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Sample records for monoclonal antibodies demonstrate

  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.

    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)

  3. Monoclonal Antibodies.

    PubMed

    Geskin, Larisa J

    2015-10-01

    Use of monoclonal antibodies (mAbs) has revolutionized cancer therapy. Approaches targeting specific cellular targets on the malignant cells and in tumor microenvironment have been proved to be successful in hematologic malignancies, including cutaneous lymphomas. mAb-based therapy for cutaneous T-cell lymphoma has demonstrated high response rates and a favorable toxicity profile in clinical trials. Several antibodies and antibody-based conjugates are approved for use in clinical practice, and many more are in ongoing and planned clinical trials. In addition, these safe and effective drugs can be used as pillars for sequential therapies in a rational stepwise manner.

  4. Demonstration of the salmonid humoral response to Renibacterium salmoninarum using a monoclonal antibody against salmonid immunoglobulin

    USGS Publications Warehouse

    Bartholomew, J.L.; Arkoosh , M.R.; Rohovec, J.S.

    1991-01-01

    The specificity of the antibody response of salmonids to Renibacterium salmoninarum antigens was demonstrated by western blotting techniques that utilized a monoclonal antibody against salmonid immunoglobulin. In this study, the specificity of the response in immunized chinook salmon Oncorhynchus tshawytschawas compared with the response in naturally infected chinook salmon and coho salmon O. kisutch, and immunized rabbits. The antibody response in immunized salmon and rabbits and the naturally infected fish was primarily against the 57–58kilodalton protein complex. In addition to recognizing these proteins in the extracellular fraction and whole-cell preparations, antibody from the immunized salmon and rabbits detected four proteins with lower molecular masses. Western blotting techniques allow identification of the specific antigens recognized and are a useful tool for comparing the immunogenicity of different R. salmoninarumpreparations. Immunofluorescent techniques with whole bacteria were less sensitive than western blotting in detecting salmonid anti-R. salmoninarumantibody.

  5. Demonstration of a surface antigen of Clostridium tyrobutyricum by use of immunoblotting with a monoclonal antibody.

    PubMed

    Gueguen, F; Robreau, G; Talbot, F; Malcoste, R

    1990-01-01

    A monoclonal antibody, prepared against whole cells of Clostridium tyrobutyricum, recognized a surface antigen extracted by heat treatment or by hot phenol-water treatment. This antigen, after analysis by polyacrylamide gel electrophoresis and immunoblotting, has been shown to present a regularly-spaced ladder pattern similar to those shown by the lipopolysaccharide of many gram-negative bacteria. The proteinase K has been shown to have no effect on the recognition of this epitope by the monoclonal antibody. On the contrary, the inhibition of the antigen reactivity to the monoclonal antibody after a mild periodate oxidation suggests the involvement of a carbohydrate moiety in the epitope. Moreover, the SDS-PAGE analysis of phenol-water extracts has shown an additional compound, detected by silver staining but not recognized by the monoclonal antibody.

  6. 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…

  7. 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…

  8. 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.

  9. Heterogeneity of monoclonal antibodies.

    PubMed

    Liu, Hongcheng; Gaza-Bulseco, Georgeen; Faldu, Dinesh; Chumsae, Chris; Sun, Joanne

    2008-07-01

    Heterogeneity of monoclonal antibodies is common due to the various modifications introduced over the lifespan of the molecules from the point of synthesis to the point of complete clearance from the subjects. The vast number of modifications presents great challenge to the thorough characterization of the molecules. This article reviews the current knowledge of enzymatic and nonenzymatic modifications of monoclonal antibodies including the common ones such as incomplete disulfide bond formation, glycosylation, N-terminal pyroglutamine cyclization, C-terminal lysine processing, deamidation, isomerization, and oxidation, and less common ones such as modification of the N-terminal amino acids by maleuric acid and amidation of the C-terminal amino acid. In addition, noncovalent associations with other molecules, conformational diversity and aggregation of monoclonal antibodies are also discussed. Through a complete understanding of the heterogeneity of monoclonal antibodies, strategies can be employed to better identify the potential modifications and thoroughly characterize the molecules.

  10. 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.

  11. In situ demonstration of tissue proliferative activity using anti-bromo-deoxyuridine monoclonal antibody.

    PubMed Central

    Veronese, S; Gambacorta, M; Falini, B

    1989-01-01

    Immunohistochemical staining with anti-bromo-deoxyuridine (BrdU) monoclonal antibody was performed on a variety of human tissues following in vitro incubation with BrdU. The effect of different fixatives and DNA denaturation techniques on the reactivity with anti-BrdU was investigated. Optimal preservation of the antigenicity of BrdU incorporated into the DNA of proliferating cells was seen in tissues fixed in Bouin's fluid, while samples which had been fixed with cross-linking reagents, such as formalin, were usually unreactive. Positivity for BrdU was restored in formalin fixed tissues after digestion with pepsin, but this was usually associated with loss of morphological details. Acid and thermal DNA denaturation techniques gave similar results. It is concluded that Bouin fixation followed by acid or thermal denaturation of DNA is the method of choice for the in situ detection of cells in S-phase using anti-BrdU monoclonal antibody. Images Fig 1 Fig 1 PMID:2475528

  12. 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

  13. Monoclonal antibodies against bacteria.

    PubMed

    Macario, A J; Conway de Macario, E

    1988-01-01

    Highlights are presented of most recent work in which monoclonal antibodies have been instrumental in the study of bacteria and their products. Topics summarized pertain to human and veterinary medicines, dentistry, phytopathology, ichthyology, and bacterial ecophysiology, differentiation, evolution and methanogenic biotechnology.

  14. Recombinant monoclonal antibody technology.

    PubMed

    Siegel, D L

    2002-01-01

    With the development of murine hybridoma technology over a quarter century ago, the ability to produce large quantities of well-characterized monoclonal antibody preparations revolutionized diagnostic and therapeutic medicine. For many applications in transfusion medicine, however, the production of serological reagents in mice has certain biological limitations relating to the difficulty in obtaining murine monoclonal antibodies specific for many human blood group antigens. Furthermore, for therapeutic purposes, the efficacy of murine-derived immunoglobulin preparations is limited by the induction of anti-mouse immune responses. Technical difficulties inherent in human hybridoma formation have led to novel molecular approaches that facilitate the isolation and production of human antibodies without the need for B-cell transformation, tissue culture, or even immunized individuals. These technologies, referred to as 'repertoire cloning' or 'Fab/phage display', involve the rapid cloning of immunoglobulin gene segments to create immune libraries from which antibodies with desired specificities can be selected. The use of such recombinant methods in transfusion medicine is anticipated to play an important role in the development and production of renewable supplies of low-cost reagents for diagnostic and therapeutic applications.

  15. 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.

  16. Monoclonal antibodies to Actinobacillus actinomycetemcomitans.

    PubMed Central

    Place, D A; Scidmore, N C; McArthur, W P

    1988-01-01

    Murine hybridoma cell lines were developed which synthesized monoclonal antibodies against Actinobacillus actinomycetemcomitans-associated antigens. Monoclonal antibodies specific for an antigen(s) common to all A. actinomycetemcomitans isolates tested but not detected on other gram-negative oral plaque microorganisms or other Actinobacillus species were identified. Monoclonal antibodies specific for each serotype group of A. actinomycetemcomitans which did not bind to other Actinobacillus species or oral plaque microorganisms were also identified. PMID:3356470

  17. Immunologic diversity among serogroup 1 Legionella pneumophila urinary antigens demonstrated by monoclonal antibody enzyme-linked immunosorbent assays.

    PubMed Central

    Kohler, R B; Wilde, C; Johnson, W; Joly, J; Wheat, L J; Baker, R; Misfeldt, M

    1988-01-01

    We tested urine specimens from 222 patients with serogroup 1 Legionella pneumophila pneumonia in two enzyme-linked immunosorbent assays (ELISAs) which used different monoclonal antibodies (A and B) as detector antibodies. Of 171 specimens which contained enough antigen to be detected in the ELISAs, 169 reacted in only one of the two assays. A total of 25 patients whose infections were acquired in any of three Indianapolis hospitals excreted antigen reactive with monoclonal antibody B, but 18 patients who were treated for infections acquired elsewhere reacted with monoclonal antibody A. The urinary antigen ELISA reactivity patterns correlated with the reactivity patterns of L. pneumophila isolates when a separate panel of seven monoclonal antibodies was used. The isolate patterns, in turn, correlated well with environmental isolate patterns from two of the hospitals with nosocomial cases. We conclude that at least two different epitopes exist on the antigen molecules in urine from patients with serogroup 1 L. pneumophila pneumonia and that the subtyping of urinary antigens can be useful epidemiologically. PMID:2460492

  18. 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.

  19. Fragmentation of monoclonal antibodies

    PubMed Central

    Vlasak, Josef

    2011-01-01

    Fragmentation is a degradation pathway ubiquitously observed in proteins despite the remarkable stability of peptide bond; proteins differ only by how much and where cleavage occurs. The goal of this review is to summarize reports regarding the non-enzymatic fragmentation of the peptide backbone of monoclonal antibodies (mAbs). The sites in the polypeptide chain susceptible to fragmentation are determined by a multitude of factors. Insights are provided on the intimate chemical mechanisms that can make some bonds prone to cleavage due to the presence of specific side-chains. In addition to primary structure, the secondary, tertiary and quaternary structures have a significant impact in modulating the distribution of cleavage sites by altering local flexibility, accessibility to solvent or bringing in close proximity side chains that are remote in sequence. This review focuses on cleavage sites observed in the constant regions of mAbs, with special emphasis on hinge fragmentation. The mechanisms responsible for backbone cleavage are strongly dependent on pH and can be catalyzed by metals or radicals. The distribution of cleavage sites are different under acidic compared to basic conditions, with fragmentation rates exhibiting a minimum in the pH range 5–6; therefore, the overall fragmentation pattern observed for a mAb is a complex result of structural and solvent conditions. A critical review of the techniques used to monitor fragmentation is also presented; usually a compromise has to be made between a highly sensitive method with good fragment separation and the capability to identify the cleavage site. The effect of fragmentation on the function of a mAb must be evaluated on a case-by-case basis depending on whether cleavage sites are observed in the variable or constant regions, and on the mechanism of action of the molecule. PMID:21487244

  20. 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.

  1. 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.

  2. Microtubule-associated protein 2: monoclonal antibodies demonstrate the selective incorporation of certain epitopes into Alzheimer neurofibrillary tangles.

    PubMed Central

    Kosik, K S; Duffy, L K; Dowling, M M; Abraham, C; McCluskey, A; Selkoe, D J

    1984-01-01

    Neurofibrillary tangles (NFT) are the principal structural alteration of neuronal cell bodies in Alzheimer disease as well as in normal aging of the human brain. While the ultrastructure of these intraneuronal lesions has been extensively studied, the biochemical composition of the fibers comprising the NFT is unknown. We report the production of three monoclonal antibodies against the microtubule-associated protein 2 (MAP-2), one of which intensely labels Alzheimer NFT. All three antibodies specifically recognize MAP-2 on immunoblots and stain brain tissue in a characteristic dendritic pattern. The three antibodies are directed against at least two different antigenic sites on the MAP-2 molecule, and one appears to recognize a phosphorylation site on MAP-2. That only one of the three antibodies immunolabels NFT suggests that the formation of the tangle involves some modification of the MAP-2 molecule. Our findings suggest that one aspect of Alzheimer-type neurofibrillary pathology is an aggregation of MAP-2 or MAP-2 fragments with altered neurofilamentous elements present in NFT. Normal interactive function, which putatively occurs between neurofilaments and MAP-2, may thus be disrupted in Alzheimer disease. Images PMID:6083566

  3. Monoclonal antibodies to leukotoxin of Actinobacillus actinomycetemcomitans.

    PubMed Central

    DiRienzo, J M; Tsai, C C; Shenker, B J; Taichman, N S; Lally, E T

    1985-01-01

    Hybridoma cell lines which produce monoclonal antibodies to a leukotoxin from Actinobacillus actinomycetemcomitans were prepared. The monoclonal antibodies were selected for their ability to neutralize the cytotoxic activity of the leukotoxin and recognize the toxin on nitrocellulose blots. The antibodies belonged to either the immunoglobulin G1 (IgG1) or IgG2 subclass and differed in their ability to bind to the leukotoxin on nitrocellulose blots. However, only slight differences in neutralization titers were observed. Use of the monoclonal antibodies revealed that polymyxin B-extracted or osmotic shock-released leukotoxin could be separated into several high-molecular-weight polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblot analysis with the monoclonal antibodies also demonstrated that the leukotoxin was present in eight oral strains of A. actinomycetemcomitans that had been previously classified by a biological assay as leukotoxic. The availability of these monoclonal antibodies should facilitate and expand studies concerning the role of the leukotoxin in the pathogenicity of A. actinomycetemcomitans. Images PMID:3965404

  4. 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.

  5. 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.

  6. Monoclonal antibodies for treating cancer

    SciTech Connect

    Dillman, R.O. )

    1989-10-01

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

  7. 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.

  8. Monoclonal Antibodies in Diagnosis and Therapy

    NASA Astrophysics Data System (ADS)

    Waldmann, Thomas A.

    1991-06-01

    Monoclonal antibodies have been applied clinically to the diagnosis and therapy of an array of human disorders, including cancer and infectious diseases, and have been used for the modulation of immune responses. Effective therapy using unmodified monoclonal antibodies has, however, been elusive. Recently, monoclonal antibody-mediated therapy has been revolutionized by advances such as the definition of cell-surface structures on abnormal cells as targets for effective monoclonal antibody action, genetic engineering to create less immunogenic and more effective monoclonal antibodies, and the arming of such antibodies with toxins or radionuclides to enhance their effector function.

  9. Application of monoclonal antibodies in tumor pathology

    SciTech Connect

    Ruiter, D.J. ); Fleuren, G.J.; Warnaar, S.O. )

    1987-01-01

    This book contains papers under the following three section headings: Basic and technical aspects; Tumor associated antigens; and Practical application and case presentations. Some of the paper titles are: Monoclonal antibodies to oncofetal antigens; Monoclonal antibodies against ovarian cancer; and Tumor associated antigens and oncogene products defined by monoclonal antibodies.

  10. Monoclonal antibody technologies and rapid detection assays

    USDA-ARS?s Scientific Manuscript database

    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...

  11. 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.

  12. Immunohistochemical Analysis Using Antipodocalyxin Monoclonal Antibody PcMab-47 Demonstrates Podocalyxin Expression in Oral Squamous Cell Carcinomas.

    PubMed

    Itai, Shunsuke; Yamada, Shinji; Kaneko, Mika K; Harada, Hiroyuki; Kato, Yukinari

    2017-09-05

    Podocalyxin is a CD34-related type I transmembrane protein that is highly glycosylated with N-glycan, O-glycan, and keratan sulfate. Podocalyxin was originally found in the podocytes of rat kidney and is reportedly expressed in many types of tumors, including brain tumors, colorectal cancers, and breast cancers. Overexpression of podocalyxin is an independent predictor of progression, metastasis, and poor outcome. We recently immunized mice with recombinant human podocalyxin, which was produced using LN229 glioblastoma cells, and produced a novel antipodocalyxin monoclonal antibody (mAb), PcMab-47, which reacts with endogenous podocalyxin-expressing cancer cell lines and normal cell lines independent of glycosylation in Western blot, flow cytometry, and immunohistochemical analyses. In this study, we performed immunohistochemical analysis against oral cancers using PcMab-47. PcMab-47-stained oral squamous cell carcinoma cells in a cytoplasmic pattern and detected 26/38 (68.4%) of oral squamous cell carcinoma cells on tissue microarrays. These results indicate that PcMab-47 is useful in detecting podocalyxin of oral cancers for immunohistochemical analysis.

  13. [Evolution of monoclonal antibodies in cancer treatment].

    PubMed

    Kubczak, Małgorzata; Rogalińska, Małgorzata

    2016-01-01

    Since late 90s of last century the new age of directed therapy began using mainly biological constructs produced in rodents called monoclonal antibodies. The side effects of monoclonal antibodies were a challenge for pharmaceutical companies to improve the biological properties of these biological drugs. The humanization of monoclonal constructs was an idea to improve monoclonal antibodies next generation activity cancer cell reduction in humans. Moreover for some other patients sensitive for monoclonal antibodies therapy could also potentially induce immunological differences that might imply on human health. The new idea related to monoclonal antibodies was to design a small molecule constructs of nanoantibodies with ability to enter into cells. Such small molecules could find their targets inside human cells, even in nuclei leading to differences in cancer cells expression. The existing knowledge on monoclonal antibodies as well as directed activity of nanoantibodies could improve anticancer treatment efficancy of diseases.

  14. [The pharmacokinetics of monoclonal antibodies].

    PubMed

    Keizer, R J; Huitema, A D R; Damen, C W N; Schellens, J H M; Beijnen, J H

    2007-03-24

    Monoclonal antibodies (MOABs) are, due to their specificity, increasingly being deployed for therapeutic purposes. MOABs are derived from immunoglobulins and are fully or partially of murine or human origin. They are administered parenterally: mostly intravenously, but subcutaneous or intramuscular administration is also possible, in which case absorption probably occurs through the lymphatic system. The distribution of MOABs from the bloodstream into the tissues is slow and is hampered by the high molecular size of the MOABs, which is a lesser problem for fragments of antibodies (Fab fragments). MOABs are metabolised to peptides and amino acids. This process takes place in many tissues of the body, but probably predominantly in epithelial cells. As a consequence of the saturable binding of the MOAB to its target, a dose-dependent (non-linear) elimination is often observed. Immune reactions can accelerate the elimination of antibodies, partially depending on the degree ofhumanisation of the antibody. Antibodies and endogenous immunoglobulins are protected from elimination by binding to protective receptors (neonatal Fc-receptor; FcRn), which explains their long half-lives (up to 4 weeks). Metabolic pharmacokinetic interactions with other drugs have not been reported and are not expected. It is expected that in the years to come, new MOABs directed towards new targets will appear on the market, as well as existing antibodies with improved pharmacokinetic properties.

  15. KTN0158, a Humanized Anti-KIT Monoclonal Antibody, Demonstrates Biologic Activity against both Normal and Malignant Canine Mast Cells.

