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Sample records for nanoparticle monoclonal antibody

  1. Monoclonal Antibodies.

    ERIC Educational Resources Information Center

    Killington, R. A.; Powell, K. L.

    1984-01-01

    Monoclonal antibodies have provided an exciting addition to the "armory" of the molecular biologist and immunologist. This article discusses briefly the concept of, techniques available for, production of, and possible uses of monoclonal antibodies. (Author)

  2. Monoclonal antibodies.

    PubMed

    2009-01-01

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

  3. Magnetic nanoparticle based purification and enzyme-linked immunosorbent assay using monoclonal antibody against enrofloxacin.

    PubMed

    Kim, Nam-Gun; Kim, Myeong-Ae; Park, Young-Il; Jung, Tae-Sung; Son, Seong-Wan; So, ByungJae; Kang, Hwan-Goo

    2015-01-01

    Monoclonal anti-enrofloxacin antibody was prepared for a direct competitive enzyme-linked immunosorbent assay (ELISA) and purification system using monoclonal antibody (mAb) coupled magnetic nanoparticles (MNPs). The IC50 values of the developed mAb for enrofloxacin (ENR), ciprofloxacin, difloxacin, sarafloxacin, pefloxacin, and norfloxacin were 5.0, 8.3, 9.7, 21.7, 36.0, and 63.7 ng/mL, respectively. The lowest detectable level of ENR was 0.7 ng/mL in the prepared ELISA system. To validate the developed ELISA in the food matrix, known amounts of ENR were spiked in meat and egg samples at 10, 20 and 30 ng/mL. Recoveries for ENR ranged from 72.9 to 113.16% with a coefficient of variation (CV) of 2.42 to 10.11%. The applicability of the mAb-MNP system was verified by testing the recoveries for ENR residue in three different matrices. Recoveries for ENR ranged from 75.16 to 86.36%, while the CV ranged from 5.08 to 11.53%. Overall, ENR-specific monoclonal antibody was prepared and developed for use in competitive to ELISAs for the detection of ENR in animal meat samples. Furthermore, we suggest that a purification system for ENR using mAb-coupled MNPs could be useful for determination of ENR residue in food. PMID:26040610

  4. Magnetic nanoparticle based purification and enzyme-linked immunosorbent assay using monoclonal antibody against enrofloxacin

    PubMed Central

    Kim, Nam-Gun; Kim, Myeong-Ae; Park, Young-Il; Jung, Tae-Sung; Son, Seong-Wan; So, ByungJae

    2015-01-01

    Monoclonal anti-enrofloxacin antibody was prepared for a direct competitive enzyme-linked immunosorbent assay (ELISA) and purification system using monoclonal antibody (mAb) coupled magnetic nanoparticles (MNPs). The IC50 values of the developed mAb for enrofloxacin (ENR), ciprofloxacin, difloxacin, sarafloxacin, pefloxacin, and norfloxacin were 5.0, 8.3, 9.7, 21.7, 36.0, and 63.7 ng/mL, respectively. The lowest detectable level of ENR was 0.7 ng/mL in the prepared ELISA system. To validate the developed ELISA in the food matrix, known amounts of ENR were spiked in meat and egg samples at 10, 20 and 30 ng/mL. Recoveries for ENR ranged from 72.9 to 113.16% with a coefficient of variation (CV) of 2.42 to 10.11%. The applicability of the mAb-MNP system was verified by testing the recoveries for ENR residue in three different matrices. Recoveries for ENR ranged from 75.16 to 86.36%, while the CV ranged from 5.08 to 11.53%. Overall, ENR-specific monoclonal antibody was prepared and developed for use in competitive to ELISAs for the detection of ENR in animal meat samples. Furthermore, we suggest that a purification system for ENR using mAb-coupled MNPs could be useful for determination of ENR residue in food. PMID:26040610

  5. Therapeutic Recombinant Monoclonal Antibodies

    ERIC Educational Resources Information Center

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  6. Monoclonal antibody "gold rush".

    PubMed

    Maggon, Krishan

    2007-01-01

    The market, sales and regulatory approval of new human medicines, during the past few years, indicates increasing number and share of new biologics and emergence of new multibillion dollar molecules. The global sale of monoclonal antibodies in 2006 were $20.6 billion. Remicade had annual sales gain of $1 billion during the past 3 years and five brands had similar increase in 2006. Rituxan with 2006 sales of $4.7 billion was the best selling monoclonal antibody and biological product and the 6th among the top selling medicinal brand. It may be the first biologic and monoclonal antibody to reach $10 billion annual sales in the near future. The strong demand from cancer and arthritis patients has surpassed almost all commercial market research reports and sales forecast. Seven monoclonal antibody brands in 2006 had sales exceeding $1 billion. Humanized or fully human monoclonal antibodies with low immunogenicity, enhanced antigen binding and reduced cellular toxicity provide better clinical efficacy. The higher technical and clinical success rate, overcoming of technical hurdles in large scale manufacturing, low cost of market entry and IND filing, use of fully human and humanized monoclonal antibodies has attracted funds and resources towards R&D. Review of industry research pipeline and sales data during the past 3 years indicate a real paradigm shift in industrial R&D from pharmaceutical to biologics and monoclonal antibodies. The antibody bandwagon has been joined by 200 companies with hundreds of new projects and targets and has attracted billions of dollars in R&D investment, acquisitions and licensing deals leading to the current Monoclonal Antibody Gold Rush. PMID:17691940

  7. Monoclonal antibodies and cancer therapy

    SciTech Connect

    Reisfeld, R.A.; Sell, S.

    1985-01-01

    These proceedings collect papers on the subject of monoclonal antibodies. Topics include: Monoclonal antibody, biochemical effects and cancer therapeutic potential of tunicamycin, use of monoclonal antibodies for detection of lymph node metastases, active specific immunotherapy, and applications of monoclonal antibodies to investigations of growth factors.

  8. PEGylated gold nanoparticles conjugated to monoclonal F19 antibodies as targeted labeling agents for human pancreatic carcinoma tissue.

    PubMed

    Eck, Wolfgang; Craig, Gary; Sigdel, Aruna; Ritter, Gerd; Old, Lloyd J; Tang, Laura; Brennan, Murray F; Allen, Peter J; Mason, Michael D

    2008-11-25

    In this study, we describe optical detection of antibody-conjugated nanoparticles bound to surgically resected human pancreatic cancer tissue. Gold nanoparticles stabilized by heterobifunctional polyethylene glycol (PEG) were prepared using approximately 15 nm spherical gold cores and covalently coupled to F19 monoclonal antibodies. The heterobifunctional PEG ligands contain a dithiol group for stable anchoring onto the gold surface and a terminal carboxy group for coupling of antibodies to the outside of the PEG shell. The nanoparticle-antibody bioconjugates form highly stable dispersions and exhibit long-term resistance to agglomeration. This has been demonstrated by dynamic light scattering, size exclusion chromatography, and transmission electron microscopy. The nanoparticle bioconjugates were used to label tumor stroma in approximately 5 mum thick sections of resected human pancreatic adenocarcinoma. After rinsing away nonbound nanoparticles and fixation, the tissue samples were imaged by darkfield microscopy near the nanoparticle resonance scattering maximum (approximately 560 nm). The images display pronounced tissue features and suggest that this novel labeling method could provide for facile identification of cancer tissue. Tumor samples treated with gold nanoparticles conjugated to nonspecific control antibodies and noncancerous pancreatic tissue treated with mAb-F19-conjugated gold nanoparticles both exhibited correctly negative results and showed no tissue staining. PMID:19206392

  9. Monoclonal Antibodies against Pectin

    PubMed Central

    Liners, Françoise; Letesson, Jean-Jacques; Didembourg, Christian; Van Cutsem, Pierre

    1989-01-01

    Monoclonal antibodies have been produced that recognize a conformation of homopolygalacturonic acid (pectic acid) induced by an optimum concentration of calcium and sodium of about 1 and 150 millinormal, respectively. The epitope recognized is probably part of the dimers of pectin chains associated according to the `egg box' model. Images Figure 2 PMID:16667195

  10. Monoclonal antibodies to gonadotropin subunits

    SciTech Connect

    Ehrlich, P.H.; Moyle, W.R.; Canfield, R.E.

    1985-01-01

    The production of monoclonal antibodies to peptide hormones, with their unifocal binding sites, can provide tools for understanding hormone structure and function. The paper focuses on techniques that are important for the study of monoclonal antibodies to chorionic gonadotropin (hCG), including hybridoma production, methods of screening for desired clones, properties of the monoclonal antibodies, effect of antibodies on hormone-receptor interaction, inhibition of binding of radiolabeled hCG, inhibition of hCG induced steroidogenesis, determination of relative orientation of epitopes, and synergistic actions of monoclonal antibodies to hCG.

  11. [Targeted therapy by monoclonal antibodies].

    PubMed

    Ohnuma, Kei; Morimoto, Chikao

    2010-10-01

    Human monoclonal antibodies are virtually indispensable for immunotherapy of cancer, infectious diseases, autoimmune diseases, or organ transplantation. The hybridoma technique, developed by Georges Köhler and César Milstein in 1975, has been shown to be most and highly producible method for generating murine monoclonal antibodies. However, poor results were obtained when it was administered in human bodies. With development of biotechnology, human monoclonal antibodies have been manufactured with higher efficiency. A major hindrance of producing therapeutic human monoclonal antibodies is the lack of an appropriate strategy for determining and selecting the antibodies that would be effective in vivo. In this review, we give an overview of the present techniques on therapeutic monoclonal antibodies. PMID:20954327

  12. Production of monoclonal antibodies.

    PubMed

    Freysd'ottir, J

    2000-01-01

    The discovery of monoclonal antibodies (mAbs) produced by "hybridoma technology" by George Köhler and Cesar Milstein in 1975 has had a great impact both on basic biological research and on clinical medicine. However, this impact was not immediately recognized. It took around 10 years to appreciate the importance of using these mAbs in various fields of science other than immunology, such as cell biology, biochemistry, microbiology, virology, para-sitology, physiology, genetics, and molecular biology; and also in areas of clinical medicine, such as pathology, hematology, oncology, and infectious disease. The contribution of mAbs to science and clinical medicine was recognized in 1984 by the award of the Nobel Prize for Medicine to Köhler and Milstein. PMID:21337095

  13. Monoclonal antibodies in myeloma.

    PubMed

    Sondergeld, Pia; van de Donk, Niels W C J; Richardson, Paul G; Plesner, Torben

    2015-09-01

    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 addition to the therapeutic armamentarium. The incorporation of mAbs into current treatment strategies is hoped to enable more effective and targeted treatment, resulting in improved outcomes for patients. A number of targets have been identified, including molecules on the surface of the myeloma cell and components of the bone marrow microenvironment. Our review focuses on a small number of promising mAbs directed against molecules on the surface of myeloma cells, including CS1 (elotuzumab), CD38 (daratumumab, SAR650984, MOR03087), CD56 (lorvotuzumab mertansine), and CD138/syndecan-1 (BT062/indatuximab ravtansine). PMID:26452191

  14. Antibodies and Selection of Monoclonal Antibodies.

    PubMed

    Hanack, Katja; Messerschmidt, Katrin; Listek, Martin

    2016-01-01

    Monoclonal antibodies are universal binding molecules with a high specificity for their target and are indispensable tools in research, diagnostics and therapy. The biotechnological generation of monoclonal antibodies was enabled by the hybridoma technology published in 1975 by Köhler and Milstein. Today monoclonal antibodies are used in a variety of applications as flow cytometry, magnetic cell sorting, immunoassays or therapeutic approaches. First step of the generation process is the immunization of the organism with appropriate antigen. After a positive immune response the spleen cells are isolated and fused with myeloma cells in order to generate stable, long-living antibody-producing cell lines - hybridoma cells. In the subsequent identification step the culture supernatants of all hybridoma cells are screened weekly for the production of the antibody of interest. Hybridoma cells producing the antibody of interest are cloned by limited dilution till a monoclonal hybridoma is found. This is a very time-consuming and laborious process and therefore different selection strategies were developed since 1975 in order to facilitate the generation of monoclonal antibodies. Apart from common automation of pipetting processes and ELISA testing there are some promising approaches to select the right monoclonal antibody very early in the process to reduce time and effort of the generation. In this chapter different selection strategies for antibody-producing hybridoma cells are presented and analysed regarding to their benefits compared to conventional limited dilution technology. PMID:27236550

  15. Monoclonal antibodies and neuroblastoma

    SciTech Connect

    Miraldi, F. )

    1989-10-01

    Several antineuroblastoma monoclonal antibodies (MoAbs) have been described and two have been used in radioimmunoimaging and radioimmunotherapy in patients. MoAb 3F8 is a murine IgG3 antibody specific for the ganglioside GD2. Radioiodine-labeled 3F8 has been shown to specifically target human neuroblastoma in patients, and radioimmunoimaging with this agent has provided consistently high uptakes with tumor-to-background ratios of greater than or equal to 10:1. Radioimmunotherapy has been attempted with both MoAb 3F8 and MoAb UJ13A, and although encouraging results have been obtained, dosimetry data and tissue dose response information for these agents is lacking, which impedes the development of such therapy. 124I, a positron emitter, can be used with 3F8 in positron emission tomography (PET) scanning to provide dosimetry information for radioimmunotherapy. The tumor radiation dose response from radiolabeled MoAb also can be followed with PET images with fluorodeoxyglucose (FDG) scanning of neuroblastoma tumors. Results to date indicate that radioimmunoimaging has clinical use in the diagnosis of neuroblastoma and the potential for radioimmunotherapy for this cancer remains high.48 references.

  16. The therapeutic monoclonal antibody market

    PubMed Central

    Ecker, Dawn M; Jones, Susan Dana; Levine, Howard L

    2015-01-01

    Since the commercialization of the first therapeutic monoclonal antibody product in 1986, this class of biopharmaceutical products has grown significantly so that, as of November 10, 2014, forty-seven monoclonal antibody products have been approved in the US or Europe for the treatment of a variety of diseases, and many of these products have also been approved for other global markets. At the current approval rate of ∼ four new products per year, ∼70 monoclonal antibody products will be on the market by 2020, and combined world-wide sales will be nearly $125 billion. PMID:25529996

  17. Immunotoxicity of monoclonal antibodies

    PubMed Central

    2009-01-01

    Monoclonal antibodies (mAbs) are large molecules intended to bind to specific targets often expressed on the immune system, and to treat various immunopathological conditions. Therefore, mAbs can be considered to have a high potential for immunotoxicity, which is reflected in the clinical experience accumulated on mAbs-induced adverse effects related to immunosuppression, immunostimulation and hypersensitivity (immunogenicity). So far, non clinical immunotoxicity studies have been inadequate to address all safety issues in relation to the possible immunotoxicity of mAbs, because they are fraught with limitations and pitfalls primarily related to the lack of relevant animal species. In addition, clinical studies rarely include validated end-points dedicated to the prediction of immunotoxicity. With the ongoing development of mAbs as novel therapeutic strategies for a wide variety of diseases, efforts should be paid to improve our understanding of mAbs-induced immunotoxic effects and design dedicated strategies to assess their immunological safety, both non clinically and clinically. PMID:20061816

  18. Monoclonal Antibody Interactions with Micro- and Nanoparticles: Adsorption, Aggregation and Accelerated Stress Studies

    PubMed Central

    Bee, Jared S.; Chiu, David; Sawicki, Suzanne; Stevenson, Jennifer L.; Chatterjee, Koustuv; Freund, Erwin; Carpenter, John F.; Randolph, Theodore W.

    2009-01-01

    Therapeutic proteins are exposed to various wetted surfaces that could shed sub-visible particles. In this work we measured the adsorption of a monoclonal antibody (mAb) to various microparticles, characterized the adsorbed mAb secondary structure, and determined the reversibility of adsorption. We also developed and used a front-face fluorescence quenching method to determine that the mAb tertiary structure was near-native when adsorbed to glass, cellulose and silica. Initial adsorption to each of the materials tested was rapid. During incubation studies, exposure to the air-water interface was a significant cause of aggregation but acted independently of the effects of microparticles. Incubations with glass, cellulose, stainless steel or Fe2O3 microparticles gave very different results. Cellulose preferentially adsorbed aggregates from solution. Glass and Fe2O3 adsorbed the mAb but did not cause aggregation. Adsorption to stainless steel microparticles was irreversible, and caused appearance of soluble aggregates upon incubation. The secondary structure of mAb adsorbed to glass and cellulose was near-native. We suggest that the protocol described in this work could be a useful preformulation stress screening tool to determine the sensitivity of a therapeutic protein to exposure to common surfaces encountered during processing and storage. PMID:19492408

  19. Uses of monoclonal antibody 8H9

    DOEpatents

    Cheung, Nai-Kong V.

    2013-04-09

    This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides different uses of the monoclonal antibody 8H9 or its derivative.

  20. Uses of monoclonal antibody 8H9

    DOEpatents

    Cheung, Nai-Kong V.

    2010-06-22

    This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides different uses of the monoclonal antibody 8H9 or its derivative.

  1. [Monoclonal antibody for cancer treatment].

    PubMed

    Achiwa, Hiroyuki; Sato, Shigeki; Ueda, Ryuzo

    2002-04-01

    Antibodies have for many decades been viewed as ideal molecules for cancer therapy. Although promising from the start, it has taken much of more than two decades to reach the level of clinical application. Genetic engineering of antibodies; that is novel technologies for chimeric or humanizing monoclonal antibodies, has greatly advanced their utility in molecular targeting therapies, and in the past four years some therapeutic monoclonal antibodies for hematologic malignancies and solid tumors, such as Rituximab for B-cell lymphoma and Trastuzumab for metastatic breast cancer, have provided sufficient efficacy and safety to support regulatory approval from the U.S. Food and Drug Administration. They were subsequently approved by the Japanese Ministry of Health, Labour and Welfare in 2001. Many molecular biological and immunological studies have revealed the targeting properties of the host immune system and the biological mechanism of cancer cells for a more specific anticancer effect. Many clinical trials of monoclonal antibodies as a single agent, or in combination protocol with current standard chemotherapy or immunoconjugates have shown promise in the treatment of specific diseases. Furthermore, novel antibody designs and improved understanding of the mode of action of current antibodies lend great hope to the future of this therapeutic approach. The accumulating results from many basic, clinical and translational studies may lead to more individualized therapeutic strategies using these agent directed at specific genetic and immunologic targets. PMID:11977531

  2. Detection of Campylobacter species using monoclonal antibodies

    NASA Astrophysics Data System (ADS)

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

    1999-01-01

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

  3. Monoclonal Antibodies for Cancer Immunotherapy

    PubMed Central

    Weiner, Louis M.; Dhodapkar, Madhav V.; Ferrone, Soldano

    2008-01-01

    Monoclonal antibodies have emerged as effective therapeutic agents for many human malignancies. However, the ability of antibodies to initiate tumor antigen-specific immune responses has not received as much attention as other mechanisms of antibody action. Here we describe the rationale and evidence for developing anti-cancer antibodies that can stimulate host tumor antigen-specific immune responses. This may be accomplished by inducing antibody-dependent cellular cytotoxicity, by promoting antibody-targeted cross-presentation of tumor antigens or by triggering the idiotypic network. Future treatment modifications or combinations should be able to prolong, amplify and shape these immune responses to increase the clinical benefits of antibody therapy of human cancer. PMID:19304016

  4. A monoclonal antibody against leptin.

    PubMed

    Mahmoudian, Jafar; Jeddi-Tehrani, Mahmood; Bayat, Ali Ahmad; Mahmoudi, Ahmad Reza; Vojgani, Yasaman; Tavangar, Banafsheh; Hadavi, Reza; Zarei, Saeed

    2012-10-01

    Leptin is an important protein that regulates energy storage and homeostasis in humans and animals. Leptin deficiency results in various abnormalities such as diabetes, obesity, and infertility. Producing a high affinity monoclonal antibody against human leptin provides an important tool to monitor and trace leptin function in different biological fluids. In this study, recombinant human leptin was conjugated to KLH and injected into mice. After immunization, mouse myeloma SP2/0 cells were fused with murine splenocytes followed by selection of antibody-producing hybridoma cells. After screening of different hybridoma colonies by ELISA, a high affinity antibody was selected and purified by affinity chromatography. The affinity constant of the antibody was measured by ELISA. Western blot, immunocytochemistry, and flow cytometry experiments were used to characterize the antibody. The anti-leptin antibody had a high affinity (around 1.13 × 10(-9) M) for its antigen. The saturation of the antibody with leptin (20 moles leptin per 1 mole antibody) in Western blot analysis proved that the antibody had specific binding to its antigen. Immunocytochemistry and flow cytometry on JEG-3 (human placental choriocarcinoma cell) cells revealed that the anti-leptin antibody recognized intracellular leptin. In conclusion, we report here the production and characterization of a murine anti-leptin antibody with high affinity for human leptin. PMID:23098305

  5. Advances in monoclonal antibody application in myocarditis*

    PubMed Central

    Han, Li-na; He, Shuang; Wang, Yu-tang; Yang, Li-ming; Liu, Si-yu; Zhang, Ting

    2013-01-01

    Monoclonal antibodies have become a part of daily preparation technologies in many laboratories. Attempts have been made to apply monoclonal antibodies to open a new train of thought for clinical treatments of autoimmune diseases, inflammatory diseases, cancer, and other immune-associated diseases. This paper is a prospective review to anticipate that monoclonal antibody application in the treatment of myocarditis, an inflammatory disease of the heart, could be a novel approach in the future. In order to better understand the current state of the art in monoclonal antibody techniques and advance applications in myocarditis, we, through a significant amount of literature research both domestic and abroad, developed a systematic elaboration of monoclonal antibodies, pathogenesis of myocarditis, and application of monoclonal antibodies in myocarditis. This paper presents review of the literature of some therapeutic aspects of monoclonal antibodies in myocarditis and dilated cardiomyopathy to demonstrate the advance of monoclonal antibody application in myocarditis and a strong anticipation that monoclonal antibody application may supply an effective therapeutic approach to relieve the severity of myocarditis in the future. Under conventional therapy, myocarditis is typically associated with congestive heart failure as a progressive outcome, indicating the need for alternative therapeutic strategies to improve long-term results. Reviewing some therapeutic aspects of monoclonal antibodies in myocarditis, we recently found that monoclonal antibodies with high purity and strong specificity can accurately act on target and achieve definite progress in the treatment of viral myocarditis in rat model and may meet the need above. However, several issues remain. The technology on how to make a higher homologous and weak immunogenic humanized or human source antibody and the treatment mechanism of monoclonal antibodies may provide solutions for these open issues. If we are to

  6. Assessment of the enhancement of PLGA nanoparticle uptake by dendritic cells through the addition of natural receptor ligands and monoclonal antibody.

    PubMed

    Walters, Adam A; Somavarapu, Satyanarayana; Riitho, Victor; Stewart, Graham R; Charleston, Bryan; Steinbach, Falko; Graham, Simon P

    2015-11-27

    Targeting of specific receptors on antigen-presenting cells is an appealing prospect in the production of novel nanoparticulate vaccines. In particular, the targeting of vaccines to dendritic cell (DC) subsets has been shown in models to significantly improve the induction of immune responses. This paper describes the evaluation of natural ligands, mannan and chitosan, and monoclonal antibodies as targeting motifs to enhance uptake of PLGA nanoparticle carriers by bovine DCs. To assess enhancement of uptake after the addition of natural ligands a bovine monocyte derived DC (MoDC) model was used. For the assessment of monoclonal antibody targeting, the model was expanded to include afferent lymph DCs (ALDCs) in a competitive uptake assay. Mannan, proved unsuccessful at enhancing uptake or targeting by MoDCs. Chitosan coated particle uptake could be impeded by the addition of mannan suggesting uptake may be mediated through sugar receptors. Inclusion of monoclonal antibodies specific for the DEC-205 (CD205) receptor increased the number of receptor expressing DCs associated with particles as well as the number of particles taken up by individual cells. These results support the further evaluation of active targeting of nanovaccines to DCs to enhance their immunogenicity in cattle and other large mammalian species including humans. PMID:26529067

  7. Natural monoclonal antibodies and cancer.

    PubMed

    Vollmers, Peter H; Brändlein, Stephanie

    2008-06-01

    Immunity is responsible for recognition and elimination of infectious particles and for removal of cellular waste, modified self structures and transformed cells. Innate or natural immunity acts as a first line defense and is also the link to acquired immunity and memory. By using the human hybridoma technology, a series of monoclonal antibodies and several new tumor-specific targets could be identified. A striking phenomenon of immunity against malignant cells is that all so far isolated tumor-specific antibodies were germ-line coded natural IgM antibodies. And neither in animals nor in humans affinity-maturated tumor-specific IgG antibodies have been detected so far. These IgM's preferentially bind to carbohydrate epitopes on post-transcriptionally modified surface receptors, which are recently patented and preferentially remove malignant cells by inducing apoptosis to avoid inflammatory processes. Our "biology-" or "function-driven" method represents a unique yet powerful approach compared to the typical approaches on screening compounds or antibodies against non-validated targets (mostly differentially expressed). Moreover, the approach creates a competitive patenting strategy of creating proprietary antibodies and validated targets at the same time, which has the potential of further streamlining the discovery of new cancer therapies. PMID:18537750

  8. Monoclonal antibody purification with hydroxyapatite.

    PubMed

    Gagnon, Pete

    2009-06-01

    Hydroxyapatite (HA) has been used for IgG purification since its introduction in the 1950s. Applications expanded to include IgA and IgM in the 1980s, along with elucidation of its primary binding mechanisms and the development of ceramic HA media. With the advent of recombinant monoclonal antibodies, HA was demonstrated to be effective for removal of antibody aggregates, as well as host cell proteins and leached protein A. HA's inherent abilities have been enhanced by the development of elution strategies that permit differential control of its primary binding mechanisms: calcium metal affinity and phosphoryl cation exchange. These strategies support reduction of antibody aggregate content from greater than 60% to less than 0.1%, in conjunction with enhanced removal of DNA, endotoxin, and virus. HA also has a history of discriminating various immunological constructs on the basis of differences in their variable regions, or discriminating Fab fragments from Fc contaminants in papain digests of purified monoclonal IgG. Continuing development of novel elution strategies, alternative forms of HA, and application of robotic high throughput screening systems promise to expand HA's utility in the field. PMID:19491046

  9. Monoclonal Antibodies for Lipid Management.

    PubMed

    Feinstein, Matthew J; Lloyd-Jones, Donald M

    2016-07-01

    In recent years, biochemical and genetic studies have identified proprotein convertase subtilisin/kexin type 9 (PCSK9) as a major mediator of low-density lipoprotein cholesterol (LDL-c) levels and thereby a potential novel target for reducing risk of coronary heart disease (CHD). These observations led to the development of PCSK9 inhibitors, which lower LDL-c levels more than any other non-invasive lipid-lowering therapy presently available. The PCSK9 inhibitors furthest along in clinical trials are subcutaneously injected monoclonal antibodies. These PCSK9 inhibitors have demonstrated LDL-c-lowering efficacy with acceptable safety in phase III clinical trials and may offer a useful therapy in addition to maximally tolerated HMG-CoA reductase inhibitors (statins) in certain patient groups. Longer-term data are required to ensure sustained efficacy and safety of this new class of medications. This review provides an overview of the biology, genetics, development, and clinical trials of monoclonal antibodies designed to inhibit PCSK9. PMID:27221501

  10. Uses of monoclonal antibody 8H9

    DOEpatents

    Cheung, Nai-Kong V

    2013-08-06

    This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides a method of inhibiting the growth of tumor cells comprising contacting said tumor cells with an appropriate amount of monoclonal antibody 8H9 or a derivative thereof.

  11. Uses of monoclonal antibody 8H9

    DOEpatents

    Cheung, Nai-Kong V.

    2010-06-15

    This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides a method of inhibiting the growth of tumor cells comprising contacting said tumor cells with an appropriate amount of monoclonal antibody 8H9 or a derivative thereof.

  12. Monoclonal antibodies against Vibrio cholerae lipopolysaccharide.

    PubMed Central

    Gustafsson, B; Rosén, A; Holme, T

    1982-01-01

    A cell line producing monoclonal antibodies directed against the core region of Vibrio cholerae lipopolysaccharide has been established. These antibodies were inhibited by lipopolysaccharide preparations of both O-group 1 vibrios and some non-O-group 1 vibrios as detected in enzyme-linked immunosorbent assay-inhibition experiments. Coagglutination experiments with monoclonal and polyclonal antibodies adsorbed to protein A-carrying staphylococci were performed. All V. cholerae strains tested, regardless of serotype, were agglutinated when mixed with staphylococci coated with the monoclonal antibodies, whereas staphylococci coated with group-specific (O1) polyclonal antibodies only agglutinated with O-group 1 vibrios. Images PMID:6183214

  13. Improved monoclonal antibodies to halodeoxyuridine

    DOEpatents

    Vanderlaan, M.; Dolbeare, F.A.; Gray, J.W.; Thomas, C.B.

    1983-10-18

    The development, method of production, characterization and methods of use of two hybridomas, CIdU-1 (ATCC Accession No. HB-8321) and CIdU-2 (ATCC Accession No. HB-8320), are described. These secrete IgG/sub 1/(K) immunoglobulins that react with halodeoxyuridine (HdU or halodU) such as bromo, chloro, fluoro and iodo deoxyuridine (BrdU, CldU, FdU and IdU), whether these are free in solution or incorporated into single stranded DNA in whole cells. The antibodies do not react with naturally occurring free nucleic acids or with deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) polymers. These antibodies are suitable for use in enzyme immunoassays for free CldU, FdU, IdU and BrdU and for detecting cells with these nucleotides incorporated into them. The monoclonal antibodies are useful in the detection of the sensitivity of tumor cells to specific chemotherapeutic agents, in the measurement of the rate of cellular DNA synthesis, in the measurement of the rate of proliferation of normal and malignant cells and in the detection of HPRT deficiency in cells. 1 tab.

  14. Monoclonal antibodies that detect live salmonellae.

    PubMed Central

    Torensma, R; Visser, M J; Aarsman, C J; Poppelier, M J; van Beurden, R; Fluit, A C; Verhoef, J

    1992-01-01

    Nine immunoglobulin G and nine immunoglobulin M murine monoclonal antibody-producing hybridomas reactive with live Salmonella bacteria were obtained from several fusions of immune spleen cells and Sp2/0 myeloma cells. The antibodies were selected by the magnetic immunoluminescence assay. The monoclonal antibodies were reactive with serogroups A, B, C1, C2, D, E, and K and Salmonella choleraesuis subsp. diarizonae. Each monoclonal antibody proved to be reactive with a distinct serotype. Clinical isolates belonging to these Salmonella serogroups could be detected. Reactivity with non-Salmonella bacteria proved to be minor. Images PMID:1476430

  15. Monoclonal Antibodies for the Treatment of Cancer

    PubMed Central

    Shuptrine, Casey; Surana, Rishi; Weiner, Louis M.

    2012-01-01

    Over the past decade, the clinical utility of monoclonal antibodies has been realized and antibodies are now a mainstay for the treatment of cancer. Antibodies have the unique capacity to target and kill tumor cells while simultaneously activating immune effectors to kill tumor cells through the complement cascade or antibody-dependent cellular cytotoxicity (ADCC). This multifaceted mechanism of action combined with target specificity underlies the capacity of antibodies to elicit anti-tumor responses while minimizing the frequency and magnitude of adverse events. This review will focus on mechanisms of action, clinical applications and putative mechanisms of resistance to monoclonal antibody therapy in the context of cancer. PMID:22245472

  16. Production of monoclonal antibodies against avidin.

    PubMed

    Ashorn, R; Ashorn, P; Kulomaa, M; Tuohimaa, P; Krohn, K

    1985-01-01

    Monoclonal antibodies of the IgG1 subclass were generated against chicken avidin. These antibodies were shown to be as sensitive as polyclonal antiserum in detecting avidin by radioimmunoassay (RIA) and enzyme linked immunosorbent assay (ELISA) methods. Furthermore, the monoclonal antibodies were considerably more specific. Our results with a monoclonal anti-avidin RIA support previous findings that in inflammatory conditions avidin is synthesized also in other organs than the oviduct, although in the liver a major part of the activity detected by polyclonal anti-avidin RIA or biotin-bentonite assay was not due to avidin. PMID:4053566

  17. Monoclonal antibodies in the treatment of cancer

    SciTech Connect

    Dillman, R.O.

    1984-01-01

    Potential uses of monoclonal antibodies in anti-cancer treatment include passive serotherapy, radioisotope conjugates, toxin-linked conjugates, and chemotherapy-monoclonal antibody conjugates. The bases for these applications have been founded in research with heterologous antisera, and in some cases with monoclonal antibodies in animal tumor models. Human trials with passive serotherapy have already begun in both hematopoietic and solid tumor malignancies. Promising results have been reported in cutaneous T cell lymphoma with anti-T cell monoclonal antibody, and in nodular lymphoma with anti-idiotype monoclonal antibody. Radioisotope conjugate work appears promising for imaging in both animals and humans, and this work will lay the foundation for possible therapeutic application of radio-immunotherapy. Toxin-linked conjugates are promising in vitro and may have application in autologous bone marrow transplantation. Research with chemotherapy conjugates is also underway. Preliminary results suggest that murine monoclonal antibodies will be well tolerated clinically except in the setting of circulating cells which bear the target antigen, where rapid infusions may be associated with intolerable side effects. In certain diseases, production of endogenous anti-mouse antibodies may also limit application. Advances in the technology for human-human hybridoma production may help solve some of these problems. 132 references.

  18. Preparation of astatine-labeled monoclonal antibodies

    SciTech Connect

    Milesz, S.; Norseev, Yu.V.; Szucs, Z. |

    1995-07-01

    In the cationic state astatine forms a stable complex with diethylenetriaminepentaacetic acid. Thanks to this complex, astatine can be bound to monoclonal antibodies of the RYa{sub 1} type. The most favorable conditions for preparing astatine-labeled antibodies are established. The chromatographic analysis and electromigration experiments showed that astatine is firmly linked to a biomolecule in vitro and it did not escape from labeled monoclonal antibodies even under treatment with such highly effective astatine-complexing agent as thiourea. The immune activity of astatine-labeled antibodies did not change even after 20 h.

  19. Monoclonal Antibody That Defines Human Myoepithelium

    NASA Astrophysics Data System (ADS)

    Dairkee, Shahnaz Hashmi; Blayney, Carlene; Smith, Helene S.; Hackett, Adeline J.

    1985-11-01

    We have isolated a mouse monoclonal antibody that, upon immunohistochemical localization in frozen sections, displays specificity for human myoepithelial cells in the resting mammary gland, sweat glands, and salivary glands. Furthermore, this antibody was strongly and homogeneously reactive with frozen sections of 3 of 60 breast carcinoma specimens. Using immunolocalization techniques in conjunction with polyacrylamide gel electrophoresis, we have determined that the reactivity of this monoclonal antibody is directed toward a 51,000-dalton keratin polypeptide. The potential uses of this antibody in the prognosis of human mammary carcinoma and in understanding the role of the myoepithelium in development and differentiation are discussed.

  20. Mouse monoclonal antibodies against estrogen receptor.

    PubMed

    De Rosa, Caterina; Rossi, Valentina; Abbondanza, Ciro

    2014-01-01

    The production of monoclonal antibodies, by cloning hybridoma derived from the fusion of myeloma cells and spleen lymphocytes, has allowed to obtain great advances in many fields of biological knowledge. The use of specific antibodies to the estrogen receptor, in fact, has been an invaluable method to bring out its mechanisms of action and its effects, both genomic and extra-genomic. Here we describe, step by step, the production of monoclonal antibodies, starting from protocol for antigen preparation to the selection of antibody-secreting hybridoma. PMID:25182770

  1. Polyclonal and monoclonal antibodies in clinic.

    PubMed

    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. PMID:24037837

  2. Trends in Malignant Glioma Monoclonal Antibody Therapy

    PubMed Central

    Chekhonin, Ivan; Gurina, Olga

    2015-01-01

    Although new passive and active immunotherapy methods are emerging, unconjugated monoclonal antibodies remain the only kind of biological preparations approved for high-grade glioma therapy in clinical practice. In this review, we combine clinical and experimental data discussion. As antiangiogenic therapy is the standard of care for recurrent glioblastoma multiforme (GBM), we analyze major clinical trials and possible therapeutic combinations of bevacizumab, the most common monoclonal antibody to vascular endothelial growth factor (VEGF). Another humanized antibody to gain recognition in GBM is epidermal growth factor (EGFR) antagonist nimotuzumab. Other antigens (VEGF receptor, platelet-derived growth factor receptor, hepatocyte growth factor and c-Met system) showed significance in gliomas and were used to create monoclonal antibodies applied in different malignant tumors. We assess the role of genetic markers (isocitrate dehydrogenase, O6-methylguanine-DNA methyltransnsferase) in GBM treatment outcome prediction. Besides antibodies studied in clinical trials, we focus on perspective targets and briefly list other means of passive immunotherapy.

  3. Development of a monoclonal antibody against ochratoxin A and its application in enzyme-linked immunosorbent assay and gold nanoparticle immunochromatographic strip.

    PubMed

    Liu, Biing-Hui; Tsao, Zih-Jay; Wang, Jing-Jhih; Yu, Feng-Yih

    2008-09-15

    A monoclonal antibody (mAb) specific to ochratoxin A (OTA) was produced from a stable hybridoma cell line, 9C9H9, generated by the fusion of P3/NS1/1-AG4-1 myeloma cells with spleen cells isolated from a BALB/c mouse immunized with OTA-keyhole limpet hemocyanin. The 9C9H9 mAb belongs to the immunoglobulin G1 (kappa chain) isotype. A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a competitive indirect ELISA were established for antibody characterization. The concentrations causing 50% inhibition of binding of OTA-horseradish peroxidase to the antibody by OTA, OTB, and OTC were found to be 0.32, 0.17, and 0.28 ng/mL, respectively, in the cdELISA. A sensitive and rapid mAb-based gold nanoparticle immunochromatographic strip was also developed using this mAb. This strip has a detection limit of 5 ng/mL for OTA and can be completed in 10 min. Analysis of OTA in coffee samples revealed that data obtained from immunochromatographic strip were in a good agreement with those obtained from cdELISA. The mAb-based cdELISA and immunochromatographic strip assay established in this study were sensitive and accurate for rapid screening of OTA in coffee samples. PMID:18698802

  4. Cold denaturation of monoclonal antibodies

    PubMed Central

    Lazar, Kristi L; Patapoff, Thomas W

    2010-01-01

    The susceptibility of monoclonal antibodies (mAbs) to undergo cold denaturation remains unexplored. In this study, the phenomenon of cold denaturation was investigated for a mAb, mAb1, through thermodynamic and spectroscopic analyses. tryptophan fluorescence and circular dichroism (CD) spectra were recorded for the guanidine hydrochloride (GuHCl)-induced unfolding of mAb1 at pH 6.3 at temperatures ranging from −5 to 50°C. A three-state unfolding model incorporating the linear extrapolation method was fit to the fluorescence data to obtain an apparent free energy of unfolding, ΔGu, at each temperature. CD studies revealed that mAb1 exhibited polyproline II helical structure at low temperatures and at high GuHCl concentrations. the Gibbs-Helmholtz expression fit to the ΔGu versus temperature data from fluorescence gave a ΔCp of 8.0 kcal mol−1 K−1, a maximum apparent stability of 23.7 kcal mol−1 at 18°C, and an apparent cold denaturation temperature (TCD) of −23°C. ΔGu values for another mAb (mAb2) with a similar framework exhibited less stability at low temperatures, suggesting a depressed protein stability curve and a higher relative TCD. Direct experimental evidence of the susceptibility of mAb1 and mAb2 to undergo cold denaturation in the absence of denaturant was confirmed at pH 2.5. thus, mAbs have a potential to undergo cold denaturation at storage temperatures near −20°C (pH 6.3), and this potential needs to be evaluated independently for individual mAbs. PMID:20093856

  5. Monoclonal Antibody Therapy for Advanced Neuroblastoma

    Cancer.gov

    NCI is sponsoring two clinical trials of a monoclonal antibody called ch14.18, in combination with other drugs, to see if the antibody may be helpful for children or young adults (up to age 21) with relapsed or refractory neuroblastoma.

  6. Monoclonal antibodies in acute lymphoblastic leukemia

    PubMed Central

    O’Brien, Susan; Ravandi, Farhad; Kantarjian, Hagop

    2015-01-01

    With modern intensive combination polychemotherapy, the complete response (CR) rate in adults with acute lymphoblastic leukemia (ALL) is 80% to 90%, and the cure rate is 40% to 50%. Hence, there is a need to develop effective salvage therapies and combine novel agents with standard effective chemotherapy. ALL leukemic cells express several surface antigens amenable to target therapies, including CD20, CD22, and CD19. Monoclonal antibodies target these leukemic surface antigens selectively and minimize off-target toxicity. When added to frontline chemotherapy, rituximab, an antibody directed against CD20, increases cure rates of adults with Burkitt leukemia from 40% to 80% and those with pre-B ALL from 35% to 50%. Inotuzumab ozogamicin, a CD22 monoclonal antibody bound to calicheamicin, has resulted in marrow CR rates of 55% and a median survival of 6 to 7 months when given to patients with refractory-relapsed ALL. Blinatumomab, a biallelic T cell engaging the CD3-CD19 monoclonal antibody, also resulted in overall response rates of 40% to 50% and a median survival of 6.5 months in a similar refractory-relapsed population. Other promising monoclonal antibodies targeting CD20 (ofatumumab and obinutuzumab) or CD19 or CD20 and bound to different cytotoxins or immunotoxins are under development. Combined modalities of chemotherapy and the novel monoclonal antibodies are under investigation. PMID:25999456

  7. Production of Monoclonal Antibody against Human Nestin.

    PubMed

    Hadavi, Reza; Zarnani, Amir Hassan; Ahmadvand, Negah; Mahmoudi, Ahmad Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Sadeghi, Mohammad-Reza; Soltanghoraee, Haleh; Akhondi, Mohammad Mehdi; Tarahomi, Majid; Jeddi-Tehrani, Mahmood; Rabbani, Hodjattallah

    2010-04-01

    We have employed a peptide-based antibody generation protocol for producing antibody against human nestin. Using a 12-mer synthetic peptide from repetitive region of human nestin protein devoid of any N- or O-glyco-sylation sequences, we generated a mouse monoclonal antibody capable of recognizing human, mouse, bovine, and rat nestin. A wide variety of nestin proteins ranging from 140-250 kDa was detected by this antibody. This antibody is highly specific and functional in applications such as ELISA, flow cytometry, immunocytochemistry, and Western blot assays. PMID:23407796

  8. Production of Monoclonal Antibody against Human Nestin

    PubMed Central

    Hadavi, Reza; Zarnani, Amir Hassan; Ahmadvand, Negah; Mahmoudi, Ahmad Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Sadeghi, Mohammad-Reza; Soltanghoraee, Haleh; Akhondi, Mohammad Mehdi; Tarahomi, Majid; Jeddi-Tehrani, Mahmood; Rabbani, Hodjattallah

    2010-01-01

    We have employed a peptide-based antibody generation protocol for producing antibody against human nestin. Using a 12-mer synthetic peptide from repetitive region of human nestin protein devoid of any N- or O-glyco-sylation sequences, we generated a mouse monoclonal antibody capable of recognizing human, mouse, bovine, and rat nestin. A wide variety of nestin proteins ranging from 140–250 kDa was detected by this antibody. This antibody is highly specific and functional in applications such as ELISA, flow cytometry, immunocytochemistry, and Western blot assays. PMID:23407796

  9. 5th Annual Monoclonal Antibodies Conference

    PubMed Central

    2009-01-01

    The conference, which was organized by Visiongain and held at the BSG Conference Center in London, provided an excellent opportunity for participants to exchange views on the development, production and marketing of therapeutic antibodies, and discuss the current business environment. The conference included numerous interactive panel and group discussions on topics such as isotyping for therapeutic antibodies (panel chair: Nick Pullen, Pfizer), prospects for fully human monoclonal antibodies (chair: Christian Rohlff, Oxford BioTherapeutics), perspectives on antibody manufacturing and development (chair: Bo Kara, Avecia), market impact and post-marketing issues (chair: Keith Rodgers, Bodiam Consulting) and angiogenesis inhibitors (chair: David Blakey, AstraZeneca). PMID:20073132

  10. [Current situations and the future prospect of monoclonal antibody products].

    PubMed

    Yamaguchi, Teruhide

    2014-01-01

    Monoclonal antibody products and monoclonal antibody-based biopharmaceuticals have shown considerable effectiveness in the treatment for variety of diseases; cancer, auto-immune/auto-inflammation diseases and so on. Significant advance in monoclonal antibody products for cancer treatments was made with antibody-drug conjugates (ADC), and antibodies for blockade of immune checkpoints. Already 3 ADCs and 2 anti-immune-checkpoint antibodies products have been approved, and these monoclonal antibody-related product pipelines reach over 30. On the other hand, EU approved first monoclonal-antibody biosimilar, RemsimaTM (infliximab), suggesting that other monoclonal-antibody biosmilars will follow to the market. In this paper, several new issues about monoclonal antibody products will be discussed. PMID:25707201

  11. Monoclonal antibodies reactive with chicken interleukin-17

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In our previous study chicken interleukin -17 (chIL-17) gene was cloned from the expressed sequence tag (EST) cDNA library and initially analyzed. To further investigate biological properties of chicken IL-17, six monoclonal antibodies (mAbs) against bacterially expressed protein were produced and c...

  12. Monoclonal antibody technologies and rapid detection assays

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Novel methodologies and screening strategies will be outlined on the use of hybridoma technology for the selection of antigen specific monoclonal antibodies. The development of immunoassays used for diagnostic detection of prions and bacterial toxins will be discussed and examples provided demonstr...

  13. Monoclonal antibodies against chicken interleukin-6

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Monoclonal antibodies (mAb) were produced against a recombinant (r) chicken interleukin-6 (IL-6). Eight mAbs that were produced were tested for isotype; ability to inhibit recombinant forms of chicken (ch), human (h) and murine (m) IL-6; and recognition of rchIL-6 by Western immunoblotting. The mA...

  14. Generation of a monoclonal antibody against Mycoplasma spp. following accidental contamination during production of a monoclonal antibody against Lawsonia intracellularis.

    PubMed

    Hwang, Jeong-Min; Lee, Ji-Hye; Yeh, Jung-Yong

    2012-03-01

    This report describes Mycoplasma contamination of Lawsonia intracellularis cultures that led to the unintended acquisition of a monoclonal antibody against Mycoplasma spp. during the attempted generation of a monoclonal antibody against L. intracellularis. PMID:22247145

  15. Phase Separation in Solutions of Monoclonal Antibodies

    NASA Astrophysics Data System (ADS)

    Benedek, George; Wang, Ying; Lomakin, Aleksey; Latypov, Ramil

    2012-02-01

    We report the observation of liquid-liquid phase separation (LLPS) in a solution of humanized monoclonal antibodies, IgG2, and the effects of human serum albumin, a major blood protein, on this phase separation. We find a significant reduction of phase separation temperature in the presence of albumin, and a preferential partitioning of the albumin into the antibody-rich phase. We provide a general thermodynamic analysis of the antibody-albumin mixture phase diagram and relate its features to the magnitude of the effective inter-protein interactions. Our analysis suggests that additives (HSA in this report), which have moderate attraction with antibody molecules, may be used to forestall undesirable protein condensation in antibody solutions. Our findings are relevant to understanding the stability of pharmaceutical solutions of antibodies and the mechanisms of cryoglobulinemia.

  16. SPECT assay of radiolabeled monoclonal antibodies

    SciTech Connect

    Jaszczak, R.J.

    1992-02-01

    The accurate determination of the biodistribution of radiolabeled monoclonal antibodies (MoAbs) is important for calculation of dosimetry and evaluation of pharmacokinetic variables such as antibody dose and route of administration. The hypothesis of this application is that the biodistribution of radiolabeled monoclonal antibodies (MoAbs) can be quantitatively determined using single photon emission computed tomography (SPECT). The major thrusts during the third year include the continued development and evaluation of improved 3D SPECT acquisition and reconstruction approaches to improve quantitative imaging of radiolabeled monoclonal antibodies (MoAbs), and the implementation and evaluation of algorithms to register serial SPECT image data sets, or to register 3D SPECT images with 3D image data sets acquired from positron emission tomography (PEI) and magnetic resonance images (MRI). The research has involved the investigation of statistical models and iterative reconstruction algorithms that accurately account for the physical characteristics of the SPECT acquisition system. It is our belief that SPECT quantification can be improved by accurately modeling the physical processes such as attenuation, scatter, geometric collimator response, and other factors that affect the measured projection data.

  17. Chemoenzymatic Glyco-engineering of Monoclonal Antibodies.

    PubMed

    Giddens, John P; Wang, Lai-Xi

    2015-01-01

    Monoclonal antibodies (mAbs) are an important class of therapeutic glycoproteins widely used for the treatment of cancer, inflammation, and infectious diseases. Compelling data have shown that the presence and fine structures of the conserved N-glycans at the Fc domain can profoundly affect the effector functions of antibodies. However, mAbs are usually produced as mixtures of Fc glycoforms and the control of glycosylation to a favorable, homogeneous status in various host expression systems is still a challenging task. In this chapter, we describe a detailed procedure of chemoenzymatic glyco-engineering of monoclonal antibodies, using rituximab (a therapeutic monoclonal antibody) as a model system. The protocol includes the deglycosylation of a mAb by an endoglycosidase (such as wild type EndoS) to remove the heterogeneous Fc N-glycans, leaving only the innermost GlcNAc or the core-fucosylated GlcNAc at the glycosylation site. Then the deglycosylated IgG serves as an acceptor for an endoglycosidase-catalyzed transglycosylation to add a desired N-glycan to the GlcNAc acceptor to reconstitute a defined, homogeneous natural glycoform of IgG, using a glycosynthase mutant as the enzyme and activated glycan oxazoline as the donor substrate. A semi-synthesis of sialylated and asialylated biantennary N-glycan oxazolines is also described. This detailed procedure can be used for the Fc glycosylation remodeling of other mAbs to provide homogeneous Fc glycoforms for various applications. PMID:26082235

  18. Monoclonal antibodies: the promise and the reality.

    PubMed

    Coons, T

    1995-01-01

    Monoclonal antibodies, or "MoAbs," have revolutionized clinical approaches to diagnostic imaging and therapy of many diseases. The use of MoAbs for diagnosing and treating cancer has been especially promising. However, the full potential of these "magic bullets" has yet to be realized. This article examines the current and potential uses of MoAbs, describes problems with the technology and looks at potential solutions. Along with descriptions of how MoAbs are made and prepared for use in the clinic, the article provides examples of the ways in which MoAbs can be used to complement and expand the information obtained from standard diagnostic imaging modalities. Specific examples of the use of monoclonal antibodies for treating cancer and other diseases also are provided. PMID:7491408

  19. Next generation and biosimilar monoclonal antibodies

    PubMed Central

    2011-01-01

    The Next Generation and Biosimilar Monoclonal Antibodies: Essential Considerations Towards Regulatory Acceptance in Europe workshop, organized by the European Centre of Regulatory Affairs Freiburg (EUCRAF), was held February 3–4, 2011 in Freiburg, Germany. The workshop attracted over 100 attendees from 15 countries, including regulators from 11 agencies, who interacted over the course of two days. The speakers presented their authoritative views on monoclonal antibodies (mAbs) as attractive targets for development, the experience to date with the regulatory process for biosimilar medicinal products, the European Medicines Agency draft guideline on biosimilar mAbs, as well as key elements in the development of mAbs. Participants engaged in many lively discussions, and much speculation on the nature of the quality, non-clinical and clinical requirements for authorization of biosimilar mAbs. PMID:21487235

  20. Innovative Monoclonal Antibody Therapies in Multiple Sclerosis

    PubMed Central

    Kieseier, Bernd C.

    2008-01-01

    The recent years have witnessed great efforts in establishing new therapeutic options for multiple sclerosis (MS), especially for relapsing–remitting disease courses. In particular, the application of monoclonal antibodies provide innovative approaches allowing for blocking or depleting specific molecular targets, which are of interest in the pathogenesis of MS. While natalizumab received approval by the US Food and Drug Administration and the European Medicines Agency in 2006 as the first monoclonal antibody in MS therapy, rituximab, alemtuzumab, and daclizumab were successfully tested for relapsing-remitting MS in small cohorts in the meantime. Here, we review the data available from these recent phase II trials and at the same time critically discuss possible pitfalls which may be relevant for clinical practice. The results of these studies may not only broaden our therapeutic options in the near future, but also provide new insights into disease pathogenesis. PMID:21180564

  1. Recent developments in monoclonal antibody radiolabeling techniques

    SciTech Connect

    Srivastava, S.C.; Mease, R.C.

    1989-01-01

    Monoclonal antibodies (MAbs) have shown the potential to serve as selective carriers of radionuclides to specific in vivo antigens. Accordingly, there has been an intense surge of research activity in an effort to develop and evaluate MAb-based radiopharmaceuticals for tumor imaging (radioimmunoscintigraphy) and therapy (radioimmunotherapy), as well as for diagnosing nonmalignant diseases. A number of problems have recently been identified, related to the MAbs themselves and to radiolabeling techniques, that comprise both the selectivity and the specificity of the in vivo distribution of radiolabeled MAbs. This paper will address some of these issues and primarily discuss recent developments in the techniques for radiolabeling monoclonal antibodies that may help resolve problems related to the poor in vivo stability of the radiolabel and may thus produce improved biodistribution. Even though many issues are identical with therapeutic radionuclides, the discussion will focus mainly on radioimmunoscintigraphic labels. 78 refs., 6 tabs.

  2. Monoclonal antibodies specific for sickle cell hemoglobin

    SciTech Connect

    Jensen, R.H.; Vanderlaan, M.; Grabske, R.J.; Branscomb, E.W.; Bigbee, W.L.; Stanker, L.H.

    1985-01-01

    Two mouse hybridoma cell lines were isolated which produce monoclonal antibodies that bind hemoglobin S. The mice were immunized with peptide-protein conjugates to stimulate a response to the amino terminal peptide of the beta chain of hemoglobin S, where the single amino acid difference between A and S occurs. Immunocharacterization of the antibodies shows that they bind specifically to the immunogen peptide and to hemoglobin S. The specificity for S is high enough that one AS cell in a mixture with a million AA cells is labeled by antibody, and such cells can be analyzed by flow cytometry. Immunoblotting of electrophoretic gels allows definitive identification of hemoglobin S as compared with other hemoglobins with similar electrophoretic mobility. 12 references, 4 figures.

  3. Monoclonal antibodies and method for detecting dioxins and dibenzofurans

    DOEpatents

    Vanderlaan, Martin; Stanker, Larry H.; Watkins, Bruce E.; Bailey, Nina R.

    1989-01-01

    Compositions of matter are described which include five monoclonal antibodies that react with dioxins and dibenzofurans, and the five hybridomas that produce these monoclonal antibodies. In addition, a method for the use of these antibodies in a sensitive immunoassay for dioxins and dibenzofurans is given, which permits detection of these pollutants in samples at concentrations in the range of a few parts per billion.

  4. Labeling of monoclonal antibodies with radionuclides

    SciTech Connect

    Bhargava, K.K.; Acharya, S.A. )

    1989-07-01

    Antibodies, specifically monoclonal antibodies, are potentially very useful and powerful carriers of therapeutic agents to target tissues and diagnostic agents. The loading or charging of antibodies with agents, especially radiotracers, is reviewed here. The choice of radioisotope for immunodetection and/or immunotherapy is based on its availability, half-life, nature of the radiation emitted, and the metabolic pathways of the radionuclide in the body. Most important of all are the derivatization techniques available for labeling the antibody with the given radionuclide. Isotopes of iodine and divalent metal ions are the most commonly used radionuclides. Antibodies labeled with iodine at tyrosine residues are metabolized rapidly in vivo. This leads to the incorporation of metabolized radioactive iodine into various tissues, mainly the thyroid gland and stomach, and to the accumulation of high levels of circulating iodine in the blood, which masks tumor uptake considerably. To overcome these limitations, the use of iodohippurate as an iodine-anchoring molecule to the protein should be considered. When divalent or multivalent metal ions are used as the preferred radionuclide, bifunctional chelating reagents such as EDTA or DTPA are first coupled to the protein or antibody. These chelating molecules are attached to the protein by formation of an isopeptide linkage between the carboxylate of the chelating reagent and the amino group of the protein. Several procedures are available to generate the isopeptide linkage. When the anchoring of the chelating agent through isopeptide linkage results in the inactivation of the antibody, periodate oxidation of the carbohydrate moiety of the antibody, followed by reductive coupling of chelator, could be considered as an alternative. There is still a need for better, simpler, and more direct methods for labeling antibodies with radionuclides. 78 references.

  5. Taxonomic investigation of Legionella pneumophila using monoclonal antibodies.

    PubMed

    Brindle, R J; Bryant, T N; Draper, P W

    1989-03-01

    A panel of 19 monoclonal antibodies was used to produce patterns of immunofluorescent staining of 468 isolates of Legionella pneumophila. Twelve monoclonal antibodies were selected that divided L. pneumophila into 17 phenons which, in the majority of cases, conform to serogroup divisions. These phenons are more easily defined than the present serogroups, and isolates can be placed in them with little ambiguity. The standardized set of monoclonal antibodies was also used to define the subgroups of serogroup 1. PMID:2654183

  6. The Role of Monoclonal Antibodies in the Management of Leukemia

    PubMed Central

    Al-Ameri, Ali; Cherry, Mohamad; Al-Kali, Aref; Ferrajoli, Alessandra

    2010-01-01

    This article will review the monoclonal antibodies more commonly used in leukemias. In the last three decades, scientists have made considerable progress understanding the structure and the functions of various surface antigens, such as CD20, CD33. The introduction of rituximab, an anti CD20 monoclonal antibody, had a great impact in the treatment of lymphoproliferative disorders. Gemtuzumab, an anti CD 33 conjugated monoclonal antibody has activity in acute mylegenous leukemia (AML). As this field is undergoing a rapid growth, the years will see an increasing use of monoclonal antibodies in hematological malignancies.

  7. A novel monoclonal antibody specific for cocaine.

    PubMed

    Nakayama, Hiroshi; Kenjyou, Noriko; Shigetoh, Nobuyuki

    2013-08-01

    Detection systems for the illegal drug cocaine need to have a high sensitivity and specificity for cocaine and to be relatively easy to use. In the current study, a monoclonal antibody (MAb) with a high specificity for cocaine was produced. Enzyme-linked immunosorbent assay and fluorescence quenching immunoassay were used to screen the hybridomas. The MAb S27Y (IgG1) was shown to be sensitive and specific for cocaine and quenched fluorescence. Thus, S27Y has the potential to be used in screening assays for the rapid and sensitive detection of cocaine. PMID:23909419

  8. Anaphylaxis to chemotherapy and monoclonal antibodies.

    PubMed

    Castells, Mariana C

    2015-05-01

    Hypersensitivity reactions are increasingly prevalent, although underrecognized and underreported. Platins induce immunoglobulin E-mediated sensitization; taxenes and some monoclonal antibodies can induce reactions at first exposure. Severe hypersensitivity can preclude first-line therapy. Tryptase level at the time of a reaction is a useful diagnostic tool. Skin testing provides a specific diagnosis. Newer tests are promising diagnostic tools to help identify patients at risk before first exposure. Safe management includes rapid drug desensitization. This review provides information regarding the scope of hypersensitivity and anaphylactic reactions induced by chemotherapy and biological drugs, as well as diagnosis, management, and treatment options. PMID:25841555

  9. Monoclonal antibody disulfide reduction during manufacturing

    PubMed Central

    Hutterer, Katariina M.; Hong, Robert W.; Lull, Jonathon; Zhao, Xiaoyang; Wang, Tian; Pei, Rex; Le, M. Eleanor; Borisov, Oleg; Piper, Rob; Liu, Yaoqing Diana; Petty, Krista; Apostol, Izydor; Flynn, Gregory C.

    2013-01-01

    Manufacturing-induced disulfide reduction has recently been reported for monoclonal human immunoglobulin gamma (IgG) antibodies, a widely used modality in the biopharmaceutical industry. This effect has been tied to components of the intracellular thioredoxin reduction system that are released upon cell breakage. Here, we describe the effect of process parameters and intrinsic molecule properties on the extent of reduction. Material taken from cell cultures at the end of production displayed large variations in the extent of antibody reduction between different products, including no reduction, when subjected to the same reduction-promoting harvest conditions. Additionally, in a reconstituted model in which process variables could be isolated from product properties, we found that antibody reduction was dependent on the cell line (clone) and cell culture process. A bench-scale model using a thioredoxin/thioredoxin reductase regeneration system revealed that reduction susceptibility depended on not only antibody class but also light chain type; the model further demonstrates that the trend in reducibility was identical to DTT reduction sensitivity following the order IgG1λ > IgG1κ > IgG2λ > IgG2κ. Thus, both product attributes and process parameters contribute to the extent of antibody reduction during production. PMID:23751615

  10. Monoclonal antibodies to Pseudomonas aeruginosa ferripyochelin-binding protein.

    PubMed Central

    Sokol, P A; Woods, D E

    1986-01-01

    Hybridomas secreting specific monoclonal antibodies against the Pseudomonas aeruginosa ferripyochelin-binding protein (FBP) were isolated. These monoclonal antibodies reacted with FBP in immunoblots of outer membrane preparations from all serotypes of P. aeruginosa. Two of the monoclonal antibodies also reacted with FBP in strains of P. putida, P. fluorescens, and P. stutzeri. These antibodies did not react with outer membranes of P. cepacia, "P. multivorans," P. maltophilia, or other gram-negative organisms. The monoclonal antibodies were opsonophagocytic and blocked the binding of [59Fe]ferripyochelin to isolated outer membranes of strain PAO. By indirect immunofluorescence techniques, the monoclonal antibodies were used to demonstrate that FBP is present on the cell surface of P. aeruginosa cells grown in low-iron but not high-iron medium. These observations were confirmed by using 125I in surface-labeling techniques. Images PMID:3091506

  11. Kinetics of intralymphatically delivered monoclonal antibodies

    SciTech Connect

    Wahl, R.L.; Geatti, O.; Liebert, M.; Beers, B.; Jackson, G.; Laino, L.; Kronberg, S.; Wilson, B.S.; Beierwaltes, W.H.

    1985-05-01

    Radiolabeled monoclonal antibody (MoAb) administration subcutaneously (sq), so that preferential uptake is to the lymphatics, holds significant promise for the detection of lymph node metastases. Only limited information is available about clearance rates of intralymphatically administered MoAbs. I-131 labeled intact IgG (225.28S), F(ab's)2 (225.28S) or IgM (FT162) were administered sq to anesthetized Balb/C mice. Eight mice were studied with each MoAb, 4 with a foot-pad injection, 4 with an anterior abdominal injection. Gamma camera images were collected into a computer, over the first 6 hrs after injection with the animals anesthetized and immobile. Animals were then allowed to move about freely. Additional images were then acquired out to 48 hrs. Regions of interest wre selected over the injection site and the kinetics of antibody egress determined. Clearance rates from local sq injection sites are influenced by motion and somewhat by location. The class and fragment status of the MoAb appear relatively less important in determining clearance rates from sq injections than they are in determining whole-body clearance after iv injections. Additional studies using Fab fragments and additional monoclonals will be useful in extending these observations.

  12. Production of monoclonal antibodies against canine leukocytes.

    PubMed

    Aguiar, Paulo Henrique Palis; Borges dos Santos, Roberto Robson; Lima, Carla Andrade; Rios de Sousa Gomes, Hilton; Larangeira, Daniela Farias; Santos, Patrícia Meira; Barrouin-Melo, Stella Maria; Conrado dos-Santos, Washington Luis; Pontes-de-Carvalho, Lain

    2004-04-01

    A panel of anti-canine leukocyte monoclonal antibodies (MAbs) was produced by immunizing BALB/c mice with canine peripheral blood mononuclear cells (PBMC), either resting or stimulated with concanavalin A (ConA). Three out of 28 clones-IH1, AB6, and HG6-screened by ELISA and producing antibody with the highest specificity for canine cell immunostaining, were subjected to three subsequent subcloning steps by limiting dilution, and selected for further characterization. These MAbs belonged to IgG1 (HG6 and IH1) and IgG2a (AB6) isotypes. The distribution of cell populations expressing the antigen recognized by the antibodies was identified by indirect immunoflorescence on canine PBMC and on tissue sections of lymph node, spleen, liver and skin. The possible crossreactivity with human PBMC was also examined in immunocytochemistry. One of the antibodies specifically recognized macrophages. The MAbs presented here can be foreseen as possible valuable diagnostic and research tools to study immune functions in dogs. PMID:15165486

  13. A Monoclonal Antibody Toolkit for C. elegans

    PubMed Central

    Hadwiger, Gayla; Dour, Scott; Arur, Swathi; Fox, Paul; Nonet, Michael L.

