thi Cam Hoa; Duong Van Dong; Mai Phuoc Tho

2003-01-01

The polystyrene tubes are coated with T3/ T4 antibodies by γ-globulin, second antibody and specific antibodies. They are immobilized on the solid at a suitably dilution and incubation for 24 h, pH 9.6. The variation of the binding capacity values (obtained for 10 consecutive preparations) was less than 10%. NSB <3%, Binding 30-50%. Using dried tubes coated either with anti-T3 or anti-T4 antibody according to the developed coating approach for the determination of total T3 and total T4 in human serum. The recovery of T3 was found to be between 85.5% and 104% while the recovery of T4 ranged between 90.9% and 119%. The cross-reactivity for T4 in the T3 assay was 0.22%. Both assays were sensitive, the detection limit of the RIA for total T3 assay was 0.15 ng/ml while the detection limit of the RIA for total T4 assay was 5 ng/ml. (author)

Prevalence of rotavirus antibodies in breast milk and inhibitory effects to rotavirus vaccines.

Science.gov (United States)

Trang, Nguyen V; Braeckman, Tessa; Lernout, Tinne; Hau, Vu T B; Anh, Le T K; Luan, Le T; Van Damme, Pierre; Anh, Dang D

2014-01-01

Rotavirus (RV) is the most common cause of childhood diarrhea worldwide, and several vaccines have been successfully developed to reduce the burden of disease. However, lower vaccine immunogenicity and efficacy in developing countries might be related to the virus-neutralizing activity of breast milk. We examined possible differences in breast milk antibody levels (total IgA antibody, RV-specific antibodies, and RV-neutralizing antibodies) between healthy mothers living in a rural area (n=145) and mothers living in an urban area (n=147) of Vietnam. Total IgA concentration was significantly higher in samples from mothers in the rural region than in samples from mothers in the urban region, whereas urban mothers had significantly higher RV-specific IgA antibody titers than did rural mothers. Neutralizing antibodies against RV strain G1P[8] were undetected in nearly one-half of the breast milk samples (45-48%), whereas the majority of the remaining samples had low antibody titers (2-16). Despite these low titers, the breast milk still reduced vaccine strain titers (2×10(6) plaque forming units/mL) up to 80% or more, even at a milk-to-virus ratio of 1:8. An increase in neutralizing anti-G1P[8] antibody titers (Pvaccine efficacy and immunogenicity in Vietnamese infants.

A three-layer immunoradiometric assay for antibodies in different immunoglobulin classes and its application to the detection of chicken thyroglobulin autoantibodies and of antibodies to sheep erythrocytes

International Nuclear Information System (INIS)

Carvalho, L.C.P. de; Roitt, I.M.; Wick, G.

1980-01-01

A versatile solid-phase assay for detection of antibodies in different immunoglobulin classes is described. The assay has been applied to: (1) the detection of IgG and IgM antithyroglobulin autoantibodies in chickens with spontaneous autoimmune thyroiditis, and (2) the detection of anti-sheep cell antibodies in normal chickens. Thyroglobulin-coated plastic tubes or formaldehyde-fixed sheep erythrocytes were used as the solid phase. Antisera were added in succession to the solid-phase antigen so as to form 3 antibody layers: (1) chicken antibody against the solid-phase antigen; (2) heavy-chain-specific rabbit anti-chicken immunoglobulin; and (3) 125 I-labelled goat anti-rabbit immunoglobulin. The assay is suitable for routine determinations on large numbers of samples; its sensitivity enables small volumes of serum to be tested and allows considerable economy in the use of valuable class-specific antisera. The radiolabelled reagent can be readily applied to other assays employing rabbit antisera. (Auth.)

Multiplex serology of paraneoplastic antineuronal antibodies.

Science.gov (United States)

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

2013-05-31

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

Monoclonal antibody

International Nuclear Information System (INIS)

Oyamada, Hiyoshimaru

1987-01-01

Some aspects of monoclonal antibodies are described, centering on studies made by the author and those presented at the Second International Conference on Monoclonal Antibody Immunoconjugates for Cancer held in March this year (1987). The history of immuno-nuclear medicine and procedures for producing monoclonal antibodies are briefly outlined. Monoclonal antibodies are immunoglobulins. Here, the structure of IgG, which is used most frequently, is described. An IgG is composed of two antigen binding fragments (Fab) and one crystallizable fragment (Fc). The end portion of a Fab reacts with an antigen. One of the major applications of immuno-nuclear medicine is the diagnosis of cancer. As label nucleides, 131 I and 111 I were selected in most cases in the past while 123 I and 99m Tc are currently used more often. Advantages and disadvantages of this diagnosis method is discussed citing studies presented at the First (1986) and Second (1987) International Conference on Monoclonal Antibody Immunoconjugates for Cancer. The present status of the application of monoclonal antibodies to treatment of cancer is also described. (Nogami, K.)

Monoclonal antibodies for use in an immunoradiometric assay for α-foetoprotein

International Nuclear Information System (INIS)

Hunter, W.M.; Bennie, J.G.

1982-01-01

The advantages offered by a mouse IgG 1 monoclonal antibody to human α-foetoprotein (AFP) for the preparation of [ 125 I]antibody for use in an immunoradiometric assay (IRMA) have been investigated. The antibody was isolated from ascites fluid by sodium sulphate precipitation followed by gel filtration on Sephadex G-200. The freeze-dried powder and solutions thereof were stable and were used for iodination to 1 atom 125 I/molecule antibody by the chloramine-T procedure. At high antigen concentrations 70-80% of the added [ 125 ]Ab was present in the sandwich. Linear response curves in the range 1-100 μg antigen/l incubate were obtained when [ 125 I]Ab was in slight excess. In this region an Ag : Ab ratio 1.9 : 1 was obtained which is consistent with the saturation of a bifunctional antibody. Although non-specific binding (in the absence of antigen) was consistently 125 I]Ab, this was the main factor in determining assay detection limits. The serum AFP levels from both non-pregnant and pregnant subjects as measured by the IRMA using the [ 125 I]monoclonal Ab and by radioimmunoassay (RIA) using a sheep antiserum to AFP were in excellent agreement. The IRMA was manipulatively simple, employed a shorter incubation time (2h), required shorter counting times than the RIA and gave a much wider working range. The provision of a monoclonal antibody for labelling removes the one major practicability barrier which otherwise limits the development and use of the potentially superior IRMA system. (Auth.)

Identification and typing of herpes simplex viruses with monoclonal antibodies.

OpenAIRE

Balachandran, N; Frame, B; Chernesky, M; Kraiselburd, E; Kouri, Y; Garcia, D; Lavery, C; Rawls, W E

1982-01-01

Monoclonal antibodies which reacted with type-specific antigens of herpes simplex virus type 2 or with antigens shared by herpes simplex virus types 1 and 2 were used in an indirect immunofluorescence assay to type virus isolates and to detect viral antigens in cells obtained from herpetic lesions. Complete concordance was obtained for 42 isolates typed by endonuclease restriction analysis of viral DNA and by indirect immunofluorescence with monoclonal antibodies. Examination of a limited num...

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.

A systematic analysis of commonly used antibodies in cancer diagnostics.

Science.gov (United States)

Gremel, Gabriela; Bergman, Julia; Djureinovic, Dijana; Edqvist, Per-Henrik; Maindad, Vikas; Bharambe, Bhavana M; Khan, Wasif Ali Z A; Navani, Sanjay; Elebro, Jacob; Jirström, Karin; Hellberg, Dan; Uhlén, Mathias; Micke, Patrick; Pontén, Fredrik

2014-01-01

Immunohistochemistry plays a pivotal role in cancer differential diagnostics. To identify the primary tumour from a metastasis specimen remains a significant challenge, despite the availability of an increasing number of antibodies. The aim of the present study was to provide evidence-based data on the diagnostic power of antibodies used frequently for clinical differential diagnostics. A tissue microarray cohort comprising 940 tumour samples, of which 502 were metastatic lesions, representing tumours from 18 different organs and four non-localized cancer types, was analysed using immunohistochemistry with 27 well-established antibodies used in clinical differential diagnostics. Few antibodies, e.g. prostate-specific antigen and thyroglobulin, showed a cancer type-related sensitivity and specificity of more than 95%. A majority of the antibodies showed a low degree of sensitivity and specificity for defined cancer types. Combinations of antibodies provided limited added value for differential diagnostics of cancer types. The results from analysing 27 diagnostic antibodies on consecutive sections of 940 defined tumours provide a unique repository of data that can empower a more optimal use of clinical immunohistochemistry. Our results highlight the benefit of immunohistochemistry and the unmet need for novel markers to improve differential diagnostics of cancer. © 2013 John Wiley & Sons Ltd.

Detection of IgG antibodies against Bordetella pertussis with 125I-protein A

International Nuclear Information System (INIS)

Wirsing von Koenig, C.H.; Finger, H.

1981-01-01

A method for the detection of IgG antibodies against Bordetella pertussis is described, based on the principle of 'sandwich' radioimmunoassay. 125 I protein A is used as radioactive tracer. The influence of amounts of antigen, antibody, radioactive tracer, incubation time and temperature were tested and the optimal conditions for the assay are described. The procedure offers a simple, quick, and sensitive method for detecting antibodies against B. pertussis. Application and limitation of the test are discussed. (orig.)

Expression and purification of a major allergen, Pla a 1, from Platanus acerifolia pollen and the preparation of its monoclonal antibody.

Science.gov (United States)

Ni, Wei-Wei; Huang, Wen; Wu, De-Qin; Zhou, Yan-Jun; Ji, Chun-Mei; Cao, Meng-Da; Guo, Miao; Sun, Jin-Lu; Wei, Ji-Fu

2017-09-01

Platanus acerifolia pollen is considered an important source of airborne allergens in numerous cities. Pla a 1 is a major allergen from P. acerifolia pollen. The present study aimed to express and purify Pla a 1, and to prepare its monoclonal antibody. In the present study, the Pla a 1 gene was subcloned into a pET‑28a vector and transformed into the ArcticExpress™ (DE3) RP Escherichia coli host strain. The purified Pla a 1 was then used to immunize BALB/c mice. When serum detection was positive, spleen cells were isolated from the mice and fused with SP2/0 myeloma cells at a ratio of 10:1. Hybridoma cells were screened by indirect ELISA and limiting dilution. Positive cells were used to induce the formation of antibody‑containing ascites fluid, and the antibodies were purified using protein A‑agarose. The results of the present study demonstrated that recombinant Pla a 1 was successfully expressed and purified, and exhibited positive immunoglobulin E‑binding to serum from patients allergic to P. acerifolia. A total of 11 hybridomas that steadily secreted anti‑Pla a 1 antibody were obtained and an immunoblotting analysis indicated that all of these monoclonal antibodies specifically recognized the Pla a 1 protein. These results suggested that specific anti‑Pla a 1 antibodies may be obtained, which can be used for the rapid detection of Pla a 1 allergens and in the preparation of vaccines against P. acerifolia pollen.

Generation of human antibody fragments against Streptococcus mutans using a phage display chain shuffling approach

Directory of Open Access Journals (Sweden)

Barth Stefan

2005-01-01

Full Text Available Abstract Background Common oral diseases and dental caries can be prevented effectively by passive immunization. In humans, passive immunotherapy may require the use of humanized or human antibodies to prevent adverse immune responses against murine epitopes. Therefore we generated human single chain and diabody antibody derivatives based on the binding characteristics of the murine monoclonal antibody Guy's 13. The murine form of this antibody has been used successfully to prevent Streptococcus mutans colonization and the development of dental caries in non-human primates, and to prevent bacterial colonization in human clinical trials. Results The antibody derivatives were generated using a chain-shuffling approach based on human antibody variable gene phage-display libraries. Like the parent antibody, these derivatives bound specifically to SAI/II, the surface adhesin of the oral pathogen S. mutans. Conclusions Humanization of murine antibodies can be easily achieved using phage display libraries. The human antibody fragments bind the antigen as well as the causative agent of dental caries. In addition the human diabody derivative is capable of aggregating S. mutans in vitro, making it a useful candidate passive immunotherapeutic agent for oral diseases.

Antibodies against deamidated gliadin peptides identify adult coeliac disease patients negative for antibodies against endomysium and tissue transglutaminase.

Science.gov (United States)

Dahle, C; Hagman, A; Ignatova, S; Ström, M

2010-07-01

This study was done to evaluate the diagnostic utility of antibodies against deamidated gliadin peptides compared to traditional markers for coeliac disease. To evaluate diagnostic utility of antibodies against deamidated gliadin peptide (DGP). Sera from 176 adults, referred for endoscopy without previous analysis of antibodies against tissue transglutaminase (tTG) or endomysium (EmA), were retrospectively analysed by ELISAs detecting IgA/IgG antibodies against DGP or a mixture of DGP and tTG, and compared with IgA-tTG and EmA. Seventy-nine individuals were diagnosed with coeliac disease. Receiver operating characteristic analyses verified the manufacturers' cut-off limits except for IgA/IgG-DGP/tTG. In sera without IgA deficiency, the sensitivity was higher for IgA/IgG-DGP (0.85-0.87) compared with IgA-tTg (0.76) and EmA (0.61). All tests showed high specificity (0.95-1.00). Eighteen coeliac disease-sera were negative regarding IgA-tTG, nine of which were positive for IgA/IgG-DGP. Sera from coeliac disease-patients >70 years were more often negative for IgA-tTG (50%) and IgA/IgG-DGP (36%) than younger patients (15% and 8% respectively) (P adult coeliac disease patients negative for antibodies against endomysium and tissue transglutaminase. Serology is often negative in elderly patients with coeliac disease; a small bowel biopsy should therefore be performed generously before coeliac disease is excluded.

Activity and longevity of antibody in paper-based blood typing diagnostics

Science.gov (United States)

Henderson, Clare A.; McLiesh, Heather; Then, Whui L.; Garnier, Gil

2018-05-01

Paper-based diagnostics provide a low-cost, reliable and easy to use mode of blood typing. The shelf-life of such products, however, can be limited due to the reduced activity of reagent antibodies sorbed on the paper cellulose fibres. This study explores the effects of ageing on antibody activity for periods up to twelve months on paper and in solution under different ageing and drying conditions - air-dried, lyophilised and kept as a liquid. Paper kept wet with undiluted antibody is shown to have the longest shelf-life and the clearest negatives. Antibody diluted with bovine serum albumin (BSA) protects against the lyophilisation process, however, beyond nine months ageing, false positives are seen. Paper with air-dried antibodies is not suitable for use after one month ageing. These results inform preparation and storage conditions for the development of long shelf-life blood grouping paper-based diagnostics.

Activity and Longevity of Antibody in Paper-Based Blood Typing Diagnostics

Directory of Open Access Journals (Sweden)

Clare A. Henderson

2018-05-01

Full Text Available Paper-based diagnostics provide a low-cost, reliable and easy to use mode of blood typing. The shelf-life of such products, however, can be limited due to the reduced activity of reagent antibodies sorbed on the paper cellulose fibers. This study explores the effects of aging on antibody activity for periods up to 12 months on paper and in solution under different aging and drying conditions—air-dried, lyophilized, and kept as a liquid. Paper kept wet with undiluted antibody is shown to have the longest shelf-life and the clearest negatives. Antibody diluted with bovine serum albumin (BSA protects against the lyophilization process, however, beyond 9 months aging, false positives are seen. Paper with air-dried antibodies is not suitable for use after 1 month aging. These results inform preparation and storage conditions for the development of long shelf-life blood grouping paper-based diagnostics.

Addressing the Immunogenicity of the Cargo and of the Targeting Antibodies with a Focus on Deimmunized Bacterial Toxins and on Antibody-Targeted Human Effector Proteins

OpenAIRE

Grinberg, Yehudit; Benhar, Itai

2017-01-01

Third-generation immunotoxins are composed of a human, or humanized, targeting moiety, usually a monoclonal antibody or an antibody fragment, and a non-human effector molecule. Due to the non-human origin of the cytotoxic domain, these molecules stimulate potent anti-drug immune responses, which limit treatment options. Efforts are made to deimmunize such immunotoxins or to combine treatment with immunosuppression. An alternative approach is using the so-called ?human cytotoxic fusion protein...

Screening individual hybridomas by microengraving to discover monoclonal antibodies

Science.gov (United States)

Ogunniyi, Adebola O; Story, Craig M; Papa, Eliseo; Guillen, Eduardo; Love, J Christopher

2014-01-01

The demand for monoclonal antibodies (mAbs) in biomedical research is significant, but the current methodologies used to discover them are both lengthy and costly. Consequently, the diversity of antibodies available for any particular antigen remains limited. Microengraving is a soft lithographic technique that provides a rapid and efficient alternative for discovering new mAbs. This protocol describes how to use microengraving to screen mouse hybridomas to establish new cell lines producing unique mAbs. Single cells from a polyclonal population are isolated into an array of microscale wells (~105 cells per screen). The array is then used to print a protein microarray, where each element contains the antibodies captured from individual wells. The antibodies on the microarray are screened with antigens of interest, and mapped to the corresponding cells, which are then recovered from their microwells by micromanipulation. Screening and retrieval require approximately 1–3 d (9–12 d including the steps for preparing arrays of microwells). PMID:19528952

Monoclonal antibodies and cancer

International Nuclear Information System (INIS)

Haisma, H.J.

1987-01-01

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

Anti-phosphatidylserine/prothrombin antibodies: an additional diagnostic marker for APS?

Science.gov (United States)

Pregnolato, Francesca; Chighizola, Cecilia B; Encabo, Susan; Shums, Zakera; Norman, Gary L; Tripodi, Armando; Chantarangkul, Veena; Bertero, Tiziana; De Micheli, Valeria; Borghi, Maria Orietta; Meroni, Pier Luigi

2013-07-01

Among the diagnostic assays for anti-phospholipid syndrome (APS), lupus anticoagulant (LA) is the strongest predictor of thrombosis; however, it presents several limitations as interference with anticoagulant therapy and poor inter-laboratory agreement. Two-thirds of LA activity is apparently due to antibodies against prothrombin (PT), usually detectable by ELISA. Binding of PT to phosphatidylserine (PS) has been shown to enhance solid-phase anti-PT assay sensitivity. To determine the prevalence of antibodies against PS/PT (aPS/PT) in APS, we tested the semiquantitative QUANTA Lite(®) aPS/PT ELISA in a cohort of 80 APS patients. The prevalence of aPS/PT was 81.3%, rising to 87.6% when considering LA-positive subjects only. We observed a strong correlation between aPS/PT and LA (p = 0.006). To note, APS patients with thrombotic manifestations displayed significantly higher IgG aPS/PT titers compared to 20 aPL asymptomatic carriers (p = 0.012). To rule out a possible cross-reactivity of anti-β2 glycoprotein I antibodies (aβ2GPI) with PS/PT complex, we tested two monoclonal aβ2GPI antibodies and an affinity-purified (AP) polyclonal aβ2GPI IgG obtained from the serum of a patient reacting against both β2GPI and PS/PT. The two monoclonal antibodies did not show any reactivity against PS/PT complex, similarly the AP IgGs did not react toward PS/PT antigen while preserved their aβ2GPI activity. Our findings suggest that aPS/PT are a definite antibody population in APS. Moreover, the good correlation between aPS/PT ELISA and LA may support its use as a surrogate test for LA, particularly useful to overcome the technical limitations of the functional assay.

Use of monoclonal-antibodies for the detection of fecal bacteria in water

CSIR Research Space (South Africa)

Kfir, R

1993-01-01

Full Text Available Monoclonal antibodies (MAbs) against heat-killed Escherichia coli and Klebsiella oxytoca originating from wastewater effluent were raised in BALB/C mice. The fusion was highly successful and three hybridomas cloned were selected to study...

Human anti-luteinizing hormone-releasing hormone antibodies in patients treated with synthetic luteinizing hormone-releasing hormone

International Nuclear Information System (INIS)

Meakin, J.L.; Keogh, E.J.; Martin, C.E.

1985-01-01

One hundred sixty-three patients who were given synthetic LH-RH therapeutically underwent monitoring of serum IgG anti-LH-RH antibodies. Five of the patients showed specific binding to antibodies. Development of anti-LH-RH antibodies was not limited to those patients with a congenital deficiency of LH-RH. Urticarial responses occurred in four patients, only one of whom had IgG antibodies. Patients who had IgG antibodies or an urticarial response underwent monitoring of their serum IgE anti-LH-RH antibodies, but none had a positive binding response. The refractory state which has been reported in patients in whom similar antibodies to LH-RH develop was not invariably observed among these patients

Discovery of functional monoclonal antibodies targeting G-protein-coupled receptors and ion channels.

Science.gov (United States)

Wilkinson, Trevor C I

2016-06-15

The development of recombinant antibody therapeutics is a significant area of growth in the pharmaceutical industry with almost 50 approved monoclonal antibodies on the market in the US and Europe. Despite this growth, however, certain classes of important molecular targets have remained intractable to therapeutic antibodies due to complexity of the target molecules. These complex target molecules include G-protein-coupled receptors and ion channels which represent a large potential target class for therapeutic intervention with monoclonal antibodies. Although these targets have typically been addressed by small molecule approaches, the exquisite specificity of antibodies provides a significant opportunity to provide selective modulation of these target proteins. Given this opportunity, substantial effort has been applied to address the technical challenges of targeting these complex membrane proteins with monoclonal antibodies. In this review recent progress made in the strategies for discovery of functional monoclonal antibodies for these challenging membrane protein targets is addressed. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

DARPA Antibody Technology Program. Standardized Test Bed for Antibody Characterization: Characterization of an MS2 ScFv Antibody Produced by Illumina

Science.gov (United States)

2016-08-01

ECBC-TR-1395 DARPA ANTIBODY TECHNOLOGY PROGRAM STANDARDIZED TEST BED FOR... ANTIBODY CHARACTERIZATION: CHARACTERIZATION OF AN MS2 SCFV ANTIBODY PRODUCED BY ILLUMINA Patricia E. Buckley Alena M. Calm Heather Welsh Roy...4. TITLE AND SUBTITLE DARPA Antibody Technology Program Standardized Test Bed for Antibody Characterization: Characterization of an MS2 ScFv

Maintenance plasma exchange treatment for muscle specific kinase antibody positive myasthenia gravis patients.

Science.gov (United States)

Yamada, Chisa; Teener, James W; Davenport, Robertson D; Cooling, Laura

2015-10-01

Anti-muscle specific kinase antibody positive myasthenia gravis (MuSK MG) is often characterized by a relatively severe and progressive course, refractoriness to standard myasthenia gravis (MG) medications, and an increased risk of myasthenic crisis. We report here successful management of three MuSK MG patients using maintenance therapeutic plasma exchange (TPE) treatment for up to 4.5 years. The study was a 5-year retrospective review of all MG patients treated with TPE between 2008 and 2013 at University of Michigan. Inclusion criteria of MuSK MG were positive for anti-MuSK antibodies and a diagnosis of MuSK MG by staff neurologists. Patient data included age, gender, diagnostic testing results, medications, and the dates and response to TPE treatments. A total of 153 MG patients underwent at least one course of TPE between 2008 and 2013. A total of 12 patients (7.8%) were positive for anti-MuSK antibodies. Patients were predominantly female (83.3%) and a median age of onset was 46-years old. Three MuSK MG patients were successfully managed with maintenance TPE. Maintenance TPE may be an effective option for MuSK MG patients. The key of successful maintenance treatment at our institution has been to tailor the TPE frequency for each individual, and to modify the treatment interval in conjunction with medical management. © 2014 Wiley Periodicals, Inc.

Monoclonal Antibodies to Intracellular Stages of Cryptosporidium parvum Define Life Cycle Progression In Vitro.

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Wilke, Georgia; Ravindran, Soumya; Funkhouser-Jones, Lisa; Barks, Jennifer; Wang, Qiuling; VanDussen, Kelli L; Stappenbeck, Thaddeus S; Kuhlenschmidt, Theresa B; Kuhlenschmidt, Mark S; Sibley, L David

2018-06-27

Among the obstacles hindering Cryptosporidium research is the lack of an in vitro culture system that supports complete life development and propagation. This major barrier has led to a shortage of widely available anti- Cryptosporidium antibodies and a lack of markers for staging developmental progression. Previously developed antibodies against Cryptosporidium were raised against extracellular stages or recombinant proteins, leading to antibodies with limited reactivity across the parasite life cycle. Here we sought to create antibodies that recognize novel epitopes that could be used to define intracellular development. We identified a mouse epithelial cell line that supported C. parvum growth, enabling immunization of mice with infected cells to create a bank of monoclonal antibodies (MAbs) against intracellular parasite stages while avoiding the development of host-specific antibodies. From this bank, we identified 12 antibodies with a range of reactivities across the parasite life cycle. Importantly, we identified specific MAbs that can distinguish different life cycle stages, such as trophozoites, merozoites, type I versus II meronts, and macrogamonts. These MAbs provide valuable tools for the Cryptosporidium research community and will facilitate future investigation into parasite biology. IMPORTANCE Cryptosporidium is a protozoan parasite that causes gastrointestinal disease in humans and animals. Currently, there is a limited array of antibodies available against the parasite, which hinders imaging studies and makes it difficult to visualize the parasite life cycle in different culture systems. In order to alleviate this reagent gap, we created a library of novel antibodies against the intracellular life cycle stages of Cryptosporidium We identified antibodies that recognize specific life cycle stages in distinctive ways, enabling unambiguous description of the parasite life cycle. These MAbs will aid future investigation into Cryptosporidium biology and

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

International Nuclear Information System (INIS)

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

2009-01-01

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

Antiparietal cell antibody test

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APCA; Anti-gastric parietal cell antibody; Atrophic gastritis - anti-gastric parietal cell antibody; Gastric ulcer - anti-gastric parietal cell antibody; Pernicious anemia - anti-gastric parietal cell antibody; ...

Fusion proteins of HIV-1 envelope glycoprotein gp120 with CD4-induced antibodies showed enhanced binding to CD4 and CD4 binding site antibodies

Energy Technology Data Exchange (ETDEWEB)

Chen, Weizao, E-mail: chenw3@mail.nih.gov [Protein Interactions Group, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); Feng, Yang [Protein Interactions Group, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); Wang, Yanping [Protein Interactions Group, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); The Basic Research Program, Science Applications International Corporation-Frederick, Inc., National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); Zhu, Zhongyu; Dimitrov, Dimiter S. [Protein Interactions Group, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States)

2012-09-07

Highlights: Black-Right-Pointing-Pointer Some recombinant HIV-1 gp120s do not preserve their conformations on gp140s. Black-Right-Pointing-Pointer We hypothesize that CD4i antibodies could induce conformational changes in gp120. Black-Right-Pointing-Pointer CD4i antibodies enhance binding of CD4 and CD4bs antibodies to gp120. Black-Right-Pointing-Pointer CD4i antibody-gp120 fusion proteins could have potential as vaccine immunogens. -- Abstract: Development of successful AIDS vaccine immunogens continues to be a major challenge. One of the mechanisms by which HIV-1 evades antibody-mediated neutralizing responses is the remarkable conformational flexibility of its envelope glycoprotein (Env) gp120. Some recombinant gp120s do not preserve their conformations on gp140s and functional viral spikes, and exhibit decreased recognition by CD4 and neutralizing antibodies. CD4 binding induces conformational changes in gp120 leading to exposure of the coreceptor-binding site (CoRbs). In this study, we test our hypothesis that CD4-induced (CD4i) antibodies, which target the CoRbs, could also induce conformational changes in gp120 leading to better exposed conserved neutralizing antibody epitopes including the CD4-binding site (CD4bs). We found that a mixture of CD4i antibodies with gp120 only weakly enhanced CD4 binding. However, such interactions in single-chain fusion proteins resulted in gp120 conformations which bound to CD4 and CD4bs antibodies better than the original or mutagenically stabilized gp120s. Moreover, the two molecules in the fusion proteins synergized with each other in neutralizing HIV-1. Therefore, fusion proteins of gp120 with CD4i antibodies could have potential as components of HIV-1 vaccines and inhibitors of HIV-1 entry, and could be used as reagents to explore the conformational flexibility of gp120 and mechanisms of entry and immune evasion.