    PubMed

    London, Cheryl A; Gardner, Heather L; Rippy, Sarah; Post, Gerald; La Perle, Krista; Crew, Linda; Lopresti-Morrow, Lori; Garton, Andrew J; McMahon, Gerald; LaVallee, Theresa M; Gedrich, Richard

    2016-11-04

    Purpose: KTN0158 is a novel anti-KIT antibody that potently inhibits wild-type and mutant KIT. This study evaluated the safety, biologic activity, and pharmacokinetic/pharmacodynamics profile of KTN0158 in dogs with spontaneous mast cell tumors (MCT) as a prelude to human clinical applications.Experimental Design: Cell proliferation, KIT phosphorylation, and mast cell degranulation were evaluated in vitro KTN0158 was administered to 4 research dogs to assess clinical effects and cutaneous mast cell numbers. Thirteen dogs with spontaneous MCT were enrolled into a prospective phase I dose-escalating open-label clinical study of KTN0158 evaluating 3 dose levels and 2 schedules and with weekly assessments for response and clinical toxicities.Results: KTN0158 was a potent inhibitor of human and dog KIT activation and blocked mast cell degranulation in vitro In dogs, KTN0158 was well tolerated and reduced cutaneous mast cell numbers in a dose-dependent manner. Clinical benefit of KTN0158 administration in dogs with MCT (n = 5 partial response; n = 7 stable disease) was observed regardless of KIT mutation status, and decreased KIT phosphorylation was demonstrated in tumor samples. Histopathology after study completion demonstrated an absence of neoplastic cells in the primary tumors and/or metastatic lymph nodes from 4 dogs. Reversible hematologic and biochemical adverse events were observed at doses of 10 and 30 mg/kg. The MTD was established as 10 mg/kg.Conclusions: KTN0158 inhibits KIT phosphorylation, demonstrates an acceptable safety profile in dogs, and provides objective responses in canine MCT patients with and without activating KIT mutations, supporting future clinical evaluation of KTN0158 in people. Clin Cancer Res; 1-10. ©2016 AACR.

  16. 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.

  17. 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.

  18. Expression of Ia-like antigens by human vascular endothelial cells is inducible in vitro: demonstration by monoclonal antibody binding and immunoprecipitation.

    PubMed Central

    Pober, J S; Gimbrone, M A

    1982-01-01

    The expression of Ia-like antigens by cultured human endothelial cells has been investigated by means of monoclonal antibody binding to intact cells and by immunoprecipitation of radioiodinated membrane proteins. Primary growing and confluent cultures of human umbilical vein endothelium express little, if any, detectable Ia-like antigens under standard culture conditions. However, treatment of primary cultures with the lectin phytohemagglutinin induces the expression of Ia-like antigens. This action of the lectin uniformly affects all the endothelial cells in a culture, does not depend on cell division, and is associated with a cell shape change. The data presented in this report provide unequivocal serological and biochemical demonstration of Ia-like antigens on human vascular endothelial cells. The fact that the expression of Ia-like antigens by endothelium can be induced may have important implications for organ transplantation and for regulation of the immunological response. Images PMID:6815654

  19. Monoclonal antibodies in chronic lymphocytic leukemia.

    PubMed

    Ferrajoli, Alessandra; Faderl, Stefan; Keating, Michael J

    2006-09-01

    Multiple options are now available for the treatment of chronic lymphocytic leukemia. Over the last 10 years, monoclonal antibodies have become an integral part of the management of this disease. Alemtuzumab has received approval for use in patients with fludarabine-refractory chronic lymphocytic leukemia. Rituximab has been investigated extensively in chronic lymphocytic leukemia both as a single agent and in combination with chemotherapy and other monoclonal antibodies. Epratuzumab and lumiliximab are newer monoclonal antibodies in the early phase of clinical development. This article will review the monoclonal antibodies more commonly used to treat chronic lymphocytic leukemia, the results obtained with monoclonal antibodies as single agents and in combination with chemotherapy, and other biological agents and newer compounds undergoing clinical trials.

  20. 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.

  1. Tumor detection using radiolabeled monoclonal antibodies

    SciTech Connect

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

    1987-01-01

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

  2. 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.

  3. Monoclonal Antibodies for Multiple Sclerosis Treatment.

    PubMed

    Palavra, Filipe

    2015-01-01

    Since their introduction in medical therapy, in the last quarter of the 20th century, monoclonal antibodies have gained an increasing importance in the treatment of various diseases. Neurology has been one of the medical specialties benefiting of the therapeutic potential of these monoclonal antibodies and certain neurological conditions may now contain such drugs in their therapeutic algorithms. Multiple sclerosis is one of these diseases and, in addition to the monoclonal antibodies already licensed for clinical use, several others are in development for future utilization in this specific area. The future will certainly pass through this kind of drugs and, in this article, a review of the most relevant data related to monoclonal antibodies already in use and also in clinical development for multiple sclerosis treatment will be performed.

  4. [Demonstration of β-1,2 mannan structures expressed on the cell wall of Candida albicans yeast form but not on the hyphal form by using monoclonal antibodies].

    PubMed

    Aydın, Cevahir; Ataoğlu, Haluk

    2015-01-01

    Candida albicans is a polymorphic fungus that may be observed as both commensal and opportunistic pathogen in humans. As one of the major components of Candida cell wall structure, mannan plays an important role in the fungus-host cell interaction and in virulence. The ability to switch from yeast to hypha form of microorganism is crutial in the development of C.albicans infections. Hyphal form has different antigenic properties compared to yeast form and structural changes occur in the yeast cell wall during transition from yeast to hypha form. Although there are several factors associated with this transition process, sufficient information is not available. The aim of this study was to investigate the change of configuration in mannan structure found in C.albicans cell wall by using monoclonal antibodies. C.albicans (NIHA 207) serotype A strains were used as test strains throughout the study, together with Salmonella choleraesuis 211 and Salmonella infantis as controls with similar cell wall structures to that of C.albicans. Cultures were maintained on YPD-agar medium by incubating at 28°C for yeast forms, and on YPD-broth medium in a shaking incubator at 37°C for 3-4 hours for the growth of hyphal forms. Cells were harvested in the exponential phase, and after being washed, the mannan content from C.albicans were extracted from pellet by heating in 20 mM sodium citrate buffer for 90 minutes at 125°C. Hybridoma technique was used for the production of monoclonal antibodies. After immunizing the Balb/C mice with antigen, the splenocytes were harvested and fusion was performed between spleen cells and F0 myeloma cells. The clones grown in HAT medium were screened for the presence of antibody producing hybrid cells by ELISA method. The antibody isotypes were determined by using a commercial kit (Pierce Biotechnology, ABD). The culture supernatants which contained monoclonal antibodies were collected and purified according to the ammonium sulphate method

  5. Expression of Surface Protein LapB by a Wide Spectrum of Listeria monocytogenes Serotypes as Demonstrated with Anti-LapB Monoclonal Antibodies

    PubMed Central

    Boivin, Teela; Elmgren, Cathie; Brooks, Brian W.; Huang, Hongsheng; Pagotto, Franco

    2016-01-01

    ABSTRACT Protein antigens expressed on the surface of all strains of Listeria monocytogenes and absent from nonpathogenic Listeria spp. are presumably useful targets for pathogen identification, detection, and isolation using specific antibodies (Abs). To seek such surface proteins expressed in various strains of L. monocytogenes for diagnostic applications, we focused on a set of surface proteins known to be involved or putatively involved in L. monocytogenes virulence and identified Listeria adhesion protein B (LapB) as a candidate based on the bioinformatics analysis of whole-genome sequences showing that the gene coding for LapB was present in L. monocytogenes strains and absent from strains of other Listeria spp. Immunofluorescence microscopy (IFM), performed with rabbit polyclonal antibodies against the recombinant LapB protein (rLapB) of L. monocytogenes serotype 4b strain L10521, confirmed expression of LapB on the surface. A panel of 48 mouse monoclonal antibodies (MAbs) to rLaB was generated, and 7 of them bound strongly to the surface of L. monocytogenes cells as demonstrated using IFM. Further characterization of these 7 anti-LapB MAbs, using an enzyme-linked immunosorbent assay (ELISA), revealed that 6 anti-LapB MAbs (M3484, M3495, M3500, M3509, M3517, and M3519) reacted strongly with 46 (86.8%) of 53 strains representing 10 of the 12 serotypes tested (1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4ab, 4b, 4d, and 4e). These results indicate that LapB, together with companion anti-LapB MAbs, can be targeted as a biomarker for the detection and isolation of various L. monocytogenes strains from contaminated foods. IMPORTANCE Strains of L. monocytogenes are traditionally grouped into serotypes. Identification of a surface protein expressed in all or the majority of at least 12 serotypes would aid in the development of surface-binding monoclonal antibodies (MAbs) for detection and isolation of L. monocytogenes from foods. Bioinformatics analysis revealed that the gene

  6. 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.

  7. 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.

  8. Monoclonal Antibodies against the Drosophila Nervous System

    NASA Astrophysics Data System (ADS)

    Fujita, Shinobu C.; Zipursky, Stephen L.; Benzer, Seymour; Ferrus, Alberto; Shotwell, Sandra L.

    1982-12-01

    A panel of 148 monoclonal antibodies directed against Drosophila neural antigens has been prepared by using mice immunized with homogenates of Drosophila tissue. Antibodies were screened immunohistochemically on cryostat sections of fly heads. A large diversity of staining patterns was observed. Some antigens were broadly distributed among tissues; others were highly specific to nerve fibers, neuropil, muscle, the tracheal system, cell nuclei, photoreceptors, or other structures. The antigens for many of the antibodies have been identified on immunoblots. Monoclonal antibodies that identify specific molecules within the nervous system should prove useful in the study of the molecular genetics of neural development.

  9. From rabbit antibody repertoires to rabbit monoclonal antibodies

    PubMed Central

    Weber, Justus; Peng, Haiyong; Rader, Christoph

    2017-01-01

    In this review, we explain why and how rabbit monoclonal antibodies have become outstanding reagents for laboratory research and increasingly for diagnostic and therapeutic applications. Starting with the unique ontogeny of rabbit B cells that affords highly distinctive antibody repertoires rich in in vivo pruned binders of high diversity, affinity and specificity, we describe the generation of rabbit monoclonal antibodies by hybridoma technology, phage display and alternative methods, along with an account of successful humanization strategies. PMID:28336958

  10. 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

  11. Heterogeneous distribution of Candida albicans cell-surface antigens demonstrated with an Als1-specific monoclonal antibody

    PubMed Central

    Coleman, David A.; Oh, Soon-Hwan; Zhao, Xiaomin; Hoyer, Lois L.

    2010-01-01

    Despite an abundance of data describing expression of genes in the Candida albicans ALS (agglutinin-like sequence) gene family, little is known about the production of Als proteins on individual cells, their spatial localization or stability. Als proteins are most commonly discussed with respect to function in adhesion of C. albicans to host and abiotic surfaces. Development of a mAb specific for Als1, one of the eight large glycoproteins encoded by the ALS family, provided the opportunity to detect Als1 during growth of yeast and hyphae, both in vitro and in vivo, and to demonstrate the utility of the mAb in blocking C. albicans adhesion to host cells. Although most C. albicans yeast cells in a saturated culture are Als1-negative by indirect immunofluorescence, Als1 is detected on the surface of nearly all cells shortly after transfer into fresh growth medium. Als1 covers the yeast cell surface, with the exception of bud scars. Daughters of the inoculum cells, and sometimes granddaughters, also have detectable Als1, but Als1 is not detectable on cells from subsequent generations. On germ tubes and hyphae, most Als1 is localized proximal to the mother yeast. Once deposited on yeasts or hyphae, Als1 persists long after the culture has reached saturation. Growth stage-dependent production of Als1, coupled with its persistence on the cell surface, results in a heterogeneous population of cells within a C. albicans culture. Anti-Als1 immunolabelling patterns vary depending on the source of the C. albicans cells, with obvious differences between cells recovered from culture and those from a murine model of disseminated candidiasis. Results from this work highlight the temporal parallels for ALS1 expression and Als1 production in yeasts and germ tubes, the specialized spatial localization and persistence of Als1 on the C. albicans cell surface, and the differences in Als1 localization that occur in vitro and in vivo. PMID:20705663

  12. 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.

  13. 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)

  14. Production of monoclonal antibodies to plant pathogens.

    PubMed

    Thornton, Christopher R

    2009-01-01

    The use of monoclonal antibodies in plant pathology has improved the quality and specificity of detection methods for diseases. Hybridoma technology allows the limitless production of highly specific antibodies which can be used to identify pathogens to the species or even sub-species level.

  15. 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.

  16. Binding and transepithelial transport of immunoglobulins by intestinal M cells: demonstration using monoclonal IgA antibodies against enteric viral proteins

    PubMed Central

    1989-01-01

    M cells of intestinal epithelia overlying lymphoid follicles endocytose luminal macromolecules and microorganisms and deliver them to underlying lymphoid tissue. The effect of luminal secretory IgA antibodies on adherence and transepithelial transport of antigens and microorganisms by M cells is unknown. We have studied the interaction of monoclonal IgA antibodies directed against specific enteric viruses, or the hapten trinitrophenyl (TNP), with M cells. To produce monospecific IgA antibodies against mouse mammary tumor virus (MMTV) and reovirus type 1, Peyer's patch cells from mucosally immunized mice were fused with myeloma cells, generating hybridomas that secreted virus-specific IgA antibodies in monomeric and polymeric forms. One of two anti-MMTV IgA antibodies specifically bound the viral surface glycoprotein gp52, and 3 of 10 antireovirus IgA antibodies immunoprecipitated sigma 3 and mu lc surface proteins. 35S-labeled IgA antibodies injected intravenously into rats were recovered in bile as higher molecular weight species, suggesting that secretory component had been added on passage through the liver. Radiolabeled or colloidal gold-conjugated mouse IgA was injected into mouse, rat, and rabbit intestinal loops containing Peyer's patches. Light microscopic autoradiography and EM showed that all IgA antibodies (antivirus or anti-TNP) bound to M cell luminal membranes and were transported in vesicles across M cells. IgA-gold binding was inhibited by excess unlabeled IgA, indicating that binding was specific. IgG-gold also adhered to M cells and excess unlabeled IgG inhibited IgA-gold binding; thus binding was not isotype-specific. Immune complexes consisting of monoclonal anti-TNP IgA and TNP-ferritin adhered selectively to M cell membranes, while TNP-ferritin alone did not. These results suggest that selective adherence of luminal antibody to M cells may facilitate delivery of virus-antibody complexes to mucosal lymphoid tissue, enhancing subsequent

  17. Fixed Dosing of Monoclonal Antibodies in Oncology.

    PubMed

    Hendrikx, Jeroen J M A; Haanen, John B A G; Voest, Emile E; Schellens, Jan H M; Huitema, Alwin D R; Beijnen, Jos H

    2017-07-28

    Most monoclonal antibodies in oncology are administered in body-size-based dosing schedules. This is believed to correct for variability in both drug distribution and elimination between patients. However, monoclonal antibodies typically distribute to the blood plasma and extracellular fluids only, which increase less than proportionally with the increase in body weight. Elimination takes place via proteolytic catabolism, a nonspecific immunoglobulin G elimination pathway, and intracellular degradation after binding to the target. The latter is the primary route of elimination and is related to target expression levels rather than body size. Taken together, the minor effects of body size on distribution and elimination of monoclonal antibodies and their usually wide therapeutic window do not support body-size-based dosing. We evaluated effects of body weight on volume of distribution and clearance of monoclonal antibodies in oncology and show that a fixed dose for most of these drugs is justified based on pharmacokinetics. A survey of the savings after fixed dosing of monoclonal antibodies at our hospital showed that fixed dosing can reduce costs of health care, especially when pooling of preparations is not possible (which is often the case in smaller hospitals). In conclusion, based on pharmacokinetic parameters of monoclonal antibodies, there is a rationale for fixed dosing of these drugs in oncology. Therefore, we believe that fixed dosing is justified and can improve efficiency of the compounding. Moreover, drug spillage can be reduced and medication errors may become less likely. The currently available knowledge of elimination of monoclonal antibodies combined with the publicly available data from clinical trials and extensive population pharmacokinetic (PopPK) modeling justifies fixed dosing. Interpatient variation in exposure is comparable after body weight and fixed dosing and most monoclonal antibodies show relatively flat dose-response relationships

  18. Immunocytochemical demonstration of chicken hypophyseal thyrotropes and development of a radioimmunological indicator for chicken TSH using monoclonal and polyclonal homologous antibodies in a subtractive strategy.

    PubMed

    Berghman, L R; Darras, V M; Chiasson, R B; Decuypere, E; Kühn, E R; Buyse, J; Vandesande, F

    1993-11-01

    The production and characterization of monoclonal antibodies (mAbs) toward the chicken pituitary glycoproteins are described. MAbs were first subdivided on an immunocytochemical (ICC) basis: one category of mAbs reacts with both thyrotropic and gonadotropic cells whereas another stains exclusively gonadotropic cells. The hypothesis that the former are anti-glycoprotein alpha and that the latter are anti-beta LH and/or anti-beta FSH mAbs was further elaborated in a homologous tracer binding assay. The putative anti-beta gonadotropin mAbs exclusively recognize iodinated USDA-cLH-I-1, whereas the supposed anti-alpha mAbs react with both USDA-cLH-I-1 and USDA-cFSH-I-1. The ICC localization of pituitary thyrotropes has been achieved in a double-staining protocol using an anti-LH mAb to subtract specifically the gonadotropic cells from the complete alpha-subunit-containing cell population. The result is a strictly cephalic cell population that is densely granulated in hyperthyroid and hardly detectable in hypothyroid birds. A similar subtractive approach was used to develop a thyroid-stimulating hormone (TSH)-indicator in RIA. The total titer of alpha-containing molecules is first quantified with an anti-alpha mAb. The contribution of both LH and FSH to this titer is then measured by homologous USDA-RIA and subtracted from the total alpha-immunoreactivity to give an indication of the TSH level of the sample. The direct effect of thyroid-releasing hormone on pituitary TSH release in vitro in juvenile chicks and the effect of hypo- and hyperthyroidism on circulating TSH levels in vivo in juvenile broilers have been demonstrated.

  19. Use of monoclonal antibodies prepared against Schistosoma mansoni hatching fluid antigens for demonstration of Schistosoma haematobium circulating egg antigens in urine.

    PubMed

    Nibbeling, H A; Kahama, A I; Van Zeyl, R J; Deelder, A M

    1998-05-01

    A panel of 17 monoclonal antibodies (MAbs) against Schistosoma soluble egg antigens (SEAs) was produced from BALB/c mice immunized with antigens secreted/excreted by Schistosoma mansoni eggs. In this study, we demonstrate that 16 MAbs were reactive with S. haematobium SEA in addition to S. mansoni SEA. The MAbs were tested as potential immunodiagnostic reagents in a homologous sandwich ELISA format to detect circulating soluble egg antigens (CSEAs) in serum and urine samples of S. mansoni- or S. haematobium-infected individuals. When samples of S. mansoni-infected individuals were tested, none of these MAbs performed as good as the previously described S. mansoni-specific 114-5B1-A and 114-4D12-A MAbs. However, 11 MAbs (of the IgM isotype) detected CSEA in urine samples of S. haematobium-infected individuals. Three MAbs, 290-2E6-A, 291-3D5-A, and 291-5D5-A, were selected for a pilot study with 47 urine samples of S. haematobium-infected individuals from Kenya. The CSEA levels detected with each of these ELISAs showed a significant correlation with urinary egg counts (Spearman rho > 0.37, P < 0.01) and with each other (Spearman rho > 0.74, P < 0.001). Based on the 92% specificity and 90% sensitivity of the assay, the ELISA using MAb 290-2E6-A was found to be the most promising assay for immunodiagnosis of S. haematobium infections.