    2010-01-01

    Background Antibodies are critical tools in many avenues of biological research. Though antibodies can be produced in the research laboratory setting, most research labs working with vertebrates avail themselves of the wide array of commercially available reagents. By contrast, few such reagents are available for work with model organisms. Methodology/Principal Findings We report the production of monoclonal antibodies directed against a wide range of proteins that label specific subcellular and cellular components, and macromolecular complexes. Antibodies were made to synaptobrevin (SNB-1), a component of synaptic vesicles; to Rim (UNC-10), a protein localized to synaptic active zones; to transforming acidic coiled-coil protein (TAC-1), a component of centrosomes; to CENP-C (HCP-4), which in worms labels the entire length of their holocentric chromosomes; to ORC2 (ORC-2), a subunit of the DNA origin replication complex; to the nucleolar phosphoprotein NOPP140 (DAO-5); to the nuclear envelope protein lamin (LMN-1); to EHD1 (RME-1) a marker for recycling endosomes; to caveolin (CAV-1), a marker for caveolae; to the cytochrome P450 (CYP-33E1), a resident of the endoplasmic reticulum; to β-1,3-glucuronyltransferase (SQV-8) that labels the Golgi; to a chaperonin (HSP-60) targeted to mitochondria; to LAMP (LMP-1), a resident protein of lysosomes; to the alpha subunit of the 20S subcomplex (PAS-7) of the 26S proteasome; to dynamin (DYN-1) and to the α-subunit of the adaptor complex 2 (APA-2) as markers for sites of clathrin-mediated endocytosis; to the MAGUK, protein disks large (DLG-1) and cadherin (HMR-1), both of which label adherens junctions; to a cytoskeletal linker of the ezrin-radixin-moesin family (ERM-1), which localized to apical membranes; to an ERBIN family protein (LET-413) which localizes to the basolateral membrane of epithelial cells and to an adhesion molecule (SAX-7) which localizes to the plasma membrane at cell-cell contacts. In addition to working

  14. Characterization of Tritrichomonas foetus antigens by use of monoclonal antibodies.

    PubMed Central

    Hodgson, J L; Jones, D W; Widders, P R; Corbeil, L B

    1990-01-01

    The specificity for and function of monoclonal antibodies against Tritrichomonas foetus were characterized. Four monoclonal antibodies generated by immunization of mice with live T. foetus were selected on the basis of enzyme-linked immunosorbent assay reactions. The approximate molecular masses of the predominant proteins were determined by Western blotting (immunoblotting). Monoclonal antibody TF3.8 recognized a predominant band at approximately 155 kilodaltons, whereas TF3.2 reacted with several bands. Monoclonal antibodies TF1.17 and TF1.15 recognized broad bands between 45 and 75 kilodaltons. The first two antibodies (TF3.8 and TF3.2) did not react with the surface of T. foetus, as determined by live-cell immunofluorescence, agglutination, and immobilization, whereas two other monoclonal antibodies (TF1.17 and TF1.15) did react with surface epitopes, as determined by these criteria. The latter two monoclonal antibodies also mediated complement-dependent killing of T. foetus and prevented of adherence of organisms to bovine vaginal epithelial cells. One antibody, TF1.15, also killed in the absence of complement. Since these functions are in vitro correlates of protection, the antigens recognized by these monoclonal antibodies may induce protective immunity. Images PMID:2201645

  15. A new tool for monoclonal antibody analysis

    PubMed Central

    An, Yan; Zhang, Ying; Mueller, Hans-Martin; Shameem, Mohammed; Chen, Xiaoyu

    2014-01-01

    Monoclonal antibody (mAb) products are extraordinarily heterogeneous due to the presence of a variety of enzymatic and chemical modifications, such as deamidation, isomerization, oxidation, glycosylation, glycation, and terminal cyclization. The modifications in different domains of the antibody molecule can result in different biological consequences. Therefore, characterization and routine monitoring of domain-specific modifications are essential to ensure the quality of the therapeutic antibody products. For this purpose, a rapid and informative methodology was developed to examine the heterogeneity of individual domains in mAb products. A recently discovered endopeptidase, IdeS, cleaves heavy chains below the hinge region, producing F(ab')2 and Fc fragments. Following reduction of disulfide bonds, three antibody domains (LC, Fd, and Fc/2) can be released for further characterization. Subsequent analyses by liquid chromatography/mass spectrometry, capillary isoelectric focusing, and glycan mapping enable domain-specific profiling of oxidation, charge heterogeneity, and glycoform distribution. When coupled with reversed phase chromatography, the unique chromatographic profile of each molecule offers a simple strategy for an identity test, which is an important formal test for biopharmaceutical quality control purposes. This methodology is demonstrated for a number of IgGs of different subclasses (IgG1, IgG2, IgG4), as well as an Fc fusion protein. The presented technique provides a convenient platform approach for scientific and formal therapeutic mAb product characterization. It can also be applied in regulated drug substance batch release and stability testing of antibody and Fc fusion protein products, in particular for identity and routine monitoring of domain-specific modifications. PMID:24927271

  16. Detection of enterovirus 70 with monoclonal antibodies.

    PubMed

    Anderson, L J; Hatch, M H; Flemister, M R; Marchetti, G E

    1984-09-01

    To improve the ability to identify enterovirus-70 (EV-70) from patients with acute hemorrhagic conjunctivitis, we developed four monoclonal antibodies (MAbs) to EV-70. We reacted the four MAbs against nine previously characterized strains of EV-70 and heterologous viruses by virus neutralization, indirect immunofluorescence, and enzyme-linked immunosorbent assay (ELISA). Two of the MAbs neutralized all nine strains of EV-70 and none of the other enterovirus types tested. Two of the MAbs gave a positive reaction with all nine strains by indirect immunofluorescence, and three reacted with all nine strains by ELISA. None of the MAbs gave a positive reaction with heterologous viruses, including those associated with eye disease, by indirect immunofluorescence or ELISA. The two neutralizing MAbs failed to give a positive reaction with some of the strains of EV-70 by indirect immunofluorescence and ELISA, yet they neutralized these viruses. By ELISA with a polyclonal serum as capture antibody and a mixture of MAbs as detector antibody, we were able to detect from 10(2.2) to 10(5.8) 50% tissue culture infective doses of virus and to type lyophilized isolates of EV-70 sent from Taiwan from which we could not recover infectious virus. By choosing the appropriate MAb, or mixture of MAbs, we could construct a test which had the type specificity and strain sensitivity needed to type isolates of EV-70. PMID:6092426

  17. Detection of enterovirus 70 with monoclonal antibodies.

    PubMed Central

    Anderson, L J; Hatch, M H; Flemister, M R; Marchetti, G E

    1984-01-01

    To improve the ability to identify enterovirus-70 (EV-70) from patients with acute hemorrhagic conjunctivitis, we developed four monoclonal antibodies (MAbs) to EV-70. We reacted the four MAbs against nine previously characterized strains of EV-70 and heterologous viruses by virus neutralization, indirect immunofluorescence, and enzyme-linked immunosorbent assay (ELISA). Two of the MAbs neutralized all nine strains of EV-70 and none of the other enterovirus types tested. Two of the MAbs gave a positive reaction with all nine strains by indirect immunofluorescence, and three reacted with all nine strains by ELISA. None of the MAbs gave a positive reaction with heterologous viruses, including those associated with eye disease, by indirect immunofluorescence or ELISA. The two neutralizing MAbs failed to give a positive reaction with some of the strains of EV-70 by indirect immunofluorescence and ELISA, yet they neutralized these viruses. By ELISA with a polyclonal serum as capture antibody and a mixture of MAbs as detector antibody, we were able to detect from 10(2.2) to 10(5.8) 50% tissue culture infective doses of virus and to type lyophilized isolates of EV-70 sent from Taiwan from which we could not recover infectious virus. By choosing the appropriate MAb, or mixture of MAbs, we could construct a test which had the type specificity and strain sensitivity needed to type isolates of EV-70. PMID:6092426

  18. Monoclonal antibodies based on hybridoma technology.

    PubMed

    Yagami, Hisanori; Kato, Hiroshi; Tsumoto, Kanta; Tomita, Masahiro

    2013-03-01

    Based on the size and scope of the present global market for medicine, monoclonal antibodies (mAbs) have a very promising future, with applications for cancers through autoimmune ailments to infectious disease. Since mAbs recognize only their target antigens and not other unrelated proteins, pinpoint medical treatment is possible. Global demand is dramatically expanding. Hybridoma technology, which allows production of mAbs directed against antigens of interest is therefore privileged. However, there are some pivotal points for further development to generate therapeutic antibodies. One is selective generation of human mAbs. Employment of transgenic mice producing human antibodies would overcome this problem. Another focus is recognition sites and conformational epitopes in antigens may be just as important as linear epitopes, especially when membrane proteins such as receptors are targeted. Recognition of intact structures is of critical importance for medical purposes. In this review, we describe patent related information for therapeutic mAbs based on hybridoma technology and also discuss new advances in hybridoma technology that facilitate selective production of stereospecific mAbs. PMID:24237029

  19. SPECT assay of radiolabeled monoclonal antibodies

    SciTech Connect

    Jaszczak, R.J.

    1992-02-01

    The long-term goal of this research project is to develop methods to improve the utility of single photon emission computed tomography (SPECI) to quantify the biodistribution of monoclonal antibodies (MoAbs) labeled with clinically relevant radionuclides ({sup 123}I, {sup 131}I, and {sup 111}In) and with another radionuclide,{sup 211}At, recently used in therapy. We describe here our progress in developing quantitative SPECT methodology for {sup 111}In and {sup 123}I. We have focused our recent research thrusts on the following aspects of SPECT: (1) The development of improved SPECT hardware, such as improved acquisition geometries. (2) The development of better reconstruction methods that provide accurate compensation for the physical factors that affect SPECT quantification. (3) The application of carefully designed simulations and experiments to validate our hardware and software approaches.

  20. The birth pangs of monoclonal antibody therapeutics

    PubMed Central

    2012-01-01

    This paper examines the development and termination of nebacumab (Centoxin®), a human IgM monoclonal antibody (mAb) drug frequently cited as one of the notable failures of the early biopharmaceutical industry. The non-approval of Centoxin in the United States in 1992 generated major concerns at the time about the future viability of any mAb therapeutics. For Centocor, the biotechnology company that developed Centoxin, the drug posed formidable challenges in terms of safety, clinical efficacy, patient selection, the overall economic costs of health care, as well as financial backing. Indeed, Centocor's development of the drug brought it to the brink of bankruptcy. This article shows how many of the experiences learned with Centoxin paved the way for the current successes in therapeutic mAb development. PMID:22531443

  1. Monitoring therapeutic monoclonal antibodies in brain tumor

    PubMed Central

    Ait-Belkacem, Rima; Berenguer, Caroline; Villard, Claude; Ouafik, L’Houcine; Figarella-Branger, Dominique; Beck, Alain; Chinot, Olivier; Lafitte, Daniel

    2014-01-01

    Bevacizumab induces normalization of abnormal blood vessels, making them less leaky. By binding to vascular endothelial growth factor, it indirectly attacks the vascular tumor mass. The optimal delivery of targeted therapies including monoclonal antibodies or anti-angiogenesis drugs to the target tissue highly depends on the blood-brain barrier permeability. It is therefore critical to investigate how drugs effectively reach the tumor. In situ investigation of drug distribution could provide a better understanding of pharmacological agent action and optimize chemotherapies for solid tumors. We developed an imaging method coupled to protein identification using matrix-assisted laser desorption/ionization mass spectrometry. This approach monitored bevacizumab distribution within the brain structures, and especially within the tumor, without any labeling. PMID:25484065

  2. A humanized monoclonal antibody targeting Staphylococcus aureus.

    PubMed

    Patti, Joseph M

    2004-12-01

    This current presentation describes the in vitro and in vivo characterization of Aurexis (tefibazumab), a humanized monoclonal antibody that exhibits a high affinity and specificity and for the Staphylococcus aureus MSCRAMM (Microbial Surface Components Recognizing Adhesive Matrix Molecules) protein ClfA. Aurexis inhibited ClfA binding to human fibrinogen, and enhanced the opsonophagocytic uptake of ClfA-coated beads. Preclinical in vivo testing revealed that a single administration of Aurexis significantly protected against an IV challenge with a methicillin resistant S. aureus (MRSA) strain in murine septicemia and rabbit infective endocarditis (IE) models. Safety and pharmacokinetic data from a 19-patient phase I study support continued evaluation of Aurexis in phase II studies. PMID:15576200

  3. Monoclonal antibodies in treatment of multiple sclerosis

    PubMed Central

    Rommer, P S; Dudesek, A; Stüve, O; Zettl, UK

    2014-01-01

    Monoclonal antibodies (mAbs) are used as therapeutics in a number of disciplines in medicine, such as oncology, rheumatology, gastroenterology, dermatology and transplant rejection prevention. Since the introduction and reintroduction of the anti-alpha4-integrin mAb natalizumab in 2004 and 2006, mAbs have gained relevance in the treatment of multiple sclerosis (MS). At present, numerous mAbs have been tested in clinical trials in relapsing–remitting MS, and in progressive forms of MS. One of the agents that might soon be approved for very active forms of relapsing–remitting MS is alemtuzumab, a humanized mAb against CD52. This review provides insights into clinical studies with the mAbs natalizumab, alemtuzumab, daclizumab, rituximab, ocrelizumab and ofatumumab. PMID:24001305

  4. Building better monoclonal antibody-based therapeutics

    PubMed Central

    Weiner, George J.

    2015-01-01

    For 20 years, monoclonal antibodies (mAbs) have been a standard component of cancer therapy, yet there is still much room for improvement. Efforts continue to build better cancer therapeutics based on mAbs. Anti-cancer mAbs function via a variety of mechanisms including directly targeting the malignant cells, modifying the host response to the malignant cells, delivering cytotoxic moieties to the malignant cells or retargeting cellular immunity towards the malignant cells. Characteristics of mAbs that affect their efficacy include antigen specificity, overall structure, affinity for the target antigen and how a mAb component is incorporated into a construct that can trigger target cell death. This article reviews the various approaches to using mAb-based therapeutics to treat cancer, the strategies used to take advantage of the unique potential of each approach, and provides examples of current mAb-based treatments. PMID:25998715

  5. Drug Development of Therapeutic Monoclonal Antibodies.

    PubMed

    Mould, Diane R; Meibohm, Bernd

    2016-08-01

    Monoclonal antibodies (MAbs) have become a substantial part of many pharmaceutical company portfolios. However, the development process of MAbs for clinical use is quite different than for small-molecule drugs. MAb development programs require careful interdisciplinary evaluations to ensure the pharmacology of both the MAb and the target antigen are well-understood. Selection of appropriate preclinical species must be carefully considered and the potential development of anti-drug antibodies (ADA) during these early studies can limit the value and complicate the performance and possible duration of preclinical studies. In human studies, many of the typical pharmacology studies such as renal or hepatic impairment evaluations may not be needed but the pharmacokinetics and pharmacodynamics of these agents is complex, often necessitating more comprehensive evaluation of clinical data and more complex bioanalytical assays than might be used for small molecules. This paper outlines concerns and strategies for development of MAbs from the early in vitro assessments needed through preclinical and clinical development. This review focuses on how to develop, submit, and comply with regulatory requirements for MAb therapeutics. PMID:27342605

  6. Monoclonal antibody therapy for Junin virus infection.

    PubMed

    Zeitlin, Larry; Geisbert, Joan B; Deer, Daniel J; Fenton, Karla A; Bohorov, Ognian; Bohorova, Natasha; Goodman, Charles; Kim, Do; Hiatt, Andrew; Pauly, Michael H; Velasco, Jesus; Whaley, Kevin J; Altmann, Friedrich; Gruber, Clemens; Steinkellner, Herta; Honko, Anna N; Kuehne, Ana I; Aman, M Javad; Sahandi, Sara; Enterlein, Sven; Zhan, Xiaoguo; Enria, Delia; Geisbert, Thomas W

    2016-04-19

    Countermeasures against potential biothreat agents remain important to US Homeland Security, and many of these pharmaceuticals could have dual use in the improvement of global public health. Junin virus, the causative agent of Argentine hemorrhagic fever (AHF), is an arenavirus identified as a category A high-priority agent. There are no Food and Drug Administration (FDA) approved drugs available for preventing or treating AHF, and the current treatment option is limited to administration of immune plasma. Whereas immune plasma demonstrates the feasibility of passive immunotherapy, it is limited in quantity, variable in quality, and poses safety risks such as transmission of transfusion-borne diseases. In an effort to develop a monoclonal antibody (mAb)-based alternative to plasma, three previously described neutralizing murine mAbs were expressed as mouse-human chimeric antibodies and evaluated in the guinea pig model of AHF. These mAbs provided 100% protection against lethal challenge when administered 2 d after infection (dpi), and one of them (J199) was capable of providing 100% protection when treatment was initiated 6 dpi and 92% protection when initiated 7 dpi. The efficacy of J199 is superior to that previously described for all other evaluated drugs, and its high potency suggests that mAbs like J199 offer an economical alternative to immune plasma and an effective dual use (bioterrorism/public health) therapeutic. PMID:27044104

  7. Monoclonal antibody therapy for Junin virus infection

    PubMed Central

    Zeitlin, Larry; Geisbert, Joan B.; Deer, Daniel J.; Fenton, Karla A.; Bohorov, Ognian; Bohorova, Natasha; Goodman, Charles; Kim, Do; Hiatt, Andrew; Pauly, Michael H.; Velasco, Jesus; Whaley, Kevin J.; Altmann, Friedrich; Gruber, Clemens; Steinkellner, Herta; Honko, Anna N.; Kuehne, Ana I.; Aman, M. Javad; Sahandi, Sara; Enterlein, Sven; Zhan, Xiaoguo; Enria, Delia; Geisbert, Thomas W.

    2016-01-01

    Countermeasures against potential biothreat agents remain important to US Homeland Security, and many of these pharmaceuticals could have dual use in the improvement of global public health. Junin virus, the causative agent of Argentine hemorrhagic fever (AHF), is an arenavirus identified as a category A high-priority agent. There are no Food and Drug Administration (FDA) approved drugs available for preventing or treating AHF, and the current treatment option is limited to administration of immune plasma. Whereas immune plasma demonstrates the feasibility of passive immunotherapy, it is limited in quantity, variable in quality, and poses safety risks such as transmission of transfusion-borne diseases. In an effort to develop a monoclonal antibody (mAb)-based alternative to plasma, three previously described neutralizing murine mAbs were expressed as mouse-human chimeric antibodies and evaluated in the guinea pig model of AHF. These mAbs provided 100% protection against lethal challenge when administered 2 d after infection (dpi), and one of them (J199) was capable of providing 100% protection when treatment was initiated 6 dpi and 92% protection when initiated 7 dpi. The efficacy of J199 is superior to that previously described for all other evaluated drugs, and its high potency suggests that mAbs like J199 offer an economical alternative to immune plasma and an effective dual use (bioterrorism/public health) therapeutic. PMID:27044104

  8. Licensed monoclonal antibodies and associated challenges.

    PubMed

    Khan, Amjad Hayat; Sadroddiny, Esmaeil

    2015-12-23

    Monoclonal antibodies (mAbs) are the leading class of targeted therapeutics and remarkably effective in addressing autoimmune diseases, inflammations, infections, and various types of cancer. Several mAbs approved by US food and drug administration (FDA), are available on the market and a number are pending for approval. Luckily, FDA approved mAbs have played a pivotal role in the treatment and prevention of lethal diseases. However, claiming that licensed mAbs are 100% safe is still debatable, because infections, malignancies, anaphylactoid, and anaphylactic reactions are the more frequently associated adverse events. To evaluate benefit to risk ratio of mAbs, it is important for the clinical research staff or physicians to monitor and follow-up the patients who are receiving mAbs dozes. It is recommended that patients, physicians, biopharmaceutical companies, and researchers should keep in touch to highlight and resolve antibody-based adverse events. In this review we underscore the associated challenges of mAbs, approved by FDA from 2007-2014. PMID:27472864

  9. Sub-Nanogram Detection of RDX Explosive by Monoclonal Antibodies

    PubMed Central

    Hutchinson, Alistair P.; Nicklin, Stephen

    2015-01-01

    Polyclonal and monoclonal antibodies were raised to protein carrier molecules haptenized with RDX, a major component of many plastic explosives including Semtex. Sera from immunized mice detected RDX protein conjugates in standard ELISA. Clonally purified monoclonal antibodies had detection limits in the sub-ng/mL range for underivatized RDX in competition ELISA. The monoclonal antibodies are not dependent on the presence of taggants added during the manufacturing process, and are likely to have utility in the detection of any explosive containing RDX, or RDX contamination of environmental sites. PMID:26252765

  10. Sub-Nanogram Detection of RDX Explosive by Monoclonal Antibodies.

    PubMed

    Ulaeto, David O; Hutchinson, Alistair P; Nicklin, Stephen

    2015-08-01

    Polyclonal and monoclonal antibodies were raised to protein carrier molecules haptenized with RDX, a major component of many plastic explosives including Semtex. Sera from immunized mice detected RDX protein conjugates in standard ELISA. Clonally purified monoclonal antibodies had detection limits in the sub-ng/mL range for underivatized RDX in competition ELISA. The monoclonal antibodies are not dependent on the presence of taggants added during the manufacturing process, and are likely to have utility in the detection of any explosive containing RDX, or RDX contamination of environmental sites. PMID:26252765

  11. Monoclonal antibody specific for a pigmentation associated antigen

    SciTech Connect

    Thomson, T.M.; Mattes, M.J.; Old, L.J.; Lloyd, K.O

    1989-01-17

    Monoclonal antibody TA99, which specifically binds to a pigmentation associated antigen present on melanoma cells is described. Additionally, the hybridoma cell line deposited with the ATCC under Accession Number HB 8704 from which the antibody is derived, as well as methods for using the antibody are described.

  12. Monoclonal Antibodies Targeting Tumor Growth | NCI Technology Transfer Center | TTC

    Cancer.gov

    The NCI Nanobiology Program, Protein Interaction Group is seeking parties to license or co-develop, evaluate, or commercialize monoclonal antibodies against the insulin-like growth factor for the treatment of cancer.

  13. Technological progresses in monoclonal antibody production systems.

    PubMed

    Rodrigues, Maria Elisa; Costa, Ana Rita; Henriques, Mariana; Azeredo, Joana; Oliveira, Rosário

    2010-01-01

    Monoclonal antibodies (mAbs) have become vitally important to modern medicine and are currently one of the major biopharmaceutical products in development. However, the high clinical dose requirements of mAbs demand a greater biomanufacturing capacity, leading to the development of new technologies for their large-scale production, with mammalian cell culture dominating the scenario. Although some companies have tried to meet these demands by creating bioreactors of increased capacity, the optimization of cell culture productivity in normal bioreactors appears as a better strategy. This review describes the main technological progresses made with this intent, presenting the advantages and limitations of each production system, as well as suggestions for improvements. New and upgraded bioreactors have emerged both for adherent and suspension cell culture, with disposable reactors attracting increased interest in the last years. Furthermore, the strategies and technologies used to control culture parameters are in constant evolution, aiming at the on-line multiparameter monitoring and considering now parameters not seen as relevant for process optimization in the past. All progresses being made have as primary goal the development of highly productive and economic mAb manufacturing processes that will allow the rapid introduction of the product in the biopharmaceutical market at more accessible prices. PMID:20043321

  14. Preparation of Monoclonal Antibodies Against Bovine Haptoglobin

    PubMed Central

    Wang, Caihong; Gu, Cheng; Guo, Donghua; Gao, Jing; Li, Chunqiu; Liu, Na; Geng, Yufei; Su, Mingjun; Wang, Xinyu

    2014-01-01

    Female, 8-week-old BALB/c mice were immunized with purified recombinant proteins of the predicted immunodominant region of bovine haptoglobin (pirBoHp). Two monoclonal antibodies (MAbs), named 1B3 and 6D6, were prepared by conventional B lymphocyte hybridoma technique. Titers of ascitic fluid and cell culture supernatant of MAb 1B3 were 1:9.6×108 and 1:8.2×104, respectively, and that of MAb 6D6 were 1:4.4×105 and 1:1.0×104, respectively. The subtype of MAbs 1B3 and 6D6 was IgG1κ. In Western blot analysis, MAbs 1B3 and 6D6 could recognize the α-chain of native BoHp from plasma of dairy cows. These data indicated that MAbs 1B3 and 6D6 have a potential use for developing diagnostic reagents of BoHp. PMID:25358005

  15. Monoclonal Antibody Purification (Nicotiana benthamiana Plants)

    PubMed Central

    Husk, Adam; Hamorsky, Krystal Teasley; Matoba, Nobuyuki

    2016-01-01

    Plant-based expression systems provide an alternative biomanufacturing platform for recombinant proteins (Matoba et al., 2011). In particular, plant virus-based vectors can overexpress proteins within days in the leaf tissue of Nicotiana benthamiana (N. benthamiana). To overcome the issues of genetic instability and limited infectivity of recombinant viruses, Agrobacterium-mediated delivery of “deconstructed” virus vectors has become the mainstay for the production of large and/or multicomponent proteins, such as immunoglobulin (Ig)G monoclonal antibodies (mAbs). Here, we describe a method of producing human IgG mAbs in N. benthamiana using the tobamoviral replicon vector magnICON®. The vector can express up to a few hundred mg of a mAb per kg of leaf material in 7 days. A representative case for the broadly neutralizing anti-HIV and anti-influenza mAbs, VRC01 and CR6261 respectively, is shown (Hamorsky et al., 2013). Leaf tissue is homogenized and the extract is clarified by filtration and centrifugation. The mAb is purified by fast protein liquid chromatography (FPLC) using Protein A affinity and Phenyl HP hydrophobic interection resins.

  16. [Monoclonal antibodies from neurological and neuropsychological perspective].

    PubMed

    Piusińska-Macoch, Renata

    2013-05-01

    The role of monoclonal antibodies and other proinflammatory cytokines in the regulatory processes of the central and peripheral nervous system is not yet fully understood. Clinical studies show that they are involved in the pathogenesis of Alzheimer's disease, Parkinson's disease or other neurodegenerative disabilities with cognitive impairments. Genetic basis of these disorders is still in research. In the past few years it has been shown that increased levels of TNF-alpha and IL-6 in plasma play role in patients with ischemic stroke in the acute phase as well as transient ischemic episodes. Also the negative impact of TNF-alpha has been demonstrated on neck and coronary vessels, including the composition of plaques in the carotid arteries. A few reports indicate the involvement of tumor necrosis factor in such complex processes such as emotions, behavior or personality. Recent studies point to the important role of proinflammatory cytokines in the pathogenesis of sleep disorders such as narcolepsy, cataplexy and sleep paralysis. TNF-alpha can also activate nociceptive pathways, causing the intensity of neuropathic pain. However discloses asymmetric subtypes share TNF-1, TNF-2 in the induction and the maintenance of pain. The phenomenon of complex neurohormonal control mechanism support the proinflammatory cytokines is not fully understood and needs further empirical verification. PMID:23894773

  17. Monoclonal antibodies against plant cell wall polysaccharides

    SciTech Connect

    Hahn, M.G.; Bucheli, E.; Darvill, A.; Albersheim, P. )

    1989-04-01

    Monoclonal antibodies (McAbs) are useful tools to probe the structure of plant cell wall polysaccharides and to localize these polysaccharides in plant cells and tissues. Murine McAbs were generated against the pectic polysaccharide, rhamnogalacturonan I (RG-I), isolated from suspension-cultured sycamore cells. The McAbs that were obtained were grouped into three classes based upon their reactivities with a variety of plant polysaccharides and membrane glycoproteins. Eleven McAbs (Class I) recognize epitope(s) that appear to be immunodominant and are found in RG-I from sycamore and maize, citrus pectin, polygalacturonic acid, and membrane glycoproteins from suspension-cultured cells of sycamore, maize, tobacco, parsley, and soybean. A second group of five McAbs (Class II) recognize epitope(s) present in sycamore RG-I, but do not bind to any of the other polysaccharides or glycoproteins recognized by Class I. Lastly, one McAb (Class III) reacts with sycamore RG-I, sycamore and tamarind xyloglucan, and sycamore and rice glucuronoarabinoxylan, but does not bind to maize RG-I, polygalacturonic acid or the plant membrane glycoproteins recognized by Class I. McAbs in Classes II and III are likely to be useful in studies of the structure, biosynthesis and localization of plant cell wall polysaccharides.

  18. Therapeutic Monoclonal Antibodies and Fragments: Bevacizumab.

    PubMed

    Klein, Ainat; Loewenstein, Anat

    2016-01-01

    Bevacizumab (Avastin) is a recombinant humanized monoclonal immunoglobulin antibody that has two antigen-binding domains and blocks all active forms of vascular endothelial growth factor-A. It was originally designed and is still in use as antitumor agent (for colorectal and non-small cell lung cancers). Besides inhibiting vessel growth and neovascularization, the drug promotes the regression of existing microvessels and induces 'normalization' of surviving mature vasculature, stabilizes vessels and prevents leakage. Its molecular weight is 149 kDa and its estimated terminal half-life is approximately 20 days for both men and women. The effectiveness and safety of bevacizumab was proven in retrospective and prospective controlled clinical trials for the treatment of neovascular age-related macular degeneration, neovascularization in proliferative diabetic retinopathy, diabetic macular edema, retinal vein occlusion and retinopathy of prematurity, especially for zone I. Uncontrolled trials have shown its effectiveness in various other conditions as myopic and uveitic choroidal neovascularization and neovascular glaucoma. There are no absolute contraindications to intravitreal injection though it is recommended to withhold treatment in patients who have recently suffered from a cardiovascular or cerebrovascular event and during pregnancy. Ocular complications from intravitreal use are usually mild and transient (corneal abrasion, chemosis, subconjunctival hemorrhage and vitreous hemorrhage). Bacterial endophthalmitis is rare (about 0.1%). New or progressive subretinal hemorrhages, tears of the retinal pigment epithelium and an increased incidence of geographic atrophy have also been reported. PMID:26502311

  19. Murine monoclonal antibodies specific for virulent Treponema pallidum (Nichols).

    PubMed Central

    Robertson, S M; Kettman, J R; Miller, J N; Norgard, M V

    1982-01-01

    Murine anti-Treponema pallidum (Nichols) lymphocyte hybridoma cell lines secreting monoclonal antibodies against a variety of treponemal antigens have been generated. Hybridomas isolated were of three major types: those that were directed specifically against T. pallidum antigens, those that were directed against treponemal group antigens (as evidenced by their cross-reactivity with T. phagedenis biotype Reiter antigens), and those that cross-reacted with both treponemal as well as rabbit host testicular tissue antigens. The majority (31 of 39 clones) of these anti-T. pallidum hybridomas, which produced monoclonal antibodies of mouse isotypes immunoglobulin G1 (IgG1), IgG2a, IgG2b, IgG3 or IgM, were directed specifically against T. pallidum and not other treponemal or rabbit antigens tested by radioimmunoassay. Four of these T. pallidum-specific hybridomas secreted monoclonal antibodies with greater binding affinity for "aged" rather than freshly isolated intact T. pallidum cells, suggesting a possible specificity for "unmasked" surface antigens of T. pallidum. Six anti-T. pallidum hybridomas produced complement-fixing monoclonal antibodies (IgG2a, IgG2b, or IgM) that were capable of immobilizing virulent treponemes in the T. pallidum immobilization (TPI) test; these may represent biologically active monoclonal antibodies against treponemal surface antigens. Three other hybridomas secreted monoclonal antibodies which bound to both T. pallidum and T. phagedenis biotype Reiter antigens, thus demonstrating a possible specificity for treponemal group antigens. Five hybridoma cell lines were also isolated which produced IgM monoclonal antibodies that cross-reacted with all treponemal and rabbit host testicular tissue antigens employed in the radioimmunoassays. This report describes the construction and characteristics of these hybridoma cell lines. The potential applications of the anti-T. pallidum monoclonal antibodies are discussed. PMID:7047388

  20. Considerations for the development of therapeutic monoclonal antibodies.

    PubMed

    Swann, Patrick G; Tolnay, Mate; Muthukkumar, Subramanian; Shapiro, Marjorie A; Rellahan, Barbara L; Clouse, Kathleen A

    2008-08-01

    An increasing number of Investigational New Drug (IND) applications for therapeutic monoclonal antibodies (mAbs) have been submitted to US FDA over the past several years. Monoclonal antibodies and related products are under development for a wide range of indications. In addition, the diversity of antibody-related products is increasing including IgG2/IgG4 subclasses and engineered Fc regions to enhance or reduce antibody effector functionality. Recent findings highlight the need to more fully characterize these products and their activity. Advances in product characterization tools, immunogenicity assessments, and other bioanalytical assays can be used to better understand product performance and facilitate development. PMID:18586093

  1. Monoclonal antibodies: new agents for cancer detection and targeted therapy

    SciTech Connect

    Baldwin, R.W.; Byers, V.S. )

    1991-01-01

    Antibodies directed against markers on cancer cells are gaining in importance for the purpose of targeting diagnostic and therapeutic agents. In the past, this approach has had very limited success principally because the classical methods for producing antibodies from blood serum of animals immunized with cancer cells or extracts were unsatisfactory. The situation has changed dramatically since 1975 following the design of procedures for 'immortalizing' antibody-producing cells (lymphocytes) by fusing them with cultured myeloma cells to form hybridomas which continuously secrete antibodies. Since these hybridomas produce antibodies coded for by a single antibody-producing cell, the antibodies are called monoclonal. Building on these advances in biomedical research, it is now possible to reproducibly manufacture monoclonal antibodies on a scale suitable for use in cancer detection and therapy.

  2. Boronated monoclonal antibody conjugates for neutron capture therapy

    SciTech Connect

    Borg, D.C.; Elmore, J.J. Jr.; Ferrone, S.

    1986-01-01

    This paper describes the effectiveness of /sup 10/B-labeled monoclonal antibodies against Colo-38 human melanoma in vitro. The authors obtained high boron to antibody ratios while maintaining antibody activity by using dextran intermediate carriers to link /sup 10/B to the antibody. They developed a double cell quasi-competitive binding bioassay to minimize the effects of nonspecific binding of boronated complexes to cells. 1 fig., 2 tabs.

  3. The Architecture and Function of Monoclonal Antibody-Functionalized Mesoporous Silica Nanoparticles Loaded with Mifepristone: Repurposing Abortifacient for Cancer Metastatic Chemoprevention.

    PubMed

    Gao, Yu; Gu, Songen; Zhang, Yingying; Xie, Xiaodong; Yu, Ting; Lu, Yusheng; Zhu, Yewei; Chen, Wenge; Zhang, Huijuan; Dong, Haiyan; Sinko, Patrick J; Jia, Lee

    2016-05-01

    The circulating tumor cells (CTCs) existing in cancer survivors are considered the root cause of cancer metastasis. To prevent the devastating metastasis cascade from initiation, we hypothesize that a biodegradable nanomaterial loaded with the abortifacient mifepristone (MIF) and conjugated with the epithelial cell adhesion molecule antibody (aEpCAM) may serve as a safe and effective cancer metastatic preventive agent by targeting CTCs and preventing their adhesion-invasion to vascular intima. It is demonstrated that MIF-loaded mesoporous silica nanoparticles (MSN) coated with aEpCAM (aE-MSN-M) can specifically target and bind colorectal cancer cells in either cell medium or blood through EpCAM recognition proven by quantitative flow cytometric detection and free aEpCAM competitive assay. The specific binding results in downregulation of the captured cells and drives them into G0/G1 phase primarily attributed to the effect of aEpCAM. The functional nanoparticles significantly inhibit the heteroadhesion between cancer cells and endothelial cells, suggesting the combined inhibition effects of aEpCAM and MIF on E-selectin and ICAM-1 expression. The functionalized nanoparticles circulate in mouse blood long enough to deliver MIF and inhibit lung metastasis. The present proof-of-concept study shows that the aE-MSN-M can prevent cancer metastasis by restraining CTC activity and their adhesion-invasion to vascular intima. PMID:27027489

  4. Characterization and utilization of a monoclonal antibody against pancreatic carcinoma

    SciTech Connect

    Kurtzman, S.H.; Sindelar, W.F.; Atcher, R.W.; Mitchell, J.B.; DeGraff, W.G.; Gamson, J.; Russo, A.; Friedman, A.M.; Hines, J.J.

    1994-10-01

    A monoclonal antibody was produced against a human pancreatic adenocarcinoma line and was found to react with several different human carcinomas by immunoperoxidase staining of fixed tissues. The original cells used to generate the monoclonal antibody were treated with detergent to lyse the cell membrane. A membrane associated protein of molecular weight 35kD was isolated from this detergent lysed preparation and found to be recognized by the monoclonal antibody. The binding constant of the antigen antibody reaction on the cells is 5 x 10{sup {minus}5}. It was further determined that there are 700,000 binding sites per cell. Kinetics of the antigen-antibody reaction under several conditions were also explored.

  5. Palladium-109 labeled anti-melanoma monoclonal antibodies

    DOEpatents

    Srivastava, S.C.; Fawwaz, R.A.; Ferrone, S.

    1984-04-30

    The invention consists of new monoclonal antibodies labelled with Palladium 109, a beta-emitting radionuclide, the method of preparing this material, and its use in the radiotherapy of melanoma. The antibodies are chelate-conjugated and demonstrate a high uptake in melanomas. (ACR)

  6. CD147 monoclonal antibody mediated by chitosan nanoparticles loaded with α-hederin enhances antineoplastic activity and cellular uptake in liver cancer cells

    PubMed Central

    Zhu, Rong; Zhang, Chun-ge; Liu, Yang; Yuan, Zhi-qiang; Chen, Wei-liang; Yang, Shu-di; Li, Ji-zhao; Zhu, Wen-jing; Zhou, Xiao-feng; You, Ben-gang; Zhang, Xue-nong

    2015-01-01

    An antibody that specifically interacts with an antigen could be applied to an active targeting delivery system. In this study, CD147 antibody was coupled with α-hed chitosan nanoparticles (α-Hed-CS-NPs). α-Hed-CS-CD147-NPs were round and spherical in shape, with an average particle size of 148.23 ± 1.75 nm. The half-maximum inhibiting concentration (IC50) of α-Hed-CS-CD147-NPs in human liver cancer cell lines HepG2 and SMMC-7721 was lower than that of free α-Hed and α-Hed-CS-NPs. α-Hed-induced cell death was mainly triggered by apoptosis. The increase in intracellular accumulation of α-Hed-CS-CD147-NPs was also related to CD147-mediated internalization through the Caveolae-dependent pathway and lysosomal escape. The higher targeting antitumor efficacy of α-Hed-CS-CD147-NPs than that α-Hed-CS-NPs was attributed to its stronger fluorescence intensity in the tumor site in nude mice. PMID:26639052

  7. CD147 monoclonal antibody mediated by chitosan nanoparticles loaded with α-hederin enhances antineoplastic activity and cellular uptake in liver cancer cells.

    PubMed

    Zhu, Rong; Zhang, Chun-ge; Liu, Yang; Yuan, Zhi-qiang; Chen, Wei-liang; Yang, Shu-di; Li, Ji-zhao; Zhu, Wen-jing; Zhou, Xiao-feng; You, Ben-gang; Zhang, Xue-nong

    2015-01-01

    An antibody that specifically interacts with an antigen could be applied to an active targeting delivery system. In this study, CD147 antibody was coupled with α-hed chitosan nanoparticles (α-Hed-CS-NPs). α-Hed-CS-CD147-NPs were round and spherical in shape, with an average particle size of 148.23 ± 1.75 nm. The half-maximum inhibiting concentration (IC50) of α-Hed-CS-CD147-NPs in human liver cancer cell lines HepG2 and SMMC-7721 was lower than that of free α-Hed and α-Hed-CS-NPs. α-Hed-induced cell death was mainly triggered by apoptosis. The increase in intracellular accumulation of α-Hed-CS-CD147-NPs was also related to CD147-mediated internalization through the Caveolae-dependent pathway and lysosomal escape. The higher targeting antitumor efficacy of α-Hed-CS-CD147-NPs than that α-Hed-CS-NPs was attributed to its stronger fluorescence intensity in the tumor site in nude mice. PMID:26639052

  8. A perspective of monoclonal antibodies: Past, present, and future

    SciTech Connect

    DeLand, F.H. )

    1989-07-01

    In 1975, the development of the technique to produce monoclonal antibodies revolutionized the approach to cancer detection and therapy. Hundreds of monoclonal antibodies to the epitopes of tumor cells have been produced, providing more specific tools for probing the cellular elements of cancer. At the same time, these tools have disclosed greater complexity in the character of these cells and stimulated further investigation. Although there are antibodies to specific epitopes of neoplastic cells, this purity has not provided the improved detection and therapy of cancer first expected. Technical manipulations have provided limited improvement in results, but more sophisticated techniques, such as biologic response modifiers, may be required to attain clinical results that can be universally applied. The intense research in monoclonal antibodies and their application does offer promise that the goal of improved cancer detection and therapy will be forthcoming. 58 references.

  9. Complete De Novo Assembly of Monoclonal Antibody Sequences.

    PubMed

    Tran, Ngoc Hieu; Rahman, M Ziaur; He, Lin; Xin, Lei; Shan, Baozhen; Li, Ming

    2016-01-01

    De novo protein sequencing is one of the key problems in mass spectrometry-based proteomics, especially for novel proteins such as monoclonal antibodies for which genome information is often limited or not available. However, due to limitations in peptides fragmentation and coverage, as well as ambiguities in spectra interpretation, complete de novo assembly of unknown protein sequences still remains challenging. To address this problem, we propose an integrated system, ALPS, which for the first time can automatically assemble full-length monoclonal antibody sequences. Our system integrates de novo sequencing peptides, their quality scores and error-correction information from databases into a weighted de Bruijn graph to assemble protein sequences. We evaluated ALPS performance on two antibody data sets, each including a heavy chain and a light chain. The results show that ALPS was able to assemble three complete monoclonal antibody sequences of length 216-441 AA, at 100% coverage, and 96.64-100% accuracy. PMID:27562653

  10. Complete De Novo Assembly of Monoclonal Antibody Sequences

    PubMed Central

    Tran, Ngoc Hieu; Rahman, M. Ziaur; He, Lin; Xin, Lei; Shan, Baozhen; Li, Ming

    2016-01-01

    De novo protein sequencing is one of the key problems in mass spectrometry-based proteomics, especially for novel proteins such as monoclonal antibodies for which genome information is often limited or not available. However, due to limitations in peptides fragmentation and coverage, as well as ambiguities in spectra interpretation, complete de novo assembly of unknown protein sequences still remains challenging. To address this problem, we propose an integrated system, ALPS, which for the first time can automatically assemble full-length monoclonal antibody sequences. Our system integrates de novo sequencing peptides, their quality scores and error-correction information from databases into a weighted de Bruijn graph to assemble protein sequences. We evaluated ALPS performance on two antibody data sets, each including a heavy chain and a light chain. The results show that ALPS was able to assemble three complete monoclonal antibody sequences of length 216–441 AA, at 100% coverage, and 96.64–100% accuracy. PMID:27562653

  11. 90Y-labeled monoclonal antibodies for cancer therapy.

    PubMed

    Washburn, L C; Hwa Sun, T T; Crook, J E; Byrd, B L; Carlton, J E; Hung, Y W; Steplewski, Z S

    1986-01-01

    Monoclonal antibody 17-1A, which has specificity for colorectal carcinoma, was labeled with 90Y (10-20% radiolabeling yield). Tissue distribution studies in tumor-bearing nude mice were carried out. 90Y-labeled 17-1A showed good uptake in the SW 948 colon carcinoma cell line. However, 90Y-labeled A5C3, a monoclonal antihepatitis virus antibody studied as a control, showed similar uptake in this tumor. Neither antibody was taken up well by a WM-9 melanoma. It is believed that the loss of specificity observed is due to the low specific activity of the 90Y-labeled monoclonal antibody preparations used. This hypothesis is supported by radioimmunoassay data. PMID:3793501

  12. Coarse grained modeling of transport properties in monoclonal antibody solution

    NASA Astrophysics Data System (ADS)

    Swan, James; Wang, Gang

    Monoclonal antibodies and their derivatives represent the fastest growing segment of the bio pharmaceutical industry. For many applications such as novel cancer therapies, high concentration, sub-cutaneous injections of these protein solutions are desired. However, depending on the peptide sequence within the antibody, such high concentration formulations can be too viscous to inject via human derived force alone. Understanding how heterogenous charge distribution and hydrophobicity within the antibodies leads to high viscosities is crucial to their future application. In this talk, we explore a coarse grained computational model of therapeutically relevant monoclonal antibodies that accounts for electrostatic, dispersion and hydrodynamic interactions between suspended antibodies to predict assembly and transport properties in concentrated antibody solutions. We explain the high viscosities observed in many experimental studies of the same biologics.

  13. Polymorphism of normal factor IX detected by mouse monoclonal antibodies.

    PubMed Central

    Wallmark, A; Ljung, R; Nilsson, I M; Holmberg, L; Hedner, U; Lindvall, M; Sjögren, H O

    1985-01-01

    Hemophilia B is an X-chromosomal recessive disease due to deficiency of coagulation factor IX. Three monoclonal antibodies against factor IX were prepared and used to develop immunoradiometric assays (IRMAs) of factor IX antigen (IX-Ag). IX-Ag was measured in 65 normal individuals with one IRMA based on polyclonal anti-IX antibodies and two IRMAs based on three monoclonal anti-IX antibodies. One of the monoclonal antibodies differed in specificity since it neutralized less than 50% of the clotting activity of factor IX (IX-C), whereas the other two monoclonal antibodies neutralized 80-95%. When the former antibody was used as the solid phase in IRMA, two groups of normal individuals were distinguished: group A with measurable IX-Ag, and group B without demonstrable IX-Ag. There were no differences between the groups either in IX-C or in IX-Ag measured with polyclonal antibodies. A subgroup comprising only women could be distinguished in group A, in whom intermediate IX-Ag concentrations were found. Family studies showed the group B variant of normal factor IX to be transmitted according to the pattern of X-linked recessive inheritance. The allelic frequency of group A was 0.66, and that of group B was 0.34. PMID:3873655

  14. Improved iodine radiolabels for monoclonal antibody therapy.

    PubMed

    Stein, Rhona; Govindan, Serengulam V; Mattes, M Jules; Chen, Susan; Reed, Linda; Newsome, Guy; McBride, Bill J; Griffiths, Gary L; Hansen, Hans J; Goldenberg, David M

    2003-01-01

    A major disadvantage of (131)iodine (I)-labeled monoclonal antibodies (MAbs) for radioimmunotherapy has been the rapid diffusion of iodotyrosine from target cells after internalization and catabolism of the radioiodinated MAbs. We recently reported that a radioiodinated, diethylenetriaminepentaacetic acid-appended peptide, designated immunomedics' residualizing peptide 1 (IMP-R1), was a residualizing iodine label that overcame many of the limitations that had impeded the development of residualizing iodine for clinical use. To determine the factors governing the therapeutic index of the labeled MAb, as well as the factors required for production of radioiodinated MAb in high yield and with high specific activity, variations in the peptide structure of IMP-R1 were evaluated. A series of radioiodinated, diethylenetriaminepentaacetic acid-appended peptide moieties (IMP-R1 through IMP-R8) that differed in overall hydrophilicity and charge were compared. Radioiodinations of the peptides followed by conjugations to disulfide-reduced RS7 (an anti-epithelial glycoprotein-1 MAb) furnished radioimmunoconjugates in good overall incorporations, with immunoreactivities comparable to that of directly radioiodinated RS7. Specific activities of up to 8 mCi/mg and yields > 80% have been achieved. In vitro processing experiments showed marked increases in radioiodine retention with all of the adducts; radioiodine retention at 45 h was up to 86% greater in cells than with directly iodinated RS7. Each of the (125)I-peptide-RS7 conjugates was compared with (131)I-RS7 (labeled by the chloramine-T method) in paired-label biodistribution studies in nude mice bearing human lung tumor xenografts. All of the residualizing substrates exhibited significantly enhanced retention in tumor in comparison to directly radioiodinated RS7, but the nontarget uptakes differed significantly among the residualizing labels. The best labels were IMP-R4 and IMP-R8, showing superior tumor-to-non-tumor ratios

  15. Veterinary sources of nonrodent monoclonal antibodies: interspecific and intraspecific hybridomas.

    PubMed

    Groves, D J; Morris, B A

    2000-06-01

    The generation of monoclonal antibodies from species other than rats and mice has developed slowly over the last 20 years. The advent of antibody engineering and realization of the advantages of nonmurine antibodies, in terms of their superior affinities and specificities, and their potential as components of human and veterinary therapeutics has increased their relevance recently. There have been significant advances in the development of myeloma and heteromyeloma fusion partners. This is an opportune moment to consolidate experiences of MAb production across the range of species of veterinary interest and place it into context with other developments in the field of monoclonal antibodies. The background to the development of antibodies from species other than the mouse is discussed. The species and antigens used to date are reviewed, as are the methods and results reported. A suggested protocol is provided for first attempts to exploit the huge potential of this aspect of hybridoma technology and suggestions are made for its further expansion. PMID:10952409

  16. Choriocarcinoma: blocking factor and monoclonal antibody iodine 131 imaging

    SciTech Connect

    Pattillo, R.A.; Khazaeli, M.B.; Ruckert, A.C.; Hussa, R.O.; Collier, B.D.; Beierwaltes, W.; Mattingly, R.F.

    1984-04-01

    Postoperative iodine 131 monoclonal antibody localization in metastatic choriocarcinoma was accomplished in this study. The monoclonal antibody was prepared to male choriocarcinoma which cross reacted with gestational choriocarcinoma. The antibody was raised against whole choriocarcinoma cells and human chorionic gonadotropin (hCG) cross reactivity was excluded. The purified antibody was iodinated with /sup 131/I and successfully imaged BeWo choriocarcinoma transplanted in nude mice; however, imaging of choriocarcinoma in a patient was verified only after resection. It is our belief that failure to sufficiently concentrate the antibody in the tumor before operation was due to blocking factor in the serum of the patient. Blocking factor and hCG dropped postoperatively. Blocking factor activity in 15 patients with metastatic trophoblastic disease was monitored and, like hCG, was found to be a sensitive indicator of the presence of disease. Its efficacy may be in the small number of patients without hCG but with persistent disease.

  17. Preparation and identification of anti-rabies virus monoclonal antibodies.

    PubMed

    Wang, Wen-juan; Li, Xiong; Wang, Li-hua; Shan, Hu; Cao, Lei; Yu, Peng-cheng; Tang, Qing; Liang, Guo-dong

    2012-06-01

    To provide a foundation for the development of rapid and specific methods for the diagnosis of rabies virus infection, anti-rabies virus monoclonal antibodies were prepared and rabies virus nucleoprotein and human rabies virus vaccine strain (PV strain) were used as immunogens to immunize 6-8 week old female BALB/c mice. Spleen cells and SP2/0 myeloma cells were fused according to conventional methods: the monoclonal cell strains obtained were selected using the indirect immunofluorescence test; this was followed by preparation of monoclonal antibody ascitic fluid; and finally, systematic identification of subclass, specificity and sensitivity was carried out. Two high potency and specific monoclonal antibodies against rabies virus were obtained and named 3B12 and 4A12, with ascitic fluid titers of 1:8000 and 1:10000, respectively. Both belonged to the IgG2a subclass. These strains secrete potent, stable and specific anti-rabies virus monoclonal antibodies, which makes them well suited for the development of rabies diagnosis reagents. PMID:22684471

  18. Monoclonal antibodies and Fc fragments for treating solid tumors.

    PubMed

    Eisenbeis, Andrea M; Grau, Stefan J

    2012-01-01

    Advances in biotechnology, better understanding of pathophysiological processes, as well as the identification of an increasing number of molecular markers have facilitated the use of monoclonal antibodies and Fc fragments in various fields in medicine. In this context, a rapidly growing number of these substances have also emerged in the field of oncology. This review will summarize the currently approved monoclonal antibodies used for the treatment of solid tumors with a focus on their clinical application, biological background, and currently ongoing trials. PMID:22291463

  19. Monoclonal antibodies and Fc fragments for treating solid tumors

    PubMed Central

    Eisenbeis, Andrea M; Grau, Stefan J

    2012-01-01

    Advances in biotechnology, better understanding of pathophysiological processes, as well as the identification of an increasing number of molecular markers have facilitated the use of monoclonal antibodies and Fc fragments in various fields in medicine. In this context, a rapidly growing number of these substances have also emerged in the field of oncology. This review will summarize the currently approved monoclonal antibodies used for the treatment of solid tumors with a focus on their clinical application, biological background, and currently ongoing trials. PMID:22291463

  20. Identification of mutant monoclonal antibodies with increased antigen binding.

    PubMed Central

    Pollock, R R; French, D L; Gefter, M L; Scharff, M D

    1988-01-01

    Sib selection and an ELISA have been used to isolate hybridoma subclones producing mutant antibodies that bind antigen better than the parental monoclonal antibody. Such mutants arise spontaneously in culture at frequencies of 2.5-5 X 10(-5). The sequences of the heavy and light chain variable regions of the mutant antibodies are identical to that of the parent and the Ka values of the mutants and the parent are the same. The increase in binding is associated with abnormalities of the constant region polypeptide and probably reflect changes in avidity of these antibodies. Images PMID:3267219

  1. MONOCLONAL ANTIBODIES IDENTIFY CONSERVED EPITOPES ON THE POLYHEDRIN OF 'HELIOTHIS ZEA' NUCLEAR POLYHEDROSIS VIRUS

    EPA Science Inventory

    Recent advances in monoclonal antibody techniques have provided an opportunity to simplify the procedures of serological identification of microorganisms. Because monoclonal antibodies are raised against individual antigenic determinants (epitopes), they can be used to screen wit...

  2. Monoclonal Antibodies Attached to Carbon Nanotube Transistors for Paclitaxel Detection

    NASA Astrophysics Data System (ADS)

    Lee, Wonbae; Lau, Calvin; Richardson, Mark; Rajapakse, Arith; Weiss, Gregory; Collins, Philip; UCI, Molecular Biology; Biochemistry Collaboration; UCI, Departments of Physics; Astronomy Collaboration

    Paclitaxel is a naturally-occurring pharmaceutical used in numerous cancer treatments, despite its toxic side effects. Partial inhibition of this toxicity has been demonstrated using weakly interacting monoclonal antibodies (3C6 and 8A10), but accurate monitoring of antibody and paclitaxel concentrations remains challenging. Here, single-molecule studies of the kinetics of antibody-paclitaxel interactions have been performed using single-walled carbon nanotube field-effect transistors. The devices were sensitized with single antibody attachments to record the single-molecule binding dynamics of paclitaxel. This label-free technique recorded a range of dynamic interactions between the antibody and paclitaxel, and it provided sensitive paclitaxel detection for pM to nM concentrations. Measurements with two different antibodies suggest ways of extending this working range and uncovering the mechanistic differences among different antibodies.

  3. Targeted therapeutics for severe refractory asthma: monoclonal antibodies.

    PubMed

    Grainge, Christopher L; Maltby, Steven; Gibson, Peter G; Wark, Peter A B; McDonald, Vanessa M

    2016-07-01

    Severe asthma is a complex multifactorial disease that requires specialist multidisciplinary input for optimal clinical outcomes. Following multidimensional assessment for optimisation of current therapy, self-management skills and comorbidities, all patients should be accurately phenotyped. Only after this assessment has been completed should new monoclonal antibody therapies be considered. In this review, we summarise the new antibody approaches targeting identified pathological pathways in severe refractory asthma. PMID:27018798

  4. Generation of monoclonal antibodies to recombinant vascular endothelial growth factor.

    PubMed

    Shein, S A; Gurina, O I; Leopol'd, A V; Baklaushev, V P; Korchagina, A A; Grinenko, N F; Ivanova, N V; Volgina, N E; Ryabukhin, I A; Chekhonin, V P

    2012-05-01

    Female BALB/c mice were subcutaneously immunized with recombinant VEGF-164. After 3 immunization cycles, splenic B cells from immunized mouse were fused with immortalized myeloma culture SP2/0-Ag14 cells. Screening of hybrid cells producing anti-VEGF antibodies was performed by ELISA and immunocytochemical analysis on cultured C6 glioma cells. Subsequent cloning yielded hybridoma stably expressing monoclonal anti-VEGF antibodies recognizing recombinant and native VEGF. PMID:22808513

  5. Mechanisms of monoclonal antibody stabilization and release from silk biomaterials

    PubMed Central

    Guziewicz, Nicholas A.; Massetti, Andrew J.; Perez-Ramirez, Bernardo J.; Kaplan, David L.

    2013-01-01

    The availability of stabilization and sustained delivery systems for antibody therapeutics remains a major clinical challenge, despite the growing development of antibodies for a wide range of therapeutic applications due to their specificity and efficacy. A mechanistic understanding of protein-matrix interactions is critical for the development of such systems and is currently lacking as a mode to guide the field. We report mechanistic insight to address this need by using well-defined matrices based on silk gels, in combination with a monoclonal antibody. Variables including antibody loading, matrix density, charge interactions, hydrophobicity and water access were assessed to clarify mechanisms involved in the release of antibody from the biomaterial matrix. The results indicate that antibody release is primarily governed by hydrophobic interactions and hydration resistance, which are controlled by silk matrix chemistry, peptide domain distribution and protein density. Secondary ionic repulsions are also critical in antibody stabilization and release. Matrix modification by free methionine incorporation was found to be an effective strategy for mitigating encapsulation induced antibody oxidation. Additionally, these studies highlight a characterization approach to improve the understanding and development of other protein sustained delivery systems, with broad applicability to the rapidly developing monoclonal antibody field. PMID:23859659

  6. Identification of two antigenic determinants in pseudomurein by monoclonal antibodies

    SciTech Connect

    Conway de Macario, E.; Macario, A.J.L.; Kandler, O.; Wolin, M.J.

    1982-01-01

    Pseudomurein is a unique peptidoglycan found only in the wall of methanogenic bacteria (MB) of the family Methanobacteriaceae. Although its chemical composition has recently been determined, its immunologic properties have not been elucidated. Methanobacteriaceae elicit antibodies in rabbits and mice. The authors have produced monoclonal antibodies against the bacteria. Antigenic determinants on the MB's surface were resolved with the monoclonal antibodies by means of inhibition-blocking procedures combined with immunoenzymatic assays devised for the structural analysis of bacterial antigens. One monoclonal antibody against Methanobrevibacter arboriphilus DHl recognized a determinant involving the ..gamma..-Glu-Ala end of the pseudomurein peptide. A second antibody did not react with the above determinant but with another involving N-acetylglucosamine. The latter antibody reacted with the immunizing MB, i.e. Methanobacterium thermoautotrophicum ..delta..H and with another strain of this species, GGl, but it did not react with the rest of the pseudomurein-containing bacteria. The data show that pseudomurein possess at least two different determinants, one in the C-terminus of the peptide moiety and the other in the backbone structure and indicate that the spatial arrangement of the peptidoglycan components is distinctive for the species examined and plays a role in antigenicity.

  7. Humanization of a chicken anti-IL-12 monoclonal antibody.

    PubMed

    Tsurushita, Naoya; Park, Minha; Pakabunto, Kanokwan; Ong, Kelly; Avdalovic, Anamarija; Fu, Helen; Jia, Audrey; Vásquez, Max; Kumar, Shankar

    2004-12-01

    Chicken anti-IL-12 monoclonal antibodies were isolated by phage display using spleen cells from a chicken immunized with human and mouse IL-12 as a source for library construction. One of the chicken monoclonal antibodies, DD2, exhibited binding to both human and mouse IL-12 in the single-chain Fv form and also after conversion to chicken-human chimeric IgG1/lambda antibody. The chicken DD2 variable regions were humanized by transferring their CDRs and several framework amino acids onto human acceptor variable regions. In the Vlambda, six chicken framework amino acids were identified to be important for the conformation of the CDR structure by computer modeling and therefore were retained in the humanized form; likewise, five chicken amino acids in the VH framework regions were retained in the humanized VH. The affinities of humanized DD2 IgG1/lambda to human and mouse IL-12 measured by competitive binding were nearly identical to those of chicken-human chimeric DD2 IgG1/lambda. This work demonstrates that humanization of chicken monoclonal antibodies assisted by computer modeling is possible, leading to a new way to generate therapeutic humanized antibodies against antigens to which the rodent immune system may fail to efficiently raise high affinity antibodies. PMID:15627607

  8. Development and evaluation of monoclonal antibodies for paxilline

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Paxilline (PAX) is a tremorgenic mycotoxin that has been found in perennial ryegrass infected with Acremonium lolii. To facilitate screening for this toxin, four murine monoclonal antibodies (mAbs) were developed. In competitive indirect enzyme-linked immunosorbent assays (CI-ELISAs) the concentrati...

  9. Indium-111 labeled anti-melanoma monoclonal antibodies

    DOEpatents

    Srivastava, S.C.; Fawwaz, R.A.; Ferrone, S.

    1984-04-30

    A monoclonal antibody to a high molecular weight melanoma-associated antigen was chelated and radiolabeled with indium-111. This material shows high affinity for melanoma and thus can be used in the detection, localization and imaging of melanoma. 1 figure.

  10. Bacterial surface antigens defined by monoclonal antibodies: the methanogens

    SciTech Connect

    Conway de Macario, E.; Macario, A.J.L.; Magarinos, M.C.; Jovell, R.J.; Kandler, O.

    1982-01-01

    The methanogens (MB) are unique microbes of great evolutionary interest with applications in biotechnology-bioengineerings and are important in digestive processes. Their cell-wall composition is distinctively different from that of Eubacteria, e.g. the Methanobacteriaceae possess the peptidoglycan pseudomurein rather than murein. The range of cell-wall compositions among MB and their evolutionary and functional significance is not well known. The authors undertook a systematic study of the MB's surface structure using monoclonal antibodies through the following steps: (1) generation of hybridomas that produce antibody to several MB from 3 of their 4 families; (2) development of immunoenzymatic assays for MB's antigens and antibodies; (3) determination of the fine specificity of monoclonal antibodies by inhibition-blocking tests using cell-wall extracts and compounds of known structure; thus a set of monoclonal probes of predetermined specificity was assembled; and (4) resolution of surface determinants of MB representative of the Methanobacteriaceae using the monoclonal probes. Specific markers of MB strains were characterized. Two epitopes were identified within the pseudomurein molecule.

  11. Characterization of monoclonal antibodies produced against Avian metapneumovirus Sybtype C

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Monoclonal antibodies (MAbs) were prepared against avian metapneumovirus (aMPV) subtype C (aMPV/Minnesota/turkey/1a/97). Six MAbs were selected based on ELISA activities and characterized by isotyping, neutralization test, Western blot analysis, and immunohistochemistry (IHC) assay. The results show...

  12. 78 FR 7438 - Prospective Grant of Exclusive License: Development of Human Monoclonal Antibodies Against DR4

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-01

    ... Human Monoclonal Antibodies Against DR4 AGENCY: National Institutes of Health, Public Health Service... Monoclonal Antibodies Against DR4'' (HHS Ref. No. E-158-2010/0) to Customized Biosciences, Inc., which is... relates to the development of two human monoclonal antibodies (mAbs) that bind to death receptor 4...

  13. Monoclonal antibodies produced by muscle after plasmid injection and electroporation.

    PubMed

    Tjelle, Torunn Elisabeth; Corthay, Alexandre; Lunde, Elin; Sandlie, Inger; Michaelsen, Terje E; Mathiesen, Iacob; Bogen, Bjarne

    2004-03-01

    Antibodies are useful for the treatment of a variety of diseases. We here demonstrate that mouse muscle produced monoclonal antibodies (mAb) after a single injection of immunoglobulin genes as plasmid DNA. In vivo electroporation of muscle greatly enhanced antibody production. For chimeric antibodies, levels of 50-200 ng mAb/ml serum were obtained but levels declined after 7-14 days due to an immune response against the xenogeneic parts of the antibody. By contrast, fully mouse antibodies persisted in serum for at least 7 months. mAb produced by the muscle had correct structure, specificity, and biological effector functions. The findings were extended to a larger animal, the sheep, in which mAb serum levels of 30-50 ng/ml were obtained. Sustained levels of serum mAb, induced by single injection of Ig genes and electroporation of muscle cells, may offer significant advantages in the treatment of human diseases. PMID:15006599

  14. Nucleotide sequences of five anti-lysozyme monoclonal antibodies.

    PubMed Central

    Darsley, M J; Rees, A R

    1985-01-01

    The nucleotide sequences of the heavy and light chain immunoglobulin mRNAs derived from five hybridomas (Gloop 1-5) secreting IgGs specific for the loop region of hen egg lysozyme were determined. These monoclonal antibodies recognise three distinct but overlapping epitopes within the loop region. The sequences of two pairs of antibodies with indistinguishable fine specificities were similar in both chains whereas the sequences of antibodies of non-identical specificities were very different. It is proposed that the D-segments expressed in two of the antibodies (Gloop3 and Gloop4) are the products of one, or perhaps two, previously unidentified germ line D-genes. Gloop1 and Gloop2 use a D-segment previously identified in antibodies specific for the hapten 2-phenyloxazolone; however it is recombined in a different reading frame in the anti-lysozyme antibodies, producing a different amino acid sequence. PMID:2410256

  15. Initial Characterization of Monoclonal Antibodies against Human Monocytes

    NASA Astrophysics Data System (ADS)

    Ugolini, Valentina; Nunez, Gabriel; Smith, R. Graham; Stastny, Peter; Capra, J. Donald

    1980-11-01

    Three monoclonal antibodies against human monocytes have been produced by somatic cell fusion. Extensive specificity analysis suggests that these antibodies react with most if not all human peripheral blood monocytes and not with highly purified T or B cells. Initial chemical characterization of the monocyte antigen recognized by two of these antibodies is presented. The molecule is a single polypeptide chain with an apparent molecular weight of 200,000. These reagents should prove useful in the clinical definition of disorders of monocyte differentiation, in studies of monocyte function, and in the elucidation of the genetics and structure of monocyte cell surface antigens.

  16. Monoclonal Antibody Cross-Reactions between Drosophila and Human Brain

    NASA Astrophysics Data System (ADS)

    Miller, Carol A.; Benzer, Seymour

    1983-12-01

    A panel of 146 monoclonal antibodies (MAbs), obtained with Drosophila melanogaster tissue as primary immunogen, was tested for cross-reactivity with the human central nervous system. Sites examined included spinal cord, cerebellum, hippocampus, and optic nerve. Nonnervous tissues tested were liver and lymph node. Approximately half of the antibodies reacted with one or more sites in the human central nervous system, identifying regional, cell class, and subcellular antigens. Some recognized neuronal, glial, or axonal subsets. Immunoblot analysis revealed that some antibodies reacted with similar antigen patterns in both species.

  17. Monkey-derived monoclonal antibodies against Plasmodium falciparum.

    PubMed Central

    Stanley, H A; Reese, R T

    1985-01-01

    A system has been developed that allows efficient production of monkey monoclonal antibodies from owl monkeys. Splenocytes or peripheral blood lymphocytes from monkeys immune to the human malarial parasite, Plasmodium falciparum, were fused with P3X63 Ag8.653 mouse myelomas. The resulting hybridomas were screened by an indirect fluorescent antibody test for the production of monkey monoclonal antibodies (mAb) reactive with P. falciparum. Most of the mAb reacted with the P. falciparum merozoites and immunoprecipitated a parasite-derived glycoprotein having a relative molecular weight of 185,000. These mAb gave a minimum of five different immunoprecipitation patterns, thus demonstrating that a large number of polypeptides obtained when parasitized erythrocytes are solubilized share epitopes with this large glycoprotein. In addition, mAb were obtained that reacted with antigens associated with the infected erythrocyte membrane. One of these mAb bound a Mr 95,000 antigen. Images PMID:3898084

  18. Monoclonal antibodies to the alternative oxidase of higher plant mitochondria

    SciTech Connect

    Elthon, T.E.; Nickels, R.L.; McIntosh, L. )

    1989-04-01

    The higher plant mitochondrial electron transport chain contains, in addition to the cytochrome chain which terminates with cytochrome oxidase, an alternative pathway that terminates with an alternative oxidase. The alternative oxidase of Sauromatum guttatum Schott has recently been identified as a cluster of proteins with apparent M{sub r} of 37, 36, and 35 kilodaltons (kD). Monoclonal antibodies have now been prepared to these proteins and designated as AOA (binding all three proteins of the alternative oxidase cluster), AOU (binding the upper or 37 kD protein), and AOL (binding the lower or 36 and 35 kD proteins). All three antibodies bind to their respective alternative oxidase proteins whether the proteins are in their native or denatured states. AOA and AOU inhibit alternative oxidase activity around 49%, whereas AOL inhibits activity only 14%. When coupled individually to Sepharose 4B, all three monoclonal resins were capable of retaining the entire cluster of alternative oxidase proteins, suggesting that these proteins are physically associated in some manner. The monoclonals were capable of binding similar mitochondrial proteins in a number of thermogenic and nonthermogenic species, indicating that they will be useful in characterizing and purifying the alternative oxidase of different systems. The ability of the monoclonal-Sepharose 4B resins to retain the cluster of previously identified alternative oxidase proteins, along with the inhibition of alternative oxidase activity by these monoclonals, supports the role of these proteins in constituting the alternative oxidase.