Stoichiometric constraints do not limit successful invaders: zebra mussels in Swedish lakes.

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Naddafi, Rahmat; Eklöv, Peter; Pettersson, Kurt

2009-01-01

Elemental imbalances of carbon (C): nitrogen (N): phosphorus (P) ratios in food resources can constrain the growth of grazers owning to tight coupling between growth rate, RNA allocation and biomass P content in animals. Testing for stoichiometric constraints among invasive species is a novel challenge in invasion ecology to unravel how a successful invader tackles ecological barriers in novel ecosystems. We examined the C:P and N:P ratios and the condition factor of a successful invader in lakes, the zebra mussel (Dreissena polymorpha), collected from two Swedish lakes. Concurrently, we analyzed the elemental composition of the food (seston) and tissue of the mussels in which nutrient composition of food and mussels varied over time. Zebra mussel condition factor was weakly related to the their own tissue N:P and C:P ratios, although the relation with the later ratio was not significant. Smaller mussels had relatively lower tissue N:P ratio and higher condition factor. There was no difference in C:P and N:P ratios between seston and mussels' tissues. Our results indicated that the variation in nutrient stoichiometry of zebra mussels can be explained by food quality and quantity. Our study suggests that fitness of invasive zebra mussels is not constrained by nutrient stoichiometry which is likely to be important for their proliferation in novel ecosystems. The lack of imbalance in C:P and N:P ratios between seston and mussels along with high tissue C:P ratio of the mussel allow them to tolerate potential P limitation and maintain high growth rate. Moreover, zebra mussels are able to change their tissue C:P and N:P ratios in response to the variation in elemental composition of their food. This can also help them to bypass potential nutrient stoichiometric constraints. Our finding is an important step towards understanding the mechanisms contributing to the success of exotic species from stoichiometric principles.

Stoichiometric constraints do not limit successful invaders: zebra mussels in Swedish lakes.

Directory of Open Access Journals (Sweden)

Rahmat Naddafi

Full Text Available Elemental imbalances of carbon (C: nitrogen (N: phosphorus (P ratios in food resources can constrain the growth of grazers owning to tight coupling between growth rate, RNA allocation and biomass P content in animals. Testing for stoichiometric constraints among invasive species is a novel challenge in invasion ecology to unravel how a successful invader tackles ecological barriers in novel ecosystems.We examined the C:P and N:P ratios and the condition factor of a successful invader in lakes, the zebra mussel (Dreissena polymorpha, collected from two Swedish lakes. Concurrently, we analyzed the elemental composition of the food (seston and tissue of the mussels in which nutrient composition of food and mussels varied over time. Zebra mussel condition factor was weakly related to the their own tissue N:P and C:P ratios, although the relation with the later ratio was not significant. Smaller mussels had relatively lower tissue N:P ratio and higher condition factor. There was no difference in C:P and N:P ratios between seston and mussels' tissues. Our results indicated that the variation in nutrient stoichiometry of zebra mussels can be explained by food quality and quantity.Our study suggests that fitness of invasive zebra mussels is not constrained by nutrient stoichiometry which is likely to be important for their proliferation in novel ecosystems. The lack of imbalance in C:P and N:P ratios between seston and mussels along with high tissue C:P ratio of the mussel allow them to tolerate potential P limitation and maintain high growth rate. Moreover, zebra mussels are able to change their tissue C:P and N:P ratios in response to the variation in elemental composition of their food. This can also help them to bypass potential nutrient stoichiometric constraints. Our finding is an important step towards understanding the mechanisms contributing to the success of exotic species from stoichiometric principles.

Anti-Idiotypic Antibodies Specific to prM Monoantibody Prevent Antibody Dependent Enhancement of Dengue Virus Infection

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Miao Wang

2017-05-01

Full Text Available Dengue virus (DENV co-circulates as four serotypes (DENV1-4. Primary infection only leads to self-limited dengue fever. But secondary infection with another serotype carries a higher risk of increased disease severity, causing life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS. Serotype cross-reactive antibodies facilitate DENV infection in Fc-receptor-bearing cells by promoting virus entry via Fcγ receptors (FcγR, a process known as antibody dependent enhancement (ADE. Most studies suggested that enhancing antibodies were mainly specific to the structural premembrane protein (prM of DENV. However, there is still no effective drugs or vaccines to prevent ADE. In this study, we firstly confirmed that both DENV-2 infected human sera (anti-DENV-2 and DENV-2 prM monoclonal antibody (prM mAb could significantly enhance DENV-1 infection in K562 cells. Then we developed anti-idiotypic antibodies (prM-AIDs specific to prM mAb by immunizing of Balb/c mice. Results showed that these polyclonal antibodies can dramatically reduce ADE phenomenon of DENV-1 infection in K562 cells. To further confirm the anti-ADE effect of prM-AIDs in vivo, interferon-α and γ receptor-deficient mice (AG6 were used as the mouse model for DENV infection. We found that administration of DENV-2 prM mAb indeed caused a higher DENV-1 titer as well as interleukin-10 (IL-10 and alaninea minotransferase (ALT in mice infected with DENV-1, similar to clinical ADE symptoms. But when we supplemented prM-AIDs to DENV-1 challenged AG6 mice, the viral titer, IL-10 and ALT were obviously decreased to the negative control level. Of note, the number of platelets in peripheral blood of prM-AIDs group were significantly increased at day 3 post infection with DENV-1 compared that of prM-mAb group. These results confirmed that our prM-AIDs could prevent ADE not only in vitro but also in vivo, suggested that anti-idiotypic antibodies might be a new choice to be considered to

Anti-Idiotypic Antibodies Specific to prM Monoantibody Prevent Antibody Dependent Enhancement of Dengue Virus Infection.

Science.gov (United States)

Wang, Miao; Yang, Fan; Huang, Dana; Huang, Yalan; Zhang, Xiaomin; Wang, Chao; Zhang, Shaohua; Zhang, Renli

2017-01-01

Dengue virus (DENV) co-circulates as four serotypes (DENV1-4). Primary infection only leads to self-limited dengue fever. But secondary infection with another serotype carries a higher risk of increased disease severity, causing life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Serotype cross-reactive antibodies facilitate DENV infection in Fc-receptor-bearing cells by promoting virus entry via Fcγ receptors (FcγR), a process known as antibody dependent enhancement (ADE). Most studies suggested that enhancing antibodies were mainly specific to the structural premembrane protein (prM) of DENV. However, there is still no effective drugs or vaccines to prevent ADE. In this study, we firstly confirmed that both DENV-2 infected human sera (anti-DENV-2) and DENV-2 prM monoclonal antibody (prM mAb) could significantly enhance DENV-1 infection in K562 cells. Then we developed anti-idiotypic antibodies (prM-AIDs) specific to prM mAb by immunizing of Balb/c mice. Results showed that these polyclonal antibodies can dramatically reduce ADE phenomenon of DENV-1 infection in K562 cells. To further confirm the anti-ADE effect of prM-AIDs in vivo , interferon-α and γ receptor-deficient mice (AG6) were used as the mouse model for DENV infection. We found that administration of DENV-2 prM mAb indeed caused a higher DENV-1 titer as well as interleukin-10 (IL-10) and alaninea minotransferase (ALT) in mice infected with DENV-1, similar to clinical ADE symptoms. But when we supplemented prM-AIDs to DENV-1 challenged AG6 mice, the viral titer, IL-10 and ALT were obviously decreased to the negative control level. Of note, the number of platelets in peripheral blood of prM-AIDs group were significantly increased at day 3 post infection with DENV-1 compared that of prM-mAb group. These results confirmed that our prM-AIDs could prevent ADE not only in vitro but also in vivo , suggested that anti-idiotypic antibodies might be a new choice to be considered to treat

Immunity War: A Novel Therapy for Lymphoma Using T-cell Bispecific Antibodies.

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Prakash, Ajay; Diefenbach, Catherine S

2018-06-08

The activity of T cell mediated immunotherapies in B cell lymphoma has been limited to date. The novel bi-specific antibody CD20-TCB, has a 2:1 antibody design to maximize T cell engagement, and demonstrates activity in preclinical models. This may represent a novel therapeutic approach for patients with relapsed/refractory NHL. Copyright ©2018, American Association for Cancer Research.

One target, different effects: a comparison of distinct therapeutic antibodies against the same targets.

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Shim, Hyunbo

2011-10-31

To date, more than 30 antibodies have been approved worldwide for therapeutic use. While the monoclonal antibody market is rapidly growing, the clinical use of therapeutic antibodies is mostly limited to treatment of cancers and immunological disorders. Moreover, antibodies against only five targets (TNF-α, HER2, CD20, EGFR, and VEGF) account for more than 80 percent of the worldwide market of therapeutic antibodies. The shortage of novel, clinically proven targets has resulted in the development of many distinct therapeutic antibodies against a small number of proven targets, based on the premise that different antibody molecules against the same target antigen have distinct biological and clinical effects from one another. For example, four antibodies against TNF-α have been approved by the FDA -- infliximab, adalimumab, golimumab, and certolizumab pegol -- with many more in clinical and preclinical development. The situation is similar for HER2, CD20, EGFR, and VEGF, each having one or more approved antibodies and many more under development. This review discusses the different binding characteristics, mechanisms of action, and biological and clinical activities of multiple monoclonal antibodies against TNF-α, HER-2, CD20, and EGFR and provides insights into the development of therapeutic antibodies.

Generation of single domain antibody fragments derived from camelids and generation of manifold constructs.

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Vincke, Cécile; Gutiérrez, Carlos; Wernery, Ulrich; Devoogdt, Nick; Hassanzadeh-Ghassabeh, Gholamreza; Muyldermans, Serge

2012-01-01

Immunizing a camelid (camels and llamas) with soluble, properly folded proteins raises an affinity-matured immune response in the unique camelid heavy-chain only antibodies (HCAbs). The peripheral blood lymphocytes of the immunized animal are used to clone the antigen-binding antibody fragment from the HCAbs in a phage display vector. A representative aliquot of the library of these antigen-binding fragments is used to retrieve single domain antigen-specific binders by successive rounds of panning. These single domain antibody fragments are cloned in tandem to generate manifold constructs (bivalent, biparatopic or bispecific constructs) to increase their functional affinity, to increase specificity, or to connect two independent antigen molecules.

Anti-insulin antibody test

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Insulin antibodies - serum; Insulin Ab test; Insulin resistance - insulin antibodies; Diabetes - insulin antibodies ... Normally, there are no antibodies against insulin in your blood. ... different laboratories. Some labs use different measurements or ...

Characterization of Tumor-Avid Antibody Fragments Genetically Engineered for Mono-Specific Radionuclide Chelation

International Nuclear Information System (INIS)

Quinn, T.P.

2003-01-01

The successful clinical application of targeted-radiopharmaceuticals depends on the development of molecules that optimize tumor specific radionuclide deposition and minimize non-specific organ irradiation. To this end, this proposal outlines a research effort to identify and evaluate novel antibodies and antibody fragments that bind breast tumors. The tumor-avid antibodies will be investigated for as imaging and therapeutic agents and to gain a better understanding of the pharmacokinetics and metabolism of radiolabeled tumor-avid antibody fragments through the use of site-specifically labeled molecules. Antibodies or antibody fragments, that bind breast carcinoma carbohydrate antigens, will be obtained from hybridoma or bacteriophage library screening. More specifically, antibody fragments that bind the carcinoma-associated Thomsen-Friedenreich (T) antigen will be radiolabeled with 99m Tc and 188 Re at a natural amino acid chelation site and will be investigated in vivo for their abilities to target human breast tumors. In addition, site-specific radiolabeled antibody fragments will be biosynthesized using misacylated suppressor tRNAs. Homogeneously radiolabeled populations of antibody fragments will be used to investigate the effects of radionuclide location and chelation chemistries on their biodistribution and metabolism. It is hypothesized that site-specifically radiolabeled antibody fragments will possess enhanced tumor imaging and therapeutic properties due to optimal label location and conjugation chemistries. New insights into the factors that govern antibody metabolism in vivo are also expected from this work. Results from these studies should enhance our ability to design and synthesize radiolabeled antibody fragments that have improved pharmacokinetic properties. The studies in this proposal involve basic research into the development of antibody-based radiopharmaceuticals, with the ultimate goal of application in humans. This type of basic nuclear

A Model System for Concurrent Detection of Antigen and Antibody Based on Immunological Fluorescent Method

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Yuan-Cheng Cao

2015-01-01

Full Text Available This paper describes a combined antigen/antibody immunoassay implemented in a 96-well plate using fluorescent spectroscopic method. First, goat anti-human IgG was used to capture human IgG (model antigen; goat anti-human IgG (Cy3 or FITC was used to detect the model antigen; a saturating level of model antigen was then added followed by unlabelled goat anti-human IgG (model antibody; finally, Cy3 labelled rabbit anti-goat IgG was used to detect the model antibody. Two approaches were applied to the concomitant assay to analyze the feasibility. The first approach applied FITC and Cy3 when both targets were present at the same time, resulting in 50 ng/mL of the antibody detection limit and 10 ng/mL of antigen detection limit in the quantitative measurements of target concentration, taking the consideration of FRET efficiency of 68% between donor and acceptor. The sequential approach tended to lower the signal/noise (S/N ratio and the detection of the model antigen (lower than 1 ng/mL had better sensitivity than the model antibody (lower than 50 ng/mL. This combined antigen/antibody method might be useful for combined detection of antigens and antibodies. It will be helpful to screen for both antigen and antibody particularly in the situations of the multiserotype and high-frequency mutant virus infections.

Novel HIT antibody detection method using Sonoclot® coagulation analyzer.

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Wanaka, Keiko; Asada, Reiko; Miyashita, Kumiko; Kaneko, Makoto; Endo, Hirokazu; Yatomi, Yutaka

2015-01-01

Since heparin-induced thrombocytopenia (HIT), caused by the generation of antibodies against platelet factor 4 (PF4)/heparin complexes (HIT antibodies), may induce serious complications due to thrombosis, a prompt diagnosis is desirable. Functional tests with platelet activation to detect HIT antibodies are useful for diagnosis of HIT, in particular (14)C-selotonin release assay (SRA). However, they are complicated and so can be performed only in limited laboratories. We tested if a blood coagulation test using Sonoclot® analyzer can serve for the detection of HIT antibodies. A murine monoclonal antibody (HIT-MoAb) against PF4/heparin complexes was used as an alternative to human HIT antibodies. To the mixture of HIT-MoAb and heparin (0.5 U/mL, final), whole blood obtained from a healthy volunteer was added, and then the activated clotting time (ACT), clot rate (CR), and area under the curve (AUC) were measured with Sonoclot® analyzer for 30minutes. The HIT-MoAb (30 to 100μg/mL, final) concentration dependently suppressed the anticoagulation activity (prolongation of ACT and decrease of CR and AUC) of heparin. The suppression of anticoagulation effect of heparin by HIT-MoAb was demonstrated by measurements using Sonoclot® analyzer. This method may provide a new tool for screening of HIT antibodies. Copyright © 2014 Elsevier Ltd. All rights reserved.

Hepatitis A virus antibody

International Nuclear Information System (INIS)

Novak, J.; Kselikova, M.; Urbankova, J.

1980-01-01

A description is presented of a radioimmunoassay designed to prove the presence of the antibody against the hepatitis A virus (HA Ab, anti-Ha) using an Abbott HAVAB set. This proof as well as the proof of the antibody against the nucleus of the hepatitis B virus is based on competition between a normal antibody against hepatitis A virus and a 125 I-labelled antibody for the binding sites of a specific antigen spread all over the surface of a tiny ball; this is then indirect proof of the antibody under investigation. The method is described of reading the results from the number of impulses per 60 seconds: the higher the titre of the antibody against the hepatitis A virus in the serum examined, the lower the activity of the specimen concerned. The rate is reported of incidence of the antibody against the hepatitis A virus in a total of 68 convalescents after hepatitis A; the antibody was found in 94.1%. The immunoglobulin made from the convalescents' plasma showed the presence of antibodies in dilutions as high as 1:250 000 while the comparable ratio for normal immunoglobulin Norga was only 1:2500. Differences are discussed in the time incidence of the antibodies against the hepatitis A virus, the antibodies against the surface antigen of hepatitis B, and the antibody against the nucleus of the hepatitis V virus. (author)

Neuronal antibodies in patients with suspected or confirmed sporadic Creutzfeldt-Jakob disease.

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Rossi, Meghan; Mead, Simon; Collinge, John; Rudge, Peter; Vincent, Angela

2015-06-01

There have been reports of patients with antibodies to neuronal antigens misdiagnosed as sporadic Creutzfeldt-Jakob disease (sCJD). Conversely, low levels of antibodies to neuronal proteins have been reported in patients with sCJD. However, the frequency of misdiagnoses, or of antibodies in patients with subsequently confirmed sCJD, is not clear. We reviewed 256 consecutive cases of sCJD seen in the National Prion Clinic, of whom 150 had sera previously referred for selected antibody tests. Eighty-two available samples were retested for antibodies to N-methyl-d-aspartate receptor (NMDAR), the glycine receptor (GlyR), voltage-gated potassium channel (VGKC)-complex and the associated proteins, leucine-rich glioma inactivated 1 (LGI1) and contactin-associated protein 2 (CASPR2). Four of the initial 150 sera referred were positive; two had antibodies to NMDAR, and two to the VGKC-complex, one of which was also positive for GlyR antibodies. Of the 82 sCJD sera retested, one had VGKC-complex antibodies confirming the previous result, two had CASPR2 and GlyR antibodies and one had CASPR2 and NMDAR antibodies; all antibodies were at low levels. Over the same period three patients with autoimmune encephalitis and high VGKC-complex antibodies were initially referred as sCJD. This study indicates that VGKC-complex/LGI1 antibodies. Low titres of neuronal antibodies occur only rarely in suspected patients with sCJD and when present should be interpreted with caution. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Secondary antibodies as tools to improve tumor to non tumor ratio at radioimmunolocalisation and radioimmunotherapy

International Nuclear Information System (INIS)

Ullen, A.; Riklund Aalstroem, K.; Hietala, S.O.; Nilsson, B.; Aerlestig, L.; Stigbrand, T.

1996-01-01

One way of selectively improving the efficiency of radioimmunolocalization and radioimmunotherapy is to eliminate redundant, circulating, non-targeting radiolabeled antibodies after saturation of the target sites. Secondary antibodies of different types have been proposed as clearing agents for such purposes. The conceptually different approaches of the 'secondary antibody' strategy including its advantages and limitations are discussed. This mini-review also presents a model describing the kinetics of the components (the antigen, the primary and secondary antibodies) and approaches required to improve the efficacy of both radioimmunolocalization and radioimmunotherapy. (orig.)

Donor-derived HLA antibody production in patients undergoing SCT from HLA antibody-positive donors.

Science.gov (United States)

Taniguchi, K; Yoshihara, S; Maruya, E; Ikegame, K; Kaida, K; Hayashi, K; Kato, R; Inoue, T; Fujioka, T; Tamaki, H; Okada, M; Onuma, T; Fujii, N; Kusunoki, Y; Soma, T; Saji, H; Ogawa, H

2012-10-01

Pre-existing donor-specific HLA antibodies in patients undergoing HLA-mismatched SCT have increasingly been recognized as a risk factor for primary graft failure. However, the clinical implications of the presence of HLA antibodies in donors remain unknown. We prospectively examined 123 related donors for the presence of HLA antibodies by using a Luminex-based single antigen assay. Of these, 1/57 (1.8%) male, 6/27 (22%) parous female and 0/39 (0%) nonparous female donors were HLA antibody-positive. Then, we determined the presence of HLA antibodies in seven patients who received SCT from antibody-positive donors. Of these, four became HLA antibody-positive after SCT. The specificities of the antibodies that emerged in the patients closely resembled those of the antibodies found in the donors, indicating their production by donor-derived plasma cells. Moreover, the kinetics of the HLA antibody levels were similar in all four patients: levels started increasing within 1 week after SCT and peaked at days 10-21, followed by a gradual decrease. These results suggest that donor-derived HLA antibody production frequently occurs in patients undergoing SCT from antibody-positive donors. Further studies are warranted for clarifying the clinical significance of donor-derived HLA antibodies, including the role of these antibodies in post transplant platelet transfusion refractoriness.

A novel lentiviral scFv display library for rapid optimization and selection of high affinity antibodies.

Science.gov (United States)

Qudsia, Sehar; Merugu, Siva B; Mangukiya, Hitesh B; Hema, Negi; Wu, Zhenghua; Li, Dawei

2018-04-30

Antibody display libraries have become a popular technique to screen monoclonal antibodies for therapeutic purposes. An important aspect of display technology is to generate an optimization library by changing antibody affinity to antigen through mutagenesis and screening the high affinity antibody. In this study, we report a novel lentivirus display based optimization library antibody in which Agtuzumab scFv is displayed on cell membrane of HEK-293T cells. To generate an optimization library, hotspot mutagenesis was performed to achieve diverse antibody library. Based on sequence analysis of randomly selected clones, library size was estimated approximately to be 1.6 × 10 6 . Lentivirus display vector was used to display scFv antibody on cell surface and flow cytometery was performed to check the antibody affinity to antigen. Membrane bound scFv antibodies were then converted to secreted antibody through cre/loxP recombination. One of the mutant clones, M8 showed higher affinity to antigen in flow cytometery analysis. Further characterization of cellular and secreted scFv through western blot showed that antibody affinity was increased by three fold after mutagenesis. This study shows successful construction of a novel antibody library and suggests that hotspot mutagenesis could prove a useful and rapid optimization tool to generate similar libraries with various degree of antigen affinity. Copyright © 2018 Elsevier Inc. All rights reserved.

Hemolytic disease of the fetus and newborn due to multiple maternal antibodies.

Science.gov (United States)

Markham, Kara Beth; Rossi, Karen Q; Nagaraja, Haikady N; O'Shaughnessy, Richard W

2015-07-01

The objective of the study was to determine whether women with combinations of red blood cell antibodies are more likely to develop significant hemolytic disease of the fetus and newborn than those with single antibodies. A retrospective exposure cohort study was conducted of pregnant women with red blood cell antibodies. The development of significant hemolytic disease of the fetus and newborn was then compared between patients with single antibodies and those with multiple antibodies. Data analysis was limited to pregnancies delivering since the year 2000. Thirteen percent of the patients referred to our program had multiple red blood cell antibodies. Odds of developing significant hemolytic disease of the fetus and newborn for patients with anti-Rh(D) combined with at least 1 additional red blood cell antibody were 3.65 times the odds for women with anti-Rh(D) antibodies in isolation (95% confidence interval, 1.84-7.33). In the setting of multiple antibodies including anti-Rh(D), Rh-positive fetuses/neonates have an increased odds of developing significant hemolytic disease even if the fetus is negative for the other corresponding red blood cell antigen. Women with multiple red blood cell antibodies are more likely to develop significant hemolytic disease of the fetus and newborn than those with a single antibody especially in the presence of anti-(Rh)D. This pathophysiology may suggest a more aggressive immune response in women who develop more than 1 red blood cell antibody. Copyright © 2015 Elsevier Inc. All rights reserved.

The self-nonself discrimination and the nature and acquisition of the antibody repertoire.

Science.gov (United States)

Coutinho, A

1980-01-01

Network ideas are confronted with current hypotheses for the origin of antibody diversity and self-nonself discrimination. The difficulties of reconciling the promethean evolution of the antibody system with "germ line" theories are discussed, as well as the problems of "somatic" hypotheses to explain the completeness of the antibody repertoire. The formal incompatibility of the network theory with ideas basing self-nonself discrimination on the elimination of self-reactive cells is demonstrated, as well as the difficulties of these and other environment-dependent hypotheses for lymphocyte activation, to encompass the internal activity in the immune system. It is argued, on the other hand, that the limitations of the network theory in providing a functional basis for the idiotypic network and in accounting for self-nonself discrimination, can be solved by finding in a complete repertoire of antibody-combining sites the complementary structures to growth receptors on B lymphocytes, and by using these as internal mitogens in the expansion of the precursor cell pools and in the maintenance of the mature steady states. Letting self-nonself discrimination be accounted for by such growth receptors, both the integrity of the antibody repertoire and the internal activity in the system can also be ensured. Moreover, by postulating a germ line origin for the antireceptor antibodies and by accepting idiotypic cross-reactivity between growth receptors and other germ line antibodies, the possibilities are set for a phylogenetically and ontogenically autonomous immune system embodied with the capabilities for self-expansion, diversification and selection of available repertoires. Its promethean characteristics are explained by its completeness, and this is achieved by idiotypic interactions between growth receptors and a limited number of complementary or cross-reactive germ line antibodies, naturally selected on the basis of their structural relationships with growth receptors.

Aggregate complexes of HIV-1 induced by multimeric antibodies.

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Stieh, Daniel J; King, Deborah F; Klein, Katja; Liu, Pinghuang; Shen, Xiaoying; Hwang, Kwan Ki; Ferrari, Guido; Montefiori, David C; Haynes, Barton; Pitisuttithum, Punnee; Kaewkungwal, Jaranit; Nitayaphan, Sorachai; Rerks-Ngarm, Supachai; Michael, Nelson L; Robb, Merlin L; Kim, Jerome H; Denny, Thomas N; Tomaras, Georgia D; Shattock, Robin J

2014-10-02

Antibody mediated viral aggregation may impede viral transfer across mucosal surfaces by hindering viral movement in mucus, preventing transcytosis, or reducing inter-cellular penetration of epithelia thereby limiting access to susceptible mucosal CD4 T cells and dendritic cells. These functions may work together to provide effective immune exclusion of virus from mucosal tissue; however little is known about the antibody characteristics required to induce HIV aggregation. Such knowledge may be critical to the design of successful immunization strategies to facilitate viral immune exclusion at the mucosal portals of entry. The potential of neutralizing and non-neutralizing IgG and IgA monoclonals (mAbs) to induce HIV-1 aggregation was assessed by Dynamic light scattering (DLS). Although neutralizing and non-neutralizing IgG mAbs and polyclonal HIV-Ig efficiently aggregated soluble Env trimers, they were not capable of forming viral aggregates. In contrast, dimeric (but not monomeric) IgA mAbs induced stable viral aggregate populations that could be separated from uncomplexed virions. Epitope specificity influenced both the degree of aggregation and formation of higher order complexes by dIgA. IgA purified from serum of uninfected RV144 vaccine trial responders were able to efficiently opsonize viral particles in the absence of significant aggregation, reflective of monomeric IgA. These results collectively demonstrate that dIgA is capable of forming stable viral aggregates providing a plausible basis for testing the effectiveness of aggregation as a potential protection mechanism at the mucosal portals of viral entry.