  20. Monoclonal antibodies as diagnostics; an appraisal.

    PubMed

    Siddiqui, M Z

    2010-01-01

    Ever since the development of Hybridoma Technology in 1975 by Kohler and Milstein, our vision for antibodies as tools for research for prevention, detection and treatment of diseases, vaccine production, antigenic characterization of pathogens and in the study of genetic regulation of immune responses and disease susceptibility has been revolutionized. The monoclonal antibodies being directed against single epitopes are homogeneous, highly specific and can be produced in unlimited quantities. In animal disease diagnosis, they are very useful for identification and antigenic characterization of pathogens. Monoclonal antibodies have tremendous applications in the field of diagnostics, therapeutics and targeted drug delivery systems, not only for infectious diseases caused by bacteria, viruses and protozoa but also for cancer, metabolic and hormonal disorders. They are also used in the diagnosis of lymphoid and myeloid malignancies, tissue typing, enzyme linked immunosorbent assay, radio immunoassay, serotyping of microorganisms, immunological intervention with passive antibody, antiidiotype inhibition, or magic bullet therapy with cytotoxic agents coupled with anti mouse specific antibody. Recombinant deoxyribonucleic acid technology through genetic engineering has successfully led to the possibility of reconstruction of monoclonal antibodies viz. chimeric antibodies, humanized antibodies and complementarily determining region grafted antibodies and their enormous therapeutic use.

  1. 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.

  2. 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

  3. Monoclonal Antibodies Against Xenopus Greatwall Kinase

    PubMed Central

    Wang, Ling; Fisher, Laura A.; Wahl, James K.

    2011-01-01

    Mitosis is known to be regulated by protein kinases, including MPF, Plk1, Aurora kinases, and so on, which become active in M-phase and phosphorylate a wide range of substrates to control multiple aspects of mitotic entry, progression, and exit. Mechanistic investigations of these kinases not only provide key insights into cell cycle regulation, but also hold great promise for cancer therapy. Recent studies, largely in Xenopus, characterized a new mitotic kinase named Greatwall (Gwl) that plays essential roles in both mitotic entry and maintenance. In this study, we generated a panel of mouse monoclonal antibodies (MAbs) specific for Xenopus Gwl and characterized these antibodies for their utility in immunoblotting, immunoprecipitation, and immunodepletion in Xenopus egg extracts. Importantly, we generated an MAb that is capable of neutralizing endogenous Gwl. The addition of this antibody into M-phase extracts results in loss of mitotic phosphorylation of Gwl, Plk1, and Cdk1 substrates. These results illustrate a new tool to study loss-of-function of Gwl, and support its essential role in mitosis. Finally, we demonstrated the usefulness of the MAb against human Gwl/MASTL. PMID:22008075

  4. Syngeneic anti-idiotypic monoclonal antibodies to an anti-NeuGc-containing ganglioside monoclonal antibody.

    PubMed

    Vázquez, A M; Pérez, A; Hernández, A M; Macías, A; Alfonso, M; Bombino, G; Pérez, R

    1998-12-01

    An IgM monoclonal antibody (MAb), named P3, has the characteristic to react specifically with a broad battery of N-glycolyl containing-gangliosides and with antigens expressed on breast tumors. When this MAb was administered alone in syngeneic mice, an specific IgG anti-idiotypic antibody (Ab2) response was induced, this Ab2 response was increased when P3 MAb was injected coupled to a carrier protein and in the presence of Freund's adjuvant. Spleen cells from these mice were used in somatic-cell hybridization experiments, using the murine myeloma cell line P3-X63-Ag8.653 as fusion partner. Five Ab2 MAbs specific to P3 MAb were selected. These IgG1 Ab2 MAbs were able to block the binding of P3 MAb to GM3(NeuGc) ganglioside and to a human breast carcinoma cell line. Cross-blocking experiments demonstrated that these Ab2 MAbs are recognizing the same or very close sites on the Abl MAb. The five Ab2 MAbs were injected into syngeneic mice and four of them produced strong anti-anti-idiotypic antibody (Ab3) response. While these Ab2 MAbs were unable to generate Ab3 antibodies with the same antigenic specificity than P3 MAb, three of them induced antibodies bearing P3 MAb idiotopes (Ag-Id+ Ab3). These results demonstrated that these Ab2 MAbs are not "internal image" antibodies, but they could define "regulatory idiotopes."

  5. Breast cancer immunotherapy: monoclonal antibodies and peptide-based vaccines.

    PubMed

    Mohit, Elham; Hashemi, Atieh; Allahyari, Mojgan

    2014-07-01

    Recently, immunotherapy has emerged as a treatment strategy in the adjuvant setting of breast cancer. In this review, monoclonal antibodies in passive and peptide-based vaccines, as one of the most commonly studied in active immunotherapy approaches, are discussed. Trastuzumab, a monoclonal antibody against HER-2/neu, has demonstrated considerable efficacy. However, resistance to trastuzumab has led to development of many targeted therapies which have been examined in clinical trials. Monoclonal antibodies against immune-checkpoint molecules that are dysregulated by tumors as an immune resistance mechanism are also explained in this review. Additionally, monoclonal antibodies with the ability to target breast cancer stem cells that play a role in cancer recurrence are mentioned. Here, clinical trials of HER-2/neu B and T cells, MUC1 and hTERT cancer peptide vaccines are also presented. In addition, various strategies for enhancing vaccine efficacy including combination with monoclonal antibodies and using different delivery systems for peptide/protein-based vaccine are described.

  6. Quantitative analysis of monoclonal antibodies by cation-exchange chromatofocusing.

    PubMed

    Rozhkova, Anna

    2009-08-07

    A robust cation-exchange chromatofocusing method was developed for the routine analysis of a recombinant humanized monoclonal IgG antibody. We compare the chromatofocusing method to the conventional cation-exchange chromatography (CEX) employing a salt gradient and show clear advantages of chromatofocusing over CEX. We demonstrate the suitability of the present chromatofocusing method for its intended purpose by testing the validation characteristics. To our knowledge, this is the first chromatofocusing method developed for the routine analysis of monoclonal antibody charge species.

  7. Novel monoclonal antibodies to study tissue regeneration in planarians.

    PubMed

    Ross, Kelly G; Omuro, Kerilyn C; Taylor, Matthew R; Munday, Roma K; Hubert, Amy; King, Ryan S; Zayas, Ricardo M

    2015-01-21

    Planarians are an attractive model organism for studying stem cell-based regeneration due to their ability to replace all of their tissues from a population of adult stem cells. The molecular toolkit for planarian studies currently includes the ability to study gene function using RNA interference (RNAi) and observe gene expression via in situ hybridizations. However, there are few antibodies available to visualize protein expression, which would greatly enhance analysis of RNAi experiments as well as allow further characterization of planarian cell populations using immunocytochemistry and other immunological techniques. Thus, additional, easy-to-use, and widely available monoclonal antibodies would be advantageous to study regeneration in planarians. We have created seven monoclonal antibodies by inoculating mice with formaldehyde-fixed cells isolated from dissociated 3-day regeneration blastemas. These monoclonal antibodies can be used to label muscle fibers, axonal projections in the central and peripheral nervous systems, two populations of intestinal cells, ciliated cells, a subset of neoblast progeny, and discrete cells within the central nervous system as well as the regeneration blastema. We have tested these antibodies using eight variations of a formaldehyde-based fixation protocol and determined reliable protocols for immunolabeling whole planarians with each antibody. We found that labeling efficiency for each antibody varies greatly depending on the addition or removal of tissue processing steps that are used for in situ hybridization or immunolabeling techniques. Our experiments show that a subset of the antibodies can be used alongside markers commonly used in planarian research, including anti-SYNAPSIN and anti-SMEDWI, or following whole-mount in situ hybridization experiments. The monoclonal antibodies described in this paper will be a valuable resource for planarian research. These antibodies have the potential to be used to better understand

  8. 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.

  9. 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.

  10. A mouse monoclonal antibody against Alexa Fluor 647.

    PubMed

    Wuethrich, Irene; Guillen, Eduardo; Ploegh, Hidde L

    2014-04-01

    Fluorophores are essential tools in molecular and cell biology. However, their application is mostly confined to the singular exploitation of their fluorescent properties. To enhance the versatility and expand the use of the fluorophore Alexa Fluor 647 (AF647), we generated a mouse monoclonal antibody against it. We demonstrate its use of AF647 for immunoblot, immunoprecipitation, and cytofluorimetry.

  11. HLAMatchmaker-based analysis of human monoclonal antibody reactivity demonstrates the importance of an additional contact site for specific recognition of triplet-defined epitopes.

    PubMed

    Duquesnoy, René J; Mulder, Arend; Askar, Medhat; Fernandez-Vina, Marcelo; Claas, Frans H J

    2005-07-01

    Five HLA-A3 reactive human monoclonal antibodies (mAbs) originating from a parous woman were screened against HLA-typed panels by means of complement-dependent lymphocytotoxicity, high-definition ELISA, and flow cytometry with single antigen beads. Antibody reactivity profiles were compared with triplet amino acid sequence polymorphisms identified by HLAMatchmaker, and a three-dimensional structural modeling program (Cn3D of the National Center for Biotechnology Information) was used to determine the topography of epitopes recognized by each mAb. These mAbs originated from a woman who during pregnancy developed antibodies to the paternal HLA-A3 antigen of her child. Each mAb was specific for one mismatched triplet on HLA-A3, and the reactivity patterns of these IgM-type mAbs were practically the same in lymphocytotoxicity and antigen-binding assays. One mAb was specific for 163dT, a unique triplet present only on A3. The other mAbs reacted with 62Qe, 142mI, or 144tKr; these triplets are present on different groups of HLA-A alleles, some of which, however, did not react. Topographic modeling of triplet-defined epitopes identified clusters of polymorphic surface residues that were shared between reactive alleles. These clusters may serve as primary contact sites for the specificity-determining complementarity-determining region (CDR) loops of antibody. The reactivity with these mAbs required also the presence of self-sequence elsewhere on the HLA molecular surface as a critical secondary contact site for antibody, likely through another CDR loop. For instance, the reactivity of the 62Qe-specific mAb required the presence of a glycine residue in position 56 and the reactivity of the 142mI-specific mAb required the presence of the GTLRG sequence in positions 79-83. Conversely, there were many other amino acid differences between the mAb-reactive alleles and HLA-A3 that did not prevent antibody binding. For instance, the 62Qe-specific mAb-reactive alleles had 35 and the

  12. Pathological inclusion bodies in tauopathies contain distinct complements of tau with three or four microtubule-binding repeat domains as demonstrated by new specific monoclonal antibodies.

    PubMed

    de Silva, R; Lashley, T; Gibb, G; Hanger, D; Hope, A; Reid, A; Bandopadhyay, R; Utton, M; Strand, C; Jowett, T; Khan, N; Anderton, B; Wood, N; Holton, J; Revesz, T; Lees, A

    2003-06-01

    Pathological inclusions containing fibrillar aggregates of hyperphosphorylated tau protein are a characteristic feature in the tauopathies, which include Alzheimer's disease, frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy, corticobasal degeneration and Pick's disease. Tau isoform composition and cellular and regional distribution as well as morphology of these inclusions vary in each disorder. Recently, several pathological missense and exon 10 splice-donor site mutations of the tau gene were identified in FTDP-17. Exon 10 codes for the second of four microtubule-binding repeat domains. The splice-site mutations result in increased inclusion of exon 10 which causes a relative increase in tau isoforms containing four microtubule-binding repeat domains over those containing three repeat domains. This could be a central aetiological mechanism in FTDP-17 and, perhaps, other related tauopathies. We have investigated changes in the ratio and distribution of three-repeat and four-repeat tau in the different tauopathies as a basis of the phenotypic range of these disorders and the selective vulnerability of different subsets of neurones. In this study, we have developed two monoclonal antibodies, RD3 and RD4 that effectively distinguish these closely related tau isoforms. These new isoform-specific antibodies are useful tools for analysing tau isoform expression and distribution as well as pathological changes in the human brain.

  13. Chemoenzymatic glyco-engineering of monoclonal antibodies

    PubMed Central

    Giddens, John P.; Wang, Lai-Xi

    2016-01-01

    Summary 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

  14. [Preparation of monoclonal antibody against phosphinothricin acetyltransferase].

    PubMed

    Gao, Xudong; Wang, Yongzhi; Shi, Shengfeng; Li, Zhongpeng; Li, Xiaoyu; Xu, Wenjing; Zhang, Zhengkun; Lu, Yang; Zhang, Jiashi; Li, Qiyun; Wang, Jingang

    2013-05-01

    To express phosphinothricin acetyltransferase (PAT) with biological activity and prepare monoclonal antibodies against PAT. The full length bar gene was cloned by PCR and inserted into prokaryotic expression vector pET28a⁺. The recombinant plasmid pET28-bar was transformed into E.coli BL21(DE3), and under the induction of IPTG, PAT was expressed. The expressed protein was purified by Ni⁺; affinity chromatography to analyze its activity. The purified PAT was used to immunize BALB/c mice, and then the spleen cells from the immunized mice were fused with Sp2/0 cells. The hybridoma clones secreting antibodies against PAT were isolated by indirect ELISA and then subcloned. Soluble PAT was expressed in E.coli. The purified PAT had the activity of acetyltransferase. We totally prepared 9 hybridoma cell lines which secreted specific anti-PAT monoclonal antibodies. The expressed recombinant PAT can be used for biological reagent to prevent and relieve herbicide damage. Monoclonal antibodies against PAT may be used to detect the transgenic products.

  15. Monoclonal antibodies reacting with murine teratocarcinoma cells.

    PubMed Central

    Goodfellow, P N; Levinson, J R; Williams, V E; McDevitt, H O

    1979-01-01

    Monoclonal antibodies were produced in vitro by fusing mouse myeloma cells with spleen cells from a rat immunized with the C3H mouse teratocarcinoma C86-S1. After the fusion two clones were chosen for further analysis. The first clone, 3C4-10, produced an antibody recognizing an antigen with a distribution restricted to teratocarcinoma cell lines, an endoderm cell line, and a neuroblastoma. The second clone, 4A1-9, produced an antibody that reacted with all cultured murine cells tested and adult brain. Neither antibody reacted with preimplantation embryos. The 3C4-10 antibody recognized an antigen associated with proteins. The apparent molecular weight of the 3C4-10 antigen was greater than 100,000. PMID:284353

  16. Monoclonal antibodies to endogenous galactose-specific tumor cell lectins.

    PubMed Central

    Raz, A; Meromsky, L; Carmi, P; Karakash, R; Lotan, D; Lotan, R

    1984-01-01

    A monoclonal antibody, 5D7, was obtained after immunization of syngeneic mice with B16 melanoma cell extracts enriched for endogenous lectin activity and screening for inhibition of lectin-mediated hemagglutination. Binding of this antibody to affinity-purified B16 melanoma galactoside-specific lectin was revealed by solid-phase radioimmunoassay and binding to the surface of viable B16 cells was demonstrated by indirect immunofluorescence. Inhibition of lectin activity and cell surface labeling by 5D7 antibody were also found with several types of cultured human and murine cells including melanoma, sarcoma and carcinoma. This monoclonal antibody should be useful for evaluating the role of tumor cell surface lectins in intercellular interactions and metastasis. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:6084591

  17. Immunoglobulin VH determinants defined by monoclonal antibodies.

    PubMed

    Kubagawa, H; Mayumi, M; Kearney, J F; Cooper, M D

    1982-10-01

    Hybridoma clones secreting antibodies against common VH determinants were readily produced by fusion of cells from mice immunized with isolated V mu fragments of human immunoglobulins (Ig), but not with intact Ig molecules or isolated heavy chains. Four monoclonal antibodies to the V mu fragments of different IgM paraproteins were selected for analysis: MH-44 (mu kappa), GB-24 (mu kappa), NF-11 (gamma 1 kappa), and SA-44 (gamma 1 kappa). Each antibody reacted with the homologous V mu fragment, homologous mu chain, and normal gamma chains, but not with the intact IgM molecules, intact IgG, or isolated light chains, as determined by radioimmunoassay. The VH reaction spectra with a panel of myeloma heavy chains showed overlapping but distinctive patterns for the four antibodies. Each of the four monoclonal anti-VH antibodies appeared to react with a different "hidden" VH determinant that is not exposed on undenatured, intact Ig molecules and differs from conventional VH subgroup determinants. In immunofluorescence studies, the monoclonal anti-VH antibodies did not bind to surface Ig on viable B lymphocytes, but visibly stained subpopulations of fixed B lymphocytes, pre-B cells, and normal plasma cells. The mean frequencies of VH+ plasma cells were 30% (MH-44), 17% (GB-24), 13% (NF-11), and 3% (SA-44), and similar frequencies were obtained for the VH+ B cell subpopulations. While subpopulations of B cells could be identified at all stages in differentiation by immunofluorescence with the anti-VH antibodies, neither resting nor activated T cells expressed these VH determinants in detectable amounts.

  18. Immunoglobulin VH determinants defined by monoclonal antibodies

    PubMed Central

    1982-01-01

    Hybridoma clones secreting antibodies against common VH determinants were readily produced by fusion of cells from mice immunized with isolated V mu fragments of human immunoglobulins (Ig), but not with intact Ig molecules or isolated heavy chains. Four monoclonal antibodies to the V mu fragments of different IgM paraproteins were selected for analysis: MH-44 (mu kappa), GB-24 (mu kappa), NF-11 (gamma 1 kappa), and SA-44 (gamma 1 kappa). Each antibody reacted with the homologous V mu fragment, homologous mu chain, and normal gamma chains, but not with the intact IgM molecules, intact IgG, or isolated light chains, as determined by radioimmunoassay. The VH reaction spectra with a panel of myeloma heavy chains showed overlapping but distinctive patterns for the four antibodies. Each of the four monoclonal anti-VH antibodies appeared to react with a different "hidden" VH determinant that is not exposed on undenatured, intact Ig molecules and differs from conventional VH subgroup determinants. In immunofluorescence studies, the monoclonal anti-VH antibodies did not bind to surface Ig on viable B lymphocytes, but visibly stained subpopulations of fixed B lymphocytes, pre-B cells, and normal plasma cells. The mean frequencies of VH+ plasma cells were 30% (MH-44), 17% (GB-24), 13% (NF-11), and 3% (SA-44), and similar frequencies were obtained for the VH+ B cell subpopulations. While subpopulations of B cells could be identified at all stages in differentiation by immunofluorescence with the anti-VH antibodies, neither resting nor activated T cells expressed these VH determinants in detectable amounts. PMID:6185604

  19. Pulmonary monoclonal antibody delivery via a portable microfluidic nebulization platform

    PubMed Central

    Cortez-Jugo, Christina; Qi, Aisha; Rajapaksa, Anushi; Friend, James R.