  19. Monoclonal IgA Antibodies for Aflatoxin Immunoassays

    PubMed Central

    Ertekin, Özlem; Pirinçci, Şerife Şeyda; Öztürk, Selma

    2016-01-01

    Antibody based techniques are widely used for the detection of aflatoxins which are potent toxins with a high rate of occurrence in many crops. We developed a murine monoclonal antibody of immunoglobulin A (IgA) isotype with a strong binding affinity to aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2) and aflatoxin M1 (AFM1). The antibody was effectively used in immunoaffinity column (IAC) and ELISA kit development. The performance of the IACs was compatible with AOAC performance standards for affinity columns (Test Method: AOAC 991.31). The total binding capacity of the IACs containing our antibody was 111 ng, 70 ng, 114 ng and 73 ng for AFB1, AFB2, and AFG1 andAFG2, respectively. Furthermore, the recovery rates of 5 ng of each AF derivative loaded to the IACs were determined as 104.9%, 82.4%, 85.5% and 70.7% for AFB1, AFB2, AFG1 and AFG2, respectively. As for the ELISA kit developed using non-oriented, purified IgA antibody, we observed a detection range of 2–50 µg/L with 40 min total test time. The monoclonal antibody developed in this research is hitherto the first presentation of quadruple antigen binding IgA monoclonal antibodies in mycotoxin analysis and also the first study of their utilization in ELISA and IACs. IgA antibodies are valuable alternatives for immunoassay development, in terms of both sensitivity and ease of preparation, since they do not require any orientation effort. PMID:27187470

  20. Monoclonal IgA Antibodies for Aflatoxin Immunoassays.

    PubMed

    Ertekin, Özlem; Pirinçci, Şerife Şeyda; Öztürk, Selma

    2016-01-01

    Antibody based techniques are widely used for the detection of aflatoxins which are potent toxins with a high rate of occurrence in many crops. We developed a murine monoclonal antibody of immunoglobulin A (IgA) isotype with a strong binding affinity to aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2) and aflatoxin M1 (AFM1). The antibody was effectively used in immunoaffinity column (IAC) and ELISA kit development. The performance of the IACs was compatible with AOAC performance standards for affinity columns (Test Method: AOAC 991.31). The total binding capacity of the IACs containing our antibody was 111 ng, 70 ng, 114 ng and 73 ng for AFB1, AFB2, and AFG1 andAFG2, respectively. Furthermore, the recovery rates of 5 ng of each AF derivative loaded to the IACs were determined as 104.9%, 82.4%, 85.5% and 70.7% for AFB1, AFB2, AFG1 and AFG2, respectively. As for the ELISA kit developed using non-oriented, purified IgA antibody, we observed a detection range of 2-50 µg/L with 40 min total test time. The monoclonal antibody developed in this research is hitherto the first presentation of quadruple antigen binding IgA monoclonal antibodies in mycotoxin analysis and also the first study of their utilization in ELISA and IACs. IgA antibodies are valuable alternatives for immunoassay development, in terms of both sensitivity and ease of preparation, since they do not require any orientation effort. PMID:27187470

  1. Treatment of leukemia with radiolabeled monoclonal antibodies.

    PubMed

    Sgouros, G; Scheinberg, D A

    1993-01-01

    In contrast to radioimmunotherapy of solid disease, wherein the primary obstacle to success is access of radiolabeled antibody to antigen-positive cells, in the treatment of leukemia delivering a lethal absorbed dose to the isolated cell appears to be the primary obstacle. The isolated cell is defined as one that is exposed only to self-irradiation (from internalized or surface-bound radiolabeled antibody) and to irradiation from free antibody in the blood. It is isolated in the sense that the particulate (beta, electron, alpha) emissions from its nearest neighboring antigen-positive cell do not contribute to its absorbed dose. Disease in the bone marrow and other tissues, since it is confined to a smaller volume, is more easily eradicated because the absorbed dose to a given cell nucleus is enhanced by emissions from adjacent cells (a smaller fraction of the emission energy is 'wasted'). The optimization simulations presented above for the M195 antibody suggest that the optimum dose of antibody that should be administered is that required to yield a concentration within the distribution volume of the antibody that is approximately equal to the concentration of antigen sites as determined by the tumor burden. Although not specifically considered in the modeling example presented above, antibody internalization and catabolism may be expected to play an important role in radioimmunotherapy treatment planning of leukemia. Depending upon the kinetics of internalization and catabolism, the absorbed dose to the red marrow and to antigen-positive cells may be reduced considerably, since catabolism, assuming that it is followed by rapid extrusion of the radioactive label, would decrease the cells' exposure time considerably. The recently demonstrated effectiveness of radioimmunotherapy in certain cases of B-cell lymphoma and in reducing tumor burden in acute myelogenous leukemia suggests that radioimmunotherapy is beginning to fulfill the promise held when it was initially

  2. Cysteinylation of a monoclonal antibody leads to its inactivation.

    PubMed

    McSherry, Troy; McSherry, Jennifer; Ozaeta, Panfilo; Longenecker, Kenton; Ramsay, Carol; Fishpaugh, Jeffrey; Allen, Steven

    2016-01-01

    Post-translational modifications can have a signification effect on antibody stability. A comprehensive approach is often required to best understand the underlying reasons the modification affects the antibody's potency or aggregation state. Monoclonal antibody 001 displayed significant variation in terms of potency, as defined by surface plasmon resonance testing (Biacore), from lot to lot independent of any observable aggregation or degradation, suggesting that a post-translational modification could be driving this variability. Analysis of different antibody lots using analytical hydrophobic interaction chromatography (HIC) uncovered multiple peaks of varying size. Electrospray ionization mass spectrometry (ESI-MS) indicated that the antibody contained a cysteinylation post-translational modification in complementarity-determining region (CDR) 3 of the antibody light chain. Fractionation of the antibody by HIC followed by ESI-MS and Biacore showed that the different peaks were antibody containing zero, one, or two cysteinylation modifications, and that the modification interferes with the ability of the modified antibody arm to bind antigen. Molecular modeling of the modified region shows that this oxidation of an unpaired cysteine in the antibody CDR would block a potential antigen binding pocket, suggesting an inhibition mechanism. PMID:27050640

  3. Molecular specificities of monoclonal antibodies directed against virulent Treponema pallidum.

    PubMed Central

    Marchitto, K S; Selland-Grossling, C K; Norgard, M V

    1986-01-01

    Radioimmunoprecipitation (RIP) and Western blot analyses with specific anti-Treponema pallidum subsp. pallidum monoclonal antibodies were used to identify antigens with apparent masses of 102, 84, 54, 53, 52, 47, 32, 29, and 24 kilodaltons (kDa). Cross-reactivity of these antibodies with T. pallidum subsp. pertenue antigens and lack of cross-reactivity with T. phagedenis biotype Reiter, T. vincentii, T. refringens, T. scoliodontum, and T. denticola were also demonstrated by RIP and Western blot analyses. Reactivities in the T. pallidum immobilization test, along with the RIP of lactoperoxidase-catalyzed iodination products, suggested that the identified antigens were surface associated. The abundance and surface association of the 47- and 84-kDa antigens were supported by reactivity in the microhemagglutination test for T. pallidum and by strong reactivity of monoclonal antibodies upon indirect immunofluorescence assays with rabbit-cultivated T. pallidum subsp. pallidum, respectively, but not with T. phagedenis biotype Reiter. Anti-47-kDa and anti-84-kDa monoclonal antibodies were also reactive in indirect immunofluorescence assays using treponemes found in dark-field-positive smears of human genital ulcers. Images PMID:3510168

  4. The Use of Monoclonal Antibodies in Human Prion Disease

    NASA Astrophysics Data System (ADS)

    Bodemer, Walter

    Detection of PrP and its pathological isoform(s) is the key to understanding the etiology and pathogenesis of transmissible spongiform encephalopathy. There is ample evidence that PrP isoforms constitute a major component of an unknown and perhaps unconventional infectious agent. An etiological relationship between human and zoonotic transmissible spongiform encephalopathies may be revealed with monoclonal antibodies. Knowledge of the conformational transition rendering a nonpathogenic, almost ubiquitous cellular protein into a pathogenic one is crucial to defining pathomechanisms. The stepwise or even continuous formation of pathogenic molecules can be monitored. Any improvement in the early diagnosis could help to conceive new therapeutic measures which are not currently available. Determination of PrP isoforms in tissue, cells, or body fluids may be of prognostic value. Many experimental approaches in molecular medicine and molecular biology of the prion protein already rely on monoclonal antibodies. Recombinant antibodies such as the single-chain Fv may soon replace traditional hybridoma techniques. Binding affinity can easily be manipulated by a number of techniques, including in vitro mutagenesis - a step which could never be carried out using the traditional hybridoma technology. Monoclonal antibodies are and will remain an essential support for ongoing research on the prion protein in general and on the unconventional infectious prions.

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

    PubMed

    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

  6. Human antiglioma monoclonal antibodies from patients with astrocytic tumors.

    PubMed

    Dan, M D; Schlachta, C M; Guy, J; McKenzie, R G; Dorscheid, D R; Sandor, V A; Villemure, J G; Price, G B

    1992-04-01

    The current management of malignant gliomas is unsatisfactory compared to that of other solid tumors; the expected median survival period is less than 1 year with the patient undergoing conventional surgery, radiotherapy, and chemotherapy treatment. Immunological reagents could be a useful adjunct. Human monoclonal antibodies derived from patients with astrocytic tumors might recognize subtle antigenic specificities that would differ from those recognized by xenogeneic (murine) systems. Five hybridomas, designated as BT27/1A2, BT27/2A3, BT32/A6, BT34/A5, and BT54/B8, were produced from the fusion of peripheral blood lymphocytes of four patients with astrocytic tumors to the human myeloma-like cell line TM-H2-SP2. This cell line has a 46, XX karyotype and is negative for hypoxanthine guanine phosphoribosyltransferase. All five human monoclonal antibodies produced 2.4 to 44 micrograms/ml of immunoglobulin M, had a similar but not identical pattern of reactivity against a panel of human tumor cell lines, and failed to react with normal human astrocytes. Labeling of four neuroectodermal tumor explant cultures by BT27/2A3 was demonstrated by flow cytometry. Karyotyping of three of the five hybridomas demonstrated that two were pseudodiploid (2-3n) and one hypodiploid (less than 2n). The monoclonality of the hybridomas was evaluated by Southern blot analysis of JH gene rearrangements, revealing two types of rearrangements for each hybridoma, both consistent with monoclonality. Preliminary antigen characterization indicated that at least four of the five human monoclonal antibodies were directed to cell-surface glycolipids. PMID:1545260

  7. Monoclonal Antibody Drugs for Systemic Lupus Erythematosus.

    PubMed

    Kamenarska, Zornitsa G; Hristova, Maria H; Vinkov, Anton I; Dourmishev, Lyubomir A

    2015-01-01

    Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease which engages most of the immune cells in its development. Various studies concerning the application of antibodies against TNF-α, BlyS, CD20, CD22, IL-6R and complement factors in treatment of SLE have been recently conducted and in spite of the good results reported by some of them, no definite conclusion on their risk-benefit profile can be drawn. The current review summarizes the results obtained in the field and reveals the perspectives for the development of new and more effective strategies for SLE treatment in combination with other immunomodulating drugs. PMID:26933777

  8. Adverse Events of Monoclonal Antibodies Used for Cancer Therapy

    PubMed Central

    Guan, Mei; Zhou, Yan-Ping; Sun, Jin-Lu; Chen, Shu-Chang

    2015-01-01

    In 1997, the first monoclonal antibody (MoAb), the chimeric anti-CD20 molecule rituximab, was approved by the US Food and Drug administration for use in cancer patients. Since then, the panel of MoAbs that are approved by international regulatory agencies for the treatment of hematopoietic and solid malignancies has continued to expand, currently encompassing a stunning amount of 20 distinct molecules for 11 targets. We provide a brief scientific background on the use of MoAbs in cancer therapy, review all types of monoclonal antibodies-related adverse events (e.g., allergy, immune-related adverse events, cardiovascular adverse events, and pulmonary adverse events), and discuss the mechanism and treatment of adverse events. PMID:26075239

  9. [Increases in pharmaceutical expenditures of PHI by monoclonal antibodies].

    PubMed

    Wild, F

    2013-06-01

    The dynamics of one of the most innovative segments of health care and its impact on pharmaceutical expenditure of private health insurance (PHI) is examined on the basis of drug prescription data from private health insurance companies. The study shows that the increase in pharmaceutical expenditure can be explained partly by the new treatment possibilities available with monoclonal antibodies. The per capita expenditure on drugs with monoclonal antibodies increased by 255% from 2006 to 2010 in private health insurance, while the corresponding expenditure of all pharmaceuticals has risen by only 19% in the same period. In the coming years, growth on this scale will be a challenge for all payers in the health system. PMID:23926705

  10. Adverse events of monoclonal antibodies used for cancer therapy.

    PubMed

    Guan, Mei; Zhou, Yan-Ping; Sun, Jin-Lu; Chen, Shu-Chang

    2015-01-01

    In 1997, the first monoclonal antibody (MoAb), the chimeric anti-CD20 molecule rituximab, was approved by the US Food and Drug administration for use in cancer patients. Since then, the panel of MoAbs that are approved by international regulatory agencies for the treatment of hematopoietic and solid malignancies has continued to expand, currently encompassing a stunning amount of 20 distinct molecules for 11 targets. We provide a brief scientific background on the use of MoAbs in cancer therapy, review all types of monoclonal antibodies-related adverse events (e.g., allergy, immune-related adverse events, cardiovascular adverse events, and pulmonary adverse events), and discuss the mechanism and treatment of adverse events. PMID:26075239

  11. Biosimilar monoclonal antibodies in lymphoma: a critical appraisal.

    PubMed

    Rioufol, Catherine; Salles, Gilles

    2015-05-01

    Rituximab, an anti-CD20 monoclonal antibody, revolutionized the treatment of lymphoma. Although newer generation anti-CD20 monoclonal antibodies are being examined, patent expiries and patient demand have fueled the development of rituximab biosimilars. The development of such agents is both an important and difficult undertaking. By definition, although they aim to have safety and efficacy comparable with their reference agents, biosimilars are not exact replicas of those agents, and small changes in nonclinical and preclinical properties may ultimately affect in vivo activity. Consideration must be given to the complex mechanisms of action, sensitive patient populations that may be treated, and appropriate clinical trial endpoints. Furthermore, extrapolation of indications is multifaceted, deserving close examination. This review represents a critical look at biosimilars in lymphoma and their safety, efficacy and long-term effects on patient outcomes. PMID:25818308

  12. Immunohistochemical identification of cytotoxic lymphocytes using human perforin monoclonal antibody.

    PubMed Central

    Hameed, A.; Olsen, K. J.; Cheng, L.; Fox, W. M.; Hruban, R. H.; Podack, E. R.

    1992-01-01

    Perforin is a potent cytolytic pore-forming protein expressed in cytoplasmic granules of cytotoxic T lymphocytes and natural killer cells. A new monoclonal antibody raised against human perforin was used to detect both in vitro and in vivo perforin expression in cytotoxic cells. Immunohistochemical analysis of human peripheral blood mononuclear cells cultured in recombinant interleukin-2 (rIL-2) showed strong granular cytoplasmic staining of the IL-2 activated cytotoxic cells. Fresh-frozen tissue sections from patients with heart allograft rejection were also stained. Strong granular cytoplasmic staining of the mononuclear inflammatory infiltrate characteristic for perforin in cardiac allograft rejection was observed. The detection and quantitative analysis of perforin-associated cytotoxic cells by the human anti-perforin monoclonal antibody will help to evaluate the significance of these functionally distinct cytotoxic cells in human tissue. Images Figure 1 PMID:1374586

  13. Human tumor antigens identified with monoclonal antibodies

    SciTech Connect

    AlSedairy, S.T.

    1987-01-01

    MoAbLc1 (IgM) and MoAbLc2 (IgG/sub 2a/) were produced against human lung carcinoma cell line (ChaGo). Lc1 recognizes a approx. = 330-kd/approx. = 310-kd glycoprotein complexes, and Lc2 recognizes a approx. = 60-kd/approx. = 47-kd protein complex. With a panel of cell lines of different tissue origin, Lc1 showed a more restricted reactivity to ChaGo; it cross-reacted with another lung carcinoma cell line (SK-Lc-2) and two breast carcinoma cell lines, but failed to react with cell lines of fetal lung, of colon, esophageal, prostate, stomach, and ovarian carcinomas, of B and T lymphoblastoid cells, neuroblastomas, glioblastoma, astrocytoma, and human peripheral blood lymphocytes. New and improved methods were developed for the production of indium-111-labeled MoAbs for tumor imaging. To facilitate the application of bicyclic anhydride diethylenetriaminepentaacetic acid (BADTPA) to In-111 labeling of antibodies, we have modified the original method by using C-14-labeled BADTPA, which allows precise quantitation of DTPA molecules incorporated. A new heterobifunctional reagent, 2,6-dioxo-N-(carboxyl)morpholine (DCM) was synthesized for chelating In-111 to MoAbs, and demonstrated higher retention of immunoreactivity of the labeled antibody.

  14. Monoclonal antibodies to the two most basic papaya proteinases.

    PubMed

    Goodenough, P W; Kilshaw, P J; McEwan, F; Owen, A J

    1986-08-01

    The proteinases from Carica papaya include papain, isoenzymes of chymopapain and two proteinases A and B distinguished by their unusually high pI. The identity of one of the most basic proteinases has been questioned. The present report describes the preparation and characterisation of two monoclonal antibodies that react specifically with papaya proteinases A and B respectively and a third that identifies a common structural feature found in papain and proteinase A. PMID:3545314

  15. Monoclonal antibodies directed against surface molecules of multicell spheroids

    NASA Technical Reports Server (NTRS)

    Martinez, Andrew O.

    1993-01-01

    The objective of this project is to generate a library of monoclonal antibodies (MAbs) to surface molecules of mammalian tumor and transformed cells grown as multicell spheroids (MCS). These MCS are highly organized, three dimensional multicellular structures which exhibit many characteristics of in vivo organized tissues not found in conventional monolayer or suspension culture; therefore, MCS make better in vitro model systems to study the interactions of mammalian cells. Additionally, they provide a functional assay for surface adhesion molecules.

  16. Positron emission tomographic imaging of tumors using monoclonal antibodies

    SciTech Connect

    Zalutsky, M.R.

    1992-08-01

    This research project is developing methods for utilizing positron emission tomography (PET) to increase the clinical potential of radiolabeled monoclonal antibodies (MAbs). This report describes the development of methods for labeling MAbs and their fragments with positron-emitting halogen nuclides, fluorine-18 and iodine-124. These nulides were selected because of the widespread availability of F-18 and because of our extensive experience in the development of new protein radiohalogenation methods.

  17. Recovery and purification process development for monoclonal antibody production

    PubMed Central

    Ma, Junfen; Winter, Charles; Bayer, Robert

    2010-01-01

    Hundreds of therapeutic monoclonal antibodies (mAbs) are currently in development, and many companies have multiple antibodies in their pipelines. Current methodology used in recovery processes for these molecules are reviewed here. Basic unit operations such as harvest, Protein A affinity chromatography and additional polishing steps are surveyed. Alternative processes such as flocculation, precipitation and membrane chromatography are discussed. We also cover platform approaches to purification methods development, use of high throughput screening methods, and offer a view on future developments in purification methodology as applied to mAbs. PMID:20647768

  18. Current status of tumor imaging with monoclonal antibodies.

    PubMed

    Blend, M J

    1991-12-01

    Although the full potential of MoAb imaging has yet to be realized, technologic advances continue with great intensity at a number of academic and industrial research institutions. Continuous production of MoAbs will eventually yield a variety of highly specific antibodies and novel approaches for improving cancer detection. As new diagnostic and therapeutic methods continue to be developed, MoAbs will begin to play a major role as targeted carriers, provided adequate funding from industry and government can be readily obtained. At present, the future of monoclonal antibodies in diagnosis and therapy for cancer patients appears promising. PMID:1790666

  19. Monoclonal antibodies for medical oncology: a few critical perspectives.

    PubMed

    Belda-Iniesta, Cristóbal; Ibáñez de Cáceres, Inmaculada; de Castro, Javier

    2011-02-01

    Incorporation of antibodies as weapons for cancer therapy has meant a turning point in the survival, clinical and radiological response of many oncology patients. These drugs are effective, well designed missiles that either alone or in combination with chemotherapy are unavoidable weapons for breast, lung and colon cancer as well as for haematological tumours. In addition, incoming monoclonal antibodies (mAbs) and folder-like proteins will be incorporated into clinical practice in the near future. This review aims to discuss a few imminent indications of current mAbs that are used for solid tumours and to briefly introduce future mAbs to the reader. PMID:21324795

  20. Characterization of rabbit cells by monoclonal and polyclonal antibodies.

    PubMed Central

    Ponsard, D C; Cinader, B; Chou, C T; Dubiski, S

    1986-01-01

    Reagents for the identification of rabbit cell markers have been developed at a relatively slow rate. In this paper, rabbit cells are being characterized by polyclonal antibodies against a T-cell antigen (RTLA), a B-cell antigen (RABELA) and an analogue of murine Ia antigen. A number of monoclonal antibodies, specific for lymphocytes and/or bone marrow and/or polymorphonuclear leucocytes, have been used for the analysis of cells with identifiable membrane antigens. Populations that have cells with two of the above antigens in the membranes were identified. To these ends, complement-mediated cell kill by antisera alone and in mixtures was employed. PMID:3489667

  1. Production of Monoclonal Antibodies in Plants for Cancer Immunotherapy

    PubMed Central

    Moussavou, Ghislain; Ko, Kisung; Lee, Jeong-Hwan; Choo, Young-Kug

    2015-01-01

    Plants are considered as an alternative platform for recombinant monoclonal antibody (mAb) production due to the improvement and diversification of transgenic techniques. The diversity of plant species offers a multitude of possibilities for the valorization of genetic resources. Moreover, plants can be propagated indefinitely, providing cheap biomass production on a large scale in controlled conditions. Thus, recent studies have shown the successful development of plant systems for the production of mAbs for cancer immunotherapy. However, their several limitations have to be resolved for efficient antibody production in plants. PMID:26550566

  2. [Continuous perfusion culture hybridoma cells for production of monoclonal antibody].

    PubMed

    Mi, Li; Li, Ling; Feng, Qiang; Yu, Xiao-Ling; Chen, Zhi-Nan

    2002-05-01

    Hybridoma cells were cultured by continuous perfusion in Fibra-Cel of 5L packed-bed bioreactor for 22 days in low serum or serum-free media. The corresponded amino acids were fed and serum concentration was decreased by analyzing glucose concentration, oxygen uptake rate, secretary antibody amount and amino acids concentration in culture supernatant. Comparing with continuous perfusion culture that amino acids were not fed, antibody amount of production was increased about 2-3 times. The inoculated cell density was 2.5 x 10(5) cells/mL, while the final cell density was 8.79 x 10(8) cells/mL. Antibody production was reached 295 mg/L/d at average level, and the highest level was reached 532 mg/L/d. These results provided a primary mode of enlarge culture for monoclonal antibody industralization. PMID:12192875

  3. Monoclonal Antibodies to Shigella Lipopolysaccharide Are Useful for Vaccine Production.

    PubMed

    Lin, Jisheng; Smith, Mark A; Benjamin, William H; Kaminski, Robert W; Wenzel, Heather; Nahm, Moon H

    2016-08-01

    There is a significant need for an effective multivalent Shigella vaccine that targets the most prevalent serotypes. Most Shigella vaccines under development utilize serotype-specific lipopolysaccharides (LPSs) as a major component based on protection and epidemiological data. As vaccine formulations advance from monovalent to multivalent, assays and reagents need to be developed to accurately and reproducibly quantitate the amount of LPSs from multiple serotypes in the final product. To facilitate this effort, we produced 36 hybridomas that secrete monoclonal antibodies (MAbs) against the O antigen on the LPS from Shigella flexneri 2a, Shigella flexneri 3a, and Shigella sonnei We used six of these monoclonal antibodies for an inhibition enzyme-linked immunosorbent assay (iELISA), measuring LPSs with high sensitivity and specificity. It was also demonstrated that the Shigella serotype-specific MAbs were useful for bacterial surface staining detected by flow cytometry. These MAbs are also useful for standardizing the serum bactericidal assay (SBA) for Shigella Functional assays, such as the in vitro bactericidal assay, are necessary for vaccine evaluation and may serve as immunological correlates of immunity. An S. flexneri 2a-specific monoclonal antibody killed S. flexneri 2b isolates, suggesting that S. flexneri 2a LPS may induce cross-protection against S. flexneri 2b. Overall, the Shigella LPS-specific MAbs described have potential utility to the vaccine development community for assessing multivalent vaccine composition and as a reliable control for multiple immunoassays used to assess vaccine potency. PMID:27280622

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

    PubMed

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

    2016-08-25

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

  5. Isolation of human monoclonal antibodies from peripheral blood B cells.

    PubMed

    Huang, Jinghe; Doria-Rose, Nicole A; Longo, Nancy S; Laub, Leo; Lin, Chien-Li; Turk, Ellen; Kang, Byong H; Migueles, Stephen A; Bailer, Robert T; Mascola, John R; Connors, Mark

    2013-10-01

    Isolation of monoclonal antibodies is an important technique for understanding the specificities and characteristics of antibodies that underlie the humoral immune response to a given antigen. Here we describe a technique for isolating monoclonal antibodies from human peripheral blood mononuclear cells. The protocol includes strategies for the isolation of switch-memory B cells from peripheral blood, the culture of B cells, the removal of the supernatant for screening and the lysis of B cells in preparation for immunoglobulin heavy-chain and light-chain amplification and cloning. We have observed that the addition of cytokines IL-2, IL-21 and irradiated 3T3-msCD40L feeder cells can successfully stimulate switch-memory B cells to produce high concentrations of IgG in the supernatant. The supernatant may then be screened by appropriate assays for binding or for other functions. This protocol can be completed in 2 weeks. It is adaptable to use in other species and enables the efficient isolation of antibodies with a desired functional characteristic without prior knowledge of specificity. PMID:24030440

  6. Monkey-derived monoclonal antibodies against Plasmodium falciparum

    SciTech Connect

    Stanley, H.A.; Reese, R.T.

    1985-09-01

    A system has been developed that allows efficient production of monkey monoclonal antibodies from owl monkeys. Splenocytes or peripheral blood lymphocytes from monkeys immune to the human malarial parasite, Plasmodium falciparum, were fused with P3X63 Ag8.653 mouse myelomas. The resulting hybridomas were screened by an indirect fluorescent antibody test for the production of monkey monoclonal antibodies (mAb) reactive with P. falciparum. Most of the mAb reacted with the P. falciparum merozoites and immunoprecipitated a parasite-derived glycoprotein having a relative molecular weight of 185,000. These mAb gave a minimum of five different immunoprecipitation patterns, thus demonstrating that a large number of polypeptides obtained when parasitized erythrocytes are solubilized share epitopes with this large glycoprotein. In addition, mAb were obtained that reacted with antigens associated with the infected erythrocyte membrane. One of these mAb bound a M/sub r/ 95,000 antigen. Radioimmunoprecipitation assays using /sup 125/T-antibodies were done.

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

    PubMed Central

    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. PMID:25996084

  8. Analysis of acetylcholine receptor phosphorylation sites using antibodies to synthetic peptides and monoclonal antibodies.

    PubMed Central

    Safran, A; Neumann, D; Fuchs, S

    1986-01-01

    Three peptides corresponding to residues 354-367, 364-374, 373-387 of the acetylcholine receptor (AChR) delta subunit were synthesized. These peptides represent the proposed phosphorylation sites of the cAMP-dependent protein kinase, the tyrosine-specific protein kinase and the calcium/phospholipid-dependent protein kinase respectively. Using these peptides as substrates for phosphorylation by the catalytic subunit of cAMP-dependent protein kinase it was shown that only peptides 354-367 was phosphorylated whereas the other two were not. These results verify the location of the cAMP-dependent protein kinase phosphorylation site within the AChR delta subunit. Antibodies elicited against these peptides reacted with the delta subunit. The antipeptide antibodies and two monoclonal antibodies (7F2, 5.46) specific for the delta subunit were tested for their binding to non-phosphorylated receptor and to receptor phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. Antibodies to peptide 354-367 were found to react preferentially with non-phosphorylated receptor whereas the two other anti-peptide antibodies bound equally to phosphorylated and non-phosphorylated receptors. Monoclonal antibody 7F2 reacted preferentially with the phosphorylated form of the receptor whereas monoclonal antibody 5.46 did not distinguish between the two forms. Images Fig. 2. Fig. 4. Fig. 5. PMID:3816758

  9. Using monoclonal antibodies to prevent mucosal transmission of epidemic infectious diseases.

    PubMed Central

    Zeitlin, L.; Cone, R. A.; Whaley, K. J.

    1999-01-01

    Passive immunization with antibodies has been shown to prevent a wide variety of diseases. Recent advances in monoclonal antibody technology are enabling the development of new methods for passive immunization of mucosal surfaces. Human monoclonal antibodies, produced rapidly, inexpensively, and in large quantities, may help prevent respiratory, diarrheal, and sexually transmitted diseases on a public health scale. PMID:10081672

  10. [Progress in preparation of small monoclonal antibodies of knock out technique].

    PubMed

    Liu, Jing; Mao, Xin-min; Li, Lin-lin; Li, Xin-xia; Wang, Ye; Lan, Yi

    2015-10-01

    With the application of monoclonal antibody technology more and more widely, its production technology is becoming more and more perfect. Small molecule monoclonal antibody technology is becoming a hot research topic for people. The application of traditional Chinese medicine small molecule monoclonal antibody technology has been more and more widely, the technology for effective Chinese medicine component knockout provide strong technical support. The preparation of monoclonal antibodies and small molecule knockout technology are reviewed in this paper. The preparation of several steps, such as: in the process of preparation of antigen, hapten carrier coupling, coupling ratio determination and identification of artificial antigen and establishment of animal immunization and hybridoma cell lines of monoclonal antibody, the large-scale preparation; small molecule monoclonal antibody on Immune in affinity chromatography column method is discussed in detail. The author believes that this technology will make the traditional Chinese medicine research on a higher level, and improve the level of internationalization of Chinese medicine research. PMID:26975094

  11. Macaque Monoclonal Antibodies Targeting Novel Conserved Epitopes within Filovirus Glycoprotein

    PubMed Central

    Keck, Zhen-Yong; Enterlein, Sven G.; Howell, Katie A.; Vu, Hong; Shulenin, Sergey; Warfield, Kelly L.; Froude, Jeffrey W.; Araghi, Nazli; Douglas, Robin; Biggins, Julia; Lear-Rooney, Calli M.; Wirchnianski, Ariel S.; Lau, Patrick; Wang, Yong; Herbert, Andrew S.; Dye, John M.; Glass, Pamela J.; Holtsberg, Frederick W.; Foung, Steven K. H.

    2015-01-01

    ABSTRACT Filoviruses cause highly lethal viral hemorrhagic fever in humans and nonhuman primates. Current immunotherapeutic options for filoviruses are mostly specific to Ebola virus (EBOV), although other members of Filoviridae such as Sudan virus (SUDV), Bundibugyo virus (BDBV), and Marburg virus (MARV) have also caused sizeable human outbreaks. Here we report a set of pan-ebolavirus and pan-filovirus monoclonal antibodies (MAbs) derived from cynomolgus macaques immunized repeatedly with a mixture of engineered glycoproteins (GPs) and virus-like particles (VLPs) for three different filovirus species. The antibodies recognize novel neutralizing and nonneutralizing epitopes on the filovirus glycoprotein, including conserved conformational epitopes within the core regions of the GP1 subunit and a novel linear epitope within the glycan cap. We further report the first filovirus antibody binding to a highly conserved epitope within the fusion loop of ebolavirus and marburgvirus species. One of the antibodies binding to the core GP1 region of all ebolavirus species and with lower affinity to MARV GP cross neutralized both SUDV and EBOV, the most divergent ebolavirus species. In a mouse model of EBOV infection, this antibody provided 100% protection when administered in two doses and partial, but significant, protection when given once at the peak of viremia 3 days postinfection. Furthermore, we describe novel cocktails of antibodies with enhanced protective efficacy compared to individual MAbs. In summary, the present work describes multiple novel, cross-reactive filovirus epitopes and innovative combination concepts that challenge the current therapeutic models. IMPORTANCE Filoviruses are among the most deadly human pathogens. The 2014-2015 outbreak of Ebola virus disease (EVD) led to more than 27,000 cases and 11,000 fatalities. While there are five species of Ebolavirus and several strains of marburgvirus, the current immunotherapeutics primarily target Ebola virus

  12. Structural Basis of Human Parechovirus Neutralization by Human Monoclonal Antibodies

    PubMed Central

    Shakeel, Shabih; Westerhuis, Brenda M.; Ora, Ari; Koen, Gerrit; Bakker, Arjen Q.; Claassen, Yvonne; Wagner, Koen; Beaumont, Tim; Wolthers, Katja C.

    2015-01-01

    ABSTRACT Since it was first recognized in 2004 that human parechoviruses (HPeV) are a significant cause of central nervous system and neonatal sepsis, their clinical importance, primarily in children, has started to emerge. Intravenous immunoglobulin treatment is the only treatment available in such life-threatening cases and has given moderate success. Direct inhibition of parechovirus infection using monoclonal antibodies is a potential treatment. We have developed two neutralizing monoclonal antibodies against HPeV1 and HPeV2, namely, AM18 and AM28, which also cross-neutralize other viruses. Here, we present the mapping of their epitopes using peptide scanning, surface plasmon resonance, fluorescence-based thermal shift assays, electron cryomicroscopy, and image reconstruction. We determined by peptide scanning and surface plasmon resonance that AM18 recognizes a linear epitope motif including the arginine-glycine-aspartic acid on the C terminus of capsid protein VP1. This epitope is normally used by the virus to attach to host cell surface integrins during entry and is found in 3 other viruses that AM18 neutralizes. Therefore, AM18 is likely to cause virus neutralization by aggregation and by blocking integrin binding to the capsid. Further, we show by electron cryomicroscopy, three-dimensional reconstruction, and pseudoatomic model fitting that ordered RNA interacts with HPeV1 VP1 and VP3. AM28 recognizes quaternary epitopes on the capsid composed of VP0 and VP3 loops from neighboring pentamers, thereby increasing the RNA accessibility temperature for the virus-AM28 complex compared to the virus alone. Thus, inhibition of RNA uncoating probably contributes to neutralization by AM28. IMPORTANCE Human parechoviruses can cause mild infections to severe diseases in young children, such as neonatal sepsis, encephalitis, and cardiomyopathy. Intravenous immunoglobulin treatment is the only treatment available in such life-threatening cases. In order to develop more

  13. Poliovirus neutralization epitopes: analysis and localization with neutralizing monoclonal antibodies.

    PubMed Central

    Emini, E A; Jameson, B A; Lewis, A J; Larsen, G R; Wimmer, E

    1982-01-01

    Two hybridomas (H3 and D3) secreting monoclonal neutralizing antibody to intact poliovirus type 1 (Mahoney strain) were established. Each antibody bound to a site qualitatively different from that to which the other antibody bound. The H3 site was located on intact virions and, to a lesser extent, on 80S naturally occurring empty capsids and 14S precursor subunits. The D3 site was found only on virions and empty capsids. Neither site was expressed on 80S heat-treated virions. The antibodies did not react with free denatured or undenatured viral structural proteins. Viral variants which were no longer capable of being neutralized by either one or the other antibody were obtained. Such variants arose during normal cell culture passage of wild-type virus and were present in the progeny viral population on the order of 10(-4) variant per wild-type virus PFU. Toluene-2,4-diisocyanate, a heterobifunctional covalent cross-linking reagent, was used to irreversibly bind the F(ab) fragments of the two antibodies to their respective binding sites. In this way, VP1 was identified as the structural protein containing both sites. PMID:6183443

  14. Labeling of cerebral amyloid in vivo with a monoclonal antibody.

    PubMed

    Walker, L C; Price, D L; Voytko, M L; Schenk, D B

    1994-07-01

    We assessed the ability of a murine monoclonal antibody to bind selectively to beta-amyloid in the brains of living nonhuman primates. To circumvent the blood-brain barrier, we injected unlabeled antibody 10D5 (murine whole IgG1 and/or Fab fragments) into the cerebrospinal fluid of the cisterna magna in three aged monkeys. A control animal was given an intracisternal injection of nonimmune mouse whole IgG plus Fab. Twenty-four hours later, the animals were perfused and prepared for immunohistochemical detection of bound murine immunoglobulin in brain. All three experimental animals showed selective binding of 10D5 to approximately 5-15% of amyloid deposits in cerebral cortex, primarily near the cortical surface. There was no labeling in the control animal. In vivo-labeled deposits were confirmed to be beta-amyloid by electron microscopy and by in vitro immunohistochemistry in adjacent sections. The animals tolerated the injection well, although some polymorphonuclear leukocytes infiltrated portions of the subarachnoid space and superficial neocortex. These results provide the first demonstration that it may be feasible to selectively direct a tagged monoclonal antibody to beta-amyloid in the brain for therapeutic or diagnostic purposes. With enhancement of labeling efficiency, the method also may be useful for studying the progression of beta-amyloidosis in experimental animals using emission tomography. PMID:8021711

  15. Endotoxin reduction in monoclonal antibody preparations using arginine.

    PubMed

    Ritzén, Ulrika; Rotticci-Mulder, Joke; Strömberg, Patrik; Schmidt, Stefan R

    2007-09-01

    A monoclonal antibody preparation was found to be contaminated with endotoxin. Several commercial endotoxin removal steps were attempted but failed to produce a significant reduction due to the fact that the endotoxin was associated with the antibody. Here, several methods for endotoxin removal based on immobilizing monoclonal antibodies to chromatographic media have been evaluated. A crucial step in this process was to dissociate the endotoxin from the protein surface for subsequent removal. This was accomplished by introducing different buffer additives in the mobile phase. In agreement with previous reports, non-ionic detergents efficiently removed endotoxin, but it was also found that 0.5M arginine performed equally well. Since arginine is a non-toxic common amino acid that can be readily removed, it was selected and successfully used in large-scale experiments. With this method, endotoxin could be reduced to <0.2 EU mg(-1) with recovery of the target protein being >95%. Since this procedure is easily integrated into the existing processes of mAb purification, it offers advantages in speed, cost and effort. PMID:17644450

  16. Immunolocalization of neuroblastoma using radiolabeled monoclonal antibody UJ13A

    SciTech Connect

    Goldman, A.; Vivian, G.; Gordon, I.; Pritchard, J.; Kemshead, J.

    1984-08-01

    The monoclonal antibody UJ13A, raised after immunization of mice with human fetal brain, recognized an antigen expressed on human neuroblastoma cell lines and fresh tumors. Antibody was purified and radiolabeled with iodine isotopes using chloramine-T. In preclinical studies, 125I-labeled UJ13A was injected intravenously into nude mice bearing xenografts of human neuroblastoma. Radiolabeled UJ13A uptake by the tumors was four to 23 times greater than that by blood. In control animals, injected with a similar quantity of a monoclonal antibody known not to bind to neuroblastoma cells in vitro (FD44), there was no selective tumor uptake. Nine patients with histologically confirmed neuroblastoma each received 100 to 300 micrograms UJ13A radiolabeled with 1 to 2.8 mCi 123I or 131I. Sixteen positive sites were visible on gamma scans 1 to 7 days after injection: 15 were primary or secondary tumor sites, and one was a false positive; there were two false negatives. In two of the 15 positive sites, tumor had not been demonstrated by other imaging techniques; these were later confirmed as areas of malignant infiltration. No toxicity was encountered.

  17. Development and application of a monoclonal antibody against Thiothrix spp.

    PubMed Central

    Brigmon, R L; Bitton, G; Zam, S G; O'Brien, B

    1995-01-01

    Historically, methods used to identify Thiothrix spp. in environmental samples have been inadequate because isolation and identification procedures are time-consuming and often fail to separate Thiothrix spp. from other filamentous microorganisms. We described a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) procedure which was used to identify Thiothrix spp. in wastewater, artesian springs, groundwater, and underwater subterranean samples. The ELISA utilized monoclonal antibody T3511 to a species-specific carbohydrate epitope of Thiothrix spp. No cross-reactions were observed among non-Thiothrix strains consisting of 12 species and nine genera. In field trials, the ELISA identified 100% of 20 biochemically and cytologically confirmed Thiothrix spp.-containing samples with no false positives. Indirect immunofluorescent microscopy utilizing T3511 was effective for wastewater samples but not for those from natural spring water because of background fluorescence in the latter. In addition, electron micrographs of Thiothrix spp. labeled with T3511-biotin-anti-mouse antibody-gold showed that epitope T3511 was intracellular both in laboratory strains and environmental isolates. The minimum level of detection of the ELISA was 0.10 microgram/ml. PMID:7887596

  18. Comparison of type 2 and type 6 fimbriae of Bordetella pertussis by using agglutinating monoclonal antibodies.

    PubMed

    Li, Z M; Brennan, M J; David, J L; Carter, P H; Cowell, J L; Manclark, C R

    1988-12-01

    Two types of fimbriae have been identified on the pathogenic gram-negative organism Bordetella pertussis. Monoclonal antibodies to these fimbriae were produced to better understand the role of fimbriae as serotype-specific agglutinogens and to investigate the antigenic relationship between these fimbriae. Three monoclonal antibodies were identified that specifically agglutinated B. pertussis cells containing the U.S. Reference Factor 2 agglutinogen, and six monoclonal antibodies were produced that agglutinated only those strains containing the U.S. Reference Factor 6 agglutinogen. Indirect immunofluorescence studies and immunogold electron microscopy demonstrated that these monoclonal antibodies bind to an outer membrane component on serotype-specific strains of B. pertussis. All of the monoclonal antibodies reacted with native or partially assembled type-specific fimbriae but not with monomeric fimbrial subunits as indicated by Western blot (immunoblot) analysis. The fimbrial agglutinogens recognized by the monoclonal antibodies were also uniquely reactive with either U.S. Reference Factor 2 or 6 antiserum (Eldering agglutinogen 2 or 6 polyclonal antiserum) in an indirect ELISA. No cross-reactivity of the monoclonal antibodies with the unrelated fimbriae was observed in any of the comparative immunological studies. Some of the monoclonal antibodies agglutinated certain strains of B. bronchiseptica, suggesting that this closely related species can contain antigenically similar fimbriae. These monoclonal antibodies should prove useful for further structural and functional analysis of Bordetella fimbriae and for studies on the role that these antigens play in prevention of infection and disease. PMID:2903125

  19. Comparison of type 2 and type 6 fimbriae of Bordetella pertussis by using agglutinating monoclonal antibodies.

    PubMed Central

    Li, Z M; Brennan, M J; David, J L; Carter, P H; Cowell, J L; Manclark, C R

    1988-01-01

    Two types of fimbriae have been identified on the pathogenic gram-negative organism Bordetella pertussis. Monoclonal antibodies to these fimbriae were produced to better understand the role of fimbriae as serotype-specific agglutinogens and to investigate the antigenic relationship between these fimbriae. Three monoclonal antibodies were identified that specifically agglutinated B. pertussis cells containing the U.S. Reference Factor 2 agglutinogen, and six monoclonal antibodies were produced that agglutinated only those strains containing the U.S. Reference Factor 6 agglutinogen. Indirect immunofluorescence studies and immunogold electron microscopy demonstrated that these monoclonal antibodies bind to an outer membrane component on serotype-specific strains of B. pertussis. All of the monoclonal antibodies reacted with native or partially assembled type-specific fimbriae but not with monomeric fimbrial subunits as indicated by Western blot (immunoblot) analysis. The fimbrial agglutinogens recognized by the monoclonal antibodies were also uniquely reactive with either U.S. Reference Factor 2 or 6 antiserum (Eldering agglutinogen 2 or 6 polyclonal antiserum) in an indirect ELISA. No cross-reactivity of the monoclonal antibodies with the unrelated fimbriae was observed in any of the comparative immunological studies. Some of the monoclonal antibodies agglutinated certain strains of B. bronchiseptica, suggesting that this closely related species can contain antigenically similar fimbriae. These monoclonal antibodies should prove useful for further structural and functional analysis of Bordetella fimbriae and for studies on the role that these antigens play in prevention of infection and disease. Images PMID:2903125

  20. Characterization of group II avian adenoviruses with a panel of monoclonal antibodies.

    PubMed Central

    van den Hurk, J V; van Drunen Littel-van den Hurk, S

    1988-01-01

    The interaction between a panel of ten monoclonal antibodies and hemorrhagic enteritis virus, a group II avian adenovirus, was determined. The monoclonal antibodies reacted with all nine isolates of group II avian adenoviruses, but not with any of five types of group I avian adenoviruses. All ten monoclonal antibodies recognized antigenic determinants on the hexon protein of hemorrhagic enteritis virus when analyzed by immunoprecipitation and immunoblotting. They reacted only with the native hexon protein and not with protein denatured by sodium dodecyl sulfate or guanidine-HCl/urea treatment combined with reduction and carboxymethylation. Based on the results of competitive binding assays, the panel of monoclonal antibodies could be subdivided into two groups, which recognized different antigenic domains of the hemorrhagic enteritis virus hexon protein. The monoclonal antibodies in group 1 neutralized hemorrhagic enteritis virus infectivity while the monoclonal antibodies of group 2 did not. Group 1 consisted of eight monoclonal antibodies which could be further subdivided into subgroups 1A, 1B, 1C and 1D. The subdivision of the monoclonal antibodies was based on the degree of blocking in the competitive binding assays and differences in their ability to induce enhancement. In general, the monoclonal antibodies had a higher avidity for the virulent isolate of hemorrhagic enteritis virus than for the avirulent hemorrhagic enteritis virus isolate. Images Fig. 1. Fig. 2. Fig. 4. PMID:2461793

  1. Monoclonal antibody that preferentially binds polylysine, polyarginine, and histones and selectively decorates nuclei and chromosomes.

    PubMed Central

    Morgan, J L; Dennis, D D

    1984-01-01

    A monoclonal antibody, designated J-57, selectively and uniformly decorates the interphase nuclei and mitotic chromosomes of a variety of eucaryotic cells as determined by indirect immunofluorescence. As determined by enzyme-linked immunosorbent assay, however, this monoclonal antibody is not monospecific. It reacts weakly with cytochrome c, RNase A, and brain tubulin. By these tests monoclonal antibody J-57 has broad cross-reactivity similar to that of antisera directed against polylysine. The differential reactions of this monoclonal antibody suggest that it may be a useful immunohistochemical probe for nuclei and chromosomes in whole cells. Images PMID:6490815

  2. High-throughput assay for measuring monoclonal antibody self-association and aggregation in serum.

    PubMed

    Li, Xiaoning; Geng, Steven B; Chiu, Mark L; Saro, Dorina; Tessier, Peter M

    2015-03-18

    Subcutaneous delivery is one of the preferred administration routes for therapeutic monoclonal antibodies (mAbs). High antibody dosing requirements and small injection volumes necessitate formulation and delivery of highly concentrated mAb solutions. Such elevated antibody concentrations can lead to undesirable solution behaviors such as mAb self-association and aggregation, which are relatively straightforward to detect using various biophysical methods because of the high purity and concentration of antibody formulations. However, the biophysical properties of mAbs in serum can also impact antibody activity, but these properties are less well understood because of the difficulty characterizing mAbs in such a complex environment. Here we report a high-throughput assay for directly evaluating mAb self-association and aggregation in serum. Our approach involves immobilizing polyclonal antibodies specific for human mAbs on gold nanoparticles, and then using these conjugates to capture human antibodies at a range of subsaturating to saturating mAb concentrations in serum. Antibody aggregation is detected at subsaturating mAb concentrations via blue-shifted plasmon wavelengths due to the reduced efficiency of capturing mAb aggregates relative to monomers, which reduces affinity cross-capture of mAbs by multiple conjugates. In contrast, antibody self-association is detected at saturating mAb concentrations via red-shifted plasmon wavelengths due to attractive interparticle interactions between immobilized mAbs. The high-throughput nature of this assay along with its compatibility with unusually dilute mAb solutions (0.1-10 μg per mL) should make it useful for identifying antibody candidates with high serum stability during early antibody discovery. PMID:25714504

  3. Positron emission tomographic imaging of tumors using monoclonal antibodies

    SciTech Connect

    Zalutsky, M.R. . Dept. of Radiology)

    1989-12-01

    The overall objective of this research project is to develop methods for utilizing positron emission tomography (PET) to increase the clinical potential of radiolabeled monoclonal antibodies (MAbs). Both diagnostic and therapeutic applications of labeled MAbs could be improved as a result of knowledge obtained through the exploitation of the advantageous imaging characteristics associated with PET. By labeling MAbs with positron-emitting nuclides, it should be possible to quantitate the dynamics of their three-dimensional distribution in vivo. Our long-term goals are to apply this approach. 3 tabs.

  4. Monoclonal antibodies to Nocardia asteroides and Nocardia brasiliensis antigens.

    PubMed Central

    Jiménez, T; Díaz, A M; Zlotnik, H

    1990-01-01

    Nocardia asteroides and Nocardia brasiliensis whole-cell extracts were used as antigens to generate monoclonal antibodies (MAbs). Six stable hybrid cell lines secreting anti-Nocardia spp. MAbs were obtained. These were characterized by enzyme-linked immunosorbent assay, Western blot (immunoblot), and immunofluorescence assay. Although all the MAbs exhibited different degrees of cross-reactivity with N. asteroides and N. brasiliensis antigens as well as with culture-filtrate antigens from Mycobacteria spp., they have the potential for use as reagents in the purification of Nocardia antigens. Images PMID:2405017

  5. [Production of the monoclonal antibodies to the rabies virus nucleoprotein].

    PubMed

    Gribencha, S V; Kozlov, A Iu; Kostina, L V; Elakov, A L; Losich, M A; Tsibezov, V V; Zaberezhnyĭ, A D; Aliper, T I

    2013-01-01

    Five hybridomas secreting monoclonal antibodies (MAbs) for the nucleocapsid protein of the rabies virus were obtained through the fusion of the SP2/0 murine myeloma cells with splenocytes of BALB/c mice immunized with fixed rabies virus (CVS strain). All hybridomas secret MAbs of the IgG class that display different specificity to the nucleocapsids of rabies and rabies-related viruses. MAbs 2ell showed the specificity for the prevalent in Russia rabies viruses that are similar to commercially available anti-rabies conjugate. PMID:24640170

  6. Immunosuppression associated with novel chemotherapy agents and monoclonal antibodies.

    PubMed

    Morrison, Vicki A

    2014-11-15

    The introduction of novel agents to the therapeutic armamentarium for oncologic, rheumatologic, and neurologic disorders has resulted in major clinical advances. These agents impact immune function, resulting in a discrete spectrum of infectious complications. Purine analogues and alemtuzumab alter cell-mediated immunity, resulting in opportunistic viral/fungal infections. Herpes zoster incidence increases with bortezomib. Hepatitis B reactivation may occur with rituximab. Cases of progressive multifocal leukoencephalopathy have occurred following monoclonal antibody therapy. Tumor necrosis factor-α inhibitor therapy is complicated by tuberculosis reactivation and fungal infections. We summarize the impact of these therapies on pathogenesis and spectrum of infection complicating their usage. PMID:25352632

  7. Rapid diagnosis of whooping cough using monoclonal antibody.

    PubMed Central

    Boreland, P C; Gillespie, S H; Ashworth, L A

    1988-01-01

    A counterimmunoelectrophoresis (CIE) method for antigen detection using monoclonal antibody was assessed for its ability to aid in the rapid diagnosis of Bordetella pertussis in 59 patients. A positive diagnosis from a combination of results from tests of serum and urine was obtained in 51 (87%) of cases. For sera, CIE had a sensitivity of 85% and a specificity of 94%; for urine samples the sensitivity was 81% and a specificity of 100%. Antigen detection by CIE is simple to perform and yields results on the same day, thus allowing treatment to begin at an early stage. PMID:2898488

  8. Development of monoclonal antibodies in China: overview and prospects.

    PubMed

    Zhang, Mao-Yu; Lu, Jin-Jian; Wang, Liang; Gao, Zi-Chao; Hu, Hao; Ung, Carolina Oi Lam; Wang, Yi-Tao

    2015-01-01

    Monoclonal antibodies (mAbs) have become increasingly important as human therapeutic agents. Yet, current research concentrates on technology itself and pays attention to developed countries. This paper aims to provide a comprehensive review of mAbs development in China through systematic analysis of drug registry, patent applications, clinical trials, academic publication, and ongoing R&D projects. The trends in therapeutic areas and industrialization process are also highlighted. Development and research trends of mAbs are analyzed to provide a future perspective of mAbs as therapeutic agents in China. PMID:25811022

  9. Development of Monoclonal Antibodies in China: Overview and Prospects

    PubMed Central

    Zhang, Mao-Yu; Lu, Jin-Jian; Wang, Liang; Gao, Zi-Chao; Ung, Carolina Oi Lam; Wang, Yi-Tao

    2015-01-01

    Monoclonal antibodies (mAbs) have become increasingly important as human therapeutic agents. Yet, current research concentrates on technology itself and pays attention to developed countries. This paper aims to provide a comprehensive review of mAbs development in China through systematic analysis of drug registry, patent applications, clinical trials, academic publication, and ongoing R&D projects. The trends in therapeutic areas and industrialization process are also highlighted. Development and research trends of mAbs are analyzed to provide a future perspective of mAbs as therapeutic agents in China. PMID:25811022

  10. Boronated monoclonal antibody conjugates for neutron capture therapy

    SciTech Connect

    Borg, D.C.; Elmore, J.J. Jr.; Ferrone, S.

    1986-01-01

    Monoclonal antibodies (MoAbs) to tumor-associated antigens are attractive for concentrating /sup 10/B in cancer tissue, in part because neutron capture therapy (NCT) is not disadvantaged by the hours to days required to optimize tumor:background concentration ratios of MoAbs or their F(ab')/sub 2/ or Fab fragments. Since direct coupling of /sup 10/B compounds in amounts sufficient for radiotherapy appears to inactivate MoAbs, the authors used dextran intermediate carriers to provide high levels of /sup 10/B per MoAb while modifying fewer amino acid residues.

  11. Monoclonal Antibodies in Cancer Therapy: Mechanisms, Successes and Limitations

    PubMed Central

    Coulson, A; Levy, A; Gossell-Williams, M

    2014-01-01

    ABSTRACT Rituximab was the first chemotherapeutic monoclonal antibody (CmAb) approved for clinical use in cancer therapeutics in 1997 and has significantly improved the clinical outcomes in non-Hodgkin's lymphoma. Since then, numerous CmAbs have been developed and approved for the treatment of various haematologic and solid human cancers. In this review, the classification, efficacy and significantly reduced toxicity of CmAbs available for use in the United States of America are presented. Finally, the limitations of CmAbs and future considerations are explored. PMID:25803383

  12. Monoclonal Antibody Shows Promise as Potential Therapeutic for MERS | Poster

    Cancer.gov

    A monoclonal antibody has proven effective in preventing Middle Eastern Respiratory Syndrome (MERS) in lab animals, suggesting further development as a potential intervention for the deadly disease in humans, according to new research. MERS is a newly emerged coronavirus first detected in humans in 2012. Most cases have occurred in the Middle East, but the disease has appeared elsewhere. In all, MERS has infected more than 1,700 individuals and killed more than 600, according to the World Health Organization. No vaccines or antiviral therapies currently exist. Several candidate vaccines are being developed, and some have been tested in animal models, a prerequisite to human clinical trials.

  13. Therapeutic monoclonal antibodies and derivatives: Historical perspectives and future directions.

    PubMed

    Rodgers, Kyla R; Chou, Richard C

    2016-11-01

    Biologics, both monoclonal antibodies (mAbs) and fusion proteins, have revolutionized the practice of medicine. This year marks the 30th anniversary of the Food and Drug Administration approval of the first mAb for human use. In this review, we examine the biotechnological breakthroughs that spurred the explosive development of the biopharmaceutical mAb industry, as well as how critical lessons learned about human immunology informed the development of improved biologics. We also discuss the most common mechanisms of action of currently approved biologics and the indications for which they have been approved to date. PMID:27460206

  14. Monoclonal antibodies directed against surface molecules of multicell spheroids

    NASA Technical Reports Server (NTRS)

    Martinez, Andrew O.

    1993-01-01

    The objective of this project is to generate a library of monoclonal antibodies (MAb's) to surface molecules involved in the cell-cell interactions of mammalian cells grown as multicell spheroids (MCS). MCS are highly organized 3-dimensional multicellular structures which exhibit many characteristics in vivo tissues not found in conventional monolayer or suspension culture. They also provide a functional assay for surface adhesion molecules. In brief, MCS combine the relevance of organized tissues with the accuracy of in vitro methodology. Further, one can manipulate these MCS experimentally to discern important information about their biology.

  15. Mass Spectrometry for the Biophysical Characterization of Therapeutic Monoclonal Antibodies

    PubMed Central

    Zhang, Hao; Cui, Weidong; Gross, Michael L.

    2014-01-01

    Monoclonal antibodies (mAbs) are powerful therapeutics, and their characterization has drawn considerable attention and urgency. Unlike small-molecular drugs (150-600 Da) that have rigid structures, mAbs (~150 kDa) are engineered proteins that undergo complicated folding and can exist in a number of low-energy structures, posing a challenge for traditional methods in structural biology. Mass spectrometry (MS)-based biophysical characterization approaches can provide structural information, bringing high sensitivity, fast turnaround, and small sample consumption. This review outlines various MS-based strategies for protein biophysical characterization and then reviews how these strategies provide structural information of mAbs at the protein level (intact or top-down approaches), peptide, and residue level (bottom-up approaches), affording information on higher order structure, aggregation, and the nature of antibody complexes. PMID:24291257

  16. A review of monoclonal antibody therapies in lymphoma.

    PubMed

    Teo, Esmeralda Chi-yuan; Chew, Yveline; Phipps, Colin

    2016-01-01

    Monoclonal antibodies (moAb) represent a novel way of delivering therapy through specific target antigens expressed on lymphoma cells and minimizes the collateral damage that is common with conventional chemotherapy. The paradigm of this approach is the targeting of CD20 by rituximab. Since its FDA approval in 1997, rituximab has become the standard of care in almost every line of therapy in most B-cell lymphomas. This review will briefly highlight some of the key rituximab trials while looking more closely at the evidence that is bringing other antibodies, including next generation anti-CD20 moAbs, and anti-CD30 moAbs, among others to the forefront of lymphoma therapy. PMID:26318093

  17. Internal radiation dosimetry for clinical testing of radiolabeled monoclonal antibodies

    SciTech Connect

    Fisher, D.R.; Durham, J.S.; Hui, T.E.; Hill, R.L.

    1990-11-01

    In gauging the efficacy of radiolabeled monoclonal antibodies in cancer treatment, it is important to know the amount of radiation energy absorbed by tumors and normal tissue per unit administered activity. This paper describes methods for estimating absorbed doses to human tumors and normal tissues, including intraperitoneal tissue surfaces, red marrow, and the intestinal tract from incorporated radionuclides. These methods use the Medical Internal Radiation Dose (MIRD) scheme; however, they also incorporate enhancements designed to solve specific dosimetry problems encountered during clinical studies, such as patient-specific organ masses obtained from computerized tomography (CT) volumetrics, estimates of the dose to tumor masses within normal organs, and multicellular dosimetry for studying dose inhomogeneities in solid tumors. Realistic estimates of absorbed dose are provided within the short time requirements of physicians so that decisions can be made with regard to patient treatment and procurement of radiolabeled antibodies. Some areas in which further research could improve dose assessment are also discussed. 16 refs., 3 figs.

  18. Discovery and characterization of hydroxylysine in recombinant monoclonal antibodies.

    PubMed

    Xie, Qing; Moore, Benjamin; Beardsley, Richard L

    2016-01-01

    Tryptic peptide mapping analysis of a Chinese hamster ovary (CHO)-expressed, recombinant IgG1 monoclonal antibody revealed a previously unreported +16 Da modification. Through a combination of MS(n) experiments, and preparation and analysis of known synthetic peptides, the possibility of a sequence variant (Ala to Ser) was ruled out and the presence of hydroxylysine was confirmed. Post-translational hydroxylation of lysine was found in a consensus sequence (XKG) known to be the site of modification in other proteins such as collagen, and was therefore presumed to result from the activity of the CHO homolog of the lysyl hydroxylase complex. Although this consensus sequence was present in several locations in the antibody sequence, only a single site on the heavy-chain Fab was found to be modified. PMID:26651858

  19. Monoclonal antibody-based immunotherapy for multiple myeloma.

    PubMed

    Danylesko, Ivetta; Beider, Katia; Shimoni, Avichai; Nagler, Arnon

    2012-09-01

    Multiple myeloma (MM) is a life-threatening hematological malignancy. High-dose chemotherapy followed by autologous stem cell transplantation is a relatively effective treatment, but disease recurrence remains a major obstacle. Allogeneic transplantation may result in durable responses and cure due to antitumor immunity mediated by donor lymphocytes. However, morbidity and mortality related to graft-versus-host disease remain a challenge. Recent advances in understanding the interaction between the immune system of the patient and the malignant cells are influencing the design of clinically more efficient study protocols for MM. This review will focus on MM antigens and their specific antibodies. These monoclonal antibodies are an attractive therapeutic tool for MM humoral immunotherapy, with most promising preclinical results. PMID:23046236

  20. NCI Requests Targets for Monoclonal Antibody Production and Characterization - Office of Cancer Clinical Proteomics Research

    Cancer.gov

    In an effort to provide well-characterized monoclonal antibodies to the scientific community, NCI's Antibody Characterization Program requests cancer-related protein targets for affinity production and distribution.

  1. Monoclonal antibodies to native noncollagenous bone-specific proteins.

    PubMed Central

    Stenner, D D; Romberg, R W; Tracy, R P; Katzmann, J A; Riggs, B L; Mann, K G

    1984-01-01

    Hybridoma technology was used for preparation of murine monoclonal antibodies of high titer against bone-Gla protein and osteonectin. A procedure of immunization and hybridization similar to that already described [Katzmann, J.A., Nesheim, M.E., Hibbard, L.S. & Mann, K.G. (1981) Proc. Natl. Acad. Sci. USA 78, 162-166; and Foster, W.B., Katzmann, J.A., Miller, R.S., Nesheim, M.E. & Mann, K.G. (1982) Thromb. Res. 28, 649-661] was used. However, in contrast to earlier studies, mice were immunized with an unfractionated protein mixture that had been extracted from bone under nondenaturing conditions. The extract was labeled with 125I by the chloramine-T method. After fusion and initial hybrid growth, screening was accomplished by a solid-phase radioimmunoassay with total 125I-labeled bovine bone protein extract as the tracer. The identities of antibody-bound 125I-labeled proteins were assessed by dissolution of the solid-phase immune complex in NaDodSO4 and subsequent electrophoresis and autoradiography. Clones producing specific antibody to a single protein were selected by limiting dilution. The identity of the proteins against which the specific antibodies were produced was confirmed by immunoprecipitation, electrophoresis, and autoradiography. From two fusions, 30 positive hybrids to bone-Gla protein were identified; 7 of these were subcloned and 1 has been expanded as an ascites tumor. One hybrid population was positive for osteonectin, a Mr 15,000 peptide, and for bone-Gla protein. By limiting dilution, the osteonectin clone was selected and subsequently expanded as an ascites tumor. Titration curves made using the respective 125I-labeled purified proteins show the ascites tumors to be producing antibody of high titer (I50 = 10(-6) for anti-bone-Gla protein and (I50 = 10(-5) for antiosteonectin. Both of the antibovine antibodies are cross-reactive with the corresponding human protein. Immobilized specific anti-bone-Gla protein has been used to isolate human bone

  2. Neutralizing determinants defined by monoclonal antibodies on polypeptides specified by bovine herpesvirus 1.

    PubMed Central

    Collins, J K; Butcher, A C; Riegel, C A; McGrane, V; Blair, C D; Teramoto, Y A; Winston, S

    1984-01-01

    Monoclonal antibodies were used to study neutralizing determinants on polypeptides of bovine herpesvirus 1. Two of three monoclonal antibodies which recognized nonoverlapping epitopes on a glycoprotein of 82,000 daltons were found to neutralize. A second group of monoclonal antibodies that individually precipitated five viral glycopolypeptides ranging in size from 102,000 to 55,000 daltons also neutralized. Two monoclonal antibodies which were the most efficient in neutralization recognized a non-glycosylated protein of 115,000 daltons which was the major polypeptide on the virus. A fourth group of monoclonal antibodies precipitated a non-glycosylated polypeptide of 91,000 daltons and several smaller polypeptides, but these antibodies demonstrated only limited neutralizing activity. Images PMID:6208375

  3. Monoclonal Antibodies Directed to Fucoidan Preparations from Brown Algae

    PubMed Central

    Torode, Thomas A.; Marcus, Susan E.; Jam, Murielle; Tonon, Thierry; Blackburn, Richard S.; Hervé, Cécile; Knox, J. Paul

    2015-01-01

    Cell walls of the brown algae contain a diverse range of polysaccharides with useful bioactivities. The precise structures of the sulfated fucan/fucoidan group of polysaccharides and their roles in generating cell wall architectures and cell properties are not known in detail. Four rat monoclonal antibodies, BAM1 to BAM4, directed to sulfated fucan preparations, have been generated and used to dissect the heterogeneity of brown algal cell wall polysaccharides. BAM1 and BAM4, respectively, bind to a non-sulfated epitope and a sulfated epitope present in the sulfated fucan preparations. BAM2 and BAM3 identified additional distinct epitopes present in the fucoidan preparations. All four epitopes, not yet fully characterised, occur widely within the major brown algal taxonomic groups and show divergent distribution patterns in tissues. The analysis of cell wall extractions and fluorescence imaging reveal differences in the occurrence of the BAM1 to BAM4 epitopes in various tissues of Fucus vesiculosus. In Ectocarpus subulatus, a species closely related to the brown algal model Ectocarpus siliculosus, the BAM4 sulfated epitope was modulated in relation to salinity levels. This new set of monoclonal antibodies will be useful for the dissection of the highly complex and yet poorly resolved sulfated polysaccharides in the brown algae in relation to their ecological and economic significance. PMID:25692870

  4. Monoclonal antibody-directed radioimmunoassay of specific cytochromes P-450

    SciTech Connect

    Song, B.J.; Fujino, T.; Park, S.S.; Friedman, F.K.; Gelboin, H.V.