CD44 antibodies and immune thrombocytopenia in the amelioration of murine inflammatory arthritis.

Directory of Open Access Journals (Sweden)

Patrick J Mott

Full Text Available Antibodies to CD44 have been used to successfully ameliorate murine models of autoimmune disease. The most often studied disease model has been murine inflammatory arthritis, where a clear mechanism for the efficacy of CD44 antibodies has not been established. We have recently shown in a murine passive-model of the autoimmune disease immune thrombocytopenia (ITP that some CD44 antibodies themselves can induce thrombocytopenia in mice, and the CD44 antibody causing the most severe thrombocytopenia (IM7, also is known to be highly effective in ameliorating murine models of arthritis. Recent work in the K/BxN serum-induced model of arthritis demonstrated that antibody-induced thrombocytopenia reduced arthritis, causing us to question whether CD44 antibodies might primarily ameliorate arthritis through their thrombocytopenic effect. We evaluated IM7, IRAWB14.4, 5035-41.1D, KM201, KM114, and KM81, and found that while all could induce thrombocytopenia, the degree of protection against serum-induced arthritis was not closely related to the length or severity of the thrombocytopenia. CD44 antibody treatment was also able to reverse established inflammation, while thrombocytopenia induced by an anti-platelet antibody targeting the GPIIbIIIa platelet antigen, could not mediate this effect. While CD44 antibody-induced thrombocytopenia may contribute to some of its therapeutic effect against the initiation of arthritis, for established disease there are likely other mechanisms contributing to its efficacy. Humans are not known to express CD44 on platelets, and are therefore unlikely to develop thrombocytopenia after CD44 antibody treatment. An understanding of the relationship between arthritis, thrombocytopenia, and CD44 antibody treatment remains critical for continued development of CD44 antibody therapeutics.

Emerging psychiatric syndromes associated with antivoltage-gated potassium channel complex antibodies.

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Prüss, Harald; Lennox, Belinda R

2016-11-01

Antibodies against the voltage-gated potassium channel (VGKC) were first recognised as having a potential pathogenic role in disorders of the central nervous system in 2001, with VGKC antibodies described in patients with limbic encephalitis, and the subsequent seminal paper describing the clinical phenotype and immunotherapy treatment responsiveness in 13 patients with VGKC antibodies and limbic encephalitis in 2004. These initial case descriptions were of a progressive neuropsychiatric syndrome with abnormalities of mood, sleep and cognition recognised alongside the neurological symptoms of seizures and autonomic instability. The clinical syndromes associated with VGKC complex (VGKCC) antibodies have broadened considerably over the last 15 years, with multiple cases of more restricted 'formes fruste' presentations associated with VGKCC antibodies being described. However, the relevance of antibodies in these cases has remained controversial. The understanding of the pathogenic nature of VGKC antibodies has further advanced since 2010 with the discovery that VGKC antibodies are not usually antibodies against the VGKC subunits themselves, but instead to proteins that are complexed with the potassium channel, in particular leucine-rich, glioma-inactivated protein 1 (LGI1) and contactin-associated protein 2 (Caspr2). Antibodies against these proteins have been associated with particular, although overlapping, clinical phenotypes, each also including neuropsychiatric features. Our aim is to critically review the association between VGKCC, LGI1 and Caspr2 antibodies with isolated psychiatric presentations-with a focus on cognitive impairment, mood disorders and psychosis. We recommend that screening for VGKCC, LGI1 and Caspr2 antibodies be considered for those with neuropsychiatric presentations. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

Radioimmunodetection of hepatocellular carcinoma with a radiolabeled antibody to alpha fetoprotein

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Ishii, N; Nakata, K; Muro, T [Nagasaki Univ. (Japan). School of Medicine

1982-03-01

The present study was undertaken to investigate whether or not radiolabeled antibody to Alpha-fetoprotein (AFP) intravenously administered accumulates in AFP-producing tumors. Rats with subcutaneously transplanted hepatoma or with heptoma produced in liver by 3'-Me-DAB, and 12 patients with hepatocellular carcinoma (HCC) were studied. In animal experiments, the tumor localization was clearly demonstrated by total body scintigraphy 48 to 168 hours after the injection of the radioiodinated antibody. The rats were sacrificed and radioactivity in tissues was counted. Tumor tissues showed the highest counts. In patients with HCC, the imaging was made with a gamma camera usually 24 and 48 hours after injection of I-131-labeled antibody. In order to make the contrast of tumor image clear, nonspecific background images obtained by administration of Tc-99m-labeled serum albumin were subtracted from the images obtained by I-131 labeled antibody with a computer. In 6 of 12 patients with HCC, the tumor location could successfully demonstrated. However, tumor with sizes below 2 cm in diameter which were able to be detected by the celiac angiography were not detectable. Interestingly the images were more clear in patients with relatively higher serum AFP levels. In the blood of patient given injection of AFP radioantibody, both immune complex and free antibody were able to be detected at least for 120 hours so that the presence of free antigen (AFP) did not appear to prevent tumor radioimmunodetection. The information obtained concerning the biochemical factors which influence antibody localization in the tumor would provide better method for radioimmunodetection with antitumor antibodies.

Use and limitations of radiolabelled anti-CEA antibodies and their fragments for photoscanning detection of human colorectal carcinomas

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Mach, J P; Forni, M; Ritschard, J; Buchegger, F; Carrel, S; Widgren, S; Donath, A; Alberto, P [Lausanne Univ. (Switzerland)

1980-08-01

Fifty-three patients with histologically proven carcinoma were injected with highly purified (/sup 131/I)-labelled goat antibodies or fragments of antibodies against carcinoembryonic antigen (CEA). Each patient was tested by external photoscanning 4, 24, 36, 48h after injection. In 22 patients (16 of 38 injected with intact antibodies, 5 of 13 with F(ab')/sub 2/ fragments and 1 of 2 with Fab' fragments), an increased concentration of /sup 131/I radioactivity corresponding to the previously known tumor location was detected by photoscanning 36-48 h after injection. Blood pool and secreted radioactivity was determined in all patients by injecting 15 min after scanning, (sup(99m)Tc)-labeled normal serum albumin and free sup(99m)TcO/sub 4//sup -/. The computerized subtraction of sup(99m)Tc from /sup 131/I radioactivity enhanced the definition of tumor localization in the 22 positive patients. However, in spite of the computerized subtraction, interpretation of the scans remained doubtful for 12 patients and was entirely negative for 19 additional patients.

CEACAM1 induces B-cell survival and is essential for protective antiviral antibody production

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Khairnar, Vishal; Duhan, Vikas; Maney, Sathish Kumar; Honke, Nadine; Shaabani, Namir; Pandyra, Aleksandra A.; Seifert, Marc; Pozdeev, Vitaly; Xu, Haifeng C.; Sharma, Piyush; Baldin, Fabian; Marquardsen, Florian; Merches, Katja; Lang, Elisabeth; Kirschning, Carsten; Westendorf, Astrid M.; Häussinger, Dieter; Lang, Florian; Dittmer, Ulf; Küppers, Ralf; Recher, Mike; Hardt, Cornelia; Scheffrahn, Inka; Beauchemin, Nicole; Göthert, Joachim R.; Singer, Bernhard B.; Lang, Philipp A.; Lang, Karl S.

2015-01-01

B cells are essential for antiviral immune defence because they produce neutralizing antibodies, present antigen and maintain the lymphoid architecture. Here we show that intrinsic signalling of CEACAM1 is essential for generating efficient B-cell responses. Although CEACAM1 exerts limited influence on the proliferation of B cells, expression of CEACAM1 induces survival of proliferating B cells via the BTK/Syk/NF-κB-axis. The absence of this signalling cascade in naive Ceacam1−/− mice limits the survival of B cells. During systemic infection with cytopathic vesicular stomatitis virus, Ceacam1−/− mice can barely induce neutralizing antibody responses and die early after infection. We find, therefore, that CEACAM1 is a crucial regulator of B-cell survival, influencing B-cell numbers and protective antiviral antibody responses. PMID:25692415

A study of labelling of monoclonal antibody C50 with 99Tcm and using for radioimmunoimaging

International Nuclear Information System (INIS)

Huang Biao; Yang Min; Wang Yang; Lu Zhongwei; Guan Liang; Guo Wanhua; Zhu Chengmo

2002-01-01

Objective: To provide an accurate and scientific method of radionuclide imaging with 99 Tc m -C50. Methods: The labelled C50 was derived by imino-thiolane and ligand exchange from 99 Tc m O(GH) 2 - . HPLC ws used to analyse the modified antibody and labelled antibody, labelling efficiency was measured by paper chromatography. Radioimmunoimaging was performed on human colon tumor bearing nude mice. Results: The labelling efficiency of the labelled antibody was 97%, with 4% colloidal 99 Tc m in it; the in vitro competition test showed that 99 Tc m being bound up with the antibody at high affinity. The stability of the imino-thiolane modified antibody could be kept at 4 degree C for 3 months. The biodistribution study showed that the tumor radioactivity uptake at 24 h postinjection was the highest, the ratio of tumor to muscle was 4.03. Conclusion: The labelling method applied to the preparation of 99 Tc m labelled antibody C50 was successful, and appears to be equally applicable for labelling other antibodies

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

Science.gov (United States)

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

2014-11-15

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

Locking the Elbow: Improved Antibody Fab Fragments as Chaperones for Structure Determination.

Science.gov (United States)

Bailey, Lucas J; Sheehy, Kimberly M; Dominik, Pawel K; Liang, Wenguang G; Rui, Huan; Clark, Michael; Jaskolowski, Mateusz; Kim, Yejoon; Deneka, Dawid; Tang, Wei-Jen; Kossiakoff, Anthony A

2018-02-02

Antibody Fab fragments have been exploited with significant success to facilitate the structure determination of challenging macromolecules as crystallization chaperones and as molecular fiducial marks for single particle cryo-electron microscopy approaches. However, the inherent flexibility of the "elbow" regions, which link the constant and variable domains of the Fab, can introduce disorder and thus diminish their effectiveness. We have developed a phage display engineering strategy to generate synthetic Fab variants that significantly reduces elbow flexibility, while maintaining their high affinity and stability. This strategy was validated using previously recalcitrant Fab-antigen complexes where introduction of an engineered elbow region enhanced crystallization and diffraction resolution. Furthermore, incorporation of the mutations appears to be generally portable to other synthetic antibodies and may serve as a universal strategy to enhance the success rates of Fabs as structure determination chaperones. Copyright © 2017 Elsevier Ltd. All rights reserved.

The Role of Anti-Drug Antibodies in the Pharmacokinetics, Disposition, Target Engagement, and Efficacy of a GITR Agonist Monoclonal Antibody in Mice.

Science.gov (United States)

Brunn, Nicholas D; Mauze, Smita; Gu, Danling; Wiswell, Derek; Ueda, Roanna; Hodges, Douglas; Beebe, Amy M; Zhang, Shuli; Escandón, Enrique

2016-03-01

Administration of biologics to enhance T-cell function is part of a rapidly growing field of cancer immunotherapy demonstrated by the unprecedented clinical success of several immunoregulatory receptor targeting antibodies. While these biologic agents confer significant anti-tumor activity through targeted immune response modulation, they can also elicit broad immune responses potentially including the production of anti-drug antibodies (ADAs). DTA-1, an agonist monoclonal antibody against GITR, is a highly effective anti-tumor treatment in preclinical models. We demonstrate that repeated dosing with murinized DTA-1 (mDTA-1) generates ADAs with corresponding reductions in drug exposure and engagement of GITR on circulating CD3(+) CD4(+) T cells, due to rapid hepatic drug uptake and catabolism. Mice implanted with tumors after induction of preexisting mDTA-1 ADA show no anti-tumor efficacy when given 3 mg/kg mDTA-1, an efficacious dose in naive mice. Nonetheless, increasing mDTA-1 treatment to 30 mg/kg in ADA-positive mice restores mDTA-1 exposure and GITR engagement on circulating CD3(+) CD4(+) T cells, thereby partially restoring anti-tumor efficacy. Formation of anti-mDTA-1 antibodies and changes in drug exposure and disposition does not occur in GITR(-/-) mice, consistent with a role for GITR agonism in humoral immunity. Finally, the administration of muDX400, a murinized monoclonal antibody against the checkpoint inhibitor PD-1, dosed alone or combined with mDTA-1 did not result in reduced muDX400 exposure, nor did it change the nature of the anti-mDTA-1 response. This indicates that anti-GITR immunogenicity may not necessarily impact the pharmacology of coadministered monoclonal antibodies, supporting combination immunomodulatory strategies. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Production of a Human Antibody Library in the Phage-Display Vector pSEX81.

Science.gov (United States)

Welschof, M; Little, M; Dörsam, H

1998-01-01

Human monoclonal antibodies (MAbs) are more suitable than MAbs of animal origin for clinical applications because of lower hypersensitivity reactions, less formation of circulating immune complexes and lower anti-immunoglobulin responses The classical production of human MAbs via the hybridoma technique or Epstein-Barr virus (EBV) transformation is limited by the instability of cell lines, low antibody production, and the problems of imununizing humans with certain antigens (1,2). A promising alternative 1s the production of human recombinant antibodies (3). Recombinant DNA technology has made it possible to clone human antibody genes in vectors and to generate antibody expression libraries (4-7). One approach has been to amplify and recombine the IgG repertoire of an "immunized" donor. This has been used to isolate several antibodies related to diseases (8,9). In order to obtain more universal antibody libraries the naive IgM repertoire of several "unimmunized" donors were pooled (10,12). The complexity of the combinatorial libraries has been further increased by creating the so-called "semisynthetic" antibody libraries (22-14).

Six Sigma: Problems, Limitations, Critical Success Factors and Future Prospects

Directory of Open Access Journals (Sweden)

Paulo Mazieiro Pohlmann

2015-12-01

Full Text Available Six Sigma is a business strategy based on objective decision making and problem solving in order to achieve, maintain and maximize business success through understanding and meeting the needs of customers. The visualization of this methodology as a powerful tool in reducing variability and improving quality led to the interest in performing this bibliographical study, whose purpose was to assess the critical success factors and future prospects of this managerial system. A survey was conducted in order to discover the main critical success factors of the implementation of the methodology in organizations, among which stood out the proper selection of projects, connecting the project with the business strategy, customer focus, financial, human and infrastructure resources, the involvement of senior management, professional training, and cultural change.

Engineering cholesterol-based fibers for antibody immobilization and cell capture

Science.gov (United States)

Cohn, Celine

abilities of the CSS fibers were compared to that of hydrophobic polycaprolactone (PCL) fibers and hydrophilic plasma-treated PCL fibers. Electrospun CSS fibers were found to immobilize equivalent amounts of protein as hydrophobically immobilized proteins. However, these proteins captured 6 times more cells, indicative of retained protein function. The second key concept was the design and fabrication of a hybridized lipid fiber. Lipid fibers provide improved protein function but fabrication difficulties have limited their adoption. Thus, we sought to fabricate a lipid-polymer hybrid that is easily fabricated while maintaining protein function. The hybrid fiber consists of a PCL backbone with conjugated CSS. The hybrid lipid fibers showed improved protein function. In addition, higher lipid concentrations were directly correlated to higher cell capture efficiencies. The third key concept was on the development of dually functionalized lipid fibers and understanding the resulting cell capture efficiencies. Many platforms are unable to simultaneously search for heterogeneous populations of CTCs -- the ability to dually functionalize cell-capturing platforms would address this technological weakness. Studies indicated that dually functionalizing the lipid fibers did not compromise the platforms' abilities to capture the cells of interest. Such dually functionalized fibers allow for a single cell-capture platform to successfully detect heterogeneous populations of CTCs. The body of work encompassed herein describes the use of lipid fibers for antibody immobilization and cell capture. Data from various projects indicate that the use of cholesterol-based fibers produced from electrospun CSS are well suited for protein immobilization. The CSS fibers are able to immobilize equivalent amounts of protein as compared to other immobilization techniques. However, the benefit of these fibers is illustrated by the strong cell-capturing efficiencies, indicating that the immobilized

GABARAPL1 antibodies: target one protein, get one free!

Science.gov (United States)

Le Grand, Jaclyn Nicole; Chakrama, Fatima Zahra; Seguin-Py, Stéphanie; Fraichard, Annick; Delage-Mourroux, Régis; Jouvenot, Michèle; Risold, Pierre-Yves; Boyer-Guittaut, Michaël

2011-11-01

Atg8 is a yeast protein involved in the autophagic process and in particular in the elongation of autophagosomes. In mammals, several orthologs have been identified and are classed into two subfamilies: the LC3 subfamily and the GABARAP subfamily, referred to simply as the LC3 or GABARAP families. GABARAPL1 (GABARAP-like protein 1), one of the proteins belonging to the GABARAP (GABA(A) receptor-associated protein) family, is highly expressed in the central nervous system and implicated in processes such as receptor and vesicle transport as well as autophagy. The proteins that make up the GABARAP family demonstrate conservation of their amino acid sequences and protein structures. In humans, GABARAPL1 shares 86% identity with GABARAP and 61% with GABARAPL2 (GATE-16). The identification of the individual proteins is thus very limited when working in vivo due to a lack of unique peptide sequences from which specific antibodies can be developed. Actually, and to our knowledge, there are no available antibodies on the market that are entirely specific to GABARAPL1 and the same may be true of the anti-GABARAP antibodies. In this study, we sought to examine the specificity of three antibodies targeted against different peptide sequences within GABARAPL1: CHEM-CENT (an antibody raised against a short peptide sequence within the center of the protein), PTG-NTER (an antibody raised against the N-terminus of the protein) and PTG-FL (an antibody raised against the full-length protein). The results described in this article demonstrate the importance of testing antibody specificity under the conditions for which it will be used experimentally, a caution that should be taken when studying the expression of the GABARAP family proteins.

Localisation of lung cancer by a radiolabelled monoclonal antibody against the c-myc oncogene product

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Chan, S Y.T.; Evan, G I; Ritson, A; Watson, J; Wraight, P; Sikora, K

1986-11-01

A set of mouse monoclonal antibodies against the c-myc oncogene product, a 62,000 dalton nuclear binding protein involved in cell cycle control, has been constructed by immunisation with synthetic peptide fragments. One such antibody, CT14, was radiolabelled with /sup 131/I and administered to 20 patients with different malignant diseases. Good tumour localisation was observed in 12 out of 14 patients with primary bronchial carcinoma but not in patients with pulmonary metastases from primary tumours elsewhere. Successfully localised tumours were all 3 cm or more in diameter. Monoclonal antibodies against oncogene products may provide novel selective tools for the diagnosis and therapy of cancer.

Long-term outcome of anti-glomerular basement membrane antibody disease treated with immunoadsorption.

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Biesenbach, Peter; Kain, Renate; Derfler, Kurt; Perkmann, Thomas; Soleiman, Afschin; Benharkou, Alexandra; Druml, Wilfred; Rees, Andrew; Säemann, Marcus D

2014-01-01

Anti-glomerular basement membrane (GBM) antibody disease may lead to acute crescentic glomerulonephritis with poor renal prognosis. Current therapy favours plasma exchange (PE) for removal of pathogenic antibodies. Immunoadsorption (IAS) is superior to PE regarding efficiency of antibody-removal and safety. Apart from anecdotal data, there is no systemic analysis of the long-term effects of IAS on anti-GBM-disease and antibody kinetics. To examine the long-term effect of high-frequency IAS combined with standard immunosuppression on patient and renal survival in patients with anti-GBM-disease and to quantify antibody removal and kinetics through IAS. Retrospective review of patients treated with IAS for anti-GBM-antibody disease confirmed by biopsy and/or anti-GBM-antibodies. University Hospital of Vienna, Austria. 10 patients with anti-GBM-disease treated with IAS. Patient and renal survival, renal histology, anti-GBM-antibodies. Anti-GBM-antibodies were reduced by the first 9 IAS treatments (mean number of 23) to negative levels in all patients. Renal survival was 40% at diagnosis, 70% after the end of IAS, 63% after one year and 50% at the end of observation (mean 84 months, range 9 to 186). Dialysis dependency was successfully reversed in three of six patients. Patient survival was 90% at the end of observation. IAS efficiently eliminates anti-GBM-antibodies suggesting non-inferiority to PE with regard to renal and patient survival. Hence IAS should be considered as a valuable treatment option for anti-GBM-disease, especially in patients presenting with a high percentage of crescents and dialysis dependency due to an unusual high proportion of responders.

Long-term outcome of anti-glomerular basement membrane antibody disease treated with immunoadsorption.

Directory of Open Access Journals (Sweden)

Peter Biesenbach

Full Text Available Anti-glomerular basement membrane (GBM antibody disease may lead to acute crescentic glomerulonephritis with poor renal prognosis. Current therapy favours plasma exchange (PE for removal of pathogenic antibodies. Immunoadsorption (IAS is superior to PE regarding efficiency of antibody-removal and safety. Apart from anecdotal data, there is no systemic analysis of the long-term effects of IAS on anti-GBM-disease and antibody kinetics.To examine the long-term effect of high-frequency IAS combined with standard immunosuppression on patient and renal survival in patients with anti-GBM-disease and to quantify antibody removal and kinetics through IAS.Retrospective review of patients treated with IAS for anti-GBM-antibody disease confirmed by biopsy and/or anti-GBM-antibodies.University Hospital of Vienna, Austria.10 patients with anti-GBM-disease treated with IAS.Patient and renal survival, renal histology, anti-GBM-antibodies.Anti-GBM-antibodies were reduced by the first 9 IAS treatments (mean number of 23 to negative levels in all patients. Renal survival was 40% at diagnosis, 70% after the end of IAS, 63% after one year and 50% at the end of observation (mean 84 months, range 9 to 186. Dialysis dependency was successfully reversed in three of six patients. Patient survival was 90% at the end of observation.IAS efficiently eliminates anti-GBM-antibodies suggesting non-inferiority to PE with regard to renal and patient survival. Hence IAS should be considered as a valuable treatment option for anti-GBM-disease, especially in patients presenting with a high percentage of crescents and dialysis dependency due to an unusual high proportion of responders.

Annual literature review of donor-specific HLA antibodies after organ transplantation.

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Kaneku, Hugo

2011-01-01

The literature review of post-transplant DSA published in 2011 shows: Observations after kidney and lung transplant in non-sensitized transplant recipients show that monitoring post-transplant HLA antibodies offers limited benefit in predicting acute rejection episodes. It remains to be seen if a different monitoring schedule and/ or studying other organs may show otherwise. Nevertheless, others have shown that monitoring post-transplant antibodies does identify patients at higher risk for chronic rejection. Studies in kidney, heart, and liver patients transplanted in the presence of preformed DSA show that detecting these antibodies early after transplant identifies a group of patients with greater risk for allograft dysfunction. New and larger studies using bortezomib and eculizumab to treat acute antibody-mediated rejection confirm earlier observations that these two therapies are effective in treating and preventing rejections. In general, identification of HLAantibodies and DSA after transplant is associated with higher rates of rejection and poor allograft survival in all organs examined. IgM antibodies appear to play an important role after lung transplants.

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

Science.gov (United States)

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

2014-11-13

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

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

Science.gov (United States)

Li, Lei; Lyu, Dan

2017-06-01

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

Monoclonal antibodies for use in an immunoradiometric assay for. cap alpha. -foetoprotein

Energy Technology Data Exchange (ETDEWEB)

Hunter, W.M.; Bennie, J.G. (Medical Research Council, Edinburgh (UK). Immunoassay Team); Brock, D.J.H.; Heyningen, V. van (Western General Hospital, Edinburgh (UK))

1982-04-29

The advantages offered by a mouse IgG/sub 1/ monoclonal antibody to human ..cap alpha..-foetoprotein (AFP) for the preparation of (/sup 125/I)antibody for use in an immunoradiometric assay (IRMA) have been investigated. The antibody was isolated from ascites fluid by sodium sulphate precipitation followed by gel filtration on Sephadex G-200. The freeze-dried powder and solutions thereof were stable and were used for iodination to 1 atom /sup 125/I/molecule antibody by the chloramine-T procedure. At high antigen concentrations 70-80% of the added (/sup 125/)Ab was present in the sandwich. Linear response curves in the range 1-100 ..mu..g antigen/l incubate were obtained when (/sup 125/I)Ab was in slight excess. In this region an Ag : Ab ratio 1.9 : 1 was obtained which is consistent with the saturation of a bifunctional antibody. Although non-specific binding (in the absence of antigen) was consistently <0.1% of added (/sup 125/I)Ab, this was the main factor in determining assay detection limits. The serum AFP levels from both non-pregnant and pregnant subjects as measured by the IRMA using the (/sup 125/I)monoclonal Ab and by radioimmunoassay (RIA) using a sheep antiserum to AFP were in excellent agreement. The IRMA was manipulatively simple, employed a shorter incubation time (2h), required shorter counting times than the RIA and gave a much wider working range. The provision of a monoclonal antibody for labelling removes the one major practicability barrier which otherwise limits the development and use of the potentially superior IRMA system.

C4d-negative antibody-mediated rejection with high anti-angiotensin II type I receptor antibodies in absence of donor-specific antibodies.

Science.gov (United States)

Fuss, Alexander; Hope, Christopher M; Deayton, Susan; Bennett, Greg Donald; Holdsworth, Rhonda; Carroll, Robert P; Coates, P Toby H

2015-07-01

Acute antibody-mediated rejection can occur in absence of circulating donor-specific antibodies. Agonistic antibodies targeting the anti-angiotensin II type 1 receptor (anti-AT1 R) are emerging as important non-human leucocyte antigen (HLA) antibodies. Elevated levels of anti-angiotensin II receptor antibodies were first observed in kidney transplant recipients with malignant hypertension and allograft rejection. They have now been studied in three separate kidney transplant populations and associate to frequency of rejection, severity of rejection and graft failure. We report 11 cases of biopsy-proven, Complement 4 fragment d (C4d)-negative, acute rejection occurring without circulating donor-specific anti-HLA antibodies. In eight cases, anti-angiotensin receptor antibodies were retrospectively examined. The remaining three subjects were identified from our centre's newly instituted routine anti-angiotensin receptor antibody screening. All subjects fulfilled Banff 2013 criteria for antibody-mediated rejection and all responded to anti-rejection therapy, which included plasma exchange and angiotensin receptor blocker therapy. These cases support the routine assessment of anti-AT1 R antibodies in kidney transplant recipients to identify subjects at risk. Further studies will need to determine optimal assessment protocol and the effectiveness of pre-emptive treatment with angiotensin receptor blockers. © 2015 Asian Pacific Society of Nephrology.

Raising an Antibody Specific to Breast Cancer Subpopulations Using Phage Display on Tissue Sections

DEFF Research Database (Denmark)

Larsen, Simon Asbjørn; Meldgaard, Theresa; Fridriksdottir, Agla Jael Rubner

2016-01-01

BACKGROUND/AIM: Primary tumors display a great level of intra-tumor heterogeneity in breast cancer. The current lack of prognostic and predictive biomarkers limits accurate stratification and the ability to predict response to therapy. The aim of the present study was to select recombinant antibody...... fragments specific against breast cancer subpopulations, aiding the discovery of novel biomarkers. MATERIALS AND METHODS: Recombinant antibody fragments were selected by phage display. A novel shadowstick technology enabled the direct selection using tissue sections of antibody fragments specific against...