    2015-01-01

    Nebulizers have considerable advantages over conventional inhalers for pulmonary drug administration, particularly because they do not require coordinated breath actuation to generate and deliver the aerosols. Nevertheless, besides being less amenable to miniaturization and hence portability, some nebulizers are prone to denature macromolecular drugs due to the large forces generated during aerosolization. Here, we demonstrate a novel portable acoustomicrofluidic device capable of nebulizing epidermal growth factor receptor (EGFR) monoclonal antibodies into a fine aerosol mist with a mass median aerodynamic diameter of approximately 1.1 μm, optimal for deep lung deposition via inhalation. The nebulized monoclonal antibodies were tested for their stability, immunoactivity, and pharmacological properties, which confirmed that nebulization did not cause significant degradation of the antibody. In particular, flow cytometry demonstrated that the antigen binding capability of the antibody is retained and able to reduce phosphorylation in cells overexpressing the EGFR, indicating that the aerosols generated by the device were loaded with stable and active monoclonal antibodies. The delivery of antibodies via inhalation, particularly for the treatment of lung cancer, is thus expected to enhance the efficacy of this protein therapeutic by increasing the local concentration where they are needed. PMID:25945147

  20. 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.

  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. 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

  3. 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

  4. Monoclonal antibodies as blood grouping reagents.

    PubMed

    Voak, D

    1990-04-01

    The large volume requirements for high quality ABO and Rh(D) typing reagents can now be supplied by selected monoclonal antibodies. Superior anti-A and anti-B monoclonal reagents can be prepared, from blends of at least two antibodies, to optimize the intensity of agglutination for slide tests and the potency for the detection of the weaker sub-groups, including Ax and Bw, by tube techniques. New quality control steps have been described for some highly sensitive anti-A/anti-B antibodies to avoid the detection of traces of A on B cells or traces of B on A1 cells, which results from the non-specific activity of A and B transferases. Excellent anti-A,B reagents may also be made by blends of at least two antibodies to optimize both A and B reactions, but the need for their continued use is now debatable. The development of high titre IgM monoclonal anti-D reagents offers simple rapid saline Rh(D) typing of both patients and donors, but they cannot reliably detect weak D (Du) and some D variants, e.g. the epitopes on D category VI cells. However, this can be achieved by blending an IgM anti-D with IgG (polyclonal) anti-D which can detect these types after conversion of negative saline tests to an antiglobulin phase. In addition, high grade Du, D categories and variants can be reliably detected (for typing donors) by selected monoclonal IgM and IgG anti-Ds by use of suitably enhanced tests without the use of an antiglobulin test.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. Monoclonal antibodies against metallothioneins and metalloproteins.

    PubMed

    Talbot, B G; Bilodeau, G; Thirion, J P

    1986-10-01

    Monoclonal antibodies against rabbit metallothioneins (MT) were prepared by in vitro immunization of mouse lymphocytes with a mixture of the two forms of metallothionein MT1 and MT2. Six IgM antibodies (TN1,3,4,5,6,7) which bind to metallothionein were characterized. Antibody TN3 is specific for rabbit MT1 and does not react with any other MT's tested. TN5 is specific for both rabbit MT1 and MT2. TN7 is specific for rabbit MT2 but not MT1 and cross-reacts also with Chinese hamster, mouse and rat metallothioneins. The antibodies TN1, TN4 and TN6 bind not only to rabbit MT1 and MT2 but also to other metal binding proteins like alcohol dehydrogenase and carbonic anhydrase.

  6. Probing myosin head structure with monoclonal antibodies.

    PubMed

    Winkelmann, D A; Lowey, S

    1986-04-20

    Monoclonal antibodies that react with defined regions of the heavy and light chains of chicken skeletal muscle myosin have been used to provide a correlation between the primary and the tertiary structures of the head. Electron microscopy of rotary shadowed antibody-myosin complexes shows that the sites for three epitopes in the 25,000 Mr tryptic fragment (25k) of subfragment-1, including one within 4000 Mr of the amino terminus of the myosin heavy chain, are clustered 145(+/- 20) A from the head-rod junction. An epitope in the 50,000 Mr fragment maps even further out on the head. These antibodies bind to the head in several orientations, suggesting that each of the heads can rotate can rotate 180 degrees about the head-rod junction. The epitopes are accessible on subfragment-1 bound to actin when they were probed with Fab fragments; therefore, none of these heavy chain sites is is on the contact surface between the head and actin. Two of the anti-25k antibodies affect the K+-EDTA-and Ca2+-ATPase activities of myosin in a manner that mimics the effect on activity of the modification of the reactive thiol, SH-1. These two antibodies also inhibit the actin-activated ATPase non-competitively with respect to actin. None of the other eight antibodies tested had any marked effect on activity. A monoclonal antibody that reacts with an epitope in the amino-terminal third of myosin light chain 2 maps close to the head-rod junction. A polyclonal antibody specific for the amino terminus of light chain 3 binds further up in the "neck region" of the head, indicating that these portions of the two classes of light chains are located at different sites.

  7. Monoclonal antibodies as catalysts for cyanide removal

    SciTech Connect

    Cook, C.E.; Whisnant, C.C.; Miller, D.B.; Allen, D.A.; Basta, P.V.

    1993-05-13

    We have shown that hydrogen cyanide reacts with alpha, beta-unsaturated ketones to form stable compounds under physiological conditions (temperature, pH). Although spontaneous reaction is too slow for protection against cyanide intoxication, rate enhancement in the presence of a suitable catalyst would permit the use of alpha, beta-unsaturated ketones (enones) as prophylactics for cyanide exposure. Based on the accepted mechanism for this 1,4-addition reaction, we have designed and synthesized sized a transition state analog (TSA), conjugated it to protein and used the conjugate to produce more than 300 monoclonal antibodies which bind the TSA. Approximately 10% of these antibodies have been purified from ascites and tested for catalysis of the addition reaction of cyanide to enone. Product formation was measured by HPLC. Four antibodies have been found which significantly enhance the initial velocity of the reaction. The TSA markedly diminishes the reaction velocity, indicating the involvement of the antibody binding site in the observed enhancement. Preliminary kinetic analysis on one antibody gave values of K sub (enone) and K sub KCN 51 uM and 9.6 mM, respectively. The value of k sub (cat) was 2.33 hr-1. The data suggest a rate enhancement of 2 x 10 to the 4th power for the encounter of the enone with the antibody-cyanide complex, whereas the rate enhancement for encounter of cyanide with the antibody-enone complex is 70. To utilize the potential of genetic engineering for modifying the proper-lies of anti-TSA monoclonal antibodies, we are cloning heavy and light chain genes for sequencing and subsequent site-specific mutagenesis.

  8. Therapeutic monoclonal antibody for sporotrichosis

    PubMed Central

    Almeida, Sandro R.

    2012-01-01

    Sporotrichosis is a chronic subcutaneous mycosis that affects both humans and animals worldwide. This subcutaneous mycosis had been attributed to a single etiological agent, Sporothrix schenckii. S. schenckii exhibits considerable genetic variability, and recently, it was suggested that this taxon consists of a complex of species. Sporotrichosis is caused by traumatic inoculation of the fungus, which is a ubiquitous environmental saprophyte that can be isolated from soil and plant debris. The infection is limited to cutaneous forms, but recently, more severe clinical forms of this mycosis have been described, especially among immunocompromised individuals. The immunological mechanisms involved in the prevention and control of sporotrichosis are not well understood. Some studies suggest that cell-mediated immunity plays an important role in protecting the host against S. schenckii. In contrast, the role of the humoral immune response in protection against this fungus has not been studied in detail. In a previous study, we showed that antigens secreted by S. schenckii induced a specific humoral response in infected animals, primarily against a 70-kDa molecule, indicating a possible role of specific antibodies against this molecule in infection control. In another study by our group, we produced a mAb against a 70-kDa glycoprotein of S. schenckii to better understand the effect of the passive immunization of mice infected with S. schenckii. The results showed a significant reduction in the number of CFUs in various mice organs when the mAb was injected before or during S. schenckii infection. Similar results were observed when T-cell-deficient mice were used. The drugs of choice in the treatment of sporotrichosis require long periods, and relapses are frequently observed, primarily in immunocompromised patients. The strong protection induced by the mAb against a 70-kDa glycoprotein makes it a strong candidate as a therapeutic vaccine against sporotrichosis. PMID

  9. Microbials for the production of monoclonal antibodies and antibody fragments

    PubMed Central

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

    2014-01-01

    Monoclonal antibodies (mAbs) and antibody fragments represent the most important biopharmaceutical products today. Because full length antibodies are glycosylated, mammalian cells, which allow human-like N-glycosylation, are currently used for their production. However, mammalian cells have several drawbacks when it comes to bioprocessing and scale-up, resulting in long processing times and elevated costs. By contrast, antibody fragments, that are not glycosylated but still exhibit antigen binding properties, can be produced in microbial organisms, which are easy to manipulate and cultivate. In this review, we summarize recent advances in the expression systems, strain engineering, and production processes for the three main microbials used in antibody and antibody fragment production, namely Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli. PMID:24183828

  10. Aggregates in monoclonal antibody manufacturing processes.

    PubMed

    Vázquez-Rey, María; Lang, Dietmar A

    2011-07-01

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

  11. 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.

  12. 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

  13. Idiotypic analysis of a monoclonal anti-Sm antibody.

    PubMed

    Pisetsky, D S; Lerner, E A

    1982-10-01

    Among murine models of autoimmunity, MRL mice are unique in their expression of antibodies to the nuclear antigen Sm. To assess genetic mechanisms in the control of this response, the idiotypes borne by a monoclonal anti-Sm antibody of MRL-Ipr/Ipr origin were investigated. Rabbit antisera were prepared against Y2, a hybridoma product with anti-Sm activity, and were rendered specific for idiotype by extensive absorption with normal globulins from BALB/c mice. In assays of idiotype by an inhibition ELISA, Y2 was shown to share idiotypes with Y12, another monoclonal anti-Sm derived from the same fusion as Y2; other monoclonal autoantibodies of MRL origin but different antigenic specificity failed to display idiotype activity in this assay. The presence of other anti-idiotypic specificities was revealed by absorption and elution of the anti-idiotype from an MRL globulin column; sera from both anti-Sm-positive and negative mice demonstrated these idiotypes. These results suggest that the predominant specificities detected by the anti-idiotype were unique to the monoclonal antibodies of the same animal, although there was also activity to idiotypes not related to anti-Sm binding molecules.

  14. 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.

  15. 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.

  16. Dissecting monoclonal antibody mega-deals

    PubMed Central

    Villiger, Ralph

    2009-01-01

    Monoclonal antibody (mAb) deals notable in size have made headlines recently. While deals with over US$ 1 bio in milestones were highly unusual in the past, even preclinical agreements, including some with very attractive co-promotion or profit-sharing clauses for the licensor, now reach this mark. This article presents an analysis of the structure of high-value mAb deals and the impact of increasingly sophisticated terms. Strategies of licensees and the impact of strategy on a deal's size and structure are also examined. PMID:20061828

  17. Effects of the orientation of anti-BMP2 monoclonal antibody immobilized on scaffold in antibody-mediated osseous regeneration

    PubMed Central

    Ansari, Sahar; Freire, Marcelo; Choi, Moon G; Tavari, Azadeh; Almohaimeed, Mohammad; Moshaverinia, Alireza; Zadeh, Homayoun H

    2015-01-01

    Recently, we have shown that anti-BMP2 monoclonal antibodies (mAbs) can trap endogenous osteogenic BMP ligands, which can in turn mediate osteodifferentiation of progenitor cells. The effectiveness of this strategy requires the availability of the anti-BMP-2 monoclonal antibodies antigen-binding sites for anti-BMP-2 monoclonal antibodies to bind to the scaffold through a domain that will leave its antigen-binding region exposed and available for binding to an osteogenic ligand. We examined whether antibodies bound to a scaffold by passive adsorption versus through Protein G as a linker will exhibit differences in mediating bone formation. In vitro anti-BMP-2 monoclonal antibodies was immobilized on absorbable collagen sponge (ACS) with Protein G as a linker to bind the antibody through its Fc region and implanted into rat calvarial defects. The biomechanical strength of bone regenerated by absorbable collagen sponge/Protein G/anti-BMP-2 monoclonal antibodies immune complex was compared to ACS/anti-BMP-2 monoclonal antibodies or ACS/Protein G/isotype mAb control group. Results demonstrated higher binding of anti-BMP-2 monoclonal antibodies/BMPs to C2C12 cells, when the mAb was initially attached to recombinant Protein G or Protein G-coupled microbeads. After eight weeks, micro-CT and histomorphometric analyses revealed increased bone formation within defects implanted with absorbable collagen sponge/Protein G/anti-BMP-2 monoclonal antibodies compared with defects implanted with absorbable collagen sponge/anti-BMP-2 monoclonal antibodies (p < 0.05). Confocal laser scanning microscopy (CLSM) confirmed increased BMP-2, -4, and -7 detection in sites implanted with absorbable collagen sponge/Protein G/anti-BMP-2 monoclonal antibodies in vivo. Biomechanical analysis revealed the regenerated bone in sites with Protein G/anti-BMP-2 monoclonal antibodies had higher mechanical strength in comparison to anti-BMP-2 monoclonal antibodies. The negative control group, Protein G

  18. 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.

  19. Use of commercially available rabbit monoclonal antibodies for immunofluorescence double staining.

    PubMed

    Bzorek, Michael; Stamp, Inger Merete; Petersen, Bodil Laub; Frederiksen, Lisbeth

    2008-07-01

    Immunohistochemistry, that is, the use of polyclonal and monoclonal antibodies to detect cell and tissue antigens at a microscopical level is a powerful tool for both research and diagnostic purposes. Especially in the field of hematologic disease, there is often a need to detect several antigens synchronously, and we report here a fast and easy technique for demonstrating more than 1 antigen in 1 slide using immunofluorescence. We have used commercially available rabbit monoclonal antibodies (Cyclin D1, CD3, CD5, CD23, etc.) paired with mouse monoclonal antibodies (CD7, CD20, CD79a, Pax-5, etc.) for double immunofluorescence labeling on paraffin-embedded tissue sections. Commercially available rabbit monoclonal antibodies in combination with mouse monoclonal antibodies proved useful in double immunofluorescence labeling on paraffin-embedded tissue, and all combinations used yielded excellent results.

  20. Cellular Mechanisms of CNS Repair by Natural Autoreactive Monoclonal Antibodies

    PubMed Central

    Wright, Brent R.; Warrington, Arthur E.; Edberg, Dale E.; Rodriguez, Moses

    2009-01-01

    Natural autoreactive monoclonal IgMs have demonstrated potential as therapeutic agents for CNS disease. These antibodies bind surface antigens on specific CNS cells activating intracellular repair-promoting signals. IgMs that bind to surface antigens on oligodendrocytes enhanced remyelination in animal models of multiple sclerosis. IgMs that bind to neurons stimulate neurite outgrowth and prevent neuron apoptosis. The neuron-binding IgMs may have utility in CNS axon- or neuron-damaging diseases such as amyotrophic lateral sclerosis, stroke, spinal cord injury or secondary progressive multiple sclerosis. Recombinant remyelination-promoting IgMs have been generated for formal toxicology studies and, after FDA approval, a Phase I clinical trial. Natural autoreactive monoclonal antibodies directed against CNS cells represent novel therapeutic molecules to induce repair of the nervous system. PMID:20008649

  1. 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

  2. Selection of Ceratitis capitata (Diptera: Tephritidae) specific recombinant monoclonal phage display antibodies for prey detection analysis.

    PubMed

    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.

  3. Purification and identification of cell surface antigens using lamprey monoclonal antibodies

    PubMed Central

    Yu, Cuiling; Ali, Shabab; St. Germain, Jonathan; Liu, Yanling; Yu, Xuecong; Jaye, David L.; Moran, Michael F.; Cooper, Max D.; Ehrhardt, Götz R.A.

    2013-01-01

    Variable lymphocyte receptor (VLR) B antibodies of the evolutionary distant sea lamprey are structurally distinct from conventional mammalian antibodies. The different protein architecture and large evolutionary distance of jawless vertebrates suggest that VLR antibodies may represent promising tools for biomarker discovery. Here we report the generation of panels of monoclonal VLR antibodies from lamprey larvae immunized with human T cells and the use of a recombinant monoclonal VLR antibody for antigen purification and mass spectrometric identification. We demonstrate that despite predicted low affinity of individual VLR antigen binding units to the antigen, the high avidity resulting from decameric assembly of secreted VLR antibodies allows for efficient antigen capture and subsequent identification by mass spectometry. We show that VLR antibodies detect their antigens with high specificity and can be used in various standard laboratory application techniques. The lamprey antibodies are novel reagents that can complement conventional monoclonal antibodies in multiple scientific research disciplines. PMID:22964555

  4. Structure and specificity of lamprey monoclonal antibodies.

    PubMed

    Herrin, Brantley R; Alder, Matthew N; Roux, Kenneth H; Sina, Christina; Ehrhardt, Götz R A; Boydston, Jeremy A; Turnbough, Charles L; Cooper, Max D

    2008-02-12

    Adaptive immunity in jawless vertebrates (lamprey and hagfish) is mediated by lymphocytes that undergo combinatorial assembly of leucine-rich repeat (LRR) gene segments to create a diverse repertoire of variable lymphocyte receptor (VLR) genes. Immunization with particulate antigens induces VLR-B-bearing lymphocytes to secrete antigen-specific VLR-B antibodies. Here, we describe the production of recombinant VLR-B antibodies specific for BclA, a major coat protein of Bacillus anthracis spores. The recombinant VLR-B antibodies possess 8-10 uniform subunits that collectively bind antigen with high avidity. Sequence analysis, mutagenesis, and modeling studies show that antigen binding involves residues in the beta-sheets lining the VLR-B concave surface. EM visualization reveals tetrameric and pentameric molecules having a central core and highly flexible pairs of stalk-region "arms" with antigen-binding "hands." Remarkable antigen-binding specificity, avidity, and stability predict that these unusual LRR-based monoclonal antibodies will find many biomedical uses.

  5. Recombinant genetic libraries and human monoclonal antibodies.

    PubMed

    Adams, Jarrett J; Nelson, Bryce; Sidhu, Sachdev S

    2014-01-01

    In order to comprehensively manipulate the human proteome we require a vast repertoire of pharmacological reagents. To address these needs we have developed repertoires of synthetic antibodies by phage display, where diversified oligonucleotides are used to modify the complementarity-determining regions (CDRs) of a human antigen-binding fragment (Fab) scaffold. As diversity is produced outside the confines of the mammalian immune system, synthetic antibody libraries allow us to bypass several limitations of hybridoma technology while improving the experimental parameters under which pharmacological reagents are produced. Here we describe the methodologies used to produce synthetic antibody libraries from a single human framework with diversity restricted to four CDRs. These synthetic repertoires can be extremely functional as they produce highly selective, high affinity Fabs to the majority of soluble human antigens. Finally we describe selection methodologies that allow us to overcome immuno-dominance in our selections to target a variety of epitopes per antigen. Together these methodologies allow us to produce human monoclonal antibodies to manipulate the human proteome.

  6. 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

  7. [Progress in monoclonal antibody-based immunotherapy for cancer treatment].