    1984-02-10

    A rapid solid phase radioimmunoassay (RIA) for cytochromes P-450 has been developed utilizing specific monoclonal antibodies to major forms of rat liver cytochrome P-450 that are induced by 3-methylcholanthrene (MC-P-450) and phenobarbital (PB-P-450). Monoclonal antibodies (MAbs) that were endogenously labeled with (/sup 35/S)methionine were used to detect MAb-specific cytochromes P-450 in liver microsomes from untreated rats and rats pretreated with 3-methylcholanthrene (MC) or phenobarbital. The competitive binding assays are rapid and can detect cytochrome P-450 in less than 100 ng of microsomal protein. Tthe RIA was used to examine the distribution of MAb-specific cytochromes P-450 in extrahepatic tissues of MC-treated rats; an approximately 30- to 50-fold greater amount of MC-P-450 in liver relative to lung and kidney was observed, which corresponds well with aryl hydrocarbon hydroxylase activity in these tissues. The inducibility of MAb-specific cytochromes P-450 were observed in MC-treated rats, guinea pigs, and C57BL/6 mice, all highly inducible for aryl hydrocarbon hydroxylase; little increase was observed for the relatively noninducible DBA/2 mouse strain.

  5. Monoclonal antibodies directed to fucoidan preparations from brown algae.

    PubMed

    Torode, Thomas A; Marcus, Susan E; Jam, Murielle; Tonon, Thierry; Blackburn, Richard S; Hervé, Cécile; Knox, J Paul

    2015-01-01

    Cell walls of the brown algae contain a diverse range of polysaccharides with useful bioactivities. The precise structures of the sulfated fucan/fucoidan group of polysaccharides and their roles in generating cell wall architectures and cell properties are not known in detail. Four rat monoclonal antibodies, BAM1 to BAM4, directed to sulfated fucan preparations, have been generated and used to dissect the heterogeneity of brown algal cell wall polysaccharides. BAM1 and BAM4, respectively, bind to a non-sulfated epitope and a sulfated epitope present in the sulfated fucan preparations. BAM2 and BAM3 identified additional distinct epitopes present in the fucoidan preparations. All four epitopes, not yet fully characterised, occur widely within the major brown algal taxonomic groups and show divergent distribution patterns in tissues. The analysis of cell wall extractions and fluorescence imaging reveal differences in the occurrence of the BAM1 to BAM4 epitopes in various tissues of Fucus vesiculosus. In Ectocarpus subulatus, a species closely related to the brown algal model Ectocarpus siliculosus, the BAM4 sulfated epitope was modulated in relation to salinity levels. This new set of monoclonal antibodies will be useful for the dissection of the highly complex and yet poorly resolved sulfated polysaccharides in the brown algae in relation to their ecological and economic significance. PMID:25692870

  6. Production of a diagnostic monoclonal antibody in perennial alfalfa plants.

    PubMed

    Khoudi, H; Laberge, S; Ferullo, J M; Bazin, R; Darveau, A; Castonguay, Y; Allard, G; Lemieux, R; Vézina, L P

    1999-07-20

    The increasing use of monoclonal antibodies (mAbs) in diagnostic reagents necessitates efficient and cost-effective mAb production methods. In blood banks, one of the most routinely used reagents is the anti-human IgG reagent used for the detection of non-agglutinating antibodies. Here we report the production of a functional, purified anti-human IgG, through the expression of its encoding genes in perennial transgenic alfalfa. Transgenic plants expressing the light- and heavy-chain encoding mRNAs were obtained, and plants from crosses were found to express fully assembled C5-1. The purification procedure yielded mainly the H2L2 form with specificity and affinity identical to those of hybridoma-derived C5-1. The ability to accumulate the antibody was maintained both in parental F1 lines during repeated harvesting and in clonal material; the antibody was stable in the drying hay as in extracts made in pure water. Also, plant and hybridoma-derived C5-1 had similar in vivo half-lives in mice. These results indicate that plant C5-1 could be used in a diagnostic reagent as effectively as hybridoma-derived C5-1, and demonstrates the usefulness of perennial systems for the cost-effective, stable, and reliable production of large amounts of mAbs. PMID:10397849

  7. Examination of HER3 targeting in cancer using monoclonal antibodies.

    PubMed

    Gaborit, Nadège; Abdul-Hai, Ali; Mancini, Maicol; Lindzen, Moshit; Lavi, Sara; Leitner, Orith; Mounier, Lucile; Chentouf, Myriam; Dunoyer, Sai; Ghosh, Manjusha; Larbouret, Christel; Chardès, Thierry; Bazin, Hervé; Pèlegrin, André; Sela, Michael; Yarden, Yosef

    2015-01-20

    The human EGF receptor (HER/EGFR) family of receptor tyrosine kinases serves as a key target for cancer therapy. Specifically, EGFR and HER2 have been repeatedly targeted because of their genetic aberrations in tumors. The therapeutic potential of targeting HER3 has long been underestimated, due to relatively low expression in tumors and impaired kinase activity. Nevertheless, in addition to serving as a dimerization partner of EGFR and HER2, HER3 acts as a key player in tumor cells' ability to acquire resistance to cancer drugs. In this study, we generated several monoclonal antibodies to HER3. Comparisons of their ability to degrade HER3, decrease downstream signaling, and inhibit growth of cultured cells, as well as recruit immune effector cells, selected an antibody that later emerged as the most potent inhibitor of pancreatic cancer cells grown as tumors in animals. Our data predict that anti-HER3 antibodies able to intercept autocrine and stroma-tumor interactions might strongly inhibit tumor growth, in analogy to the mechanism of action of anti-EGFR antibodies routinely used now to treat colorectal cancer patients. PMID:25564668

  8. Examination of HER3 targeting in cancer using monoclonal antibodies

    PubMed Central

    Gaborit, Nadège; Abdul-Hai, Ali; Mancini, Maicol; Lindzen, Moshit; Lavi, Sara; Leitner, Orith; Mounier, Lucile; Chentouf, Myriam; Dunoyer, Sai; Ghosh, Manjusha; Larbouret, Christel; Chardès, Thierry; Bazin, Hervé; Pèlegrin, André; Sela, Michael; Yarden, Yosef

    2015-01-01

    The human EGF receptor (HER/EGFR) family of receptor tyrosine kinases serves as a key target for cancer therapy. Specifically, EGFR and HER2 have been repeatedly targeted because of their genetic aberrations in tumors. The therapeutic potential of targeting HER3 has long been underestimated, due to relatively low expression in tumors and impaired kinase activity. Nevertheless, in addition to serving as a dimerization partner of EGFR and HER2, HER3 acts as a key player in tumor cells’ ability to acquire resistance to cancer drugs. In this study, we generated several monoclonal antibodies to HER3. Comparisons of their ability to degrade HER3, decrease downstream signaling, and inhibit growth of cultured cells, as well as recruit immune effector cells, selected an antibody that later emerged as the most potent inhibitor of pancreatic cancer cells grown as tumors in animals. Our data predict that anti-HER3 antibodies able to intercept autocrine and stroma–tumor interactions might strongly inhibit tumor growth, in analogy to the mechanism of action of anti-EGFR antibodies routinely used now to treat colorectal cancer patients. PMID:25564668

  9. Screening individual hybridomas by microengraving to discover monoclonal antibodies

    PubMed Central

    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

  10. Screening individual hybridomas by microengraving to discover monoclonal antibodies.

    PubMed

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

    2009-01-01

    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 (approximately 10(5) 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

  11. Characterization of a monoclonal antibody to thymidine glycol monophosphate

    SciTech Connect

    Chen, B.X.; Hubbard, K.; Ide, H.; Wallace, S.S.; Erlanger, B.F. )

    1990-11-01

    A monoclonal antibody specific for thymine glycol (TG) in irradiated or OsO4-treated DNA was obtained by immunizing with thymidine glycol monophosphate (TMP-glycol) conjugated to bovine serum albumin by a carbodiimide procedure. Screening by dot-immunobinding and enzyme-linked immunosorbant assay (ELISA) procedures gave eight clones that bound OsO4- treated DNA. One of them, 2.6F.6B.6C, an IgG2a kappa, was characterized further. Hapten inhibition studies with OsO4-treated DNA showed that the antibody was specific for TMP-glycol. Among the various inhibitors tested, inhibition was in the order TMP-glycol greater than 5,6-dihydrothymidine phosphate greater than TMP greater than thymidine glycol greater than TG. Inhibition by 5,6-dihydrothymidine, thymidine, thymine, AMP, and CMP was negligible. In OsO4-treated DNA, as few as 0.5 TG per 10,000 bp were detectable by direct ELISA. Inhibition assays could detect as few as 1.5 TG per 10,000 bp. The antibody was equally reactive with native or denatured DNA containing TG. Among the X-irradiated homopolymers dC, dA, dG, and dT, only dT reacted with the antibody. Using an ELISA, the antibody could detect damage in irradiated DNA at the level of 20 Gy. Thus the antibody is of potential use in assays for DNA damage caused by X rays or other agents that damage DNA by free radical interactions.

  12. Polyclonal and monoclonal antibody therapy for experimental Pseudomonas aeruginosa pneumonia.

    PubMed Central

    Pennington, J E; Small, G J; Lostrom, M E; Pier, G B

    1986-01-01

    A human immunoglobulin G preparation, enriched in antibodies to lipopolysaccharide (LPS) Pseudomonas aeruginosa antigens (PA-IGIV) and murine monoclonal antibodies (MAb) to P. aeruginosa Fisher immunotype-1 (IT-1) LPS antigen and outer membrane protein F (porin), were evaluated for therapeutic efficacy in a guinea pig model of P. aeruginosa pneumonia. The concentration of antibodies to IT-1 LPS was 7.6 micrograms/ml in PA-IGIV and 478 micrograms/ml in the IT-1 MAb preparation. No antibody to IT-1 was detected in MAb to porin. For study, animals were infected by intratracheal instillation of IT-1 P. aeruginosa and then treated 2 h later with intravenous infusions of PA-IGIV, IT-1 MAb, or porin MAb. Control groups received intravenous albumin, and routinely died from pneumonia. Both PA-IGIV (500 mg/kg) and IT-1 MAb (greater than or equal to 2.5 mg/kg) treatment resulted in increased survival (P less than 0.01 to 0.001), and also improved intrapulmonary killing of bacteria. Porin MAb failed to protect from fatal pneumonia. IT-1 MAb treatment produced more survivals than did PA-IGIV treatment but only at dosages of MAb resulting in serum antibody concentrations greater than those achieved with PA-IGIV. PA-IGIV and IT-1 MAb demonstrated in vitro and in vivo (posttreatment guinea pig serum) opsonophagocytic activity for the IT-1 challenge strain. However, the polyclonal preparation required complement, whereas the MAb did not. We conclude that passive immunization with polyclonal hyperimmune P. aeruginosa globulin or with MAb to LPS antigens may be useful in the treatment of acute P. aeruginosa pneumonia. The relative efficacies of such preparations may be limited, however, by their type-specific LPS antibody concentrations. PMID:3093385

  13. Structural Characterization of a Monoclonal Antibody-Maytansinoid Immunoconjugate.

    PubMed

    Luo, Quanzhou; Chung, Hyo Helen; Borths, Christopher; Janson, Matthew; Wen, Jie; Joubert, Marisa K; Wypych, Jette

    2016-01-01

    Structural characterization was performed on an antibody-drug conjugate (ADC), composed of an IgG1 monoclonal antibody (mAb), mertansine drug (DM1), and a noncleavable linker. The DM1 molecules were conjugated through nonspecific modification of the mAb at solvent-exposed lysine residues. Due to the nature of the lysine conjugation process, the ADC molecules are heterogeneous, containing a range of species that differ with respect to the number of DM1 per antibody molecule. The DM1 distribution profile of the ADC was characterized by electrospray ionization mass spectrometry (ESI-MS) and capillary isoelectric focusing (cIEF), which showed that 0-8 DM1s were conjugated to an antibody molecule. By taking advantage of the high-quality MS/MS spectra and the accurate mass detection of diagnostic DM1 fragment ions generated from the higher-energy collisional dissociation (HCD) approach, we were able to identify 76 conjugation sites in the ADC, which covered approximately 83% of all the putative conjugation sites. The diagnostic DM1 fragment ions discovered in this study can be readily used for the characterization of other ADCs with maytansinoid derivatives as payload. Differential scanning calorimetric (DSC) analysis of the ADC indicated that the conjugation of DM1 destabilized the C(H)2 domain of the molecule, which is likely due to conjugation of DM1 on lysine residues in the C(H)2 domain. As a result, methionine at position 258 of the heavy chain, which is located in the C(H)2 domain of the antibody, is more susceptible to oxidation in thermally stressed ADC samples when compared to that of the naked antibody. PMID:26629796

  14. Tau Monoclonal Antibody Generation Based on Humanized Yeast Models

    PubMed Central

    Rosseels, Joëlle; Van den Brande, Jeff; Violet, Marie; Jacobs, Dirk; Grognet, Pierre; Lopez, Juan; Huvent, Isabelle; Caldara, Marina; Swinnen, Erwin; Papegaey, Anthony; Caillierez, Raphaëlle; Buée-Scherrer, Valerie; Engelborghs, Sebastiaan; Lippens, Guy; Colin, Morvane; Buée, Luc; Galas, Marie-Christine; Vanmechelen, Eugeen; Winderickx, Joris

    2015-01-01

    A link between Tau phosphorylation and aggregation has been shown in different models for Alzheimer disease, including yeast. We used human Tau purified from yeast models to generate new monoclonal antibodies, of which three were further characterized. The first antibody, ADx201, binds the Tau proline-rich region independently of the phosphorylation status, whereas the second, ADx215, detects an epitope formed by the Tau N terminus when Tau is not phosphorylated at Tyr18. For the third antibody, ADx210, the binding site could not be determined because its epitope is probably conformational. All three antibodies stained tangle-like structures in different brain sections of THY-Tau22 transgenic mice and Alzheimer patients, and ADx201 and ADx210 also detected neuritic plaques in the cortex of the patient brains. In hippocampal homogenates from THY-Tau22 mice and cortex homogenates obtained from Alzheimer patients, ADx215 consistently stained specific low order Tau oligomers in diseased brain, which in size correspond to Tau dimers. ADx201 and ADx210 additionally reacted to higher order Tau oligomers and presumed prefibrillar structures in the patient samples. Our data further suggest that formation of the low order Tau oligomers marks an early disease stage that is initiated by Tau phosphorylation at N-terminal sites. Formation of higher order oligomers appears to require additional phosphorylation in the C terminus of Tau. When used to assess Tau levels in human cerebrospinal fluid, the antibodies permitted us to discriminate patients with Alzheimer disease or other dementia like vascular dementia, indicative that these antibodies hold promising diagnostic potential. PMID:25540200

  15. Agglutinating monoclonal antibodies that specifically recognize lipooligosaccharide A of Bordetella pertussis.

    PubMed Central

    Li, Z M; Cowell, J L; Brennan, M J; Burns, D L; Manclark, C R

    1988-01-01

    Monoclonal antibodies that specifically agglutinate strains of Bordetella pertussis having serotype 1 agglutinogen were uniquely reactive with the electrophoretically slow-migrating A form of lipooligosaccharide. These monoclonal antibodies should be useful for the structural analysis of B. pertussis lipooligosaccharide and for the establishment of a better-defined serogroup for Bordetella species. Images PMID:2893776

  16. Agglutinating monoclonal antibodies that specifically recognize lipooligosaccharide A of Bordetella pertussis.

    PubMed

    Li, Z M; Cowell, J L; Brennan, M J; Burns, D L; Manclark, C R

    1988-03-01

    Monoclonal antibodies that specifically agglutinate strains of Bordetella pertussis having serotype 1 agglutinogen were uniquely reactive with the electrophoretically slow-migrating A form of lipooligosaccharide. These monoclonal antibodies should be useful for the structural analysis of B. pertussis lipooligosaccharide and for the establishment of a better-defined serogroup for Bordetella species. PMID:2893776

  17. Monoclonal antibodies to cyclodiene insecticides and method for detecting the same

    DOEpatents

    Stanker, L.H.; Vanderlaan, M.; Watkins, B.E.

    1994-08-02

    Methods are described for making specific monoclonal antibodies useful for detection of cyclodienes in foods and environmental samples. Monoclonal antibodies specifically reactive with cyclodienes can detect accumulated pesticides in food, tissue or environmental samples. Extraction and preparation of organic samples for immunoassay in a polar-nonpolar reaction medium permits detection of halogenated organic ring structures at concentrations in samples. 13 figs.

  18. Monoclonal antibodies to cyclodiene insecticides and method for detecting the same

    DOEpatents

    Stanker, Larry H.; Vanderlaan, Martin; Watkins, Bruce E.

    1994-01-01

    Methods are described for making specific monoclonal antibodies useful for detection of cyclodienes in foods and environmental samples. Monoclonal antibodies specifically reactive with cyclodienes can detect accumulated pesticides in food, tissue or environmental samples. Extraction and preparation of organic samples for immunoassay in a polar-nonpolar reaction medium permits detection of halogenated organic ring structures at concentrations in samples.

  19. Method of rapid production of hybridomas expressing monoclonal antibodies on the cell surface

    DOEpatents

    Meagher, Richard B.; Laterza, Vince

    2006-12-12

    The present invention relates to genetically altered hybridomas, myelomas and B cells. The invention also relates to utilizing genetically altered hybridomas, myelomas and B cells in methods of making monoclonal antibodies. The present invention also provides populations of hybridomas and B cells that can be utilized to make a monoclonal antibody of interest.

  20. Monoclonal antibody typing of Chlamydia psittaci strains derived from avian and mammalian species.

    PubMed Central

    Fukushi, H; Nojiri, K; Hirai, K

    1987-01-01

    A total of 77 Chlamydia psittaci strains of avian, human, and mammalian origin were grouped into four serovars with 11 monoclonal antibodies recognizing the lipopolysaccharide and the major outer membrane protein antigens. The avian and human strains, which were closely related to each other, were distinct from the mammalian strains. Immunological typing of C. psittaci with monoclonal antibodies seems practical. PMID:3667918

  1. Development and characterization of mouse monoclonal antibodies specific for chicken interleukin 18

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Four mouse monoclonal antibodies (mAbs) which are specific for chicken interleukin 18 (chIL18) were produced and characterized by enzyme-linked immunosorbent assay (ELISA), Western blotting, quantitative real-time PCR and neutralization assays. Monoclonal antibodies specific for chIL18 identified a ...

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

    PubMed

    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. PMID:27284048

  3. Selection of Ceratitis capitata (Diptera: Tephritidae) Specific Recombinant Monoclonal Phage Display Antibodies for Prey Detection Analysis

    PubMed Central

    Monzó, César; Urbaneja, Alberto; Ximénez-Embún, Miguel; García-Fernández, Julia; García, José Luis; Castañera, Pedro

    2012-01-01

    Several recombinant antibodies against the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), one of the most important pests in agriculture worldwide, were selected for the first time from a commercial phage display library of human scFv antibodies. The specificity and sensitivity of the selected recombinant antibodies were compared with that of a rabbit polyclonal serum raised in parallel using a wide range of arthropod species as controls. The selected recombinant monoclonal antibodies had a similar or greater specificity when compared with classical monoclonal antibodies. The selected recombinant antibodies were successfully used to detect the target antigen in the gut of predators and the scFv antibodies were sequenced and compared. These results demonstrate the potential for recombinant scFv antibodies to be used as an alternative to the classical monoclonal antibodies or even molecular probes in the post-mortem analysis studies of generalist predators. PMID:23272105

  4. Therapy of a murine sarcoma using syngeneic monoclonal antibody

    SciTech Connect

    Kennel, S.J.; Lankford, T.; Flynn, K.M.

    1983-01-01

    Syngeneic monoclonal antibodies (MoAb) to Moloney sarcoma cells were produced by fusion of spleen cells from MSC regressor mice to myeloma SP2/0. MoAb 244-19A, an immunoglobulin G2b, bound to MSC cells and did not bind to two other sarcomas (K-BALB and Ha2), a carcinoma (Line 1), a fibroblast (A31) or a fibroblast infected with C-type virus (A31) or a fibroblast infected with C-type virus (A31-Moloney leukemia virus). In contrast, MoAb 271-1A bound to the MSC and Ha2 sarcoma and line 1 carcinoma as well as to the normal and infected fibroblast cultures. Antibodies were tested for therapeutic effect using three schedules of antibody injection. Injection i.p. of ascites fluid containing 244-19A MoAb given on Days -1, 0, and +1 relative to tumor cell injection increased life span significantly over that of control animals given injections (P3, immunoglobulin G, or MoAb 271-1A) and produced some seven of 19, one of five, and one of five long-term survivors in three separate experiments. Antibody given to animals with established tumors (4 days after implantation) also prolonged life span significantly and produced three of nine long-term survivors. Antibody given to animals with very large tumor burdens (10 days after implantation) did not prolong life span significantly. Optimal dose, schedule, and mechanism studies concerning this therapy are in progress.

  5. Legionella micdadei and Legionella dumoffii monoclonal antibodies for laboratory diagnosis of Legionella infections.

    PubMed Central

    Cercenado, E; Edelstein, P H; Gosting, L H; Sturge, J C

    1987-01-01

    Two different monoclonal antibodies directed against Legionella micdadei and L. dumoffii (Genetic Systems Corp., Seattle, Wash.) were evaluated for their specificity and ability to detect L. micdadei and L. dumoffii in human and animal clinical samples and bacterial isolates in an indirect immunofluorescence assay. All three frozen sputum samples and all three Formalin-fixed sputum and liver samples from patients with culture-documented L. micdadei pneumonia were positive when tested with the L. micdadei monoclonal antibody. A Formalin-preserved lung sample from a patient with culture-documented L. dumoffii pneumonia was positive with its homologous monoclonal antibody. No cross-staining reactions were found with either monoclonal antibody on any of 25 human sputum samples tested from patients without Legionella infections. A total of 66 Legionella strains and 56 non-Legionella strains including 22 Pseudomonas strains and 34 other bacterial strains were studied. No cross-staining reactions were found except in Staphylococcus aureus Cowan 1 ATCC 12598. The lower limit of detection in seeded sputum samples was about 7 X 10(4) cells per ml for both monoclonal antibodies. Lung and tracheal lavage specimens from L. micdadei- or L. dumoffii-infected guinea pigs showed specific staining only with their respective monoclonal antibodies. The monoclonal antibodies stained homologous bacteria slightly less intensely than did the polyclonal antisera, but the signal-to-noise ratio was considerably higher for the monoclonal antibodies. No differences in sensitivity of staining of clinical specimens or bacterial isolates were noted between the monoclonal antibodies and the polyclonal reagents for L. micdadei and L. dumoffii (Centers for Disease Control, Atlanta, Ga., and BioDx, Denville, N.J. These monoclonal antibodies ae sensitive and specific, making them good candidates for laboratory diagnostic purposes. PMID:3320084

  6. Production and characterization of monoclonal antibodies to Newcastle Disease Virus.

    PubMed

    Kumar, G Ravi; Saxena, Shikha; Sahoo, A P; Chaturvedi, Uttara; Kumar, Satish; Santra, Lakshman; Desai, G S; Singh, Lakshyaveer; Tiwari, Ashok K

    2016-03-01

    Newcastle Disease (ND) is one of the major causes of economic loss in the poultry industry. Newcastle Disease Virus (NDV) is a single-stranded, negative-sense enveloped RNA virus (Fam. Paramyxoviridae; Order Mononegavirales). In the present study three monoclonal antibodies (MAbs) were produced by polyethyleneglycol (PEG)-mediated fusion of lymphocytes sensitized to NDV Bareilly strain and myeloma cells. NDV possesses ability to agglutinate erythrocytes of avian species. All the three MAbs designated as 2H7, 3E9 and 3G6 caused hemagglutination inhibition of NDV by specifically binding to NDV. The reactivity for all the 3 MAbs on indirect ELISA was found to be significantly higher than the antibody and antigen controls. On flowcytometry of HeLa cells infected with NDV using the MAbs as primary antibodies, there was a significant difference in the percentage of cells showing positive fluorescence compared to the mock control. One of the MAbs (3E9) was found to react with hemagglutinin-neuraminidase (HN) protein on western blot. PMID:27145631

  7. Removal of drugs from the circulation using immobilized monoclonal antibodies

    SciTech Connect

    Brizgys, M.V.

    1987-01-01

    High-affinity monoclonal antidigoxin antibodies (dig-Ab) were immobilized to a pellicular microbead and characterized in terms of antibody affinity, specificity for other glycosides, and binding capacity. Determination of digoxin binding revealed that the binding capacity decreased to 25% of theoretical capacity. Attempts to improve the binding capacity were ineffective. A guinea pig animal model was developed to determine the efficacy of removing digoxin in vivo from the circulation using an antibody column. Male guinea pigs were hemoperfused with either a dig-Ab or bovine Y-globulin control column 16 h after a single i.v. injection of digoxin. Pre- and postcolumn plasma concentrations were obtained to evaluate the extraction efficiency. Hemoperfusion continued for 3 h at flow rates of 1.0-2.0 mL/min. Bound digoxin was eluted as described earlier and concentrations determined by (/sup 125/I) digoxin RIA. Amounts of digoxin removed represented less than 1% of the total body content. After several studies with the same column, the dig-Ab had lost most of its activity. A freshly prepared dig-Ab column removed approximately 20% of the total body content. Most of the measured constituents of the blood were unaffected by the procedure.

  8. Protective activities in mice of monoclonal antibodies against pertussis toxin.

    PubMed Central

    Sato, H; Sato, Y

    1990-01-01

    Pertussis toxin (PT) protein, which is the most important protective antigen of Bordetella pertussis, has a hexameric structure composed of five subunits, designated S1 through S5. Immunoprotective activity of 20 different mouse monoclonal antibodies (MAbs) against pertussis toxin, 10 anti-S1, 1 anti-S2, 2 anti-S3, 4 anti-S23, and 3 anti-S4 antibodies, were investigated by aerosol and intracerebral challenges with virulent B. pertussis organisms in mice. Four anti-S1, named 1B7, 1D7, 3F11, and 10D6, and three anti-S23 antibodies, named 11E6, 10B5, and 10C9, showed the highest, and almost complete, protectivity against the aerosol challenge. Mouse protectivity against the intracerebral challenge was significant for these four anti-S1 MAbs but not for any of the three anti-S23 MAbs. Four anti-S1 and two anti-S4 MAbs did not protect the mice against either challenge. The other seven MAbs also showed dose-dependent moderate but significant protection against the aerosol challenge. In the aerosol challenge system, bacterial numbers and amounts of PT detected in the lung and the number of peripheral leukocytes were lower in the mice given the protective MAbs. All mice surviving 5 weeks after the infection produced high titers of antibodies against PT, filamentous hemagglutinin (FHA), and agglutinogens from the challenge organisms. A combination of the protective MAbs 1B7 and 11E6 strongly suppressed the disease and mortality of the mice at smaller amounts than with the anti-PT polyclonal antibody. Although combinations of one of the protective MAb and anti-FHA or anti-agglutinogen 2 also showed extremely high mouse protection without development of symptoms of the disease, antibody titers of the survivors against PT, FHA, and agglutinogens were significantly low. The foregoing results suggest that some important protective epitopes should be in S1 and S2 and/or S3, although there are both differences and similarities in the protective roles between anti-S1 and anti-S23

  9. Protective activities in mice of monoclonal antibodies against pertussis toxin.

    PubMed

    Sato, H; Sato, Y

    1990-10-01

    Pertussis toxin (PT) protein, which is the most important protective antigen of Bordetella pertussis, has a hexameric structure composed of five subunits, designated S1 through S5. Immunoprotective activity of 20 different mouse monoclonal antibodies (MAbs) against pertussis toxin, 10 anti-S1, 1 anti-S2, 2 anti-S3, 4 anti-S23, and 3 anti-S4 antibodies, were investigated by aerosol and intracerebral challenges with virulent B. pertussis organisms in mice. Four anti-S1, named 1B7, 1D7, 3F11, and 10D6, and three anti-S23 antibodies, named 11E6, 10B5, and 10C9, showed the highest, and almost complete, protectivity against the aerosol challenge. Mouse protectivity against the intracerebral challenge was significant for these four anti-S1 MAbs but not for any of the three anti-S23 MAbs. Four anti-S1 and two anti-S4 MAbs did not protect the mice against either challenge. The other seven MAbs also showed dose-dependent moderate but significant protection against the aerosol challenge. In the aerosol challenge system, bacterial numbers and amounts of PT detected in the lung and the number of peripheral leukocytes were lower in the mice given the protective MAbs. All mice surviving 5 weeks after the infection produced high titers of antibodies against PT, filamentous hemagglutinin (FHA), and agglutinogens from the challenge organisms. A combination of the protective MAbs 1B7 and 11E6 strongly suppressed the disease and mortality of the mice at smaller amounts than with the anti-PT polyclonal antibody. Although combinations of one of the protective MAb and anti-FHA or anti-agglutinogen 2 also showed extremely high mouse protection without development of symptoms of the disease, antibody titers of the survivors against PT, FHA, and agglutinogens were significantly low. The foregoing results suggest that some important protective epitopes should be in S1 and S2 and/or S3, although there are both differences and similarities in the protective roles between anti-S1 and anti-S23

  10. Potential of palladium-109-labeled antimelanoma monoclonal antibody for tumor therapy

    SciTech Connect

    Fawwaz, R.A.; Wang, T.S.T.; Srivastava, S.C.; Rosen, J.M.; Ferrone, S.; Hardy, M.A.; Alderson, P.O.

    1984-07-01

    Palladium-109, a beta-emitting radionuclide, was chelated to the monoclonal antibody 225.28S to the high molecular weight antigen associated with human melanoma. Injection of the radiolabeled monoclonal antibody into nude mice bearing human melanoma resulted in significant accumulation of the radiolabel in the tumors: 19% injected dose/g; 38:1 and 61:1 tumor-to-blood ratios at 24 and 48 hr, respectively. The localization of the radiolabeled antibody in liver and kidney also was high, but appreciably lower than that achieved in tumor. These results suggest Pd-109-labeled monoclonal antibody to tumor-associated antigens may have potential applications in tumor immunotherapy.

  11. Development of Human Monoclonal Antibodies Against Respiratory Syncytial Virus Using a High Efficiency Human Hybridoma Technique.

    PubMed

    Alvarado, Gabriela; Crowe, James E

    2016-01-01

    Human monoclonal antibodies against RSV have high potential for use as prophylaxis or therapeutic molecules, and they also can be used to define the structure of protective epitopes for rational vaccine design. In the past, however, isolation of human monoclonal antibodies was difficult and inefficient. Here, we describe contemporary methods for activation and proliferation of primary human memory B cells followed by cytofusion to non-secreting myeloma cells by dielectrophoresis to generate human hybridomas secreting RSV-specific monoclonal antibodies. We also provide experimental methods for screening human B cell lines to obtain RSV-specific lines, especially lines secreting neutralizing antibodies. PMID:27464688

  12. Analysis of Campylobacter jejuni antigens with monoclonal antibodies.

    PubMed Central

    Kosunen, T U; Bång, B E; Hurme, M

    1984-01-01

    To develop monoclonal reagents for antigenic analysis and serotyping of Campylobacter spp., hybridoma cell lines were produced by fusion of mouse myeloma cells and spleen cells from mice immunized with Formalin-treated Campylobacter jejuni organisms. An enzyme immunoassay was used for preliminary screening of the cell culture supernatants and ascites. Twenty-nine clones which reacted with the immunogen were obtained. Seven of these clones were positive in passive hemagglutination tests with sheep erythrocytes coated with boiled saline extract of whole bacteria; four of these reacted with the purified polysaccharide preparation and with the autoclaved saline extract, but not with lipopolysaccharide prepared from the immunogen strain. Two of the antipolysaccharide clones agglutinated live bacteria in slide tests. Four additional clones gave positive slide agglutination tests with live bacteria, but in tube testing no clones agglutinated Formalin-treated bacteria. No cross-reactions with unrelated bacteria were seen, but several clones reacted in the enzyme immunoassay with many of the 24 Campylobacter strains studied. The clone which gave the highest mean enzyme immunoassay values with Campylobacter coli and C. jejuni strains also reacted with Campylobacter fetus subsp. veneralis and C. fetus subsp. fetus strains. This clone also gave the highest enzyme immunoassay value with an acid glycine extract of the immunogen, which indicates the presence of common antigens in the extract. The results suggest that monoclonal antibodies may be used to devise serotyping schemes for Campylobacter spp. PMID:6365954

  13. [Monoclonal antibodies for the treatment of multiple sclerosis].

    PubMed

    Sánchez-Seco, Victoria Galán; Casanova Peño, Ignacio; Arroyo González, Rafael

    2014-12-01

    Until the mid 1990s, with the appearance of interferon beta and glatiramer acetate, there was no treatment for multiple sclerosis (MS). However, due to their moderate therapeutic potential in some patients, a broad search was continued to find new and more effective treatment strategies, largely concentrated on monoclonal antibodies (MOAB). Natalizumab, the first MOAB for the treatment of MS, was approved at the end of 2004, representing a major advance in the field of neuroimmunology. Today, there is broad experience with natalizumab and other MOAB (alemtuzumab, daclizumab, rituximab, ocrelizumab, ofatumumab and anti-lingo-1) that are pending commercialization or are under phase II or III of development with promising results. The present review analyzes the efficacy and safety results of all these drugs. PMID:25732947

  14. Monoclonal antibodies and the transformation of blood typing

    PubMed Central

    Marks, Lara

    2014-01-01

    Today, when monoclonal antibodies (mAbs) have become one of the most important classes of therapeutic drugs, it is easy to forget how much they have transformed our healthcare in other ways. One of the first clinical areas, as this paper shows, where mAbs made their mark was in the field of blood typing. The adoption of mAbs for this purpose was done with little public fanfare or funding. Nonetheless, it radically transformed the accuracy and cost of blood typing and shifted the procedure away from a dependence on reagents made from human blood donated by volunteers. This paper argues that the development of mAbs as reagents for blood typing laid the foundation for the first large-scale production of mAbs thereby paving the way to the advent of mAb diagnostics and therapeutics. PMID:25484059

  15. Target Therapy in Hematological Malignances: New Monoclonal Antibodies

    PubMed Central

    Szymczyk, Agnieszka; Pawlowski, Johannes

    2014-01-01

    Apart from radio- and chemotherapy, monoclonal antibodies (MoAbs) represent a new, more selective tool in the treatment of hematological malignancies. MoAbs bind with the specific antigens of the tumors. This interaction is a basis for targeted therapies which exhibit few side effects and significant antitumor activity. This review provides an overview of the functional characteristics of MoAbs, with some examples of their clinical application. The promising results in the treatment of hematological malignancies have led to the more frequent usage of MoAbs in the therapy. Development of MoAbs is a subject of extensive research. They are a promising method of cancer treatment in the future.

  16. Monoclonal antibody capture and viral clearance by cation exchange chromatography.

    PubMed

    Miesegaes, G R; Lute, S; Strauss, D M; Read, E K; Venkiteshwaran, A; Kreuzman, A; Shah, R; Shamlou, P; Chen, D; Brorson, K

    2012-08-01

    Traditionally, post-production culture harvest capture of therapeutic monoclonal antibodies (mAbs) is performed using Protein A chromatography. We investigated the efficiency and robustness of cation exchange chromatography (CEX) in an effort to evaluate alternative capture methodologies. Up to five commercially available CEX resins were systematically evaluated using an experimentally optimized buffer platform and a design-of-experiment (DoE) approach for their ability to (a) capture a model mAb with a neutral isoelectric point, (b) clear three model viruses (porcine parvovirus, CHO type-C particles, and a bacteriophage). This approach identified a narrow operating space where yield, purity, and viral clearance were optimal under a CEX capture platform, and revealed trends between viral clearance of PPV and product purity (but not yield). Our results suggest that after unit operation optimization, CEX can serve as a suitable capture step. PMID:22488719

  17. Monoclonal Antibodies for Specific Detection of Encephalitozoon cuniculi

    PubMed Central

    Mo, Lan; Drancourt, Michel

    2004-01-01

    Seven species-specific monoclonal antibodies (MAbs) were produced against Encephalitozoon cuniculi and characterized. The MAbs were immunoglobulin G, and when used for indirect microimmunofluorescence microscopy and Western immunoblot assays, they detected E. cuniculi originating from clinical samples. They did not cross-react with other Encephalitozoon species (E. intestinalis and E. hellem) or with a collection of gram-negative bacteria, yeast, and other parasites. The MAbs reacted primarily with 121-, 56-, 45-, 43-, and 41-kDa protein epitopes of E. cuniculi. These epitopes were demonstrated to be E. cuniculi species specific by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We developed MAbs to strains of E. cuniculi that can be used successfully to distinguish E. cuniculi from other microsporidial species in cultures established from clinical specimens. These MAbs may provide a specific, simple, rapid, and low-cost tool for the identification and diagnosis of infections due to microsporidia. PMID:15539506

  18. Enantioselective hydrolysis of naproxen ethyl ester catalyzed by monoclonal antibodies.

    PubMed

    Shi, Zhen-Dan; Yang, Bing-Hui; Zhao, Jing-Jing; Wu, Yu-Lin; Ji, Yong-Yong; Yeh, Ming

    2002-07-01

    This report described that a hapten of racemic phosphonate 3 designed as the mimic of the transition state of hydrolysis of naproxen ethyl ester was successfully synthesized from easily available 2-acetyl-6-methoxy-naphthalene 5. Then BALB/C mice were immunized and one of the monoclonal catalytic antibodies, N116-27, which enantioselectively accelerated the hydrolysis of the R-(-)-naproxen ethyl ester was given. The Michaelis-Menton parameter for the catalyzed reaction was K(M)=6.67 mM and k(cat)/k(uncat)=5.8 x 10(4). This enantioselective result was explained by the fact that the R-isomer of rac-hapten was more immunogenic than the S-isomer. PMID:11983513

  19. Enhancing tumor-targeting monoclonal antibodies therapy by PARP inhibitors

    PubMed Central

    Yélamos, José; Galindo, Miguel; Navarro, Judith; Albanell, Joan; Rovira, Ana; Rojo, Federico; Oliver, Javier

    2016-01-01

    ABSTRACT Monoclonal antibodies (mAbs) have become a successful therapeutic approach in cancer. However, some patients do not achieve long-term clinical benefit and most mAbs only exert modest effects as monotherapies. Therefore, combinations with chemotherapy are currently being investigated. Emerging studies have shown a synergistic therapeutic effect of PARP inhibitors and mAbs in cancer. PARP enzymes catalytically cleave β-NAD+ and transfer the ADP-ribose moiety to acceptor proteins, modifying their function. In here, we update recent data about the therapeutic effect of the combination of PARP inhibitors with mAbs in cancer treatment and discuss the molecular mechanisms involved in this synergy. PMID:26942084

  20. Cell line profiling to improve monoclonal antibody production.

    PubMed

    Kang, Sohye; Ren, Da; Xiao, Gang; Daris, Kristi; Buck, Lynette; Enyenihi, Atim A; Zubarev, Roman; Bondarenko, Pavel V; Deshpande, Rohini

    2014-04-01

    Mammalian cell culture performance is influenced by both intrinsic (genetic) and extrinsic (media and process) factors. In this study, intrinsic capacity of various monoclonal antibody-producing Chinese Hamster Ovary (CHO) cell lines was compared by exposing them to the same culture condition. Microarray-based transcriptomics and LC-MS/MS shotgun proteomics technologies were utilized to obtain expression landscape of different cell lines. Specific transcripts and proteins correlating with productivity, growth rate and cell size have been identified. The proteomics analysis results showed a strong correlation between the intracellular protein expression levels of the recombinant DHFR and productivity. In contrast, neither the light chain nor the heavy chain of the recombinant monoclonal antibody showed correlation to productivity. Other top ranked proteins which demonstrated positive correlation to productivity included the adaptor protein complex subunits AP3D1and AP2B2, DNA repair protein DDB1 and the ER translocation complex component, SRPR. The subunits of molecular chaperone T-complex protein 1 and the regulator of mitochondrial one-carbon metabolism MTHFD2 showed negative correlation to productivity. The transcriptomics analysis has identified the regulators of calcium signaling, Tmem20 and Rcan1, as the top ranked genes displaying positive and negative correlation to productivity, respectively. For the second part of the study, the principal component analysis (PCA) was generated to view the underlying global structure of the expression data. A clear division and expression polarity was observed between the two distinct clusters of cell lines, independent of link to productivity or any other traits examined. The primary component of the PCA generated from either transcriptomics or proteomics data displayed a strong correlation to cell size and doubling time, while none of the main principal components showed correlation to productivity. Our findings suggest

  1. Probing Functional Changes in Exocyst Configuration with Monoclonal Antibodies

    PubMed Central

    Inamdar, Shivangi M.; Hsu, Shu-Chan; Yeaman, Charles

    2016-01-01

    Spatial regulation of exocytosis relies on the exocyst, a hetero-octameric protein complex that tethers vesicles to fusion sites at the plasma membrane. Nevertheless, our understanding of mechanisms regulating exocyst assembly/disassembly, localization, and function are incomplete. Here, we have exploited a panel of anti-Sec6 monoclonal antibodies (mAbs) to probe possible configurational changes accompanying transitions in exocyst function in epithelial MDCK cells. Sec6 is quantitatively associated with Sec8 in high molecular weight complexes, as shown by gel filtration and co-immunoprecipitation studies. We mapped epitopes recognized by more than 20 distinct mAbs to one of six Sec6 segments. Surprisingly, mAbs that bound epitopes in each segment labeled distinct subcellular structures. In general, antibodies to epitopes in N-terminal domains labeled Sec6 in either cytosolic or nuclear pools, whereas those that bound epitopes in C-terminal domains labeled membrane-associated Sec6. In this latter group, we identified antibodies that labeled distinct Sec6 populations at the apical junctional complex, desmosomes, endoplasmic reticulum and vimentin-type intermediate filaments. That each antibody was specific was verified by both Sec6 RNAi and competition with fusion proteins containing each domain. Comparison of non-polarized and polarized cells revealed that many Sec6 epitopes either redistribute or become concealed during epithelial polarization. Transitions in exocyst configurations may be regulated in part by the actions of Ral GTPases, because the exposure of Sec6 C-terminal domain epitopes at the plasma membrane is significantly reduced upon RalA RNAi. To determine whether spatio-temporal changes in epitope accessibility was correlated with differential stability of interactions between Sec6 and other exocyst subunits, we quantified relative amounts of each subunit that co-immunoprecipitated with Sec6 when antibodies to N-terminal or C-terminal epitopes were used

  2. Probing Functional Changes in Exocyst Configuration with Monoclonal Antibodies.

    PubMed

    Inamdar, Shivangi M; Hsu, Shu-Chan; Yeaman, Charles

    2016-01-01

    Spatial regulation of exocytosis relies on the exocyst, a hetero-octameric protein complex that tethers vesicles to fusion sites at the plasma membrane. Nevertheless, our understanding of mechanisms regulating exocyst assembly/disassembly, localization, and function are incomplete. Here, we have exploited a panel of anti-Sec6 monoclonal antibodies (mAbs) to probe possible configurational changes accompanying transitions in exocyst function in epithelial MDCK cells. Sec6 is quantitatively associated with Sec8 in high molecular weight complexes, as shown by gel filtration and co-immunoprecipitation studies. We mapped epitopes recognized by more than 20 distinct mAbs to one of six Sec6 segments. Surprisingly, mAbs that bound epitopes in each segment labeled distinct subcellular structures. In general, antibodies to epitopes in N-terminal domains labeled Sec6 in either cytosolic or nuclear pools, whereas those that bound epitopes in C-terminal domains labeled membrane-associated Sec6. In this latter group, we identified antibodies that labeled distinct Sec6 populations at the apical junctional complex, desmosomes, endoplasmic reticulum and vimentin-type intermediate filaments. That each antibody was specific was verified by both Sec6 RNAi and competition with fusion proteins containing each domain. Comparison of non-polarized and polarized cells revealed that many Sec6 epitopes either redistribute or become concealed during epithelial polarization. Transitions in exocyst configurations may be regulated in part by the actions of Ral GTPases, because the exposure of Sec6 C-terminal domain epitopes at the plasma membrane is significantly reduced upon RalA RNAi. To determine whether spatio-temporal changes in epitope accessibility was correlated with differential stability of interactions between Sec6 and other exocyst subunits, we quantified relative amounts of each subunit that co-immunoprecipitated with Sec6 when antibodies to N-terminal or C-terminal epitopes were used

  3. Monoclonal Antibody Analysis of Keratin Expression in the Central Nervous System

    NASA Astrophysics Data System (ADS)

    Franko, Maryellen C.; Gibbs, Clarence J.; Rhoades, Dorothy A.; Carleton Gajdusek, D.

    1987-05-01

    A monoclonal antibody directed against a 65-kDa brain protein demonstrates an epitope found in keratin from human epidermis. By indirect immunofluorescence, the antibody decorates intracytoplasmic filaments in a subclass of astrocytes and Purkinje cells of adult hamster brain. Double-label immunofluorescence study using antibody to glial fibrillary acidic protein and this antibody reveals the 65-kDa protein to be closely associated with glial filaments in astrocytes of fetal mouse brain cultures. Immunoblot analysis of purified human epidermal keratin and hamster brain homogenate confirms the reactivity of this antibody to epidermal keratin polypeptides. All the major epidermal keratins were recognized by this antibody. It did not bind to the remaining major intermediate filament proteins. These findings suggest that monoclonal antibody 34C9 recognizes a cytoskeletal structure connected with intermediate filaments. In addition, the monoclonal antibody demonstrates that epidermal keratins share an epitope not only among themselves but also with a ``neural keratin.''

  4. Monoclonal antibody therapeutics with up to five specificities

    PubMed Central

    LaFleur, David W.; Abramyan, Donara; Kanakaraj, Palanisamy; Smith, Rodger G.; Shah, Rutul R.; Wang, Geping; Yao, Xiao-Tao; Kankanala, Spandana; Boyd, Ernie; Zaritskaya, Liubov; Nam, Viktoriya; Puffer, Bridget A.; Buasen, Pete; Kaithamana, Shashi; Burnette, Andrew F.; Krishnamurthy, Rajesh; Patel, Dimki; Roschke, Viktor V.; Kiener, Peter A.; Hilbert, David M.; Barbas III, Carlos F.

    2013-01-01

    The recognition that few human diseases are thoroughly addressed by mono-specific, monoclonal antibodies (mAbs) continues to drive the development of antibody therapeutics with additional specificities and enhanced activity. Historically, efforts to engineer additional antigen recognition into molecules have relied predominantly on the reformatting of immunoglobulin domains. In this report we describe a series of fully functional mAbs to which additional specificities have been imparted through the recombinant fusion of relatively short polypeptides sequences. The sequences are selected for binding to a particular target from combinatorial libraries that express linear, disulfide-constrained, or domain-based structures. The potential for fusion of peptides to the N- and C- termini of both the heavy and light chains affords the bivalent expression of up to four different peptides. The resulting molecules, called zybodies, can gain up to four additional specificities, while retaining the original functionality and specificity of the scaffold antibody. We explore the use of two clinically significant oncology antibodies, trastuzumab and cetuximab, as zybody scaffolds and demonstrate functional enhancements in each case. The affect of fusion position on both peptide and scaffold function is explored, and penta-specific zybodies are demonstrated to simultaneously engage five targets (ErbB2, EGFR, IGF-1R, Ang2 and integrin αvβ3). Bispecific, trastuzumab-based zybodies targeting ErbB2 and Ang2 are shown to exhibit superior efficacy to trastuzumab in an angiogenesis-dependent xenograft tumor model. A cetuximab-based bispecific zybody that targeting EGFR and ErbB3 simultaneously disrupted multiple intracellular signaling pathways; inhibited tumor cell proliferation; and showed efficacy superior to that of cetuximab in a xenograft tumor model. PMID:23575268

  5. Monoclonal antibodies specific for human monocytes, granulocytes and endothelium.

    PubMed Central

    Hogg, N; MacDonald, S; Slusarenko, M; Beverley, P C

    1984-01-01

    Four monoclonal antibodies against antigens of human myeloid cells have been produced and thoroughly characterized in terms of their reactions with peripheral blood cells, cell lines, nine lymphoid and non-lymphoid tissues and the polypeptides with which they react. UCHM1 and SmO identify antigens present on the majority of blood monocytes and a variable, but lower, proportion of tissue macrophages. From their morphology and location in tissues, these cells appear to be recirculating monocytes. SMO antigen is also present on platelets. In addition, both antibodies stained endothelial cells, SMO in all tissues examined and UCHM1 variably. Biochemical investigation indicated that the UCHM1 antigen is a protein of 52,000 MW while the SMO antigen could not be indentified. The antibodies TG1 and 28 identify antigens mainly present on granulocytes. While mAb 28 reacted with neutrophils, TG1 also stained eosinophils and stained strongly a proportion of monocytes. TG1 also reacted variably with some non-haemopoietic cell lines. Both antibodies reacted predominantly with granulocytes in tissue sections. MAb TG1 precipitated a single polypeptide of 156,000 MW from monocytes and granulocytes, while mAb 28 precipitated non-convalently associated polypeptides of 83,000 and 155,000 MW from granulocytes but only a single molecule from monocytes, corresponding to the lower MW chain of 83,000. The epitope with which mAb 28 reacts appears not to be exposed on the surface of intact monocytes. This suggests that a similar or identical 83,000 MW molecule is made by both neutrophils and monocytes, but that its expression differs according to cell type. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:6389324

  6. Development of a bispecific monoclonal antibody for use in molecular hybridisation.

    PubMed

    Auriol, J; Guesdon, J L; Mazié, J C; Nato, F

    1994-02-28

    A mouse hybrid hybridoma (tetradoma) was prepared by fusing hybridomas producing monoclonal antibody to acetyl-aminofluorene with hybridomas producing antibody against calf intestine alkaline phosphatase. The tetradoma line established secreted immunoglobulin manifesting parental and bispecific binding characteristics. Bispecific monoclonal antibody was purified and used for a one-step immunodetection assay of non-radioactive DNA and RNA probes. The immunoassay developed was able to detect 5 pg DNA within 2 h and gave low background noise. PMID:8133070

  7. Optimal activation of carboxyl-superparamagnetic iron oxide nanoparticles bioconjugated with antibody using orthogonal array design.

    PubMed

    Liu, Lin; Zhang, Xiaoqang; Zhang, Yu; Pu, Yuepu; Yin, Lihong; Tang, Meng; Liu, Hui

    2013-12-01

    This study aims to bioconjugate anti-EMMPRIN monoclonal antibody on the surface of carboxyl-SPIO nanoparticles and to optimize the activated conditions of bioconjugation. Anti-EMMPRIN monoclonal antibody bioconjugated carboxyl-SPIO nanoparticles were performed through a coupling strategy of EDC and sulfo-NHS. The procedure was comprised of two steps by activation of carboxyl-SPIO nanoparticles and conjugation with monoclonal antibody. The optimal activated parameters of bioconjugation were evaluated by single factor design and orthogonal array design. SDS-PAGE analysis and Bradford assay was used for testing and verifying the efficiency of activated conditions obtained from orthogonal array. The results show that pH value, temperature and reaction time were important factors that influence bioconjugated efficiency. The activated parameters with pH value 6.2, temperature 25 degrees C and reaction time 30 min were obviously optimal for activation of carboxyl-SPIO nanoparticles and conjugation with monoclonal EMMPEIN antibody. This coupling strategy for anti-EMMPRIN mAb bioconjugated on SPIO nanoparticles was efficient, and may be further applied in the fields of medical or biological practices. PMID:24266206

  8. Influence of unlabeled monoclonal anti-mouse antibody on the clearance rate of radiolabeled mouse monoclonal antibody

    SciTech Connect

    Wahl, R.L.; Laino, L.; Jackson, G.; Fisher, S.; Beierwaltes, W.H.

    1985-05-01

    High blood background levels of intact radiolabeled monoclonal antibody (MoAb) after intravenous (iv) injection are problematic. The injection of unlabeled polyclonal antimouse Abs following injection with labeled MoAbs produces accelerated MoAb clearance. This study evaluates a Mo antimouse Ab for efficacy of accelerating radio MoAb clearance. HB-58 is a rat/mouse MoAb which binds strongly to mouse kappa light chains present in 95% of murine monoclonals. It is unreactive with rat, rabbit or human kappa chains. Six rats were injected iv with 30 ..mu..Ci (approximately 6 ..mu..g) of I-125 UPC-10, a non-specific IgG2ak MoAb that is bound to well by HB-58. No alteration was seen in the clearance of UPC-10 in any of the animals, regardless of the injection type or amount on the second day. In addition, no increase in liver or spleen activity was seen in those rats that received HB-58. The lack of change in rate of clearance and biodistribution of UPC-10 after the iv injection of a purified, specific, anti-mouse MoAb is in marked contrast to the accelerated clearance reported following polyclonal anti-mouse antibody administration. This may be due to the inability of MoAbs to cross link. These preliminary studies suggest that Mo anti-mouse Abs, at these dose levels, are not useful in achieving increased rates of radiolabeled murine MoAb clearance.

  9. Interfacial dilatational deformation accelerates particle formation in monoclonal antibody solutions.

    PubMed

    Lin, Gigi L; Pathak, Jai A; Kim, Dong Hyun; Carlson, Marcia; Riguero, Valeria; Kim, Yoen Joo; Buff, Jean S; Fuller, Gerald G

    2016-04-14

    Protein molecules are amphiphilic moieties that spontaneously adsorb at the air/solution (A/S) interface to lower the surface energy. Previous studies have shown that hydrodynamic disruptions to these A/S interfaces can result in the formation of protein aggregates that are of concern to the pharmaceutical industry. Interfacial hydrodynamic stresses encountered by protein therapeutic solutions under typical manufacturing, filling, and shipping conditions will impact protein stability, prompting a need to characterize the contribution of basic fluid kinematics to monoclonal antibody (mAb) destabilization. We demonstrate that dilatational surface deformations are more important to antibody stability when compared to constant-area shear of the A/S interface. We have constructed a dilatational interfacial rheometer that utilizes simultaneous pressure and bubble shape measurements to study the mechanical stability of mAbs under interfacial aging. It has a distinct advantage over methods utilizing the Young-Laplace equation, which incorrectly describes viscoelastic interfaces. We provide visual evidence of particle ejection from dilatated A/S interfaces and spectroscopic data of ejected mAb particles. These rheological studies frame a molecular understanding of the protein-protein interactions at the complex-fluid interface. PMID:26891116

  10. Tumor size: effect on monoclonal antibody uptake in tumor models

    SciTech Connect

    Hagan, P.L.; Halpern, S.E.; Dillman, R.O.; Shawler, D.L.; Johnson, D.E.; Chen, A.; Krishnan, L.; Frincke, J.; Bartholomew, R.M.; David, G.S.

    1986-03-01

    Studies were performed to determine the effect of tumor size on the incorporation of radiolabeled monoclonal antitumor antibodies (MoAbs) into human tumors growing in nude mice. The colon tumors ranged in size from 0.03-1.6 g, the melanoma from 0.1 to 6.7 g, and the lymphoma from 0.06 to 10.2 g. Indium-111 was primarily used as the radiolabel, however, both 125I and 111In were used as tracers for the MoAb in one experiment. The per g radiopharmaceutical uptake by tumors was inversely proportional to tumor size when tumor specific MoAb was administered. This finding was independent of the radiolabel and was demonstrable when the mice bore two tumors of differing size. When the MoAb was not specific for the tumor, the data were less well defined and a statistically significant correlation with size did not occur. These data are strong evidence for a decrease in per g uptake of labeled tumor specific antibodies as tumors increase in size.

  11. Tregalizumab – A Monoclonal Antibody to Target Regulatory T Cells

    PubMed Central

    König, Martin; Rharbaoui, Faiza; Aigner, Silke; Dälken, Benjamin; Schüttrumpf, Jörg

    2016-01-01

    Regulatory T cells (Tregs) represent a subpopulation of CD4+ T cells, which are essential for the maintenance of immunological tolerance. The absence or dysfunction of Tregs can lead to autoimmunity and allergies. The restoration of functional Tregs and/or Treg cell numbers represents a novel and attractive approach for the treatment of autoimmune diseases, e.g., rheumatoid arthritis (RA). The CD4 cell surface receptor is a target for modulation of T cell function. Monoclonal antibodies (mAbs) against CD4 have previously been tested for the treatment of autoimmune diseases, including RA. Furthermore, in model systems, anti-CD4 antibodies are able to induce tolerance and mediate immunomodulatory effects through a variety of mechanisms. Despite the availability of innovative and effective therapies for RA, many patients still have persistently active disease or experience adverse events that can limit use. A growing body of evidence suggests that Treg modulation could offer a new therapeutic strategy in RA and other autoimmune disorders. Here, we describe tregalizumab (BT-061), which is a novel, non-depleting IgG1 mAb that binds to a unique epitope of CD4. Tregalizumab represents the first humanized anti-CD4 mAb that selectively induces Treg activation. PMID:26834751

  12. Monoclonal Antibodies for the Diagnosis of Borrelia crocidurae.

    PubMed

    Fotso Fotso, Aurélien; Mediannikov, Oleg; Nappez, Claude; Azza, Saïd; Raoult, Didier; Drancourt, Michel

    2016-01-01

    Relapsing fever borreliae, produced by ectoparasite-borne Borrelia species, cause mild to deadly bacteremia and miscarriage. In the perspective of developing inexpensive assays for the rapid detection of relapsing fever borreliae, we produced 12 monoclonal antibodies (MAbs) against Borrelia crocidurae and characterized the two exhibiting the highest titers. P3A10 MAb reacts with the 35.6-kDa flagellin B (flaB) of B. crocidurae while P6D9 MAb recognizes a 35.1-kDa variable-like protein (Vlp) in B. crocidurae and a 35.2-kDa Vlp in Borrelia duttonii. Indirect immunofluorescence assay incorporating relapsing fever and Lyme group borreliae and 11 blood-borne organisms responsible for fever in West Africa confirmed the reactivity of these two MAbs. Combining these two MAbs in indirect immunofluorescence assays detected relapsing fever borreliae including B. crocidurae in ticks and the blood of febrile Senegalese patients. Both antibodies could be incorporated into inexpensive and stable formats suited for the rapid point-of-care diagnosis of relapsing fever. These first-ever MAbs directed against African relapsing fever borreliae are available for the scientific community to promote research in this neglected field. PMID:26598566

  13. Evidence of a saturable hepatic receptor for mouse monoclonal antibodies

    SciTech Connect

    De Nardo, G.L.; De Nardo, S.J.; Peng, J.S.; O'Grady, L.F.; Mills, S.L.; Epstein, A.L.; Cardiff, R.D.

    1985-05-01

    Monoclonal antibodies (MAb) can be labeled with I-123 at high specific activities, so that large amounts of radioactivity attached to small amounts of protein can be injected for radioimmunoimaging. This conserves antibody and decreases the opportunity for foreign protein reactions and target tissue binding site saturation. In order to assess the effects on pharmacokinetics and imaging, the authors administered microgram amounts of I-123-MAb (Lyn-1, IgG2a or B6.01, IgGl with and following 4-5 milligram preloading with MAb on separate occasions to 4 patients with a target tumor (B cell lymphoma) and 2 patients without a target tumor (breast cancer). Pharmacokinetics were observed in blood and urine by counting whole samples and HPLC fractions of these samples and in organs by serial imaging. Early blood clearance and urinary excretion were faster after injection of microgram amounts of MAb, but subsequently were comparable to those obtained after preload. This paper concludes that the amount of administered MAb dramatically influences the pharmacokinetics of mouse MAb. Saturable hepatic Fc receptors are probably the source of these observations. Reports of accelerated deiodination of MAb are related to this phenomenon. Optimal imaging and treatment with MAb requires saturation of these hepatic receptors.

  14. Trimerization Dictates Solution Opalescence of a Monoclonal Antibody.

    PubMed

    Yang, Teng-Chieh; Langford, Alex Jacob; Kumar, Sandeep; Ruesch, John Carl; Wang, Wei

    2016-08-01

    Opalescence, sometimes observed in antibody solutions, is thought to be mediated by light scattering of soluble oligomers or insoluble particulates. However, mechanistic features, such as stoichiometry and self-association affinity of oligomeric species related to opalescence, are poorly understood. Here, opalescence behavior of a monoclonal antibody (mAb-1) solution was studied over a wide range of solution conditions including different protein concentrations, pH, and in the presence or absence of salt. Hydrodynamic and thermodynamic properties of mAb-1 solutions were studied by analytical ultracentrifugation and dynamic light scattering. Opalescence in mAb-1 solutions is pH and concentration dependent. The degree of opalescence correlates with reversible monomer-trimer equilibrium detected by analytical ultracentrifugation. Increased trimer formation corresponds to increased opalescence in mAb-1 solutions at higher pH and protein concentrations. Addition of NaCl shifts this equilibrium toward monomer and reduces solution opalescence. This study demonstrates that opalescence in mAb-1 solutions does not arise from the light scattering of monomer or random molecular self-associations but is strongly correlated with a specific self-association stoichiometry and affinity. Importantly, at pH 5.5 (far below isoelectric point of mAb-1), the solution is not opalescent and with nonideal behavior. This study also dissects several parameters to describe the hydrodynamic and thermodynamic nonideality. PMID:27373839

  15. Defining process design space for monoclonal antibody cell culture.

    PubMed

    Abu-Absi, Susan Fugett; Yang, LiYing; Thompson, Patrick; Jiang, Canping; Kandula, Sunitha; Schilling, Bernhard; Shukla, Abhinav A

    2010-08-15

    The concept of design space has been taking root as a foundation of in-process control strategies for biopharmaceutical manufacturing processes. During mapping of the process design space, the multidimensional combination of operational variables is studied to quantify the impact on process performance in terms of productivity and product quality. An efficient methodology to map the design space for a monoclonal antibody cell culture process is described. A failure modes and effects analysis (FMEA) was used as the basis for the process characterization exercise. This was followed by an integrated study of the inoculum stage of the process which includes progressive shake flask and seed bioreactor steps. The operating conditions for the seed bioreactor were studied in an integrated fashion with the production bioreactor using a two stage design of experiments (DOE) methodology to enable optimization of operating conditions. A two level Resolution IV design was followed by a central composite design (CCD). These experiments enabled identification of the edge of failure and classification of the operational parameters as non-key, key or critical. In addition, the models generated from the data provide further insight into balancing productivity of the cell culture process with product quality considerations. Finally, process and product-related impurity clearance was evaluated by studies linking the upstream process with downstream purification. Production bioreactor parameters that directly influence antibody charge variants and glycosylation in CHO systems were identified. PMID:20589669

  16. Hierarchical Cluster Formation in Concentrated Monoclonal Antibody Formulations

    NASA Astrophysics Data System (ADS)

    Godfrin, P. Douglas; Zarzar, Jonathan; Zarraga, Isidro Dan; Porcar, Lionel; Falus, Peter; Wagner, Norman; Liu, Yun

    Reversible cluster formation has been identified as an underlying cause of large solution viscosities observed in some concentrated monoclonal antibody (mAb) formulations. As high solution viscosity prevents the use of subcutaneous injection as a delivery method for some mAbs, a fundamental understanding of the interactions responsible for high viscosities in concentrated mAb solutions is of significant relevance to mAb applications in human health care as well as of intellectual interest. Here, we present a detailed investigation of a well-studied IgG1 based mAb to relate the short time dynamics and microstructure to significant viscosity changes over a range of pharmaceutically relevant physiochemical conditions. Using a combination of experimental techniques, it is found that upon adding Na2SO4, these antibodies dimerize in solution. Proteins form strongly bounded reversible dimers at dilute concentrations that, when concentrated, interact with each other to form loosely bounded, large, transient clusters. The combined effect of forming strongly bounded dimers and a large transient network is a significant increase in the solution viscosity. Strongly bounded, reversible dimers may exist in many IgG1 based mAb systems such that these results contribute to a more comprehensive understanding of the physical mechanisms producing high viscosities in concentrated protein solutions.

  17. Characterization of novel neutralizing monoclonal antibodies specific to human neurturin.

    PubMed

    Hongo, J A; Tsai, S P; Moffat, B; Schroeder, K A; Jung, C; Chuntharapai, A; Lampe, P A; Johnson, E M; de Sauvage, F J; Armanini, M; Phillips, H; Devaux, B

    2000-08-01

    Neurturin (NTN) a structural and functional relative of glial cell line-derived neurotrophic factor, was originally identified based on its ability to support the survival of sympathetic neurons in culture. Similar to glial cell line-derived neurotrophic factor (GDNF), Neurturin has been shown to bind to a high affinity glycosylphosphatidylinositol (GPI)-linked receptor (GFRalpha2) and induce phosphorylation of the tyrosine kinase receptor Ret, resulting in the activation of the mitogen activated protein kinase (MAPK) signalling pathway. A panel of six novel murine monoclonal antibodies (MAbs) specific to human Neurturin has been developed and characterized. Four of the MAbs tested inhibit, to varying degrees, binding of NTN to the GPI-linked GFRalpha2 receptor. Three MAbs cross-react with the murine homolog. These antibodies have been shown to be useful reagents for Western blotting, immunohistochemistry, and also for the development of a sensitive, quantitative enzyme-linked immunosorbent assay (ELISA) for human NTN. Novel, specific MAbs with varying epitope specificities and blocking activity will be valuable tools for both the in vitro and in vivo characterization of NTN and its relationship to the GFRalpha2 and Ret receptors. PMID:11001403

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

    PubMed

    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

    2016-01-01

    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. PMID:27292946

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

    PubMed Central

    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.

    2016-01-01

    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. PMID:27292946

  20. Structure of solid tumors and their vasculature: Implications for therapy with monoclonal antibodies

    SciTech Connect

    Dvorak, H.F.; Nagy, J.A.; Dvorak, A.M. )

    1991-03-01

    Delivery of monoclonal antibodies to solid tumors is a vexing problem that must be solved if these antibodies are to realize their promise in therapy. Such success as has been achieved with monoclonal antibodies is attributable to the local hyperpermeability of the tumor vasculature, a property that favors antibody extravasation at tumor sites and that is mediated by a tumor-secreted vascular permeability factor. However, leaky tumor blood vessels are generally some distance removed from target tumor cells, separated by stroma and by other tumor cells that together represent significant barriers to penetration by extravasated monoclonal antibodies. For this reason, alternative approaches may be attractive. These include the use of antibody-linked cytotoxins, which are able to kill tumor cells without immediate contact, and direction of antibodies against nontumor cell targets, for example, antigens unique to the tumor vascular endothelium or to tumor stroma. 50 refs.

  1. Radiolabeled monoclonal antibodies for imaging and therapy: Potential, problems, and prospects: Scientific highlights

    SciTech Connect

    Srivastava, S.C.; Buraggi, G.L.

    1986-01-01

    This meeting focused on areas of research on radiolabeled monoclonal antibodies. Topics covered included the production, purification, and fragmentation of monoclonal antibodies and immunochemistry of hybridomas; the production and the chemistry of radionuclides; the radiohalogenation and radiometal labeling techniques; the in-vivo pharmacokinetics of radiolabeled antibodies; the considerations of immunoreactivity of radiolabeled preparations; the instrumentation and imaging techniques as applied to radioimmunodetection; the radiation dosimetry in diagnostic and therapeutic use of labeled antibodies; the radioimmunoscintigraphy and radioimmunotherapy studies; and perspectives and directions for future research. Tutorial as well as scientific lectures describing the latest research data on the above topics were presented. Three workshop panels were convened on ''Methods for Determining Immunoreactivity of Radiolabeled Monoclonal Antibodies - Problems and Pitfalls,'' Radiobiological and Dosimetric Considerations for Immunotherapy with Labeled Antibodies,'' and ''The Human Anti-Mouse Antibody Response in Patients.''