Antibody biotechnology

African Journals Online (AJOL)

STORAGESEVER

2009-07-06

Jul 6, 2009 ... Another milestone in the history of antibodies was the work of Porter and Edelman ... transgenic animals (Lonberg et al., 1994; Green et al.,. 1994) or .... create and to screen human recombinant antibodies libraries, that is ...

A simple physiologically based pharmacokinetic model evaluating the effect of anti-nicotine antibodies on nicotine disposition in the brains of rats and humans

Energy Technology Data Exchange (ETDEWEB)

Saylor, Kyle, E-mail: saylor@vt.edu; Zhang, Chenming, E-mail: chzhang2@vt.edu

2016-09-15

Physiologically based pharmacokinetic (PBPK) modeling was applied to investigate the effects of anti-nicotine antibodies on nicotine disposition in the brains of rats and humans. Successful construction of both rat and human models was achieved by fitting model outputs to published nicotine concentration time course data in the blood and in the brain. Key parameters presumed to have the most effect on the ability of these antibodies to prevent nicotine from entering the brain were selected for investigation using the human model. These parameters, which included antibody affinity for nicotine, antibody cross-reactivity with cotinine, and antibody concentration, were broken down into different, clinically-derived in silico treatment levels and fed into the human PBPK model. Model predictions suggested that all three parameters, in addition to smoking status, have a sizable impact on anti-nicotine antibodies' ability to prevent nicotine from entering the brain and that the antibodies elicited by current human vaccines do not have sufficient binding characteristics to reduce brain nicotine concentrations. If the antibody binding characteristics achieved in animal studies can similarly be achieved in human studies, however, nicotine vaccine efficacy in terms of brain nicotine concentration reduction is predicted to meet threshold values for alleviating nicotine dependence. - Highlights: • Modelling of nicotine disposition in the presence of anti-nicotine antibodies • Key vaccine efficacy factors are evaluated in silico in rats and in humans. • Model predicts insufficient antibody binding in past human nicotine vaccines. • Improving immunogenicity and antibody specificity may lead to vaccine success.

A simple physiologically based pharmacokinetic model evaluating the effect of anti-nicotine antibodies on nicotine disposition in the brains of rats and humans

International Nuclear Information System (INIS)

Saylor, Kyle; Zhang, Chenming

2016-01-01

Physiologically based pharmacokinetic (PBPK) modeling was applied to investigate the effects of anti-nicotine antibodies on nicotine disposition in the brains of rats and humans. Successful construction of both rat and human models was achieved by fitting model outputs to published nicotine concentration time course data in the blood and in the brain. Key parameters presumed to have the most effect on the ability of these antibodies to prevent nicotine from entering the brain were selected for investigation using the human model. These parameters, which included antibody affinity for nicotine, antibody cross-reactivity with cotinine, and antibody concentration, were broken down into different, clinically-derived in silico treatment levels and fed into the human PBPK model. Model predictions suggested that all three parameters, in addition to smoking status, have a sizable impact on anti-nicotine antibodies' ability to prevent nicotine from entering the brain and that the antibodies elicited by current human vaccines do not have sufficient binding characteristics to reduce brain nicotine concentrations. If the antibody binding characteristics achieved in animal studies can similarly be achieved in human studies, however, nicotine vaccine efficacy in terms of brain nicotine concentration reduction is predicted to meet threshold values for alleviating nicotine dependence. - Highlights: • Modelling of nicotine disposition in the presence of anti-nicotine antibodies • Key vaccine efficacy factors are evaluated in silico in rats and in humans. • Model predicts insufficient antibody binding in past human nicotine vaccines. • Improving immunogenicity and antibody specificity may lead to vaccine success.

Chimeric recombinant antibody fragments in cardiac troponin I immunoassay.

Science.gov (United States)

Hyytiä, Heidi; Heikkilä, Taina; Brockmann, Eeva-Christine; Kekki, Henna; Hedberg, Pirjo; Puolakanaho, Tarja; Lövgren, Timo; Pettersson, Kim

2015-03-01

To introduce a novel nanoparticle-based immunoassay for cardiac troponin I (cTnI) utilizing chimeric antibody fragments and to demonstrate that removal of antibody Fc-part and antibody chimerization decrease matrix related interferences. A sandwich-type immunoassay for cTnI based on recombinant chimeric (mouse variable/human constant) antigen binding (cFab) antibodies and intrinsically fluorescent nanoparticles was developed. To test whether using chimeric antibody fragments helps to avoid matrix related interferences, samples (n=39) with known amounts of triglycerides, bilirubin, rheumatoid factor (RF) or human anti-mouse antibodies (HAMAs) were measured with the novel assay, along with a previously published nanoparticle-based research assay with the same antibody epitopes. The limit of detection (LoD) was 3.30ng/L. Within-laboratory precision for 29ng/L and 2819ng/L cTnI were 13.7% and 15.9%, respectively. Regression analysis with Siemens ADVIA Centaur® yielded a slope (95% confidence intervals) of 0.18 (0.17-1.19) and a y-intercept of 1.94 (-1.28-3.91) ng/L. When compared to a previously published nanoparticle-based assay, the novel assay showed substantially reduced interference in the tested interference prone samples, 15.4 vs. 51.3%. A rheumatoid factor containing sample was decreased from 241ng/L to

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

International Nuclear Information System (INIS)

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

2006-01-01

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

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

International Nuclear Information System (INIS)

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

1984-01-01

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

Success and limitations in the prevention of violence: a case study of nine Brazilian experiences

Directory of Open Access Journals (Sweden)

Romeu Gomes

2006-06-01

Full Text Available The present study analyzes the success of Brazilian experiences engaged in prevention of violence as well as their limitations, and seeks to show ways for a solution of the problem. The investigation underlying this article consisted in a case study of nine experiences, using triangulation of methods. For this purpose, we used questionnaires and interviewed individuals and groups. Successful results were mainly: informed citizenship, work preparation, involvement of children and adolescents in recreating and educative activities, and increased self-esteem of the children and teens involved in the experiences. The greatest difficulties found were the lack of funds and of articulation of proposals with other groups and institutions. These experiences are not the only successful ones carried out in the country, but they point to a new horizon by innovating and creating a different practical approach to violence.O presente estudo tem como objetivo analisar os êxitos e os limites de experiências nacionais na prevenção da violência e indicar possibilidades de enfrentamento do problema. A pesquisa que dá origem ao artigo consistiu num estudo de caso de nove experiências, seguindo princípios da triangulação de métodos. Foram aplicados questionários quantitativos e realizadas entrevistas (individuais e em grupo. Os resultados apontam que os principais êxitos relatados são: informação para a cidadania; preparação para o trabalho; envolvimento de crianças e adolescentes em atividades lúdicas e educativas; e o fortalecimento da auto-estima dos jovens e crianças participantes. As maiores dificuldades das experiências são a manutenção financeira e a articulação de suas propostas com as de outros grupos e instituições. O estudo leva a concluir que essas experiências, ainda que não sejam as únicas bem-sucedidas no país, apontam para um horizonte novo pela forma como atuam, inovando e criando uma práxis diferenciada na abordagem

Antibody informatics for drug discovery

DEFF Research Database (Denmark)

Shirai, Hiroki; Prades, Catherine; Vita, Randi

2014-01-01

to the antibody science in every project in antibody drug discovery. Recent experimental technologies allow for the rapid generation of large-scale data on antibody sequences, affinity, potency, structures, and biological functions; this should accelerate drug discovery research. Therefore, a robust bioinformatic...... 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...... for antibody rational design using computational approaches to affinity and stability improvement, as well as ab-initio and homology-based antibody modeling; (ii) resources for antibody sequences, structures, and immune epitopes and open drug discovery resources for development of antibody drugs; and (iii...

HAI-178 antibody-conjugated fluorescent magnetic nanoparticles for targeted imaging and simultaneous therapy of gastric cancer

Science.gov (United States)

Wang, Can; Bao, Chenchen; Liang, Shujing; Zhang, Lingxia; Fu, Hualin; Wang, Yutian; Wang, Kan; Li, Chao; Deng, Min; Liao, Qiande; Ni, Jian; Cui, Daxiang

2014-05-01

The successful development of safe and highly effective nanoprobes for targeted imaging and simultaneous therapy of in vivo gastric cancer is a great challenge. Herein we reported for the first time that anti-α-subunit of ATP synthase antibody, HAI-178 monoclonal antibody-conjugated fluorescent magnetic nanoparticles, was successfully used for targeted imaging and simultaneous therapy of in vivo gastric cancer. A total of 172 specimens of gastric cancer tissues were collected, and the expression of α-subunit of ATP synthase in gastric cancer tissues was investigated by immunohistochemistry method. Fluorescent magnetic nanoparticles were prepared and conjugated with HAI-178 monoclonal antibody, and the resultant HAI-178 antibody-conjugated fluorescent magnetic nanoparticles (HAI-178-FMNPs) were co-incubated with gastric cancer MGC803 cells and gastric mucous GES-1 cells. Gastric cancer-bearing nude mice models were established, were injected with prepared HAI-178-FMNPs via tail vein, and were imaged by magnetic resonance imaging and small animal fluorescent imaging system. The results showed that the α-subunit of ATP synthase exhibited high expression in 94.7% of the gastric cancer tissues. The prepared HAI-178-FMNPs could target actively MGC803 cells, realized fluorescent imaging and magnetic resonance imaging of in vivo gastric cancer, and actively inhibited growth of gastric cancer cells. In conclusion, HAI-178 antibody-conjugated fluorescent magnetic nanoparticles have a great potential in applications such as targeted imaging and simultaneous therapy of in vivo early gastric cancer cells in the near future.

Intracellular and non-neuronal targets of voltage-gated potassium channel complex antibodies.

Science.gov (United States)

Lang, Bethan; Makuch, Mateusz; Moloney, Teresa; Dettmann, Inga; Mindorf, Swantje; Probst, Christian; Stoecker, Winfried; Buckley, Camilla; Newton, Charles R; Leite, M Isabel; Maddison, Paul; Komorowski, Lars; Adcock, Jane; Vincent, Angela; Waters, Patrick; Irani, Sarosh R

2017-04-01

Autoantibodies against the extracellular domains of the voltage-gated potassium channel (VGKC) complex proteins, leucine-rich glioma-inactivated 1 (LGI1) and contactin-associated protein-2 (CASPR2), are found in patients with limbic encephalitis, faciobrachial dystonic seizures, Morvan's syndrome and neuromyotonia. However, in routine testing, VGKC complex antibodies without LGI1 or CASPR2 reactivities (double-negative) are more common than LGI1 or CASPR2 specificities. Therefore, the target(s) and clinical associations of double-negative antibodies need to be determined. Sera (n=1131) from several clinically defined cohorts were tested for IgG radioimmunoprecipitation of radioiodinated α-dendrotoxin ( 125 I-αDTX)-labelled VGKC complexes from mammalian brain extracts. Positive samples were systematically tested for live hippocampal neuron reactivity, IgG precipitation of 125 I-αDTX and 125 I-αDTX-labelled Kv1 subunits, and by cell-based assays which expressed Kv1 subunits, LGI1 and CASPR2. VGKC complex antibodies were found in 162 of 1131 (14%) sera. 90 of these (56%) had antibodies targeting the extracellular domains of LGI1 or CASPR2. Of the remaining 72 double-negative sera, 10 (14%) immunoprecipitated 125 I-αDTX itself, and 27 (38%) bound to solubilised co-expressed Kv1.1/1.2/1.6 subunits and/or Kv1.2 subunits alone, at levels proportionate to VGKC complex antibody levels (r=0.57, p=0.0017). The sera with LGI1 and CASPR2 antibodies immunoprecipitated neither preparation. None of the 27 Kv1-precipitating samples bound live hippocampal neurons or Kv1 extracellular domains, but 16 (59%) bound to permeabilised Kv1-expressing human embryonic kidney 293T cells. These intracellular Kv1 antibodies mainly associated with non-immune disease aetiologies, poor longitudinal clinical-serological correlations and a limited immunotherapy response. Double-negative VGKC complex antibodies are often directed against cytosolic epitopes of Kv1 subunits and occasionally against

Radioimmunodetection of hepatocellular carcinoma with a radiolabeled antibody to alpha fetoprotein

International Nuclear Information System (INIS)

Ishii, Nobuko; Nakata, Keisuke; Muro, Toyokichi

1982-01-01

The present study was undertaken to investigate whether or not radiolabeled antibody to Alpha-fetoprotein (AFP) intravenously administered is accumulate in AFP-producing tumors. Rats with subcutaneously transplanted hepatoma or with heptoma produced in liver by 3'-Me-DAB, and 12 patients with hepatocellular carcinoma (HCC) were studied. In animal experiments, the tumor localization was clearly demonstrated by total body scintigraphy 48 to 168 hours after the injection of the radioiodinated antibody. The rats were sacrificed and radioactivity in tissues was counted. Tumor tissues showed the highest counts. In patients with HCC, the imaging was made with a gamma camera usually 24 and 48 hours after injection of I-131-labeled antibody. In order to make the contrast of tumor image clear, nonspecific background images obtained by administration of Tc-99m-labeled serum albumin were subtracted from the images obtained by I-131 labeled antibody with a computer. In 6 of 12 patients with HCC, the tumor location could successfully demonstrated. However, tumor with sizes below 2 cm in diameter which were able to be detected by the celiac angiography were not detectable. Interes tingly the images were more clear in patients with relatively higher serum AFP levels. In the blood of patient given injection of AFP radioantibody, both immune complex and free antibody were able to be detected at least for 120 hours so that the presence of free antigen (AFP) did not appear to prevent tumor radioimmunodetection. The information obtained concerning the biochemical factors which influence antibody localization in the tumor would provide better method for radioimmunodetection with antitumor antibodies. (author)

Ribosome display: next-generation display technologies for production of antibodies in vitro.

Science.gov (United States)

He, Mingyue; Khan, Farid

2005-06-01

Antibodies represent an important and growing class of biologic research reagents and biopharmaceutical products. They can be used as therapeutics in a variety of diseases. With the rapid expansion of proteomic studies and biomarker discovery, there is a need for the generation of highly specific binding reagents to study the vast number of proteins encoded by the genome. Display technologies provide powerful tools for obtaining antibodies. Aside from the preservation of natural antibody repertoires, they are capable of exploiting diversity by DNA recombination to create very large libraries for selection of novel molecules. In contrast to in vivo immunization processes, display technologies allow selection of antibodies under in vitro-defined selection condition(s), resulting in enrichment of antibodies with desired properties from large populations. In addition, in vitro selection enables the isolation of antibodies against difficult antigens including self-antigens, and this can be applied to the generation of human antibodies against human targets. Display technologies can also be combined with DNA mutagenesis for antibody evolution in vitro. Some methods are amenable to automation, permitting high-throughput generation of antibodies. Ribosome display is considered as representative of the next generation of display technologies since it overcomes the limitations of cell-based display methods by using a cell-free system, offering advantages of screening larger libraries and continuously expanding new diversity during selection. Production of display-derived antibodies can be achieved by choosing one of a variety of prokaryotic and eukaryotic cell-based expression systems. In the near future, cell-free protein synthesis may be developed as an alternative for large-scale generation of antibodies.

Antibody profiling sensitivity through increased reporter antibody layering

Science.gov (United States)

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.

Antibody profiling sensitivity through increased reporter antibody layering

Energy Technology Data Exchange (ETDEWEB)

Apel, William A.; Thompson, Vicki S.

2017-03-28

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.

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.

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.

A robust robotic high-throughput antibody purification platform.

Science.gov (United States)

Schmidt, Peter M; Abdo, Michael; Butcher, Rebecca E; Yap, Min-Yin; Scotney, Pierre D; Ramunno, Melanie L; Martin-Roussety, Genevieve; Owczarek, Catherine; Hardy, Matthew P; Chen, Chao-Guang; Fabri, Louis J

2016-07-15

Monoclonal antibodies (mAbs) have become the fastest growing segment in the drug market with annual sales of more than 40 billion US$ in 2013. The selection of lead candidate molecules involves the generation of large repertoires of antibodies from which to choose a final therapeutic candidate. Improvements in the ability to rapidly produce and purify many antibodies in sufficient quantities reduces the lead time for selection which ultimately impacts on the speed with which an antibody may transition through the research stage and into product development. Miniaturization and automation of chromatography using micro columns (RoboColumns(®) from Atoll GmbH) coupled to an automated liquid handling instrument (ALH; Freedom EVO(®) from Tecan) has been a successful approach to establish high throughput process development platforms. Recent advances in transient gene expression (TGE) using the high-titre Expi293F™ system have enabled recombinant mAb titres of greater than 500mg/L. These relatively high protein titres reduce the volume required to generate several milligrams of individual antibodies for initial biochemical and biological downstream assays, making TGE in the Expi293F™ system ideally suited to high throughput chromatography on an ALH. The present publication describes a novel platform for purifying Expi293F™-expressed recombinant mAbs directly from cell-free culture supernatant on a Perkin Elmer JANUS-VariSpan ALH equipped with a plate shuttle device. The purification platform allows automated 2-step purification (Protein A-desalting/size exclusion chromatography) of several hundred mAbs per week. The new robotic method can purify mAbs with high recovery (>90%) at sub-milligram level with yields of up to 2mg from 4mL of cell-free culture supernatant. Copyright © 2016 Elsevier B.V. All rights reserved.

Anti-Pseudomonas aeruginosa IgY antibodies augment bacterial clearance in a murine pneumonia model

DEFF Research Database (Denmark)

Thomsen, K.; Christophersen, L.; Bjarnsholt, T.

2016-01-01

Background: Oral prophylactic therapy by gargling with pathogen-specific egg yolk immunoglobulins (IgY) may reduce the initial airway colonization with Pseudomonas aeruginosa in cystic fibrosis (CF) patients. IgY antibodies impart passive immunization and we investigated the effects of anti......-P. aeruginosa IgY antibodies on bacterial eradication in a murine pneumonia model. Methods: P. aeruginosa pneumonia was established in Balb/c mice and the effects of prophylactic IgY administration on lung bacteriology, clinical parameters and subsequent inflammation were compared to controls. Results......: Prophylactic administration of IgY antibodies targeting P. aeruginosa significantly reduced the bacterial burden by 2-log 24 h post-infection compared to controls and was accompanied by significantly reduced clinical symptom scores and successive inflammatory cytokine profile indicative of diminished lung...

Generation and Characterization of Polyclonal Antibody Against Part of Immunoglobulin Constant Heavy υ Chain of Goose

Science.gov (United States)

Zhao, Panpan; Guo, Yongli; Ma, Bo; Wang, Junwei

2014-01-01

Immunoglobulin Y (abbreviated as IgY) is a type of immunoglobulin that is the major antibody in bird, reptile, and lungfish blood. IgY consists of two light (λ) and two heavy (υ) chains. In the present study, polyclonal antibody against IgYFc was generated and evaluated. rIgYCυ3/Cυ4 was expressed in Escherichia coli, purified and utilized to raise polyclonal antibody in rabbit. High affinity antisera were obtained, which successfully detected the antigen at a dilution of 1:204,800 for ELISA assay. The antibody can specifically recognize both rIgYCυ3/Cυ4 and native IgY by Western bolt analysis. Furthermore, the serum of Grus japonensis or immunoglobulin of chicken, duck, turkey, and silkie samples and dynamic changes of serum GoIgY after immunogenicity with GPV-VP3-virus-like particles (GPV-VP3-VLPs) can be detected with the anti-GoIgYFc polyclonal antibody. These results suggested that the antibody is valuable for the investigation of biochemical properties and biological functions of GoIgY. PMID:25171010

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

Science.gov (United States)

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

2015-01-01

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

Superficial Dsg2 Expression Limits Epidermal Blister Formation Mediated by Pemphigus Foliaceus Antibodies and Exfoliative Toxins

Directory of Open Access Journals (Sweden)

Donna Brennan

2010-01-01

Full Text Available Cell-cell adhesion mediated by desmosomes is crucial for maintaining proper epidermal structure and function, as evidenced by several severe and potentially fatal skin disorders involving impairment of desmosomal proteins. Pemphigus foliaceus (PF and staphylococcal scalded skin syndrome (SSSS are subcorneal blistering diseases resulting from loss of function of the desmosomal cadherin, desmoglein 1 (Dsg1. To further study the pathomechanism of these diseases and to assess the adhesive properties of Dsg2, we employed a recently established transgenic (Tg mouse model expressing Dsg2 in the superficial epidermis. Neonatal Tg and wild type (WT mice were injected with purified ETA or PF Ig. We showed that ectopic expression of Dsg2 reduced the extent of blister formation in response to both ETA and PF Ig. In response to PF Ig, we observed either a dramatic loss or a reorganization of Dsg1-α, Dsg1-β, and, to a lesser extent, Dsg1-γ, in WT mice. The Inv-Dsg2 Tg mice showed enhanced retention of Dsg1 at the cell-cell border. Collectively, our data support the role for Dsg2 in cell adhesion and suggest that ectopic superficial expression of Dsg2 can increase membrane preservation of Dsg1 and limit epidermal blister formation mediated by PF antibodies and exfoliative toxins.

Limited naturally occurring escape in broadly neutralizing antibody epitopes in hepatitis C glycoprotein E2 and constrained sequence usage in acute infection

NARCIS (Netherlands)

Rodrigo, Chaturaka; Walker, Melanie R.; Leung, Preston; Eltahla, Auda A.; Grebely, Jason; Dore, Gregory J.; Applegate, Tanya; Page, Kimberly; Dwivedi, Sunita; Bruneau, Julie; Morris, Meghan D.; Cox, Andrea L.; Osburn, William; Kim, Arthur Y.; Schinkel, Janke; Shoukry, Naglaa H.; Lauer, Georg M.; Maher, Lisa; Hellard, Margaret; Prins, Maria; Luciani, Fabio; Lloyd, Andrew R.; Bull, Rowena A.

2017-01-01

Broadly neutralizing antibodies have been associated with spontaneous clearance of the hepatitis C infection as well as viral persistence by immune escape. Further study of neutralizing antibody epitopes is needed to unravel pathways of resistance to virus neutralization, and to identify conserved

Myostatin inhibitors in sports drug testing: Detection of myostatin-neutralizing antibodies in plasma/serum by affinity purification and Western blotting.

Science.gov (United States)

Walpurgis, Katja; Thomas, Andreas; Schänzer, Wilhelm; Thevis, Mario

2016-02-01

Myostatin is a key regulator of skeletal muscle growth and inhibition of its signaling pathway results in an increased muscle mass and function. The aim of this study was to develop a qualitative detection assay for myostatin-neutralizing antibodies for doping control purposes by using immunological approaches. To detect different types of myostatin-neutralizing antibodies irrespective of their amino acid sequence, an immunological assay specific for antibodies directed against myostatin and having a human Fc domain was established. Affinity purification and Western blotting strategies were combined to allow extracting and identifying relevant analytes from 200 μL of plasma/serum in a non-targeted approach. The assay was characterized regarding specificity, linearity, precision, robustness, and recovery. The assay was found to be highly specific, robust, and linear from 0.1 to 1 μg/mL. The precision was successfully specified at three different concentrations and the recovery of the affinity purification was 58%. Within this study, an immunological detection assay for myostatin-neutralizing antibodies present in plasma/serum specimens was developed and successfully characterized. The presented approach can easily be modified to include other therapeutic antibodies and serves as proof-of-concept for the detection of antibody-based myostatin inhibitors in doping control samples. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Monoclonal antibody form and function: manufacturing the right antibodies for treating drug abuse.

Science.gov (United States)

Peterson, Eric; Owens, S Michael; Henry, Ralph L

2006-05-26

Drug abuse continues to be a major national and worldwide problem, and effective treatment strategies are badly needed. Antibodies are promising therapies for the treatment of medical problems caused by drug abuse, with several candidates in preclinical and early clinical trials. Monoclonal antibodies can be designed that have customized affinity and specificity against drugs of abuse, and because antibodies can be designed in various forms, in vivo pharmacokinetic characteristics can be tailored to suit specific clinical applications (eg, long-acting for relapse prevention, or short-acting for overdose). Passive immunization with antibodies against drugs of abuse has several advantages over active immunization, but because large doses of monoclonal antibodies may be needed for each patient, efficient antibody production technology is essential. In this minireview we discuss some of the antibody forms that may be effective clinical treatments for drug abuse, as well as several current and emerging production systems that could bridge the gap from discovery to patient use.

Escherichia coli surface display of single-chain antibody VRC01 against HIV-1 infection

International Nuclear Information System (INIS)

Wang, Lin-Xu; Mellon, Michael; Bowder, Dane; Quinn, Meghan; Shea, Danielle; Wood, Charles; Xiang, Shi-Hua

2015-01-01

Human immunodeficiency virus type 1 (HIV-1) transmission and infection occur mainly via the mucosal surfaces. The commensal bacteria residing in these surfaces can potentially be employed as a vehicle for delivering inhibitors to prevent HIV-1 infection. In this study, we have employed a bacteria-based strategy to display a broadly neutralizing antibody VRC01, which could potentially be used to prevent HIV-1 infection. The VRC01 antibody mimics CD4-binding to gp120 and has broadly neutralization activities against HIV-1. We have designed a construct that can express the fusion peptide of the scFv-VRC01 antibody together with the autotransporter β-barrel domain of IgAP gene from Neisseria gonorrhoeae, which enabled surface display of the antibody molecule. Our results indicate that the scFv-VRC01 antibody molecule was displayed on the surface of the bacteria as demonstrated by flow cytometry and immunofluorescence microscopy. The engineered bacteria can capture HIV-1 particles via surface-binding and inhibit HIV-1 infection in cell culture. - Highlights: • Designed single-chain VRC01 antibody was demonstrated to bind HIV-1 envelope gp120. • Single-chain VRC01 antibody was successfully displayed on the surface of E. coli. • Engineered bacteria can absorb HIV-1 particles and prevent HIV-1 infection in cell culture

Bioanalysis of antibody-drug conjugates: American Association of Pharmaceutical Scientists Antibody-Drug Conjugate Working Group position paper.

Science.gov (United States)

Gorovits, Boris; Alley, Stephen C; Bilic, Sanela; Booth, Brian; Kaur, Surinder; Oldfield, Phillip; Purushothama, Shobha; Rao, Chetana; Shord, Stacy; Siguenza, Patricia

2013-05-01

Antibody-drug conjugates (ADCs) typically consist of a cytotoxic drug covalently bound to an antibody by a linker. These conjugates have the potential to substantially improve efficacy and reduce toxicity compared with cytotoxic small-molecule drugs. Since ADCs are generally complex heterogeneous mixtures of multiple species, these novel therapeutic products present unique bioanalytical challenges. The growing number of ADCs being developed across the industry suggests the need for alignment of the bioanalytical methods or approaches used to assess the multiple species and facilitate consistent interpretation of the bioanalytical data. With limited clinical data, the current strategies that can be used to provide insight into the relationship between the multiple species and the observed clinical safety and efficacy are still evolving. Considerations of the bioanalytical strategies for ADCs based on the current industry practices that take into account the complexity and heterogeneity of ADCs are discussed.

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

Science.gov (United States)

Walter, Roland B

2014-06-01

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

Breast cancer imaging with mouse monoclonal antibodies

Energy Technology Data Exchange (ETDEWEB)

Major, P.; Wang Taqui; Unger, M.; Rosenthall, L.