    PubMed

    Guo, Yajun

    2015-06-01

    More than 100 years ago, Paul Ehrlich first proposed the "magic bullets" concept in which antibody targeting disease related antigen can fight against human disease. Since then, with the development of hybridoma technology for monoclonal antibody production and cancer serum therapy, immunotherapy based monoclonal antibody bas been used in chinical practice to treat hematological and solid tumor. Up to now, more than 20 recombinant antibody drugs were approved for cancer treatment worldwide. In recent years, the next-generation antibody drug, including immune checkpoint antagonists, bi-specific antibody, and antibody drug conjugates have successfully cured various malignant tumor. This review recalled the history of monoclonal antibody as potent immunotherapy of cancer firstly, and focused on the next-generation antibody drug's mechanism of action, construction strategies, and the side effects in clinic. Lastly, the future trend of anti-tumor antibody drug was also discussed.

  8. Immunochemical Characterization of Anti-Acetylcholinesterase Inhibitory Monoclonal Antibodies

    DTIC Science & Technology

    1993-01-01

    formation. This conformation was first proposed using studies with monoclonal antibodies against a synthetic peptide mimicking the sequence of the...distinct antigenic determinants on dengue -2 virus using monoclonal antibodies, Am. J. Trop. Med. Hyg., 31 (1982) 548-555. 7 D. De la Hoz, B.P. Doctor

  9. 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.

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

    PubMed Central

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

    2016-01-01

    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

  11. 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.

  12. Monoclonal antibodies to human urinary thrombopoietin

    SciTech Connect

    McDonald, T.P.; Clift, R.; Cottrell, M.

    1986-01-01

    Monoclonal antibodies (MA) to a thrombocytopoiesis-stimulating factor (TSF or thrombopoietin) were obtained from hybridomas derived from the fusion of P3 x 63/Ag 8 cells and spleen cells from TSF-immunized BALB/c mice. Media from several hybrid cultures were tested in a microantibody detection technique that measured the binding of MA to a /sup 125/I-purified TSF preparation from human embryonic kidney (HEK) cells. Hybridized cells were injected into pristane-primed mice and the antibodies produced in the ascites fluid were also shown to bind the /sup 125/I-TSF. Compared to the results of normal mouse serum, ascites fluid containing MA was shown to bind the unlabeled TSF from HEK cells. The TSF activity was significantly reduced in the supernatant fluid after precipitating the TSF-anti-TSF immune complex by a second antibody when tested in an immunothrombocythemic mouse assay. After SDS-PAGE, the precipitate from this TSF-Ma conjugate showed that the antiserum bound a single 32,000 mol wt component, indicating the monospecificity of the MA. MA directed toward human TSF will allow studies that were not previously possible.

  13. Antibacterial monoclonal antibodies: the next generation?

    PubMed

    DiGiandomenico, Antonio; Sellman, Bret R

    2015-10-01

    There is a clear need for renewed efforts to combat the increasing incidence of antibiotic resistance. While the antibiotic resistance epidemic is due in part to the misuse of antibiotics, even proper empiric antibiotic therapy increases the selective pressure and potential for drug-resistance and spread of resistance mechanisms between bacteria. Antibiotic resistance coupled with the detrimental effects of broad-spectrum antibiotics on the healthy microbiome, have led the field to explore pathogen specific antibacterials such as monoclonal antibodies (mAbs). Medical need along with advances in mAb discovery, engineering, and production have driven significant effort developing mAb-based antibacterials. If successful, they will provide physicians with precision weapons to combat bacterial infections and can help prevent a return to a pre-antibiotic era.

  14. 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

  15. 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.

  16. 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

  17. 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

  18. Monoclonal antibodies against methionyl recombinant human prolactin.

    PubMed

    Paris, N; Robert, R; Mercier, L

    1993-02-01

    Hybridoma cell lines producing monoclonal antibody (Mab) against recombinant human prolactin (rhPrl) were established from fusion between X63-Ag8 myeloma cells and Balb/c mice splenocytes. Four Mabs numbered I to IV were selected by ELISA, purified and characterized. All these Mabs were of the Ig1 kappa isotype and able to recognize oxidized as well as reduced rhPrl. As shown by a competitive inhibition assay, Mab IV did not compete with any of the three others. Moreover, both rhPrl and hPrl extracted from human pituitaries, were recognized equally by this Mab. Properties displayed by Mab IV make it very attractive for the evaluation of prolactin levels by sandwich immunoassays.

  19. 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

  20. Clinical pharmacokinetics of therapeutic monoclonal antibodies.

    PubMed

    Keizer, Ron J; Huitema, Alwin D R; Schellens, Jan H M; Beijnen, Jos H

    2010-08-01

    Monoclonal antibodies (mAbs) have been used in the treatment of various diseases for over 20 years and combine high specificity with generally low toxicity. Their pharmacokinetic properties differ markedly from those of non-antibody-type drugs, and these properties can have important clinical implications. mAbs are administered intravenously, intramuscularly or subcutaneously. Oral administration is precluded by the molecular size, hydrophilicity and gastric degradation of mAbs. Distribution into tissue is slow because of the molecular size of mAbs, and volumes of distribution are generally low. mAbs are metabolized to peptides and amino acids in several tissues, by circulating phagocytic cells or by their target antigen-containing cells. Antibodies and endogenous immunoglobulins are protected from degradation by binding to protective receptors (the neonatal Fc-receptor [FcRn]), which explains their long elimination half-lives (up to 4 weeks). Population pharmacokinetic analyses have been applied in assessing covariates in the disposition of mAbs. Both linear and nonlinear elimination have been reported for mAbs, which is probably caused by target-mediated disposition. Possible factors influencing elimination of mAbs include the amount of the target antigen, immune reactions to the antibody and patient demographics. Bodyweight and/or body surface area are generally related to clearance of mAbs, but clinical relevance is often low. Metabolic drug-drug interactions are rare for mAbs. Exposure-response relationships have been described for some mAbs. In conclusion, the parenteral administration, slow tissue distribution and long elimination half-life are the most pronounced clinical pharmacokinetic characteristics of mAbs.

  1. A Strategy for Screening Monoclonal Antibodies for Arabidopsis Flowers

    PubMed Central

    Shi, Qian; Zhou, Lian; Wang, Yingxiang; Ma, Hong

    2017-01-01

    The flower is one of the most complex structures of angiosperms and is essential for sexual reproduction. Current studies using molecular genetic tools have made great advances in understanding flower development. Due to the lack of available antibodies, studies investigating the localization of proteins required for flower development have been restricted to use commercial antibodies against known antigens such as GFP, YFP, and FLAG. Thus, knowledge about cellular structures in the floral organs is limited due to the scarcity of antibodies that can label cellular components. To generate monoclonal antibodies that can facilitate molecular studies of the flower, we constructed a library of monoclonal antibodies against antigenic proteins from Arabidopsis inflorescences and identified 61 monoclonal antibodies. Twenty-four of these monoclonal antibodies displayed a unique band in a western blot assay in at least one of the examined tissues. Distinct cellular distribution patterns of epitopes were detected by these 24 antibodies by immunofluorescence microscopy in a flower section. Subsequently, a combination of immunoprecipitation and mass spectrometry analysis identified potential targets for three of these antibodies. These results provide evidence for the generation of an antibody library using the total plant proteins as antigens. Using this method, the present study identified 61 monoclonal antibodies and 24 of them were efficiently detecting epitopes in both western blot experiments and immunofluorescence microscopy. These antibodies can be applied as informative cellular markers to study the biological mechanisms underlying floral development in plants. PMID:28293248

  2. Production, characterization, and protective effect of monoclonal antibodies to Clostridium chauvoei flagella.

    PubMed

    Tanaka, M; Hirayama, N; Tamura, Y

    1987-08-01

    Monoclonal antibodies to flagella of Clostridium chauvoei were obtained by the fusion of murine myeloma cells (P3-X63-Ag8-U1) and spleen cells from BALB/c mice immunized with partially purified flagella of strain Okinawa. Enzyme-linked immunosorbent assay and Western blot analysis with partially purified flagella, flagellated cells, and nonflagellated mutants were used to show that five monoclonal antibodies are specific for the flagella. In the Western blot analysis, all five antiflagellar antibodies reacted strongly with the 56,000-molecular-weight protein, which corresponds to the flagellin. By using the ELISA-derived reactivity of monoclonal antibodies to the various clostridia and the competitive binding assay, we showed that the flagella of C. chauvoei had at least three epitopes. The three antiflagellar monoclonal antibodies (one immunoglobulin G and two immunoglobulin M) demonstrated passive protective effects in mice. These results strongly suggest that the flagella of C. chauvoei are important for protective immunity in mice.

  3. 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.

  4. Clinical laboratory applications of monoclonal antibodies.

    PubMed Central

    Payne, W J; Marshall, D L; Shockley, R K; Martin, W J

    1988-01-01

    Monoclonal antibody (MAb) technology is well recognized as a significant development for producing specific serologic reagents to a wide variety of antigens in unlimited amounts. These reagents have provided the means for developing a number of highly specific and reproducible immunological assays for rapid and accurate diagnosis of an extensive list of diseases, including infectious diseases. The impact that MAbs have had in characterizing infectious disease pathogens, as well as their current and future applications for use in clinical microbiology laboratories, is reviewed. In addition, the advantages (and disadvantages) of the use of MAbs in a number of immunoassays, such as particle agglutination, radioimmunoassays, enzyme-linked immunosorbent assays, immunofluorescent-antibody assays, and immunohistology, are explored, including the use of these reagents in novel test system assays. Also, nucleic acid probe technology is compared with the use of MAbs from the perspective of their respective applications in the diagnosis of infectious disease agents. There is no question that hybridoma technology has the potential to alter significantly the methods currently used in most clinical microbiology laboratories. PMID:3058298

  5. A human monoclonal antibody that binds serotype A botulinum neurotoxin.

    PubMed

    Adekar, Sharad P; Jones, R Mark; Elias, M D; Al-Saleem, Fetweh H; Root, Michael J; Simpson, Lance L; Dessain, Scott K

    2008-02-01

    Monoclonal antibodies have demonstrated significant potential as therapeutics for botulinum neurotoxin exposures. We previously described a hybridoma method for cloning native human antibodies that uses a murine myeloma cell line that ectopically expresses the human telomerase catalytic subunit gene (hTERT) and the murine interleukin-6 gene (mIL-6). Here we describe a heterohybridoma cell line that ectopically expresses mIL-6 and hTERT and has improved stability of hTERT expression. We fused this cell line to human peripheral blood B cells from a subject who had received the botulinum toxoid vaccine, cloning a high-affinity antibody (13A) specific for serotype A botulinum neurotoxin (BoNT/A). The 13A antibody is an affinity-matured, post-germinal center IgG(1) lambda antibody that has partial neutralization activity in vivo. 13A binds an epitope on BoNT/A that overlaps the binding epitope of an IgG antibody previously shown to fully neutralize a lethal dose of BoNT/A in vivo. The 13A antibody may be useful for diagnostic testing or for incorporation into an oligoclonal therapeutic to counteract BoNT/A exposure.

  6. Monoclonal antibody treatments for rheumatoid arthritis.

    PubMed

    Bossaller, Lukas; Rothe, Achim

    2013-09-01

    Rheumatoid arthritis (RA) is a systemic disease and the most prevalent of all autoimmune disorders. Here we review recent advances in the development and availability of biologic agents with a focus on monoclonal antibody or smaller formats of targeted engineered therapeutics including novel, non-antibody-based therapeutics. Today an array of biologics blocking either proinflammatory cytokines or lymphocyte activation/survival are available that enable a substantial improvement over conventional disease-modifying antirheumatic drugs (DMARDs). We review the engineering process of antibody-based biologics, their preclinical and clinical application, and current efforts to treat RA by interfering with B-cell function (notable targets covered are CD20, CD38, B-cell activating factor, transmembrane activator and calcium-modulating and cyclophilin interactor), with T-cell function (CD3, CD4, CD28), with bone erosion (RANKL), and with cytokines or growth factors (tumor necrosis factor, interleukin-1 [IL-1], IL-6, IL-17, VEGF). Future treatment choices might encompass the blockade or modulation of danger-associated molecular patterns such as HMGB1, pattern recognition receptors, messenger RNAs or noncoding RNAs, histone acetylation, and inflammasome components. Although current therapies can reduce the signs and symptoms of RA for many patients, the quest for a cure (or a more complete blockade of the structural damage) in RA is still ongoing and will need treatment approaches, which are not exclusively confined to blocking a particular cytokine, receptor, or autoreactive B or T cell involved in disease progression. To this end exciting treatment alternatives and drug targets are on the horizon that may become available to patients in the future.

  7. Characterization of oxidative carbonylation on recombinant monoclonal antibodies.

    PubMed

    Yang, Yi; Stella, Cinzia; Wang, Weiru; Schöneich, Christian; Gennaro, Lynn

    2014-05-20

    In the biotechnology industry, oxidative carbonylation as a post-translational modification of protein pharmaceuticals has not been studied in detail. Using Quality by Design (QbD) principles, understanding the impact of oxidative carbonylation on product quality of protein pharmaceuticals, particularly from a site-specific perspective, is critical. However, comprehensive identification of carbonylation sites has so far remained a very difficult analytical challenge for the industry. In this paper, we report for the first time the identification of specific carbonylation sites on recombinant monoclonal antibodies with a new analytical approach via derivatization with Girard's Reagent T (GRT) and subsequent peptide mapping with high-resolution mass spectrometry. Enhanced ionization efficiency and high quality MS(2) data resulted from GRT derivatization were observed as key benefits of this approach, which enabled direct identification of carbonylation sites without any fractionation or affinity enrichment steps. A simple data filtering process was also incorporated to significantly reduce false positive assignments. Sensitivity and efficiency of this approach were demonstrated by identification of carbonylation sites on both unstressed and oxidized antibody bulk drug substances. The applicability of this approach was further demonstrated by identification of 14 common carbonylation sites on three highly similar IgG1s. Our approach represents a significant improvement to the existing analytical methodologies and facilitates extended characterization of oxidative carbonylation on recombinant monoclonal antibodies and potentially other protein pharmaceuticals in the biotechnology industry.

  8. 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

  9. 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.

  10. Localisation of malignant glioma by a radiolabelled human monoclonal antibody.

    PubMed Central

    Phillips, J; Alderson, T; Sikora, K; Watson, J

    1983-01-01

    Human monoclonal antibodies were produced by fusing intratumoral lymphocytes from patients with malignant gliomas with a human myeloma line. One antibody was selected for further study after screening for binding activity to glioma cell lines. The patient from whom it was derived developed recurrent glioma. 1 mg of antibody was purified, radiolabelled with 131I, and administered intravenously. The distribution of antibody was determined in the blood, CSF and tumour cyst fluid and compared with that of a control human monoclonal immunoglobulin. Antibody localisation in the tumour was observed and confirmed by external scintiscanning. Images PMID:6101173

  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. Cation-exchange chromatography of monoclonal antibodies

    PubMed Central

    Urmann, Marina; Graalfs, Heiner; Joehnck, Matthias; Jacob, Lothar R

    2010-01-01

    A novel cation-exchange resin, Eshmuno™ S, was compared to Fractogel® SO3− (M) and Toyopearl GigaCap S-650M. The stationary phases have different base matrices and carry specific types of polymeric surface modifications. Three monoclonal antibodies (mAbs) were used as model proteins to characterize these chromatographic resins. Results from gradient elutions, stirred batch adsorptions and confocal laser scanning microscopic investigations were used to elucidate binding behavior of mAbs onto Eshmuno™ S and Fractogel® SO3− and the corresponding transport mechanisms on these two resins. The number of charges involved in mAb binding for Eshmuno™ S is lower than for Fractogel® SO3−, indicating a slightly weaker electrostatic interaction. Kinetics from batch uptake experiments are compared to kinetic data obtained from confocal laser scanning microscopy images. Both experimental approaches show an accelerated protein adsorption for the novel stationary phase. The influence of pH, salt concentrations and residence times on dynamic binding capacities was determined. A higher dynamic binding capacity for Eshmuno™ S over a wider range of pH values and residence times was found compared to Fractogel® SO3− and Toyopearl GigaCap S-650M. The capture of antibodies from cell culture supernatant, as well as post-protein A eluates, were analyzed with respect to their host cell protein (hcp) removal capabilities. Comparable or even better hcp clearance was observed at much higher protein loading for Eshmuno™ S than Fractogel® SO3− or Toyopearl GigaCap S-650M. PMID:20559022

  13. 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.

  14. Monoclonal Antibodies Identify Novel Neural Antigens

    NASA Astrophysics Data System (ADS)

    Hawkes, Richard; Niday, Evelyn; Matus, Andrew

    1982-04-01

    Monoclonal antibodies (Mabs) were raised against synaptic plasma membranes from rat cerebellum. The hybridomas were screened with a solid-phase immunoassay, the positive lines were characterized by their immunoperoxidase staining pattern on cerebellum, and the specific polypeptide antigens were identified on protein blots. Among the Mabs described are some that stain only neurons or only glia and others that react with specific parts of cells, such as axons, dendrites, and synapses. Many Mabs reveal novel relationships between antigens and the cells in which they occur. For example, a Mab designated 7D5 reacts with a family of > 30 proteins but stains only glial cells. Several Mabs stain punctate sites of synaptic size and distribution in the cerebellar cortex but each reacts with a different subset of polypeptides. One of the most restricted cytological staining patterns is given by 12D5, which stains punctate sites in the granular layer of the cerebellar cortex and reacts with a single polypeptide band of apparent Mr 270,000. These results illustrate the feasibility of raising Mabs that can be used to follow the expression of specific gene products during brain development.

  15. 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.

  16. A monoclonal antibody against human MUDENG protein.

    PubMed

    Wagley, Yadav; Choi, Jun-Ha; Wickramanayake, Dimuthu Dhammika; Choi, Geun-Yeol; Kim, Chang-Kyu; Kim, Tae-Hyoung; Oh, Jae-Wook

    2013-08-01

    MUDENG (mu-2-related death-inducing gene, MuD) encodes a predicted ∼54-kDa protein in humans, considered to be involved in trafficking proteins from endosomes toward other membranous compartments as well as in inducing cell death. Here we report on the generation of a mouse monoclonal antibody (MAb) against the middle domain of human (h) MuD. This IgG sub 1 MAb, named M3H9, recognizes residues 244-326 in the middle domain of the MuD protein. Thus, the MuD proteins expressed in an astroglioma cell line and primary astrocytes can be detected by the M3H9 MAb. We showed that M3H9 MAb can be useful in enzyme-linked immunosorbent assay (ELISA) and immunoblot experiments. In addition, M3H9 MAb can detect the expression of the MuD protein in formalin-fixed, paraffin-embedded mouse ovary and uterus tissues. These results indicate that the MuD MAb M3H9 could be useful as a new biomarker of hereditary spastic paraplegia and other related diseases.

  17. Viral Epitopes and Monoclonal Antibodies: Isolation of Blocking Antibodies that Inhibit Virus Neutralization

    NASA Astrophysics Data System (ADS)

    Massey, Richard J.; Schochetman, Gerald

    1981-07-01

    The inability of pathogenic animal viruses to be completely neutralized by antibodies can lead to chronic viral infections in which infectious virus persists even in the presence of excess neutralizing antibody. A mechanism that results in this nonneutralized fraction of virus was defined by the topographical relationships of viral epitopes identified with monoclonal antibodies wherein monoclonal antibodies bind to virus and sterically block the binding of neutralizing antibodies.