  2. Tumor-associated hyaluronan limits efficacy of monoclonal antibody therapy.

    PubMed

    Singha, Netai C; Nekoroski, Tara; Zhao, Chunmei; Symons, Rebecca; Jiang, Ping; Frost, Gregory I; Huang, Zhongdong; Shepard, H Michael

    2015-02-01

    Despite tremendous progress in cancer immunotherapy for solid tumors, clinical success of monoclonal antibody (mAb) therapy is often limited by poorly understood mechanisms associated with the tumor microenvironment (TME). Accumulation of hyaluronan (HA), a major component of the TME, occurs in many solid tumor types, and is associated with poor prognosis and treatment resistance in multiple malignancies. In this study, we describe that a physical barrier associated with high levels of HA (HA(high)) in the TME restricts antibody and immune cell access to tumors, suggesting a novel mechanism of in vivo resistance to mAb therapy. We determined that approximately 60% of HER2(3+) primary breast tumors and approximately 40% of EGFR(+) head and neck squamous cell carcinomas are HA(high), and hypothesized that HA(high) tumors may be refractory to mAb therapy. We found that the pericellular matrix produced by HA(high) tumor cells inhibited both natural killer (NK) immune cell access to tumor cells and antibody-dependent cell-mediated cytotoxicity (ADCC) in vitro. Depletion of HA by PEGPH20, a pegylated recombinant human PH20 hyaluronidase, resulted in increased NK cell access to HA(high) tumor cells, and greatly enhanced trastuzumab- or cetuximab-dependent ADCC in vitro. Furthermore, PEGPH20 treatment enhanced trastuzumab and NK cell access to HA(high) tumors, resulting in enhanced trastuzumab- and NK cell-mediated tumor growth inhibition in vivo. These results suggest that HA(high) matrix in vivo may form a barrier inhibiting access of both mAb and NK cells, and that PEGPH20 treatment in combination with anticancer mAbs may be an effective adjunctive therapy for HA(high) tumors. PMID:25512619

  3. A novel high affinity human monoclonal antibody to mesothelin

    PubMed Central

    Ho, Mitchell; Feng, Mingqian; Fisher, Robert J.; Rader, Christoph; Pastan, Ira

    2010-01-01

    Mesothelin is a glycosylphosphatidylinisotol-anchored glycoprotein that is highly expressed on the cell surface of mesothelioma, ovarian cancer and other malignant tumors. The interaction between mesothelin and CA125 (also called MUC16) may facilitate the implantation and metastasis of tumors in the peritoneal cavity. A desirable therapeutic agent involves finding a fully human monoclonal antibody (mAb) that binds to mesothelin or CA125 and inhibits their interaction. Here we report the identification of a novel human mAb to mesothelin. HN1, a human single chain Fv specific for mesothelin, was isolated from a naïve human scFv phage display library. To investigate HN1 as a potential therapeutic, we generated a fully human IgG with the γ 1 heavy chain and the κ light chain, and an immuntoxin by fusing the HN1 scFv to a truncated Pseudomonas exotoxin A. The HN1 IgG kills cancer cells with very strong antibody-dependent cell-mediated cytotoxicity. HN1 binds a conformation-sensitive epitope in human mesothelin with high affinity (KD = 3 nM). The HN1 epitope is different from that of SS1, a mouse Fv used to develop therapeutic antibodies that are currently in clinical trials. HN1 binds to cell surface-associated mesothelin on human mesothelioma, ovarian cancer, lung adenocarcinoma and pancreatic cancer cells. In addition, HN1 can functionally block the interaction of mesothelin and CA125 on cancer cells. Most importantly, because the HN1 immuntoxin kills mesothelin-expressing cancer cells with high cytotoxic activity, we believe that it has significant potential for mesothelin-expressing cancer treatment and diagnosis. PMID:20635390

  4. Melioidosis in a patient on monoclonal antibody therapy for psoriatic arthritis.

    PubMed

    Commons, R J; Grivas, R; Currie, B J

    2014-12-01

    Melioidosis is caused by the environmental bacterium Burkholderia pseudomallei and can present with severe sepsis. Predisposing risk factors are present in 80% of cases. Monoclonal antibodies are increasingly prescribed for varied medical conditions. This report describes the first known case of melioidosis in a patient whose only risk factor for disease is treatment with a monoclonal antibody. Prescribers of monoclonal antibodies and other immunosuppressants should ensure that their patients are aware of the potential risk of melioidosis prior to travel and the precautions that should be taken. PMID:25442759

  5. The Use of Humanized Monoclonal Antibodies for the Prevention of Respiratory Syncytial Virus Infection

    PubMed Central

    Arcuri, Santo; Galletti, Silvia; Faldella, Giacomo

    2013-01-01

    Monoclonal antibodies are widely used both in infants and in adults for several indications. Humanized monoclonal antibodies (palivizumab) have been used for many years for the prevention of respiratory syncytial virus infection in pediatric populations (preterm infants, infants with chronic lung disease or congenital heart disease) at high risk of severe and potentially lethal course of the infection. This drug was reported to be safe, well tolerated and effective to decrease the hospitalization rate and mortality in these groups of infants by several clinical trials. In the present paper we report the development and the current use of monoclonal antibodies for prophylaxis against respiratory syncytial virus. PMID:23840240

  6. Use of AN Eosinophil Specific Monoclonal Antibody in Assessing Eosinophil Function.

    NASA Astrophysics Data System (ADS)

    Minkoff, Marjorie Sue

    A monoclonal antibody to an eosinophil specific determinant is very important in assessing eosinophil function during helminthic infection. Eosinophils induced by Schistosoma mansoni infection in BALB/c mice were used to induce C57B1/6 immunocytes for production of hybridomas secreting eosinophil monoclonal antibodies. These antibodies were shown to react with an eosinophil surface epitope but not with neutrophils or macrophages as determined by ELISA, immunodiffusion, immunofluorescence, and immunoblot assay. Affinity chromatography with eosinophil chemotactic factor-sepharose consistently selected out a { rm M_ R} 67,000 protein from solubilized eosinophil membrane antigens but not from neutrophil and macrophage antigens. In vitro studies showed that the eosinophil-specific monoclonal antibodies abrogated antibody-dependent eosinophil -mediated killing of S. mansoni schistosomula using mouse, rat or human eosinophils. Neutrophil and macrophage killing activities were unaffected. The monoclonal antibodies effected complement-dependent lysis of mouse and rat eosinophils but not of human eosinophils. ECF-treated eosinophils showed enhanced killing of schistosomula which was blocked by the monoclonal antibody. Murine and human eosinophils preincubated with monoclonal antibody exhibited decreased chemotaxis to ECF at optimal chemotactic concentrations. The monoclonal antibody also blocked eosinophil binding to ECF- sepharose beads. In vivo induction of peripheral blood eosinophilia by injection of S. mansoni eggs was suppressed by injections of monoclonal antibodies 2CD13 and 2QD45 in mouse and rat experimental models. Eosinophilia induced by keyhole limpet hemocyanin- cyclophosphamide treatment was also suppressed by monoclonal antibody in both murine and rat systems. Pulmonary granulomas in mice given egg injection and monoclonal antibody were smaller and contained fewer eosinophils than those granulomas from mice given eggs only. In immuno-biochemical studies, the

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

    PubMed

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

    2012-12-01

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

  8. PRODUCTION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES DIRECTED AGAINST THE AH RECEPTOR

    EPA Science Inventory

    Six hybridomas secreting monoclonal antibodies that are specific for the N-terminal peptide sequence of the murine Ah receptor were isolated. hese antibodies bind with high specificity to the Al receptor on protein blots of Hepa 1c1c7 cytosol. hree IgG1 antibodies (Rpt1, 2, and 3...

  9. Production and characterization of monoclonal antibodies against dog immunoglobulin isotypes.

    PubMed

    Arce, C; Moreno, A; Millán, Y; Martín de las Mulas, J; Llanes, D

    2002-09-01

    A panel of six monoclonal antibodies (mAbs) recognizing antigenic determinants on canine immunoglobulin (Ig) heavy or light chains was produced and characterized. All monoclonals recognized the IgG(2) subclass, although only two were subclass-specific (CA3H1 and CA4F1). The CA3B8 mAb was found to be specific for an epitope on canine immunoglobulin G heavy chain, (IgG(1) and IgG(2) subclasses). Two mAbs (CA2E9 and CA5B2) reacted with an epitope on the heavy chain of canine IgG and IgM and another, CA4E7, bound to canine IgA, IgG and IgM isotypes; CA4E7 recognized an epitope on canine immunoglobulin light chain. CA4E7, CA4F1 and CA5B2 recognized an epitope in the Fab region. Three mAbs, CA3B8, CA4E7 and CA5B2, showed much lower reactivity with canine IgG by ELISA when IgG was periodate-treated, suggesting that they recognized a carbohydrate determinant. Cross-reactivity analysis of these mAbs with sera from horse, goat, cow, sheep, pig, cat, rabbit, hamster, rat, mouse and human indicated that two mAbs, CA3B8 and CA5B2, recognized a canine IgG-specific epitope; two others, CA3H1 and CA4E7, recognized an epitope also present in rabbit and sheep immunoglobulin respectively; and the remaining two (CA2E9 and CA4F1) recognized an epitope broadly present on the Igs of the species analyzed. This panel of antibodies will be a useful tool for future canine immunodiagnosis tests. With the exception of CA2E9, all mAbs were able to recognize plasma cells on paraffin-embedded tissues, and will thus be useful for immunohistochemical assays. PMID:12088642

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

    PubMed

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

    1991-01-01

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

  11. Detection and quantification of circulating antigen in schistosomiasis by a monoclonal antibody. I. Specificity analysis of a monoclonal antibody with immunodiagnostic capacity.

    PubMed Central

    Nogueira-Queiroz, J A; Lutsch, C; Capron, M; Dessaint, J P; Capron, A

    1986-01-01

    Monoclonal antibodies were obtained after immunization of mice with Schistosoma mansoni excretory/secretory antigen, previously shown to contain the circulating cathodic (M) antigen. Among these, the 40:B1 monoclonal antibody proved to be specific for the schistosome genus and to detect only adult worm-derived antigens as shown both by immunoprecipitation and with a two-site immunoradiometric assay using the monoclonal as both the solid-phase and the labelled antibody. The two-site immunoradiometric assay allows a sensitive measurement (detection limit: 5 ng) of circulating schistosome antigen in blood and in urine from patients with schistosomiasis. The amount of circulating schistosome M antigen is correlated with schistosome egg excretion in stool. Images Fig. 2 PMID:3098474

  12. Enzymic oxidation of monoclonal antibodies by soluble and immobilized bifunctional enzyme complexes.

    PubMed

    Solomon, B; Koppel, R; Schwartz, F; Fleminger, G

    1990-06-27

    Site-specific modification of monoclonal antibodies was achieved by oxidation of the carbohydrate moieties of antibodies which are located remote from the antigen binding sites. Sialic acid and galactose are terminal sugars of these carbohydrate chains. Concomitant treatment of the antibodies with neuraminidase and galactose oxidase generated aldehyde groups in the oligosaccharide moieties of immunoglobulins which reacted selectively with amino or hydrazide groups of the matrix. Subsequent immobilization of neuraminidase and galactose oxidase on Eupergit C-adipic dihydrazide proved to be an efficient and selective system for the enzymic oxidation of the monoclonal antibodies without impairing their immunological activity. Oriented immobilization of enzymically oxidized monoclonal antibodies on hydrazide or amino Eupergit C derivatives thus leads to the formation of antibody matrix conjugates which possess high antigen-binding activities. PMID:2119387

  13. Preparation of species-specific murine monoclonal antibodies against the yeast phase of Paracoccidioides brasiliensis.

    PubMed Central

    Figueroa, J I; Hamilton, A J; Bartholomew, M A; Harada, T; Fenelon, L; Hay, R J

    1990-01-01

    A panel of four murine monoclonal antibodies showing species specificity for the yeast phase of the pathogenic dimorphic fungus Paracoccidioides brasiliensis was produced by using a modification of the standard monoclonal antibody technology. This involved the use of the immunosuppressive drug cyclophosphamide to suppress the immune response of test animals to fungi showing cross-reactivity, i.e., to Histoplasma capsulatum. One monoclonal antibody, P4, which had a high titer by enzyme-linked immunosorbent assay, was shown to recognize a linear antigenic epitope of P. brasiliensis at a molecular size of 70,000 to 75,000 daltons by Western blot (immunoblot) analysis. The potential use of these monoclonal antibodies, which are the first species-specific probes to P. brasiliensis that have been produced, in the field of serodiagnosis is discussed. Images PMID:2394802

  14. A simple method for the production of anti-C3d monoclonal antibody.

    PubMed

    Cruz, Carlos; León, Graciela

    2007-12-01

    Production of monoclonal antibodies to C3d usually involves the purification of protein. Our method does not require C3 purification; it relies on attachment of C3b to mouse erythrocytes by activation of alternative pathways and further conversion in C3d. We prepared human complement-coated mouse red cells and sensitized mice of the same strain with our own schedule of immunization and applied the classical methods to obtain a mouse monoclonal antibody. We obtained a clone called BMS-11 which produces a monoclonal antibody of IgM class, to C3d with a title of 1:500000. The monoclonal antibody obtained has shown that it is suitable for use as an antiglobulin reagent. PMID:18158789

  15. DEMONSTRATION OF MULTIPLE ANTIGENIC DETERMINANTS ON 'MYCOPLASMA PNEUMONIAE' ATTACHMENT PROTEIN BY MONOCLONAL ANTIBODIES

    EPA Science Inventory

    Distinct multiple antigenic determinants of the attachment protein of Mycoplasma pneumoniae have been identified by limited proteolytic cleavage using specific monoclonal antibodies. Western blots prepared from the gels containing the cleaved fragments were probed with antiserum ...

  16. Anti-Mesothelin Monoclonal Antibodies for the Treatment of Cancer | NCI Technology Transfer Center | TTC

    Cancer.gov

    The National Cancer Institute, Laboratory of Molecular Biology is seeking parties interested in collaborative research to further co-develop monoclonal antibodies for the treatment of mesothelin-expressing cancers.

  17. Development of antibody arrays for monoclonal antibody Higher Order Structure analysis

    PubMed Central

    Wang, Xing; Li, Qing; Davies, Michael

    2013-01-01

    Antibody arrays were developed to probe a monoclonal antibody's three-dimensional structure (3-D structure). Peptides with overlapping regions were designed to cover the whole mAb light chain and heavy chain, respectively, and used to generate polyclonal antibodies after the conjugation of the peptides to a carrier protein, KLH. It was shown that good peptide specificity was achieved from the antibodies generated. Using more than 30 different polyclonal antibodies to measure the surface epitope distribution, it was shown that the mAb antibody array can detect epitope exposure as low as 0.1% of defined mAb populations. This ELISA-based analysis of mAb epitope exposure can be considered as a measurement of “conformational impurity” in biologics development, similar to the analysis of other product-related impurities such as different forms of glycosylation, deamidation, and oxidation. This analysis of “conformational impurity” could provide valuable information on the mAb conformational comparability for biosimilar mAbs as well as novel mAbs, especially in the area of protein immunogenicity. Furthermore, stability studies indicated that there are several conformational “hot spots” in many mAbs tested, especially in the hinge region. This antibody array technology can be used for novel mAb Higher Order Structure (HOS) analysis during process and formulation development. Another important area of application is for biosimilar mAb development where the innovator molecule and biosimilar molecule could be compared based on their systemic “fingerprint” from the 30 plus antibodies. PMID:23970865

  18. Heterobifunctional reagents: A new approach to radiolabeling of monoclonal antibodies

    SciTech Connect

    Wang, T.S.T.; Ng, A.K.; Fawwaz, R.A.; Liu, Z.; Alderson, P.O.

    1985-05-01

    The use of bifunctional chelate such as the cyclic anhydride of DTPA for radiolabeling antibodies (Abs) may lead to homopolymerization, and intra- or intermolecular cross-linking, with resulting denaturation and decrease immunoreactivity of Abs. The authors, therefore, investigated the use of heterobifunctional reagents, whereby one group selectively couples to the amino group of the Ab and the other group to the radiometal for Ab labeling. One such reagent, 2,6-Dioxo-N-(carboxymethyl)morphine (DCM) was synthesized by reacting nitrilotriacetic acid with acetic anhydride. The other agent tested was commercially available N-Succinimidyl-3-(2-pyridyldithio) propionate (SPDP). These agents were evaluated independently for their ability to label a monoclonal antibody (MoAb) to a melanoma associated antigen (Ag). Labeling proceeded at a 2mg/ml concentration of the Ab, at HEPES pH 8.2, and 7.0, respectively, at room temperature for 30 min. The conjugate subsequently was labeled with Tc-99m or In-111. For comparison, the same labeled Abs also were prepared by using the cyclic anhydride of DTPA. Binding of the Ab to melanoma cells and control cells then was assayed. The results of cell binding experiments (N=3 per agent) in the region of Ag excess (X+-SD) were as follows: 62.6 +- 2.83% for Tc-99m-DCM-MoAb and 41.3+-1.84% for Tc-99m-SPDP-MoAb vs. 28.6 +- 1.16% for Tc-99m-DTPA-MoAb (p<0.01); 56.2 +- 2.97% for In-111-DCM-MoAb vs. 28.6 +- 1.16% for In-111-DTPA-M0Ab. Binding of all agents to the control lymphoid cell line was less than 3%. These results suggest that heterobifunctional reagents can reduce the loss of immunoreactivity of labeled MoAbs.

  19. How does mild hypothermia affect monoclonal antibody glycosylation?

    PubMed

    Sou, Si Nga; Sellick, Christopher; Lee, Ken; Mason, Alison; Kyriakopoulos, Sarantos; Polizzi, Karen M; Kontoravdi, Cleo

    2015-06-01

    The application of mild hypothermic conditions to cell culture is a routine industrial practice used to improve recombinant protein production. However, a thorough understanding of the regulation of dynamic cellular processes at lower temperatures is necessary to enhance bioprocess design and optimization. In this study, we investigated the impact of mild hypothermia on protein glycosylation. Chinese hamster ovary (CHO) cells expressing a monoclonal antibody (mAb) were cultured at 36.5°C and with a temperature shift to 32°C during late exponential/early stationary phase. Experimental results showed higher cell viability with decreased metabolic rates. The specific antibody productivity increased by 25% at 32°C and was accompanied by a reduction in intracellular nucleotide sugar donor (NSD) concentrations and a decreased proportion of the more processed glycan structures on the mAb constant region. To better understand CHO cell metabolism at 32°C, flux balance analysis (FBA) was carried out and constrained with exometabolite data from stationary phase of cultures with or without a temperature shift. Estimated fluxomes suggested reduced fluxes of carbon species towards nucleotide and NSD synthesis and more energy was used for product formation. Expression of the glycosyltransferases that are responsible for N-linked glycan branching and elongation were significantly lower at 32°C. As a result of mild hypothermia, mAb glycosylation was shown to be affected by both NSD availability and glycosyltransferase expression. The combined experimental/FBA approach generated insight as to how product glycosylation can be impacted by changes in culture temperature. Better feeding strategies can be developed based on the understanding of the metabolic flux distribution. PMID:25545631

  20. Characterization of monoclonal antibodies against Naja naja oxiana neurotoxin I.

    PubMed

    Stiles, B G; Sexton, F W; Guest, S B; Olson, M A; Hack, D C

    1994-10-01

    Seven monoclonal antibodies (mAbs) were developed against neurotoxin I (NT-1), a protein from central Asian cobra (Naja naja oxiana) venom which binds specifically to nicotinic acetylcholine receptor (AchR). All of the mAbs cross-reacted with another long-chain post-synaptic neurotoxin, Bungarus multicinctus alpha-bungarotoxin (alpha-BT), but not Naja naja kaouthia alpha-cobratoxin, in an enzyme-linked immunosorbent assay (e.l.i.s.a.). Short-chain post-synaptic neurotoxins like Naja naja atra cobrotoxin, Laticauda semifasciata erabutoxin b, or N. n. oxiana neurotoxin II did not cross-react with the NT-1 mAbs, but an antigen(s) found in Dendroaspis polylepis, Acanthophis antarcticus and Pseudechis australis venoms was immunoreactive. The e.l.i.s.a. readings for dithiothreitol-reduced NT-1 and NT-1 mAbs ranged from 13 to 27% of those for native toxin but reduced alpha-BT was not immunoreactive. Synthetic NT-1 peptides were used in epitope-mapping studies and two, non-contiguous regions (Cys15-Tyr23 and Lys25-Gly33 or Pro17-Lys25 and Asp29-Lys37) were recognized by the NT-1 mAbs. The NT-1 mAbs individually inhibited 31-71% of alpha-BT binding to AchR in vitro and afforded a slight protective effect in vivo with a toxin: antibody mole ratio of 1:1.5. This report is the first to describe mAbs which recognize and protect against a heterologous, long-chain, post-synaptic neurotoxin from snake venom. PMID:7945236

  1. Characterization of monoclonal antibodies against Naja naja oxiana neurotoxin I.

    PubMed Central

    Stiles, B G; Sexton, F W; Guest, S B; Olson, M A; Hack, D C

    1994-01-01

    Seven monoclonal antibodies (mAbs) were developed against neurotoxin I (NT-1), a protein from central Asian cobra (Naja naja oxiana) venom which binds specifically to nicotinic acetylcholine receptor (AchR). All of the mAbs cross-reacted with another long-chain post-synaptic neurotoxin, Bungarus multicinctus alpha-bungarotoxin (alpha-BT), but not Naja naja kaouthia alpha-cobratoxin, in an enzyme-linked immunosorbent assay (e.l.i.s.a.). Short-chain post-synaptic neurotoxins like Naja naja atra cobrotoxin, Laticauda semifasciata erabutoxin b, or N. n. oxiana neurotoxin II did not cross-react with the NT-1 mAbs, but an antigen(s) found in Dendroaspis polylepis, Acanthophis antarcticus and Pseudechis australis venoms was immunoreactive. The e.l.i.s.a. readings for dithiothreitol-reduced NT-1 and NT-1 mAbs ranged from 13 to 27% of those for native toxin but reduced alpha-BT was not immunoreactive. Synthetic NT-1 peptides were used in epitope-mapping studies and two, non-contiguous regions (Cys15-Tyr23 and Lys25-Gly33 or Pro17-Lys25 and Asp29-Lys37) were recognized by the NT-1 mAbs. The NT-1 mAbs individually inhibited 31-71% of alpha-BT binding to AchR in vitro and afforded a slight protective effect in vivo with a toxin: antibody mole ratio of 1:1.5. This report is the first to describe mAbs which recognize and protect against a heterologous, long-chain, post-synaptic neurotoxin from snake venom. PMID:7945236

  2. Quantitative assessment of antibody internalization with novel monoclonal antibodies against Alexa fluorophores.

    PubMed

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

    2015-01-01

    Antibodies against cell surface antigens may be internalized through their specific interactions with these proteins and in some cases may induce or perturb antigen internalization. The anti-cancer efficacy of antibody-drug conjugates is thought to rely on their uptake by cancer cells expressing the surface antigen. Numerous techniques, including microscopy and flow cytometry, have been used to identify antibodies with desired cellular uptake rates. To enable quantitative measurements of internalization of labeled antibodies, an assay based on internalized and quenched fluorescence was developed. For this approach, we generated novel anti-Alexa Fluor monoclonal antibodies (mAbs) that effectively and specifically quench cell surface-bound Alexa Fluor 488 or Alexa Fluor 594 fluorescence. Utilizing Alexa Fluor-labeled mAbs against the EphA2 receptor tyrosine kinase, we showed that the anti-Alexa Fluor reagents could be used to monitor internalization quantitatively over time. The anti-Alexa Fluor mAbs were also validated in a proof of concept dual-label internalization assay with simultaneous exposure of cells to two different mAbs. Importantly, the unique anti-Alexa Fluor mAbs described here may also enable other single- and dual-label experiments, including label detection and signal enhancement in macromolecules, trafficking of proteins and microorganisms, and cell migration and morphology. PMID:25894652

  3. Adverse events to monoclonal antibodies used for cancer therapy

    PubMed Central

    Baldo, Brian A

    2013-01-01

    Fifteen monoclonal antibodies (mAbs) are currently registered and approved for the treatment of a range of different cancers. These mAbs are specific for a limited number of targets (9 in all). Four of these molecules are indeed directed against the B-lymphocyte antigen CD20; 3 against human epidermal growth factor receptor 2 (HER2 or ErbB2), 2 against the epidermal growth factor receptor (EGFR), and 1 each against epithelial cell adhesion molecule (EpCAM), CD30, CD52, vascular endothelial growth factor (VEGF), tumor necrosis factor (ligand) superfamily, member 11 (TNFSF11, best known as RANKL), and cytotoxic T lymphocyte-associated protein 4 (CTLA4). Collectively, the mAbs provoke a wide variety of systemic and cutaneous adverse events including the full range of true hypersensitivities: Type I immediate reactions (anaphylaxis, urticaria); Type II reactions (immune thrombocytopenia, neutopenia, hemolytic anemia); Type III responses (vasculitis, serum sickness; some pulmonary adverse events); and Type IV delayed mucocutaneous reactions as well as infusion reactions/cytokine release syndrome (IRs/CRS), tumor lysis syndrome (TLS), progressive multifocal leukoencephalopathy (PML) and cardiac events. Although the term “hypersensitivity” is widely used, no common definition has been adopted within and between disciplines and the requirement of an immunological basis for a true hypersensitivity reaction is sometimes overlooked. Consequently, some drug-induced adverse events are sometimes incorrectly described as “hypersensitivities” while others that should be described are not. PMID:24251081

  4. Elotuzumab: the first approved monoclonal antibody for multiple myeloma treatment.

    PubMed

    Magen, Hila; Muchtar, Eli

    2016-08-01

    Elotuzumab is a monoclonal antibody directed against the SLAMF7 receptor, expressed on normal and malignant plasma cells with a lower expression on other lymphoid cells such as natural killer (NK) cells. Elotuzumab has no significant antimyeloma activity when given as a single agent to patients with relapsed or refractory multiple myeloma (RRMM). However, when combined with other antimyeloma agents, it results in improved response and outcome. Owing to the results from the landmark ELOQUENT-2 phase III clinical trial, which compared lenalidomide and dexamethasone with or without elotuzumab in patients with RRMM, elotuzumab in combination with lenalidomide and dexamethasone was approved by the American Food and Drug Administration (FDA) in November 2015 for multiple myeloma (MM) patients who received one to three prior lines of therapy. This review will give a brief description of the signaling lymphocytic activation molecule (SLAM) family receptors, the unique SLAMF7 receptor and the mechanism of action of elotuzumab. Thereafter, we will give an overview on its antimyeloma activity in preclinical and clinical trials, including its toxicity profile and management thereof. PMID:27493709

  5. Production of radiolabeled monoclonal antibody conjugates by photoaffinity labeling

    SciTech Connect

    Volkert, W.A.; Ketring, A.R.; Kuntz, R.R.; Holmes, R.A.; Mitchell, E.P. ); Feldbush, T.L. )

    1990-06-01

    This report discusses activities and progress that has occurred since initiation of this project on September 1, 1989. We have synthesized ethyl N,N{prime}-bis(benzoylmercaptoacetyl)-2,3-diaminopropanoate, a ligand to be used as a bifunctional chelating agent (BFCA), to form {sup 186}Re or {sup 188}Re ({sup 186}Re/{sup 188}Re) complexes. {sup 186}Re/{sup 188}Re, in reducing media, reacts with this ligand to form {sup 186}Re/{sup 188}Re-CO{sub 2}DADS chelates that will be used to formulate new radiolabeled photoaffinity labels (RPALs). Initial steps have been taken to synthesize R-As-dithiol compounds. This approach will be used to produce {sup 77}As-RPALs or covalently link {sup 77}As directly to monoclonal antibodies (MAbs). The R group will contain a group that can be used for conjugation reactions. Spectral and photochemical properties of various types of photoaffinity labels (PALs) have been studied. Acrylo-azido compounds and 9-azido acridine have been studied as well as several other photoprobes. The binding characteristics of the azido-based PALs to HSA have been studied and progress has been made on developing techniques for efficiently separating of non-covalently sound PALs. The Nd-YAG laser was purchased and arrived in 1990. It has been assembled and tested and is now operational.

  6. Antigenic heterogeneity in Mycoplasma iowae demonstrated with monoclonal antibodies.

    PubMed

    Panangala, V S; Gresham, M M; Morsy, M A

    1992-01-01

    Western blots of proteins of 14 Mycoplasma iowae strains and isolates resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were probed with three monoclonal antibodies (MAbs), MI6, MI7, and MI8. MAb MI6 reacted with one or more antigens with apparent molecular weights of 60,000, 70,000, and 94,000. In three strains (N-PHN-D13, R-D2497, and K 1805), antigens located on a single peptide band were recognized, while in others additional epitopes at different molecular-weight positions were revealed. A similar pattern was observed with MAb MI7, although it reacted with fewer antigens than did MAb MI6 and failed to recognize antigens in strains N-PHN-D13 and R-D2497. MAb MI8 reacted with an antigen at an apparent molecular-weight position of 28,000 in four of the 14 strains and isolates. The diverse reaction patterns observed with the MAbs in the 14 M. iowae strains and isolates confirms the occurrence of antigenic variation within this species. Antigenic variation in M. iowae may be pivotal in determining host-parasite interactions, pathogenesis, and the outcome of disease. PMID:1373600

  7. Elotuzumab: the first approved monoclonal antibody for multiple myeloma treatment

    PubMed Central

    Magen, Hila; Muchtar, Eli

    2016-01-01

    Elotuzumab is a monoclonal antibody directed against the SLAMF7 receptor, expressed on normal and malignant plasma cells with a lower expression on other lymphoid cells such as natural killer (NK) cells. Elotuzumab has no significant antimyeloma activity when given as a single agent to patients with relapsed or refractory multiple myeloma (RRMM). However, when combined with other antimyeloma agents, it results in improved response and outcome. Owing to the results from the landmark ELOQUENT-2 phase III clinical trial, which compared lenalidomide and dexamethasone with or without elotuzumab in patients with RRMM, elotuzumab in combination with lenalidomide and dexamethasone was approved by the American Food and Drug Administration (FDA) in November 2015 for multiple myeloma (MM) patients who received one to three prior lines of therapy. This review will give a brief description of the signaling lymphocytic activation molecule (SLAM) family receptors, the unique SLAMF7 receptor and the mechanism of action of elotuzumab. Thereafter, we will give an overview on its antimyeloma activity in preclinical and clinical trials, including its toxicity profile and management thereof. PMID:27493709

  8. DNA immunization as a technology platform for monoclonal antibody induction

    PubMed Central

    Liu, Shuying; Wang, Shixia; Lu, Shan

    2016-01-01

    To combat the threat of many emerging infectious diseases, DNA immunization offers a unique and powerful approach to the production of high-quality monoclonal antibodies (mAbs) against various pathogens. Compared with traditional protein-based immunization approaches, DNA immunization is efficient for testing novel immunogen designs, does not require the production or purification of proteins from a pathogen or the use of recombinant protein technology and is effective at generating mAbs against conformation-sensitive targets. Although significant progress in the use of DNA immunization to generate mAbs has been made over the last two decades, the literature does not contain an updated summary of this experience. The current review provides a comprehensive analysis of the literature, including our own work, describing the use of DNA immunization to produce highly functional mAbs, in particular, those against emerging infectious diseases. Critical factors such as immunogen design, delivery approach, immunization schedule, use of immune modulators and the role of final boost immunization are discussed in detail. PMID:27048742

  9. Production of a Chaetomium globosum Enolase Monoclonal Antibody

    PubMed Central

    Nayak, Ajay P.; Lemons, Angela R.; Rittenour, William R.; Hettick, Justin M.; Beezhold, Donald H.

    2014-01-01

    Chaetomium globosum is a hydrophilic fungal species and a contaminant of water-damaged building materials in North America. Methods to detect Chaetomium species include subjective identification of ascospores, viable culture, or molecular-based detection methods. In this study, we describe the production and initial characterization of a monoclonal antibody (MAb) for C. globosum enolase. MAb 1C7, a murine IgG1 isotype MAb, was produced and reacted with recombinant C. globosum enolase (rCgEno) in an enzyme-linked immunosorbent assay and with a putative C. globosum enolase in a Western blot. Epitope mapping showed MAb 1C7 specific reactivity to an enolase decapeptide, LTYEELANLY, that is highly conserved within the fungal class Sordariomycetes. Cross-reactivity studies showed MAb 1C7 reactivity to C. atrobrunneum but not C. indicum. MAb 1C7 did not react with enolase from Aspergillus fumigatus, which is divergent in only two amino acids within this epitope. The results of this study suggest potential utility of MAb 1C7 in Western blot applications for the detection of Chaetomium and other Sordariomycetes species. PMID:25495488

  10. DNA immunization as a technology platform for monoclonal antibody induction.

    PubMed

    Liu, Shuying; Wang, Shixia; Lu, Shan

    2016-01-01

    To combat the threat of many emerging infectious diseases, DNA immunization offers a unique and powerful approach to the production of high-quality monoclonal antibodies (mAbs) against various pathogens. Compared with traditional protein-based immunization approaches, DNA immunization is efficient for testing novel immunogen designs, does not require the production or purification of proteins from a pathogen or the use of recombinant protein technology and is effective at generating mAbs against conformation-sensitive targets. Although significant progress in the use of DNA immunization to generate mAbs has been made over the last two decades, the literature does not contain an updated summary of this experience. The current review provides a comprehensive analysis of the literature, including our own work, describing the use of DNA immunization to produce highly functional mAbs, in particular, those against emerging infectious diseases. Critical factors such as immunogen design, delivery approach, immunization schedule, use of immune modulators and the role of final boost immunization are discussed in detail. PMID:27048742

  11. Potent neutralizing monoclonal antibodies against Ebola virus infection.

    PubMed

    Zhang, Qi; Gui, Miao; Niu, Xuefeng; He, Shihua; Wang, Ruoke; Feng, Yupeng; Kroeker, Andrea; Zuo, Yanan; Wang, Hua; Wang, Ying; Li, Jiade; Li, Chufang; Shi, Yi; Shi, Xuanling; Gao, George F; Xiang, Ye; Qiu, Xiangguo; Chen, Ling; Zhang, Linqi

    2016-01-01

    Ebola virus infections cause a deadly hemorrhagic disease for which no vaccines or therapeutics has received regulatory approval. Here we show isolation of three (Q206, Q314 and Q411) neutralizing monoclonal antibodies (mAbs) against the surface glycoprotein (GP) of Ebola virus identified in West Africa in 2014 through sequential immunization of Chinese rhesus macaques and antigen-specific single B cell sorting. These mAbs demonstrated potent neutralizing activities against both pseudo and live Ebola virus independent of complement. Biochemical, single particle EM, and mutagenesis analysis suggested Q206 and Q411 recognized novel epitopes in the head while Q314 targeted the glycan cap in the GP1 subunit. Q206 and Q411 appeared to influence GP binding to its receptor NPC1. Treatment with these mAbs provided partial but significant protection against disease in a mouse model of Ebola virus infection. These novel mAbs could serve as promising candidates for prophylactic and therapeutic interventions against Ebola virus infection. PMID:27181584

  12. Fluorescent monoclonal antibody for confirmation of Neisseria gonorrhoeae cultures.

    PubMed Central

    Laughon, B E; Ehret, J M; Tanino, T T; Van der Pol, B; Handsfield, H H; Jones, R B; Judson, F N; Hook, E W

    1987-01-01

    We evaluated a monoclonal fluorescent-antibody (FA) reagent (Neisseria gonorrhoeae Culture Confirmation Test; Syva Co., Palo Alto, Calif.) for confirmation of N. gonorrhoeae isolates obtained from clinics for sexually transmitted diseases in four cities. The FA test was performed in parallel with established confirmation procedures on all organisms growing on 773 primary culture plates of modified Thayer-Martin agar. All N. gonorrhoeae isolates reacted with the FA reagent and produced a bright, easily interpretable fluorescence. The FA test correctly identified 533 N. gonorrhoeae isolates from 474 patients and did not react with 90 N. meningitidis or with 213 non-Neisseria isolates. In one city (Baltimore), Gonochek II (Du Pont Co., Wilmington, Del.) failed to identify four N. gonorrhoeae isolates reactive with the FA reagent and confirmed as N. gonorrhoeae by Phadebact (Pharmacia Inc., Piscataway, N.J.) and acid production from sugars. The FA test was rapid and specific and could be performed directly from primary isolation plates. The test requires 1 h to perform and is applicable to mixed-flora cultures. PMID:3123514

  13. Reversible cluster formation in concentrated monoclonal antibody solutions

    NASA Astrophysics Data System (ADS)

    Godfrin, P. Douglas; Porcar, Lionel; Falus, Peter; Zarraga, Isidro; Wagner, Norm; Liu, Yun

    2015-03-01

    Protein cluster formation in solution is of fundamental interest for both academic research and industrial applications. Recently, industrial scientists are also exploring the effect of reversible cluster formation on biopharmaceutical processing and delivery. However, despite of its importance, the understanding of protein clusters at concentrated solutions remains scientifically very challenging. Using the neutron spin echo technique to study the short time dynamics of proteins in solutions, we have recently systematically studied cluster formation in a few monoclonal antibody (mAb) solutions and their relation with solution viscosity. We show that the existence of anisotropic attraction can cause the formation of finite sized clusters, which increases the solution viscosity. Interestingly, once clusters form at relatively low concentrations, the average size of clusters in solutions remains almost constant over a wide range of concentrations similar to that of micelle formation. For a different mAb we have also investigated, the attraction is mostly induced by hydrophobic patches. As a result, these mAbs form large clusters with loosely linked proteins. In both cases, the formation of clusters all increases the solution viscosity substantially. However, due to different physics origins of cluster formation, solutions viscosities for these two different types of mAbs need to be controlled by different ways.

  14. Potent neutralizing monoclonal antibodies against Ebola virus infection

    PubMed Central

    Zhang, Qi; Gui, Miao; Niu, Xuefeng; He, Shihua; Wang, Ruoke; Feng, Yupeng; Kroeker, Andrea; Zuo, Yanan; Wang, Hua; Wang, Ying; Li, Jiade; Li, Chufang; Shi, Yi; Shi, Xuanling; Gao, George F.; Xiang, Ye; Qiu, Xiangguo; Chen, Ling; Zhang, Linqi

    2016-01-01

    Ebola virus infections cause a deadly hemorrhagic disease for which no vaccines or therapeutics has received regulatory approval. Here we show isolation of three (Q206, Q314 and Q411) neutralizing monoclonal antibodies (mAbs) against the surface glycoprotein (GP) of Ebola virus identified in West Africa in 2014 through sequential immunization of Chinese rhesus macaques and antigen-specific single B cell sorting. These mAbs demonstrated potent neutralizing activities against both pseudo and live Ebola virus independent of complement. Biochemical, single particle EM, and mutagenesis analysis suggested Q206 and Q411 recognized novel epitopes in the head while Q314 targeted the glycan cap in the GP1 subunit. Q206 and Q411 appeared to influence GP binding to its receptor NPC1. Treatment with these mAbs provided partial but significant protection against disease in a mouse model of Ebola virus infection. These novel mAbs could serve as promising candidates for prophylactic and therapeutic interventions against Ebola virus infection. PMID:27181584

  15. Monoclonal antibodies directed against surface molecules of multicell spheroids

    NASA Technical Reports Server (NTRS)

    Martinez, Andrew O.

    1994-01-01

    The objective of this project is to generate a library of monoclonal antibodies (MAbs) directed against surface molecules of tumor and transformed cells grown as multicell spheroids (MCS). These MCS are highly organized, 3-dimensional multicellular structures which exhibit many characteristics of in vivo organized tissues not found in conventional monolayer or suspension culture. Therefore MCS make better in vitro model systems to study the interactions of mammalian cells, and provide a functional assay for surface adhesion molecules. This project also involves investigations of cell-cell interactions in a gravity-based environment. It will provide a base of scientific information necessary to expand the focus of the project in future years to microgravity and hypergravity-based environments. This project also has the potential to yield important materials (e.g., cellular products) which may prove useful in the diagnosis and/or treatment of certain human diseases. Moreover, this project supports the training of both undergraduate and graduate students; thus, it will assist in developing a pool of future scientists with research experience in an area (gravitational biology) of interest to NASA.

  16. Trial Watch: Immunomodulatory monoclonal antibodies for oncological indications

    PubMed Central

    Buqué, Aitziber; Bloy, Norma; Aranda, Fernando; Castoldi, Francesca; Eggermont, Alexander; Cremer, Isabelle; Fridman, Wolf Hervé; Fucikova, Jitka; Galon, Jérôme; Marabelle, Aurélien; Spisek, Radek; Tartour, Eric; Zitvogel, Laurence; Kroemer, Guido; Galluzzi, Lorenzo

    2015-01-01

    Immunomodulatory monoclonal antibodies (mAbs) differ from their tumor-targeting counterparts because they exert therapeutic effects by directly interacting with soluble or (most often) cellular components of the immune system. Besides holding promise for the treatment of autoimmune and inflammatory disorders, immunomodulatory mAbs have recently been shown to constitute a potent therapeutic weapon against neoplastic conditions. One class of immunomodulatory mAbs operates by inhibiting safeguard systems that are frequently harnessed by cancer cells to establish immunological tolerance, the so-called “immune checkpoints.” No less than 3 checkpoint-blocking mAbs have been approved worldwide for use in oncological indications, 2 of which during the past 12 months. These molecules not only mediate single-agent clinical activity in patients affected by specific neoplasms, but also significantly boost the efficacy of several anticancer chemo-, radio- or immunotherapies. Here, we summarize recent advances in the development of checkpoint-blocking mAbs, as well as of immunomodulatory mAbs with distinct mechanisms of action. PMID:26137403

  17. Trial watch: Tumor-targeting monoclonal antibodies for oncological indications

    PubMed Central

    Vacchelli, Erika; Pol, Jonathan; Bloy, Norma; Eggermont, Alexander; Cremer, Isabelle; Fridman, Wolf Hervé; Galon, Jérôme; Marabelle, Aurélien; Kohrt, Holbrook; Zitvogel, Laurence; Kroemer, Guido; Galluzzi, Lorenzo

    2015-01-01

    An expanding panel of monoclonal antibodies (mAbs) that specifically target malignant cells or intercept trophic factors delivered by the tumor stroma is now available for cancer therapy. These mAbs can exert direct antiproliferative/cytotoxic effects as they inhibit pro-survival signal transduction cascades or activate lethal receptors at the plasma membrane of cancer cells, they can opsonize neoplastic cells to initiate a tumor-targeting immune response, or they can be harnessed to specifically deliver toxins or radionuclides to transformed cells. As an indication of the success of this immunotherapeutic paradigm, international regulatory agencies approve new tumor-targeting mAbs for use in cancer patients every year. Moreover, the list of indications for previously licensed molecules is frequently expanded to other neoplastic disorders as the results of large, randomized clinical trials become available. Here, we discuss recent advances in the preclinical and clinical development of tumor-targeting mAbs for oncological indications. PMID:25949870

  18. Monoclonal antibody: the corner stone of modern biotherapeutics.

    PubMed

    Xia, Zhi-nan; Cai, Xue-ting; Cao, Peng

    2012-10-01

    Worldwide sales of biologic drugs exceeded 100 billion USD in 2011. About 32% is from therapeutic monoclonal antibody (mAb). With many blockbuster biopharmaceutical patents expiring over the next decade, there is a great opportunity for biosimilar to enter the worldwide especially emerging market. Both European Medicines Agency (EMA) and Food and Drug Administration (FDA) have introduced regulatory frameworks for the potential approval of biosimilar mAb therapeutics. Rather than providing a highly abbreviated path, as in the case for small molecule chemical drug, approval for biosimilar mAb will require clinical trial and the details will be very much on a case-by-case basis. Since mAb is the dominant category of biologic drugs, mAb will be the focus of this review. First, the United States (US) and European Union (EU) approved mAb and those in phase 3 trials will be reviewed, then strategies on how to win biosimilar competition will be reviewed. PMID:23289138

  19. Monoclonal antibody characterization of a leukoagglutinin produced by Renibacterium salmoninarum.

    PubMed

    Wiens, G D; Kaattari, S L

    1991-02-01

    Renibacterium salmoninarum causes a chronic disease of salmonid fish known as bacterial kidney disease. High concentrations of bacterially produced extracellular protein (ECP) are present in plasma, kidney, and spleen tissue of naturally and experimentally infected fish. ECP agglutinated salmonid leukocytes in vitro at concentrations which correspond to levels found in highly infected fish. Association of biological activity with the structure of the major protein constituent of ECP, p57, was accomplished by monoclonal antibody (MAb) analysis. Location of the antigenic binding sites recognized by the MAbs was determined by two-dimensional electrophoresis and Western immunoblotting of the proteolytic breakdown fragments of p57. Eight MAbs have been classified into three groups on the basis of their differential recognition of these proteolytic breakdown products. Group I MAbs bound a region proximal to the amino terminus of the protein. Two of these MAbs were also able to block leukoagglutinating activity. Group III MAbs bound to a region associated with the bacterial cell surface, while group II MAbs bound a region between group I and group III. These analyses have allowed the identification of potential structural and functional regions of p57. PMID:1987079

  20. Monoclonal antibody characterization of a leukoagglutinin produced by Renibacterium salmoninarum.

    PubMed Central

    Wiens, G D; Kaattari, S L

    1991-01-01

    Renibacterium salmoninarum causes a chronic disease of salmonid fish known as bacterial kidney disease. High concentrations of bacterially produced extracellular protein (ECP) are present in plasma, kidney, and spleen tissue of naturally and experimentally infected fish. ECP agglutinated salmonid leukocytes in vitro at concentrations which correspond to levels found in highly infected fish. Association of biological activity with the structure of the major protein constituent of ECP, p57, was accomplished by monoclonal antibody (MAb) analysis. Location of the antigenic binding sites recognized by the MAbs was determined by two-dimensional electrophoresis and Western immunoblotting of the proteolytic breakdown fragments of p57. Eight MAbs have been classified into three groups on the basis of their differential recognition of these proteolytic breakdown products. Group I MAbs bound a region proximal to the amino terminus of the protein. Two of these MAbs were also able to block leukoagglutinating activity. Group III MAbs bound to a region associated with the bacterial cell surface, while group II MAbs bound a region between group I and group III. These analyses have allowed the identification of potential structural and functional regions of p57. Images PMID:1987079

  1. Characterization of a new monoclonal antibody against mercury (II)

    SciTech Connect

    Marx, A.; Hock, B.

    1998-07-01

    Monoclonal antibodies (mabs) were produced against mercury (II) and an enzyme immunoassay was developed for the detection of mercury (II) in water. Since mercury (II) ions are too small to elicit an immune response, they were coupled via glutathione (GSH) to the immunogenic carrier protein keyhole limpet hemocyanin (KLH). Several mice were immunized with this KLH-GSH-Hg immunoconjugate. Spleen cells of immunized mice were fused with myeloma cells. The resulting hybridoma cells were screened for the production of specific anti-Hg mabs. Five positive cells were detected. The hybridoma cell line K3C6 was adjusted to protein free medium; it produced mabs with high selectivity and sensitivity. A detection limit of 2.8 {micro}g/L HgCl{sub 2} (= 2.1 {micro}g/L Hg{sup 2+}) was achieved with a non-competitive enzyme immunoassay (EIA). Cross-reactivities with other metals were below 1%. Measurement of spiked water samples with this EIA showed good correlation with results obtained by mass spectrometry with inductive coupled plasma (ICP-MS).

  2. Development and Evaluation of Monoclonal Antibodies for Paxilline.

    PubMed

    Maragos, Chris M

    2015-10-01

    Paxilline (PAX) is a tremorgenic mycotoxin that has been found in perennial ryegrass infected with Acremonium lolii. To facilitate screening for this toxin, four murine monoclonal antibodies (mAbs) were developed. In competitive indirect enzyme-linked immunosorbent assays (CI-ELISAs) the concentrations of PAX required to inhibit signal development by 50% (IC50s) ranged from 1.2 to 2.5 ng/mL. One mAb (2-9) was applied to the detection of PAX in maize silage. The assay was sensitive to the effects of solvents, with 5% acetonitrile or 20% methanol causing a two-fold or greater increase in IC50. For analysis of silage samples, extracts were cleaned up by adsorbing potential matrix interferences onto a solid phase extraction column. The non-retained extract was then diluted with buffer to reduce solvent content prior to assay. Using this method, the limit of detection for PAX in dried silage was 15 µg/kg and the limit of quantification was 90 µg/kg. Recovery from samples spiked over the range of 100 to 1000 µg/kg averaged 106% ± 18%. The assay was applied to 86 maize silage samples, with many having detectable, but none having quantifiable, levels of PAX. The results suggest the CI-ELISA can be applied as a sensitive technique for the screening of PAX in maize silage. PMID:26426046

  3. Development and Evaluation of Monoclonal Antibodies for Paxilline

    PubMed Central

    Maragos, Chris M.

    2015-01-01

    Paxilline (PAX) is a tremorgenic mycotoxin that has been found in perennial ryegrass infected with Acremonium lolii. To facilitate screening for this toxin, four murine monoclonal antibodies (mAbs) were developed. In competitive indirect enzyme-linked immunosorbent assays (CI-ELISAs) the concentrations of PAX required to inhibit signal development by 50% (IC50s) ranged from 1.2 to 2.5 ng/mL. One mAb (2-9) was applied to the detection of PAX in maize silage. The assay was sensitive to the effects of solvents, with 5% acetonitrile or 20% methanol causing a two-fold or greater increase in IC50. For analysis of silage samples, extracts were cleaned up by adsorbing potential matrix interferences onto a solid phase extraction column. The non-retained extract was then diluted with buffer to reduce solvent content prior to assay. Using this method, the limit of detection for PAX in dried silage was 15 µg/kg and the limit of quantification was 90 µg/kg. Recovery from samples spiked over the range of 100 to 1000 µg/kg averaged 106% ± 18%. The assay was applied to 86 maize silage samples, with many having detectable, but none having quantifiable, levels of PAX. The results suggest the CI-ELISA can be applied as a sensitive technique for the screening of PAX in maize silage. PMID:26426046

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

    PubMed

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

    2016-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    1995-07-01

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

  6. Quality control of murine monoclonal antibodies using isoelectric focusing affinity immunoblot analysis

    NASA Technical Reports Server (NTRS)

    Hamilton, Robert G.; Rodkey, L. Scott; Reimer, Charles B.

    1987-01-01

    The quality control of murine hybridoma secretory products has been performed using two approaches for isoelectric focusing affinity immunoblot analysis: (1) a method in which antigen-coated nitrocellulose is placed on top of an acrylamide gel containing isoelectrically focused ascites to bind the antigen specific monoclonal antibody; and (2) a method in which focused ascite proteins were passively blotted onto nitrocellulose and specific monoclonal antibodies were detected with enzyme-conjugated antigen. Analysis by both methods of batches of ascites containing antihuman IgG antibodies that were produced by six hybridomas permitted effective monitoring of immunoreactive antibodies for pI microheterogeneity.

  7. Development of a standardized subgrouping scheme for Legionella pneumophila serogroup 1 using monoclonal antibodies.

    PubMed Central

    Joly, J R; McKinney, R M; Tobin, J O; Bibb, W F; Watkins, I D; Ramsay, D

    1986-01-01

    A panel of monoclonal antibodies to Legionella pneumophila serogroup 1 and a subclassification scheme were developed in a collaborative project among three laboratories. The seven most useful monoclonal antibodies were selected from three previously developed panels on the basis of indirect fluorescent antibody patterns with 83 strains of L. pneumophila serogroup 1 that were obtained from widely distributed geographic locations. The isolates were divided into 10 major subgroups on the basis of reactivity patterns that can be readily reproduced in any laboratory and are not subject to major inconsistencies of interpretation of staining intensity. A standard protocol for the indirect fluorescent antibody procedure was also developed. PMID:3517064

  8. Sequential Antigen Panning for Selection of Broadly Cross-Reactive HIV-1-Neutralizing Human Monoclonal Antibodies

    PubMed Central

    Zhang, Mei-Yun; Dimitrov, Dimiter S.

    2012-01-01

    Summary Many phage display techniques drive selection toward the isolation of highly specific antibodies. However, the identification of monoclonal antibodies that are cross-reactive has implications for the development of diagnostics, therapeutics, and vaccines against pathogens or cancer cells that are able to rapidly generate variants and escape mutants. To identify human monoclonal antibodies with high activity against HIV and broad-spectrum activity, we developed a technique termed sequential antigen panning. This methodology could be used to isolated recombinant antibodies against any antigen that shares epitopes with other antigens. PMID:19554293

  9. Novel method for the high-throughput production of phosphorylation site-specific monoclonal antibodies.

    PubMed

    Kurosawa, Nobuyuki; Wakata, Yuka; Inobe, Tomonao; Kitamura, Haruki; Yoshioka, Megumi; Matsuzawa, Shun; Kishi, Yoshihiro; Isobe, Masaharu

    2016-01-01

    Threonine phosphorylation accounts for 10% of all phosphorylation sites compared with 0.05% for tyrosine and 90% for serine. Although monoclonal antibody generation for phospho-serine and -tyrosine proteins is progressing, there has been limited success regarding the production of monoclonal antibodies against phospho-threonine proteins. We developed a novel strategy for generating phosphorylation site-specific monoclonal antibodies by cloning immunoglobulin genes from single plasma cells that were fixed, intracellularly stained with fluorescently labeled peptides and sorted without causing RNA degradation. Our high-throughput fluorescence activated cell sorting-based strategy, which targets abundant intracellular immunoglobulin as a tag for fluorescently labeled antigens, greatly increases the sensitivity and specificity of antigen-specific plasma cell isolation, enabling the high-efficiency production of monoclonal antibodies with desired antigen specificity. This approach yielded yet-undescribed guinea pig monoclonal antibodies against threonine 18-phosphorylated p53 and threonine 68-phosphorylated CHK2 with high affinity and specificity. Our method has the potential to allow the generation of monoclonal antibodies against a variety of phosphorylated proteins. PMID:27125496

  10. Novel method for the high-throughput production of phosphorylation site-specific monoclonal antibodies

    PubMed Central

    Kurosawa, Nobuyuki; Wakata, Yuka; Inobe, Tomonao; Kitamura, Haruki; Yoshioka, Megumi; Matsuzawa, Shun; Kishi, Yoshihiro; Isobe, Masaharu

    2016-01-01

    Threonine phosphorylation accounts for 10% of all phosphorylation sites compared with 0.05% for tyrosine and 90% for serine. Although monoclonal antibody generation for phospho-serine and -tyrosine proteins is progressing, there has been limited success regarding the production of monoclonal antibodies against phospho-threonine proteins. We developed a novel strategy for generating phosphorylation site-specific monoclonal antibodies by cloning immunoglobulin genes from single plasma cells that were fixed, intracellularly stained with fluorescently labeled peptides and sorted without causing RNA degradation. Our high-throughput fluorescence activated cell sorting-based strategy, which targets abundant intracellular immunoglobulin as a tag for fluorescently labeled antigens, greatly increases the sensitivity and specificity of antigen-specific plasma cell isolation, enabling the high-efficiency production of monoclonal antibodies with desired antigen specificity. This approach yielded yet-undescribed guinea pig monoclonal antibodies against threonine 18-phosphorylated p53 and threonine 68-phosphorylated CHK2 with high affinity and specificity. Our method has the potential to allow the generation of monoclonal antibodies against a variety of phosphorylated proteins. PMID:27125496

  11. The use of monoclonal antibodies for the antigenic analysis of influenza A viruses

    PubMed Central

    Pereira, M. S.; Chakraverty, P.; Cunningham, P.; Webster, R. G.

    1985-01-01

    Monoclonal antibodies have been found to provide useful additional information for the antigenic analysis of influenza A viruses of the H3N2 and H1N1 subtypes. They have been particularly useful in the interpandemic period when multiple variants circulate concurrently. Apparently heterogeneous isolates can be placed in fairly clear-cut groups on the basis of their reactivity with certain monoclonal antibody preparations. It is thought likely that variants reacting with the least number of monoclones are the most different antigenically from the fully reactive strains. PMID:2410156

  12. [Advances in the research of anti-CD20 therapeutic monoclonal antibodies].

    PubMed

    Deng, Cheng-Lian; Zou, Jia; Song, Hai-Feng

    2013-10-01

    As targeted drugs to B-cell malignancies, anti-CD20 monoclonal antibodies have been proved to be important in therapeutic antibody field. With three generations in more than ten years' development, the structures of these drugs have been improved, and many new indications have been found. Nowadays, these kinds of antibodies are not only used in the treatment of lymphoid malignancies, but also been proved to be useful in some autoimmune diseases treatment, and their new indications are still being expanded. With the optimization of their clinical dosage regimens, drug reaction has been increased, thus, therapeutic and side effects of anti-CD20 monoclonal antibody have been further improved as well. However, the exact mechanism of action of their combination therapy with other chemical drugs is still unclear, which remains to be further studied. This article reviewed new development of anti-CD20 therapeutic monoclonal antibodies research in recent years. PMID:24417077

  13. Antibody purification using affinity chromatography: a case study with a monoclonal antibody to ractopamine.

    PubMed

    Wang, Zhanhui; Liang, Qi; Wen, Kai; Zhang, Suxia; Shen, Jianzhong

    2014-11-15

    The application of antibodies to small molecules in the field of bioanalytics requires antibodies with stable biological activity and high purity; thus, there is a growing interest in developing rapid, inexpensive and effective procedures to obtain such antibodies. In this work, a ractopamine (RAC) derivative, N-4-aminobutyl ractopamine (ABR), was synthesized for preparing new specific affinity chromatography to purify a murine monoclonal antibody (mAb) against RAC from ascites. The performance of the new specific chromatography was compared with four other purification methods in terms of recovery, purity and biological activity of mAb. These four purification methods were prepared by using specific ligands (RAC and RAC-ovalbumin) and commercial ligands (protein G and protein A), respectively. The results showed that the highest recovery (88.1%) was achieved using the new chromatography; in comparison, the recoveries from the other methods were all below 70%. The purity of the mAbs from the new chromatography was 88.3%, while, the highest purity of 97.6% was from protein G chromatography and the lowest purity of 84.7% was from protein A chromatography. The biological activity of the purified mAb from all of the chromatography methods was comparable in enzyme-linked immunosorbent immunoassay (ELISA). PMID:25261834

  14. Two monoclonal antibodies raised against different epitopes of chloroplast fructose-1. 6-bisphosphatase (FBPase)

    SciTech Connect

    Hermoso, R.; Fonolla, J.; Lopez-Gorge, J. ); Ruiz-Cabello, F.; Garrido, F. )

    1990-05-01

    Two monoclonal antibodies (GR-BP5 and GR-BP8) were obtained by fusion of spleen cells of mice immunized against pea photosynthetic FBPase with cells of myeloma NSI. Both mAbs showed by double immunodiffusion a {chi} light chain, and the GR-BP8 secreted an IgM. By Western-blotting and immunoprecipitation of the in vivo labelled pea FBPase, GR-BP5 and GR-BP8 showed specificity for the chloroplast enzyme. Competition binding of the {sup 125}I-labelled mAbs against pea FBPase showed specific binding sites to different epitopes of the enzyme molecule. Cross reaction assays between both monoclonal antibodies and pea and spinach chloroplast FBPases showed a 90-100% homology in the corresponding epitopes of both enzymes. Preliminary assays showed a moderate inhibition of FBPase by GR-BP5 monoclonal antibody, but a weak enhancement by the GR-BP8 monoclonal one.

  15. Development of new versions of anti-human CD34 monoclonal antibodies with potentially reduced immunogenicity

    SciTech Connect

    Qian Weizhu; Wang Ling; Li Bohua; Wang Hao; Hou Sheng; Hong Xueyu; Zhang Dapeng; Guo Yajun

    2008-03-07

    Despite the widespread clinical use of CD34 antibodies for the purification of human hematopoietic stem/progenitor cells, all the current anti-human CD34 monoclonal antibodies (mAbs) are murine, which have the potential to elicit human antimouse antibody (HAMA) immune response. In the present study, we developed three new mouse anti-human CD34 mAbs which, respectively, belonged to class I, class II and class III CD34 epitope antibodies. In an attempt to reduce the immunogenicity of these three murine mAbs, their chimeric antibodies, which consisted of mouse antibody variable regions fused genetically to human antibody constant regions, were constructed and characterized. The anti-CD34 chimeric antibodies were shown to possess affinity and specificity similar to that of their respective parental murine antibodies. Due to the potentially better safety profiles, these chimeric antibodies might become alternatives to mouse anti-CD34 antibodies routinely used for clinical application.

  16. Lymphocyte phosphatase-associated phosphoprotein proteoforms analyzed using monoclonal antibodies

    PubMed Central

    Filatov, Alexander; Kruglova, Natalia; Meshkova, Tatiana; Mazurov, Dmitriy

    2015-01-01

    Phosphatase CD45 regulates the activation of lymphocytes by controlling the level of receptor and signal molecule phosphorylation. However, it remains unknown which molecules mediate the phosphatase activity of CD45. A candidate for such a molecule is a small transmembrane adapter protein called lymphocyte phosphatase-associated phosphoprotein (LPAP). LPAP forms a supramolecular complex that consists of not only CD45 molecule but also CD4 and Lck kinase. The function of LPAP has not been defined clearly. In our study, we determined the pattern of LPAP expression in various cell types and characterized its proteoforms using new monoclonal antibodies generated against the intracellular portion of the protein. We show that LPAP is a pan-lymphocyte marker, and its expression in cells correlates with the expression of CD45. The majority of T, B and NK cells express high levels of LPAP, whereas monocytes, granulocytes, monocyte-derived dendritic cells, platelets and red blood cells are negative for LPAP. Using one- and two-dimensional protein gel electrophoresis, we demonstrate that LPAP has at least four sites of phosphorylation. The resting cells express at least six different LPAP phosphoforms representing mono-, di- and tri-phosphorylated LPAP. T and B cells differ in the distribution of the protein between phosphoforms. The activation of lymphocytes with PMA reduces the diversity of phosphorylated forms. Our experiments on Lck-deficient Jurkat cells show that Lck kinase is not involved in LPAP phosphorylation. Thus, LPAP is a dynamically phosphorylated protein, the function of which can be understood, when all phosphosites and kinases involved in its phosphorylation will be identified. PMID:26682052

  17. Characterizing monoclonal antibody structure by carboxyl group footprinting

    PubMed Central

    Kaur, Parminder; Tomechko, Sara E; Kiselar, Janna; Shi, Wuxian; Deperalta, Galahad; Wecksler, Aaron T; Gokulrangan, Giridharan; Ling, Victor; Chance, Mark R

    2015-01-01

    Structural characterization of proteins and their antigen complexes is essential to the development of new biologic-based medicines. Amino acid-specific covalent labeling (CL) is well suited to probe such structures, especially for cases that are difficult to examine by alternative means due to size, complexity, or instability. We present here a detailed account of carboxyl group labeling (with glycine ethyl ester (GEE) tagging) applied to a glycosylated monoclonal antibody therapeutic (mAb). The experiments were optimized to preserve the structural integrity of the mAb, and experimental conditions were varied and replicated to establish the reproducibility of the technique. Homology-based models were generated and used to compare the solvent accessibility of the labeled residues, which include aspartic acid (D), glutamic acid (E), and the C-terminus (i.e., the target probes), with the experimental data in order to understand the accuracy of the approach. Data from the mAb were compared to reactivity measures of several model peptides to explain observed variations in reactivity. Attenuation of reactivity in otherwise solvent accessible probes is documented as arising from the effects of positive charge or bond formation between adjacent amine and carboxyl groups, the latter accompanied by observed water loss. A comparison of results with previously published data by Deperalta et al using hydroxyl radical footprinting showed that 55% (32/58) of target residues were GEE labeled in this study whereas the previous study reported 21% of the targets were labeled. Although the number of target residues in GEE labeling is fewer, the two approaches provide complementary information. The results highlight advantages of this approach, such as the ease of use at the bench top, the linearity of the dose response plots at high levels of labeling, reproducibility of replicate experiments (<2% variation in modification extent), the similar reactivity of the three target probes

  18. Pharmacological effects of two anti-methamphetamine monoclonal antibodies

    PubMed Central

    Laurenzana, Elizabeth M; Stevens, Misty W; Frank, John C; Hambuchen, Michael D; Hendrickson, Howard P; White, Sarah J; Williams, D Keith; Owens, S Michael; Gentry, W Brooks

    2014-01-01

    This lead candidate selection study compared two anti-(+)-methamphetamine (METH) monoclonal antibodies (mAb) to determine their ability to reduce METH-induced locomotor effects and redistribute METH and (+)-amphetamine (AMP) in a preclinical overdose model. Both mAbs have high affinity for METH, but mAb4G9 has moderate and mAb7F9 has low affinity for AMP. In the placebo-controlled behavioral experiment, the effects of each mAb on the locomotor response to a single 1 mg/kg intravenous (IV) METH dose were determined in rats. The doses of mAb binding sites were administered such that they equaled 1, 0.56, 0.32, and 0.1 times the molar equivalent (mol-eq) of METH in the body 30 min after the METH dose. METH disposition was determined in separate animals that similarly received either a 1 or 0.32 mol-eq dose of mAb binding sites 30 min after a 1 mg/kg METH dose. Total METH-induced distance traveled was significantly reduced in rats that received the highest three doses of each mAb compared with saline. The duration of METH effects was also significantly reduced by mAb7F9 at the highest dose. The disposition of METH was altered dose-dependently by both mAbs as shown in reductions of volume of distribution and total clearance, and increases in elimination half-life. These data indicate that both mAbs are effective at reducing METH-induced behavior and favorably altering METH disposition. Both were therefore suitable for further preclinical testing as potential human medications for treating METH use; however, due to results reported here and in later studies, mAb7F9 was selected for clinical development. PMID:25483484

  19. Safety and immunotoxicity assessment of immunomodulatory monoclonal antibodies

    PubMed Central

    Morton, Laura Dill; Spindeldreher, Sebastian; Kiessling, Andrea; Allenspach, Roy; Hey, Adam; Muller, Patrick Y; Frings, Werner; Sims, Jennifer

    2010-01-01

    Most therapeutic monoclonal antibodies (mAbs) licensed for human use or in clinical development are indicated for treatment of patients with cancer and inflammatory/autoimmune disease and as such, are designed to directly interact with the immune system. A major hurdle for the development and early clinical investigation of many of these immunomodulatory mAbs is their inherent risk for adverse immune-mediated drug reactions in humans such as infusion reactions, cytokine storms, immunosuppression and autoimmunity. A thorough understanding of the immunopharmacology of a mAb in humans and animals is required to both anticipate the clinical risk of adverse immunotoxicological events and to select a safe starting dose for first-in-human (FIH) clinical studies. This review summarizes the most common adverse immunotoxicological events occurring in humans with immunomodulatory mAbs and outlines non-clinical strategies to define their immunopharmacology and assess their immunotoxic potential, as well as reduce the risk of immunotoxicity through rational mAb design. Tests to assess the relative risk of mAb candidates for cytokine release syndrome, innate immune system (dendritic cell) activation and immunogenicity in humans are also described. The importance of selecting a relevant and sensitive toxicity species for human safety assessment in which the immunopharmacology of the mAb is similar to that expected in humans is highlighted, as is the importance of understanding the limitations of the species selected for human safety assessment and supplementation of in vivo safety assessment with appropriate in vitro human assays. A tiered approach to assess effects on immune status, immune function and risk of infection and cancer, governed by the mechanism of action and structural features of the mAb, is described. Finally, the use of immunopharmacology and immunotoxicity data in determining a minimum anticipated biologic effect Level (MABEL) and in the selection of safe human

  20. Characterization of Cross-Reactive Norovirus-Specific Monoclonal Antibodies

    PubMed Central

    Kou, Baijun; Crawford, Sue E.; Ajami, Nadim J.; Czakó, Rita; Neill, Frederick H.; Tanaka, Tomoyuki N.; Kitamoto, Noritoshi; Palzkill, Timothy G.; Estes, Mary K.