1989-10-01

The localization of /sup 111/In-labelled MA5 monoclonal antibody, reactive with a breast tumor associated antigen, was studied in 17 patients. MA5 was selected because (1) it reacts with >95% of primary and metastatic lesions, (2) the recognized antigen is present on the cell surface in vivo and (3) MA5 gives excellent localization in human breast tumor xenografts. Each patient received 2 mg antibody labeled with 5 mCi /sup 111/In and in some cases, 3 mg or 18 mg unlabeled carrier antibody. No serious allergic reactions were noted. There was a large uptake in the liver, less significant uptake in the spleen and bone and minimal accumulation in the bowel. Bone lesions, primary tumors, soft tissue recurrences and lung metastases larger than 3 cm diameter were imaged, while only 1 lesion smaller than 3 cm was detected. Non specific accumulation of tracer was noted at the site of a port-a-cath, in a hematoma, in fibrocystic lesions, and at sites of previous radiation treatment. Extensive fibrosis and poor vascularization characteristic of breast tumors may explain in part the limited sensitivity of the imaging. (orig.).

Specificity of anti-phospholipid antibodies in infectious mononucleosis: a role for anti-cofactor protein antibodies

Science.gov (United States)

Sorice, M; Pittoni, V; Griggi, T; Losardo, A; Leri, O; Magno, M S; Misasi, R; Valesini, G

2000-01-01

The antigen specificity of anti-phospholipid antibodies in infectious mononucleosis (IM) was studied using ELISA for the detection of anti-β2-glycoprotein I (β2-GPI), anti-annexin V, anti-protein S and anti-prothrombin antibodies and TLC immunostaining for the detection of anti-phospholipid antibodies. This technique enabled us to look at antibodies reacting to ‘pure’ phospholipid antigens in the absence of protein contamination. Sera from 46 patients with IM, 18 with systemic lupus erythematosus (SLE), 21 with primary anti-phospholipid antibody syndrome (PAPS), 50 with Helicobacter pylori infection and 30 healthy blood donors were tested. This study highlights anti-phospholipid antibodies in patients with IM as specific ‘pure’ anti-cardiolipin antibodies, while in PAPS and SLE patients anti-phosphatidylserine and anti-phosphatidylethanolamine antibodies were also found. This investigation also shows that the anti-cardiolipin antibodies found in IM can be present with anti-cofactor protein antibodies. The higher prevalence of anti-cofactor antibodies found in IM sera than in Helicobacter pylori sera may be due to the immunostimulatory effect and/or the polyclonal activation often observed in course of Epstein–Barr virus infection. However, anti-β2-GPI and, to a lesser extent, anti-prothrombin antibodies occur with a significantly lower prevalence in IM than in PAPS patients. This finding suggests that these antibodies should be regarded as the expression of the broad autoimmune syndrome involving the phospholipid-binding plasma proteins. PMID:10792380

Limited diagnostic capacities of two commercial assays for the detection of Leptospira immunoglobulin M antibodies in Laos

NARCIS (Netherlands)

Blacksell, Stuart D.; Smythe, Lee; Phetsouvanh, Rattanaphone; Dohnt, Michael; Hartskeerl, Rudy; SymondS, Meegan; Slack, Andrew; Vongsouvath, Manivanh; Davong, Viengmone; Lattana, Olay; Phongmany, Simmaly; Keolouangkot, Valy; White, Nicholas J.; Day, Nicholas P. J.; Newton, Paul N.

2006-01-01

The diagnostic utility of immunochromatographic (Leptotek) and enzyme-linked immunosorbent assay (ELISA; Panbio) tests for the detection of Leptospira immunoglobulin M antibodies was assessed in febrile adults admitted in Vientiane, Laos. Both tests demonstrated poor diagnostic accuracy using

Radiolabeled antibodies in cancer. Oncology Overview

International Nuclear Information System (INIS)

1984-11-01

Oncology Overviews are a service of the International Cancer Research Data Bank (ICRDB) Program of the National Cancer Institute, intended to facilitate and promote the exchange of information between cancer scientists by keeping them aware of literature related to their research being published by other laboratories through the world. Each Oncology Overview represents a survey of the literature associated with a selected area of cancer research. It contains abstracts of articles which have been selected and organized by researchers associated with the field. Contents: Radiolabeled antibodies--labeling and imaging techniques; Radiolabeled antibodies--carcinoembryonic antigen; Radiolabeled antibodies--alpha-fetoprotein; Radiolabeled antibodies--human chorionic gonadotropin; Radiolabeled antibodies--ferritin; Radiolabeled antibodies--imaging of colorectal tumors; Radiolabeled antibodies--imaging of malignant melanoma; Radiolabeled antibodies--imaging of urogenital tumors; Radiolabeled antibodies--imaging of thyroid tumors; Radiolabeled antibodies--other clinical studies; Radiolabeled antibodies--selected preclinical studies; Radiolabeled antibodies--reviews

Generation and Characterization of Anti-CD34 Monoclonal Antibodies that React with Hematopoietic Stem Cells

Science.gov (United States)

Aghebati Maleki, Leili; Majidi, Jafar; Baradaran, Behzad; Movassaghpour, Aliakbar; Abdolalizadeh, Jalal

2014-01-01

CD34 is a type I membrane protein with a molecular mass of approximately 110 kDa. This antigen is associated with human hematopoietic progenitor cells and is a differentiation stage-specific leukocyte antigen. In this study we have generated and characterized monoclonal antibodies (mAbs) directed against a CD34 marker. Mice were immunized with two keyhole lympet hemocyanin (KLH)-conjugated CD34 peptides. Fused cells were grown in hypoxanthine, aminopterine and thymidine (HAT) selective medium and cloned by the limiting dilution (L.D) method. Several monoclones were isolated by three rounds of limited dilutions. From these, we chose stable clones that presented sustained antibody production for subsequent characterization. Antibodies were tested for their reactivity and specificity to recognize the CD34 peptides and further screened by enzyme-linked immunosorbent assay (ELISA) and Western blotting analyses. One of the mAbs (3D5) was strongly reactive against the CD34 peptide and with native CD34 from human umbilical cord blood cells (UCB) in ELISA and Western blotting analyses. The results have shown that this antibody is highly specific and functional in biomedical applications such as ELISA and Western blot assays. This monoclonal antibodies (mAb) can be a useful tool for isolation and purification of human hematopoietic stem cells (HSCs). PMID:24611141

Diagnostic significance of DNA and antibodies against capsid antigens of anti-Epstein–Barr virus antibodies levels in blood plasma of nasopharyngeal carcinoma patients from non-endemic region

Directory of Open Access Journals (Sweden)

V. E. Gurtsevich

2015-01-01

Full Text Available Epstein–Barr virus (EBV, a representative of the herpesvirus family, is the etiological agent for a number of benign and malignant human neoplasms. Among the latter, the nasopharyngeal carcinoma (NPC occupies a special place. In NPC development EBV plays a key role stimulating the progression of the pathological process from precancerous lesions to the cancer development. For most NPC patients, elevated levels of humoral IgG and IgA antibodies against capsid and early EBV antigens are characteristic and their antibody titers rise to high levels long before the diagnosis of cancer. Using this phenomenon, virus-specific antibodies are used for many years as markers for NPC screening, especially in cases of undiagnosed primary lesion. In recent years, in endemic for NPC regions (South China, South-East Asia a great attention has been paid to the use of quantitative determination of EBV DNA copies in the blood plasma of patients with NPC as a method of early cancer detection and monitoring.The aim of this study was to compare clinical significance of EBV DNA and humoral antibodies levels in blood plasma of NPC patients in non-endemic region, Russia. The results obtained indicate that both markers DNA / EBV and IgA antibodies against capsid EBV antigens can be successfully used for diagnosis of NPC in non-endemic region. However, in comparison with the virus-specific antibody titers, the viral DNA levels in the patients plasma are more sensitive and specific as NPC marker reflecting the efficacy of the therapy, and the state of remission or relapse.

Antibody Engineering & Therapeutics 2016: The Antibody Society's annual meeting, December 11-15, 2016, San Diego, CA.

Science.gov (United States)

Larrick, James W; Alfenito, Mark R; Scott, Jamie K; Parren, Paul W H I; Burton, Dennis R; Bradbury, Andrew R M; Lemere, Cynthia A; Messer, Anne; Huston, James S; Carter, Paul J; Veldman, Trudi; Chester, Kerry A; Schuurman, Janine; Adams, Gregory P; Reichert, Janice M

Antibody Engineering & Therapeutics, the largest meeting devoted to antibody science and technology and the annual meeting of The Antibody Society, will be held in San Diego, CA on December 11-15, 2016. Each of 14 sessions will include six presentations by leading industry and academic experts. In this meeting preview, the session chairs discuss the relevance of their topics to current and future antibody therapeutics development. Session topics include bispecifics and designer polyclonal antibodies; antibodies for neurodegenerative diseases; the interface between passive and active immunotherapy; antibodies for non-cancer indications; novel antibody display, selection and screening technologies; novel checkpoint modulators / immuno-oncology; engineering antibodies for T-cell therapy; novel engineering strategies to enhance antibody functions; and the biological Impact of Fc receptor engagement. The meeting will open with keynote speakers Dennis R. Burton (The Scripps Research Institute), who will review progress toward a neutralizing antibody-based HIV vaccine; Olivera J. Finn, (University of Pittsburgh School of Medicine), who will discuss prophylactic cancer vaccines as a source of therapeutic antibodies; and Paul Richardson (Dana-Farber Cancer Institute), who will provide a clinical update on daratumumab for multiple myeloma. In a featured presentation, a representative of the World Health Organization's INN expert group will provide a perspective on antibody naming. "Antibodies to watch in 2017" and progress on The Antibody Society's 2016 initiatives will be presented during the Society's special session. In addition, two pre-conference workshops covering ways to accelerate antibody drugs to the clinic and the applications of next-generation sequencing in antibody discovery and engineering will be held on Sunday December 11, 2016.

Identification and prevention of antibody disulfide bond reduction during cell culture manufacturing.

Science.gov (United States)

Trexler-Schmidt, Melody; Sargis, Sandy; Chiu, Jason; Sze-Khoo, Stefanie; Mun, Melissa; Kao, Yung-Hsiang; Laird, Michael W

2010-06-15

In the biopharmaceutical industry, therapeutic monoclonal antibodies are primarily produced in mammalian cell culture systems. During the scale-up of a monoclonal antibody production process, we observed excessive mechanical cell shear as well as significant reduction of the antibody's interchain disulfide bonds during harvest operations. This antibody reduction event was catastrophic as the product failed to meet the drug substance specifications and the bulk product was lost. Subsequent laboratory studies have demonstrated that cells subjected to mechanical shear release cellular enzymes that contribute to this antibody reduction phenomenon (manuscript submitted; Kao et al., 2009). Several methods to prevent this antibody reduction event were developed using a lab-scale model to reproduce the lysis and reduction events. These methods included modifications to the cell culture media with chemicals (e.g., cupric sulfate (CuSO(4))), pre- and post-harvest chemical additions to the cell culture fluid (CCF) (e.g., CuSO(4), EDTA, L-cystine), as well as lowering the pH and air sparging of the harvested CCF (HCCF). These methods were evaluated for their effectiveness in preventing disulfide bond reduction and their impact to product quality. Effective prevention methods, which yielded acceptable product quality were evaluated for their potential to be implemented at manufacturing-scale. The work described here identifies numerous effective reduction prevention measures from lab-scale studies; several of these methods were then successfully translated into manufacturing processes. 2010 Wiley Periodicals, Inc.

Seroprevalence of Anti-Dengue Virus 2 Serocomplex antibodies in ...

African Journals Online (AJOL)

Introduction: There has been a recent increase in the spread of dengue to rural areas. Rural parts of western kenya are naturally prone to mosquito-borne diseases, however, limited research has been documented on infections with dengue. This study therefore investigated the presence of antibodies against dengue virus ...

Cytosolic antibody delivery by lipid-sensitive endosomolytic peptide

Science.gov (United States)

Akishiba, Misao; Takeuchi, Toshihide; Kawaguchi, Yoshimasa; Sakamoto, Kentarou; Yu, Hao-Hsin; Nakase, Ikuhiko; Takatani-Nakase, Tomoka; Madani, Fatemeh; Gräslund, Astrid; Futaki, Shiroh

2017-08-01

One of the major obstacles in intracellular targeting using antibodies is their limited release from endosomes into the cytosol. Here we report an approach to deliver proteins, which include antibodies, into cells by using endosomolytic peptides derived from the cationic and membrane-lytic spider venom peptide M-lycotoxin. The delivery peptides were developed by introducing one or two glutamic acid residues into the hydrophobic face. One peptide with the substitution of leucine by glutamic acid (L17E) was shown to enable a marked cytosolic liberation of antibodies (immunoglobulins G (IgGs)) from endosomes. The predominant membrane-perturbation mechanism of this peptide is the preferential disruption of negatively charged membranes (endosomal membranes) over neutral membranes (plasma membranes), and the endosomolytic peptide promotes the uptake by inducing macropinocytosis. The fidelity of this approach was confirmed through the intracellular delivery of a ribosome-inactivation protein (saporin), Cre recombinase and IgG delivery, which resulted in a specific labelling of the cytosolic proteins and subsequent suppression of the glucocorticoid receptor-mediated transcription. We also demonstrate the L17E-mediated cytosolic delivery of exosome-encapsulated proteins.

The value of LGI1, Caspr2 and voltage-gated potassium channel antibodies in encephalitis.

Science.gov (United States)

van Sonderen, Agnes; Petit-Pedrol, Mar; Dalmau, Josep; Titulaer, Maarten J

2017-05-01

The discovery, in 2010, of autoantibodies against the extracellular proteins LGI1 and Caspr2 facilitated a change of view regarding the clinical importance of voltage-gated potassium channel (VGKC) antibodies. Currently, these antibodies are all classified as VGKC-complex antibodies, and are commonly considered to have a similar clinical value. However, studies from the past few years show that the immune responses mediated by these antibodies have differing clinical relevance. Here, we review the clinical importance of these immune responses in three settings: patients with anti-LGI1 antibodies, patients with anti-Caspr2 antibodies, and patients with antibodies against the VGKC complex that lack LGI1 and Caspr2 specificity. Antibodies against LGI1 and Caspr2 are associated with different but well-defined syndromes, whereas the clinical importance of VGKC-complex antibodies without LGI1 and Caspr2 specificity is questionable. We describe each of these syndromes, discuss the function of the target antigens and review the limited paediatric literature on the topic. The findings emphasize the importance of defining these disorders according to the molecular identity of the targets (LGI1 or Caspr2), and caution against the use of VGKC-complex antibodies for the diagnosis and treatment of patients without further definition of the antigen.

Antibody immobilized on Fe3O4 particles and its application to RIAs

International Nuclear Information System (INIS)

Shen Rongsen; Xing Ruiyun

1997-01-01

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

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

Science.gov (United States)

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

2016-02-01

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

Synthesis of Polyclonal Antibodies against Aflatoxin B1

Directory of Open Access Journals (Sweden)

Wiyogo Prio Wicaksono

2015-09-01

Full Text Available Polyclonal antibodies of aflatoxin B1 were successfully produced from New Zealand White female rabbits after immunization by the hapten of aflatoxin B1-carboxymethyl hydroxylamine hemihydrochloride (AFB1-CMO conjugated with bovine serum albumin (BSA as the antigen. The hapten was synthesized using the carbodiimide method with CMO as a linker. Absorption peaks at 362, 264, and 218 nm were observed as a result of characterization with UV-Vis spectroscopy, while IR spectroscopy showed peaks at 3448 cm-1 and 1642 cm-1 attributable to the hydroxyl and nitrile groups, respectively. Furthermore, mass spectrometry showed fragmentation at the m/z of 386, 368.2, and 310, which confirms that the hapten of AFB1-CMO was successfully synthesized. The hapten was then conjugated with BSA to serve as an antigen of AFB1 when it was injected into the rabbits. The specificity of the antigen towards its antibody and the confirmation of hapten-BSA conjugation were characterized using the dot blot immunoassay, which showed a BSA concentration of 1.74 mg/mL. Two weeks after the primary immunization by its antigen, agar gel precipitation testing showed that the rabbit blood serum had positive results for polyclonal antibodiest against AFB1 with the highest concentration of antibodiest of 2.19 mg/mL.

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

2010-04-01

Full Text Available 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.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.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 also highlights differences in the humoral immune response to

Identification of antigen-specific human monoclonal antibodies using high-throughput sequencing of the antibody repertoire.

Science.gov (United States)

Liu, Ju; Li, Ruihua; Liu, Kun; Li, Liangliang; Zai, Xiaodong; Chi, Xiangyang; Fu, Ling; Xu, Junjie; Chen, Wei

2016-04-22

High-throughput sequencing of the antibody repertoire provides a large number of antibody variable region sequences that can be used to generate human monoclonal antibodies. However, current screening methods for identifying antigen-specific antibodies are inefficient. In the present study, we developed an antibody clone screening strategy based on clone dynamics and relative frequency, and used it to identify antigen-specific human monoclonal antibodies. Enzyme-linked immunosorbent assay showed that at least 52% of putative positive immunoglobulin heavy chains composed antigen-specific antibodies. Combining information on dynamics and relative frequency improved identification of positive clones and elimination of negative clones. and increase the credibility of putative positive clones. Therefore the screening strategy could simplify the subsequent experimental screening and may facilitate the generation of antigen-specific antibodies. Copyright © 2016 Elsevier Inc. All rights reserved.

Metabolomics reveals distinct, antibody-independent, molecular signatures of MS, AQP4-antibody and MOG-antibody disease.

Science.gov (United States)

Jurynczyk, Maciej; Probert, Fay; Yeo, Tianrong; Tackley, George; Claridge, Tim D W; Cavey, Ana; Woodhall, Mark R; Arora, Siddharth; Winkler, Torsten; Schiffer, Eric; Vincent, Angela; DeLuca, Gabriele; Sibson, Nicola R; Isabel Leite, M; Waters, Patrick; Anthony, Daniel C; Palace, Jacqueline

2017-12-06

The overlapping clinical features of relapsing remitting multiple sclerosis (RRMS), aquaporin-4 (AQP4)-antibody (Ab) neuromyelitis optica spectrum disorder (NMOSD), and myelin oligodendrocyte glycoprotein (MOG)-Ab disease mean that detection of disease specific serum antibodies is the gold standard in diagnostics. However, antibody levels are not prognostic and may become undetectable after treatment or during remission. Therefore, there is still a need to discover antibody-independent biomarkers. We sought to discover whether plasma metabolic profiling could provide biomarkers of these three diseases and explore if the metabolic differences are independent of antibody titre. Plasma samples from 108 patients (34 RRMS, 54 AQP4-Ab NMOSD, and 20 MOG-Ab disease) were analysed by nuclear magnetic resonance spectroscopy followed by lipoprotein profiling. Orthogonal partial-least squares discriminatory analysis (OPLS-DA) was used to identify significant differences in the plasma metabolite concentrations and produce models (mathematical algorithms) capable of identifying these diseases. In all instances, the models were highly discriminatory, with a distinct metabolite pattern identified for each disease. In addition, OPLS-DA identified AQP4-Ab NMOSD patient samples with low/undetectable antibody levels with an accuracy of 92%. The AQP4-Ab NMOSD metabolic profile was characterised by decreased levels of scyllo-inositol and small high density lipoprotein particles along with an increase in large low density lipoprotein particles relative to both RRMS and MOG-Ab disease. RRMS plasma exhibited increased histidine and glucose, along with decreased lactate, alanine, and large high density lipoproteins while MOG-Ab disease plasma was defined by increases in formate and leucine coupled with decreased myo-inositol. Despite overlap in clinical measures in these three diseases, the distinct plasma metabolic patterns support their distinct serological profiles and confirm that these

A novel heavy domain antibody library with functionally optimized complementarity determining regions.

Directory of Open Access Journals (Sweden)

Ole Aalund Mandrup

Full Text Available Today a number of synthetic antibody libraries of different formats have been created and used for the selection of a large number of recombinant antibodies. One of the determining factors for successful isolation of recombinant antibodies from libraries lies in the quality of the libraries i.e. the number of correctly folded, functional antibodies contained in the library. Here, we describe the construction of a novel, high quality, synthetic single domain antibody library dubbed Predator. The library is based on the HEL4 domain antibody with the addition of recently reported mutations concerning the amino acid composition at positions critical for the folding characteristics and aggregation propensities of domain antibodies. As a unique feature, the CDR3 of the library was designed to mimic the natural human immune response by designating amino acids known to be prevalent in functional antibodies to the diversity in CDR3. CDR randomizations were performed using trinucleotide synthesis to avoid the presence of stop codons. Furthermore a novel cycle free elongation method was used for the conversion of the synthesized single stranded DNA containing the randomized CDRs into double stranded DNA of the library. In addition a modular approach has been adopted for the scaffold in which each CDR region is flanked by unique restrictions sites, allowing easy affinity maturation of selected clones by CDR shuffling. To validate the quality of the library, one round phage display selections were performed on purified antigens and highly complex antigen mixtures such as cultured eukaryotic cells resulting in several specific binders. The further characterization of some of the selected clones, however, indicates a reduction in thermodynamic stability caused by the inclusion the additional mutations to the HEL4 scaffold.

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

Science.gov (United States)

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

2013-01-01

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

Drug delivery systems--2. Site-specific drug delivery utilizing monoclonal antibodies.

Science.gov (United States)

Ranade, V V

1989-10-01

Monoclonal antibodies (MAbs) are purified antibodies produced by a single clone of cells. They are engineered to recognize and bind to a single specific antigen. Accordingly, when administered, MAbs home in on a particular circulating protein or on cells that bear the correct antigenic signature on their surfaces. It is the specificity of MAbs that has made them valuable tools for health professions. Following the discovery of Kohler and Milstein regarding the method of somatic cell hybridization, a number of investigators have successfully adopted this technique to obtain T-lymphocyte hybrid cell lines by fusion of activated T (thymus derived) lymphocytes with a T lymphoma cell line leading to an immortalization of a specific differentiated function. The hybrids thus obtained were subsequently shown to produce homogeneous effector molecules with a wide variety of immune functions such as enhancement or suppression of antibody responses, generation of helper T cells, suppressor T cells and cytotoxic T cells. Study of these regulatory molecules has been further shown to provide a greater insight into the genetic, biochemical and molecular mechanisms responsible for cellular development, and the interaction and triggering of various cell types. The successful application of hybridoma technology has now resulted into several advances in the understanding the mechanism and treatment of diseases, especially cancer and development of vaccines, promotion of organ transplantation and therapy against parasites as well. Since monoclonal antibodies could be made in unlimited supply, they have been used in genetic studies such as mRNA and gene isolation, chromosomal isolation of specific genes, immunoglobulin structure, detection of new or rare immunoglobulin gene products, structural studies of enzymes and other proteins and structural and population studies of protein polymorphisms. In some instances, the monoclonal antibodies have been found to replace conventional antisera

INTRAVENOUS IMMUNOGLOBULIN ADMINISTRATION FOR DESENSITIZATION BEFORE RENAL TRANSPLANTATION AND MANAGING ANTIBODY-MEDIATED REJECTION

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A. I. Sushkov

2011-01-01

Full Text Available Much attention has been placed recently in transplantation in highly HLA-sensitized patients. In attempts to remove these antibodies and enable successful renal transplantation, several approaches have been developed. Intravenous immunoglobulin (IVIG was found to be effective in the treatment of autoimmune and inflammatory disorders (e. g. Kawasaki disease, Guillain-Barre syndrome. Recently, a beneficial effect of IVIG on the reduc- tion of anti-HLA antibodies was described. The anti-inflammatory effect of IVIG provides hopeful opportunities in antibody-mediated rejection (AMR management. There are several protocols of IVIG administration for pre-transplant desensitization and AMR treatment: high-dose IVIG, low-dose IVIG + plasmapheresis, IVIG + plasmapheresis + rituximab. These advancements have enabled transplantation in patients previously considered untransplantable and in concert with new diagnostic techniques has resulted in new approaches to management of AMR. 

Thermodynamics of antibody-antigen interaction revealed by mutation analysis of antibody variable regions.

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Akiba, Hiroki; Tsumoto, Kouhei

2015-07-01

Antibodies (immunoglobulins) bind specific molecules (i.e. antigens) with high affinity and specificity. In order to understand their mechanisms of recognition, interaction analysis based on thermodynamic and kinetic parameters, as well as structure determination is crucial. In this review, we focus on mutational analysis which gives information about the role of each amino acid residue in antibody-antigen interaction. Taking anti-hen egg lysozyme antibodies and several anti-small molecule antibodies, the energetic contribution of hot-spot and non-hot-spot residues is discussed in terms of thermodynamics. Here, thermodynamics of the contribution from aromatic, charged and hydrogen bond-forming amino acids are discussed, and their different characteristics have been elucidated. The information gives fundamental understanding of the antibody-antigen interaction. Furthermore, the consequences of antibody engineering are analysed from thermodynamic viewpoints: humanization to reduce immunogenicity and rational design to improve affinity. Amino acid residues outside hot-spots in the interface play important roles in these cases, and thus thermodynamic and kinetic parameters give much information about the antigen recognition. Thermodynamic analysis of mutant antibodies thus should lead to advanced strategies to design and select antibodies with high affinity. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

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

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

2017-10-01

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

LIMITED ANTIBODY EVIDENCE OF EXPOSURE TO MYCOBACTERIUM BOVIS IN FERAL SWINE (SUS SCROFA) IN THE USA.

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Pedersen, Kerri; Miller, Ryan S; Anderson, Theodore D; Pabilonia, Kristy L; Lewis, Jonathan R; Mihalco, Rebecca L; Gortázar, Christian; Gidlewski, Thomas

2017-01-01

Bovine tuberculosis is a chronic disease of cattle ( Bos taurus ) caused by the bacterium Mycobacterium bovis . Efforts have been made in the US to eradicate the disease in cattle, but spillover into wildlife and subsequent spillback have impeded progress in some states. In particular, infection in white-tailed deer ( Odocoileus virginianus ) has been followed by infection in cattle in some Midwestern states. Infection has also been documented in feral swine ( Sus scrofa ) on the Hawaiian island of Molokai and in various European countries, but no large-scale survey of antibody exposure to the bacteria has been conducted in feral swine in the US. We tested 488 sera from feral swine collected near previously documented outbreaks of bovine tuberculosis in cattle and captive cervids, in addition to 2,237 feral swine sera collected across the US from 1 October 2013 to 30 September 2014. While all but one of the samples were antibody negative, the results are important for establishing baseline negative data since feral swine are capable reservoirs and could be implicated in future outbreaks of the disease.

Mouse-specific antibody responses to a monoclonal antibody during repeated immunoscintigraphy investigations: Comparison of antibody titres and imaging studies in a rat model

International Nuclear Information System (INIS)

Pimm, M.V.; Gribben, S.J.; Markham, A.J.; Perkins, A.C.