  18. 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.

  19. DEVELOPMENT OF MONOCLONAL ANTIBODIES AGAINST FATHEAD MINNOW (PIMEPHALES PROMELAS) VITELLOGENIN

    EPA Science Inventory

    We have obtained a panel of monoclonal antibodies directed against fathead minnow vitellogenin (Vtg) for use in sensitive ELISAs to quantify the response of exposure in vivo to estrogen or estrogen mimics.

  20. Adverse cardiac events to monoclonal antibodies used for cancer therapy

    PubMed Central

    Kounis, Nicholas G; Soufras, George D; Tsigkas, Grigorios; Hahalis, George

    2014-01-01

    Monoclonal antibodies are currently used in the treatment of neoplastic, hematological, or inflammatory diseases, a practice that is occasionally associated with a variety of systemic and cutaneous adverse events. Cardiac adverse events include cardiomyopathy, ventricular dysfunction, arrhythmias, arrests, and acute coronary syndromes, such as acute myocardial infarction and vasospastic angina pectoris. These events generally follow hypersensitivity reactions including cutaneous erythema, pruritus chills, and precordial pain. Recently, IgE specific for therapeutic monoclonal antibodies have been detected, pointing to the existence of hypersensitivity and Kounis hypersensitivity-associated syndrome. Therefore, the careful monitoring of cardiovascular events is of paramount importance in the course of monoclonal antibody-based therapies. Moreover, further studies are needed to elucidate the pathophysiology of cardiovascular adverse events elicited by monoclonal antibodies and to identify preventive, protective, and therapeutic measures. PMID:25340003

  1. Daratumumab: a first-in-class CD38 monoclonal antibody for the treatment of multiple myeloma.

    PubMed

    Sanchez, Larysa; Wang, Yucai; Siegel, David S; Wang, Michael L

    2016-06-30

    Daratumumab is a human monoclonal antibody that targets CD38, a cell surface protein that is overexpressed on multiple myeloma (MM) cells. Preclinical studies have shown that daratumumab induces MM cell death through several mechanisms, including complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and apoptosis. Given the encouraging efficacy and acceptable safety profile of daratumumab demonstrated in clinical trials, daratumumab has emerged as a novel treatment option for myeloma and became the first monoclonal antibody approved by the FDA for the treatment of MM.

  2. Monoclonal antibodies against type II rat brain protein kinase

    SciTech Connect

    Nakabayashi, C.H.; Huang, K.P.

    1987-05-01

    Three monoclonal antibodies (8/1, 10/10, and 25/3) against rat brain type II protein kinase C (PKC) were used to carry out the immunochemical characterization of this kinase. These antibodies immunoprecipitated the type II PKC in a dose-dependent manner but did neither to type I nor type III isozyme. Purified type II PKC has a molecular weight of 82,000 and consists of heterogeneous isoelectric point species, all of which are cross reactive with these antibodies. Immunoblot analysis of the tryptic fragments from PKC revealed that all three antibodies recognized the 33-38-KDa fragments, the phospholipid/phorbol ester-binding domain, but not the 45-48-KDa fragments, the kinase catalytic domain. The immune complexes of the kinase and the antibodies retained the kinase activity which was dependent on Ca/sup 2 +/ and phosphatidylserine (PS) and further activated by diacylglycerol. With antibody 8/1, the apparent Km values of the kinase for Ca/sup 2 +/ and PS were not influenced. The initial rate and final extent of autophosphorylation were reduced. The concentration of PS required for half-maximal (/sup 3/H)phorbol 12,13-dibutyrate (PDBu) binding was increased and the total PDBu binding was reduced. In the presence of optimum concentrations of Ca/sup 2 +/ and PS, the Kd of PDBu was unaffected by the antibody but the total binding was reduced. These results demonstrate that the PS/PDBu-binding domain contains the major epitope for the antibodies and the antibody mainly influences the PS/PDBu binding to the kinase.

  3. Use of Human Hybridoma Technology To Isolate Human Monoclonal Antibodies.

    PubMed

    Smith, Scott A; Crowe, James E

    2015-02-01

    The human hybridoma technique offers an important approach for isolation of human monoclonal antibodies. A diversity of approaches can be used with varying success. Recent technical advances in expanding the starting number of human antigen-specific B cells, improving fusion efficiency, and isolating new myeloma partners and new cell cloning methods have enabled the development of protocols that make the isolation of human monoclonal antibodies from blood samples feasible. Undoubtedly, additional innovations that could improve efficiency are possible.

  4. Specific immunofluorescent staining of pathogenic treponemes with a monoclonal antibody.

    PubMed Central

    Ito, F; Hunter, E F; George, R W; Pope, V; Larsen, S A

    1992-01-01

    Two hybrid cell lines which produced mouse monoclonal antibody to the DAL-1 street strain of Treponema pallidum subsp. pallidum were established. These monoclonal antibodies strongly reacted with T. pallidum subsp. pallidum (Nichols strain, DAL-1, and two other street strains, strains MN-1 and MN-3) and T. pallidum subsp. pertenue by indirect microimmunofluorescent antibody and enzyme-linked immunosorbent assay techniques, but they did not react with normal rabbit testicular tissue. These monoclonal antibodies did not react with nonpathogenic treponemes, such as T. phagedenis Reiter, T. denticola MRB, T. refringens Noguchi, or other spirochetes, such as Borrelia burgdorferi and Leptospira interrogans serovar pomona in microimmunofluorescent antibody smear slides or in Western blots (immunoblots). While unlabeled antibodies are useful for investigating the antigenic structures of T. pallidum, we labeled these monoclonal antibodies with fluorescein isothiocyanate and used them for diagnosing syphilis by direct staining of lesion exudate or T. pallidum subsp. pallidum in formalin-fixed tissues from patients suspected of having syphilis. Both monoclonal antibodies were directed against antigens of T. pallidum subsp. pallidum with a molecular weight of 37,000 as determined by the Western blotting technique. Images PMID:1374079

  5. 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.

  6. Invasion of erythrocytes in vitro by Plasmodium falciparum can be inhibited by monoclonal antibody directed against an S antigen.

    PubMed

    Saul, A; Cooper, J; Ingram, L; Anders, R F; Brown, G V

    1985-11-01

    A monoclonal antibody has been produced which binds to the heat stable S antigen present in the FCQ-27/PNG isolate of Plasmodium falciparum. This monoclonal antibody also inhibits the invasion in vitro of erythrocytes by malarial merozoites thus demonstrating that the S antigens of Plasmodium falciparum may be a target of protective immune responses.

  7. [Comparative studies on monoclonal antibody KM10 and anti-CEA monoclonal antibodies].

    PubMed

    Soyama, N; Yamamoto, M; Ohyanagi, H; Saitoh, Y

    1989-11-01

    The specificity of KM10 was evaluated in comparison with newly developed anti-CEA monoclonal antibodies (A10, B9, JA4, AH3). Both KM10 and all anti-CEA monoclonal antibodies reacted with CEA in ELISA system, and with adenocarcinoma of the stomach, colon, and pancreas in the immunohistochemical assay. B9, JA4, and AH3 were suggested to react with CEA related antigens, such as NCA and BGPI, whereas KM10 and A10 were suggested to recognize the distinctive part of CEA. The antigenic determinant of CEA reactive with KM10 and A10 was revealed to be protein moiety after enzyme treatment. The competitive binding inhibition assay, however, indicated that epitopes of KM10 and A10 were different each other. Enzyme immunoassay using both KM10 and A10 could detect CEA. These findings showed the possible use of both KM10 and A10 for clinical diagnosis and treatment by means of targeting for the distinctive part of CEA.

  8. 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.

  9. Anti-schistosome monoclonal antibodies of different isotypes--correlation with cytotoxicity.

    PubMed Central

    Horowitz, S; Tarrab-Hazdai, R; Eshhar, Z; Arnon, R

    1983-01-01

    Five monoclonal antibodies specific towards Schistosoma mansoni antigens were prepared by fusion of spleen cells of infected and immunized mouse with the murine myeloma NS-1 cells. Three of the five antibodies belonged to the IgG1 class, one was an IgM and the fifth one was an IgE. The IgE monoclonal antibody designated 54.10, induced antigen-specific degranulation of rat basophilic cell line, a property which served as the basis for the screening assay. Its biological function was demonstrated by a specific macrophage activation that led to killing of schistosomula; no such killing was obtained with anti-schistosome antibodies of other classes or with IgE of different antigenic specificity. The second monoclonal antibody of biological significance was an IgG1, designated 27.21 which is reactive in the immunofluorescence staining of surface antigens on intact schistosomula. All three monoclonal antibodies that belonged to the IgG1 class were effective in mediating killing of schistosomula by complement, with the highest effect exerted by 27.21. It is thus apparent that the 27.21 monoclonal antibody is directed against a densely distributed surface antigen on the schistosomula membrane which is possibly involved in the protective immunity. Preliminary data showed that immunoprecipitation with the 27.21 antibodies results in the isolation of three major protein bands, of 60 kd, 50 kd, 19 kd, respectively. Images Fig. 2. Fig. 4. PMID:11894925

  10. Characterization of monoclonal antibodies against Gnathostoma nipponicum.

    PubMed

    Ikadai, H; Fujii, T; Nagai, T; Yoshioka, K; Nagasao, J; Kudo, N; Oyamada, T

    2003-02-01

    Monoclonal antibodies (mAbs) were produced against the proteins of advanced third-stage larvae (AdL3) of Gnathostoma nipponicum. Six mAbs (Gn2C3, Gn2H3, Gn4C3, Gn4E9, GnSH1, and Gn10B7) were obtained as determined by enzyme-linked immunosorbent assay (ELISA). Gn4E9 and GnSH1 seemed to be genus-specific, as they did not cross-react with Anisakis sp., Dirofilaria immitis, Gongylonema pulchrum, Toxocara canis, Trichinella sp., Trichuris vulpis, Metagonimus sp., or Spirometra erinaceieuropaei by ELISA. Immunohistochemistry showed that Gn2C3, Gn4E9, and Gn5H1 reacted strongly with the central esophagus; Gn2H3 reacted with cuticle,muscle, intestine, and the cervical sac; and Gn4C3 and Gn10B7 reacted with cuticle, muscle, esophagus, intestine, and the cervical sac of AdL3. In Western blotting analysis, Gn2C3, Gn4E9, and Gn5H1 reacted to 60-, 53-, 46-, and 41-kDa proteins; Gn4C3 reacted to the AdL3 protein of G. nipponicum (>42 kDa). Moreover, proteins purified using a mAb Gn4E9 immunoprecipitation method (sizes 60-, 53-, 46-, and 41-kDa) were used as antigens in ELISAs. A significant difference (P < 0.01) was shown between mouse sera infected with G. nipponicum and sera infected with Trichnella sp. or not infected. These results provide a rationale for evaluating esophageal proteins for the development of diagnostic methods for detecting G. nipponicum or Gnathostoma sp. infections.

  11. Application of monoclonal antibodies for antigen mapping of male and female generative tissue.

    PubMed

    Mettler, L

    1985-01-01

    With the single experiment of nature hindering the rejection of the fetus in the mother, new basic mechanisms of this immunological phenomenon can be studied. The technique of monoclonal antibodies provides a helpful tool for the study of membrane structural and cytoplasmic molecules. Hybridoma clones offer for the first time the possibility of obtaining unlimited amounts of almost pure antibodies or very high titers and of defined characteristics. The most common application with reproductive tract monoclonals are found as follows: 1) Immuno-assays; 2) Immuno-diagnostics; 3) Imaging of tumors; 4) Immuno-therapy; 5) Induction of anti-idio-types; 6) Fertility control; 7) Application in basic studies and mechanisms. The following interesting results were obtained in this area: 1) The biological role of hormones can be critically analysed by the use of highly specific non-cross-reactive monoclonal antibodies. 2) A synthetic decapeptide earlier shown by our group to be reactive with naturally occurring human iso and autosperm antibodies demonstrated to be reactive with the monoclonal antibodies Ki VII-5 presenting potentials for an investigation of sperm as target of immunological contraception for possible diagnostic and therapeutic use in immunologic infertility. 3) A specific post fertilization-development influencing antigen was identified by Menge using monoclonal antibodies. 4) Five hybridoma clones producing monoclonals to porcine zona pellucida were established, and showed a characteristic staining pattern of oocytes from human eggs, hamsters, rabbits and mice by immunofluorescence.

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. Immunoblotting with monoclonal antibodies: importance of the blocking solution.

    PubMed

    Hauri, H P; Bucher, K

    1986-12-01

    Four commonly used blocking agents, i.e., fetal calf serum, mammalian gelatin-Nonidet-P40, fish gelatin-Nonidet-P40, and defatted powdered milk were compared with respect to their efficiency to block the nonspecific background and to promote maximal immunoreactivity of monoclonal antibodies against human intestinal sucrase-isomaltase during immunoblotting. Two of five monoclonal antibodies were found to react with the electroblotted enzyme. However, one of the reacting antibodies gave optimal results with fish gelatin-Nonidet-P40 and the other with defatted powdered milk, while fetal calf serum lead to unacceptably high backgrounds. The results suggest that some of the difficulties encountered with monoclonal antibodies in immunoblotting may be due to inappropriate blocking conditions.

  17. [Production and characteristics of monoclonal antibodies to the diphtheria toxin].

    PubMed

    Valiakina, T I; Lakhtina, O E; Komaleva, R L; Simonova, M A; Samokhvalova, L V; Shoshina, N S; Kalinina, N A; Rubina, A Iu; Filippova, M A; Vertiev, Iu V; Grishin, E V

    2009-01-01

    Monoclonal antibodies to the diphtheria toxin were produced without cross reactivity with the thermolabile toxin (LT) from Escherichia coli; ricin; choleraic toxin; the SeA, SeB, SeE, SeI, and SeG toxins of staphylococcus; the lethal factor of the anthrax toxin; and the protective antigen of the anthrax toxin. A pair of antibodies for the quantitative determination of the diphtheria toxin in the sandwich variation of enzyme-linked immunosorbent assay (ELISA) was chosen. The determination limit of the toxin was 0.7 ng/ml in plate and 1.6 ng/ml in microchip ELISA. The presence of a secretion from the nasopharynx lavage did not decrease the sensitivity of the toxin determination by sandwich ELISA. The immunization of mice with the diphtheria toxin and with a conjugate of the diphtheria toxin with polystyrene microspheres demonstrated that the conjugate immunization resulted in the formation of hybridoma clones which produced antibodies only to the epitopes of the A fragment of the diphtheria toxin. The immunization with the native toxin caused the production of hybridoma clones which predominantly produced antibodies to the epitopes of the B fragment.

  18. 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.

  19. [Preparation and Assessment of IL1RAP Monoclonal Antibody].

    PubMed

    Yin, Ling-Ling; Ruan, Su-Hong; Tian, Yu; Xu, Kai Lin; Zhao, Kai

    2015-08-01

    To prepare and identify human monoclonal antibody against IL-1 receptor accessory protein (IL1RAP), which is a new identified surface marker for leukemia stem cells (LSC). BALB/c mice were inoculated intraperitoneally with hybridoma cells (3H6E10, 10D8A7) and their ascites were collected. The monoclonal antibody against hu-IL1RAP specifically was purified from ascites, the nondenaturing-PAGE, ELISA and Western blot were used to detect the purity, titer and sensitivity of antibody. Two purified antibodies were obtained and named as 3H6E10 McAb and 10D8A7 McAb, whose purity was 95% and 94% respectively. The titer of two purified monoclonal antibodies was 1 : 81000 and specific conjugation of IL1RAP purified protein and endogenous protein from normal people and leukemia patients with purified antibodies were confirmed. The purified monoclonal antibodies which can specifically bind to hu-IL1RAP are successfully prepared, thus providing novel way to effectively clear LSC in the future.

  20. Two novel anti-von Willebrand factor monoclonal antibodies.

    PubMed

    Spadafora-Ferreira, M; Lopes, A A; Coelho, V; Guilherme, L; Kalil, J

    2000-01-15

    Von Willebrand Factor is a multimer produced by endothelial cells and megakaryocytes, being stored in intracellular organelles, such as the Weibel-Palade bodies and alpha-granules in endothelial cells and platelets, respectively. This molecule acts as a carrier protein for factor VIIIc, involved in the intrinsic pathway of blood coagulation maintaining its stability in circulation. Von Willebrand Factor also plays an important role in platelet aggregation and adhesion to injured vessel wall. It interacts with platelets through two distinct glycoproteins, GPIb and GPIIb/IIIa. We raised two monoclonal antibodies, ECA-3 and ECA-4, against human umbilical vascular endothelial cells that recognize and immunoprecipitate von Willebrand Factor. Interestingly, ECA-4 monoclonal antibody is able to completely inhibit platelet agglutination induced by ristocetin, suggesting that it binds to von Willebrand Factor close to platelet GPIb binding site. The use of monoclonal antibodies to identify von Willebrand Factor binding regions to factor VIII or platelets has been reported by others. In pulmonary hypertension, abnormalities have been detected on the multimeric structure of the molecule as well as on its proteolytic fragments, by using monoclonal antibodies. Moreover, monoclonal antibodies raised against specific regions of von Willebrand Factor molecule may allow studies of functional abnormalities of this protein in inherited and acquired disorders like subtypes of von Willebrand's disease.

  1. New monoclonal antibodies on the horizon in multiple myeloma

    PubMed Central

    O’Donnell, Elizabeth K.; Raje, Noopur S.