    2014-01-01

    Noroviruses (NoVs) commonly cause acute gastroenteritis outbreaks. Broadly reactive diagnostic assays are essential for rapid detection of NoV infections. We previously generated a panel of broadly reactive monoclonal antibodies (MAbs). We characterized MAb reactivities by use of virus-like particles (VLPs) from 16 different NoV genotypes (6 from genogroup I [GI], 9 from GII, and 1 from GIV) coating a microtiter plate (direct enzyme-linked immunosorbent assay [ELISA]) and by Western blotting. MAbs were genotype specific or recognized multiple genotypes within a genogroup and between genogroups. We next applied surface plasmon resonance (SPR) analysis to measure MAb dissociation constants (Kd) as a surrogate for binding affinity; a Kd level of <10 nM was regarded as indicating strong binding. Some MAbs did not interact with the VLPs by SPR analysis. To further assess this lack of MAb-VLP interaction, the MAbs were evaluated for the ability to identify NoV VLPs in a capture ELISA. Those MAbs for which a Kd could not be measured by SPR analysis also failed to capture the NoV VLPs; in contrast, those with a measurable Kd gave a positive signal in the capture ELISA. Thus, some broadly cross-reactive epitopes in the VP1 protruding domain may be partially masked on intact particles. One MAb, NV23, was able to detect genogroup I, II, and IV VLPs from 16 genotypes tested by sandwich ELISA, and it successfully detected NoVs in stool samples positive by real-time reverse transcription-PCR when the threshold cycle (CT) value was <31. Biochemical analyses of MAb reactivity, including SPR analysis, identified NV23 as a broadly reactive ligand for application in norovirus diagnostic assays. PMID:25428247

  1. Response of a Concentrated Monoclonal Antibody Formulation to High Shear

    PubMed Central

    Bee, Jared S.; Stevenson, Jennifer L.; Mehta, Bhavya; Svitel, Juraj; Pollastrini, Joey; Platz, Robert; Freund, Erwin; Carpenter, John F.

    2009-01-01

    There is concern that shear could cause protein unfolding or aggregation during commercial biopharmaceutical production. In this work we exposed two concentrated immunoglobulin-G1 (IgG1) monoclonal antibody (mAb, at >100 mg/mL) formulations to shear rates of between 20,000 and 250,000 s-1 for between 5 minutes and 30 ms using a parallel-plate and capillary rheometer respectively. The maximum shear and force exposures were far in excess of those expected during normal processing operations (20,000 s-1 and 0.06 pN respectively). We used multiple characterization techniques to determine if there was any detectable aggregation. We found that shear alone did not cause aggregation, but that prolonged exposure to shear in the stainless steel parallel-plate rheometer caused a very minor reversible aggregation (<0.3%). Additionally, shear did not alter aggregate populations in formulations containing 17% preformed heat-induced aggregates of a mAb. We calculate that that the forces applied to a protein by production shear exposures (<0.06 pN) are small when compared with the 140 pN force expected at the air-water interface or the 20 to 150 pN forces required to mechanically unfold proteins described in the atomic force microscope (AFM) literature. Therefore, we suggest that in many cases air-bubble entrainment, adsorption to solid surfaces (with possible shear synergy), contamination by particulates, or pump cavitation stresses could be much more important causes of aggregation than shear exposure during production. PMID:19370772

  2. Advective hydrogel membrane chromatography for monoclonal antibody purification in bioprocessing.

    PubMed

    Hou, Ying; Brower, Mark; Pollard, David; Kanani, Dharmesh; Jacquemart, Renaud; Kachuik, Bradley; Stout, James

    2015-01-01

    Protein A chromatography is widely employed for the capture and purification of monoclonal antibodies (mAbs). Because of the high cost of protein A resins, there is a significant economic driving force to seek new downstream processing strategies. Membrane chromatography has emerged as a promising alternative to conventional resin based column chromatography. However, to date, the application has been limited to mostly ion exchange flow through (FT) mode. Recently, significant advances in Natrix hydrogel membrane has resulted in increased dynamic binding capacities for proteins, which makes membrane chromatography much more attractive for bind/elute operations. The dominantly advective mass transport property of the hydrogel membrane has also enabled Natrix membrane to be run at faster volumetric flow rates with high dynamic binding capacities. In this work, the potential of using Natrix weak cation exchange membrane as a mAb capture step is assessed. A series of cycle studies was also performed in the pilot scale device (> 30 cycles) with good reproducibility in terms of yield and product purities, suggesting potential for improved manufacturing flexibility and productivity. In addition, anion exchange (AEX) hydrogel membranes were also evaluated with multiple mAb programs in FT mode. Significantly higher binding capacity for impurities (support mAb loads up to 10Kg/L) and 40X faster processing speed were observed compared with traditional AEX column chromatography. A proposed protein A free mAb purification process platform could meet the demand of a downstream purification process with high purity, yield, and throughput. PMID:26018631

  3. Monoclonal antibodies: longitudinal prescribing information analysis of hypersensitivity reactions.

    PubMed

    Kleyman, Konstantin; Weintraub, Debra S

    2012-01-01

    Monoclonal antibodies (mAbs) are known to cause hypersensitivity reactions (HSRs). The reactions pose a significant challenge to investigators, regulators, and health providers. Because HSRs cannot be predicted through the pharmacological basis of a therapy, clinical data are often relied upon to detect the reactions. Unfortunately, clinical studies are often unable to adequately characterize HSRs especially in therapies for orphan diseases. HSRs can go undetected until post-marketing safety surveillance when a large number of patients have been exposed to the therapy. The presented data demonstrates how hypersensitivity reaction warnings have changed over time in the prescribing information (PI), i.e., the drug package insert, through August 1, 2011 for 28 US-marketed mAbs. Tracking all PI revisions for each mAb over time revealed that hypersensitivity warning statements were expanded to include more severe manifestations. Over the course of a mAb therapy's life cycle, the hypersensitivity warning is twice more likely to be upgraded than downgraded in priority. Approximately 85% of hypersensitivity-associated fatality warnings were added in PI revisions as a result of post-marketing experience. Over 60% (20/33) of revisions to hypersensitivity warnings occurred within 3-4 y of product approval. While HSRs are generally recognized and described in the initial PI of mAbs, fatal HSRs are most commonly observed in post-marketing surveillance. Results of this study suggest that initial product labeling information may not describe rare but clinically significant occurrences of severe or fatal HSRs, but subsequent label revisions include rare events observed during post-marketed product use. PMID:22531444

  4. Development and Characterization of Monoclonal Antibodies and Aptamers Against Major Antigens of Mycobacterium avium subsp. paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Specific antibodies, available in unlimited quantities, have not been produced against Mycobacterium avium subsp. paratuberculosis, the bacterium that causes Johne’s disease (JD). To fill this gap in JD research, monoclonal antibodies (mAbs) against M. avium subsp. paratuberculosis were produced fr...

  5. Human Monoclonal Antibodies Targeting Glypican-2 in Neuroblastoma | NCI Technology Transfer Center | TTC

    Cancer.gov

    Researchers at the National Cancer Institute’s Laboratory of Molecular Biology (NCI LMB) have developed and isolated several single domain monoclonal human antibodies against GPC2. NCI seeks parties interested in licensing or co-developing GPC2 antibodies and/or conjugates.

  6. PRODUCTION OF MONOCLONAL ANTIBODIES TO 'LEGIONELLA PNEUMOPHILA' SEROGROUPS 1 AND 6

    EPA Science Inventory

    To better define the surface antigens of Legionella pneumophila for clinical and experimental purposes, were produced monoclonal antibodies to L. pneumophila serogroups 1 and 6. Two hybridomas were produced in serogroup 1. One antibody, LP-I-17, recognized a serogroup-common anti...

  7. New Stx2e monoclonal antibodies for immunological detection and distinction of Stx2 subtypes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background Stx2e is a primary virulence factor in STEC strains that cause edema disease in neonatal piglets. Though Stx2a and Stx2e are similar, most antibody-based Stx detection kits are designed to detect Stx2a and do not recognize the Stx2e subtype. Methods and Findings Four monoclonal antibodie...

  8. PREPARATION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES TO ENTERIC ADENOVIRUS TYPES 40 AND 41

    EPA Science Inventory

    The authors have prepared monoclonal antibodies to each of the enteric adenoviruses types 40 and 41. Three different hybridoma cell lines were selected which produced antibody found to react by radioimmunoprecipitation with adenovirus (Ad) hexon antigens. One was specific for Ad4...

  9. Prophylaxis and therapy of influenza pneumonia in mice by intratracheal instillation of monoclonal antibody

    SciTech Connect

    Ratcliffe, D.R.

    1985-01-01

    This study on passive immunity dealt principally with the following topics: pathogenesis of the pneumonia produced by influenza virus (PR8) in CF-1 mice; the distribution and retention of monoclonal antibody instilled intratracheally (IT) into the lung; and prophylaxis and therapy of influenza pneumonia using specific monoclonal antibody (IgG 2a/k anti-HA). The fate of a single 50 ul bolus of antibody instilled IT was determined by monitoring the activity of /sup 125/I-labelled monoclonal IgG in the lungs and by lavage recovery of functional antibody.Antibody was demonstrated in high concentrations for the first 3 days and was present in the lungs for a period of 7 days. For prophylaxis several trials indicated that monoclonal antibody provided significant protection from lethal effects of the virus. Antibody given to clinically ill mice on day 3 produced a highly significant reduction in mortality (P < 0.001) when compared to control mice. The treatment reversed the weight loss and apparently arrested the development of lesions in most of the mice within 2 days following antibody administration.

  10. The generation of monoclonal antibodies and their use in rapid diagnostic tests

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Antibodies are the most important component of an immunoassay. In these proceedings we outline novel methods used to generate and select monoclonal antibodies that meet performance criteria for use in rapid lateral flow and microfluidic immunoassay tests for the detection of agricultural pathogens ...

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

    NASA Astrophysics Data System (ADS)

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

    1981-05-01

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

  12. Ionizing radiation improves glioma-specific targeting of superparamagnetic iron oxide nanoparticles conjugated with cmHsp70.1 monoclonal antibodies (SPION-cmHsp70.1)

    NASA Astrophysics Data System (ADS)

    Shevtsov, Maxim A.; Nikolaev, Boris P.; Ryzhov, Vyacheslav A.; Yakovleva, Ludmila Y.; Marchenko, Yaroslav Y.; Parr, Marina A.; Rolich, Valerij I.; Mikhrina, Anastasiya L.; Dobrodumov, Anatolii V.; Pitkin, Emil; Multhoff, Gabriele

    2015-12-01

    The stress-inducible 72 kDa heat shock protein Hsp70 is known to be expressed on the membrane of highly aggressive tumor cells including high-grade gliomas, but not on the corresponding normal cells. Membrane Hsp70 (mHsp70) is rapidly internalized into tumor cells and thus targeting of mHsp70 might provide a promising strategy for theranostics. Superparamagnetic iron oxide nanoparticles (SPIONs) are contrast negative agents that are used for the detection of tumors with MRI. Herein, we conjugated the Hsp70-specific antibody (cmHsp70.1) which is known to recognize mHsp70 to superparamagnetic iron nanoparticles to assess tumor-specific targeting before and after ionizing irradiation. In vitro experiments demonstrated the selectivity of SPION-cmHsp70.1 conjugates to free and mHsp70 in different tumor cell types (C6 glioblastoma, K562 leukemia, HeLa cervix carcinoma) in a dose-dependent manner. High-resolution MRI (11 T) on T2-weighted images showed the retention of the conjugates in the C6 glioma model. Accumulation of SPION-cmHsp70.1 nanoparticles in the glioma resulted in a nearly 2-fold drop of values in comparison to non-conjugated SPIONs. Biodistribution analysis using NLR-M2 measurements showed a 7-fold increase in the tumor-to-background (normal brain) uptake ratio of SPION-cmHsp70.1 conjugates in glioma-bearing rats in comparison to SPIONs. This accumulation within Hsp70-positive glioma was further enhanced after a single dose (10 Gy) of ionizing radiation. Elevated accumulation of the magnetic conjugates in the tumor due to radiosensitization proves the combination of radiotherapy and application of Hsp70-targeted agents in brain tumors.The stress-inducible 72 kDa heat shock protein Hsp70 is known to be expressed on the membrane of highly aggressive tumor cells including high-grade gliomas, but not on the corresponding normal cells. Membrane Hsp70 (mHsp70) is rapidly internalized into tumor cells and thus targeting of mHsp70 might provide a promising strategy

  13. Production of monoclonal antibodies against Rickettsia massiliae and their use in antigenic and epidemiological studies.

    PubMed Central

    Xu, W; Raoult, D

    1997-01-01

    Rickettsiae are gram-negative, obligate intracellular bacteria which have historically been divided into three groups: the typhus group, the scrub typhus group, and the spotted fever group (SFG). Recently, several new SFG rickettsiae have been characterized, and most of these species are associated with ticks and have, as yet, no known pathogenicity toward humans. Rickettsia massiliae, which is widely distributed in Europe and Africa, is one such rickettsia. In order to investigate the antigenic relationships between R. massiliae and other rickettsial species and to develop a more convenient methodology for identifying R. massiliae, we produced monoclonal antibodies against the type strain (Mtu1T) of R. massiliae by fusing immunized splenocytes with SP2/0-Ag14 myeloma cells. A panel of 16 representatives were selected from the 163 positive hybridomas identified on initial screening, and their secreted monoclonal antibodies were further characterized. The reactivities of these 16 monoclonal antibodies with a large panel of rickettsial species were assessed by the microimmunofluorescence assay. All species of the SFG rickettsiae reacted with the monoclonal antibodies directed against epitopes on lipopolysaccharide, which is the common antigen among the SFG rickettsiae. Some closely related species of the SFG, such as Bar29, "R. aeschlimanni," and R. rhipicephali, showed strong cross-reactivities with the monoclonal antibodies directed against epitopes on the two major high-molecular-mass heat-labile proteins (106 and 120 kDa). In addition, species-specific monoclonal antibodies demonstrated that R. massiliae is antigenically different from other rickettsial species. Moreover, these species-specific monoclonal antibodies were successfully used for identifying R. massiliae in the ticks collected from southern France, and are therefore potentially useful tools in the identification and investigation of R. massiliae in ticks in large-scale field work. PMID:9196180

  14. Ionizing radiation improves glioma-specific targeting of superparamagnetic iron oxide nanoparticles conjugated with cmHsp70.1 monoclonal antibodies (SPION-cmHsp70.1).

    PubMed

    Shevtsov, Maxim A; Nikolaev, Boris P; Ryzhov, Vyacheslav A; Yakovleva, Ludmila Y; Marchenko, Yaroslav Y; Parr, Marina A; Rolich, Valerij I; Mikhrina, Anastasiya L; Dobrodumov, Anatolii V; Pitkin, Emil; Multhoff, Gabriele

    2015-12-28

    The stress-inducible 72 kDa heat shock protein Hsp70 is known to be expressed on the membrane of highly aggressive tumor cells including high-grade gliomas, but not on the corresponding normal cells. Membrane Hsp70 (mHsp70) is rapidly internalized into tumor cells and thus targeting of mHsp70 might provide a promising strategy for theranostics. Superparamagnetic iron oxide nanoparticles (SPIONs) are contrast negative agents that are used for the detection of tumors with MRI. Herein, we conjugated the Hsp70-specific antibody (cmHsp70.1) which is known to recognize mHsp70 to superparamagnetic iron nanoparticles to assess tumor-specific targeting before and after ionizing irradiation. In vitro experiments demonstrated the selectivity of SPION-cmHsp70.1 conjugates to free and mHsp70 in different tumor cell types (C6 glioblastoma, K562 leukemia, HeLa cervix carcinoma) in a dose-dependent manner. High-resolution MRI (11 T) on T(2)-weighted images showed the retention of the conjugates in the C6 glioma model. Accumulation of SPION-cmHsp70.1 nanoparticles in the glioma resulted in a nearly 2-fold drop of T*(2) values in comparison to non-conjugated SPIONs. Biodistribution analysis using NLR-M(2) measurements showed a 7-fold increase in the tumor-to-background (normal brain) uptake ratio of SPION-cmHsp70.1 conjugates in glioma-bearing rats in comparison to SPIONs. This accumulation within Hsp70-positive glioma was further enhanced after a single dose (10 Gy) of ionizing radiation. Elevated accumulation of the magnetic conjugates in the tumor due to radiosensitization proves the combination of radiotherapy and application of Hsp70-targeted agents in brain tumors. PMID:26599206

  15. Developmental regulation of cytokeratins in cells of the rat mammary gland studied with monoclonal antibodies.

    PubMed Central

    Allen, R; Dulbecco, R; Syka, P; Bowman, M; Armstrong, B

    1984-01-01

    We have isolated two monoclonal antibodies to cytokeratins and determined their cell specificities. They display interesting localization within the rat mammary gland. One (1A10) shows specificity for myoepithelial cells; the other (24B42) is specific for lumenal cells at various stages of development. These two monoclonal antibodies and three others to cytokeratin previously isolated were used in conjunction with antibodies to myosin and collagen IV to confirm and extend our previous findings on epithelial cell types and development within the mammary gland. Images PMID:6199793

  16. Diffusion and binding of monoclonal antibody TNT-1 in multicellular tumor spheroids

    SciTech Connect

    Cheng, F.M.; Hansen, E.B.; Taylor, C.R.; Epstein, A.L. )

    1991-02-06

    Tumor spheroids of HT-29 human colon adenocarcinoma and A375 melanoma were established to investigate the uptake and clearance kinetics of TNT-1, a monoclonal antibody that targets necrotic cells of tumors. Our data reveal that there was rapid uptake of TNT-1 and its F(ab')2 fragment in both spheroid models, whereas an antibody of irrelevant specificity, Lym-1, and its F(ab')2 fragment bound poorly to the spheroids. Unlike previously reported monoclonal antibodies to tumor cell-surface antigens, TNT-1 showed (1) a linear uptake that increased over time without saturation in tumor spheroids and (2) an unexpected uptake by a subpopulation of cells in the viable outer rim of the spheroids. These preclinical studies provide important information concerning the therapeutic potential of TNT monoclonal antibodies for the treatment of cancer and micrometastases.

  17. [The development of methods for obtaining monoclonal antibody-producing cells].

    PubMed

    Skowicki, Michał; Lipiński, Tomasz

    2016-01-01

    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. PMID:27117113

  18. Virus mutation frequencies can be greatly underestimated by monoclonal antibody neutralization of virions.

    PubMed Central

    Holland, J J; de la Torre, J C; Steinhauer, D A; Clarke, D; Duarte, E; Domingo, E

    1989-01-01

    Monoclonal antibody-resistant mutants have been widely used to estimate virus mutation frequencies. We demonstrate that standard virion neutralization inevitably underestimates monoclonal antibody-resistant mutant genome frequencies of vesicular stomatitis virus, due to phenotypic masking-mixing when wild-type (wt) virions are present in thousandsfold greater numbers. We show that incorporation of antibody into the plaque overlay medium (after virus penetration at 37 degrees C) can provide accurate estimates of genome frequencies of neutral monoclonal antibody-resistant mutant viruses in wt clones. By using this method, we have observed two adjacent G----A base transition frequencies in the I3 epitope to be of the order of 10(-4) in a wt glycine codon. This appears to be slightly lower than the frequencies observed at other sites for total (viable and nonviable) virus genomes when using a direct sequence approach. Images PMID:2479770

  19. Generation of Mouse Monoclonal Antibodies Specific to Chikungunya Virus Using ClonaCell-HY Hybridoma Cloning Kit.

    PubMed

    Yew, Chow Wenn; Tan, Yee Joo

    2016-01-01

    Monoclonal antibodies offer high specificity and this makes it an important tool for molecular biology, biochemistry and medicine. Typically, monoclonal antibodies are generated by fusing mouse spleen cells that have been immunized with the desired antigen with myeloma cells to create immortalized hybridomas. Here, we describe the generation of monoclonal antibodies that are specific to Chikungunya virus using ClonaCell-HY system. PMID:27233275

  20. Elicited antibody nature of human monoclonal protein with anti-streptolysin O activity--analysis with monoclonal anti-idiotype antibody.

    PubMed

    Sawada, S; Shida, M; Suenaga, R; Mizuma, H; Karasaki, M; Hashimoto, M; Kawano, K; Amaki, I

    1986-01-01

    Sera from 7 patients with multiple myeloma having antistreptolysin O (ASO) activity in high titers were detected by a streptolysin O (SLO) inhibition assay. However, activity was in low titer when assayed by a passive agglutination assay. The discrepancy between these 2 assays raised some doubts as to whether these monoclonal proteins (M.protein) bond to SLO in the same manner as elicited antibodies. Immunochemical analysis and idiotope analysis using monoclonal antibody to one of these M.proteins strongly suggest that M.protein with ASO activity bind to SLO in a manner similar to elicited antibody. The discrepancy between the 2 assays might be due to differences in the antigenic structure of different forms of the SLO molecule. PMID:2422380

  1. Radioimmunoassay for detecting antibodies against murine malarial parasite antigens: monoclonal antibodies recognizing Plasmodium yoelii antigens

    SciTech Connect

    Kim, K.J.; Taylor, D.W.; Evans, C.B.; Asofsky, R.

    1980-12-01

    A solid-phase radioimmunoassay (SPRIA) in microtiter wells was established for detecting antibodies against Plasmodium yoelii Ag. The SPRIA was found (1) to require as little as 5 ..mu..g of crude parasite Ag per well, (2) to be able to detect 0.5 ng of monoclonal Ab, and (3) to be 10/sup 4/ times more sensitive than the indirect fluorescent Ab staining technique. In a modification of the above assay using intact RBC as an Ag, hyperimmune serum showed significant binding to the surface of erythrocytes of mice infected with P. yoelii parasites but not to RBC of normal mice. Hybridomas were prepared by fusing infected mouse spleen cells with myeloma cells. Using the SPRIA, hybrids secreting Ab against P. yoelii 17XL Ag were detected.

  2. Monoclonal antibodies to coagulation factor IX define a high-frequency polymorphism by immunoassays.

    PubMed Central

    Smith, K J

    1985-01-01

    Monoclonal antibodies have been used to demonstrate a polymorphism of human plasma coagulation factor IX antigen in double antibody solid-phase immunoradiometric assays. This polymorphism is detected in an assay where a monoclonal antibody (A-1) adsorbed to microtiter wells is used to bind factor IX from diluted plasma samples. Plasma samples with the factor IX polymorphism have less than 0.2 U/ml of apparent antigen when tested with the A-1 antibody, while assays with other monoclonal antibodies and assays with goat antisera to factor IX show normal amounts of factor IX antigen. Factor IX coagulant activity was normal in samples from donors with the polymorphism. The thin-layer polyacrylamide gel isoelectric focusing pattern of factor IX purified from a donor with the factor IX polymorphism (IXp) was identical to that obtained with factor IX prepared from a donor who did not have the polymorphism (IXn). Purified radiolabeled factor IX prepared from a donor with the polymorphism showed a Ka for the A-1 antibody that was threefold less than that measured for IXn. The gene frequency of IXp in male blood donors is 0.25. This polymorphism may be useful as a marker for the X chromosome in genetic studies on plasma samples. Further studies are necessary to determine the explanation for decreased reaction of IXp with the A-1 monoclonal antibody. Images Fig. 1 PMID:9556657

  3. Evaluation of Ion Mobility-Mass Spectrometry for Comparative Analysis of Monoclonal Antibodies.

    PubMed

    Ferguson, Carly N; Gucinski-Ruth, Ashley C

    2016-05-01

    Analytical techniques capable of detecting changes in structure are necessary to monitor the quality of monoclonal antibody drug products. Ion mobility mass spectrometry offers an advanced mode of characterization of protein higher order structure. In this work, we evaluated the reproducibility of ion mobility mass spectrometry measurements and mobiligrams, as well as the suitability of this approach to differentiate between and/or characterize different monoclonal antibody drug products. Four mobiligram-derived metrics were identified to be reproducible across a multi-day window of analysis. These metrics were further applied to comparative studies of monoclonal antibody drug products representing different IgG subclasses, manufacturers, and lots. These comparisons resulted in some differences, based on the four metrics derived from ion mobility mass spectrometry mobiligrams. The use of collision-induced unfolding resulted in more observed differences. Use of summed charge state datasets and the analysis of metrics beyond drift time allowed for a more comprehensive comparative study between different monoclonal antibody drug products. Ion mobility mass spectrometry enabled detection of differences between monoclonal antibodies with the same target protein but different production techniques, as well as products with different targets. These differences were not always detectable by traditional collision cross section studies. Ion mobility mass spectrometry, and the added separation capability of collision-induced unfolding, was highly reproducible and remains a promising technique for advanced analytical characterization of protein therapeutics. Graphical Abstract ᅟ. PMID:26988372

  4. Human peripheral blood monocytes display surface antigens recognized by monoclonal antinuclear antibodies

    SciTech Connect

    Holers, V.M.; Kotzin, B.L.

    1985-09-01

    The authors used monoclonal anti-nuclear autoantibodies and indirect immunofluorescence to examine normal human peripheral blood mononuclear leukocytes for the presence of cell surface nuclear antigens. Only one monoclonal anti-histone antibody (MH-2) was found to bind to freshly isolated PBL, staining approximately 10% of large cells. However, after cells were placed into culture for 16-24 h, a high percentage (up to 60%) of large-sized cells were recognized by an anti-DNA (BWD-1) and several different antihistone monoclonal antibodies (BWH-1, MH-1, and MH-2). These antibodies recognize separate antigenic determinants on chromatin and histones extracted from chromatin. The histone antigen-positive cells were viable, and the monoclonal antibodies could be shown to be binding to the cell surface and not to the nucleus. Using monoclonal antibodies specific for monocytes and T cells, and complement-mediated cytotoxicity, the cells bearing histone antigens were shown to be primarily monocytes. The appearance of histone and DNA antigen-positive cells was nearly completely inhibited by the addition of low concentrations of cycloheximide at initiation of the cultures. In contrast, little effect on the percentage of positive cells was detected if cells were exposed to high doses of gamma irradiation before culture. These data further support the existence of cell surface nuclear antigens on selected cell subsets, which may provide insight into the immunopathogenesis of systemic lupus erythematosus and related autoimmune diseases.

  5. Evaluation of Ion Mobility-Mass Spectrometry for Comparative Analysis of Monoclonal Antibodies

    NASA Astrophysics Data System (ADS)

    Ferguson, Carly N.; Gucinski-Ruth, Ashley C.

    2016-05-01

    Analytical techniques capable of detecting changes in structure are necessary to monitor the quality of monoclonal antibody drug products. Ion mobility mass spectrometry offers an advanced mode of characterization of protein higher order structure. In this work, we evaluated the reproducibility of ion mobility mass spectrometry measurements and mobiligrams, as well as the suitability of this approach to differentiate between and/or characterize different monoclonal antibody drug products. Four mobiligram-derived metrics were identified to be reproducible across a multi-day window of analysis. These metrics were further applied to comparative studies of monoclonal antibody drug products representing different IgG subclasses, manufacturers, and lots. These comparisons resulted in some differences, based on the four metrics derived from ion mobility mass spectrometry mobiligrams. The use of collision-induced unfolding resulted in more observed differences. Use of summed charge state datasets and the analysis of metrics beyond drift time allowed for a more comprehensive comparative study between different monoclonal antibody drug products. Ion mobility mass spectrometry enabled detection of differences between monoclonal antibodies with the same target protein but different production techniques, as well as products with different targets. These differences were not always detectable by traditional collision cross section studies. Ion mobility mass spectrometry, and the added separation capability of collision-induced unfolding, was highly reproducible and remains a promising technique for advanced analytical characterization of protein therapeutics.

  6. Combination of monoclonal antibodies improves immunohistochemical diagnosis of Neospora caninum.

    PubMed

    Uzêda, R S; Schares, G; Ortega-Mora, L M; Madruga, C R; Aguado-Martinez, A; Corbellini, L G; Driemeier, D; Gondim, L F P

    2013-11-01

    Histological analysis is commonly used for a conclusive diagnosis of neosporosis. Immunohistochemistry (IHC) using monoclonal (mAb) and polyclonal (pAb) antibodies can improve diagnosis; however, the use of pAb may induce cross-reactivity with other related parasites. The aims of this study were to compare the performance of mAbs and their combinations with that of pAb in IHC and evaluate the usefulness of mAb to identify Neospora caninum infection in aborted bovine fetal tissues. For this purpose, mAbs targeting NcSRS2 (4.15.15) or NcGRA7 (4.11.5 and 1/24-12) and one pAb collected from a rabbit inoculated with N. caninum tachyzoites were tested by IHC. Artificial standardized tissue sections were prepared as positive controls using homogenized bovine brain spiked with cultured tachyzoites of N. caninum. The numbers of labeled parasites were counted in each positive control section. In addition, four equal proportional combinations of the mAbs were also analyzed in the IHC. Finally, the pAb and the best combination of mAbs obtained in the positive control experiments were tested with tissue sections of naturally-infected cattle. To confirm analytical specificity, mAbs and a pAb were tested with Toxoplasma gondii and Besnoitia besnoiti positive control slides and tissues sections from naturally infected cattle containing Sarcocystis spp. and B. besnoiti antigens. The mAb 4.15.15 detected 57% of the total parasites in sections while 4.11.5 and 1/24-12 were able to detect 49% and 41%, respectively. For the mAb combinations (I: 1/24-12+4.11.5, II: 1/24-12+4.15.15, III: 4.15.15+4.11.5, IV: 1/24-12+4.11.5+4.15.15), the detection capacity was 32.4%, 79.4%, 66.6% and 60.7% for each combination, respectively. The best mAb combination (1/24-12 and 4.15.15) and the pAb serum detected 100% (18/18) of naturally-infected animals. Sarcocystis spp. or B. besnoiti were not detected by mAb combinations in IHC, however the pAb cross-reacted with Sarcocystis spp. cysts. These results

  7. Analysis of defined combinations of monoclonal antibodies in anthrax toxin neutralization assays and their synergistic action.

    PubMed

    Ngundi, Miriam M; Meade, Bruce D; Little, Stephen F; Quinn, Conrad P; Corbett, Cindi R; Brady, Rebecca A; Burns, Drusilla L

    2012-05-01

    Antibodies against the protective antigen (PA) component of anthrax toxin play an important role in protection against disease caused by Bacillus anthracis. In this study, we examined defined combinations of PA-specific monoclonal antibodies for their ability to neutralize anthrax toxin in cell culture assays. We observed additive, synergistic, and antagonistic effects of the antibodies depending on the specific antibody combination examined and the specific assay used. Synergistic toxin-neutralizing antibody interactions were examined in more detail. We found that one mechanism that can lead to antibody synergy is the bridging of PA monomers by one antibody, with resultant bivalent binding of the second antibody. These results may aid in optimal design of new vaccines and antibody therapies against anthrax. PMID:22441391

  8. Immunodot blot assay to detect Helicobacter pylori using monoclonal antibodies against the 26 kDa protein.

    PubMed

    Amini Najafabadi, Hossein; Paknejad, Maliheh; Farshad, Shohreh; Mohammadian, Taher; Seyyed Ebrahimi, Shadi Sadat; Amini Najafabadi, Azadeh

    2012-12-01

    Development of a specific immunoassay to detect Helicobacter pylori infection in stool samples requires monoclonal antibody against the specific antigen. The aims of this study were to establish monoclonal antibodies against the 26 kDa protein of H. pylori and develop an immunodot blot for their application to recognize H. pylori infection using stool samples. Mice were immunized intraperitoneally with homogenized gel containing the 26 kDa band of cell surface proteins of H. pylori in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The monoclonal antibodies were produced using the hybridoma technique. Reactivity of monoclonal antibodies was tested with the purified 26 kDa antigen and cell surface proteins from cultured H. pylori by ELISA. Furthermore reactivity of monoclonal antibodies was tested on negative and positive stool samples for H. pylori and suspensions of several major bacteria in stool by immunodot blot assay. Five stable hybridoma monoclones were obtained. The concordant reactivity of the monoclonal antibodies with H. pylori present in the stool samples, which had been tested previously using an ACON ELISA kit for H. pylori stool antigen testing, and unreactivity with several different major fecal bacteria in immunodot blotting indicates high specificity of the immunodot blot based on the reaction of produced monoclonal antibodies with the H. pylori antigen in stools. The findings indicate that the novel immunodot blot developed based on new monoclonal antibodies for stool antigens would be useful as a noninvasive method of diagnosing H. pylori infection. PMID:23244318

  9. Development, characterization, and use of monoclonal and polyclonal antibodies against the myxosporean, Ceratomyxa shasta

    USGS Publications Warehouse

    Bartholomew, J.L.; Rohovec, J.S.; Fryer, J.L.

    1989-01-01

    Both monoclonal and polyclonal antisera were produced against Ceratomyxa shasta. Ascites containing trophozoites of the parasite was collected from infected fish and used as antigen for immunization of mice. The resulting monoclonal antibodies reacted specifically with trophozoite and sporoblast stages but did not react with C. shasta spores by either indirect fluorescent antibody techniques or in Western blots. This indicates that some C. shasta antigens are specific to certain life stages of the parasite. Polyclonal antiserum was produced in a rabbit by injecting a spore protein electro-eluted from an SDS-polyacrylamide gel. This antiserum reacted with both trophozoites and spores by indirect fluorescent antibody techniques and in Western blots. All antisera were tested for cross-reactivity to trout white blood cells, a contaminant of the ascites, and to other myxosporea. Two monoclonal antibodies reacted with white blood cells and myxosporea of the genera Sphaerospora and Myxobilatus. One hybridoma produced antibodies of high specificity for C. shasta pre-spore stages. This is the first report of a monoclonal antibody produced against a myxosporean parasite.

  10. DETECTION OF ROTAVIRUS WITH A NEW POLYCLONAL ANTIBODY ENZYME IMMUNOASSAY (ROTAZYME 2) AND A COMMERCIAL LATEX AGGLUTINATION TEXT (ROTALEX): COMPARISON WITH A MONOCLONAL ANTIBODY ENZYME IMMUNOASSAY

    EPA Science Inventory

    A total of 176 human fecal specimens were examined for the presence of rotavirus using four different assays: a monoclonal antibody enzyme immunoassay; the original polyclonal antibody enzyme immunoassay marketed by Abbott Laboratories, Chicago, IL (Rotazyme I); a modification of...

  11. Monoclonal antibody capture enzyme immunoassay for detection of Paracoccidioides brasiliensis antibodies in paracoccidioidomycosis.

    PubMed Central

    Camargo, Z P; Gesztesi, J L; Saraiva, E C; Taborda, C P; Vicentini, A P; Lopes, J D

    1994-01-01

    Four murine monoclonal antibodies (MAbs 17C, 21A, 21F, and 32B) raised against the 43-kDa glycoprotein of Paracoccidioides brasiliensis were tested in a capture enzyme immunoassay (EIA) for the detection of specific human anti-gp43 immunoglobulin G in patients with paracoccidioidomycosis (PCM). All MAbs reacted similarly in the assay. These MAbs, which detected anti-gp43 at levels of as low as 500 pg/ml, were demonstrated to specifically recognize at least two different epitopes in gp43 binding assays. Specific antibodies in the sera of patients with active PCM were detected at dilutions of as high as 1:819,200, and the reactivities of patient sera, as measured by optical densities, were found to be significantly higher than those of control sera. The comparison between classical ELISA and our capture enzyme immunoassay showed that both sensitivity and specificity were greatly improved by the latter. These MAbs represent the first specific reagents to P. brasiliensis described for use in serological tests for PCM. Images PMID:7814469

  12. Monoclonal antibody therapy in multiple myeloma: where do we stand and where are we going?

    PubMed

    Thanendrarajan, Sharmilan; Davies, Faith E; Morgan, Gareth J; Schinke, Carolina; Mathur, Pankaj; Heuck, Christoph J; Zangari, Maurizio; Epstein, Joshua; Yaccoby, Shmuel; Weinhold, Niels; Barlogie, Bart; van Rhee, Frits

    2016-01-01

    Multiple myeloma is a plasma cell malignancy that is characterized by refractory and relapsing course of disease. Despite the introduction of high-dose chemotherapy in combination with autologous stem cell transplantation and innovative agents such as proteasome inhibitors and immunomodulatory drugs, achieving cure in multiple myeloma is a challenging endeavor. In the last couple of years, enormous advances were made in implementing monoclonal antibody therapy in multiple myeloma. A large number of preclinical and clinical studies have been introduced successfully, demonstrating a safe and efficient administration of monoclonal antibodies in multiple myeloma. In particular, the application of monoclonal antibodies in combination with immunomodulatory drugs, proteasome inhibitors, corticosteroids or conventional chemotherapy seem to be promising and will expand the treatment arsenal for patients with multiple myeloma. PMID:26888183

  13. Survey of Latin American Neuroimmunologists on the Treatment of Multiple Sclerosis with Monoclonal Antibodies.

    PubMed

    Fragoso, Yara Dadalti

    2015-01-01

    Natalizumab and alemtuzumab are monoclonal antibodies approved for the treatment of relapsing-remitting multiple sclerosis (RRMS). A third monoclonal antibody, daclizumab, should soon become another alternative for RRMS therapy. A group of 26 doctors working at specific MS Units in seven different Latin American countries participated in the present study. All 26 neurologists had experience with natalizumab for the treatment of MS and were willing to discuss strategies for improving this treatment. Most neurologists had no confidence in starting a patient on natalizumab and alemtuzumab, which are new and efficient drugs approved by North American, European and most Latin American health agencies. The Latin American specialists felt they were not properly informed on daclizumab. Specific pharmacovigilance programs for each of these monoclonal antibodies were considered very important by the neurologists, who were also willing to discuss these therapeutic options with peers from other countries. PMID:25895725

  14. The effect of space flight on monoclonal antibody synthesis in a hybridoma mouse cell line

    NASA Technical Reports Server (NTRS)

    Smiley, S. A.; Gillock, E. T.; Black, M. C.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1997-01-01

    The hybridoma cell line, 3G10G5, producing a monoclonal antibody to the major capsid protein VP1 from the avian polyomavirus budgerigar fledgling disease virus, was produced from a Balb/C mouse. This cell line was used to test the effects of microgravity on cellular processes, specifically protein synthesis. A time course study utilizing incorporation of [35S]methionine into newly synthesized monoclonal antibody was performed on STS-77. After 5.5 days, it was observed that cell counts for the samples exposed to microgravity were lower than those of ground-based samples. However, radiolabel incorporation of the synthesized monoclonal antibody was similar in both orbiter and ground control samples. Overall, microgravity does not seem to have an effect on this cell line's ability to synthesize IgG protein.

  15. Localization of human tumour xenografts after i.v. administration of radiolabeled monoclonal antibodies.

    PubMed

    Moshakis, V; McIlhinney, R A; Raghavan, D; Neville, A M

    1981-07-01

    A mouse monoclonal antibody (LICR-LON/HT13) has been developed to a cell-surface antigen carried on a human germ-cell tumour xenograft (HX39). After radioiodination, the antibody localized in vivo preferentially in xenografted tumours as opposed to normal mouse tissue, whereas tumor uptake did not occur with normal mouse IgG or nonspecific monoclonal IgG. This selective localization could be abolished by simultaneous injection of an excess of the unlabelled LICR-LON/HT13. The kinetics of and factors influencing localization have been examined. Tumour weight was important in that the smaller the tumour the better the localization. LICR-LON/HT13 was found to localize also in other xenografted germ-cell tumours, but not in non-germ-cell tumour xenografts. Thus monoclonal antibodies are capable of selective in vivo localization of human tumours in an animal model, and their clinical value should now be assessed. PMID:6789857

  16. The Cloning and Expression of Human Monoclonal Antibodies: Implications for Allergen Immunotherapy.

    PubMed

    James, Louisa K

    2016-02-01

    Allergic responses are dependent on the highly specific effector functions of IgE antibodies. Conversely, antibodies that block the activity of IgE can mediate tolerance to allergen. Technologies that harness the unparalleled specificity of antibody responses have revolutionized the way that we diagnose and treat human disease. This area of research continues to advance at a rapid pace and has had a significant impact on our understanding of allergic disease. This review will present an overview of humoral responses and provide an up-to-date summary of technologies used in the generation of human monoclonal antibodies. The impact that monoclonal antibodies have on allergic disease will be discussed, with a particular focus on allergen immunotherapy, which remains the only form of treatment that can modulate the underlying immune mechanisms and induce long-term clinical tolerance. PMID:26780523

  17. Development of monoclonal antibodies against parathyroid hormone: genetic control of the immune response to human PTH

    SciTech Connect

    Nussbaum, S.R.; Lin, C.S.; Potts, J.T. Jr.; Rosenthal, A.S.; Rosenblatt, M.

    1985-01-01

    Seventeen monocloanl antibodies against the aminoterminal portion of parathyroid hormone (PTH) were generated by using BALB/c mouse for immunization fully biologically active synthetic human PTH-(1-34) and bovine PTH-(1-84) as immunogens, monoclonal antibody methods, and a solid-phase screening assay. Isotypic analysis of these monoclonal antibodies was performed using affinity purified goat antimouse immunoglobulins specific for IgG heavy chains and ..mu..(IgM). All antibodies were IgM as evidenced by 40 times greater than background activity when 25,000 cpm of /sup 125/I-labelled goat anti-mouse IgM was used as second antibody in a radioimmunoassay.

  18. Single-domain GPC-3 Monoclonal Antibodies for the Treatment of Hepatocellular Carcinoma | NCI Technology Transfer Center | TTC

    Cancer.gov

    The National Cancer Institute seeks parties to license human monoclonal antibodies and immunoconjugates and co-develop, evaluate, and/or commercialize large-scale antibody production and hepatocellular carcinoma (HCC) xenograft mouse models.

  19. Topographic antigenic determinants recognized by monoclonal antibodies on human choriogonadotropin beta-subunit

    SciTech Connect

    Bidart, J.M.; Troalen, F.; Salesse, R.; Bousfield, G.R.; Bohuon, C.J.; Bellet, D.H.

    1987-06-25

    We describe a first attempt to study the antibody-combining sites recognized by monoclonal antibodies raised against the beta-subunit of human choriogonadotropin (hCG). Two groups of antibodies were first defined by their ability to recognize only the free beta-subunit or the free and combined subunit. Antibodies FBT-11 and FBT-11-L bind only to hCG beta-subunit but not to hCG, whereas antibodies FBT-10 and D1E8 bind to both the beta-subunit and the hormone. In both cases, the antigenic determinants were localized to the core of the protein (residues 1-112), indicating the weak immunogenicity of the specific carboxyl-terminal extension of hCG-beta. Nine synthetic peptides spanning different regions of hCG-beta and lutropin-beta were assessed for their capacity to inhibit antibody binding. A synthetic peptide inclusive of the NH2-terminal region (residues 1-7) of the hCG beta-subunit was found to inhibit binding to the radiolabeled subunit of a monoclonal antibody specific for free hCG-beta (FBT-11). Further delineation of the antigenic site recognized by this antibody provided evidence for the involvement of fragment 82-92. Moreover, monoclonal antibody FBT-11 inhibited the recombination of hCG-beta to hCG-alpha, indicating that its antigenic determinant might be located nearby or in the hCG-beta portion interacting with the alpha-subunit. Binding of monoclonal antibody FBT-10, corresponding to the second antigenic determinant, was weakly inhibited by fragment 82-105 and did not impair the recombination of the hCG beta-subunit to the hCG alpha-subunit. Its combining site appeared to be located in a region of the intact native choriogonadotropin present at the surface of the hormone-receptor complex.

  20. Characterization of a humanized monoclonal antibody recognizing clumping factor A expressed by Staphylococcus aureus.

    PubMed

    Domanski, Paul J; Patel, Pratiksha R; Bayer, Arnold S; Zhang, Li; Hall, Andrea E; Syribeys, Peter J; Gorovits, Elena L; Bryant, Dawn; Vernachio, John H; Hutchins, Jeff T; Patti, Joseph M

    2005-08-01

    We report the humanization and characterization of monoclonal antibody (MAb) T1-2 or tefibazumab, a monoclonal antibody that recognizes clumping factor A expressed on the surface of Staphylococcus aureus. We demonstrate that the binding kinetics of MAb T1-2 is indistinguishable compared to that of its murine parent. Furthermore, MAb T1-2 is shown to enhance the opsonophagocytic uptake of ClfA-coated latex beads, protect against an intravenous challenge in a prophylactic model of rabbit infective endocarditis, and enhance the efficacy of vancomycin therapy in a therapeutic model of established infective endocarditis. PMID:16041045

  1. Monoclonal antibodies to antigens on human neutrophils, activated T lymphocytes, and acute leukemia blast cells

    SciTech Connect

    Miterev, G.Yu.; Burova, G.F.; Puzhitskaya, M.S.; Danilevich, S.V.; Bulycheva, T.I.

    1987-11-01

    The authors describe the production of two mouse hybridomas secreting monoclonal antibodies to antigenic determinants of the surface membranes of human neutrophils, activated T lymphocytes, and acute leukemic blast cells. The degree of lymphocyte stimulation was estimated from incorporation of /sup 3/H-thymidine with parallel microculture. Monoclonal antibodies of supernatants of hybridoma cultures shown here reacted in both immunofluorescence test and cytotoxicity test with surface membrane antigens on the majority of neutrophils and PHA-activated peripheral blood lymphocytes from healthy subjects, but did not give positive reactions with unactivated lymphocytes, adherent monocytes, erythrocytes, and alloantigen-stimulated lymphocytes.

  2. RIA of thyroglobulin using monoclonal antibodies: Minimal interference by anti-thyroglobulin autoantibodies

    SciTech Connect

    Nakashima, T.; Koizumi, M.; Sakahara, H.; Ohta, H.; Kohsaka, T.; Misaki, T.; Iida, Y.; Kasagi, K.; Endo, K.; Konishi, J.

    1985-05-01

    Thyroglobulin (Tg) is considered to be secreted from the thyroid gland with the stimulation of TSH and/or thyroid stimulating immunoglobulins. However its use as a prognostic marker for Graves' disease is hampered by anti-Tg autoantibodies in patients' serum. In order to resolve this drawback, the authors have developed monoclonal antibodies to human Tg with very little cross-reactivities with autoantiobodies. Nine monoclonal antibodies were produced by the immunization with Tg prepared from Graves' thyroid and one of them (IgGl), designated as 59A, showed the highest affinity to Tg (3.6 x 10/sup 40/M/sup -1/) and the least cross-reactivity with anti-Tg autoantibodies. The binding of I-125 labeled 59A to beads coated with Tg was not inhibited by the addition of purified IgG obtained from various thyroid diseases except a few Hashimoto's patients with very high titer of anti-Tg antibodies, although the binding of other monoclonal antibodies to Tg was greatly influenced even in the presence of Graves' IgG. The sensitivity of the assay using 59A was enough to detect 20ng Tg/ml and Tg concentrations, in patients with no detectable anti-Tg antibodies, were comparable to those determined by the conventional RIA kit (Eiken), using radioiodinated Tg and polyclonal rabbit anti-Tg antiserum. Further, the shelf-life of I-125 labeled monoclonal antibody was much longer than the radioiodinated Tg. These results indicated that RIA of Tg using monoclonal antibodies would be useful for measuring Tg values not only in patients with thyroid cancer but also in Graves' disease with anti-Tg autoantibodies.

  3. Discovery of Lung Cancer Biomarkers by Profiling the Plasma Proteome with Monoclonal Antibody Libraries*

    PubMed Central

    Guergova-Kuras, Mariana; Kurucz, István; Hempel, William; Tardieu, Nadège; Kádas, János; Malderez-Bloes, Carole; Jullien, Anne; Kieffer, Yann; Hincapie, Marina; Guttman, András; Csánky, Eszter; Dezső, Balázs; Karger, Barry L.; Takács, László

    2011-01-01

    A challenge in the treatment of lung cancer is the lack of early diagnostics. Here, we describe the application of monoclonal antibody proteomics for discovery of a panel of biomarkers for early detection (stage I) of non-small cell lung cancer (NSCLC). We produced large monoclonal antibody libraries directed against the natural form of protein antigens present in the plasma of NSCLC patients. Plasma biomarkers associated with the presence of lung cancer were detected via high throughput ELISA. Differential profiling of plasma proteomes of four clinical cohorts, totaling 301 patients with lung cancer and 235 healthy controls, identified 13 lung cancer-associated (p < 0.05) monoclonal antibodies. The monoclonal antibodies recognize five different cognate proteins identified using immunoprecipitation followed by mass spectrometry. Four of the five antigens were present in non-small cell lung cancer cells in situ. The approach is capable of generating independent antibodies against different epitopes of the same proteins, allowing fast translation to multiplexed sandwich assays. Based on these results, we have verified in two independent clinical collections a panel of five biomarkers for classifying patient disease status with a diagnostics performance of 77% sensitivity and 87% specificity. Combining CYFRA, an established cancer marker, with the panel resulted in a performance of 83% sensitivity at 95% specificity for stage I NSCLC. PMID:21947365

  4. Discovery of lung cancer biomarkers by profiling the plasma proteome with monoclonal antibody libraries.

    PubMed

    Guergova-Kuras, Mariana; Kurucz, István; Hempel, William; Tardieu, Nadège; Kádas, János; Malderez-Bloes, Carole; Jullien, Anne; Kieffer, Yann; Hincapie, Marina; Guttman, András; Csánky, Eszter; Dezso, Balázs; Karger, Barry L; Takács, László

    2011-12-01

    A challenge in the treatment of lung cancer is the lack of early diagnostics. Here, we describe the application of monoclonal antibody proteomics for discovery of a panel of biomarkers for early detection (stage I) of non-small cell lung cancer (NSCLC). We produced large monoclonal antibody libraries directed against the natural form of protein antigens present in the plasma of NSCLC patients. Plasma biomarkers associated with the presence of lung cancer were detected via high throughput ELISA. Differential profiling of plasma proteomes of four clinical cohorts, totaling 301 patients with lung cancer and 235 healthy controls, identified 13 lung cancer-associated (p < 0.05) monoclonal antibodies. The monoclonal antibodies recognize five different cognate proteins identified using immunoprecipitation followed by mass spectrometry. Four of the five antigens were present in non-small cell lung cancer cells in situ. The approach is capable of generating independent antibodies against different epitopes of the same proteins, allowing fast translation to multiplexed sandwich assays. Based on these results, we have verified in two independent clinical collections a panel of five biomarkers for classifying patient disease status with a diagnostics performance of 77% sensitivity and 87% specificity. Combining CYFRA, an established cancer marker, with the panel resulted in a performance of 83% sensitivity at 95% specificity for stage I NSCLC. PMID:21947365

  5. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants

    PubMed Central

    Hehle, Verena K.; Paul, Matthew J.; Roberts, Victoria A.; van Dolleweerd, Craig J.; Ma, Julian K.-C.

    2016-01-01

    This study examined the degradation pattern of a murine IgG1κ monoclonal antibody expressed in and extracted from transformed Nicotiana tabacum. Gel electrophoresis of leaf extracts revealed a consistent pattern of recombinant immunoglobulin bands, including intact and full-length antibody, as well as smaller antibody fragments. N-terminal sequencing revealed these smaller fragments to be proteolytic cleavage products and identified a limited number of protease-sensitive sites in the antibody light and heavy chain sequences. No strictly conserved target sequence was evident, although the peptide bonds that were susceptible to proteolysis were predominantly and consistently located within or near to the interdomain or solvent-exposed regions in the antibody structure. Amino acids surrounding identified cleavage sites were mutated in an attempt to increase resistance. Different Guy’s 13 antibody heavy and light chain mutant combinations were expressed transiently in N. tabacum and demonstrated intensity shifts in the fragmentation pattern, resulting in alterations to the full-length antibody-to-fragment ratio. The work strengthens the understanding of proteolytic cleavage of antibodies expressed in plants and presents a novel approach to stabilize full-length antibody by site-directed mutagenesis.—Hehle, V. K., Paul, M. J., Roberts, V. A., van Dolleweerd, C. J., Ma, J. K.-C. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants. PMID:26712217

  6. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants.

    PubMed

    Hehle, Verena K; Paul, Matthew J; Roberts, Victoria A; van Dolleweerd, Craig J; Ma, Julian K-C

    2016-04-01

    This study examined the degradation pattern of a murine IgG1κ monoclonal antibody expressed in and extracted from transformedNicotiana tabacum Gel electrophoresis of leaf extracts revealed a consistent pattern of recombinant immunoglobulin bands, including intact and full-length antibody, as well as smaller antibody fragments. N-terminal sequencing revealed these smaller fragments to be proteolytic cleavage products and identified a limited number of protease-sensitive sites in the antibody light and heavy chain sequences. No strictly conserved target sequence was evident, although the peptide bonds that were susceptible to proteolysis were predominantly and consistently located within or near to the interdomain or solvent-exposed regions in the antibody structure. Amino acids surrounding identified cleavage sites were mutated in an attempt to increase resistance. Different Guy's 13 antibody heavy and light chain mutant combinations were expressed transiently inN. tabacumand demonstrated intensity shifts in the fragmentation pattern, resulting in alterations to the full-length antibody-to-fragment ratio. The work strengthens the understanding of proteolytic cleavage of antibodies expressed in plants and presents a novel approach to stabilize full-length antibody by site-directed mutagenesis.-Hehle, V. K., Paul, M. J., Roberts, V. A., van Dolleweerd, C. J., Ma, J. K.-C. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants. PMID:26712217

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

    PubMed

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

    2015-01-01

    Targeted therapies of malignancies currently consist of therapeutic monoclonal antibodies and small molecule kinase inhibitors. The combination of these novel agents raises the issue of potential antagonisms. We evaluated the potential effect of 4 kinase inhibitors, including the Bruton tyrosine kinase inhibitor ibrutinib, and 3 PI3K inhibitors idelalisib, NVP-BEZ235 and LY294002, on the effects of the 3 monoclonal antibodies, rituximab and obinutuzumab (directed against CD20) and trastuzumab (directed against HER2). We found that ibrutinib potently inhibits antibody-dependent cell-mediated cytotoxicity exerted by all antibodies, with a 50% inhibitory concentration of 0.2 microM for trastuzumab, 0.5 microM for rituximab and 2 microM for obinutuzumab, suggesting a lesser effect in combination with obinutuzumab than with rituximab. The 4 kinase inhibitors were found to inhibit phagocytosis by fresh human neutrophils, as well as antibody-dependent cellular phagocytosis induced by the 3 antibodies. Conversely co-administration of ibrutinib with rituximab, obinutuzumab or trastuzumab did not demonstrate any inhibitory effect of ibrutinib in vivo in murine xenograft models. In conclusion, some kinase inhibitors, in particular, ibrutinib, are likely to exert inhibitory effects on innate immune cells. However, these effects do not compromise the antitumor activity of monoclonal antibodies in vivo in the models that were evaluated. PMID:25523586

  8. Monoclonal antibodies to the molluscan small cardioactive peptide SCPB: immunolabeling of neurons in diverse invertebrates.

    PubMed

    Masinovsky, B; Kempf, S C; Callaway, J C; Willows, A O

    1988-07-22

    We reported a development of murine monoclonal antibodies to a molluscan small cardioactive peptide (SCPB) and their application to immunolabeling of neurons in several molluscan and arthropod species. In vitro stimulations of mouse lymphocytes with SCPB conjugated to a carrier protein yielded exclusively IgM class antibodies; in vivo stimulation resulted in generation of both IgM and IgG classes of antibodies. Monoclonal antibodies of the IgM class labeled identified SCP-containing neuron B11 in the frozen sections of the buccal ganglia of Tritonia diomedia. These antibodies failed to stain any neurons in whole mount preparations. A monoclonal antibody of IgG1 subclass selectively labeled neurons in both frozen sections and whole mount preparations of diverse invertebrate species. Thus, neurons B11, B12, and GE1 and several other neurons of the buccal and gastroesophageal ganglia of T. diomedia bound the antibody, and a similar pattern of immunolabeling was found in the closely related gastropod Tritonia festiva. We also observed SCPB-like immunoreactivity in the central neurons of other nudibranch and pulmonate molluscs and in examples of insect (Acheta domesticus and Tehrmobia domestica) and crustacean (Semibalanus cariosus) classes of the Arthropoda. Our results suggest a specific pattern of distribution of SCPB-like immunoreactivity in the gastropod nervous system and a broad occurrence of SCPB-like antigenicity in the diverse invertebrates. PMID:3062048

  9. A high affinity monoclonal antibody recognizing the light chain of human coagulating factor VII.

    PubMed

    Sarial, Sheila; Asadi, Farzad; Jeddi-Tehrani, Mahmood; Hadavi, Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Taghizadeh-Jahed, Masoud; Shokri, Fazel; Rabbani, Hodjattallah

    2012-12-01

    Factor VII (FVII) is a serine protease-coagulating element responsible for the initiation of an extrinsic pathway of clot formation. Here we generated and characterized a high affinity monoclonal antibody that specifically recognizes human FVII. Recombinant human FVII (rh-FVII) was used for the production of a monoclonal antibody using BALB/c mice. The specificity of the antibody was determined by Western blot using plasma samples from human, mouse, sheep, goat, bovine, rabbit, and rat. Furthermore, the antibody was used to detect transiently expressed rh-FVII in BHK21 cell line using Western blot and sandwich ELISA. A mouse IgG1 (kappa chain) monoclonal antibody clone 1F1-B11 was produced against rh-FVII. The affinity constant (K(aff)) of the antibody was calculated to be 6.4×10(10) M(-1). The antibody could specifically recognize an epitope on the light chain of hFVII, with no reactivity with factor VII from several other animals. In addition, transiently expressed rh-FVII in BHK21 cells was recognized by 1F1-B11. The high affinity as well as the specificity of 1F1-B11 for hFVII will facilitate the affinity purification of hFVII and also production of FVII deficient plasma and minimizes the risk of bovine FVII contamination when fetal bovine serum-supplemented media are used for production and subsequent purification of rh-FVII. PMID:23244324

  10. Characterization of Two Human Monoclonal Antibodies Neutralizing Influenza A H7N9 Viruses

    PubMed Central

    Wang, Jianmin; Chen, Zhe; Bao, Linlin; Zhang, Weijia; Xue, Ying; Pang, XingHuo; Zhang, Xi

    2015-01-01

    H7N9 was a cause of significant global health concern due to its severe infection and approximately 35% mortality in humans. By screening a Fab antibody phage library derived from patients who recovered from H7N9 infections, we characterized two human monoclonal antibodies (HuMAbs), HNIgGD5 and HNIgGH8. The epitope of these two antibodies was dependent on two residues in the receptor binding site at positions V186 and L226 of the hemagglutinin glycoprotein. Both antibodies possessed high neutralizing activity. PMID:26063436

  11. Production and characterization of monoclonal antibodies to budgerigar fledgling disease virus major capsid protein VP

    NASA Technical Reports Server (NTRS)

    Fattaey, A.; Lenz, L.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    Eleven hybridoma cell lines producing monoclonal antibodies (MAbs) against intact budgerigar fledgling disease (BFD) virions were produced and characterized. These antibodies were selected for their ability to react with BFD virions in an enzyme-linked immunosorbent assay. Each of these antibodies was reactive in the immunofluorescent detection of BFD virus-infected cells. These antibodies immunoprecipitated intact virions and specifically recognized the major capsid protein, VP1, of the dissociated virion. The MAbs were found to preferentially recognize native BFD virus capsid protein when compared with denatured virus protein. These MAbs were capable of detecting BFD virus protein in chicken embryonated cell-culture lysates by dot-blot analysis.

  12. Neutralizing monoclonal antibodies against listeriolysin: mapping of epitopes involved in pore formation.

    PubMed Central

    Darji, A; Niebuhr, K; Hense, M; Wehland, J; Chakraborty, T; Weiss, S

    1996-01-01

    Six different mouse monoclonal antibodies (MAbs) and a specific rabbit polygonal antibody were raised against listeriolysin. Four of the MAbs also recognized seeligeriolysin, and five cross-reacted with ivanolysin. The hemolytic activity could be neutralized by the polygonal antibody as well as by five of the MAbs. None of the neutralizing antibodies interfered with the binding of listeriolysin to the cellular membrane. The epitopes recognized by the MAbs were localized by using overlapping synthetic peptides between positions 59 and 279, a region hitherto not implicated in mediating hemolytic activity. PMID:8675351

  13. Targeting endogenous nuclear antigens by electrotransfer of monoclonal antibodies in living cells

    PubMed Central

    Freund, Guillaume; Sibler, Annie-Paule; Desplancq, Dominique; Oulad-Abdelghani, Mustapha; Vigneron, Marc; Gannon, Julian; Van Regenmortel, Marc H.; Weiss, Etienne

    2013-01-01

    Antibodies are valuable tools for functional studies in vitro, but their use in living cells remains challenging because they do not naturally cross the cell membrane. Here, we present a simple and highly efficient method for the intracytoplasmic delivery of any antibody into cultured cells. By following the fate of monoclonal antibodies that bind to nuclear antigens, it was possible to image endogenous targets and to show that inhibitory antibodies are able to induce cell growth suppression or cell death. Our electrotransfer system allowed the cancer cells we studied to be transduced without loss of viability and may have applications for a variety of intracellular immuno-interventions. PMID:23765067

  14. Monoclonal antibodies to human hemoglobin S and cell lines for the production thereof

    DOEpatents

    Jensen, R.H.; Vanderlaan, M.; Bigbee, W.L.; Stanker, L.H.; Branscomb, E.W.; Grabske, R.J.

    1984-11-29

    The present invention provides monoclonal antibodies specific to and distinguishing between hemoglobin S and hemoglobin A and methods for their production and use. These antibodies are capable of distinguishing between two hemoglobin types which differ from each other by only a single amino acid residue. The antibodies produced according to the present method are useful as immunofluorescent markers to enumerate circulating red blood cells which have the property of altered expression of the hemoglobin gene due to somatic mutation in stem cells. Such a measurement is contemplated as an assay for in vivo cellular somatic mutations in humans. Since the monoclonal antibodies produced in accordance with the instant invention exhibit a high degree of specificity to and greater affinity for hemoglobin S, they are suitable for labeling human red blood cells for flow cytometric detection of hemoglobin genotype. 4 figs.

  15. Monoclonal antibodies to human hemoglobin S and cell lines for the production thereof

    DOEpatents

    Jensen, Ronald H.; Vanderlaan, Martin; Bigbee, William L.; Stanker, Larry H.; Branscomb, Elbert W.; Grabske, Robert J.

    1988-01-01

    The present invention provides monoclonal antibodies specific to and distinguish between hemoglobin S and hemoglobin A and methods for their production and use. These antibodies are capable of distinguishing between two hemoglobin types which differ from each other by only a single amino acid residue. The antibodies produced according to the present method are useful as immunofluorescent markers to enumerate circulating red blood cells which have the property of altered expression of the hemoglobin gene due to somatic mutation in stem cells. Such a measurement is contemplated as an assay for in vivo cellular somatic mutations in humans. Since the monoclonal antibodies produced in accordance with the instant invention exhibit a high degree of specificity to and greater affinity for hemoglobin S, they are suitable for labeling human red blood cells for flow cytometric detection of hemoglobin genotype.

  16. Measurement of free light chains with assays based on monoclonal antibodies.

    PubMed

    Te Velthuis, Henk; Drayson, Mark; Campbell, John P

    2016-06-01

    Recently, serum free light chain (FLC) assays incorporating anti-kappa (κ) and anti-lambda (λ) FLC monoclonal antibodies have become available: N Latex FLC assay (Siemens) and Seralite® (Abingdon Health). The purpose of this review is to provide an overview of these two new monoclonal antibody-based methods. In doing so, the review will outline the performance characteristics of each method, including a summary of: assay principles, antibody specificity, analytical performance and assay performance in disease. Additionally, the review will describe the potential user benefits of adopting these new generation FLC assays, which are designed to overcome the established limitations of existing polyclonal antibody based FLC assays. PMID:27010775

  17. Imaging of bone tumors using a monoclonal antibody raised against human osteosarcoma

    SciTech Connect

    Armitage, N.C.; Perkins, A.C.; Pimm, M.V.; Wastie, M.; Hopkins, J.S.; Dowling, F.; Baldwin, R.W.; Hardcastle, J.D.

    1986-07-01

    The radiolabeled monoclonal antibody 791T/36 raised against a human osteosarcoma was injected into 20 patients with known or suspected bone tumors. Gamma camera images were acquired at 48 or 72 hours after injection, and assessed for antibody localization. Positive images were obtained in all five osteosarcomas and four other primary malignant sarcomas. Two of the four other primary bone tumors gave positive images. Three patients with trauma had negative images as did one patient with Paget's disease. Two patients with suppurative disease gave positive images. The antibody localized in the majority of malignant sarcomas tested. In one tumor where tissue was available, a tumor:non-tumor ratio of 2.8:1 was measured. Repeat imaging was performed in five patients. Immunoscintigraphy using the monoclonal antibody 791T/36 has shown tumor localization in patients with bone and soft tissue sarcomas.

  18. Human Monoclonal Antibodies Against a Plethora of Viral Pathogens From Single Combinatorial Libraries

    NASA Astrophysics Data System (ADS)

    Williamson, R. Anthony; Burioni, Roberto; Sanna, Pietro P.; Partridge, Lynda J.; Barbas, Carlos F., III; Burton, Dennis R.

    1993-05-01

    Conventional antibody generation usually requires active immunization with antigen immediately prior to the preparation procedure. Combinatorial antibody library technology offers the possibility of cloning a range of antibody specificities at a single point in time and then accessing these specificities at will. Here we show that human monoclonal antibody Fab fragments against a plethora of infectious agents can be readily derived from a single library. Further examination of a number of libraries shows that whenever antibody against a pathogen can be detected in the serum of the donor, then specific antibodies can be derived from the corresponding library. We describe the generation of human Fab fragments against herpes simplex virus types 1 and 2, human cytomegalovirus, varicella zoster virus, rubella, human immunodeficiency virus type 1, and respiratory syncytial virus. The antibodies are shown to be highly specific and a number are effective in neutralizing virus in vitro.