1990-01-01

As a model for human mouse-specific antibody responses in patients undergoing immunoscintigraphy, we have investigated in rats the production of mouse-specific antibodies (MA) to the mouse monoclonal antibody 791T/36. At intervals of between 5 and 16 weeks the rats were given repeated cycles of intravenous (IV) injections of antibody with or without a simultaneous intradermal (ID) injection. The IV dose was 60 μg/kg, a dose similar to that used in many clinical immunoscintigraphy studies. The ID injection was 2 μg, which mimicks the skin test dose often given in clinical imaging protocols. The study was carried out with both 131 I-labelled antibody and with antibody labelled with 111 In by DTPA chelation. MA was measured with a passive haemagglutination assay using sheep red blood cells coated with the monoclonal antibody. Of rats given ID injections of unlabelled antibody at the same time as the IV imaging doses, 9/20 produced MA during 4 cycles of injections. In contrast, only 2/16 rats given only the IV dose produced MA. Both 131 I- and 111 In-labelled antibody appeared equally immunogenic with 5/18 and 6/18 overall responders, respectively. The production of MA was associated with a significant perturbation in the biodistribution of the IV dose of labelled antibody as seen by gamma-camera imaging of the rats given 111 In-labelled antibody. There was clearance of immune complexes to the liver, this organ accumulating up to 90% of the whole body count rate of radiolabel. MA titres of between 1/100 and 1/78000 caused equal perturbation of biodistribution, although below 1/100 the effect was more variable. (orig.)

Antibodies to poliovirus detected by immunoradiometric assay with a monoclonal antibody

International Nuclear Information System (INIS)

Spitz, M.; Fossati, C.A.; Schild, G.C.; Spitz, L.; Brasher, M.

1982-01-01

An immunoradiometric assay (IRMA) for the assay of antibodies to poliovirus antigens is described. Dilutions of the test sera or whole (finger prick) blood samples were incubated with the poliovirus antigen bound to a solid phase and the specific antibody was detected by the addition of a mouse anti-human IgG monoclonal antibody (McAb), which was itself revealed by iodinated sheep IgG antimouse F(ab). The authors have shown that this technique is suitable for the estimation of IgG anti-poliovirus antibodies induced in children following polio vaccine. The present study shows that SPRIA provides a simple and inexpensive method for serological studies with poliovirus particularly for use in large-scale surveys. (Auth.)

Antibodies to poliovirus detected by immunoradiometric assay with a monoclonal antibody

Energy Technology Data Exchange (ETDEWEB)

Spitz, M.; Fossati, C.A.; Schild, G.C.; Spitz, L.; Brasher, M. (National Inst. for Biological Standards and Control, London (UK))

1982-10-01

An immunoradiometric assay (IRMA) for the assay of antibodies to poliovirus antigens is described. Dilutions of the test sera or whole (finger prick) blood samples were incubated with the poliovirus antigen bound to a solid phase and the specific antibody was detected by the addition of a mouse anti-human IgG monoclonal antibody (McAb), which was itself revealed by iodinated sheep IgG antimouse F(ab). The authors have shown that this technique is suitable for the estimation of IgG anti-poliovirus antibodies induced in children following polio vaccine. The present study shows that SPRIA provides a simple and inexpensive method for serological studies with poliovirus particularly for use in large-scale surveys.

DNA-mediated strand displacement facilitates sensitive electronic detection of antibodies in human serums.

Science.gov (United States)

Dou, Baoting; Yang, Jianmei; Shi, Kai; Yuan, Ruo; Xiang, Yun

2016-09-15

We describe here the development of a sensitive and convenient electronic sensor for the detection of antibodies in human serums. The sensor is constructed by self-assembly formation of a mixed monolayer containing the small molecule epitope conjugated double stranded DNA probes on gold electrode. The target antibody binds the epitope on the dsDNA probe and lowers the melting temperature of the duplex, which facilitates the displacement of the antibody-linked strand of the duplex probe by an invading methylene blue-tagged single stranded DNA (MB-ssDNA) through the strand displacement reaction and leads to the capture of many MB-ssDNA on the sensor surface. Subsequent electrochemical oxidation of the methylene blue labels results in amplified current response for sensitive monitoring of the antibodies. The antibody assay conditions are optimized and the sensor exhibits a linear range between 1.0 and 25.0nM with a detection limit of 0.67nM for the target antibody. The sensor is also selective and can be employed to detect the target antibodies in human serum samples. With the advantages of using small molecule epitope as the antibody recognition element over traditional antigen, the versatile manipulability of the DNA probes and the unique properties of the electrochemical transduction technique, the developed sensor thus hold great potential for simple and sensitive detection of different antibodies and other proteins in real samples. Copyright © 2016 Elsevier B.V. All rights reserved.

Thermodynamic Mechanism for the Evasion of Antibody Neutralization in Flaviviruses

Science.gov (United States)

2015-01-01

Mutations in the epitopes of antigenic proteins can confer viral resistance to antibody-mediated neutralization. However, the fundamental properties that characterize epitope residues and how mutations affect antibody binding to alter virus susceptibility to neutralization remain largely unknown. To address these questions, we used an ensemble-based algorithm to characterize the effects of mutations on the thermodynamics of protein conformational fluctuations. We applied this method to the envelope protein domain III (ED3) of two medically important flaviviruses: West Nile and dengue 2. We determined an intimate relationship between the susceptibility of a residue to thermodynamic perturbations and epitope location. This relationship allows the successful identification of the primary epitopes in each ED3, despite their high sequence and structural similarity. Mutations that allow the ED3 to evade detection by the antibody either increase or decrease conformational fluctuations of the epitopes through local effects or long-range interactions. Spatially distant interactions originate in the redistribution of conformations of the ED3 ensembles, not through a mechanically connected array of contiguous amino acids. These results reconcile previous observations of evasion of neutralization by mutations at a distance from the epitopes. Finally, we established a quantitative correlation between subtle changes in the conformational fluctuations of the epitope and large defects in antibody binding affinity. This correlation suggests that mutations that allow viral growth, while reducing neutralization, do not generate significant structural changes and underscores the importance of protein fluctuations and long-range interactions in the mechanism of antibody-mediated neutralization resistance. PMID:24950171

Challenges to the development of vaccines to hepatitis C virus that elicit neutralizing antibodies.

Directory of Open Access Journals (Sweden)

Heidi Edelgard Drummer

2014-07-01

Full Text Available Despite 20 years of research, a vaccine to prevent hepatitis C virus (HCV infection has not been developed. A vaccine to prevent HCV will need to induce broadly reactive immunity able to prevent infection by the 7 genetically and antigenically distinct genotypes circulating world-wide. Hepatitis C virus encodes two surface exposed glycoproteins, E1 and E2 that function as a heterodimer to mediate viral entry. Neutralizing antibodies (NAbs to both E1 and E2 have been described with the major NAb target being E2. The function of E2 is to attach virions to host cells via cell surface receptors that include, but is not limited to, the tetraspanin CD81 and scavenger receptor B class I. However, E2 has developed a number of immune evasion strategies to limit the effectiveness of the NAb response and possibly limit the ability of the immune system to generate potent NAbs in natural infection. Hypervariable regions that shield the underlying core domain, subdominant neutralization epitopes and glycan shielding combine to make E2 a difficult target for the immune system. This review summarizes recent information on the role of neutralizing antibodies to prevent HCV infection, the targets of the neutralizing antibody response and structural information on glycoprotein E2 in complex with neutralizing antibodies. This new information should provide a framework for the rational design of new vaccine candidates that elicit highly potent broadly reactive NAbs to prevent HCV infection.

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...

Pre-existing neutralizing antibody mitigates B cell dysregulation and enhances the Env-specific antibody response in SHIV-infected rhesus macaques.

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Juan Pablo Jaworski

Full Text Available Our central hypothesis is that protection against HIV infection will be powerfully influenced by the magnitude and quality of the B cell response. Although sterilizing immunity, mediated by pre-formed abundant and potent antibodies is the ultimate goal for B cell-targeted HIV vaccine strategies, scenarios that fall short of this may still confer beneficial defenses against viremia and disease progression. We evaluated the impact of sub-sterilizing pre-existing neutralizing antibody on the B cell response to SHIV infection. Adult male rhesus macaques received passive transfer of a sub-sterilizing amount of polyclonal neutralizing immunoglobulin (Ig purified from previously infected animals (SHIVIG or control Ig prior to intra-rectal challenge with SHIVSF162P4 and extensive longitudinal sampling was performed. SHIVIG treated animals exhibited significantly reduced viral load and increased de novo Env-specific plasma antibody. Dysregulation of the B cell profile was grossly apparent soon after infection in untreated animals; exemplified by a ≈50% decrease in total B cells in the blood evident 2-3 weeks post-infection which was not apparent in SHIVIG treated animals. IgD+CD5+CD21+ B cells phenotypically similar to marginal zone-like B cells were highly sensitive to SHIV infection, becoming significantly decreased as early as 3 days post-infection in control animals, while being maintained in SHIVIG treated animals, and were highly correlated with the induction of Env-specific plasma antibody. These results suggest that B cell dysregulation during the early stages of infection likely contributes to suboptimal Env-specific B cell and antibody responses, and strategies that limit this dysregulation may enhance the host's ability to eliminate HIV.

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

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

2016-08-01

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

Egg yolk antibodies for detection and neutralization of Clostridium botulinum type A neurotoxin.

Science.gov (United States)

Trott, D L; Yang, M; Gonzalez, J; Larson, A E; Tepp, W H; Johnson, E A; Cook, M E

2009-05-01

The objective of this research project was to determine the usefulness of an egg antibody platform for producing materials for the detection and neutralization of botulinum type A neurotoxin. Yield estimates for detection and neutralizing antibodies produced using methods described were calculated. Antibody specific to botulinum toxoid A (aToxoid) and toxin A (aBoNT/A) was produced by immunizing hens with botulinum toxoid A (toxoid) followed by increasing amounts of botulinum neurotoxin A (BoNT/A) in Freund incomplete adjuvant. Egg yolks were extracted with polyethylene glycol (PEG) for antibody detection and neutralization experiments. A model aToxoid/toxoid immunoassay using only egg yolk antibody was developed and had a detection limit of 1 pg/ml of toxoid. In an indirect enzyme-linked immunosorbent assay of BoNT/A-specific antibody, the aBoNT/A contained more BoNT/A-specific antibody than did the aToxoid, and aBoNT/A was as effective as commercial rabbit antibody. The aToxoid provided no protection against BoNT/A in a standard mouse neutralization assay; however, 1 mg of PEG-extracted aBoNT/A neutralized 4,000 lethal doses of BoNT/A injected intraperitoneally. Based on these results, we calculated that in 1 month one hen could produce more than 100 liters of antibody detection reagents or enough antibody to neutralize approximately 11.6 million mouse lethal doses of botulinum toxin. Utilization of an egg antibody platform is potentially rapid (28 to 70 days) and scalable to kilogram quantities using current egg production facilities with as few as 1,000 hens.

Polyclonal and monoclonal antibodies in clinic.

Science.gov (United States)

Wootla, Bharath; Denic, Aleksandar; Rodriguez, Moses

2014-01-01

Immunoglobulins (Ig) or antibodies are heavy plasma proteins, with sugar chains added to amino-acid residues by N-linked glycosylation and occasionally by O-linked glycosylation. The versatility of antibodies is demonstrated by the various functions that they mediate such as neutralization, agglutination, fixation with activation of complement and activation of effector cells. Naturally occurring antibodies protect the organism against harmful pathogens, viruses and infections. In addition, almost any organic chemical induces antibody production of antibodies that would bind specifically to the chemical. These antibodies are often produced from multiple B cell clones and referred to as polyclonal antibodies. In recent years, scientists have exploited the highly evolved machinery of the immune system to produce structurally and functionally complex molecules such as antibodies from a single B clone, heralding the era of monoclonal antibodies. Most of the antibodies currently in the clinic, target components of the immune system, are not curative and seek to alleviate symptoms rather than cure disease. Our group used a novel strategy to identify reparative human monoclonal antibodies distinct from conventional antibodies. In this chapter, we discuss the therapeutic relevance of both polyclonal and monoclonal antibodies in clinic.

Radiolabelled monoclonal antibodies: magic bullets for colorectal carcinoma

International Nuclear Information System (INIS)

Slade, Linda

1997-01-01

Radiolabelled monoclonal antibodies (MoAbs) have been heralded as highly specific detection agents for many types of tumours. However, because of the many problems that have been associated with the use of these agents, their development and successes did not meet expectations. This paper discusses the use of radiolabelled MoAbs in the diagnosis and staging of colorectal cancer, the type of antibodies and radionuclides investigated over the past thirty years, and the advantages and disadvantages of each. An attempt is made to define the role of radioimmunoscintigraphy (RIS) in the investigation and management of patients with colorectal cancer. It appears that this technique can improve tumour detection, especially when used in conjunction with other imaging modalities. High sensitivities and specificities have been found using radio-labelled MoAbs for investigation of colorectal carcinoma. However, the author estimates there are a number of areas that require further research and improvement before naming radiolabelled MoAbs as 'magic bullets' for colorectal cancer. 8 refs., 3 tabs

Acetylcholine receptor antibody

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... medlineplus.gov/ency/article/003576.htm Acetylcholine receptor antibody To use the sharing features on this page, please enable JavaScript. Acetylcholine receptor antibody is a protein found in the blood of ...

Novel Phospholipid-Protein Conjugates Allow Improved Detection of Antibodies in Patients with Autoimmune Diseases

DEFF Research Database (Denmark)

Samuelsen, Simone V; Maity, Arindam; Nybo, Mads

2016-01-01

Reliable measurement of clinically relevant autoimmune antibodies toward phospholipid-protein conjugates is highly desirable in research and clinical assays. To date, the development in this field has been limited to the use of natural heterogeneous antigens. However, this approach does not take ...... on the correlation of detected autoantibodies with disease activity and manifestations. This confirms the crucial importance of antigens' composition on research and diagnostic assays, and opens up exciting perspectives for synthetic antigens in future studies of autoimmunity.......Reliable measurement of clinically relevant autoimmune antibodies toward phospholipid-protein conjugates is highly desirable in research and clinical assays. To date, the development in this field has been limited to the use of natural heterogeneous antigens. However, this approach does not take...... structural features of biologically active antigens into account and leads to low reliability and poor scientific test value. Here we describe novel phospholipid-protein conjugates for specific detection of human autoimmune antibodies. Our synthetic approach includes mild oxidation of synthetic phospholipid...

[Diagnosis of rabies infection in animals using monoclonal antibodies].

Science.gov (United States)

Akacem, O; Taril, A; Benelmouffok, A; Bemansour, A; Couillin, P; Brahimi, M; Benhassine, M

1989-01-01

Two monoclonal antibodies (M.A.), specific for viral nucleocapsid, the M.A. D-20 and the M.A. D-43 raised against a fixed strain of rabies virus (C.V.S. 11), have been tested in parallel with a standard antirabies serum (S.A.R.) in diagnosis of animal rabies virus infection. 44 brain imprints from animals which died from rabies were tested by indirect immunofluorescent technique with monoclonal antibodies. Constant correlation has been found between the M.A. D-43 and the S.A.R. in the diagnosis of animal rabies virus infection in all cases studied. For M.A. D-20, concordance of results with S.A.R. was found only in limited number of cases.

Stratification of antibody-positive subjects by antibody level reveals an impact of immunogenicity on pharmacokinetics.

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Zhou, Lei; Hoofring, Sarah A; Wu, Yu; Vu, Thuy; Ma, Peiming; Swanson, Steven J; Chirmule, Narendra; Starcevic, Marta

2013-01-01

The availability of highly sensitive immunoassays enables the detection of antidrug antibody (ADA) responses of various concentrations and affinities. The analysis of the impact of antibody status on drug pharmacokinetics (PK) is confounded by the presence of low-affinity or low-concentration antibody responses within the dataset. In a phase 2 clinical trial, a large proportion of subjects (45%) developed ADA following weekly dosing with AMG 317, a fully human monoclonal antibody therapeutic. The antibody responses displayed a wide range of relative concentrations (30 ng/mL to >13 μg/mL) and peaked at various times during the study. To evaluate the impact of immunogenicity on PK, AMG 317 concentration data were analyzed following stratification by dose group, time point, antibody status (positive or negative), and antibody level (relative concentration). With dose group as a stratifying variable, a moderate reduction in AMG 317 levels (AMG 317 levels was revealed when antibody data was stratified by both time point and antibody level. In general, high ADA concentrations (>500 ng/mL) and later time points (week 12) were associated with significantly (up to 97%) lower trough AMG 317 concentrations. The use of quasi-quantitative antibody data and appropriate statistical methods was critical for the most comprehensive evaluation of the impact of immunogenicity on PK.

Re-engineering therapeutic antibodies for Alzheimer's disease as blood-brain barrier penetrating bi-specific antibodies.

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Pardridge, William M

2016-12-01

Therapeutic antibodies are large molecule drugs that do not cross the blood-brain barrier (BBB). Therefore, drug development of therapeutic antibodies for Alzheimer's disease (AD) requires that these molecules be re-engineered to enable BBB delivery. This is possible by joining the therapeutic antibody with a transporter antibody, resulting in the engineering of a BBB-penetrating bispecific antibody (BSA). Areas covered: The manuscript covers transporter antibodies that cross the BBB via receptor-mediated transport systems on the BBB, such as the insulin receptor or transferrin receptor. Furthermore, it highlights therapeutic antibodies for AD that target the Abeta amyloid peptide, beta secretase-1, or the metabotropic glutamate receptor-1. BSAs are comprised of both the transporter antibody and the therapeutic antibody, as well as IgG constant region, which can induce immune tolerance or trigger transport via Fc receptors. Expert opinion: Multiple types of BSA molecular designs have been used to engineer BBB-penetrating BSAs, which differ in valency and spatial orientation of the transporter and therapeutic domains of the BSA. The plasma pharmacokinetics and dosing regimens of BSAs differ from that of conventional therapeutic antibodies. BBB-penetrating BSAs may be engineered in the future as new treatments of AD, as well as other neural disorders.

Antinuclear antibody panel

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... page: //medlineplus.gov/ency/article/003535.htm Antinuclear antibody panel To use the sharing features on this page, please enable JavaScript. The antinuclear antibody panel is a blood test that looks at ...

Successful Aging in the Context of the Disablement Process: Working and Volunteering as Moderators on the Association Between Chronic Conditions and Subsequent Functional Limitations.

Science.gov (United States)

Kail, Ben Lennox; Carr, Dawn C

2017-03-01

This study evaluated the successful aging model by assessing the impact of two forms of productive engagement-working and volunteering-as potential interventions in the process of disablement. The Health and Retirement Study was used to (a) estimate two-stage selection equations of (i) currently working part time and full time and (ii) currently volunteering less than 100 hours and volunteering 100 hours or more per year (net of chronic health problems) and (b) assess whether, net of selection, working, and volunteering moderate the association between chronic conditions and subsequent functional limitations. Chronic conditions were associated with elevated levels of subsequent functional limitations, whereas both working and volunteering were associated with lower levels of subsequent functional limitations. Moreover, workers and volunteers of less than 100 hours per year experienced a reduction in the association of chronic conditions on subsequent functional limitations. This research highlights the role of productive engagement as a key element in successful aging. Not only do work and volunteering have direct associations with health outcomes themselves, but they also act as potential interventions in the process of disablement by attenuating the way in which chronic conditions are translated into subsequent functional limitations. This suggests that (a) future research should apply successful aging models to health processes as well as health outcomes and (b) policy makers should support social institutions that foster late-life productive engagement. © The Author 2016. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Efficient production of Trastuzumab Fab antibody fragments in Brevibacillus choshinensis expression system.

Science.gov (United States)

Mizukami, Makoto; Onishi, Hiromasa; Hanagata, Hiroshi; Miyauchi, Akira; Ito, Yuji; Tokunaga, Hiroko; Ishibashi, Matsujiro; Arakawa, Tsutomu; Tokunaga, Masao

2018-10-01

The Brevibacillus expression system has been successfully employed for the efficient productions of a variety of recombinant proteins, including enzymes, cytokines, antigens and antibody fragments. Here, we succeeded in secretory expression of Trastuzumab Fab antibody fragments using B. choshinensis/BIC (Brevibacillus in vivocloning) expression system. In the fed-batch high-density cell culture, recombinant Trastuzumab Fab with amino-terminal His-tag (His-BcFab) was secreted at high level, 1.25 g/liter, and Fab without His-tag (BcFab) at ∼145 mg/L of culture supernatant. His-BcFab and BcFab were purified to homogeneity using combination of conventional column chromatographies with a yield of 10-13%. This BcFab preparation exhibited native structure and functions evaluated by enzyme-linked immunosorbent assay, surface plasmon resonance, circular dichroism measurements and size exclusion chromatography. To our knowledge, this is the highest production of Fab antibody fragments in gram-positive bacterial expression/secretion systems. Copyright © 2018 Elsevier Inc. All rights reserved.

Radioimmunoassay of class-specific antibodies (RIACA): chicken antibodies to DNP

International Nuclear Information System (INIS)

Viljanen, M.K.; Granfors, K.; Toivanen, P.

1977-01-01

A radioimmunological method for the quantitation of class-specific antibodies has been developed. The method allows the quantitation of nanogram per ml concentrations of IgG and IgM-anti-DNP antibodies without any physical or chemical pretreatment of the sample. DNP was coupled covalently to a cyanogen bromide activated paper disk with the augmentation of lysine molecule. Anti-DNP antibodies were allowed to react with the coupled DNP and then quantitated by their capacity to bind 125 I-labelled anti-chicken-μ or anti-chicken-γ. The inter-assay variation coefficients ranged from 8.1 to 14.7% and the mean standard deviations of duplicate determinations were about 11%. The combination of this method with the exact immunoradiometric quantitation of the total serum IgM and IgG, and with an immunoabsorption technique, makes it possible to quantitate class-specific antibodies on weight units

Distribution and pharmacokinetics of radiolabeled monoclonal antibody OC 125 after intravenous and intraperitoneal administration in gynecologic tumors

International Nuclear Information System (INIS)

Haisma, H.J.; Moseley, K.R.; Battaile, A.; Griffiths, T.C.; Knapp, R.C.

1988-01-01

Radiolabeled monoclonal antibodies may be useful for radioimmunotherapy of gynecologic tumors. Iodine 131-labeled F(ab')2 fragments of a monoclonal antibody, OC 125, with specificity for ovarian carcinoma, were used to study the distribution and pharmacokinetics of this antibody in patients with gynecologic tumors. The radiolabeled antibody was injected intravenously or intraperitoneally into 10 patients suspected of having ovarian cancer. Blood and urine samples were used for pharmacokinetic studies, and biopsy specimens were examined for the uptake of antibody. The serum half-life of the labeled antibody was 30 hours after intravenous administration, with 20% of the injected dose per liter detected at 24 hours. After intraperitoneal injection, the appearance of antibody in serum was slow, with a maximum level of 1.4% of the injected dose per liter at 24 hours. Urinary excretion of the radiolabeled antibody was similar for intravenous and intraperitoneal administration, with approximately 50% of the injected dose excreted after 48 hours. Intraperitoneal administration of the radiolabeled antibody resulted in a higher uptake of antibody in the tumor and a lower uptake of antibody in normal tissues. On the basis of this limited study, intraperitoneal administration of radiolabeled antibody is preferred over intravenous administration for radioimmunotherapy of ovarian cancer

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

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Takayoshi Matsuda

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

Egg Yolk Antibodies, State of the Art and Future Prospects.

Science.gov (United States)

Schade, Rüdiger; Hlinak, Andreas

1996-01-01

Immunization of chickens and extraction of antibodies from egg yolk belongs to the alternative methods since the animals suffering is reduced by non-invasive antibody-sampling. Also, the number of animals needed to produce a certain amount of antibody can be reduced since chickens produce a significant higher antibody quantity than rabbits. Despite its several advantages this technology (IgY-technology) is rather scarcely used. Traditional behavior as well as limited or no information at all may hamper a broader acceptance at present. However, significant arguments exist in chicken housing, the choice of appropriate IgY-extraction methods and a lack of information regarding the use of IgY-antibodies. This paper intends to give a short introduction in the IgY-technology, to briefly discuss the state of the art and to inform on recent developments and discussions in this field. The suitability of IgY for special fields of application (as a result of the structural differences between IgY and IgG) is emphasized (e.g. assays combining IgG and IgY, immunization of chickens against highly conserved anti-genes). In addition, it is stressed that the IgY-technology as an alternative method can particularly integrate requirements of animal protection (reduce, replace, refine), science (characteristics of avian immune system and resulting properties of IgY) and economy (amount of IgY produced from one chicken).

Microfluidic Production of Alginate Hydrogel Particles for Antibody Encapsulation and Release.

Science.gov (United States)

Mazutis, Linas; Vasiliauskas, Remigijus; Weitz, David A

2015-12-01

Owing to their biocompatibility and reduced side effects, natural polymers represent an attractive choice for producing drug delivery systems. Despite few successful examples, however, the production of monodisperse biopolymer-based particles is often hindered by high viscosity of polymer fluids. In this work, we present a microfluidic approach for production of alginate-based particles carrying encapsulated antibodies. We use a triple-flow micro-device to induce hydrogel formation inside droplets before their collection off-chip. The fast mixing and gelation process produced alginate particles with a unique biconcave shape and dimensions of the mammalian cells. We show slow and fast dissolution of particles in different buffers and evaluate antibody release over time. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

High-affinity monoclonal antibodies specific for deoxynucleosides structurally modified by alkylating agents: Applications for immunoanalysis

International Nuclear Information System (INIS)

Adamkiewicz, J.; Ahrens, O.; Rajewsky, M.F.

1984-01-01

So far the results of attempts to use monoclonal antibodies for the demonstration of carcinogen-DNA adducts in cells by immunostaining have been promising. Thus the authors have established a standardized procedure for the quantitation of specific alkyl-deoxynucleosides in the nuclear DNA of individual cells by direct immunofluorescence, using tetramethylrhodamine isothiocyanate-labeled monoclonal antibodies and a computer-based image analysis of electronically intensified fluorescence signals. With a fluorescent anti-(O/sup 6/-EtdGuo) monoclonal antibody, the present detection limit for O/sup 6/-Etd-Guo in the nuclei of individual cells previously exposed to an ethylating N-nitroso compound (e.g., N-ethyl-N-nitrosourea) is -- 700 O/sup 6/-EtdGuo molecules per diploid genome, i.e., similar to the detection limit for the same ethylation product in a hydrolysate of (O/sup 6/-EtdGuo)-containing DNA analyzed by competitive RIA

Lyme disease antibody

Science.gov (United States)

... JavaScript. The Lyme disease blood test looks for antibodies in the blood to the bacteria that causes ... needed. A laboratory specialist looks for Lyme disease antibodies in the blood sample using the ELISA test . ...

Evaluation of Salivary Antibodies to Detect Infection with Helicobacter pylori

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Mark B Loeb

1997-01-01

Full Text Available Helicobacter pylori infection is an important cause of peptic ulcer disease and chronic gastritis. Infection with this bacterium stimulates the production of immunoglobulin (Ig G antibody. Salivary IgG antibody tests to detect H pylori infection offer a convenient and noninvasive method of diagnosis. To evaluate an IgG salivary antibody kit, saliva was collected from 157 out-patients with dyspepsia referred for endoscopy to a tertiary centre. A salivary IgG ELISA antibody assay was performed using the Helisal Helicobacter pylori (IgG assay kit, and at least four gastric biopsies were obtained. H pylori infection was confirmed by demonstration of the organism on Warthin-Starry silver stain (sensitivity 85%, specificity 55%. The prevalence of infection with H pylori was 30%. When the analysis was redone, excluding those treated with eradication therapy, the results were similar (sensitivity 86%, specificity 58%. The positive predictive value of the assay was 45% and the negative predictive value was 90%. Despite the ease of sampling, the assay used has limited diagnostic utility, lacking the predictive value to indicate which patients referred with dyspeptic symptoms to a tertiary care setting are infected with H pylori.