    2016-01-01

    Across all cancers, monoclonal antibodies have emerged as a potential strategy for cancer therapy. Monoclonal antibodies target antigens expressed on the surface of cancer cells and accessory cells. This targeted approach uses the host’s immune system to promote the killing of cancer cells. Multiple myeloma (MM) is the second most common hematologic malignancy that remains incurable in the majority of patients. The treatment of MM has evolved dramatically over the past decade and continues to evolve with the approval of four new drugs in 2015. Most recently the United States Food and Drug Administration (US FDA) approved two monoclonal antibodies for the treatment of this disease. Monoclonal antibodies are generally well-tolerated and offer a novel method of action for treated relapsed and refractory disease and are now being studied in the upfront setting. In this article, we review the evidence for the existing approved monoclonal antibodies and discuss promising targeted therapies and innovative strategies for the treatment of MM. PMID:28203341

  2. Serological characterization of polycyclic aromatic hydrocarbon diolepoxide-DNA adducts using monoclonal antibodies.

    PubMed

    Newman, M J; Weston, A; Carver, D C; Mann, D L; Harris, C C

    1990-11-01

    Polycyclic aromatic hydrocarbons (PAHs) are a group of structurally related compounds that are present in the environment in complex mixtures as common pollutants. These compounds have been studied extensively because of their carcinogenic and toxic properties to humans. We reported previously that humans exposed to certain PAHs produce antibodies that bind to different PAH diolepoxide-DNA (PAH-DNA) adducts. The ability to detect and measure antibodies to PAH-DNA adducts in human blood samples could prove useful as a biological dosimeter for identifying persons that have been exposed to high levels of PAHs, i.e. persons who may be at high cancer risk. In our initial studies we found that it was common for persons who were exposed to PAH to produce antibodies against PAH-DNA adducts. However, we were unable to identify the actual chemical types of PAH-DNA adducts that were recognized by the serum antibodies because many serum samples contained antibody activity to more than one adduct. These data indicate that different PAH-DNA adducts may be serologically similar or that humans actually produce immune responses against more than a single PAH-DNA adduct. We have used monoclonal antibody technology to determine the extent to which different PAH-DNA adducts share serologically recognized epitopes. Monoclonal antibodies were produced against two different PAH-DNA adducts, benzo[a]pyrene diolepoxide-DNA (BPDE-DNA) and benz[a]anthracene diolepoxide-DNA (BADE-DNA). The binding of these antibodies to five PAH-DNA adduct preparations and to soluble PAHs was assessed. We found that most monoclonal antibodies bound to more than a single type of PAH-DNA adduct, documenting the serological relatedness of different PAH-DNA adducts. However, two monoclonal antibodies were produced that bound only to BPDE-DNA. Soluble non-metabolized PAHs and PAH tetraols were not recognized by these antibodies, thus demonstrating their specificity for PAH-DNA adducts and not the PAHs alone

  3. Monoclonal antibodies provide specific intramolecular markers for the study of epithelial tonofilament organization

    PubMed Central

    1982-01-01

    The tonofilament-associated protein antigens recognized in epithelial cells by a group of six monoclonal antibodies have been studied by immunofluorescence and gel immunoautoradiography. The monoclonal antibodies were generated against detergent insoluble cytoskeleton extracts from a cultured simple epithelium derived cell line, Ptk1 cells. They show various tissue specificities, and while they all recognize components at the low end of the molecular weight range for intermediate filament proteins, they confirm that single antibody species can react with multiple polypeptides of different molecular weights in the tonofilament complex. The monoclonal antibodies described here demonstrate the presence of a simple epithelium antigenic determinant associated with intermediate filaments that is not detectable in the specialized cells of squamous and keratinizing epithelia but can reappear in such cells after transformation. PMID:6177700

  4. 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

  5. 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.

  6. Impact of cell culture on recombinant monoclonal antibody product heterogeneity.

    PubMed

    Liu, Hongcheng; Nowak, Christine; Shao, Mei; Ponniah, Gomathinayagam; Neill, Alyssa

    2016-09-01

    Recombinant monoclonal antibodies are commonly expressed in mammalian cell culture and purified by several steps of filtration and chromatography. The resulting high purity bulk drug substance still contains product variants differing in properties such as charge and size. Posttranslational modifications and degradations occurring during cell culture are the major sources of heterogeneity in bulk drug substance of recombinant monoclonal antibodies. The focus of the current review is the impact of cell culture conditions on the types and levels of various modifications and degradations of recombinant monoclonal antibodies. Understanding the relationship between cell culture and product variants can help to make consistently safe and efficacious products. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1103-1112, 2016. © 2016 American Institute of Chemical Engineers.

  7. The Case for Adjunctive Monoclonal Antibody Immunotherapy in Schizophrenia.

    PubMed

    Miller, Brian J; Buckley, Peter F

    2016-06-01

    This article presents the case in favor of clinical trials of adjunctive monoclonal antibody immunotherapy in schizophrenia. Evidence for prenatal and premorbid immune risk factors for the development of schizophrenia in the offspring is highlighted. Then key evidence for immune dysfunction in patients with schizophrenia is considered. Next, previous trials of adjunctive anti-inflammatory or other immunotherapy in schizophrenia are discussed. Then evidence for psychosis as a side effect of immunotherapy for other disorders is discussed. Also presented is preliminary evidence for adjunctive monoclonal antibody immunotherapy in psychiatric disorders. Finally, important considerations in the design and implementation of clinical trials of adjunctive monoclonal antibody immunotherapy in schizophrenia are discussed. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Monoclonal antibodies specific for African swine fever virus proteins.

    PubMed Central

    Sanz, A; García-Barreno, B; Nogal, M L; Viñuela, E; Enjuanes, L

    1985-01-01

    We have obtained 60 stable hybridomas which produced immunoglobulins that recognized 12 proteins from African swine fever virus particles and African swine fever virus-infected cells. Most of the monoclonal antibodies were specific for the three major structural proteins p150, p72, and p12. The specificity of some monoclonal antibodies for the structural proteins p150 and p37 and the nonstructural proteins p220 and p60 indicated that proteins p150 and p220 are antigenically related to proteins p37 and p60. The association of some viral antigens to specific subcellular components was determined by immunofluorescence and analysis of the binding of monoclonal antibodies to infected cells. A host protein (p24) seemed to be associated with the virus particles. Images PMID:3882998

  9. 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.

  10. Belimumab: anti-BLyS human monoclonal antibody, anti-BLyS monoclonal antibody, BmAb, human monoclonal antibody to B-lymphocyte stimulator.

    PubMed

    2008-01-01

    Belimumab is a fully human monoclonal antibody that specifically recognizes and inhibits the biological activity of B-lymphocyte stimulator, or BLyS. Belimumab is in phase III trials for the treatment of systemic lupus erythematosus (SLE) and has completed a phase II trial in rheumatoid arthritis (RA); the product may also have potential in the treatment of other autoimmune disorders. In May 2001, Cambridge Antibody Technology (now MedImmune) completed its discovery programme and Human Genome Sciences identified belimumab as a candidate for clinical development. More than 1000 distinct human antibodies specific to BLyS were characterized by the collaboration.B-lymphocyte stimulator is a naturally occurring protein discovered by Human Genome Sciences that stimulates B-lymphocytes to develop into mature B cells. Laboratory studies have indicated that higher than normal levels of B-lymphocyte stimulator may contribute to the pathogenesis of autoimmune diseases, such as SLE and RA. Human Genome Sciences (HGS) and Cambridge Antibody Technology signed a collaborative agreement in August 1999 to study the B-lymphocyte stimulator as a human protein target. HGS is also developing other BLyS products. In March 2000, HGS and Cambridge Antibody Technology expanded their agreement into a 10-year collaboration and product development alliance, providing Human Genome Sciences with the right to use the antibody technology of Cambridge Antibody Technology to fully develop human antibodies for therapeutic and diagnostic purposes. Cambridge Antibody Technology will receive royalty payments on product sales from HGS, as well as the development and milestone payments it has already received. Belimumab will be manufactured in Human Genome Sciences' manufacturing facility, located in Rockville, MD, USA. HGS holds commercial rights to the drug. In July 2005, GlaxoSmithKline (GSK) exercised its co-development and co-promotion option to belimumab. In an agreement made in June 1996, HGS had

  11. Neutralizing human monoclonal antibodies prevent Zika virus infection in macaques.

    PubMed

    Magnani, Diogo M; Rogers, Thomas F; Beutler, Nathan; Ricciardi, Michael J; Bailey, Varian K; Gonzalez-Nieto, Lucas; Briney, Bryan; Sok, Devin; Le, Khoa; Strubel, Alexander; Gutman, Martin J; Pedreño-Lopez, Núria; Grubaugh, Nathan D; Silveira, Cassia G T; Maxwell, Helen S; Domingues, Aline; Martins, Mauricio A; Lee, David E; Okwuazi, Erica E; Jean, Sherrie; Strobert, Elizabeth A; Chahroudi, Ann; Silvestri, Guido; Vanderford, Thomas H; Kallas, Esper G; Desrosiers, Ronald C; Bonaldo, Myrna C; Whitehead, Stephen S; Burton, Dennis R; Watkins, David I

    2017-10-04

    Therapies to prevent maternal Zika virus (ZIKV) infection and its subsequent fetal developmental complications are urgently required. We isolated three potent ZIKV-neutralizing monoclonal antibodies (nmAbs) from the plasmablasts of a ZIKV-infected patient-SMZAb1, SMZAb2, and SMZAb5-directed against two different domains of the virus. We engineered these nmAbs with Fc LALA mutations that abrogate Fcγ receptor binding, thus eliminating potential therapy-mediated antibody-dependent enhancement. We administered a cocktail of these three nmAbs to nonhuman primates 1 day before challenge with ZIKV and demonstrated that the nmAbs completely prevented viremia in serum after challenge. Given that numerous antibodies have exceptional safety profiles in humans, the cocktail described here could be rapidly developed to protect uninfected pregnant women and their fetuses. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  12. The clinical application of monoclonal antibody therapies in renal transplantation.

    PubMed

    Dhanireddy, Kiran K; Xu, He; Mannon, Roslyn B; Hale, Douglas A; Kirk, Allan D

    2004-05-01

    Monoclonal antibodies have become valuable tools for the precise clinical manipulation of the immune system. These highly specific proteins have proven their usefulness in both the treatment and prevention of organ transplant rejection. Indeed, they are the centrepieces of many evolving regimens designed to reduce or eliminate the need for chronic immunosuppression. This manuscript will review the monoclonal antibodies that have made their way into the clinic either as experimental therapies or approved drugs. It will provide a general overview of this class of agents and their mechanisms of action. Standard therapies and potential new applications will be described.

  13. Therapeutic monoclonal antibodies in human breast milk: a case study.

    PubMed

    Ross, Elle; Robinson, Steven E; Amato, Carol; McMillan, Colette; Westcott, Jay; Wolf, Tiffany; Robinson, William A

    2014-04-01

    Recently, therapeutic monoclonal antibodies have been introduced for the treatment of advanced melanoma and other diseases. It remains unclear whether these drugs can be safely administered to women who are breast feeding because of the potential hazardous side effects for nursing infants. One such therapy for metastatic melanoma is ipilimumab, a human monoclonal antibody that blocks cytotoxic T-lymphocyte-antigen-4, and is the preferred treatment for patients with metastatic melanoma when other molecular therapies are not viable. This study measured ipilimumab levels in the breast milk of a patient undergoing treatment that were enough to raise concerns for a nursing infant exposed to ipilimumab.

  14. ERBB oncogene proteins as targets for monoclonal antibodies.

    PubMed

    Polanovski, O L; Lebedenko, E N; Deyev, S M

    2012-03-01

    General properties of the family of tyrosine kinase ERBB receptors are considered in connection with their role in the generation of cascades of signal transduction in normal and tumor cells. Causes of acquisition of oncogene features by genes encoding these receptors and their role in tumorigenesis are analyzed. Anti-ERBB monoclonal antibodies approved for therapy are described in detail, and mechanisms of their antitumor activity and development of resistance to them are reviewed. The existing and the most promising strategies for creating and using monoclonal antibodies and their derivatives for therapy of cancer are discussed.

  15. Novel Clostridium difficile Anti-Toxin (TcdA and TcdB) Humanized Monoclonal Antibodies Demonstrate In Vitro Neutralization across a Broad Spectrum of Clinical Strains and In Vivo Potency in a Hamster Spore Challenge Model

    PubMed Central

    Qiu, Hongyu; Cassan, Robyn; Johnstone, Darrell; Han, Xiaobing; Joyee, Antony George; McQuoid, Monica; Masi, Andrea; Merluza, John; Hrehorak, Bryce; Reid, Ross; Kennedy, Kieron; Tighe, Bonnie; Rak, Carla; Leonhardt, Melanie; Dupas, Brian; Saward, Laura; Berry, Jody D.; Nykiforuk, Cory L.

    2016-01-01

    Clostridium difficile (C. difficile) infection (CDI) is the main cause of nosocomial antibiotic-associated colitis and increased incidence of community-associated diarrhea in industrialized countries. At present, the primary treatment of CDI is antibiotic administration, which is effective but often associated with recurrence, especially in the elderly. Pathogenic strains produce enterotoxin, toxin A (TcdA), and cytotoxin, toxin B (TcdB), which are necessary for C. difficile induced diarrhea and gut pathological changes. Administration of anti-toxin antibodies provides an alternative approach to treat CDI, and has shown promising results in preclinical and clinical studies. In the current study, several humanized anti-TcdA and anti-TcdB monoclonal antibodies were generated and their protective potency was characterized in a hamster infection model. The humanized anti-TcdA (CANmAbA4) and anti-TcdB (CANmAbB4 and CANmAbB1) antibodies showed broad spectrum in vitro neutralization of toxins from clinical strains and neutralization in a mouse toxin challenge model. Moreover, co-administration of humanized antibodies (CANmAbA4 and CANmAbB4 cocktail) provided a high level of protection in a dose dependent manner (85% versus 57% survival at day 22 for 50 mg/kg and 20 mg/kg doses, respectively) in a hamster gastrointestinal infection (GI) model. This study describes the protective effects conferred by novel neutralizing anti-toxin monoclonal antibodies against C. difficile toxins and their potential as therapeutic agents in treating CDI. PMID:27336843

  16. Anti-interleukin-10R1 monoclonal antibody in combination with bacillus Calmette–Guérin is protective against bladder cancer metastasis in a murine orthotopic tumour model and demonstrates systemic specific anti-tumour immunity

    PubMed Central

    Newton, M R; Askeland, E J; Andresen, E D; Chehval, V A; Wang, X; Askeland, R W; O'Donnell, M A; Luo, Y

    2014-01-01

    Effective treatment of bladder cancer with bacillus Calmette–Guérin (BCG) depends on the induction of a T helper type (Th) 1 immune response. Interleukin (IL)-10 down-regulates the Th1 response and is associated with BCG failure. In this study, we investigated whether blocking IL-10 signalling could enhance the BCG-induced Th1 response and anti-tumour immunity in a murine orthotopic tumour model. Treatment with BCG and anti-IL-10 receptor 1 monoclonal antibody (anti-IL-10R1 mAb) increased the interferon (IFN)-γ to IL-10 ratio in both splenocyte cultures and urine. Mice bearing luciferase-expressing MB49 (MB49-Luc) tumours were treated and followed for tumour growth by bioluminescent imaging, bladder weight and histology. Mice treated with phosphate-buffered saline (PBS) (group 1), BCG plus control immunoglobulin (Ig)G1 (group 2) or BCG plus anti-IL-10R1 mAb (group 3) showed 0, 6 and 22% tumour regression, respectively. The mean bladder weight of group 3 mice was substantially lower than those of groups 1 and 2 mice. Remarkably, 36% of group 1 and 53% of group 2 mice but no group 3 mice developed lung metastasis (P = 0·02). To investigate the mechanisms underlying the effect of combination therapy, splenocytes were stimulated with S12 peptide (serine mutation at codon 12 of the K-ras oncogene) known to be expressed in MB49-Luc cells. Induction of ras mutation-specific IFN-γ and cytotoxicity was observed in mice treated with combination therapy. These observations indicate that BCG, in combination with anti-IL-10R1 mAb, induces enhanced anti-tumour immunity that is protective against lung metastasis. Anti-IL-10R1 mAb demonstrates systemic effects and may prove useful in clinical practice for treating bladder cancer in high-risk patients. PMID:24593764

  17. Anti-interleukin-10R1 monoclonal antibody in combination with bacillus Calmette--Guérin is protective against bladder cancer metastasis in a murine orthotopic tumour model and demonstrates systemic specific anti-tumour immunity.

    PubMed

    Newton, M R; Askeland, E J; Andresen, E D; Chehval, V A; Wang, X; Askeland, R W; O'Donnell, M A; Luo, Y

    2014-07-01

    Effective treatment of bladder cancer with bacillus Calmette-Guérin (BCG) depends on the induction of a T helper type (Th) 1 immune response. Interleukin (IL)-10 down-regulates the Th1 response and is associated with BCG failure. In this study, we investigated whether blocking IL-10 signalling could enhance the BCG-induced Th1 response and anti-tumour immunity in a murine orthotopic tumour model. Treatment with BCG and anti-IL-10 receptor 1 monoclonal antibody (anti-IL-10R1 mAb) increased the interferon (IFN)-γ to IL-10 ratio in both splenocyte cultures and urine. Mice bearing luciferase-expressing MB49 (MB49-Luc) tumours were treated and followed for tumour growth by bioluminescent imaging, bladder weight and histology. Mice treated with phosphate-buffered saline (PBS) (group 1), BCG plus control immunoglobulin (Ig)G1 (group 2) or BCG plus anti-IL-10R1 mAb (group 3) showed 0, 6 and 22% tumour regression, respectively. The mean bladder weight of group 3 mice was substantially lower than those of groups 1 and 2 mice. Remarkably, 36% of group 1 and 53% of group 2 mice but no group 3 mice developed lung metastasis (P = 0·02). To investigate the mechanisms underlying the effect of combination therapy, splenocytes were stimulated with S12 peptide (serine mutation at codon 12 of the K-ras oncogene) known to be expressed in MB49-Luc cells. Induction of ras mutation-specific IFN-γ and cytotoxicity was observed in mice treated with combination therapy. These observations indicate that BCG, in combination with anti-IL-10R1 mAb, induces enhanced anti-tumour immunity that is protective against lung metastasis. Anti-IL-10R1 mAb demonstrates systemic effects and may prove useful in clinical practice for treating bladder cancer in high-risk patients.

  18. Monoclonal Antibodies Against Human Cardiac Troponin I for Immunoassays II.

    PubMed

    Lee, Gregory; Liu, Suefay

    2015-06-01

    Human cardiac troponin I (cTnI) is one of the most specific biomarkers for detection of acute myocardial infarction (AMI). To formulate immunoassay kits for rapid immunodiagnosis of AMI, monoclonal antibodies with high affinity and specificity were generated against cTnI and subsequently tested through a series of experiments. C57BL/6 mice were immunized with cTnI as the immunogen and cell fusions with myeloma cells of BALB/c origin were performed to generate hybridomas. The supernatants of the hybridoma cell culture were routinely screened for antibody secretions against intact cTnI and synthetic peptides from the N-terminal half of cTnI (amino acid residues N1-30, N24-40, N59-79, and N80-95). Monoclonal antibodies specific to different epitope regions were then determined and selected, according to their respective affinity and specificity, for formulation of enzyme immunoassay kits. The results of this study found that most of the selected antibodies revealed comparable binding affinity to cTnI and to the corresponding synthetic peptides. Optimal sandwich enzyme immunoassays with high sensitivity could be achieved through proper combinations of the epitope-distinct monoclonal antibodies in different capture-detection pairs; signal enhancements were frequently observed when a mixture of epitope-distinct anti-cTnI monoclonal antibodies was used for coating. This indicates that a combination of epitope-distinct anti-cTnI monoclonal antibodies recognizing the N-terminal half of cTnI yield reliable detection and greater sensitivity for cTnI in AMI patients.