  19. Human monoclonal antibodies against a plethora of viral pathogens from single combinatorial libraries.

    PubMed Central

    Williamson, R A; Burioni, R; Sanna, P P; Partridge, L J; Barbas, C F; Burton, D R

    1993-01-01

    Conventional antibody generation usually requires active immunization with antigen immediately prior to the preparation procedure. Combinatorial antibody library technology offers the possibility of cloning a range of antibody specificities at a single point in time and then accessing these specificities at will. Here we show that human monoclonal antibody Fab fragments against a plethora of infectious agents can be readily derived from a single library. Further examination of a number of libraries shows that whenever antibody against a pathogen can be detected in the serum of the donor, then specific antibodies can be derived from the corresponding library. We describe the generation of human Fab fragments against herpes simplex virus types 1 and 2, human cytomegalovirus, varicella zoster virus, rubella, human immunodeficiency virus type 1, and respiratory syncytial virus. The antibodies are shown to be highly specific and a number are effective in neutralizing virus in vitro. Images Fig. 1 Fig. 2 PMID:7683424

  20. Radioimmunodetection in rhabdo- and leiomyosarcoma with sup 111 In-anti-myosin monoclonal antibody complex

    SciTech Connect

    Planting, A.; Verweij, J.; Cox, P.; Pillay, M.; Stoter, G. )

    1990-02-01

    In patients with rhabdo- and leiomyosarcoma a radioimmunodiagnostic study was performed with {sup 111}In labeled F(ab) fragments of a monoclonal antibody against myosin. Eight patients with rhabdomyosarcoma and 18 patients with leiomyosarcoma were studied. Scanning was performed at 4, 24, and 48 h after administration of 74 MBeq of the antibody complex. A high uptake with a tumor:background ratio of 10:1 was observed in several patients with rhabdomyosarcoma but the results were less accurate in leiomyosarcoma.

  1. Combination epigenetic and immunotherapy overcomes resistance to monoclonal antibodies in hematologic malignancies: A new therapeutic approach.

    PubMed

    Epner, Elliot M; Saroya, Bikramajit Singh; Hasanali, Zainul S; Loughran, Thomas P

    2016-03-01

    We recently reported that addition of epigenetic agents could overcome resistance of leukemic cells to monoclonal antibody-mediated anti-tumor effects in T-cell prolymphocytic leukemia. We also reported that epigenetic agents could induce expression of the CD30 gene, thus providing a therapeutic target for the antibody drug conjugate brentuximab vedotin. Here we discuss these findings and their generality to treatment of other hematologic and solid malignancies. PMID:26802532

  2. In vivo Therapy with Monoclonal Anti-I-A Antibody Suppresses Immune Responses to Acetylcholine Receptor

    NASA Astrophysics Data System (ADS)

    Waldor, Matthew K.; Sriram, Subramaniam; McDevitt, Hugh O.; Steinman, Lawrence

    1983-05-01

    A monoclonal antibody to I-A gene products of the immune response gene complex attenuates both humoral and cellular responses to acetylcholine receptor and appears to suppress clinical manifestations of experimental autoimmune myasthenia gravis. This demonstrates that use of antibodies against immune response gene products that are associated with susceptibility to disease may be feasible for therapy in autoimmune conditions such as myasthenia gravis.

  3. Isolation of Highly Active Monoclonal Antibodies against Multiresistant Gram-Positive Bacteria

    PubMed Central

    Rossmann, Friederike S.; Laverde, Diana; Kropec, Andrea; Romero-Saavedra, Felipe; Meyer-Buehn, Melanie; Huebner, Johannes

    2015-01-01

    Multiresistant nosocomial pathogens often cause life-threatening infections that are sometimes untreatable with currently available antibiotics. Staphylococci and enterococci are the predominant Gram-positive species associated with hospital-acquired infections. These infections often lead to extended hospital stay and excess mortality. In this study, a panel of fully human monoclonal antibodies was isolated from a healthy individual by selection of B-cells producing antibodies with high opsonic killing against E. faecalis 12030. Variable domains (VH and VL) of these immunoglobulin genes were amplified by PCR and cloned into an eukaryotic expression vector containing the constant domains of a human IgG1 molecule and the human lambda constant domain. These constructs were transfected into CHO cells and culture supernatants were collected and tested by opsonophagocytic assay against E. faecalis and S. aureus strains (including MRSA). At concentrations of 600 pg/ml, opsonic killing was between 40% and 70% against all strains tested. Monoclonal antibodies were also evaluated in a mouse sepsis model (using S. aureus LAC and E. faecium), a mouse peritonitis model (using S. aureus Newman and LAC) and a rat endocarditis model (using E. faecalis 12030) and were shown to provide protection in all models at a concentration of 4 μg/kg per animal. Here we present a method to produce fully human IgG1 monoclonal antibodies that are opsonic in vitro and protective in vivo against several multiresistant Gram-positive bacteria. The monoclonal antibodies presented in this study are significantly more effective compared to another monoclonal antibody currently in clinical trials. PMID:25706415

  4. The clinical application of monoclonal antibodies in chronic lymphocytic leukemia

    PubMed Central

    Jaglowski, Samantha M.; Alinari, Lapo; Lapalombella, Rosa; Muthusamy, Natarajan

    2010-01-01

    Chronic lymphocytic leukemia (CLL) represents the most prevalent adult leukemia. Treatment with chemotherapy over the past 3 decades has been palliative. The introduction of therapeutic antibodies has increased the number of treatment options for this disease. Despite this increase, our true understanding of the mechanism of action of antibody therapy in CLL remains limited. Rituximab, a CD20 antibody, is currently widely used in combination-based strategies for both previously untreated symptomatic CLL and as salvage therapy. Recent data suggest that the addition of rituximab to fludarabine with or without cyclophosphamide prolongs survival in younger patients with CLL. Other improved CD20 antibodies with promising clinical activity, including ofatumumab and GA-101, are coming forward. Alemtuzumab, a CD52 antibody, likewise has demonstrated benefit in both symptomatic, previously untreated CLL and in patients with relapsed disease but has less selectivity. Development of other therapeutic antibodies targeting alternative B-cell–specific antigens in CLL has been less successful, although many promising candidate antibodies and/or small modular immune pharmaceuticals (SMIPs) are coming forward. In addition, recent efforts to combine currently applied therapeutic antibodies with other biologic and targeted therapies with efficacy in CLL offers the potential to move toward alternative non–chemotherapy-based treatment approaches. PMID:20610811

  5. Review of Cisplatin and Oxaliplatin in Current Immunogenic and Monoclonal Antibody Treatments

    PubMed Central

    Mehmood, Rao Khalid

    2014-01-01

    Platinum-based chemotherapy agents initially transformed cancer treatment. However their effectiveness peaked as combined regimes showed little additional benefit in trials. New research frontiers developed with the discovery that conventional chemotherapy can induce immunological cell death by recruiting high mobility group box 1 protein through T-cell immunity. Simultaneously monoclonal antibody agents (not effective as monotherapies) showed good results in combination with conventional chemotherapy. Some of these combinations are currently in use and researchers hope to develop regimes which can offer substantial benefits. Several resistance mechanisms against platinum compounds are known, but more knowledge is still needed to gain a full understanding. It seems reasonable therefore to revisit the pharmacology of these agents, which may also lead to identify rational combinations with monoclonal agents providing regimes with less toxicity and better efficacy. This article reviews the pharmacology of cisplatin and oxaliplatin and explores their possible association with monoclonal antibody treatments. PMID:25992242

  6. Harnessing the immune system's arsenal: producing human monoclonal antibodies for therapeutics and investigating immune responses.

    PubMed

    Sullivan, Meghan; Kaur, Kaval; Pauli, Noel; Wilson, Patrick C

    2011-01-01

    Monoclonal antibody technology has undergone rapid and innovative reinvention over the last 30 years. Application of these technologies to human samples revealed valuable therapeutic and experimental insights. These technologies, each with their own benefits and flaws, have proven indispensable for immunological research and in our fight to provide new treatments and improved vaccines for infectious disease. PMID:21876728

  7. Monoclonal antibodies to synthetic pyrethroids and method for detecting the same

    DOEpatents

    Stanker, L.H.; Vanderlaan, M.; Watkins, B.E.; Van Emon, J.M.; Bigbee, C.L.

    1992-04-28

    Methods are described for making specific monoclonal antibodies which may be used in a sensitive immunoassay for detection of synthetic pyrethroids in foods and environmental samples. Appropriate sample preparation and enzyme amplification of the immunoassay for this widely-used class of pesticides permits detection at low levels in laboratory and field tested samples. 6 figs.

  8. Monoclonal antibodies to synthetic pyrethroids and method for detecting the same

    DOEpatents

    Stanker, Larry H.; Vanderlaan, Martin; Watkins, Bruce E.; Van Emon, Jeanette M.; Bigbee, Carolyn L.

    1992-01-01

    Methods are described for making specific monoclonal antibodies which may be used in a sensitive immunoassay for detection of synthetic pyrethroids in foods and environmental samples. Appropriate sample preparation and enzyme amplification of the immunoassay for this widely-used class of pesticides permits detection at low levels in laboratory and field tested samples.

  9. Correlation of monoclonal and polyclonal somatostatin receptor 5 antibodies in pancreatic neuroendocrine tumors

    PubMed Central

    Kaemmerer, Daniel; Lupp, Amelie; Peter, Luisa; Fischer, Elke; Schulz, Stefan; Klöppel, Günter; Hommann, Merten

    2013-01-01

    Aims: To evaluate the frequency of somatostatin-receptor 5 (SSTR 5) in pancreatic neuroendocrine tumors by using monoclonal and polyclonal antibodies. Material and Method: we analyzed 66 proven pancreatic neuroendocrine tumors immunohistochemically with monoclonal (clone UMB-4) and polyclonal SSTR 5-antibodies. Immunoreactive score (IRS) and DAKO-score Her2/neu were evaluated. Results: Immunohistochemistry analysis demonstrated for the IRS a significant higher staining of all specimen using the monoclonal antibodies ( IRS SSTR5 poly vs IRS SSTR 5 mono; 20.0% vs 30.3% p < 0.001) by a correlation of 0.21; p = 0.04. For the HER2 score there was also a significant higher staining in the monoclonal group (Her2 SSTR 5 poly vs Her2 SSTR 5 mono; 21.5% vs 28.8% p < 0.001) by a correlation of 0.20; p = 0.08. Conclusion: Both antibodies are useful in staining of SSTR, although UMB-4 demonstrated a 10% higher SSTR 5 staining. Due to the previous underestimated expression rate of SSTR 5, current standards in diagnostics and therapy should be reconsidered. The increasing usage of long-acting pansomatostatin receptor analogues will rise the adverse effects connected to SSTR5 binding. PMID:23236542

  10. Characterization and application of monoclonal antibodies against Shewanella marisflavi, a novel pathogen of Apostichopus japonicus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shewanella marisflavi strain AP629 was certified as a novel pathogen of the sea cucumber Apostichopus japonicus. In this study, four monoclonal antibodies (MAbs) (3C1, 3D9, 2F2, 2A8) against strain AP629 were developed by immunizing Balb/C mice. 3C1 and 3D9 recognized S. marisflavi only, showing no ...

  11. Development of highly specific monoclonal antibodies for the diagnosis of Vibrio cholerae 01.

    PubMed

    Castillo, L; Castillo, D; Silva, W; Zapata, L; Reid, M; Ulloa, M T; Seoane, M; Maldonado, A; Valenzuela, M E; Bustos, R

    1995-06-01

    We report here the development of two monoclonal antibodies, termed 5G8 and 5C12, belonging to the IgM and IgG1 class, respectively, suitable for the identification of Vibrio cholerae 01 in clinical and environmental samples. The specificities of the monoclonals were evaluated by ELISA and indirect immunofluorescent microscopy of microorganisms normally present in stool samples and with two bacterial panels. One panel included 72 potentially antigenically related bacterial strains and the second panel included 20 pathogenic bacterial strains involved in diarrhea cases. The results of these extensive analyses indicate that monoclonal antibodies 5G8 and 5C12 are highly specific and suitable for the clinical diagnosis of Vibrio cholerae 01 in human stool samples by indirect immunofluorescent microscopy. Although the antigenic sites recognized by these antibodies were not identified in this study, the observation of Western blot patterns suggested that 5G8 and 5C12 monoclonal antibodies bind to LPS epitopes, a good structural marker for the detection of V. cholerae 01 because it is present in all bacterial cell walls. PMID:7590791

  12. Ultrastructural study of Chlamydia trachomatis surface antigens by immunogold staining with monoclonal antibodies.

    PubMed Central

    Kuo, C C; Chi, E Y

    1987-01-01

    Surface antigens of Chlamydia trachomatis were studied by immunogold staining with monoclonal antibodies and by electron microscopy. The serovar- and subspecies-specific epitopes were the most surface accessible. The species- and genus-specific epitopes were the least surface exposed. Similar serological specificity as that in the microimmunofluorescence test was demonstrated by immunogold staining. Images PMID:2437035

  13. Characterization of anti-channel catfish MHC class II monoclonal antibodies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study characterizes four monoclonal antibodies (mAb) developed against the major histocompatibility complex (MHC) class II beta chain of the channel catfish, Ictalurus punctatus. Immunoprecipitations using catfish clonal B cells revealed that each of these mAbs immunoselected proteins of appro...

  14. ANTIGEN DETECTION WITH MONOCLONAL ANTIBODIES FOR THE DIAGNOSIS OF ADENOVIRUS GASTROENTERITIS

    EPA Science Inventory

    The authors have developed a monoclonal antibody-based enzyme immunoassay (EIA) for direct detection of enteric adenoviruses in stool specimens from patients with gastroenteritis. Tests specific for each of the enteric adenoviruses, adenovirus type 40 (Ad40) and type 41 (Ad41) we...

  15. Production, characterization and application of monoclonal antibody to spherulocytes: A subpopulation of coelomocytes of Apostichopus japonicus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    One monoclonal antibody (mAb 3F6) against coelomocytes of sea cucumber Apostichopus japonicus was developed by immunization of Balb/C mice. Analyzed by indirect immunofluorescence assay test (IIFAT), immunocytochemical assay (ICA),Western blotting and fluorescence-activated cell sorter (FACS), mAb 3...

  16. Survey of citrus tristeza virus populations in Central California that react with MCA13 monoclonal antibody

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Citrus Pest Detection Program (CPDP) of the Central California Tristeza Eradication Agency monitors Citrus tristeza virus (CTV) in Central California. MCA13 is a severe strain discriminating monoclonal antibody used to screen for potentially virulent CTV isolates. MCA13-reactive CTV isolates are...

  17. Modulation of p36 phosphorylation in human cells: studies using anti-p36 monoclonal antibodies.

    PubMed Central

    Isacke, C M; Trowbridge, I S; Hunter, T

    1986-01-01

    We have characterized two monoclonal antibodies which recognize human p36. These have been used to examine the sites and extent of serine and tyrosine phosphorylation of p36 in human cells treated with epidermal growth factor and platelet-derived growth factor and in human cells transformed with viruses whose oncogenes encode protein-tyrosine kinases. Images PMID:2946941

  18. Development of human neutralizing monoclonal antibodies for prevention and therapy of MERS-CoV infections

    PubMed Central

    Ying, Tianlei; Li, Haoyang; Lu, Lu; Dimitrov, Dimiter S; Jiang, Shibo

    2014-01-01

    The recent Middle East respiratory syndrome coronavirus (MERS-CoV) outbreak poses a serious threat to public health. Here, we summarize recent advances in identifying human neutralizing monoclonal antibodies (mAbs) against MERS-CoV, describe their mechanisms of action, and analyze their potential for treatment of MERS-CoV infections. PMID:25456101

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  20. INITIAL CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST THE FUNGAL HEMOLYSIN STACHYLYSIN FROM STACHYBOTRYS CHARTARUM

    EPA Science Inventory

    Stachybotrys chartarum is known to produce the hemolysin stachylysin and its detection in human serum has been proposed as a biomarker for exposure to the fungus. In this study we report the initial characterization of monoclonal antibodies (mAbs) against stachylysin and the dev...

  1. Monoclonal antibodies targeting CD38 in hematological malignancies and beyond.

    PubMed

    van de Donk, Niels W C J; Janmaat, Maarten L; Mutis, Tuna; Lammerts van Bueren, Jeroen J; Ahmadi, Tahamtan; Sasser, A Kate; Lokhorst, Henk M; Parren, Paul W H I

    2016-03-01

    CD38 is a multifunctional cell surface protein that has receptor as well as enzyme functions. The protein is generally expressed at low levels on various hematological and solid tissues, while plasma cells express particularly high levels of CD38. The protein is also expressed in a subset of hematological tumors, and shows especially broad and high expression levels in plasma cell tumors such as multiple myeloma (MM). Together, this triggered the development of various therapeutic CD38 antibodies, including daratumumab, isatuximab, and MOR202. Daratumumab binds a unique CD38 epitope and showed strong anti-tumor activity in preclinical models. The antibody engages diverse mechanisms of action, including complement-dependent cytotoxicity, antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis, programmed cell death, modulation of enzymatic activity, and immunomodulatory activity. CD38-targeting antibodies have a favorable toxicity profile in patients, and early clinical data show a marked activity in MM, while studies in other hematological malignancies are ongoing. Daratumumab has single agent activity and a limited toxicity profile, allowing favorable combination therapies with existing as well as emerging therapies, which are currently evaluated in the clinic. Finally, CD38 antibodies may have a role in the treatment of diseases beyond hematological malignancies, including solid tumors and antibody-mediated autoimmune diseases. PMID:26864107

  2. Pharmacokinetics of internally labeled monoclonal antibodies as a gold standard: comparison of biodistribution of /sup 75/Se-, /sup 111/In-, and /sup 125/I-labeled monoclonal antibodies in osteogenic sarcoma xenografts in nude mice

    SciTech Connect

    Koizumi, M.; Endo, K.; Watanabe, Y.; Saga, T.; Sakahara, H.; Konishi, J.; Yamamuro, T.; Toyama, S.

    1989-04-01

    In order to know the true biodistribution of anti-tumor monoclonal antibodies, three monoclonal antibodies (OST6, OST7, and OST15) against human osteosarcoma and control antibody were internally labeled with 75Se by incubating (75Se)methionine and hybridoma cells. 75Se-labeled monoclonal antibodies were evaluated both in vitro and in vivo using the human osteogenic sarcoma cell line KT005, and the results were compared with those of 125I- and 111In-labeled antibodies. 75Se-, 125I- and 111In-labeled monoclonal antibodies had identical binding activities to KT005 cells, and the immunoreactivity was in the decreasing order of OST6, OST7, and OST15. On the contrary, in vivo tumor uptake (% injected dose/g) of 75Se- and 125I-labeled antibodies assessed using nude mice bearing human osteosarcoma KT005 was in the order of OST7, OST6, and OST15. In the case of 111In, the order was OST6, OST7, and OST15. High liver uptake was similarly seen with 75Se- and 111In-labeled antibodies, whereas 125I-labeled antibodies showed the lowest tumor and liver uptake. These data indicate that tumor targeting of antibody conjugates are not always predictable from cell binding studies due to the difference of blood clearance of labeled antibodies. Furthermore, biodistribution of both 111In- and 125I-labeled antibodies are not identical with internally labeled antibody. Admitting that internally labeled antibody is a ''gold standard'' of biodistribution of monoclonal antibody, high liver uptake of 111In-radiolabeled antibodies may be inherent to antibodies. Little, if any, increase in tumor-to-normal tissue ratios of antibody conjugates will be expected compared to those of 111In-labeled antibodies if stably coupled conjugates are administered i.v.

  3. Prevalence, specificity and functionality of anti-ganglioside antibodies in neuropathy associated with IgM monoclonal gammopathy.

    PubMed

    Stork, Abraham C J; Jacobs, Bart C; Tio-Gillen, Anne P; Eurelings, Marijke; Jansen, Marc D; van den Berg, Leonard H; Notermans, Nicolette C; van der Pol, W-Ludo

    2014-03-15

    IgM antibodies against gangliosides and their complexes were studied in sera from 54 patients with polyneuropathy and IgM monoclonal gammopathy (IgM-PNP) without anti-MAG antibodies. Anti-ganglioside antibodies were found in 19 (35%) patients. Five (9%) patients had antibodies against ganglioside complexes. IgM antibodies against gangliosides activated complement in vitro. Light chain usage was restricted to kappa or lambda in most, but not all patients. In conclusion, anti-ganglioside antibodies in IgM-PNP are common, display pathogenic properties and do not always arise from a monoclonal B cell proliferation. PMID:24529728

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

    SciTech Connect

    Miller, M.R.; Heyneman, C.; Walker, S.; Ulrich, R.G.

    1986-03-01

    Although antibodies directed against bromodeoxyuridine (BrdU) are being used in both clinical and basic research laboratories as tools to study and monitor DNA synthesis, little is known about the epitopes with which they react. Four monoclonal antibodies directed against BrdU were produced and were characterized to learn more about the epitopes on BrdU which are important for antibody recognition, to identify compounds other than BrdU which react with the antibodies and which might interfere with immunologic assays for BrdU, and to characterize the reaction of these antibodies with BrdU-containing DNA. By radioimmunoassays, the antibodies generally reacted well with 5-iododeoxyuridine, 5-fluorodeoxyuridine, and 5-nitrouracil. However, none of the antibodies reacted well with uridine - indicating that a substituent on uridine C5 was essential for antibody reactivity - or with 5-bromo or iodo-cytosine, indicating that the region around pyrimidine C4 is important for antibody recognition. Although the antibodies reacted with 5-halogen-substituted uracil bases, the antibodies reacted much better with the corresponding halogenated nucleosides, indicating that the sugar moiety was important for recognition. The presence of a triphosphate group of C'5 of BrdU (i.e., BrdUTP) did not detectably alter antibody recognition. S/sub 1/ nuclease treatment of purified DNA suggested that all four monoclonal antibodies reacted exclusively with single-stranded regions of BrdU-containing DNA. Comparison of detecting DNA synthesis by (/sup 3/H)TdR incorporation followed by autoradiography with that by BrdU incorporation followed by indirect immunofluorescence indicated that the latter technique was both an accurate and a sensitive measure of DNA synthesis.

  5. [The protective activity of polyclonal and monoclonal antibodies to the lipopolysaccharide of Neisseria meningitidis serogroup A in in vivo experiments].

    PubMed

    Del'vig, A A; Krasnoproshina, L I; Volgareva, G M; Bobyleva, G V; Kuvakina, V I; Artem'eva, T A

    1990-10-01

    The protective activity of the sera of mice immunized with the preparations of native and detoxified N. meningitidis lipopolysaccharide (LPS), group A, as well as with monoclonal antibodies to N. meningitidis antigens, groups A and B, was studied on the mucin model of meningococcal infection. The study showed that the maximum level of anti-LPS antibodies in mice was observed on day 7 after the injection of LPS. Immune sera obtained from mice were capable of protecting the animals from fetal meningococcemia induced by N. meningitidis strains of homologous and heterologous groups. As shown by the results of this study, the alkaline treatment of N. meningitidis native LPS did not decrease the protective properties of antibodies. The monoclonal antibodies under study were found to possess high preventive activity in mice challenged with N. meningitidis, groups A and B. Anti-LPS monoclonal antibodies showed greater protective activity than antipolysaccharide monoclonal antibodies. PMID:2127501

  6. Mice are actively immunized after passive monoclonal antibody prophylaxis and ricin toxin challenge. (Reannouncement with new availability information)

    SciTech Connect

    Lemley, P.V.; Wright, D.C.

    1992-12-31

    Mice passively immunized by a protective, anti-ricin A-chain monoclonal antibody, then challenged intravenously with ricin, were protected from a subsequent ricin challenge, and were actively immunized. Two significant advantages accrued from this experiment: the monoclonal antibody neutralized the toxicity of the ricin immunogen, and active immunization was achieved with very low antigen load (approx. 0.5 micrograms/mouse). We ruled out the possibility that residual monoclonal antibody provided the protection by using three independent criteria. There was significant (four orders of magnitude) enhancement of the immune response in the presence of the monoclonal antibody; control immunizations of mice with ricin A-chain, ricin B-chain or either chain with the monoclonal antibody did not induce active immunity; and the active immunization could not be replicated when protective goat polyclonal antibody was substituted for the monoclonal antibody. Because high titers were achieved rapidly without any adjuvant, we are currently investigating haptenized ricin to determine if anti-hapten monoclonal antibodies can be produced by this refined procedure.

  7. Discovery and characterization of antibody variants using mass spectrometry-based comparative analysis for biosimilar candidates of monoclonal antibody drugs.

    PubMed

    Li, Wenhua; Yang, Bin; Zhou, Dongmei; Xu, Jun; Ke, Zhi; Suen, Wen-Chen

    2016-07-01

    Liquid chromatography mass spectrometry (LC-MS) is the most commonly used technique for the characterization of antibody variants. MAb-X and mAb-Y are two approved IgG1 subtype monoclonal antibody drugs recombinantly produced in Chinese hamster ovary (CHO) cells. We report here that two unexpected and rare antibody variants have been discovered during cell culture process development of biosimilars for these two approved drugs through intact mass analysis. We then used comprehensive mass spectrometry-based comparative analysis including reduced light, heavy chains, and domain-specific mass as well as peptide mapping analysis to fully characterize the observed antibody variants. The "middle-up" mass comparative analysis demonstrated that the antibody variant from mAb-X biosimilar candidate was caused by mass variation of antibody crystalline fragment (Fc), whereas a different variant with mass variation in antibody antigen-binding fragment (Fab) from mAb-Y biosimilar candidate was identified. Endoproteinase Lys-C digested peptide mapping and tandem mass spectrometry analysis further revealed that a leucine to glutamine change in N-terminal 402 site of heavy chain was responsible for the generation of mAb-X antibody variant. Lys-C and trypsin coupled non-reduced and reduced peptide mapping comparative analysis showed that the formation of the light-heavy interchain trisulfide bond resulted in the mAb-Y antibody variant. These two cases confirmed that mass spectrometry-based comparative analysis plays a critical role for the characterization of monoclonal antibody variants, and biosimilar developers should start with a comprehensive structural assessment and comparative analysis to decrease the risk of the process development for biosimilars. PMID:27214604

  8. Monoclonal antibodies: Principles and applications of immmunodiagnosis and immunotherapy for hepatitis C virus

    PubMed Central

    Tabll, Ashraf; Abbas, Aymn T; El-Kafrawy, Sherif; Wahid, Ahmed

    2015-01-01

    Hepatitis C virus (HCV) is a major health problem worldwide. Early detection of the infection will help better management of the infected cases. The monoclonal antibodies (mAb) of mice are predominantly used for the immunodiagnosis of several viral, bacterial, and parasitic antigens. Serological detection of HCV antigens and antibodies provide simple and rapid methods of detection but lack sensitivity specially in the window phase between the infection and antibody development. Human mAb are used in the immunotherapy of several blood malignancies, such as lymphoma and leukemia, as well as for autoimmune diseases. In this review article, we will discuss methods of mouse and human monoclonal antibody production. We will demonstrate the role of mouse mAb in the detection of HCV antigens as rapid and sensitive immunodiagnostic assays for the detection of HCV, which is a major health problem throughout the world, particularly in Egypt. We will discuss the value of HCV-neutralizing antibodies and their roles in the immunotherapy of HCV infections and in HCV vaccine development. We will also discuss the different mechanisms by which the virus escape the effect of neutralizing mAb. Finally, we will discuss available and new trends to produce antibodies, such as egg yolk-based antibodies (IgY), production in transgenic plants, and the synthetic antibody mimics approach. PMID:26464752

  9. Biodistribution and dosimetry of 3F8 neuroblastoma monoclonal antibody

    SciTech Connect

    Nelson, A.D.; Miraldi, F.; Cheung, N.K. )

    1989-01-01

    A method has been developed for quantitating radiolabeled antibody concentrations from images obtained with standard gamma cameras. The method is based on orthogonal projections and accounts both for the effective attenuation of gamma rays and the finite depth dependent resolution of a gamma camera. The method was verified in experimental phantoms and subsequently used in patient studies to quantitate radiolabeled antibody concentrations in neuroblastoma tumors. The in vivo measurements of tumor radioactivity levels were confirmed at biopsy in one patient.

  10. Inline protein A mass spectrometry for characterization of monoclonal antibodies.

    PubMed

    Prentice, Kenneth M; Wallace, Alison; Eakin, Catherine M

    2015-02-17

    Purification of antibodies is an important first step to produce material for in depth characterization of biotherapeutics. To reduce the resource burden incurred by protein purification, we developed a high throughput protein A affinity capture step coupled to inline mass spectrometry (PrA-MS). Our method enables both UV quantitation of antibodies and product characterization of an intact molecule with microgram quantities of material. When purification and analysis are coupled along with the low material demand, PrA-MS is widely applicable to protein characterization and is uniquely advantageous for moieties that rely on molecular stoichiometry. Two model systems were studied using PrA-MS and are presented here: (a) bispecific antibodies (bsAb) and (b) glycan engineered antibodies. In the bsAb samples, hetero- and homodimer species, along with partial molecule, were readily identified and quantified directly from harvested cell culture fluid (HCCF). In the glycan engineered antibodies, fully afucosylated, as well as asymmetrically and symmetrically fucosylated, glycans were identified from HCCF in experiments that utilized a small molecule inhibitor of fucosyltrasferase. The PrA-MS method represents a high throughput alternative to offline purification and product characterization that may be leveraged across the product lifecycle of engineered antibodies to enable rapid development and production of important therapeutics. PMID:25647041

  11. Cathepsin B-deficient mice as source of monoclonal anti-cathepsin B antibodies.

    PubMed

    Weber, Ekkehard; Barbulescu, Elena; Medek, Rita; Reinheckel, Thomas; Sameni, Mansoureh; Anbalagan, Arulselvi; Moin, Kamiar; Sloane, Bonnie F

    2015-03-01

    Cathepsin B has been demonstrated to be involved in several proteolytic processes that support tumor progression and metastasis and neurodegeneration. To further clarify its role, defined monoclonal antibodies are needed. As the primary structure of human cathepsin B is almost identical to that of the mouse, cathepsin B-deficient mice were used in a novel approach for generating such antibodies, providing the chance of an increased immune response to the antigen, human cathepsin B. Thirty clones were found to produce cathepsin B-specific antibodies. Seven of these antibodies were used to detect cathepsin B in MCF10-DCIS human breast cancer cells by immunocytochemistry and immunoblotting. Five different binding sites were identified by epitope mapping giving the opportunity to combine these antibodies in oligoclonal antibody mixtures for an improved detection of cathepsin B. PMID:25205719

  12. Development of VHH Antibodies against Dengue Virus Type 2 NS1 and Comparison with Monoclonal Antibodies for Use in Immunological Diagnosis

    PubMed Central

    Fatima, Aneela; Wang, Haiying; Kang, Keren; Xia, Liliang; Wang, Ying; Ye, Wei; Wang, Jufang; Wang, Xiaoning

    2014-01-01

    The possibility of using variable domain heavy-chain antibodies (VHH antibodies) as diagnostic tools for dengue virus (DENV) type 2 NS1 protein was investigated and compared with the use of conventional monoclonal antibodies. After successful expression of DENV type 2 NS1 protein, the genes of VHH antibodies against NS1 protein were biopanned from a non-immune llama library by phage display. VHH antibodies were then expressed and purified from Escherichia coli. Simultaneously, monoclonal antibodies were obtained by the conventional route. Sequence analysis of the VHH antibodies revealed novel and long complementarity determining regions 3 (CDR3). Epitope mapping was performed via a phage display peptide library using purified VHH and monoclonal antibodies as targets. Interestingly, the same region of NS1, which comprises amino acids 224HWPKPHTLW232, was conserved for both kinds of antibodies displaying the consensus motif histidine-tryptophan-tryptophan or tryptophan-proline-tryptophan. The two types of antibodies were used to prepare rapid diagnostic kits based on immunochromatographic assay. The VHH antibody immobilized rapid diagnostic kit showed better sensitivity and specificity than the monoclonal antibody immobilized rapid diagnostic kit, which might be due to the long CDR3 regions of the VHH antibodies and their ability to bind to the pocket and cleft of the targeted antigen. This demonstrates that VHH antibodies are likely to be an option for developing point-of-care tests against DENV infection. PMID:24751715

  13. Understanding the Cellular Function of TRPV2 Channel through Generation of Specific Monoclonal Antibodies

    PubMed Central

    Cohen, Matthew R.; Huynh, Kevin W.; Cawley, Daniel; Moiseenkova-Bell, Vera Y.

    2013-01-01

    Transient receptor potential vanilloid 2 (TRPV2) is a Ca2+-permeable nonselective cation channel proposed to play a critical role in a wide array of cellular processes. Although TRPV2 surface expression was originally determined to be sensitive to growth factor signaling, regulated trafficking of TRPV2 has remained controversial. TRPV2 has proven difficult to study due to the lack of specific pharmacological tools to modulate channel activity; therefore, most studies of the cellular function of TRPV2 rely on immuno-detection techniques. Polyclonal antibodies against TRPV2 have not been properly validated and characterized, which may contribute to conflicting results regarding its function in the cell. Here, we developed monoclonal antibodies using full-length TRPV2 as an antigen. Extensive characterization of these antibodies and comparison to commonly used commercially available TRPV2 antibodies revealed that while monoclonal antibodies generated in our laboratory were suitable for detection of endogenous TRPV2 by western blot, immunoprecipitation and immunocytochemistry, the commercially available polyclonal antibodies we tested were not able to recognize endogenous TRPV2. We used our newly generated and validated TRPV2 antibodies to determine the effects of insulin-like growth factor 1 (IGF-1) on TRPV2 surface expression in heterologous and endogenous expression systems. We found that IGF-1 had little to no effect on trafficking and plasma membrane expression of TRPV2. Overall, these new TRPV2 monoclonal antibodies served to dispel the controversy of the effects of IGF-1 on TRPV2 plasma membrane expression and will clarify the role TRPV2 plays in cellular function. Furthermore, our strategy of using full-length tetrameric TRP channels may allow for the generation of antibodies against other TRP channels of unclear function. PMID:24392006

  14. Clinical efficacy and management of monoclonal antibodies targeting CD38 and SLAMF7 in multiple myeloma.

    PubMed

    van de Donk, Niels W C J; Moreau, Philippe; Plesner, Torben; Palumbo, Antonio; Gay, Francesca; Laubach, Jacob P; Malavasi, Fabio; Avet-Loiseau, Hervé; Mateos, Maria-Victoria; Sonneveld, Pieter; Lokhorst, Henk M; Richardson, Paul G

    2016-02-11

    Immunotherapeutic strategies are emerging as promising therapeutic approaches in multiple myeloma (MM), with several monoclonal antibodies in advanced stages of clinical development. Of these agents, CD38-targeting antibodies have marked single agent activity in extensively pretreated MM, and preliminary results from studies with relapsed/refractory patients have shown enhanced therapeutic efficacy when daratumumab and isatuximab are combined with other agents. Furthermore, although elotuzumab (anti-SLAMF7) has no single agent activity in advanced MM, randomized trials in relapsed/refractory MM have demonstrated significantly improved progression-free survival when elotuzumab is added to lenalidomide-dexamethasone or bortezomib-dexamethasone. Importantly, there has been no significant additive toxicity when these monoclonal antibodies are combined with other anti-MM agents, other than infusion-related reactions specific to the therapeutic antibody. Prevention and management of infusion reactions is important to avoid drug discontinuation, which may in turn lead to reduced efficacy of anti-MM therapy. Therapeutic antibodies interfere with several laboratory tests. First, interference of therapeutic antibodies with immunofixation and serum protein electrophoresis assays may lead to underestimation of complete response. Strategies to mitigate interference, based on shifting the therapeutic antibody band, are in development. Furthermore, daratumumab, and probably also other CD38-targeting antibodies, interfere with blood compatibility testing and thereby complicate the safe release of blood products. Neutralization of the therapeutic CD38 antibody or CD38 denaturation on reagent red blood cells mitigates daratumumab interference with transfusion laboratory serologic tests. Finally, therapeutic antibodies may complicate flow cytometric evaluation of normal and neoplastic plasma cells, since the therapeutic antibody can affect the availability of the epitope for binding

  15. New p22-Phox Monoclonal Antibodies: Identification of a Conformational Probe for Cytochrome b558

    PubMed Central

    Campion, Yannick; Jesaitis, Algirdas J.; Nguyen, Minh Vu Chuong; Grichine, Alexei; Herenger, Yvan; Baillet, Athan; Berthier, Sylvie; Morel, Françoise; Paclet, Marie-Hélène

    2009-01-01

    The phagocyte NADPH oxidase, belonging to the NADPH oxidase family (Nox), is dedicated to the production of bactericidal reactive oxygen species. The enzyme catalytic center is the cytochrome b558, formed by 2 subunits, Nox2 (gp91-phox) and p22-phox. Cytochrome b558 activation results from a conformational change induced by cytosolic regulatory proteins (p67-phox, p47-phox, p40-phox and Rac). The catalytic subunit is Nox2, while p22-phox is essential for both Nox2 maturation and the membrane anchorage of regulatory proteins. Moreover, it has been shown to be necessary for novel Nox activity. In order to characterize both p22-phox topology and cytochrome b558 conformational change, 6 monoclonal antibodies were produced against purified cytochrome b558. Phage display epitope mapping combined with a truncation analysis of recombinant p22-phox allowed the identification of epitope regions. Some of these antibodies almost completely inhibited in vitro reconstituted NADPH oxidase activity. Data analysis identified antibodies that recognized epitopes involved in either Nox2 maturation or Nox2 activation. Moreover, flow cytometry analysis and confocal microscopy performed on stimulated neutrophils showed that the monoclonal antibody 12E6 bound preferentially active cytochrome b558. These monoclonal antibodies provided novel and unique probes to investigate maturation, activation and activity, not only of Nox2 but also of novel Nox. PMID:20375611

  16. Common epitope on the lipopolysaccharide of Legionella pneumophila recognized by a monoclonal antibody.

    PubMed Central

    Barthe, C; Joly, J R; Ramsay, D; Boissinot, M; Benhamou, N

    1988-01-01

    Serogroup-specificity of Legionella pneumophila is related to lipopolysaccharide (LPS), and few cross-reactions between serogroups have been observed with rabbit or monkey antisera. C57BL/6 mice were sequentially immunized with crude outer membrane fractions of L. pneumophila serogroups 1, 5, and 7, Legionella bozemanii, and Legionella micdadei. Spleen cells from these mice were then fused with the Sp2-0/Ag14 mouse myeloma cell line. Outer membrane-rich fractions and LPS were prepared from L. pneumophila serogroups 1 to 8 and other Legionella and non-Legionella species. Immunoblots of these extracts were performed with monoclonal antibody obtained from these fusions. One of these monoclonal antibodies recognized an epitope common to all tested serogroups of L. pneumophila and attached to the major constituent of the outer membrane, LPS. This antibody did not react with other Legionella species and numerous gram-negative rods other than Pseudomonas fluorescens CDC93. This monoclonal antibody may be useful in preliminary identification of L. pneumophila as an alternative to direct fluorescent-antibody testing. Images PMID:2454935

  17. Neutralizing monoclonal antibodies recognize antigenic variants among isolates of infectious hematopoietic necrosis virus

    USGS Publications Warehouse

    Winton, J.R.; Arakawa, C.N.; Lannan, C.N.; Fryer, J.L.

    1988-01-01

    eutralizing monoclonal antibodies were developed against strains of infectious hematopoietic necrosis virus (IHNV) from steelhead trout Salmo gairdneri in the Deschutes River of Oregon, chinook salmon Oncorhynchus tshawytscha in the Sacramento River of California, and rainbow trout Salmo gairdneri reared in the Hagerman Valley of Idaho, USA. These antibodies were tested for neutralization of 12 IHNV isolates obtained from salmonids in Japan, Alaska, Washington, Oregon, California, and Idaho. The antibodies recognized antigenic variants among the isolates and could be used to separate the viruses into 4 groups. The members of each group tended to be related by geographic area rather than by source host species, virulence, or date of isolation.

  18. Kinetic Analysis of Monoclonal Antibody Binding to HIV-1 gp120-derived Hyperglycosylated Cores

    PubMed Central

    Ingale, Jidnyasa; Wyatt, Richard T

    2015-01-01

    Kinetic analysis of antibodies is one of the important study for characterization of antibodies and screening of ligands. In our recent study1, we compared the antigenic profiles and binding characteristics of four HIV-1 envelope glycoprotein (Env) core immunogens using multiple monoclonal antibodies by Bio-Layer Light Interferometry (BLI). This technology enables real-time analysis of interactions on the surface of a fiber optic biosensor by accurately measuring kinetic constants such as Ka, Kd, and KD in a 96-well format. PMID:26855965

  19. Monoclonal antibodies for the separate detection of halodeoxyuridines and method for their use

    DOEpatents

    Vanderlaan, Martin; Watkins, Bruce E.; Stanker, Larry H.

    1991-01-01

    Monoclonal antibodies are described which have specific affinities for halogenated nucleoside analogs and are preferentially selective for one particular halogen. Such antibodies, when incorporated into immunochemical reagents, may be used to identify and independently quantify the cell division character of more than one population or subpopulation in flow cytometric measurements. Independent assessment of division activity in cell sub-populations facilitates selection of appropriate time and dose for administration of anti-proliferative agents. The hybridomas which secrete halogen selective antibodies and the method of making them are described.

  20. Monoclonal antibodies for the separate detection of halodeoxyuridines and method for their use

    DOEpatents

    Vanderlaan, M.; Watkins, B.E.; Stanker, L.H.

    1991-10-01

    Monoclonal antibodies are described which have specific affinities for halogenated nucleoside analogs and are preferentially selective for one particular halogen. Such antibodies, when incorporated into immunochemical reagents, may be used to identify and independently quantify the cell division character of more than one population or subpopulation in flow cytometric measurements. Independent assessment of division activity in cell sub-populations facilitates selection of appropriate time and dose for administration of anti-proliferative agents. The hybridomas which secrete halogen selective antibodies and the method of making them are described. 14 figures.

  1. Dashboard systems: Pharmacokinetic/pharmacodynamic mediated dose optimization for monoclonal antibodies.

    PubMed

    Mould, Diane R; Dubinsky, Marla C

    2015-03-01

    Many marketed drugs exhibit high variability in exposure and response. While these drugs are efficacious in their approved indications, finding appropriate dose regimens for individual patients is not straightforward. Similar dose adjustment problems are also seen with drugs that have a complex relationship between exposure and response and/or a narrow therapeutic window. This is particularly true for monoclonal antibodies, where prolonged dosing at a sub-therapeutic dose can also elicit anti-drug antibodies which will further compromise safety and efficacy. Thus, finding appropriate doses quickly would represent a substantial improvement in healthcare. Dashboard systems, which are decision-support tools, offer an improved, convenient means of tailoring treatment for individual patients. This article reviews the clinical need for this approach, particularly with monoclonal antibodies, the design, development, and testing of such systems, and the likely benefits of dashboard systems in clinical practice. We focus on infliximab for reference. PMID:25707964

  2. New monoclonal-antibody two-site solid-phase immunoradiometric assay for human thyrotropin evaluated

    SciTech Connect

    Pekary, A.E.; Hershman, J.M.

    1984-07-01

    The authors compared results with a commercial solid-phase two-site immunoradiometric assay kit for human thyrotropin in which monoclonal antibodies are used with those by our radioimmunoassay, which is optimized for measurement of low concentrations of thyrotropin. In the immunoradiometric assay a specific antibody to the beta subunit of human thyrotropin is immobilized on a polystyrene bead, and a radiolabeled monoclonal antibody directed against the alpha subunit provides a measure of bead-immobilized hormone. The mean thyrotropin concentrations in 70 euthyroid serum samples were similar in the two assays. Values for hypothyroid patients were clearly higher in both assays than values for euthyroid individuals. In commercial assays the major source of error in measurement of thyrotropin response to thyroliberin in terms of the increment over the basal concentration of thyrotropin has been systematic errors in the measurement of those basal concentrations. With the present assay, however, basal values are obtained with good precision and accuracy.

  3. Radiolabeled anti platelet monoclonal antibody for imaging in-vivo thrombi

    SciTech Connect

    Srivastava, S.C.; Coller, B.S.; Meinken, G.E.

    1993-07-06

    A radiolabeled monoclonal antibody radiolabeled with a halogen or metallic radionuclide selected from the group consisting of [sup 123]I, [sup 125]I, [sup 131]I, [sup 111]In-DTPA, [sup 99]mTc and [sup 97]Ru, wherein the monoclonal antibody is of the class IgG, produced by a hybridoma formed by fusion of cells from a BALB/C mouse myeloma line, X63.Ag8.653 and spleen cells from a BALB/c mouse previously immunized with human blood platelets, which antibody: (a) reacts readily with normal human platelets and with dog platelets; (b) fails to react with thrombasthenic human platelets or human platelets whose GPIIb/IIIa complex is dissociated with EDTA and (c) reacts slowly with inactivated platelets and substantially twice as rapidly with ADP activated platelets; and (d) completely blocks the interaction of fibrinogen with platelets induced by ADP.

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

    SciTech Connect

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

    1988-01-01

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

  5. Expression of CR2/EBV receptors on human thymocytes detected by monoclonal antibodies.

    PubMed

    Tsoukas, C D; Lambris, J D

    1988-08-01

    The biologic effects of the third component of complement, C3, are mediated via receptors which specifically bind the enzymatic degradation products resulting from the cleavage of C3. One of the products, C3d, has been associated with binding to the second complement receptor CR2 (CD21). This receptor, which is identical to the receptor for Epstein-Barr virus (EBV), has been primarily found on cells of the B lineage, but not on mature T cells or other cells of erythroid or myeloid lineages. In the present investigation, we report the presence of CR2 on human thymocytes. Indirect immunofluorescence analysis employing monoclonal anti-CR2 antibodies revealed a range of thymocyte reactivity from 15% to 63% in thirteen experiments using cells of different donors. Reactivity was always greater with the monoclonal anti-CR2 (CD21) antibody HB-5 than with two other antibodies which recognize distinct epitopes on the CR2 molecule. Two-color immunofluorescence analysis indicated that the brightest of the HB-5-stained thymocytes also reacted with the monoclonal anti-CD1 antibody T6 (immature thymocyte marker) while some of the duller HB-5-staining cells reacted with the monoclonal anti-CD3 antibody Leu-4 (mature thymocyte marker). Immunoprecipitation of CR2 on thymocytes with antibody HB-5 and polyacrylamide gel electrophoretic analysis revealed a protein of 145 kDa molecular mass which is consistent with the size of CR2 found on B lymphocytes. These findings raise several questions regarding the biologic role of CR2-EBV receptor on cells of the T lineage. PMID:2970972

  6. Production of mouse monoclonal antibodies which inhibit in vitro adherence of Entamoeba histolytica trophozoites.

    PubMed Central

    Ravdin, J I; Petri, W A; Murphy, C F; Smith, R D

    1986-01-01

    Adherence by axenic Entamoeba histolytica trophozoites to mammalian cells is mediated by an N-acetylgalactosamine (GalNAc)-inhibitable adhesin on the surface of the parasite. We isolated 35 hybridoma cell lines producing antibodies to E. histolytica as indicated by ELISA with sonicated amebic protein or by immunofluorescence assay with fixed whole trophozoites. Tissue culture supernatants were further screened for subcloning by the ability to bind to Chinese hamster ovary (CHO) cells which were first exposed to a partially purified soluble preparation of the amebic GalNAc-inhibitable lectin. Eight tissue culture supernatants were positive in this assay. Antibodies from four subcloned cell lines (D3-14, H8-5, I12-2, and I1-21) inhibited amebic adherence to CHO cells (P less than 0.01). Of the original 35 tissue culture supernatants, 3 also inhibited amebic adherence (P less than 0.01; F1, F14, and J10); monoclonal antibodies in these supernatants did not bind to lectin-exposed CHO cells. Three purified monoclonal antibodies (H8-5, I12-2, and I1-21) inhibited amebic adherence at greater than or equal to 2 micrograms/10(4) amebae (P less than 0.05). None of these inhibitory monoclonal antibodies immunoprecipitated with a soluble amebic protein preparation following sodium dodecyl sulfate-polyacrylamide gel electrophoresis under denaturing conditions. Monoclonal antibodies which inhibit in vitro adherence by E. histolytica will be useful in purification of the GalNAc-inhibitable lectin. PMID:2873102

  7. A technetium-labeled monoclonal antibody for imaging metastatic melanoma

    SciTech Connect

    Frytak, S.; Creagan, E.T.; Brown, M.L.; Salk, D.; Nelp, W. )

    1991-04-01

    Twenty patients with histologically proven metastatic melanoma were scanned with a 99mtechnetium ({sup 99}mTc)-labeled melanoma antibody to determine the detection rate of known malignant lesions and to evaluate the antibody's ability to discover occult metastases. Isotope localization in different organs was as follows: liver 100%, bone 100%, subcutaneous lesions 80%, lymph nodes 54%, and lung 33%. Four unsuspected bone lesions and 16 occult subcutaneous lesions were found. False positive lesions were noted in two instances--one benign thyroid adenoma, and one arthritic bone lesion. One patient developed an atypical serum sickness reaction with a rash and arthralgias that responded rapidly to treatment. The {sup 99}mTc antimelanoma antibody is a safe and effective method to detect metastatic melanoma. It has potential use for screening newly diagnosed melanomas that carry an increased risk of recurrence.

  8. [Influence of different products of platelet membrane glycoprotein monoclonal antibodies used internationally on tests for monoclonal antibody-specific immobilization of platelet antigens].

    PubMed

    Tang, Qiu-Min; Shen, Wei-Dong; Zhong, Zhou-Lin; Zhou, Yan; Wu, Guo-Guang

    2009-08-01

    This study was aimed to investigate the influence of different platelet membrane glycoprotein monoclonal antibodies (McAb) which are common used in laboratories on the monoclonal antibody-specific immobilization of platelet antigens (MAIPA) technique according to the request of 14th International Society of Blood Transfusion Platelet Immunology Workshop. 30 participant laboratories were provided with 10 known human platelet antigen (HPA) antibodies, 1 normal serum, 9 different McAbs (against GPIIb/IIIa, GPIa/IIa, GPIb/IX and GPIV respectively), and the same protocol. Each participant laboratory carried out the test as the protocol to compare the results of different McAbs against the same glycoprotein and submitted the data to organizer. The results indicated that in McAbs against GPIIb/IIIa, AP2, Gi-5 and PL2-73 showed higher mean S/CO than that of others; in GPIa/IIa, MBC202.2 and 143.1 showed higher mean S/CO than that of others; in GPIb/IX, 142.11 and CLB-MB45 (CD42b) showed higher mean S/CO than that of others; as to GPIV, 131.4 showed higher mean S/CO. In conclusion, capture effects of various McAbs are different, so that different products of McAbs exert influences on the sensitivity of MAIPA. To use a panel of McAbs against the same glycoprotein may avoid the false negative results. PMID:19698264

  9. The mechanism of hepatic uptake of a radiolabelled monoclonal antibody.

    PubMed

    Boyle, C C; Paine, A J; Mather, S J

    1992-04-01

    Clinical and experimental scintigraphic studies have found that radiolabelled antibodies are not only taken up by tumour(s) but also by normal liver. The accumulation of radionuclides in this organ poses a major problem to the use of radiolabelled antibodies as diagnostic and therapeutic tools. In an attempt to understand the mechanism of hepatic uptake and clearance of radiolabelled antibodies, the intrahepatic biodistribution of an 111In-labelled MAb (HMFG1), was determined following i.v. administration to normal male rats. Two hours after administration the liver contained 15% of the injected dose, with most of the remaining radioactivity in the blood. The hepatic burden of the 111In MAb remained constant over the next 72 hr in the face of decreasing blood levels of radioactivity as well as its urinary and faecal excretion. At 2 and 72 hr after injection, 50% and 10% respectively of the hepatic radiolabel was due to blood borne antibody. Following a collagenase-cell isolation procedure, only 23% of the amount remaining in the liver at 2 hr was found to be cell-associated; 66% was lost during the cell isolation and purification procedure. Cellular uptake increased with time so that, by 72 hr after administration, 58% was cell-associated and 29% freely removable. At all timepoints, the parenchymal cells contained more activity than non-parenchymal cells. No evidence of antibody-receptor interactions could be obtained either in vivo or in cultures of hepatic parenchymal and non-parenchymal cells. Our data suggest that the bulk of the hepatic burden of 111In MAb results from extravascular pooling of the antibody. PMID:1555890

  10. Monoclonal Antibodies to the Apical Chloride Channel in Necturus Gallbladder Inhibit the Chloride Conductance

    NASA Astrophysics Data System (ADS)

    Finn, Arthur L.; Tsai, Lih-Min; Falk, Ronald J.

    1989-10-01

    Monoclonal antibodies raised by injecting Necturus gallbladder cells into mice were tested for their ability to inhibit the apical chloride conductance induced by elevation of cellular cAMP. Five of these monoclonal antibodies bound to the apical cells, as shown by indirect immunofluorescence microscopy, and inhibited the chloride conductance; one antibody that bound only to subepithelial smooth muscle, by indirect immunofluorescence microscopy, showed no inhibition of chloride transport. The channel or a closely related molecule is present in the membrane whether or not the pathway is open, since, in addition to inhibiting the conductance of the open channel, the antibody also bound to the membrane in the resting state and prevented subsequent opening of the channel. The antibody was shown to recognize, by ELISA, epitopes from the Necturus gallbladder and small intestine. Finally, by Western blot analysis of Necturus gallbladder homogenates, the antibody was shown to recognize two protein bands of Mr 219,000 and Mr 69,000. This antibody should permit isolation and characterization of this important ion channel.

  11. Sequences of 12 monoclonal anti-dinitrophenyl spin-label antibodies for NMR studies

    SciTech Connect

    Leahy, D.J.; Rule, G.S.; Whittaker, M.M.; McConnell, H.M. )

    1988-06-01

    Eleven monoclonal antibodies specific for a spin-labeled dinitrophenyl hapten (DNP-SL) have been produces for use in NMR studies. They have been named AN01 and ANO3-AN12. The stability constants for the association of these antibodies with DNP-SL and related haptens were measured by fluorescence quenching. cDNA clones coding for the heavy and light chains of each antibody and of an additional anti-DNP-SL monoclonal antibody, ANO2, have been isolated. The nucleic acid sequence of the 5{prime} end of each clone has been determined, and the amino acid sequence of the variable regions of each antibody has been deduced from the cDNA sequence. The sequences are relatively heterogeneous, but both the heavy and the light chains of ANO1 and ANO3 are derived from the same variable-region gene families as those of the ANO2 antibody. ANO7 has a heavy chain that is related to that of ANO2, and ANO9 has a related light chain. ANO5 and ANO6 are unrelated to ANO2 but share virtually identical heavy and light chains. Preliminary NMR difference spectra comparing related antibodies show that sequence-specific assignment of resonances is possible. Such spectra also provide a measure of structural relatedness.

  12. Biokinetics of radiolabeled monoclonal antibodies in heterotransplanted nude rats: Evaluation of corrected specific tissue uptake

    SciTech Connect

    Ingvar, C.; Norrgren, K.; Strand, S.E.; Brodin, T.; Joensson, P.E.S.; Sjoegren, H.O. )

    1989-07-01

    A tumor model is presented to study the biokinetics and localization of radiolabeled monoclonal antibodies (MAb) in the nude rat (Rowett RNu/RNu) heterotransplanted with human melanoma metastases. The nude rat is larger, less sensitive, and lives longer than the nude mouse. It is, therefore, well suited for in vivo studies of tumor localization with radiolabeled monoclonal antibodies. The tumor-to-host weight ratio was closer to the human situation for the nude rat than for the mouse, and quantitative imaging could be performed with a parallel hole collimator. We followed the antibody biokinetics for as long as 8 days, with repeated blood sampling and imaging. Specific uptake of MAb was higher in tumor tissue than in all other tissues except blood. Initial high uptake was also recorded in the bone marrow. The lymph glands showed a slow uptake of specific and control antibody. A simple in vitro correction procedure is described to calculate the corrected specific tissue uptake (STUcorr) that takes the blood activity into account. Thus it was shown that 80% of the tissue uptake in the dissected liver at 30 hr was due to labeled antibodies circulating in the blood. The specific tissue uptake ratio of antibodies 96.5 and OKT3 (nonspecific control) was unity for all other organs except for tumor tissue, where the ratio was greater than two and even higher when correction for blood content of labeled antibody was made.

  13. Serrumab: a human monoclonal antibody that counters the biochemical and immunological effects of Tityus serrulatus venom.

    PubMed

    Pucca, Manuela Berto; Zoccal, Karina Furlan; Roncolato, Eduardo Crosara; Bertolini, Thaís Barboza; Campos, Lucas Benício; Cologna, Camila Takeno; Faccioli, Lúcia Helena; Arantes, Eliane Candiani; Barbosa, José Elpidio

    2012-01-01

    In Brazil, the species Tityus serrulatus is responsible for the most severe cases of scorpion envenomation. There is currently a need for new scorpion anti-venoms that are more effective and less harmful. This study attempted to produce human monoclonal antibodies capable of inhibiting the activity of T. serrulatus venom (TsV), using the Griffin.1 library of human single-chain fragment-variable (scFv) phage antibodies. Four rounds of phage antibody selection were performed, and the round with the highest phage antibody titer was chosen for the production of monoclonal phage antibodies and for further analysis. The scFv 2A, designated serrumab, was selected for the production and purification of soluble antibody fragments. In a murine peritoneal macrophage cell line (J774.1), in vitro assays of the cytokines interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IL-10 were performed. In male BALB/c mice, in vivo assays of plasma urea, creatinine, aspartate transaminase, and glucose were performed, as well as of neutrophil recruitment and leukocyte counts. It was found that serrumab inhibited the TsV-induced increases in the production of IL-6, TNFα, and IL-10 in J774.1 cells. The in vivo inhibition assay showed that serrumab also prevented TsV-induced increases in the plasma levels of urea, creatinine, aspartate transaminase, and glucose, as well as preventing the TsV-induced increase in neutrophil recruitment. The results indicate that the human monoclonal antibody serrumab is a candidate for inclusion in a mixture of specific antibodies to the various toxins present in TsV. Therefore, serrumab shows promise for use in the production of new anti-venom. PMID:22424317

  14. Data on the characterization of follicle-stimulating hormone monoclonal antibodies and localization in Japanese eel pituitary.

    PubMed

    Kim, Dae-Jung; Park, Chae-Won; Byambaragchaa, Munkhzaya; Kim, Shin-Kwon; Lee, Bae-Ik; Hwang, Hyung-Kyu; Myeong, Jeong-In; Hong, Sun-Mee; Kang, Myung-Hwa; Min, Kwan-Sik

    2016-09-01

    Monoclonal antibodies were generated against recombinant follicle-stimulating hormone (rec-FSH) from Japanese eel Anguilla japonica; rec-FSH was produced in Escherichia coli and purified using Ni-NTA Sepharose column chromatography. In support of our recent publication, "Production and characterization of monoclonal antibodies against recombinant tethered follicle-stimulating hormone from Japanese eel Anguilla japonica" [1], it was important to characterize the specificity of eel follicle-stimulating hormone antibodies. Here, the production and ELISA system of these monoclonal antibodies are presented. The affinity-purified monoclonal antibodies specifically detected eel rec-FSH in ELISA and on western blots of rec-FSH produced from CHO cells. Immunohistochemical analysis revealed that FSH staining was specifically localized in the eel pituitary. PMID:27331121

  15. Detection of experimental myocarditis by monoclonal antimyosin antibody, Fab fragment

    SciTech Connect

    Rezkalla, S.; Kloner, R.A.; Khaw, B.A.; Haber, E.; Fallon, J.T.; Smith, F.E.; Khatib, R.

    1989-02-01

    The purpose of this study was to determine whether monoclonal antimyosin Fab (antigen binding fragment) was capable of labeling hearts with experimental coxsackievirus myocarditis, and to determine whether Fab could be used for detecting myocardial damage in either early or chronic phases of the disease. Sixty-five, 3-week-old cesarean-derived 1 (CD 1) mice were divided into two groups: group I (noninfected animals) and group II (infected with coxsackievirus B3). Mice from each group were killed on days 7, 17, 30, or 90 of infection. Forty-eight hours before killing, mice were injected with monoclonal I-125 antimyosin, Fab (25 microCi/injection) and radioactivity was counted in the heart. Selected heart sections were also examined by autoradiography. Heart radioactivity, count/m/mg (m +/- SEM) on days 7, 17, 30, and 90 of infection was 10.8 +/- 1.7, 21.3 +/- 1.1, 11.2 +/- 3.4, and 12.4 +/- 1.5 for group I, versus 36.7 +/- 8.0 (p less than 0.01), 50.0 +/- 4.5 (p less than 0.001), 33.4 +/- 16.1 (p = NS), and 40.6 +/- 8.5 (p less than 0.01) for group II, respectively. Autoradiography revealed focal uptake within areas of necrotic myocardium. We conclude that I125 Fab may be useful in detecting myocardial damage in the experimental model of murine myocarditis up to day 90 of infection.

  16. Abnormal fucosylation of-ileal mucus in cystic fibrosis: II. A histochemical study using monoclonal antibodies to fucosyl oligosaccharides.

    PubMed Central

    King, A; McLeish, M; Thiru, S

    1990-01-01

    Abnormal fucosylation of cystic fibrosis mucin was previously shown using peroxidase conjugated lectins on ileal tissue sections. These abnormally fucosylated glycoproteins were investigated further using monoclonal antibodies to fucosyl oligosaccharides based on type 1 and type 2 blood group precursor chains. The results of this study, using monoclonal antibodies to blood group glycoproteins in cystic fibrosis, were negative, yet abnormal fucosylation had been found using lectin histochemistry. Using monoclonal antibodies, lectins, and appropriate enzymes, such as glycosyl hydrolases, it should be possible to delineate further the abnormality found in glycoproteins in cystic fibrosis on appropriately fixed ileal sections, obtained from infants at term presenting with meconium ileus. Images PMID:2266174

  17. Species- and infective stage-specific monoclonal antibodies to Leishmania major produced by an in vitro immunization method.

    PubMed

    Wu, S J; Rowton, E D; Ma, M; Andre, R G

    1990-12-01

    Monoclonal antibodies specific to the infective-stage promastigotes of Leishmania major are needed for developing rapid diagnostic assays of infected sand flies. An in vitro immunization protocol was applied for the production of monoclonal antibodies using small amounts of L. major. Infective-stage promastigotes were isolated from sand flies (Phlebotomus papatasi) 7-10 days after infection and used as antigen for immunization. Two weeks after a primary immunization, murine splenocytes were removed and immunized in vitro with antigen in murine EL-4 thymoma cell conditioned medium. Three fusions were performed using X63-Ag.653 myeloma cells as fusion partners and two fusions were performed using FOX-NY cells. Antibodies specific to promastigotes were detected using an indirect enzyme-linked immunosorbent assay (ELISA). Initially 56 monoclonal antibodies were selected, and their species and stage specificity were determined using both an ELISA and an indirect fluorescent antibody assay (IFA). Twelve monoclonal antibodies showed species specificity to L. major when tested against four sympatric species of Leishmania. Four other monoclonal antibodies showed species and infective-stage specificity to L. major promastigotes. When tested in immunoblots, all four species- and stage-specific monoclonal antibodies bound to five protein bands that were unique to the infective-stage promastigotes. PMID:2087235

  18. Monoclonal antibody that inhibits infection of HeLa and rhabdomyosarcoma cells by selected enteroviruses through receptor blockade

    SciTech Connect

    Crowell, R.L.; Field, A.K.; Schleif, W.A.; Long, W.L.; Colonno, R.J.; Mapoles, J.E.; Emini, E. A.

    1986-02-01

    BALB/c mice were immunized with HeLa cells, and their spleen cells were fused with myeloma cells to produce hybridomas. Initial screening of culture fluids from 800 fusion products in a cell protection assay against coxsackievirus B3 (CB3) and the CB3-RD virus variant yielded five presumptive monoclonal antibodies with three specificities: (i) protection against CB3 on HeLa, (ii) protection against CB3-RD on rhabdomyosarcoma (RD) cells, and (iii) protection against both viruses on the respective cells. Only one of the monoclonal antibodies (with dual specificity) survived two subclonings and was studied in detail. The antibody was determined to have an immunoglobulin G2a isotype and protected cells by blockade of cellular receptors, since attachment of (/sup 35/S)methionine-labeled CB3 was inhibited by greater than 90%. The monoclonal antibody protected HeLa cells against infection by CB1, CB3, CB5, echovirus 6, and coxsackievirus A21 and RD cells against CB1-RD, CB3-RD, and CB5-Rd virus variants. The monoclonal antibody did not protect either cell type against 16 other immunotypes of picornaviruses. The monoclonal antibody produced only positive fluorescence on those cells which were protected against infection, and /sup 125/I-labeled antibody confirmed the specific binding to HeLa and RD cells. The results suggest that this monoclonal antibody possesses some of the receptor specificity of the group B coxsackieviruses.

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

    SciTech Connect

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

    1982-02-01

    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.

  20. Anti-Semaphorin 3A neutralization monoclonal antibody prevents sepsis development in lipopolysaccharide-treated mice.