Cellular aging (the Hayflick limit) and species longevity: a unification model based on clonal succession.

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Juckett, D A

1987-03-01

A model is presented which proposes a specific cause-and-effect relationship between a limited cell division potential and the maximum lifespan of humans and other mammals. It is based on the clonal succession hypothesis of Kay which states that continually replicating cell beds (e.g. bone marrow, intestinal crypts, epidermis) could be composed of cells with short, well-defined division potentials. In this model, the cells of these beds are proposed to exist in an ordered hierarchy which establishes a specific sequence for cell divisions throughout the organism's lifespan. The depletion of division potential at all hierarchical levels leads to a loss of bed function and sets an intrinsic limit to species longevity. A specific hierarchy for cell proliferation is defined which allows the calculation of time to bed depletion and, ultimately, to organism mortality. The model allows the existence of a small number (n) of critical cell beds within the organism and defines organism death as the inability of any one of these beds to produce cells. The model is consistent with all major observations related to cellular and organismic aging. In particular, it links the PDLs (population doubling limit) observed for various species to their mean lifespan; it explains the slow decline in PDL as a function of age of the donor; it establishes a thermodynamically stable maximum lifespan for a disease-free population; and it can explain why tissue transplants outlive donors or hosts.

Development and immunochemical evaluation of a novel chicken IgY antibody specific for KLK6

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Sotiropoulou Georgia

2012-12-01

Full Text Available Abstract Background Human kallikrein-related peptidase 6 (KLK6 has been implicated in various types of cancer and in neurodegenerative and demyelinating diseases including multiple sclerosis. Further, anti-KLK6 antibodies attenuated disease manifestations in the mouse model of multiple sclerosis. Availability of specific antibodies against KLK6 is fundamental to the development of improved diagnostic and/or immunotherapeutic applications. Here, we exploited the enhanced immunogenicity of mammalian proteins in avian species to generate a polyclonal antibody against KLK6. Results Chicken were immunized with recombinant KLK6 and antibodies Y (IgYs were purified from egg yolk with a simple procedure and evaluated for KLK6 detection by ELISA and Western blot using recombinant proteins and human cell lysates and supernatants. The anti-KLK6 Y polyclonal exhibited high affinity for KLK6 with a detection limit of 30 fmol. On the other hand, the widely used rabbit polyclonal antibody that was raised against the same recombinant KLK6 had a detection limit of 300 fmol. Moreover, the IgYs did not display any crossreactivity with recombinant KLKs or endogenous KLKs and other cellular proteins. Conclusions Based on its high specificity and sensitivity the developed anti-KLK6 IgY is expected to aid the development of improved diagnostic tools for the detection of KLK6 in biological and clinical samples.

Antibody-cytokine fusion proteins for treatment of cancer: engineering cytokines for improved efficacy and safety.

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Young, Patricia A; Morrison, Sherie L; Timmerman, John M

2014-10-01

The true potential of cytokine therapies in cancer treatment is limited by the inability to deliver optimal concentrations into tumor sites due to dose-limiting systemic toxicities. To maximize the efficacy of cytokine therapy, recombinant antibody-cytokine fusion proteins have been constructed by a number of groups to harness the tumor-targeting ability of monoclonal antibodies. The aim is to guide cytokines specifically to tumor sites where they might stimulate more optimal anti-tumor immune responses while avoiding the systemic toxicities of free cytokine therapy. Antibody-cytokine fusion proteins containing interleukin (IL)-2, IL-12, IL-21, tumor necrosis factor (TNF)α, and interferons (IFNs) α, β, and γ have been constructed and have shown anti-tumor activity in preclinical and early-phase clinical studies. Future priorities for development of this technology include optimization of tumor targeting, bioactivity of the fused cytokine, and choice of appropriate agents for combination therapies. This review is intended to serve as a framework for engineering an ideal antibody-cytokine fusion protein, focusing on previously developed constructs and their clinical trial results. Copyright © 2014 Elsevier Inc. All rights reserved.

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

Science.gov (United States)

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

2014-01-01

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

Iodine-131 labeled anti-CEA polyclonal antibody detection of gastrointestinal cancer

International Nuclear Information System (INIS)

Nabi, H.A.; Hinkle, G.H.; Olsen, J.O.; Haagensen, D.A.; Thurston, M.O.; Mojzisik, C.; Houchens, D.; Martin, E.W. Jr.

1984-01-01

To localize gastrointestinal tumor, 31 patients were injected with 1.7-2.1 mCi I-131 anti-CEA baboon polyclonal antibody. Whole body imaging at 48, 72, and occasionally 96 hrs was performed with a Signa Camera (Technicare) peaked at 364 keV with 20% window. Additional spot views were usually obtained. No subtraction methods were used. All patients had surgical and pathological confirmation of the nuclear medicine studies. Labeled antibody images were positive in 15 (8 recurrent or metastatic colorectal, 2 gastric, 1 pancreatic, 1 primary colon, and 1 breast metastatic to chest wall). In 1, antibody images were positive for metastatic deposits in para-aortic lymph nodes, but negative for primary rectal tumor. True negative images were observed in 6; false negative images in 9 (4 liver metastases, 2 rectal, 1 pancreatic, 1 mesenteric lymph node metastasis, 1 bone metastasis). In all cases, no correlation existed between preoperative CEA serum levels and imaging. I-131 labeled anti-CEA polyclonal antibody imaging proved highly efficient in detecting gastric cancer (2/2) and moderately efficient in detecting recurrent colorectal cancer (8/15). On the other hand, the I-131 labeled polyclonal anti-CEA antibody imaging was of limited value in detecting colon cancer (1/9), pancreatic cancer (1/4) and metastatic liver disease

Echinococcus granulosus: the potential use of specific radiolabelled antibodies in diagnosis by immunoscintigraphy

International Nuclear Information System (INIS)

Rogan, M.T.; Morris, D.L.; Pritchard, D.I.; Perkins, A.C.

1990-01-01

Diagnosis of hydatid disease in man is frequently dependent on the imaging of cysts in situ by techniques such as ultrasonography and CAT scans. Such methods are useful but are not specific and can lead to errors in diagnosis. The present work reports preliminary experiments on the development of a specific imaging technique for hydatid cysts using radiolabelled antibodies. A purified preparation of antigen B of hydatid fluid was used to raise polyclonal antisera in rabbits and the resulting affinity-purified IgG labelled with 131 I. Gerbils with an established Echinococcus granulosus infection were injected intraperitoneally with the labelled antibody and imaged 48 h later with a gamma camera. Hydatid cysts could be identified within the peritoneal cavity and post-mortem assessment of activity showed the cysts to contain approximately four times as much activity as the surrounding organs thereby indicating successful targeting of the antibody to the cysts. (author)

Biological Characterization of a Stable Effector Functionless (SEFL) Monoclonal Antibody Scaffold in Vitro*

Science.gov (United States)

Liu, Ling; Jacobsen, Frederick W.; Everds, Nancy; Zhuang, Yao; Yu, Yan Bin; Li, Nianyu; Clark, Darcey; Nguyen, Mai Phuong; Fort, Madeline; Narayanan, Padma; Kim, Kei; Stevenson, Riki; Narhi, Linda; Gunasekaran, Kannan; Bussiere, Jeanine L.

2017-01-01

The stable effector functionLess (SEFL) antibody was designed as an IgG1 antibody with a constant region that lacks the ability to interact with Fcγ receptors. The engineering and stability and pharmacokinetic assessments of the SEFL scaffold is described in the accompanying article (Jacobsen, F. W., Stevenson, R., Li, C., Salimi-Moosavi, H., Liu, L., Wen, J., Luo, Q., Daris, K., Buck, L., Miller, S., Ho, S-Y., Wang, W., Chen, Q., Walker, K., Wypych, J., Narhi, L., and Gunasekaran, K. (2017) J. Biol. Chem. 292). The biological properties of these SEFL antibodies were assessed in a variety of human and cynomolgus monkey in vitro assays. Binding of parent molecules and their SEFL variants to human and cynomolgus monkey FcγRs were evaluated using flow cytometry-based binding assays. The SEFL variants tested showed decreased binding affinity to human and cynomolgus FcγRs compared with the wild-type IgG1 antibody. In addition, SEFL variants demonstrated no antibody-dependent cell-mediated cytotoxicity in vitro against Daudi cells with cynomolgus monkey peripheral blood mononuclear cells, and had minimal complement-dependent cytotoxicity activity similar to that of the negative control IgG2 in a CD20+ human Raji lymphoma cell line. SEFL mutations eliminated off-target antibody-dependent monocyte phagocytosis of cynomolgus monkey platelets, and cynomolgus platelet activation in vitro. These experiments demonstrate that the SEFL modifications successfully eliminated Fc-associated effector binding and functions. PMID:27994063

Development of broad-spectrum human monoclonal antibodies for rabies post-exposure prophylaxis

International Nuclear Information System (INIS)

Benedictis, P. de; Minola, A.; Rota, E.; Aiello, R.; Zecchin, B.; Salomoni, A.; Foglierini, M.; Agatic, G.; Vanzetta, F.; Lavenir, R.; Lepelletier, A.; Bentley, E.; Weiss, R.; Cattoli, G.

2016-01-01

Full text: Currently available rabies post-exposure prophylaxis (PEP) for use in humans includes equine or human rabies immunoglobulins (RIG). The replacement of RIG with an equally or more potent and safer product is strongly encouraged due to the high costs and limited availability of existing RIG. In this study, we identified two broadly neutralizing human monoclonal antibodies that represent a valid and affordable alternative to RIG in rabies PEP. Memory B cells from four selected vaccinated donors were immortalized and monoclonal antibodies were tested for neutralizing activity and epitope specificity. Two antibodies, identified as RVC20 and RVC58 (binding to antigenic site I and III, respectively), were selected for their potency and broad-spectrum reactivity. In vitro, RVC20 and RVC58 were able to neutralize all 35 rabies virus (RABV) and 25 non-RABV lyssaviruses. They showed higher potency and breath compared to antibodies under clinical development (namely CR57, CR4098, and RAB1) and commercially available human RIG. In vivo, the RVC20–RVC58 cocktail protected Syrian hamsters from a lethal RABV challenge and did not affect the endogenous hamster post-vaccination antibody response. (author)

Polysaccharide microarray technology for the detection of Burkholderia pseudomallei and Burkholderia mallei antibodies.

Science.gov (United States)

Parthasarathy, Narayanan; DeShazer, David; England, Marilyn; Waag, David M

2006-11-01

A polysaccharide microarray platform was prepared by immobilizing Burkholderia pseudomallei and Burkholderia mallei polysaccharides. This polysaccharide array was tested with success for detecting B. pseudomallei and B. mallei serum (human and animal) antibodies. The advantages of this microarray technology over the current serodiagnosis of the above bacterial infections were discussed.

Anti-PDGF receptor β antibody-conjugated squarticles loaded with minoxidil for alopecia treatment by targeting hair follicles and dermal papilla cells.

Science.gov (United States)

Aljuffali, Ibrahim A; Pan, Tai-Long; Sung, Calvin T; Chang, Shu-Hao; Fang, Jia-You

2015-08-01

This study developed lipid nanocarriers, called squarticles, conjugated with anti-platelet-derived growth factor (PDGF)-receptor β antibody to determine whether targeted Minoxidil (MXD) delivery to the follicles and dermal papilla cells (DPCs) could be achieved. Squalene and hexadecyl palmitate (HP) were used as the matrix of the squarticles. The PDGF-squarticles showed a mean diameter and zeta potential of 195 nm and -46 mV, respectively. Nanoparticle encapsulation enhanced MXD porcine skin deposition from 0.11 to 0.23 μg/mg. The antibody-conjugated nanoparticles ameliorated follicular uptake of MXD by 3-fold compared to that of the control solution in the in vivo mouse model. Both vertical and horizontal skin sections exhibited a wide distribution of nanoparticles in the follicles, epidermis, and deeper skin strata. The encapsulated MXD moderately elicited proliferation of DPCs and vascular endothelial growth factor (VEGF) expression. The active targeting of PDGF-squarticles may be advantageous to improving the limited success of alopecia therapy. Topical use of minoxidil is only one of the very few treatment options for alopecia. Nonetheless, the current delivery method is far from ideal. In this article, the authors developed lipid nanocarriers with anti-platelet-derived growth factor receptor ? antibody to target dermal papilla cells, and showed enhanced uptake of minoxidil. Copyright © 2015 Elsevier Inc. All rights reserved.

Anti-transferrin receptor antibody and antibody-drug conjugates cross the blood-brain barrier

International Nuclear Information System (INIS)

Friden, P.M.; Walus, L.R.; Musso, G.F.; Taylor, M.A.; Malfroy, B.; Starzyk, R.M.

1991-01-01

Delivery of nonlipophilic drugs to the brain is hindered by the tightly apposed capillary endothelial cells that make up the blood-brain barrier. The authors have examined the ability of a monoclonal antibody (OX-26), which recognizes the rat transferrin receptor, to function as a carrier for the delivery of drugs across the blood-brain barrier. This antibody, which was previously shown to bind preferentially to capillary endothelial cells in the brain after intravenous administration, labels the entire cerebrovascular bed in a dose-dependent manner. The initially uniform labeling of brain capillaries becomes extremely punctate ∼ 4 hr after injection, suggesting a time-dependent sequestering of the antibody. Capillary-depletion experiments, in which the brain is separated into capillary and parenchymal fractions, show a time-dependent migration of radiolabeled antibody from the capillaries into the brain parenchyma, which is consistent with the transcytosis of compounds across the blood-brain barrier. Antibody-methotrexate conjugates were tested in vivo to assess the carrier ability of this antibody. Immunohistochemical staining for either component of an OX-26-methotrexate conjugate revealed patterns of cerebrovascular labeling identical to those observed with the unaltered antibody. Accumulation of radiolabeled methotrexate in the brain parenchyma is greatly enhanced when the drug is conjugated to OX-26

KADAR ANTIBODI-TIROPEROKSIDASE DAN ANTIBODI-TIROGLOBULIN PADA WANITA USIA SUBUR DI DAERAH ENDEMlS GAKI

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Agus Wibowo

2012-10-01

Full Text Available Background: Thyroid hormones play a critical role in human. Disorders of the thyroid gland result primary from autoimmune processes that either stimulate the over production of thyroid hormones (hyperthyroid or causes glandular destruction and hormones deficiency (hypothyroid. Autoimmune Thyroid Disease (AITD a common organ specific autoimmune disorder is seen mostly in women. AITD are complex disease that are caused by an interaction between susceptibility genes and environmental trigger such dietary iodine. The development of antibodies to Thyroid peroxidase (TPO and Thyroglobulin (TG is the main hall mark of AITD. Method: 'Thirty respondents from were analyzed. The blood were collected for TSH, FreeT4, Tyroglobulin Antibody and Tyroperoxidase Antibody analyzed and DNA isolation. Circulating TSH, FreeT4, autoantibodies to TPO and TG are measured by ELISA. Result: 50% respondent in normal thyroid hormones and 50% in hypothyroid and hyperthyroid status. TPO antibodies  and thyroglobulin antibodies found in all of respondent with thyroid disorder. Conclusion: Antibodies to TPO and TG is seen in respondent with thyroid disorder   Keywords: AITD, TSH, FreeT4, TPO antibodies, TG antibodies.

Efficient generation of monoclonal antibodies against peptide in the context of MHCII using magnetic enrichment.

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Spanier, Justin A; Frederick, Daniel R; Taylor, Justin J; Heffernan, James R; Kotov, Dmitri I; Martinov, Tijana; Osum, Kevin C; Ruggiero, Jenna L; Rust, Blake J; Landry, Samuel J; Jenkins, Marc K; McLachlan, James B; Fife, Brian T

2016-06-13

Monoclonal antibodies specific for foreign antigens, auto-antigens, allogeneic antigens and tumour neo-antigens in the context of major histocompatibility complex II (MHCII) are highly desirable as novel immunotherapeutics. However, there is no standard protocol for the efficient generation of monoclonal antibodies that recognize peptide in the context of MHCII, and only a limited number of such reagents exist. In this report, we describe an approach for the generation and screening of monoclonal antibodies specific for peptide bound to MHCII. This approach exploits the use of recombinant peptide:MHC monomers as immunogens, and subsequently relies on multimers to pre-screen and magnetically enrich the responding antigen-specific B cells before fusion and validation, thus saving significant time and reagents. Using this method, we have generated two antibodies enabling us to interrogate antigen presentation and T-cell activation. This methodology sets the standard to generate monoclonal antibodies against the peptide-MHCII complexes.

Monospecific antibody against Bordetella pertussis Adenylate Cyclase protects from Pertussis

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Yasmeen Faiz Kazi

2012-06-01

Full Text Available Objectives: Acellular pertussis vaccines has been largely accepted world-wide however, there are reports about limitedantibody response against these vaccines suggesting that multiple antigens should be included in acellular vaccinesto attain full protection. The aim of present study was to evaluate the role of Bordetella pertussis adenylate cyclase as aprotective antigen.Materials and methods: Highly mono-specific antibody against adenylate cyclase (AC was raised in rabbits usingnitrocellulose bound adenylate cyclase and the specificity was assessed by immuoblotting. B.pertussis 18-323, wasincubated with the mono-specific serum and without serum as a control. Mice were challenged intra-nasally and pathophysiolgicalresponses were recorded.Results: The production of B.pertussis adenylate cyclase monospecific antibody that successfully recognized on immunoblotand gave protection against fatality (p< 0.01 and lung consolidation (p <0.01. Mouse weight gain showedsignificant difference (p< 0.05.Conclusion: These preliminary results highlight the role of the B.pertussis adenylate cyclase as a potential pertussisvaccine candidate. B.pertussis AC exhibited significant protection against pertussis in murine model. J Microbiol InfectDis 2012; 2(2: 36-43Key words: Pertussis; monospecific; antibody; passive-protection

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

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Xinwei Wang

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

Conducting polymer film-based immunosensors using carbon nanotube/antibodies doped polypyrrole

Energy Technology Data Exchange (ETDEWEB)

Tam, Phuong Dinh, E-mail: phuongdinhtam@gmail.com [Advanced Institute for Science and Technology (AIST), Hanoi University of Science and Technology (Viet Nam); Hieu, Nguyen Van [International Training Institute for Materials Science (ITIMS), Hanoi University of Science and Technology (Viet Nam)

2011-09-15

Carbon nanotube/polypyrrole/antibodies polymer films were synthesized successfully on microelectrodes by electrochemical deposition. Electropolymerization was performed at optimal range between -0.8 and +0.8 V at a scan rate of 50 mV s{sup -1} in an electrochemical mini-cell containing monomer pyrroles, carbon nanotubes, and goat IgGs. The conducting polymer films were characterized by Fourier transform infrared spectrometry, Raman spectra, and Field emission scanning electron microscopy. And then, it was prepared for immunosensor application to determine anti-goat IgGs. The results show that a linear range between 0.05 and 0.7 {mu}g ml{sup -1} for anti-goat IgGs detection was observed for immunosensor, a detection limit as low as 0.05 {mu}g ml{sup -1} and a response time of 1 min. The effect parameters of electropolymerization process on immunosensor response are also studied. It found that the immunosensor well active in 1.5 mg ml{sup -1} CNT concentration, 2.5 mM pyrrole, 10 {mu}g ml{sup -1} goat IgGs.

Tabhu: tools for antibody humanization

DEFF Research Database (Denmark)

Olimpieri, Pier Paolo; Marcatili, Paolo; Tramontano, Anna

2015-01-01

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...... elicit unwanted and dangerous immunogenic responses. Antibody humanization methods are designed to produce molecules with a better safety profile still maintaining their ability to bind the antigen. This can be accomplished by grafting the non-human regions determining the antigen specificity...... 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...

What Is Antiphospholipid Antibody Syndrome?

Science.gov (United States)

... Back To Health Topics / Antiphospholipid Antibody Syndrome Antiphospholipid Antibody Syndrome Also known as What Is Antiphospholipid (AN-te-fos-fo-LIP-id) antibody syndrome (APS) is an autoimmune disorder. Autoimmune disorders ...

Antibody engineering: methods and protocols

National Research Council Canada - National Science Library

Chames, Patrick

2012-01-01

"Antibody Engineering: Methods and Protocols, Second Edition was compiled to give complete and easy access to a variety of antibody engineering techniques, starting from the creation of antibody repertoires and efficient...

Assay dependence of Brucella antibody prevalence in a declining Alaskan harbor seal (Phoca vitulina population

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Hueffer Karsten

2013-01-01

Full Text Available Abstract Background Brucella is a group of bacteria that causes brucellosis, which can affect population health and reproductive success in many marine mammals. We investigated the serological prevalence of antibodies against Brucella bacteria in a declining harbor seal population in Glacier Bay National Park, Alaska. Results Prevalence ranged from 16 to 74 percent for those tests detecting antibodies, indicating that harbor seals in Glacier Bay have been exposed to Brucella bacteria. However, the actual level of serological prevalence could not be determined because results were strongly assay-dependent. Conclusions This study reinforces the need to carefully consider assay choice when comparing different studies on the prevalence of anti–Brucella antibodies in pinnipeds and further highlights the need for species- or taxon-specific assay validation for both pathogen and host species.

Designing two-in-one antibodies.

Science.gov (United States)

Valladares, Ignacio Garcia; Espinoza, Luis R

2009-09-01

Evaluation of: Bostrom J, Shang-Fan Y, Kan D et al.: Variants of the antibody Herceptin that interact with HER2 and VEGF at the antigen binding site. Science 323, 1610-1614 (2009). The longstanding held notion that one antibody equals one antigen and, hence, one function has been challenged in recent years. Improved technology in antibody production, especially the accumulation of sequence data of immunoglobulin genes and the advent of PCR have made it possible to clone antibody gene repertoires. The current paper provides further challenge to the notion of one antibody = one antigen by developing 'two-in-one' antibodies with an antigen-binding site that binds two distinct proteins with high affinity. A therapeutic variant antibody of Herceptin (Genentech, CA, USA) was isolated that binds the human EGF receptor (HER)2 and also to VEGF. This development may represent a breakthrough discovery and may have significant implications in the therapy of malignant, infectious, allergic and autoimmune disorders.

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

International Nuclear Information System (INIS)

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

1990-01-01

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

An improved yeast transformation method for the generation of very large human antibody libraries.

Science.gov (United States)

Benatuil, Lorenzo; Perez, Jennifer M; Belk, Jonathan; Hsieh, Chung-Ming

2010-04-01

Antibody library selection by yeast display technology is an efficient and highly sensitive method to identify binders to target antigens. This powerful selection tool, however, is often hampered by the typically modest size of yeast libraries (approximately 10(7)) due to the limited yeast transformation efficiency, and the full potential of the yeast display technology for antibody discovery and engineering can only be realized if it can be coupled with a mean to generate very large yeast libraries. We describe here a yeast transformation method by electroporation that allows for the efficient generation of large antibody libraries up to 10(10) in size. Multiple components and conditions including CaCl(2), MgCl(2), sucrose, sorbitol, lithium acetate, dithiothreitol, electroporation voltage, DNA input and cell volume have been tested to identify the best combination. By applying this developed protocol, we have constructed a 1.4 x 10(10) human spleen antibody library essentially in 1 day with a transformation efficiency of 1-1.5 x 10(8) transformants/microg vector DNA. Taken together, we have developed a highly efficient yeast transformation method that enables the generation of very large and productive human antibody libraries for antibody discovery, and we are now routinely making 10(9) libraries in a day for antibody engineering purposes.

CD133 antibody conjugation to decellularized human heart valves intended for circulating cell capture.

Science.gov (United States)

Vossler, John D; Min Ju, Young; Williams, J Koudy; Goldstein, Steven; Hamlin, James; Lee, Sang Jin; Yoo, James J; Atala, Anthony

2015-09-03

The long term efficacy of tissue based heart valve grafts may be limited by progressive degeneration characterized by immune mediated inflammation and calcification. To avoid this degeneration, decellularized heart valves with functionalized surfaces capable of rapid in vivo endothelialization have been developed. The aim of this study is to examine the capacity of CD133 antibody-conjugated valve tissue to capture circulating endothelial progenitor cells (EPCs). Decellularized human pulmonary valve tissue was conjugated with CD133 antibody at varying concentrations and exposed to CD133 expressing NTERA-2 cl.D1 (NT2) cells in a microflow chamber. The amount of CD133 antibody conjugated on the valve tissue surface and the number of NT2 cells captured in the presence of shear stress was measured. Both the amount of CD133 antibody conjugated to the valve leaflet surface and the number of adherent NT2 cells increased as the concentration of CD133 antibody present in the surface immobilization procedure increased. The data presented in this study support the hypothesis that the rate of CD133(+) cell adhesion in the presence of shear stress to decellularized heart valve tissue functionalized by CD133 antibody conjugation increases as the quantity of CD133 antibody conjugated to the tissue surface increases.

Lack of antibodies to NMDAR or VGKC-complex in GAD and cardiolipin antibody-positive refractory epilepsy.

Science.gov (United States)

Liimatainen, Suvi; Peltola, Jukka; Hietaharju, Aki; Sabater, Lidia; Lang, Bethan

2014-03-01

Over the last few years autoantibodies against neuronal proteins have been identified in several forms of autoimmune encephalitis and epilepsy. NMDA receptor (NMDAR) and voltage gated potassium channel (VGKC) complex antibodies are mainly associated with limbic encephalitis (LE) whereas glutamic acid decarboxylase antibodies (GADA) and anticardiolipin (ACL) antibodies are more commonly detected in patients with chronic epilepsy. Clinical features vary between these antibodies suggesting the specificity of different neuronal antibodies in seizures. Serum samples of 14 GADA positive and 24 ACL positive patients with refractory epilepsy were analyzed for the presence of VGKC or NMDAR antibodies. No positive VGKC or NMDAR antibodies were found in these patients. The results confirm the different significance of these neuronal antibodies in seizure disorders. Different autoantibodies have different significance in seizures and probably have different pathophysiological mechanisms of actions. Copyright © 2014 Elsevier B.V. All rights reserved.

HIV-1 subtype C superinfected individuals mount low autologous neutralizing antibody responses prior to intrasubtype superinfection

Directory of Open Access Journals (Sweden)

Basu Debby

2012-09-01

Full Text Available Abstract Background The potential role of antibodies in protection against intra-subtype HIV-1 superinfection remains to be understood. We compared the early neutralizing antibody (NAb responses in three individuals, who were superinfected within one year of primary infection, to ten matched non-superinfected controls from a Zambian cohort of subtype C transmission cases. Sequence analysis of single genome amplified full-length envs from a previous study showed limited diversification in the individuals who became superinfected with the same HIV-1 subtype within year one post-seroconversion. We hypothesized that this reflected a blunted NAb response, which may have made these individuals more susceptible to superinfection. Results Neutralization assays showed that autologous plasma NAb responses to the earliest, and in some cases transmitted/founder, virus were delayed and had low to undetectable titers in all three superinfected individuals prior to superinfection. In contrast, NAbs with a median IC50 titer of 1896 were detected as early as three months post-seroconversion in non-superinfected controls. Early plasma NAbs in all subjects showed limited but variable levels of heterologous neutralization breadth. Superinfected individuals also exhibited a trend toward lower levels of gp120- and V1V2-specific IgG binding antibodies but higher gp120-specific plasma IgA binding antibodies. Conclusions These data suggest that the lack of development of IgG antibodies, as reflected in autologous NAbs as well as gp120 and V1V2 binding antibodies to the primary infection virus, combined with potentially competing, non-protective IgA antibodies, may increase susceptibility to superinfection in the context of settings where a single HIV-1 subtype predominates.