    PubMed

    Yamashita, Naoya; Jitsuki-Takahashi, Aoi; Ogawara, Miyuki; Ohkubo, Wataru; Araki, Tomomi; Hotta, Chie; Tamura, Tomohiko; Hashimoto, Shu-ichi; Yabuki, Takashi; Tsuji, Toru; Sasakura, Yukie; Okumura, Hiromi; Takaiwa, Aki; Koyama, Chika; Murakami, Koji; Goshima, Yoshio

    2015-09-01

    Semaphorin 3A (Sema3A), originally identified as a potent growth cone collapsing factor in developing sensory neurons, is now recognized as a key player in immune, cardiovascular, bone metabolism and neurological systems. Here we established an anti-Sema3A monoclonal antibody that neutralizes the effects of Sema3A both in vitro and in vivo. The anti-Sema3A neutralization chick IgM antibodies were screened by combining an autonomously diversifying library selection system and an in vitro growth cone collapse assay. We further developed function-blocking chick-mouse chimeric and humanized anti-Sema3A antibodies. We found that our anti-Sema3A antibodies were effective for improving the survival rate in lipopolysaccharide-induced sepsis in mice. Our antibody is a potential therapeutic agent that may prevent the onset of or alleviate symptoms of human diseases associated with Sema3A. PMID:25855660

  1. Inhomogeneous translational diffusion of monoclonal antibodies on phospholipid Langmuir-Blodgett films.

    PubMed Central

    Wright, L L; Palmer, A G; Thompson, N L

    1988-01-01

    The translational mobility of fluorescent-labeled monoclonal antibodies specifically bound to supported phospholipid bilayers containing hapten-conjugated phospholipids has been measured as a function of the surface concentration of bound antibodies using fluorescence recovery after photobleaching. Fluorescence recovery curves are fit well by a model that assumes the presence of two populations of antibodies with different lateral diffusion coefficients. The larger diffusion coefficient equals 3.5 x 10(-9) cm2/s, the smaller diffusion coefficient ranges from 1.5 x 10(-9) cm2/s to 2.5 x 10(-10) cm2/s, and the fractional fluorescence recovery associated with the smaller coefficient increases from approximately 0 to approximately 0.7 with increasing concentration of bound antibody. These results suggest that complexes of haptenated phospholipids and antibodies in phospholipid Langmuir-Blodgett films form clusters or domains in a concentration-dependent fashion. PMID:3207834

  2. Discovery and Characterization of Phage Display-Derived Human Monoclonal Antibodies against RSV F Glycoprotein

    PubMed Central

    Tang, Aimin; Callahan, Cheryl; Pristatsky, Pavlo; Swoyer, Ryan; Cejas, Pedro; Nahas, Debbie; Galli, Jennifer; Cosmi, Scott; DiStefano, Daniel; Hoang, Van M.; Bett, Andrew; Casimiro, Danilo

    2016-01-01

    Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in infants, the elderly and in immunosuppressed populations. The vast majority of neutralizing antibodies isolated from human subjects target the RSV fusion (F) glycoprotein, making it an attractive target for the development of vaccines and therapeutic antibodies. Currently, Synagis® (palivizumab) is the only FDA approved antibody drug for the prevention of RSV infection, and there is a great need for more effective vaccines and therapeutics. Phage display is a powerful tool in antibody discovery with the advantage that it does not require samples from immunized subjects. In this study, Morphosys HuCAL GOLD® phage libraries were used for panning against RSV prefusion and postfusion F proteins. Panels of human monoclonal antibodies (mAbs) against RSV F protein were discovered following phage library panning and characterized. Antibodies binding specifically to prefusion or postfusion F proteins and those binding both conformations were identified. 3B1 is a prototypic postfusion F specific antibody while 2E1 is a prototypic prefusion F specific antibody. 2E1 is a potent broadly neutralizing antibody against both RSV A and B strains. Epitope mapping experiments identified a conformational epitope spanning across three discontinuous sections of the RSV F protein, as well as critical residues for antibody interaction. PMID:27258388

  3. Discovery and Characterization of Phage Display-Derived Human Monoclonal Antibodies against RSV F Glycoprotein.

    PubMed

    Chen, Zhifeng; Zhang, Lan; Tang, Aimin; Callahan, Cheryl; Pristatsky, Pavlo; Swoyer, Ryan; Cejas, Pedro; Nahas, Debbie; Galli, Jennifer; Cosmi, Scott; DiStefano, Daniel; Hoang, Van M; Bett, Andrew; Casimiro, Danilo; Vora, Kalpit A

    2016-01-01

    Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in infants, the elderly and in immunosuppressed populations. The vast majority of neutralizing antibodies isolated from human subjects target the RSV fusion (F) glycoprotein, making it an attractive target for the development of vaccines and therapeutic antibodies. Currently, Synagis® (palivizumab) is the only FDA approved antibody drug for the prevention of RSV infection, and there is a great need for more effective vaccines and therapeutics. Phage display is a powerful tool in antibody discovery with the advantage that it does not require samples from immunized subjects. In this study, Morphosys HuCAL GOLD® phage libraries were used for panning against RSV prefusion and postfusion F proteins. Panels of human monoclonal antibodies (mAbs) against RSV F protein were discovered following phage library panning and characterized. Antibodies binding specifically to prefusion or postfusion F proteins and those binding both conformations were identified. 3B1 is a prototypic postfusion F specific antibody while 2E1 is a prototypic prefusion F specific antibody. 2E1 is a potent broadly neutralizing antibody against both RSV A and B strains. Epitope mapping experiments identified a conformational epitope spanning across three discontinuous sections of the RSV F protein, as well as critical residues for antibody interaction. PMID:27258388

  4. Characterization and epitope mapping of a new panel of monoclonal antibodies to estradiol receptor.

    PubMed

    Abbondanza, C; de Falco, A; Nigro, V; Medici, N; Armetta, I; Molinari, A M; Moncharmont, B; Puca, G A

    1993-01-01

    A new panel of monoclonal antibodies to the calf uterus estrogen receptor was prepared. Thirteen antibodies were characterized for their isotype and for the affinity for the antigen. These antibodies recognize the human receptor and can be used in Western blot analysis. The location of the epitopes was mapped on the antigen structure using synthetic fragments of estrogen receptor, and it was possible to group the antibodies in five groups. Many antibodies were useful for the purification of estrogen receptor from tissue extracts by immunoaffinity chromatography. The reciprocal inhibition of the antibodies for the antigen binding was measured with an immunoadsorption assay. This was maximal and symmetrical for antibody pairs within the same group, but was incomplete and, in some instances, asymmetrical between pairs of antibodies from different groups. One antibody was able to inhibit the estrogen receptor-DNA interaction, whereas two others were unable to recognize the receptor-DNA complexes. This new panel of antibodies is a useful addition to the existing tools for studying structure and function of the estrogen receptor. PMID:7679226

  5. Tumor immunotherapy in the mouse with the use of 131I-labeled monoclonal antibodies

    SciTech Connect

    Zalcberg, J.R.; Thompson, C.H.; Lichtenstein, M.; McKenzie, I.F.

    1984-03-01

    This report describes the use of 131I-labeled monoclonal antibodies in two experimental models for tumor immunotherapy. In vitro treatment of the radiation-induced murine thymoma ITT-1-75NS with radiolabeled anti-Ly-2.1 significantly impaired subsequent tumor growth in vivo. However, in vivo treatment of this tumor, which previously had been injected into C57BL/6 mice, was unsuccessful. By contrast, in vitro treatment of a human colorectal tumor cell line (COLO 205) with 131I-labeled 250-30.6--a monoclonal antibody directed against a secretory component of normal and malignant gastrointestinal epithelium--completely inhibited subsequent tumor growth in BALB/c nude (nu/nu) mice. Furthermore, in vivo treatment of preexisting human colorectal tumor xenografts significantly impaired progressive tumor growth. Although some tumor inhibition was also produced by unlabeled 250-30.6 antibody, this response was considerably amplified by treatment with (131I)-labeled 250-30.6 (P less than .05), suggesting that in vivo treatment of human tumors with the use of 131I-labeled monoclonal antibodies may be clinically beneficial. The antithyroid drug propylthiouracil was used to reduce dehalogenation of the radiolabeled immunoglobulins in an attempt to improve their therapeutic efficacy.

  6. Preparation and characterization of a new monoclonal antibody against CXCR4 using lentivirus vector.

    PubMed

    Li, Xinyi; Kuang, Yu; Huang, Xiaojun; Zou, Linlin; Huang, Liuye; Yang, Ting; Li, Wanyi; Yang, Yuan

    2016-07-01

    CXCR4 is a member of chemokine receptors and plays a vital role in numerous diseases and cancer processes, which makes the CXCR4/CXCL12 chemotactic axis a potential therapeutic target. In this study, we used lentiviral vectors as a novel technology to produce a monoclonal antibody against CXCR4. Lentivirus vector pLV-CXCR4-Puro was successfully constructed and a hybridoma cell line 1A4 was generated. The CXCR4 monoclonal antibody (MAb) 1A4 had high titer and affinity, and the isotype was identified as IgG1a. The recombinant lentivirus vector could effectively stimulate the production of 39kDa CXCR4 antibody in vivo after immunization. Western blot analysis showed that the MAb could recognize the CXCR4 antigen expressed on transfected 293T cells as well as various human cancer cell lines. Immunofluorescence assays showed that MAb 1A4 mainly localized and strongly stained on the membrane of transfected 293T cells. Immunohistochemistry assays demonstrated that 1A4 could recognize strong expression of CXCR4 on the hepatocellular carcinoma (HCC). Thus, the method using lentiviral vectors may have application on effective and large-scale production of the CXCR4 monoclonal antibody, which will be a potential tool for the diagnosis and treatment of human cancers. PMID:27124560

  7. Rapid evaluation of artesunate quality with a specific monoclonal antibody-based lateral flow dipstick.

    PubMed

    Guo, Suqin; Zhang, Wei; He, Lishan; Tan, Guiyu; Min, Myo; Kyaw, Myat Phone; Wang, Baomin; Cui, Liwang

    2016-09-01

    Artesunate is a frontline antimalarial drug for treating Plasmodium falciparum malaria. To produce specific antibodies to artesunate, the carboxyl group of artesunate was directly conjugated to carrier protein as the immunogen. A specific monoclonal antibody (mAb) 3D82G6 against artesunate was obtained by high-throughput screening of positive hybridoma clones. This monoclonal antibody had 4.0, 0.5, and 0.9 % cross reactivities with artemisinin, dihydroartemisinin, and artemether, respectively. A dipstick immunoassay was developed, and the indicator range for artesunate was 1000-2000 ng mL(-1). No interference was observed with artemisinin, dihydroartemisinin, artemether, and other commonly used antimalarial drugs for up to 20,000 ng mL(-1). The dipsticks were used for determination of artesunate contents in commercial drugs, and the results were agreeable with those determined by high-performance liquid chromatography. This dipstick, with its specificity and sensitivity for artesunate and simplicity to use, makes it a potential point-of-care device for rapid quality evaluation of artesunate-containing antimalarial drugs. Graphical Abstract Specific monoclonal antibody-based lateral flow dipstick for artesunate. PMID:26873200

  8. Capillary ion-exchange chromatography with nanogram sensitivity for the analysis of monoclonal antibodies.

    PubMed

    Rea, Jennifer C; Freistadt, Benny S; McDonald, Daniel; Farnan, Dell; Wang, Yajun Jennifer

    2015-12-11

    Ion-exchange chromatography (IEC) is widely used for profiling the charge heterogeneity of proteins, including monoclonal antibodies (mAbs). Despite good resolving power and robustness, ionic strength-based ion-exchange separations are generally product specific and can be time consuming to develop. In addition, conventional analytical scale ion-exchange separations require tens of micrograms of mAbs for each injection, amounts that are often unavailable in sample-limited applications. We report the development of a capillary IEC (c-IEC) methodology for the analysis of nanogram amounts of mAb charge variants. Several key modifications were made to a commercially available liquid chromatography system to perform c-IEC for charge variant analysis of mAbs with nanogram sensitivity. We demonstrate the method for multiple monoclonal antibodies, including antibody fragments, on different columns from different manufacturers. Relative standard deviations of <10% were achieved for relative peak areas of main peak, acidic and basic regions, which are common regions of interest for quantifying monoclonal antibody charge variants using IEC. The results herein demonstrate the excellent sensitivity of this c-IEC characterization method, which can be used for analyzing charge variants in sample-limited applications, such as early-stage candidate screening and in vivo studies. PMID:26596872

  9. SPECT assay of radiolabeled monoclonal antibodies. Third yearly progress report, September 1991--February 1992

    SciTech Connect

    Jaszczak, R.J.

    1992-02-01

    The accurate determination of the biodistribution of radiolabeled monoclonal antibodies (MoAbs) is important for calculation of dosimetry and evaluation of pharmacokinetic variables such as antibody dose and route of administration. The hypothesis of this application is that the biodistribution of radiolabeled monoclonal antibodies (MoAbs) can be quantitatively determined using single photon emission computed tomography (SPECT). The major thrusts during the third year include the continued development and evaluation of improved 3D SPECT acquisition and reconstruction approaches to improve quantitative imaging of radiolabeled monoclonal antibodies (MoAbs), and the implementation and evaluation of algorithms to register serial SPECT image data sets, or to register 3D SPECT images with 3D image data sets acquired from positron emission tomography (PEI) and magnetic resonance images (MRI). The research has involved the investigation of statistical models and iterative reconstruction algorithms that accurately account for the physical characteristics of the SPECT acquisition system. It is our belief that SPECT quantification can be improved by accurately modeling the physical processes such as attenuation, scatter, geometric collimator response, and other factors that affect the measured projection data.

  10. Surface plasmon resonance for monitoring the interaction of Potato virus Y with monoclonal antibodies.

    PubMed

    Gutiérrez-Aguirre, Ion; Hodnik, Vesna; Glais, Laurent; Rupar, Matevž; Jacquot, Emmanuel; Anderluh, Gregor; Ravnikar, Maja

    2014-02-15

    Surface plasmon resonance (SPR)-based biosensors have been widely utilized for measuring interactions of a variety of molecules. Fewer examples include higher biological entities such as bacteria and viruses, and even fewer deal with plant viruses. Here, we describe the optimization of an SPR sensor chip for evaluation of the interaction of the economically relevant filamentous Potato virus Y (PVY) with monoclonal antibodies. Different virus isolates were efficiently and stably bound to a previously immobilized polyclonal antibody surface, which remained stable over subsequent injection regeneration steps. The ability of the biosensor to detect and quantify PVY particles was compared with ELISA and RT-qPCR. Stably captured virus surfaces were successfully used to explore kinetic parameters of the interaction of a panel of monoclonal antibodies with two PVY isolates representing the main viral serotypes N and O. In addition, the optimized biosensor proved to be suitable for evaluating whether two given monoclonal antibodies compete for the same epitope within the viral particle surface. The strategy proposed in this work can help to improve existing serologic diagnostic tools that target PVY and will allow investigation of the inherent serological variability of the virus and exploration for new interactions of PVY particles with other proteins. PMID:24220292

  11. Epitope-mapped monoclonal antibodies against the HPV16E1--E4 protein.

    PubMed

    Doorbar, J; Ely, S; Coleman, N; Hibma, M; Davies, D H; Crawford, L

    1992-03-01

    The human papillomavirus (HPV) E1--E4 protein is the only nonstructural late protein encoded by the virus. We have isolated three hybridomas producing monoclonal antibodies to the E1--E4 protein of HPV16, which is the HPV type most frequently associated with cervical cancer. The three antibodies (TVG 401, 402, and 403) detect adjacent epitopes within the major seroreactive region of the molecule and show no reactivity against the E4 proteins of HPV1, HPV2, HPV4, or HPV6. The E1--E4 protein migrates as a 10K species on SDS-gel electrophoresis and forms cytoplasmic inclusion granules in infected cells in vitro similar in appearance to those produced by HPV1 in benign warts. In naturally occurring HPV16-induced tumors the E1--E4 protein was detected in the cytoplasm of cells in the upper layers of the lesion in areas in which HPV16 DNA replication was occurring, as determined by in situ hybridization. Although the epitopes recognized by these monoclonal antibodies survive brief fixation in 5% formaldehyde, reactivity was destroyed by prolonged fixation. These monoclonal antibodies represent the first against HPV16 E1--E4 and should complement those already available to E7 and L1 for the screening of frozen sections of clinical biopsies and will be of value in monitoring the progression of HPV infection from benign lesions to invasive cancer. PMID:1371027

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

    PubMed

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

    2013-01-01

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

  13. Anti-idiotypic antibody to an anti-alprenolol monoclonal antibody: promotion of ligand binding to the idiotype

    SciTech Connect

    Sawutz, G.; Homcy, C.J.

    1986-03-01

    The authors previously described the production of four monoclonal antibodies to the beta-adrenergic receptor antagonist alprenolol. One of these antibodies, 5B7 (IgG/sub 2a/k), was used to generate polyclonal anti-idiotypic antisera in rabbits. In contrast to the predicted results, the anti-idiotypic antisera (R-9) promoted (/sup 125/I)-CYP binding to the monoclonal antibody 5B7. In the presence of R-9 antisera (1:300 dilution), the binding affinity improved with the dissociation constant decreasing 100-fold from 10nM to 0.1nM. This effect could not be reproduced with pre-immune, rabbit anti-mouse, or antiidiotypic antisera generated to monoclonal antibodies of different specificity. Furthermore, R-9 alone did not bind ligand. The R-9 IgG fraction was isolated by DEAE-cellulose chromatography then adsorbed to a 5B7-immunoaffinity resin and eluted with 0.5 M NaCl. This yielded a fraction with enhanced binding activity. F(ab) fragments of 5B7 and R-9 produced the same effect indicating that polyvalency was not necessary for the enhanced ligand binding. Finally, the ligand alprenolol promoted the binding of (/sup 125/I)-5B7 to %-9 as assessed by fractionating the resulting soluble complex by size exclusion chromatography on a TSK-3000 HPLC column. These results demonstrate that anti-idiotypic antibodies can be generated which will promote the binding of antigen to the original idiotype.

  14. 77 FR 9678 - Prospective Grant of Exclusive License: The Development of Human Anti-CD22 Monoclonal Antibodies...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-17

    ... Human Anti-CD22 Monoclonal Antibodies for the Treatment of Human Cancers and Autoimmune Disease AGENCY... autoimmune disease. The Licensed Field of Use includes the use of the antibodies in the form of an... disease such as lupus and Sjogren's syndrome. The specific antibodies covered by this technology...

  15. Dengue Virus Envelope Dimer Epitope Monoclonal Antibodies Isolated from Dengue Patients Are Protective against Zika Virus

    PubMed Central

    Swanstrom, J. A.; Plante, J. A.; Plante, K. S.; Young, E. F.; McGowan, E.; Gallichotte, E. N.; Widman, D. G.; Heise, M. T.; de Silva, A. M.

    2016-01-01

    ABSTRACT Zika virus (ZIKV) is a mosquito-borne flavivirus responsible for thousands of cases of severe fetal malformations and neurological disease since its introduction to Brazil in 2013. Antibodies to flaviviruses can be protective, resulting in lifelong immunity to reinfection by homologous virus. However, cross-reactive antibodies can complicate flavivirus diagnostics and promote more severe disease, as noted after serial dengue virus (DENV) infections. The endemic circulation of DENV in South America and elsewhere raises concerns that preexisting flavivirus immunity may modulate ZIKV disease and transmission potential. Here, we report on the ability of human monoclonal antibodies and immune sera derived from dengue patients to neutralize contemporary epidemic ZIKV strains. We demonstrate that a class of human monoclonal antibodies isolated from DENV patients neutralizes ZIKV in cell culture and is protective in a lethal murine model. We also tested a large panel of convalescent-phase immune sera from humans exposed to primary and repeat DENV infection. Although ZIKV is most closely related to DENV compared to other human-pathogenic flaviviruses, most DENV immune sera (73%) failed to neutralize ZIKV, while others had low (50% effective concentration [EC50], <1:100 serum dilution; 18%) or moderate to high (EC50, >1:100 serum dilution; 9%) levels of cross-neutralizing antibodies. Our results establish that ZIKV and DENV share epitopes that are targeted by neutralizing, protective human antibodies. The availability of potently neutralizing human monoclonal antibodies provides an immunotherapeutic approach to control life-threatening ZIKV infection and also points to the possibility of repurposing DENV vaccines to induce cross-protective immunity to ZIKV. PMID:27435464

  16. Comparison of different monoclonal antibodies against immunosuppressive proteins of Ascaris suum.

    PubMed

    Oshiro, T M; Rafael, A; Enobe, C S; Fernandes, I; Macedo-Soares, M F

    2004-02-01

    The extract of Ascaris suum suppresses the humoral and cellular immune responses to unrelated antigens in the mouse. In order to further characterize the suppressive components of A. suum, we produced specific monoclonal antibodies which can provide an important tool for the identification of these proteins. The A. suum immunosuppressive fractions isolated by gel filtration from an extract of adult worms were used to immunize BALB/c mice. Popliteal lymph node cells taken from the immunized animals were fused with SP2/O myeloma cells and the cloned hybrid cells obtained were screened to determine the specificity of secreted antibodies. Three monoclonal antibodies named MAIP-1, MAIP-2 and MAIP-3 were selected and were shown to react with different epitopes of high molecular weight proteins from the A. suum extract. All antibody molecules have kappa-type light chains but differ in heavy chain isotype. MAIP-1 is a mouse IgM, MAIP-2 is an IgA immunoglobulin and MAIP-3 is an IgG1 immunoglobulin and they recognize the antigen with affinity constants of 1.3 x 10(10) M-1, 7.1 x 10(9) M-1 and 3.8 x 10(7) M-1, respectively. The proteins recognized by these monoclonal antibodies (PAS-1, PAS-2 and PAS-3) were purified from the crude extract by affinity chromatography and injected with ovalbumin in BALB/c mice in order to determine their suppressive activity on heterologous antibody production. It was demonstrated that these three proteins are able to significantly suppress anti-ovalbumin antibody secretion, with PAS-1 being more efficient than the others. PMID:14762577

  17. In situ detection of acetylaminofluorene-DNA adducts in human cells using monoclonal antibodies.

    PubMed

    Iwamoto, Taka-aki; Kobayashi, Nobuhiko; Imoto, Kyoko; Yamamoto, Aya; Nakamura, Yu; Yamauchi, Yukika; Okumura, Hiromi; Tanaka, Akiko; Hanaoka, Fumio; Shibutani, Shinya; Miyagawa, Sachiko; Mori, Toshio

    2004-11-01

    The present study was performed to generate monoclonal antibodies capable of detecting N-acetoxy-2-acetylaminofluorene (NA-AAF)-derived DNA adducts in human cells in situ. As an immunogen, we employed NA-AAF-modified single-stranded DNA coupled electrostatically to methylated protein and we produced five different monoclonal antibodies. All of them showed strong binding to NA-AAF-modified DNA, but had undetectable or minimal binding to undamaged DNA. Competitive inhibition experiments revealed that the epitope recognized by these antibodies is N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF) in DNA, although deacetylated N-(deoxyguanosin-8-yl)-2-aminofluorene in DNA is also recognized with slightly less efficiency. In contrast, these antibodies did not bind to 3-(deoxyguanosin-N(2)-yl)-2-acetylaminofluorene in DNA or to UV-induced lesions in DNA. Interestingly, they showed only minimal binding to small AAF-nucleoside adducts (dG-C8-AAF), indicating that DNA regions flanking a DNA-bound adduct, in addition to the adduct itself, are essential for the stable binding of the antibodies. Using an enzyme-linked immunosorbent assay with the most promising antibody (AAF-1), we detected the concentration-dependent induction of NA-AAF-modified adducts in DNA from repair deficient xeroderma pigmentosum (XP) cells treated with physiological concentrations of NA-AAF. Moreover, the assay enabled to confirm that normal human cells efficiently repaired NA-AAF-induced DNA adducts but not XP-A cells. Most importantly, the formation of NA-AAF-induced DNA adducts in individual nuclei of XP cells could be clearly visualized using indirect immunofluorescence. Thus, we succeeded in establishing novel monoclonal antibodies capable of the in situ detection of NA-AAF-induced DNA adducts in human cells. PMID:15380103

  18. Molecular modeling of the affinity chromatography of monoclonal antibodies.

    PubMed

    Paloni, Matteo; Cavallotti, Carlo

    2015-01-01

    Molecular modeling is a methodology that offers the possibility of studying complex systems such as protein-ligand complexes from an atomistic point of view, making available information that can be difficultly obtained from experimental studies. Here, a protocol for the construction of molecular models of the interaction between antibodies and ligands that can be used for an affinity chromatography process is presented. The outlined methodology focuses mostly on the description of a procedure that may be adopted to determine the structure and free energy of interaction between the antibody and the affinity ligand. A procedure to extend the proposed methodology to include the effect of the environment (buffer solution, spacer, support matrix) is also briefly outlined. PMID:25749965

  19. Function and glycosylation of plant-derived antiviral monoclonal antibody

    PubMed Central

    Ko, Kisung; Tekoah, Yoram; Rudd, Pauline M.; Harvey, David J.; Dwek, Raymond A.; Spitsin, Sergei; Hanlon, Cathleen A.; Rupprecht, Charles; Dietzschold, Bernhard; Golovkin, Maxim; Koprowski, Hilary

    2003-01-01

    Plant genetic engineering led to the production of plant-derived mAb (mAbP), which provides a safe and economically feasible alternative to the current methods of antibody production in animal systems. In this study, the heavy and light chains of human anti-rabies mAb were expressed and assembled in planta under the control of two strong constitutive promoters. An alfalfa mosaic virus untranslated leader sequence and Lys-Asp-Glu-Leu (KDEL) endoplasmic reticulum retention signal were linked at the N and C terminus of the heavy chain, respectively. mAbP was as effective at neutralizing the activity of the rabies virus as the mammalian-derived antibody (mAbM) or human rabies Ig (HRIG). The mAbP contained mainly oligomannose type N-glycans (90%) and had no potentially antigenic α(1,3)-linked fucose residues. mAbP had a shorter half-life than mAbM. The mAbP was as efficient as HRIG for post-exposure prophylaxis against rabies virus in hamsters, indicating that differences in N-glycosylation do not affect the efficacy of the antibody in this model. PMID:12799460

  20. Pegfilgrastim Enhances the Antitumor Effect of Therapeutic Monoclonal Antibodies.

    PubMed

    Cornet, Sébastien; Mathé, Doriane; Chettab, Kamel; Evesque, Anne; Matera, Eva-Laure; Trédan, Olivier; Dumontet, Charles

    2016-06-01

    Therapeutic mAbs exert antitumor activity through various mechanisms, including apoptotic signalization, complement-dependent cytotoxicity, and antibody-dependent cellular cytotoxicity (ADCC) or phagocytosis (ADCP). G-CSF and GM-CSF have been reported to increase the activity of antibodies in preclinical models and in clinical trials. To determine the potential role of pegfilgrastim as an enhancer of anticancer antibodies, we performed a comparative study of filgrastim and pegfilgrastim. We found that pegfilgrastim was significantly more potent than filgrastim in murine xenograft models treated with mAbs. This was observed with rituximab in CD20(+) models and with trastuzumab in HER2(+) models. Stimulation with pegfilgrastim was associated with significant enhancement of leukocyte content in spleen as well as mobilization of activated monocytes/granulocytes from the spleen to the tumor bed. These results suggest that pegfilgrastim could constitute a potent adjuvant for immunotherapy with mAbs possessing ADCC/ADCP properties. Mol Cancer Ther; 15(6); 1238-47. ©2016 AACR. PMID:26988998

  1. Function and glycosylation of plant-derived antiviral monoclonal antibody.

    PubMed

    Ko, Kisung; Tekoah, Yoram; Rudd, Pauline M; Harvey, David J; Dwek, Raymond A; Spitsin, Sergei; Hanlon, Cathleen A; Rupprecht, Charles; Dietzschold, Bernhard; Golovkin, Maxim; Koprowski, Hilary

    2003-06-24

    Plant genetic engineering led to the production of plant-derived mAb (mAbP), which provides a safe and economically feasible alternative to the current methods of antibody production in animal systems. In this study, the heavy and light chains of human anti-rabies mAb were expressed and assembled in planta under the control of two strong constitutive promoters. An alfalfa mosaic virus untranslated leader sequence and Lys-Asp-Glu-Leu (KDEL) endoplasmic reticulum retention signal were linked at the N and C terminus of the heavy chain, respectively. mAbP was as effective at neutralizing the activity of the rabies virus as the mammalian-derived antibody (mAbM) or human rabies Ig (HRIG). The mAbP contained mainly oligomannose type N-glycans (90%) and had no potentially antigenic alpha(1,3)-linked fucose residues. mAbP had a shorter half-life than mAbM. The mAbP was as efficient as HRIG for post-exposure prophylaxis against rabies virus in hamsters, indicating that differences in N-glycosylation do not affect the efficacy of the antibody in this model. PMID:12799460

  2. Immunochemical identification of three proteinic determinants on the archaebacterium Methanococcus vannielii by monoclonal antibodies

    SciTech Connect

    de Macario, E.C.; Macario, A.J.L.; Magarinos, M.C.

    1983-01-01

    Archaebacteria evolved independently from Eubacteria and Eukaryotes and present unique biochemical features. One group, the methanogens, show a variety of cell-walls paralleling their own evolutionary diversity. Chemical analyses revealed proteins in the cell-wall of Methanococcacea but no murein or pseudomurein. Immunochemical studies were begun using a panel of monoclonal antibodies against M. vannielii SB and quantitative micro-immunoenzymatic methods developed for this purpose. Direct binding and inhibition-blocking assays using sugars and aminosugars also indicated absence of these residues in the SB's cell-wall. Three antibodies, however, recognized three different antigenic determinants involving amino acids: Lys, Phe and Thr (antibody 5A); Ser, Try and Tyr (antibody 5B); and Arg, Lys and Phe (antibody 5F). Each site was recognized by one antibody only, and vice versa. These results agree with those provided by well established chemical methods and support the notion that SB possesses a peculiar cell-wall with proteins only. The data also show the resolution power and reliability of monoclonal probes for structural analysis of the bacterial envelope.

  3. Relationship between in vitro binding activity and in vivo tumor accumulation of radiolabeled monoclonal antibodies

    SciTech Connect

    Sakahara, H.; Endo, K.; Koizumi, M.; Nakashima, T.; Kunimatsu, M.; Watanabe, Y.; Kawamura, Y.; Nakamura, T.; Tanaka, H.; Kotoura, Y.

    1988-02-01

    The relationship between in vitro cell binding and in vivo tumor accumulation of radiolabeled antibodies was studied using /sup 125/I- and /sup 111/In-labeled monoclonal antibodies to human osteosarcoma, and a human osteosarcoma xenograft (KT005) in nude mice. Three monoclonal antibodies--OST6, OST7, and OST15--raised against human osteosarcoma recognize the same antigen molecule. Although the binding of both /sup 125/I- and /sup 111/In-labeled OST6 to KT005 cells was higher than that of radiolabeled OST7 in vitro, /sup 125/I-labeled OST6 showed a faster clearance from the circulation and a lower accumulation in the transplanted tumor than /sup 125/I-labeled OST7. In contrast to the radioiodinated antibodies, the in vivo tumor accumulation of /sup 111/In-labeled OST6 was higher, although not significantly, than that of /sup 111/In-labeled OST7. OST15 showed the lowest binding in vitro, and its in vivo tumor localization was also lower than the others. The discrepancy in tumor uptake between OST6 and OST7 labeled with either /sup 125/I or /sup 111/In may have been a result of differing blood clearance. These results suggest that binding studies can be used to exclude from in vivo use those antibodies which show very poor binding in vitro, while in vivo serum clearance may be a better test for choosing antibodies with similar binding.

  4. Specific biodetection of B16 mouse melanoma in vivo by syngeneic monoclonal antibody

    SciTech Connect

    Yamasaki, T.; Wakabayashi, S.; Inoue, O.; Ando, K.; Kusakabe, K.; Kawasaki, Y.; Okamoto, S.; Taniguchi, M.

    1987-09-01

    The specific detection of tumors in vivo using a radiolabeled syngeneic monoclonal antibody made by fusion of P3U1 (BALB/c myeloma cells) and C57BL/6 spleen cells primed with syngeneic B16 melanoma cells was investigated by color imaging, autoradiography, and biodistribution. The radiolabeled antimelanoma antibody specifically accumulated only in the tumor lesions, whereas no radioactivity was observed in normal tissues or organs. The distribution patterns of the radioactive antibody in the tumor lesions depended on the sizes of the tumor. Almost the entire region of the small metastatic tumor in lymph nodes was labeled, whereas the radioactive antibody was irregularly localized mainly in the center of the medium-sized tumor. However, only the peripheral region of the large primary tumor was labeled. The highest uptake of radioactivity (tumor:blood ratio) was observed in the small lymph node metastatic tumor lesions rather than in the large primary tumor. Furthermore, high resolution color imaging of B16 melanoma was also obtained by using /sup 125/I-labeled monoclonal antibody. Tumor location was specifically visible without subtraction or enhancement methods 3-5 days after injection of the radiolabeled antibody.

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

    NASA Astrophysics Data System (ADS)

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

    1980-12-01

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

  6. Identification of human plasma cells with a lamprey monoclonal antibody

    PubMed Central

    Yu, Cuiling; Liu, Yanling; Chan, Justin Tze Ho; Tong, Jiefei; Li, Zhihua; Shi, Mengyao; Davani, Dariush; Parsons, Marion; Khan, Srijit; Zhan, Wei; Kyu, Shuya; Grunebaum, Eyal; Campisi, Paolo; Propst, Evan J.; Jaye, David L.; Trudel, Suzanne; Moran, Michael F.; Ostrowski, Mario; Herrin, Brantley R.; Lee, F. Eun-Hyung; Sanz, Ignacio; Cooper, Max D.; Ehrhardt, Götz R.A.

    2016-01-01

    Ab-producing plasma cells (PCs) serve as key participants in countering pathogenic challenges as well as being contributors to autoimmune and malignant disorders. Thus far, only a limited number of PC–specific markers have been identified. The characterization of the unique variable lymphocyte receptor (VLR) Abs that are made by evolutionarily distant jawless vertebrates prompted us to investigate whether VLR Abs could detect novel PC antigens that have not been recognized by conventional Abs. Here, we describe a monoclonal lamprey Ab, VLRB MM3, that was raised against primary multiple myeloma cells. VLRB MM3 recognizes a unique epitope of the CD38 ectoenzyme that is present on plasmablasts and PCs from healthy individuals and on most, but not all, multiple myelomas. Binding by the VLRB MM3 Ab coincides with CD38 dimerization and NAD glycohydrolase activity. Our data demonstrate that the lamprey VLRB MM3 Ab is a unique reagent for the identification of plasmablasts and PCs, with potential applications in the diagnosis and therapeutic intervention of PC or autoimmune disorders. PMID:27152361

  7. Optimization of monoclonal antibody delivery via the lymphatics: the dose dependence

    SciTech Connect

    Steller, M.A.; Parker, R.J.; Covell, D.G.; Holton, O.D. 3d.; Keenan, A.M.; Sieber, S.M.; Weinstein, J.N.

    1986-04-01

    After interstitial injection in mice, antibody molecules enter local lymphatic vessels, flow with the lymph to regional lymph nodes, and bind to target antigens there. Compared with i.v. administration, delivery via the lymphatics provides a more efficient means for localizing antibody in lymph nodes. An IgG2a (36-7-5) directed against the murine class I major histocompatibility antigen H-2Kk has proved useful for studying the pharmacology of lymphatic delivery. At very low doses, most of the antibody remains at the injection site in Kk-positive animals. As the dose is progressively increased, most effective labeling occurs first in nodes proximal to the injection site and then in the next group of nodes along the lymphatic chain. At higher doses, antibody overflows the lymphatic system and enters the blood-stream via the thoracic duct and other lymphatic-venous connections. Once in the blood, antibody is rapidly cleared, apparently by binding to Kk-bearing cells. These findings indicate that the single-pass distribution of monoclonal antibodies in the lymphatics can be strongly dose dependent, a principle which may be of clinical significance in the improvement of immunolymphoscintigraphic imaging, especially with antibodies directed against normal and malignant lymphoid cells. Monoclonal antibodies directed against normal cell types in the lymph node may be useful for assessing the integrity of lymphatic chains by immunolymphoscintigraphy or, more speculatively, for altering the status of regional immune function. The results presented here indicate that a low or intermediate antibody dose may optimize the signal:noise ratio for imaging. In Kk-negative animals, the percentage of dose taken up in the major organs was essentially independent of the dose administered; there was no evidence for saturable sites of nonspecific binding.

  8. Novel conformation-specific monoclonal antibodies against amyloidogenic forms of transthyretin

    PubMed Central

    Higaki, Jeffrey N.; Chakrabartty, Avi; Galant, Natalie J.; Hadley, Kevin C.; Hammerson, Bradley; Nijjar, Tarlochan; Torres, Ronald; Tapia, Jose R.; Salmans, Joshua; Barbour, Robin; Tam, Stephen J.; Flanagan, Ken; Zago, Wagner; Kinney, Gene G.

    2016-01-01

    Abstract Introduction: Transthyretin amyloidosis (ATTR amyloidosis) is caused by the misfolding and deposition of the transthyretin (TTR) protein and results in progressive multi-organ dysfunction. TTR epitopes exposed by dissociation and misfolding are targets for immunotherapeutic antibodies. We developed and characterized antibodies that selectively bound to misfolded, non-native conformations of TTR. Methods: Antibody clones were generated by immunizing mice with an antigenic peptide comprising a cryptotope within the TTR sequence and screened for specific binding to non-native TTR conformations, suppression of in vitro TTR fibrillogenesis, promotion of antibody-dependent phagocytic uptake of mis-folded TTR and specific immunolabeling of ATTR amyloidosis patient-derived tissue. Results: Four identified monoclonal antibodies were characterized. These antibodies selectively bound the target epitope on monomeric and non-native misfolded forms of TTR and strongly suppressed TTR fibril formation in vitro. These antibodies bound fluorescently tagged aggregated TTR, targeting it for phagocytic uptake by macrophage THP-1 cells, and amyloid-positive TTR deposits in heart tissue from patients with ATTR amyloidosis, but did not bind to other types of amyloid deposits or normal tissue. Conclusions: Conformation-specific anti-TTR antibodies selectively bind amyloidogenic but not native TTR. These novel antibodies may be therapeutically useful in preventing deposition and promoting clearance of TTR amyloid and in diagnosing TTR amyloidosis. PMID:26981744

  9. A case of Waldenstroem's disease with a monoclonal IgM antiphospholipid antibody.

    PubMed

    von Landenberg, Philipp; Schölmerich, Jürgen; Andreesen, Reinhard; Vogelhuber, Martin; Lackner, Karl J

    2002-07-01

    The antiphospholipid syndrome (APS) was described in 1983 as a clinical entity characterized by venous and arterial thrombosis, thrombocytopenia, and recurrent fetal loss. The serological markers of APS are antiphospholipid antibodies (APLA) directed mainly against anionic phospholipids, usually cardiolipin but also phosphatidylserine. Some APLA exhibit lupus anticoagulant activity. Monoclonal gammopathy sometimes occurs with the presence of autoantibodies. In this paper, we describe a patient with the diagnosis of immunocytoma with an IgM, kappa paraprotein with apparent specificity against anionic phospholipids, and lupus anticoagulant activity, but no clinical signs of APS. We describe in this patient the presence of a high titer of monoclonal APLA, which does apparently not induce the clinical symptoms of APS. This might be indicative for the presence of pathogenic and nonpathogenic antiphospholipid antibodies. PMID:12111091

  10. Application of a monoclonal antibody to a comparative study of alpha-lactalbumins from various species

    SciTech Connect

    Kaminogawa, S.; Shimoda, M.; Kurisaki, J.; Yamauchi, K.

    1989-05-01

    A monoclonal antibody to bovine alpha-lactalbumin was prepared and purified. The binding ability of alpha-lactalbumin from different species (cow, goat, giraffe, horse, pig, human, monkey, and guinea pig) was examined by a competitive radioimmunoassay. The order in strength of the binding affinity was cow goat, giraffe, horse, cynomolgus monkey and human, pig, and guinea pig. The order of evolutional divergence calculated from the amino acid composition was cow, goat, giraffe, horse, pig, guinea pig and human, and monkey. The orders in both cases were similar. Hence, it is suggested that immunological divergence as deduced by a monoclonal antibody is likely to be close to the evolutional divergence of alpha-lactalbumin.

  11. Improving impurities clearance by amino acids addition to buffer solutions for chromatographic purifications of monoclonal antibodies.

    PubMed

    Ishihara, Takashi; Hosono, Mareto

    2015-07-15

    The performance of amino acids in Protein A affinity chromatography, anion exchange chromatography and cation exchange chromatography for monoclonal antibody purification was investigated. Glycine, threonine, arginine, glutamate, and histidine were used as buffer components in the equilibration, washing, and elution steps of these chromatographies. Improved clearance of impurity, high molecular weight species (HMW) and host cell proteins (HCP) was observed in the purification processes when using the amino acids as base-buffer constituents, additives or eluents compared with that of buffers without these amino acids. In addition, we designed a buffer system in which the mobile phases were composed of only a single amino acid, histidine, and applied it to the above three chromatographies. Effective HMW and HCP clearance was also obtained in this manner. These results suggest that amino acids may enhance impurity clearance during the purification of monoclonal antibodies. PMID:26057847

  12. Monocyte/macrophage-specific monoclonal antibody Ki-M1 recognizes interdigitating reticulum cells.

    PubMed Central

    Radzun, H. J.; Parwaresch, M. R.; Feller, A. C.; Hansmann, M. L.

    1984-01-01

    A monoclonal antibody, Ki-M1, was produced, and its immunoreactivity was tested by light and electron-microscopic immunohistochemistry. Ki-M1 was found to react with monocytes, cells of the mononuclear phagocyte system (MPS), interdigitating reticulum cells (IDC), and the so-called indeterminate dendritic cells of lymphoid tissue. No reactivity was seen in other human tissues or other hematopoietic cells, including granulocytes and cells of the unstimulated promyelocyte cell line HL-60. Thus, Ki-M1 is the first of the monoclonal antibodies to MPS cells to react with both human IDC and MPS cells. This suggests that IDC and MPS cells may have a common cytogenesis. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:6391190

  13. Differentiation of Trypanosoma cruzi, T. cruzi marinkellei, T. dionisii and T. vespertilionis by monoclonal antibodies.

    PubMed

    Petry, K; Baltz, T; Schottelius, J

    1986-03-01

    Anti-T. dionisii and anti-T. vespertilionis monoclonal antibodies secreted by 17 hybridoma clones were tested against various strains of T. dionisii, T. vespertilionis, T. cruzi and T. cruzi marinkellei. Strain and species specific antigens were detected for the homologous immunizing strains. The common antigenic determinants of the tested trypanosome species include a component of the flagellum and different cell structures. Seventeen T. cruzi strains could be classified into two groups when tested with anti-T. dionisii monoclonal antibodies. The cross reactions between T. dionisii and T. cruzi demonstrate a strong correlation between T. dionisii and T. cruzi group 2. On the other hand T. cruzi group 1 and T. cruzi marinkellei show very similar antigenic character. PMID:2424290

  14. Legionella pneumophila serogroup 1 subgrouping by monoclonal antibodies--an epidemiological tool.

    PubMed Central

    Watkins, I. D.; Tobin, J. O.; Dennis, P. J.; Brown, W.; Newnham, R.; Kurtz, J. B.

    1985-01-01

    A panel of 10 monoclonal antibodies was used to subgroup 326 strains of Legionella pneumophila serogroup 1. All but two strains could be classified into three major subgroups named after their representative strains Pontiac 1, Olda and Bellingham 1. Of the 50 isolates from patients, 44 representing 32 separate incidents were of the Pontiac subgroup. This subgroup was also found in 16 of 18 buildings epidemiologically associated with Legionnaires' Disease. In contrast, strains of the Olda subgroup predominated in buildings where no infections had occurred. In 9 of the 11 incidents where isolates were available from at least one patient as well as from the suspected environmental source, the monoclonal antibody reaction patterns of strains from patients were identical to those of one or more of their environmental counterparts. PMID:3905954

  15. Production and characterisation of monoclonal antibodies specific for Escherichia coli O157:H7.

    PubMed

    Luciani, M; Armillotta, G; Magliulo, M; Portanti, O; Di Febo, T; Di Giannatale, E; Roda, A; Lelli, R

    2006-01-01

    Seven monoclonal antibodies (MAbs) specific for Escherichia coli O157:H7, one of the major causes of haemorrhagic colitis in humans, were produced by immunising Balb/c mice with the strain E. coli O157:H7. These monoclonal antibodies do not cross-react with other bacteria such as Salmonella enterica serovar Typhimurium, E. coli O14, E. coli JM109, S. enterica serovar Enteritidis, S. panama, S. saintpaul, S. derby, S. muenchen, S. bredeney, S. hadar, Yersinia enterocolitica, Proteus vulgaris, Shigella flexneri, Listeria ivanovii, L. monocytogenes 13M, L. innocua, Enterobacter cloacae, E. agglomerans, E. amnigenus, Citrobacter freundii, Escherichia fergussoni or Klebsiella pneumoniae. Of the seven MAbs obtained, MAb 8B8C3 was selected to prepare a high-sensitivity sandwich ELISA method specific for O157:H7. PMID:20429059

  16. Study of Leishmania major-infected macrophages by use of lipophosphoglycan-specific monoclonal antibodies.

    PubMed

    Handman, E

    1990-07-01

    Leishmania major infection of macrophages is followed by a time-dependent appearance of lipophosphoglycan (LPG) that can be detected on the surface of infected cells by monoclonal antibodies. The origin of these LPG epitopes is probably the intracellular amastigote. LPG epitopes could be detected on the amastigote and the infected macrophage by a number of monoclonal antibodies directed to several distinct determinants on the phosphoglycan moiety. The macrophage-expressed LPG may be modified because, unlike the parasite LPG as expressed on promastigotes or amastigotes, it could not be radiolabeled by galactose oxidase or periodate treatment of infected cells followed by reduction with 3H-labeled sodium borohydride. Some LPG epitopes displayed on the macrophage may be anchored with glycosylphosphatidylinositol, and some may be in the water-soluble phosphoglycan form bound to macrophage integrins involved in its specific recognition. The water-soluble population could be released from the infected macrophage by gentle protease treatment. PMID:1694823

  17. Distribution of monoclonal antibody-defined monosialoganglioside in normal and cancerous human tissues: an immunoperoxidase study.

    PubMed

    Arends, J W; Verstynen, C; Bosman, F T; Hilgers, J; Steplewski, Z

    1983-01-01

    The immunoreactivity of a monosialoganglioside antigen defined by monoclonal antibody 116NS19-9 (19-9) was studied in neoplastic and normal glandular and mucosal epithelia using an indirect immunoperoxidase method. In neoplastic mucosae, the antigen was detected in the majority of colorectal and endometrial carcinomas, predominantly in a focal staining pattern. A substantial proportion of gastric and pancreatic tumors and an occasional breast carcinoma also reacted with the monoclonal antibody. Expression of the monosialoganglioside in normal colonic mucosa appeared to be restricted to areas adjacent to tumor tissue. In gastric mucosa, the antigen was confined to some areas showing intestinal metaplasia. The antigen was also detected in the epithelium of normal mucosa of the gall bladder and endocervix, as well as in some ductal epithelia of the pancreas and salivary glands. Most other mucosae were negative for antigen expression. PMID:6381289

  18. Combining phage display with de novo protein sequencing for reverse engineering of monoclonal antibodies.

    PubMed

    Rickert, Keith W; Grinberg, Luba; Woods, Robert M; Wilson, Susan; Bowen, Michael A; Baca, Manuel

    2016-04-01

    The enormous diversity created by gene recombination and somatic hypermutation makes de novo protein sequencing of monoclonal antibodies a uniquely challenging problem. Modern mass spectrometry-based sequencing will rarely, if ever, provide a single unambiguous sequence for the variable domains. A more likely outcome is computation of an ensemble of highly similar sequences that can satisfy the experimental data. This outcome can result in the need for empirical testing of many candidate sequences, sometimes iteratively, to identity one which can replicate the activity of the parental antibody. Here we describe an improved approach to antibody protein sequencing by using phage display technology to generate a combinatorial library of sequences that satisfy the mass spectrometry data, and selecting for functional candidates that bind antigen. This approach was used to reverse engineer 2 commercially-obtained monoclonal antibodies against murine CD137. Proteomic data enabled us to assign the majority of the variable domain sequences, with the exception of 3-5% of the sequence located within or adjacent to complementarity-determining regions. To efficiently resolve the sequence in these regions, small phage-displayed libraries were generated and subjected to antigen binding selection. Following enrichment of antigen-binding clones, 2 clones were selected for each antibody and recombinantly expressed as antigen-binding fragments (Fabs). In both cases, the reverse-engineered Fabs exhibited identical antigen binding affinity, within error, as Fabs produced from the commercial IgGs. This combination of proteomic and protein engineering techniques provides a useful approach to simplifying the technically challenging process of reverse engineering monoclonal antibodies from protein material. PMID:26852694

  19. Characterization and immunotherapeutic potential of a monoclonal antibody against a ras oncogene transformed cell line

    SciTech Connect

    Ames, R.S. Jr.

    1986-01-01

    Transformed cells express cell surface antigens not present, or present in diminished amounts on normal cells. Monoclonal antibodies can be used to identify and biochemically characterize tumor-associated antigens. Monoclonal antibody (MoAb) 45-2D9 was produced by immunization of BALB/c mice with a transformed cell line (45-2D9) induced by transfection of NIH 3T3 cells with a c-H-ras oncogene in DNA isolated from a human lung carcinoma. By immunoperoxidase staining, this antibody binds to the 45-342 cells as well as to the ras transformed primary and 3 secondary transfectants, including the one used to induce 45-342, but not to other ras transformed cell lines. Murine tumors as well as human fetal and most normal adult tissues are not stained. This antibody does bind to a variety of human tumors, including lung adenocarcinomas, as well as breast, colon and esophageal carcinomas. The ability of MoAb 45-2D9 to target ricin toxin A chain (RTA) and radio-isotopes to gp74 expressing cells was investigated. An immunotoxin generated by conjugating RTA to MoAb 45-2D9 inhibits protein and DNA synthesis by the 45-342 cells. Radiolabeled antibody specifically localizes to and can be used to image subcutaneous and pulmonary gp74 expressing tumors in nu/nu mice. Monoclonal antibodies against oncogene transformed cell lines may be useful for the detection and characterization of tumor-associated antigens as well as for the development of new tumor therapeutic and diagnostic reagents.

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

    PubMed

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

    2016-01-01

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

  1. Preparation of Recombinant Human Monoclonal Antibody Fab Fragments Specific for Entamoeba histolytica

    PubMed Central

    Tachibana, Hiroshi; Cheng, Xun-Jia; Watanabe, Katsuomi; Takekoshi, Masataka; Maeda, Fumiko; Aotsuka, Satoshi; Kaneda, Yoshimasa; Takeuchi, Tsutomu; Ihara, Seiji

    1999-01-01

    Genes coding for human antibody Fab fragments specific for Entamoeba histolytica were cloned and expressed in Escherichia coli. Lymphocytes were separated from the peripheral blood of a patient with an amebic liver abscess. Poly(A)+ RNA was isolated from the lymphocytes, and then genes coding for the light chain and Fd region of the heavy chain were amplified by a reverse transcriptase PCR. The amplified DNA fragments were ligated with a plasmid vector and were introduced into Escherichia coli. Three thousand colonies were screened for the production of antibodies to E. histolytica HM-1:IMSS by an indirect fluorescence-antibody (IFA) test. Lysates from five Escherichia coli clones were positive. Analysis of the DNA sequences of the five clones showed that three of the five heavy-chain sequences and four of the five light-chain sequences differed from each other. When the reactivities of the Escherichia coli lysates to nine reference strains of E. histolytica were examined by the IFA test, three Fab fragments with different DNA sequences were found to react with all nine strains and another Fab fragment was found to react with seven strains. None of the four human monoclonal antibody Fab fragments reacted with Entamoeba dispar reference strains or with other enteric protozoan parasites. These results indicate that the bacterial expression system reported here is effective for the production of human monoclonal antibodies specific for E. histolytica. The recombinant human monoclonal antibody Fab fragments may be applicable for distinguishing E. histolytica from E. dispar and for use in the serodiagnosis of amebiasis. PMID:10225840

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

    PubMed Central

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

    2016-01-01

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

  3. Should Therapeutic Drug Monitoring for Monoclonal Antibodies Remain the Exception or Become the Norm?

    PubMed

    Stroh, M; Lum, B L

    2016-09-01

    Therapeutic drug monitoring (TDM) aims to maintain circulating drug concentrations at a desired level to optimize clinical outcome. The vast majority of marketed drugs do not require TDM, suggesting the clinical benefit of TDM has not been sufficiently demonstrated in most cases. With the continued emergence and prominence of monoclonal antibodies (mAbs) as drugs, especially in inflammation and cancer therapeutic areas, we are at a juncture to consider applicability of TDM for mAbs. PMID:26971373

  4. 75 FR 3244 - Prospective Grant of Exclusive License: Monoclonal Antibodies Against Smallpox/Orthopoxviruses

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-01-20

    ...This is notice, in accordance with 35 U.S.C. 209(c)(1) and 37 CFR 404.7(a)(1)(i), that the National Institutes of Health (NIH), Department of Health and Human Services (HHS), is contemplating the grant of a an exclusive license to practice the following invention as embodied in the following patent applications: E-145-2004/0,1,2,3,4, Purcell et al., ``Monoclonal Antibodies Against......

  5. Murine Monoclonal Antibodies against Escherichia coli O4 Lipopolysaccharide and H5 Flagellin

    PubMed Central

    Rivera-Betancourt, Mildred; Keen, James E.

    2001-01-01

    Two murine monoclonal antibodies (MAb), 2C5-F10 and 8D1-H10, reactive with Escherichia coli O4 and H5 antigens, respectively, were generated and characterized. Enzyme immunoassays and immunoblots demonstrated that MAb 2C5-F10 reacted specifically with lipopolysaccharide O antigen of E. coli O4 isolates, while MAb 8D1-H10 reacted with E. coli strains expressing H5 flagella. PMID:11526192

  6. High-throughput identification of monoclonal antibodies after compounding by UV spectroscopy coupled to chemometrics analysis.

    PubMed

    Jaccoulet, Emmanuel; Boccard, Julien; Taverna, Myriam; Azevedos, Andrea Santos; Rudaz, Serge; Smadja, Claire

    2016-08-01

    Monoclonal antibodies (mAbs) compounded into the hospital pharmacy are widely used nowadays. Their fast identification after compounding and just before administration to the patient is of paramount importance for quality control at the hospital. This remains challenging due to the high similarity of the structure between mAbs. Analysis of the ultraviolet spectral data of four monoclonal antibodies (cetuximab, rituximab, bevacizumab, and trastuzumab) using unsupervised principal component analysis led us to focus exclusively on the second-derivative spectra. Partial least squares-discriminant analysis (PLS-DA) applied to these data allowed us to build models for predicting which monoclonal antibody was present in a given infusion bag. The calibration of the models was obtained from a k-fold validation. A prediction set from another batch was used to demonstrate the ability of the models to predict well. PLS-DA models performed on the spectra of the region of aromatic amino acid residues presented high ability to predict mAb identity. The region corresponding to the tyrosine residue reached the highest score of good classification with 89 %. To improve the score, standard normal variate (SNV) preprocessing was applied to the spectral data. The quality of the optimized PLS-DA models was enhanced and the region from the tyrosine/tryptophan residues allowed us excellent classification (100 %) of the four mAbs according to the matrix of confusion. The sensitivity and specificity performance parameters assessed this excellent classification. The usefulness of the combination of UV second-derivative spectroscopy to multivariate analysis with SNV preprocessing demonstrated the unambiguous identification of commercially available monoclonal antibodies. Graphical abstract PLS-DA models on the spectra of the region of aromatic amino acid residues allows mAb identification with high prediction. PMID:27334717

  7. Radioimmunodetection in rhabdo- and leiomyosarcoma with 111In-anti-myosin monoclonal antibody complex.

    PubMed

    Planting, A; Verweij, J; Cox, P; Pillay, M; Stoter, G

    1990-02-01

    In patients with rhabdo- and leiomyosarcoma a radioimmunodiagnostic study was performed with 111In labeled F(ab) fragments of a monoclonal antibody against myosin. Eight patients with rhabdomyosarcoma and 18 patients with leiomyosarcoma were studied. Scanning was performed at 4, 24, and 48 h after administration of 74 MBeq of the antibody complex. A high uptake with a tumor:background ratio of 10:1 was observed in several patients with rhabdomyosarcoma but the results were less accurate in leiomyosarcoma. PMID:2297748

  8. Imaging of pharyngeal and laryngeal carcinomas with indium-111-labeled monoclonal anti-CEA antibodies

    SciTech Connect

    Kairemo, K.J.; Hopsu, E.V. )

    1990-10-01

    Localization of primary tumors, metastases, or recurrences in 13 consecutive patients with histological verification of squamous cell or adenocarcinoma was made with radioimmunodetection using monoclonal radiolabeled anti-CEA antibody. All surgical specimens stained immunohistochemically, except one, were positive for CEA. Of the known 19 tumor sites 17 were visualized in antibody scans. There were two positive findings that did not prove to be positive during 12 month follow-up. The scintigram findings did not correlate with CEA serum concentrations that, with one exception, were normal in all patients.

  9. Radiometric assay for direct quantitation of rat liver cytochrome P-450b using monoclonal antibodies.

    PubMed

    Rothwell, C E; Khazaeli, M B; Bernstein, I A

    1985-08-15

    A simple and sensitive assay has been developed that is capable of detecting as little as 0.2 ng of the major isozyme of cytochrome P-450 (P-450b) isolated from the livers of phenobarbital-induced rats. This assay employs monoclonal antibodies generated against cytochrome P-450b to directly quantify the levels of this enzyme in various tissues. Separation of bound from free labeled antibody is achieved by using 6,9-diaminoacridine lactate (Rivanol). The useful range of the assay is between 1 and 100 ng of P-450b. PMID:3935002

  10. Development of a monoclonal antibody against a tumor-associated antigen

    SciTech Connect

    Peng, W.W.; Bressler, J.P.; Tiffany-Castiglioni, E.; De Vellis, J.

    1982-02-26

    A monoclonal antibody-producing hybrid cell line was obtained by fusing mouse myeloma cells with spleen cells from a mouse immunized with C6 glioma cells. This antibody binds to a specific cell-surface antigen that is present on C6 rat glioma cells, tranformed astrocytes and oligodendrocytes, and a human glioma cell line but is absent on a normal glial cell line, fibroblasts, and primary cultures of astrocytes and oligodendrocytes. The antigen also appears on tumor tissue of transformed oligodendrocytes but not on normal brain tissue.

  11. Development of a Monoclonal Antibody against a Tumor-Associated Antigen

    NASA Astrophysics Data System (ADS)

    Peng, W. W.; Bressler, J. P.; Tiffany-Castiglioni, E.; de Vellis, J.

    1982-02-01

    A monoclonal antibody-producing hybrid cell line was obtained by fusing mouse myeloma cells with spleen cells from a mouse immunized with C6 glioma cells. This antibody binds to a specific cell-surface antigen that is present on C6 rat glioma cells, transformed astrocytes and oligodendrocytes, and a human glioma cell line but is absent on a normal glial cell line, fibroblasts, and primary cultures of astrocytes and oligodendrocytes. The antigen also appears on tumor tissue of transformed oligodendrocytes but not on normal brain tissue.

  12. A monoclonal antibody that recognizes B cells and B cell precursors in mice

    SciTech Connect

    Coffman, R.L.; Weissman, I.L.

    1981-02-01

    The monoclonal antibody, RA3-2C2, appears to be specific for cells within the B cell lineage. This antibody does not recognize thymocytes, peripheral T cells, or nonlymphoid hematopoietic cells in the spleen or bone marrow. Nor does it recognize the pluripotent hematopoietic stem cells, the spleen colony-forming unit, All sIg+ B cells and most plasma cells are RA3-2C2+. In addition, approximately 20% of nucleated bone marrow cells are RA3-2C2+ but sIg-. This population contains B cell precursors that can give rise to sIg+ cells within 2 d in vitro.

  13. Selective cytotoxicity of an oxygen-radical-generating enzyme conjugated to a monoclonal antibody.

    PubMed Central

    Battelli, M G; Abbondanza, A; Tazzari, P L; Dinota, A; Rizzi, S; Grassi, G; Gobbi, M; Stirpe, F

    1988-01-01

    The monoclonal antibody 8A, which recognizes a human plasma cell-associated antigen, was covalently linked to xanthine oxidase in a conjugate maintaining both immunological and enzymatic properties. A significant degree of target cell lysis was obtained at an enzyme concentration that was ineffective on non-target cells and on myeloid staminal cells (CFU-GM). The cytotoxic activity was abolished by an excess of antibody, by allopurinol and by superoxide dismutase and catalase. A possible use of the conjugate for bone marrow purging in multiple myeloma patients is suggested. PMID:3262464

  14. Selective cytotoxicity of an oxygen-radical-generating enzyme conjugated to a monoclonal antibody.

    PubMed

    Battelli, M G; Abbondanza, A; Tazzari, P L; Dinota, A; Rizzi, S; Grassi, G; Gobbi, M; Stirpe, F

    1988-07-01

    The monoclonal antibody 8A, which recognizes a human plasma cell-associated antigen, was covalently linked to xanthine oxidase in a conjugate maintaining both immunological and enzymatic properties. A significant degree of target cell lysis was obtained at an enzyme concentration that was ineffective on non-target cells and on myeloid staminal cells (CFU-GM). The cytotoxic activity was abolished by an excess of antibody, by allopurinol and by superoxide dismutase and catalase. A possible use of the conjugate for bone marrow purging in multiple myeloma patients is suggested. PMID:3262464

  15. Improved immunobinding test using monoclonal antibodies for detection of Mycoplasma bovis in milk

    PubMed Central

    Infante, F.; Infante, F.; Flores-Gutiérrez, G. H.

    2002-01-01

    The aim of this study was to evaluate an improved immunobinding test (IBT) using monoclonal antibodies to identify Mycoplasma bovis in naturally infected milk. The IBT and the improved IBT were highly specific and had an immunologic sensitivity of 5 × 103 colony-forming units per milliliter. The results for the 2 methods agreed in the 130 milk samples tested. However, the IBT required 158 min, whereas the improved IBT required only 110 min. In addition, the improved IBT used smaller quantities of antibodies and conjugates. PMID:12418785

  16. Automated pipeline for rapid production and screening of HIV-specific monoclonal antibodies using pichia pastoris.

    PubMed

    Shah, Kartik A; Clark, John J; Goods, Brittany A; Politano, Timothy J; Mozdzierz, Nicholas J; Zimnisky, Ross M; Leeson, Rachel L; Love, J Christopher; Love, Kerry R

    2015-12-01

    Monoclonal antibodies (mAbs) that bind and neutralize human pathogens have great therapeutic potential. Advances in automated screening and liquid handling have resulted in the ability to discover antigen-specific antibodies either directly from human blood or from various combinatorial libraries (phage, bacteria, or yeast). There remain, however, bottlenecks in the cloning, expression and evaluation of such lead antibodies identified in primary screens that hinder high-throughput screening. As such, "hit-to-lead identification" remains both expensive and time-consuming. By combining the advantages of overlap extension PCR (OE-PCR) and a genetically stable yet easily manipulatable microbial expression host Pichia pastoris, we have developed an automated pipeline for the rapid production and screening of full-length antigen-specific mAbs. Here, we demonstrate the speed, feasibility and cost-effectiveness of our approach by generating several broadly neutralizing antibodies against human immunodeficiency virus (HIV). PMID:26032261

  17. Functional analysis of the effect of monoclonal antibodies on monkey liver phenylalanine hydroxylase.

    PubMed Central

    Jennings, I G; Russell, R G; Armarego, W L; Cotton, R G

    1986-01-01

    An analysis of the effect of eleven monoclonal antibodies on the functional characteristics of monkey liver phenylalanine hydroxylase is presented. These eleven antibodies have been found to react with eight distinct regions on the phenylalanine hydroxylase protein. PH1 antibody inhibits enzyme activity, is dependent on phenylalanine for its binding, and appears to be related to structural changes occurring during phenylalanine activation of the enzyme activity. PH2 and PH3 antibodies stimulate enzyme activity, their binding is inhibited by lysolecithin and this group apparently is recognizing structures involved in lysolecithin activation of the enzyme activity. PH5, PH10, PH12 and PH6 recognise sites on phenylalanine hydroxylase affected by lysolecithin activation. PMID:2427069

  18. Rapid generation of a human monoclonal antibody to combat Middle East respiratory syndrome.

    PubMed

    Corti, Davide; Passini, Nadia; Lanzavecchia, Antonio; Zambon, Maria

    2016-01-01

    The last century has witnessed the emergence of several previously unknown viruses as life-threatening human pathogens. Several examples include HIV, Ebola, Lujo, and, most recently, the Middle East respiratory syndrome (MERS) and Ebola. In this study, we describe a method for the swift generation of a human-derived monoclonal antibody, known as LCA60, as a treatment for MERS infections. LCA60 antibody was generated using the Cellclone Technology from the immortalized B cells of a human donor recovering from MERS. Only four months were required from the initial screening of B cells to the development of a stable CHO cell line suitable for the production of clinical grade antibody, thereby delineating a rapid pathway for the development of antiviral therapies against emerging viruses. Currently, the LCA60 antibody is being considered for clinical development, which includes prophylaxis in individuals at risk and a treatment for severe MERS-CoV infections. PMID:27102927

  19. The properties of monoclonal antibody against sepiapterin reductase from fat body of the silkworm, Bombyx mori.

    PubMed

    Iino, T; Sawada, H; Gyure, W L; Tsusué, M

    1992-12-01

    A specific monoclonal antibody prepared for the 29-kDa a subunit of silkworm fat body sepiapterin reductase (SPR) was able to recognize the subunit in crude extract of fat body after SDS treatment. Although SPR from the silkworm fat body has biochemical properties similar to those reported for SPR from mammalian sources, especially rat erythrocytes, the antibody failed to recognize the 28-kDa subunit of rat erythrocyte SPR. This result indicates that SPR from silkworm fat body has a different amino-acid sequence from that of the rat erythrocyte enzyme. Sepiapterin reductase activity has not been found in crude extract of fat body from the silkworm mutant lemon. Although the antibody recognized only 29-kDa protein in the crude extract of silkworm fat body from normal strain after SDS-treatment, the antibody recognized only an approximately 80-kDa protein in the crude extract of the lemon mutant after SDS-treatment. PMID:1292509

  20. Clearance of persistent hepatitis C virus infection using a claudin-1-targeting monoclonal antibody

    PubMed Central

    Mailly, Laurent; Wilson, Garrick K.; Aubert, Philippe; Duong, François H. T.; Calabrese, Diego; Leboeuf, Céline; Fofana, Isabel; Thumann, Christine; Bandiera, Simonetta; Lütgehetmann, Marc; Volz, Tassilo; Davis, Christopher; Harris, Helen J.; Mee, Christopher J.; Girardi, Erika; Chane-Woon-Ming, Béatrice; Ericsson, Maria; Fletcher, Nicola; Bartenschlager, Ralf; Pessaux, Patrick; Vercauteren, Koen; Meuleman, Philip; Villa, Pascal; Kaderali, Lars; Pfeffer, Sébastien; Heim, Markus H.; Neunlist, Michel; Zeisel, Mirjam B.; Dandri, Maura; McKeating, Jane A.; Robinet, Eric; Baumert, Thomas F.

    2015-01-01

    Hepatitis C virus (HCV) infection is a leading cause of liver cirrhosis and cancer1. Cell entry of HCV2 and other pathogens3-5 is mediated by tight junction (TJ) proteins, but successful therapeutic targeting of TJ proteins has not been reported yet. Using a human liver-chimeric mouse model6 we show that a monoclonal antibody specific for TJ protein claudin-17 eliminates chronic HCV infection without detectable toxicity. This antibody inhibits HCV entry, cell-cell transmission and virus-induced signaling events. Antibody treatment reduces the number of HCV-infected hepatocytes in vivo, highlighting the need for de novo infection via host entry factors to maintain chronic infection. In summary, we demonstrate that an antibody targeting a virus receptor can cure chronic viral infection and uncover TJ proteins as targets for antiviral therapy. PMID:25798937