Mechanisms of resistance to HER family targeting antibodies

Energy Technology Data Exchange (ETDEWEB)

Kruser, Tim J. [Department of Human Oncology, University of Wisconsin School of Medicine and Public Health, Madison, WI (United States); Wheeler, Deric L., E-mail: dlwheeler@wisc.edu [Department of Human Oncology, University of Wisconsin School of Medicine and Public Health, Madison, WI (United States)

2010-04-15

The epidermal growth factor (EGF) family of receptor tyrosine kinases consists of four members: EGFR (HER1/ErbB1), HER2/neu (ErbB2), HER3 (ErbB3) and HER4 (ErbB4). Receptor activation via ligand binding leads to downstream signaling that influence cell proliferation, angiogenesis, invasion and metastasis. Aberrant expression or activity of EGFR and HER2 have been strongly linked to the etiology of several human epithelial cancers including but not limited to head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC), colorectal cancer (CRC), and breast cancer. With this, intense efforts have been made to inhibit the activity of the EGFR and HER2 by designing antibodies against the ligand binding domains (cetuximab, panitumumab and trastuzumab) or small molecules against the tyrosine kinase domains (erlotinib, gefitinib, and lapatinib). Both approaches have shown considerable clinical promise. However, increasing evidence suggests that the majority of patients do not respond to these therapies, and those who show initial response ultimately become refractory to treatment. While mechanisms of resistance to tyrosine kinase inhibitors have been extensively studied, resistance to monoclonal antibodies is less well understood, both in the laboratory and in the clinical setting. In this review, we discuss resistance to antibody-based therapies against the EGFR and HER2, similarities between these resistance profiles, and strategies to overcome resistance to HER family targeting monoclonal antibody therapy.

An efficient method for isolating antibody fragments against small peptides by antibody phage display

DEFF Research Database (Denmark)

Duan, Zhi; Siegumfeldt, Henrik

2010-01-01

We generated monoclonal scFv (single chain variable fragment) antibodies from an antibody phage display library towards three small synthetic peptides derived from the sequence of s1-casein. Key difficulties for selection of scFv-phages against small peptides were addressed. Small peptides do....... The scFvs were sequenced and characterized, and specificity was characterized by ELISA. The methods developed in this study are universally applicable for antibody phage display to efficiently produce antibody fragments against small peptides....

Designer genes. Recombinant antibody fragments for biological imaging

Energy Technology Data Exchange (ETDEWEB)

Wu, A.M.; Yazaki, P.J. [Beckman Research Institute of the City of Hope, Duarte, CA (United States). Dept. of Molecular Biology

2000-09-01

Monoclonal antibodies (MAbs), with high specificity and high affinity for their target antigens, can be utilized for delivery of agents such as radionuclides, enzymes, drugs or toxins in vivo. However, the implementation of radiolabeled antibodies as magic bullets for detection and treatment of diseases such as cancer has required addressing several shortcomings of murine MAbs. These include their immunogenicity, sub-optimal targeting and pharmacokinetic properties, and practical issues of production and radiolabeling. Genetic engineering provides a powerful approach for redesigning antibodies for use in oncologic applications in vivo. Recombinant fragments have been produced that retain high affinity for target antigens, and display a combination of rapid, high-level tumor targeting with concomitant clearance from normal tissues and the circulation in animal models. An important first step was cloning and engineering of antibody heavy and light chain variable domains into single-chain Fvs (molecular weight, 25-17 kDa), in which the variable regions are joined via a synthetic linker peptide sequence. Although scFvs themselves showed limited tumor uptake in preclinical and clinical studies, they provide a useful building block for intermediate sized recombinant fragments. Covalently linked dimers or non-covalent dimers of scFvs (also known as diabodies) show improved targeting and clearance properties due to their higher molecular weight (55kDa) and increased avidity. Further gains can be made by generation of larger recombinant fragments, such as the minibody, an scFv-C{sub H}3 fusion protein that self-assembles into a bivalent dimer of 80 kDa. A systematic evaluation of scFv, diabody, minibody, and intact antibody (based on comparison of tumor uptakes, tumor: blood activity ratios, and calculation of an Imaging Figure of Merit) can form the basis for selection of combinations of recombinant fragments and radionuclides for imaging applications. Ease of engineering

Designer genes. Recombinant antibody fragments for biological imaging

International Nuclear Information System (INIS)

Wu, A.M.; Yazaki, P.J.

2000-01-01

Monoclonal antibodies (MAbs), with high specificy and high affinity for their target antigens, can be utilized for delivery of agents such as radionuclides, enzymes, drugs or toxins in vivo. However, the implementation of radiolabeled antibodies as magic bullets for detection and treatment of diseases such as cancer has required addressing several shortcomings of murine MAbs. These include their immunogenicity, sub-optimal targeting and pharmacokinetic properties, and practical issues of production and radiolabeling. Genetic engineering provides a powerful approach for redesigning antibodies for use in oncologic applications in vivo. Recombinant fragments have been produced that retain high affinity for target antigens, and display a combination of rapid, high-level tumor targeting with concomitant clearance from normal tissues and the circulation in animal models. An important first step was cloning and engineering of antibody heavy and light chain variable domains into single-chain Fvs (molecular weight, 25-17 kDa), in which the variable regions are joined via a synthetic linker peptide sequence. Although scFvs themselves showed limited tumor uptake in preclinical and clinical studies, they provide a useful building block for intermediate sized recombinant fragments. Covalently linked dimers or non-covalent dimers of scFvs (also known as diabodies) show improved targeting and clearance properties due to their higher molecular weight (55kDa) and increased avidity. Further gains can be made by generation of larger recombinant fragments, such as the minibody, an scFv-C H 3 fusion protein that self-assembles into a bivalent dimer of 80 kDa. A systematic evaluation of scFv, diabody, minibody, and intact antibody (based on comparison of tumor uptakes, tumor: blood activity ratios, and calculation of an Imaging Figure of Merit) can form the basis for selection of combinations of recombinant fragments and radionuclides for imaging applications. Ease of engineering and

Immunogenicity of anti-tumor necrosis factor antibodies-toward improved methods of anti-antibody measurement.

Science.gov (United States)

Aarden, Lucien; Ruuls, Sigrid R; Wolbink, Gertjan

2008-08-01

To date, millions of people have been treated with therapeutic monoclonal antibodies (TmAbs) for various indications. It is becoming increasingly clear that TmAbs can be immunogenic, which may reduce efficacy or induce adverse effects. Over the years, the importance of antibody formation has been questioned and sometimes minimized, as few antibody responses to TmAbs (HACA or HAHA) were reported. However, the methods to detect and quantify such antibodies used in the past have been problematic. Only recently, methods have been developed that have adequate sensitivity and are not seriously disturbed by false-positive reactions caused by rheumatoid factors, natural antibodies to Fab or F(ab')2 fragments, or Fc interactions of IgG4. The large number of treated patients, in combination with these new assays, presents a unique opportunity to study the anti-antibody immune response in man, possibly allowing us to manipulate immunogenicity in the future.

Review on comparative efficacy of bevacizumab, panitumumab and cetuximab antibody therapy with combination of FOLFOX-4 in KRAS-mutated colorectal cancer patients.

Science.gov (United States)

Pathak, Surajit; S, Sushmitha; Banerjee, Antara; Marotta, Francesco; Gopinath, Madhumala; Murugesan, Ramachandran; Zhang, Hong; B, Bhavani; Girigoswami, Agnishwar; Sollano, Jose; Sun, Xiao-Feng

2018-01-26

Colorectal cancer, fourth leading form of cancer worldwide and is increasing in alarming rate in the developing countries. Treating colorectal cancer has become a big challenge worldwide and several antibody therapies such as bevacizumab, panitumumab and cetuximab are being used with limited success. Moreover, mutation in KRAS gene which is linked with the colorectal cancer initiation and progression further interferes with the antibody therapies. Considering median progression free survival and overall survival in account, this review focuses to identify the most efficient antibody therapy in combination with chemotherapy (FOLFOX-4) in KRAS mutated colorectal cancer patients. The bevacizumab plus FOLFOX-4 therapy shows about 9.3 months and 8.7 months of progression free survival for KRAS wild and mutant type, respectively. The overall survival is about 34.8 months for wild type whereas for the mutant it is inconclusive for the same therapy. In comparison, panitumumab results in better progression-free survival which is about (9.6 months) and overall survival is about (23.9 months) for the wild type KRAS and the overall survival is about 15.5 months for the mutant KRAS . Cetuximab plus FOLFOX-4 therapy shows about 7.7 months and 5.5 months of progression-free survival for wild type KRAS and mutant type, respectively. Thus, panitumumab shows significant improvement in overall survival rate for wild type KRAS , validating as a cost effective therapeutic for colorectal cancer therapy. This review depicts that panitumumab along with FOLFOX-4 has a higher response in colorectal cancer patients than the either of the two monoclonal antibodies plus FOLFOX-4.

Adaptive antibody diversification through N-linked glycosylation of the immunoglobulin variable region.

Science.gov (United States)

van de Bovenkamp, Fleur S; Derksen, Ninotska I L; Ooijevaar-de Heer, Pleuni; van Schie, Karin A; Kruithof, Simone; Berkowska, Magdalena A; van der Schoot, C Ellen; IJspeert, Hanna; van der Burg, Mirjam; Gils, Ann; Hafkenscheid, Lise; Toes, René E M; Rombouts, Yoann; Plomp, Rosina; Wuhrer, Manfred; van Ham, S Marieke; Vidarsson, Gestur; Rispens, Theo

2018-02-20

A hallmark of B-cell immunity is the generation of a diverse repertoire of antibodies from a limited set of germline V(D)J genes. This repertoire is usually defined in terms of amino acid composition. However, variable domains may also acquire N -linked glycans, a process conditional on the introduction of consensus amino acid motifs ( N -glycosylation sites) during somatic hypermutation. High levels of variable domain glycans have been associated with autoantibodies in rheumatoid arthritis, as well as certain follicular lymphomas. However, the role of these glycans in the humoral immune response remains poorly understood. Interestingly, studies have reported both positive and negative effects on antibody affinity. Our aim was to elucidate the role of variable domain glycans during antigen-specific antibody responses. By analyzing B-cell repertoires by next-generation sequencing, we demonstrate that N -glycosylation sites are introduced at positions in which glycans can affect antigen binding as a result of a specific clustering of progenitor glycosylation sites in the germline sequences of variable domain genes. By analyzing multiple human monoclonal and polyclonal (auto)antibody responses, we subsequently show that this process is subject to selection during antigen-specific antibody responses, skewed toward IgG4, and positively contributes to antigen binding. Together, these results highlight a physiological role for variable domain glycosylation as an additional layer of antibody diversification that modulates antigen binding.

Generalized Platform for Antibody Detection using the Antibody Catalyzed Water Oxidation Pathway

OpenAIRE

Welch, M. Elizabeth; Ritzert, Nicole L.; Chen, Hongjun; Smith, Norah L.; Tague, Michele E.; Xu, Youyong; Baird, Barbara A.; Abru?a, H?ctor D.; Ober, Christopher K.

2014-01-01

Infectious diseases, such as influenza, present a prominent global problem including the constant threat of pandemics that initiate in avian or other species and then pass to humans. We report a new sensor that can be specifically functionalized to detect antibodies associated with a wide range of infectious diseases in multiple species. This biosensor is based on electrochemical detection of hydrogen peroxide generated through the intrinsic catalytic activity of all antibodies: the antibody ...

Generation and characterization of a monoclonal antibody to the cytoplasmic tail of MUC16

DEFF Research Database (Denmark)

Gipson, Ilene K; Mandel, Ulla; Menon, Balaraj

2017-01-01

of the biological relevance of the C-terminal domain of MUC16 has been limited by lack of availability of monoclonal antibodies that recognize the native CT. Here, we report the development of a novel monoclonal antibody to the CT region of the molecule that recognizes native MUC16 and its enzymatically released CT...... for the disease and it is considered a promising target for immunotherapeutic intervention. Immunodetection of the mucin has to date been through antibodies that recognize its exceptionally large ectodomain. Similar to other membrane anchored mucins, MUC16 has a short cytoplasmic tail (CT), but studies...... region. The antibody is useful for immunoprecipitation of the released CT domain as demonstrated with the OVCAR3 ovarian cancer cell line and can be used for detailed cytolocalization in cells as well as in frozen sections of ocular surface and uterine epithelium....

Lichen planus, liver kidney microsomal (LKM1) antibodies and hepatitis C virus antibodies.

Science.gov (United States)

Divano, M C; Parodi, A; Rebora, A

1992-01-01

No anti-liver kidney microsomal (LKM1) antibodies were detected in 46 patients with LP, 16 of whom had also a chronic liver disease (CLD). In contrast, anti-hepatitis C virus (HCV) antibodies were found in 10% of patients with LP and in 50% of those with LP and CLD. Anti-HCV antibodies may be considered as a false-positive reaction in 56% of cases, especially when anti-LKM1 antibodies are present. Our findings do not support such a hypothesis, but suggest that CLD in LP patients is, at least in Italy, mostly a postviral chronic active hepatitis.

Spleen vagal denervation inhibits the production of antibodies to circulating antigens.

Directory of Open Access Journals (Sweden)

Ruud M Buijs

Full Text Available BACKGROUND: Recently the vagal output of the central nervous system has been shown to suppress the innate immune defense to pathogens. Here we investigated by anatomical and physiological techniques the communication of the brain with the spleen and provided evidence that the brain has the capacity to stimulate the production of antigen specific antibodies by its parasympathetic autonomic output. METHODOLOGY/PRINCIPAL FINDINGS: This conclusion was reached by successively demonstrating that: 1. The spleen receives not only sympathetic input but also parasympathetic input. 2. Intravenous trinitrophenyl-ovalbumin (TNP-OVA does not activate the brain and does not induce an immune response. 3. Intravenous TNP-OVA with an inducer of inflammation; lipopolysaccharide (LPS, activates the brain and induces TNP-specific IgM. 4. LPS activated neurons are in the same areas of the brain as those that provide parasympathetic autonomic information to the spleen, suggesting a feed back circuit between brain and immune system. Consequently we investigated the interaction of the brain with the spleen and observed that specific parasympathetic denervation but not sympathetic denervation of the spleen eliminates the LPS-induced antibody response to TNP-OVA. CONCLUSIONS/SIGNIFICANCE: These findings not only show that the brain can stimulate antibody production by its autonomic output, it also suggests that the power of LPS as adjuvant to stimulate antibody production may also depend on its capacity to activate the brain. The role of the autonomic nervous system in the stimulation of the adaptive immune response may explain why mood and sleep have an influence on antibody production.

[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.

Anti-idiotypic antibodies to poliovirus antibodies in commercial immunoglubulin preparations, human serum and milk.

NARCIS (Netherlands)

M. Hahn-Zoric; B. Carlsson; S. Jeansson; H.P. Ekre; A.D.M.E. Osterhaus (Albert); D. Roberton; L.A. Hanson

1993-01-01

textabstractOur previous studies have suggested that fetal antibody production can be induced by maternal antiidiotypic antibodies transferred to the fetus via the placenta. We tested commercial Ig, sera, and milk for the presence of anti-idiotypic antibodies to poliovirus type 1, using affinity

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

Science.gov (United States)

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

2016-09-22

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

Antibody-Induced Internalization of the Human Respiratory Syncytial Virus Fusion Protein.

Science.gov (United States)

Leemans, A; De Schryver, M; Van der Gucht, W; Heykers, A; Pintelon, I; Hotard, A L; Moore, M L; Melero, J A; McLellan, J S; Graham, B S; Broadbent, L; Power, U F; Caljon, G; Cos, P; Maes, L; Delputte, P

2017-07-15

Respiratory syncytial virus (RSV) infections remain a major cause of respiratory disease and hospitalizations among infants. Infection recurs frequently and establishes a weak and short-lived immunity. To date, RSV immunoprophylaxis and vaccine research is mainly focused on the RSV fusion (F) protein, but a vaccine remains elusive. The RSV F protein is a highly conserved surface glycoprotein and is the main target of neutralizing antibodies induced by natural infection. Here, we analyzed an internalization process of antigen-antibody complexes after binding of RSV-specific antibodies to RSV antigens expressed on the surface of infected cells. The RSV F protein and attachment (G) protein were found to be internalized in both infected and transfected cells after the addition of either RSV-specific polyclonal antibodies (PAbs) or RSV glycoprotein-specific monoclonal antibodies (MAbs), as determined by indirect immunofluorescence staining and flow-cytometric analysis. Internalization experiments with different cell lines, well-differentiated primary bronchial epithelial cells (WD-PBECs), and RSV isolates suggest that antibody internalization can be considered a general feature of RSV. More specifically for RSV F, the mechanism of internalization was shown to be clathrin dependent. All RSV F-targeted MAbs tested, regardless of their epitopes, induced internalization of RSV F. No differences could be observed between the different MAbs, indicating that RSV F internalization was epitope independent. Since this process can be either antiviral, by affecting virus assembly and production, or beneficial for the virus, by limiting the efficacy of antibodies and effector mechanism, further research is required to determine the extent to which this occurs in vivo and how this might impact RSV replication. IMPORTANCE Current research into the development of new immunoprophylaxis and vaccines is mainly focused on the RSV F protein since, among others, RSV F-specific antibodies are

25-years three-fluid modeling-experience: successes and limits

International Nuclear Information System (INIS)

Kolev, N.I.

2004-01-01

The paper presents many examples of successful use of the three fluid processes modeling in 1D-networks, 3D-single volumes and 3D-boundary fitted volumes in the nuclear engineering. Fig. 1 shows 2700K hot molten aluminum oxide dropped in water and 3000K uranium oxide dropped in water as a demonstration for mathematical modeling of very complex real processes. (author)

9 CFR 113.452 - Erysipelothrix Rhusiopathiae Antibody.

Science.gov (United States)

2010-01-01

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

Future of antibody purification.

Science.gov (United States)

Low, Duncan; O'Leary, Rhona; Pujar, Narahari S

2007-03-15

Antibody purification seems to be safely ensconced in a platform, now well-established by way of multiple commercialized antibody processes. However, natural evolution compels us to peer into the future. This is driven not only by a large, projected increase in the number of antibody therapies, but also by dramatic improvements in upstream productivity, and process economics. Although disruptive technologies have yet escaped downstream processes, evolution of the so-called platform is already evident in antibody processes in late-stage development. Here we perform a wide survey of technologies that are competing to be part of that platform, and provide our [inherently dangerous] assessment of those that have the most promise.

Broad antibody mediated cross-neutralization and preclinical immunogenicity of new codon-optimized HIV-1 clade CRF02_AG and G primary isolates.

Directory of Open Access Journals (Sweden)

Simon M Agwale

Full Text Available Creation of an effective vaccine for HIV has been an elusive goal of the scientific community for almost 30 years. Neutralizing antibodies are assumed to be pivotal to the success of a prophylactic vaccine but previous attempts to make an immunogen capable of generating neutralizing antibodies to primary "street strain" isolates have resulted in responses of very limited breadth and potency. The objective of the study was to determine the breadth and strength of neutralizing antibodies against autologous and heterologous primary isolates in a cohort of HIV-1 infected Nigerians and to characterize envelopes from subjects with particularly broad or strong immune responses for possible use as vaccine candidates in regions predominated by HIV-1 CRF02_AG and G subtypes. Envelope vectors from a panel of primary Nigerian isolates were constructed and tested with plasma/sera from the same cohort using the PhenoSense HIV neutralizing antibody assay (Monogram Biosciences Inc, USA to assess the breadth and potency of neutralizing antibodies. The immediate goal of this study was realized by the recognition of three broadly cross-neutralizing sera: (NG2-clade CRF02_AG, NG3-clade CRF02_AG and NG9- clade G. Based on these findings, envelope gp140 sequences from NG2 and NG9, complemented with a gag sequence (Clade G and consensus tat (CRF02_AG and G antigens have been codon-optimized, synthesized, cloned and evaluated in BALB/c mice. The intramuscular administration of these plasmid DNA constructs, followed by two booster DNA immunizations, induced substantial specific humoral response against all constructs and strong cellular responses against the gag and tat constructs. These preclinical findings provide a framework for the design of candidate vaccine for use in regions where the HIV-1 epidemic is driven by clades CRF02_AG and G.

Broad antibody mediated cross-neutralization and preclinical immunogenicity of new codon-optimized HIV-1 clade CRF02_AG and G primary isolates.

Science.gov (United States)

Agwale, Simon M; Forbi, Joseph C; Notka, Frank; Wrin, Terri; Wild, Jens; Wagner, Ralf; Wolf, Hans

2011-01-01

Creation of an effective vaccine for HIV has been an elusive goal of the scientific community for almost 30 years. Neutralizing antibodies are assumed to be pivotal to the success of a prophylactic vaccine but previous attempts to make an immunogen capable of generating neutralizing antibodies to primary "street strain" isolates have resulted in responses of very limited breadth and potency. The objective of the study was to determine the breadth and strength of neutralizing antibodies against autologous and heterologous primary isolates in a cohort of HIV-1 infected Nigerians and to characterize envelopes from subjects with particularly broad or strong immune responses for possible use as vaccine candidates in regions predominated by HIV-1 CRF02_AG and G subtypes. Envelope vectors from a panel of primary Nigerian isolates were constructed and tested with plasma/sera from the same cohort using the PhenoSense HIV neutralizing antibody assay (Monogram Biosciences Inc, USA) to assess the breadth and potency of neutralizing antibodies. The immediate goal of this study was realized by the recognition of three broadly cross-neutralizing sera: (NG2-clade CRF02_AG, NG3-clade CRF02_AG and NG9- clade G). Based on these findings, envelope gp140 sequences from NG2 and NG9, complemented with a gag sequence (Clade G) and consensus tat (CRF02_AG and G) antigens have been codon-optimized, synthesized, cloned and evaluated in BALB/c mice. The intramuscular administration of these plasmid DNA constructs, followed by two booster DNA immunizations, induced substantial specific humoral response against all constructs and strong cellular responses against the gag and tat constructs. These preclinical findings provide a framework for the design of candidate vaccine for use in regions where the HIV-1 epidemic is driven by clades CRF02_AG and G.

Immunosuppressive T-cell antibody induction for heart transplant recipients

DEFF Research Database (Denmark)

Penninga, Luit; Møller, Christian H; Gustafsson, Finn

2013-01-01

Heart transplantation has become a valuable and well-accepted treatment option for end-stage heart failure. Rejection of the transplanted heart by the recipient's body is a risk to the success of the procedure, and life-long immunosuppression is necessary to avoid this. Clear evidence is required...... to identify the best, safest and most effective immunosuppressive treatment strategy for heart transplant recipients. To date, there is no consensus on the use of immunosuppressive antibodies against T-cells for induction after heart transplantation....

Echinococcus granulosus: the potential use of specific radiolabelled antibodies in diagnosis by immunoscintigraphy

Energy Technology Data Exchange (ETDEWEB)

Rogan, M.T.; Morris, D.L.; Pritchard, D.I.; Perkins, A.C. (Nottingham Univ. (UK))

1990-05-01

Diagnosis of hydatid disease in man is frequently dependent on the imaging of cysts in situ by techniques such as ultrasonography and CAT scans. Such methods are useful but are not specific and can lead to errors in diagnosis. The present work reports preliminary experiments on the development of a specific imaging technique for hydatid cysts using radiolabelled antibodies. A purified preparation of antigen B of hydatid fluid was used to raise polyclonal antisera in rabbits and the resulting affinity-purified IgG labelled with {sup 131}I. Gerbils with an established Echinococcus granulosus infection were injected intraperitoneally with the labelled antibody and imaged 48 h later with a gamma camera. Hydatid cysts could be identified within the peritoneal cavity and post-mortem assessment of activity showed the cysts to contain approximately four times as much activity as the surrounding organs thereby indicating successful targeting of the antibody to the cysts. (author).

[Screening serum response special antibodies of U251 cell line from surface display phage antibody library].

Science.gov (United States)

Yu, Min; Tan, De-Yong; Qian, Wei; Lai, Jian-Hua; Sun, Gui-Lin

2004-05-01

U251 cell is a sensitive cell line to serum, which stops at G0 phase of cell cycle in no-serum medium, and recovers growth when the serum is added into no-serum medium. The cell can express corresponding proteins in different phase of cell cycle. Therefore it is very signification for the study of cell cycle regulation mechanism that explores these proteins. In this paper, the mouse antibody phage display library was added into the bottle in which the serum starvation U251 cells had been cultured, and the special antibody phages were absorbed. Then the absorbed antibody phages were amplified by adding E. coli TG1 and helper phage M13K07. Amplified antibody phages were added into bottle in which the serum cultured cell after serum starvation (follow named as serum recovered cells) were incubated, so that the cell absorbed the no-special antibody phages for the serum starvation cell and the special antibody phages were in supernatant. The remaining no-special antibody phages in the supernatant were discarded by repeating above program 3-4 times. The pure special antibody phages were gotten, and amplified by adding the host cell E. coli TG1 and helper phage M13K07. Then the host bacterium infected special antibody phage was spread on the plate medium with ampicillin, and the monoclonal antibody phages were gotten. Using same as above program, the monoclonal antibody phages absorbed specially for serum recovered U251 cells were obtained when the serum recovered cells instead of serum starvation cells and serum starvation cells instead of serum recovered cells. In this study, ninety-six positive monoclonal antibody phages that absorbed specially the serum starvation cells and eighty-two positive monoclonal antibody phages that absorbed specially the serum recovered cells were obtained. By using cell immunochemistry assay, two special signification antibodies were obtained. one (No.11) was the strong response in serum starvation cells, the other (No.2) was the strong

Kinetic analysis of a monoclonal therapeutic antibody and its single-chain homolog by surface plasmon resonance.

Science.gov (United States)

Patel, Rekha; Andrien, Bruce A

2010-01-01

Monoclonal antibodies (mAbs) and antibody fragments have become an emerging class of therapeutics since 1986. Their versatility enables them to be engineered for optimal efficiency and decreased immunogenicity, and the path to market has been set by recent regulatory approvals. One of the initial criteria for success of any protein or antibody therapeutic is to understand its binding characteristics to the target antigen. Surface plasmon resonance (SPR) has been widely used and is an important tool for ligand-antigen binding characterization. In this work, the binding kinetics of a recombinant mAb and its single-chain antibody homolog, single-chain variable fragment (scFv), was analyzed by SPR. These two proteins target the same antigen. The binding kinetics of the mAb (bivalent antibody) and scFv (monovalent scFv) for this antigen was analyzed along with an assessment of the thermodynamics of the binding interactions. Alternative binding configurations were investigated to evaluate potential experimental bias because theoretically experimental binding configuration should have no impact on binding kinetics. Self-association binding kinetics in the proteins' respective formulation solutions and antigen epitope mapping were also evaluated. Functional characterization of monoclonal and single-chain antibodies has become just as important as structural characterization in the biotechnology field.