  19. 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

  20. Characterization of monoclonal antibodies against human apolipoprotein E.

    PubMed Central

    Milne, R W; Douste-Blazy, P; Marcel, Y L; Retegui, L

    1981-01-01

    From a single cell fusion, five stable hybridomas secreting antiapolipoprotein E (apo E) were obtained. The immunoglobulin (Ig)G subclasses containing the respective monoclonal antibodies were isolated and were used as the antibody component in a solid-phase radioimmunoassay. The binding of 125I-apo E to the insolubilized antibody was inhibited by unlabeled apo E but not by unlabeled apoproteins A-I, A-II, C-II, and C-III, or by low density lipoprotein immunodepleted of endogenous apo E. Competition curves were obtained with lipoprotein subfractions that had the same shape as those obtained with purified apo E. Apo E levels in normal and hyperlipidemic plasma were well correlated when measured by the five monoclonal antibodies and polyclonal anti-apo E, although differences in absolute values were observed. In normal subjects 34, 10, 20, and 36% of apo E was recovered in the very low density lipoprotein, low density lipoprotein, high density lipoprotein, and the d greater than 1.21-gl/ml fractions, respectively, whereas these values were 34, 7, 12, and 47%, respectively, in type III patients. All antibodies indicated the same subfraction distribution of apo E. The monoclonal antibodies reacted with all of the isomorphs of apo E. One of the antibodies could be clearly distinguished by its reactivity with chemically modified very low density lipoprotein. Images PMID:6788802

  1. Recent Advances in Monoclonal Antibody Therapies for Multiple Sclerosis.

    PubMed

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

    2016-06-01

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

  2. Recent Advances in Monoclonal Antibody Therapies for Multiple Sclerosis

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    1988-04-01

    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.

  4. Reversal of intracellular toxicity of the trichothecene mycotoxin T-2 with monoclonal antibody.

    PubMed

    Hunter, K W; Brimfield, A A; Knower, A T; Powell, J A; Feuerstein, G Z

    1990-12-01

    The trichothecene mycotoxin T-2 is a potent inhibitor of intracellular protein synthesis. We have previously shown that a mouse immunoglobulin G1 monoclonal antibody (15H6) specific for T-2 toxin can neutralize the in vitro protein synthesis inhibitory effect of the toxin in human B-lymphoblastoid cultures, and protect rats from lethal toxemia. We now report that these monoclonal antibodies can induce the net efflux of [3H]-T-2 toxin from poisoned human B-lymphoblastoid cells in vitro, and restore protein synthesis. Administration of the monoclonal antibodies (250 mg/kg) 30 min before infusion of a lethal dose (1 mg/kg) of T-2 toxin causes the sequestration of the toxin in the plasma compartment. When administered 35 min after T-2 toxin, a time when the bulk of toxin is in the tissues, the monoclonal antibodies facilitate the migration of toxin back into the plasma compartment. These data demonstrate that monoclonal antibodies can be of therapeutic value against an intracellular toxin.

  5. Brachytherapy attains abscopal effects when combined with immunostimulatory monoclonal antibodies.

    PubMed

    Rodriguez-Ruiz, María E; Rodriguez, Inmaculada; Barbes, Benigno; Mayorga, Lina; Sanchez-Paulete, Alfonso Rodriguez; Ponz-Sarvise, Mariano; Pérez-Gracia, José Luis; Melero, Ignacio

    2017-08-21

    Preclinical and clinical evidence indicate that the proimmune effects of radiotherapy can be synergistically augmented with immunostimulatory monoclonal antibodies (mAb) to act both on irradiated tumor lesions and on tumors at distant, nonirradiated sites. We have recently reported that external beam radiotherapy achieves abscopal effects when combined with antagonist anti-PD1 mAbs and agonist anti-CD137 (4-1BB) mAbs. The goal of this work is to study the abscopal effects of radiotherapy instigated by brachytherapy techniques. Mice bearing a subcutaneous colorectal carcinoma, MC38 (colorectal cancer), in both flanks were randomly assigned to receive brachytherapy or not (8 Gy × three fractions) to only one of the two grafted tumors, in combination with intraperitoneal immunostimulatory monoclonal antibodies (anti-PD1, anti-CD137, and/or their respective isotype controls). To study the abscopal effects of brachytherapy, we established an experimental set up that permits irradiation of mouse tumors sparing a distant site resembling metastasis. Such second nonirradiated tumor was used as indicator of abscopal effect. Tumor size was monitored every 2 days. Abscopal effects on distant nonirradiated subcutaneous tumor lesions of transplanted MC38-derived tumors only took place when brachytherapy was combined with immunostimulatory anti-PD1 and/or anti-CD137 mAbs. Our results demonstrate that immunotherapy-potentiated abscopal effects can be attained by brachytherapy. Accordingly, immunotherapy plus brachytherapy combinations are suitable for clinical translation. Copyright © 2017 American Brachytherapy Society. Published by Elsevier Inc. All rights reserved.

  6. Prediction and reduction of the aggregation of monoclonal antibodies.

    PubMed

    van der Kant, Rob; Karow-Zwick, Anne R; Van Durme, Joost; Blech, Michaela; Gallardo, Rodrigo; Seeliger, Daniel; Aßfalg, Kerstin; Baatsen, Pieter; Compernolle, Griet; Gils, Ann; Studts, Joey M; Schulz, Patrick; Garidel, Patrick; Schymkowitz, Joost; Rousseau, Frederic

    2017-03-17

    Protein aggregation remains a major area of focus in the production of monoclonal antibodies. Improving the intrinsic properties of antibodies can improve manufacturability, attrition rates, safety, formulation, titers, immunogenicity and solubility. Here, we explore the potential of predicting and reducing the aggregation propensity of monoclonal antibodies, based on the identification of aggregation-prone regions (APRs) and their contribution to the thermodynamic stability of the protein. Although APRs are thought to occur in the antigen binding region to drive hydrophobic binding with antigen, we were able to rationally design variants that display a marked decrease in aggregation propensity while retaining antigen binding through the introduction of artificial aggregation gatekeeper residues. The reduction in aggregation propensity was accompanied by an increase in expression titer, showing that reducing protein aggregation is beneficial throughout the development process. The data presented show that this approach can significantly reduce liabilities in novel therapeutic antibodies and proteins, leading to a more efficient path to clinical studies.

  7. Antibody discovery: sourcing of monoclonal antibody variable domains.

    PubMed

    Strohl, William R

    2014-03-01

    Historically, antibody variable domains for therapeutic antibodies have been sourced primarily from the mouse IgG repertoire, and typically either chimerized or humanized. More recently, human antibodies from transgenic mice producing human IgG, phage display libraries, and directly from human B lymphocytes have been used more broadly as sources of antibody variable domains for therapeutic antibodies. Of the total 36 antibodies approved by major maket regulatory agencies, the variable domain sequences of 26 originate from the mouse. Of these, four are marketed as murine antibodies (of which one is a mouse-rat hybrid IgG antibody), six are mouse-human chimeric antibodies, and 16 are humanized. Ten marketed antibodies have originated from human antibody genes, three isolated from phage libraries of human antibody genes and seven from transgenic mice producing human antibodies. Five antibodies currently in clinical trials have been sourced from camelids, as well as two from non-human primates, one from rat, and one from rabbit. Additional sources of antibody variable domains that may soon find their way into the clinic are potential antibodies from sharks and chickens. Finally, the various methods for retrieval of antibodies from humans, mouse and other sources, including various display technologies and amplification directly from B cells, are described.

  8. Monoclonal Antibodies to Prevent Use of Mycotoxins as Biological Weapons

    DTIC Science & Technology

    2007-07-01

    conjugate to immunize rats. We found that vaccination by either the intraperitoneal or subcutaneous route induced very high aflatoxin B1 -binding antibody...titers that were completely competed by free aflatoxin BB1 in solution. We will make and characterize monoclonal antibodies to aflatoxins B1 and G1...their potential for use in the event of bioterrorism has been highlighted (1). Immunization with an aflatoxin B1 (AF- B1 )-BSA conjugate previously

  9. A monoclonal antibody for G protein-coupled receptor crystallography.

    PubMed

    Day, Peter W; Rasmussen, Søren G F; Parnot, Charles; Fung, Juan José; Masood, Asna; Kobilka, Tong Sun; Yao, Xiao-Jie; Choi, Hee-Jung; Weis, William I; Rohrer, Daniel K; Kobilka, Brian K

    2007-11-01

    G protein-coupled receptors (GPCRs) constitute the largest family of signaling proteins in mammals, mediating responses to hormones, neurotransmitters, and senses of sight, smell and taste. Mechanistic insight into GPCR signal transduction is limited by a paucity of high-resolution structural information. We describe the generation of a monoclonal antibody that recognizes the third intracellular loop (IL3) of the native human beta(2) adrenergic (beta(2)AR) receptor; this antibody was critical for acquiring diffraction-quality crystals.

  10. Cooperative Immunoassays: Ultrasensitive Assays with Mixed Monoclonal Antibodies

    NASA Astrophysics Data System (ADS)

    Ehrlich, Paul H.; Moyle, William R.

    1983-07-01

    Mixtures of certain monoclonal antibodies appear to bind human chorionic gonadotropin in a ``cooperative'' fashion because they form circular complexes with the hormone. Experiments illustrate how this property might be exploited to develop very sensitive immunoassays for human chorionic gonadotropin or any other antigen. Since the assays are not based on competitive inhibition between radiolabeled and unlabeled antigen, they are much more sensitive than a traditional radioimmunoassay in which either one of the same antibodies is used alone.

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. [Neutralizing Monoclonal and Chimeric Antibodies to Human IFN-γ].

    PubMed

    Larina, M V; Aliev, T K; Solopova, O N; Pozdnyakova, L P; Korobova, S V; Yakimov, S A; Sveshnikov, P G; Dolgikh, D A; Kirpichnikov, M P

    2015-01-01

    Autoiminune disorders are chronic diseases characterized by abnormal immune response directed against self-antigens that leads to tissue damage and violation of its normal functioning. Such diseases often result in disability or even death of patients. Nowadays a number of monoclonal antibodies to pro-inflammatory cytokines and their receptors are successfully used for the targeted treatment of autoimmune diseases. One of the perspective targets in autoimmune disease therapy is interferon gamma, a key cytokine in Th1 cells differentiation, activation of macrophages, and inflammation. In the present work, 5 monoclonal antibodies to human IFN-γ were obtained. For the development of potential therapeutic agent, we have performed neutralizing activity and affinity analysis of the antibodies. Based on the data obtained, the monoclonal antibody F1 was selected. This antibody has a dissociation constant 1.7 x 10(-9) M and IC90 = 8.9 ± 2.0 nM measured upon antibody inhibition of the IFN-γ-induced HLA-DR expression on the surface of U937 cells. We have constructed a bicistronic vector for the production of recombinant chimeric Fab fragment F1 chim in E. coli cells. The recombinant chimeric Fab fragment Fl chim neutralizes IFN-γ activity in vitro and has a dissociation constant 1.8 x 10(-9) M.

  17. Heterogeneity of monoclonal antibodies revealed by charge-sensitive methods.

    PubMed

    Vlasak, J; Ionescu, R

    2008-12-01

    The expanding field of monoclonal antibody-based pharmaceuticals has triggered increased interest in analytical characterization of these large proteins and in understanding of their heterogeneity and degradation pathways. As a result, a large number of enzymatic modifications as well as chemical and physical degradations have been reported in monoclonal antibodies in recent years. Most heterogeneity is related to changes in the surface charge of the antibody, either directly, as a change in the number of charged residues, or indirectly as a chemical or physical alteration that changes surface-charge distribution. This review presents an overview of the sources of charge-related heterogeneity in monoclonal antibodies and the methods used for their detection. A detailed section is dedicated to deamidation of asparagine and isomerization of aspartic acid residues, two ubiquitous degradation pathways detected in antibodies and other proteins as well. Finally, kinetic modeling of the accumulation of antibody variants is presented as a tool to determine the expected fraction of molecules that have undergone one or more degradation reactions.

  18. 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.

  19. 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.

  20. Binding properties of monoclonal antibodies to rabies virus.

    PubMed

    Cusi, M G; Valensin, P E; Tollis, M; Bracci, L; Petreni, S; Soldani, P

    1991-07-01

    The monoclonal antibodies (mAbs) obtained by immunizing mice with a tetradecapeptide corresponding to the 190-203 region of rabies virus glycoprotein, involved in binding to the acetylcholine receptor (AchR), displayed different specificities to different rabies virus strains. These mAbs, when used in immunofluorescence tests, allowed differentiation of wild rabies viruses from the attenuated ones.

  1. Development and evaluation of monoclonal antibodies for paxilline

    USDA-ARS?s Scientific Manuscript database

    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...

  2. A Fungicidal Monoclonal Antibody Protects against Murine Invasive Candidiasis

    PubMed Central

    Sevilla, María J.; Robledo, Beatriz; Rementeria, Aitor; Moragues, María D.; Pontón, José

    2006-01-01

    Mice infected by Candida albicans and treated with monoclonal antibody C7 survived longer than saline-treated animals. A prozone-like effect was observed. The in vitro candidacidal activity of macrophages was strongly enhanced when C. albicans was opsonized by C7 and complete murine serum was present. PMID:16622248

  3. Production of Monoclonal Antibodies Directed against the Microsporidium Enterocytozoon bieneusi

    PubMed Central

    Accoceberry, Isabelle; Thellier, Marc; Desportes-Livage, Isabelle; Achbarou, Abderrahim; Biligui, Sylvestre; Danis, Martin; Datry, Annick

    1999-01-01

    Several hybridomas producing antibodies detected by indirect immunofluorescence antibody test (IFAT) were established by fusion of mouse myeloma SP2/O with spleen cells from BALB/c mice immunized against whole spores (protocol 1) or chitinase-treated spores (protocol 2) of Enterocytozoon bieneusi and were cloned twice by limiting dilutions. Two monoclonal antibodies (MAbs), 3B82H2 from protocol 1, isotyped as immunoglobulin M (IgM), and 6E52D9 from protocol 2, isotyped as IgG, were expanded in both ascites and culture. IFAT with the MAbs showed that both MAbs reacted exclusively with the walls of the spores of E. bieneusi, strongly staining the surface of mature spores, and produced titers of greater than 4,096. Immunogold electron microscopy confirmed the specific reactivities of both antibodies. No cross-reaction, either with the spores of the other intestinal microsporidium species Encephalitozoon intestinalis or with yeast cells, bacteria, or any other intestinal parasites, was observed. The MAbs were used to identify E. bieneusi spores in fecal specimens from patients suspected of having intestinal microsporidiosis. The IFAT was validated against standard staining methods (Chromotrope 2R and Uvitex 2B) and PCR. We report here the first description and characterization of two MAbs specific for the spore wall of E. bieneusi. These MAbs have great potential for the demonstration and species determination of E. bieneusi, and their application in immunofluorescence identification of E. bieneusi in stool samples could offer a new diagnostic tool for clinical laboratories. PMID:10565939

  4. Analysis of viral clearance unit operations for monoclonal antibodies.

    PubMed

    Miesegaes, George; Lute, Scott; Brorson, Kurt

    2010-06-01

    Demonstration of viral clearance is a critical step in assuring the safety of biotechnology products. We generated a viral clearance database that contains product information, unit operation process parameters, and viral clearance data from monoclonal antibody and antibody-related regulatory submissions to FDA. Here we present a broad overview of the database and resulting analyses. We report that the diversity of model viruses tested expands as products transition to late-phase. We also present averages and ranges of viral clearance results by Protein A and ion exchange chromatography steps, low pH chemical inactivation, and virus filtration, focusing on retro- and parvoviruses. For most unit operations, an average log reduction value (LRV, a measure of clearance power) for retrovirus of >4 log(10) were measured. Cases where clearance data fell outside of the anticipated range (i.e., outliers) were rationally explained. Lastly, a historical analysis did not find evidence of any improvement trend in viral clearance over time. The data collectively suggest that many unit operations in general can reliably clear viruses.

  5. Profiling formulated monoclonal antibodies by (1)H NMR spectroscopy.

    PubMed

    Poppe, Leszek; Jordan, John B; Lawson, Ken; Jerums, Matthew; Apostol, Izydor; Schnier, Paul D

    2013-10-15

    Nuclear magnetic resonance (NMR) is arguably the most direct methodology for characterizing the higher-order structure of proteins in solution. Structural characterization of proteins by NMR typically utilizes heteronuclear experiments. However, for formulated monoclonal antibody (mAb) therapeutics, the use of these approaches is not currently tenable due to the requirements of isotope labeling, the large size of the proteins, and the restraints imposed by various formulations. Here, we present a new strategy to characterize formulated mAbs using (1)H NMR. This method, based on the pulsed field gradient stimulated echo (PGSTE) experiment, facilitates the use of (1)H NMR to generate highly resolved spectra of intact mAbs in their formulation buffers. This method of data acquisition, along with postacquisition signal processing, allows the generation of structural and hydrodynamic profiles of antibodies. We demonstrate how variation of the PGSTE pulse sequence parameters allows proton relaxation rates and relative diffusion coefficients to be obtained in a simple fashion. This new methodology can be used as a robust way to compare and characterize mAb therapeutics.

  6. Delineation of a conserved B cell epitope on bonnet monkey (Macaca radiata) and human zona pellucida glycoprotein-B by monoclonal antibodies demonstrating inhibition of sperm-egg binding.

    PubMed

    Govind, C K; Hasegawa, A; Koyama, K; Gupta, S K

    2000-01-01

    To circumvent autoimmune oophoritis after immunization with zona pellucida (ZP) glycoproteins, synthetic peptides encompassing B cell epitope(s) and devoid of oophoritogenic T cell epitopes as immunogens have been proposed. In this study, bonnet monkey (Macaca radiata) ZP glycoprotein-B (bmZPB) was expressed as polyhistidine fusion protein in Escherichia coli. Rabbit polyclonal antibodies against recombinant bmZPB (r-bmZPB) significantly inhibited human sperm-oocyte binding. To map B cell epitopes on ZPB, a panel of 7 murine monoclonal antibodies (mAbs) was generated against r-bmZPB. All 7 mAbs, when tested in an indirect immunofluorescence assay, reacted with bonnet monkey ZP, and only 6 recognized human zonae. Monoclonal antibodies MA-809, -811, -813, and -825 showed significant inhibition in the binding of human spermatozoa to human ZP in a hemizona assay. Epitope-mapping studies using multipin peptide synthesis strategy revealed that these 4 mAbs recognized a common epitope corresponding to amino acids (aa) 136-147 (DAPDTDWCDSIP). Competitive binding studies revealed that the synthetic peptide corresponding to the identified epitope (aa 136-147) inhibited the binding of MA-809, -811, -813, and -825 to r-bmZPB in an ELISA and to bonnet monkey ZP in an indirect immunofluorescence assay. The epitopic domain corresponding to aa 136-147 of bmZPB was completely conserved in human ZPB. These studies will further help in designing ZP-based synthetic peptide immunogens incorporating relevant B cell epitope for fertility regulation in humans.

  7. The use of combinations of monoclonal antibodies in clinical oncology.

    PubMed

    Henricks, Linda M; Schellens, Jan H M; Huitema, Alwin D R; Beijnen, Jos H

    2015-12-01

    Treatment with monoclonal antibodies is becoming increasingly important in clinical oncology. These antibodies specifically inhibit signaling pathways in tumor growth and/or induce immunological responses against tumor cells. By combining monoclonal antibodies several pathways may be targeted simultaneously, potentially leading to additive or synergistic effects. Theoretically, antibodies are very suitable for use in combination therapy, because of limited overlapping toxicity and lack of pharmacokinetic interactions. In this article an overview is given of preclinical and clinical data on twenty-five different combinations of antibodies in oncology. Some of these combinations have proven clinical benefit, for example the combination of trastuzumab and pertuzumab in HER2-positive breast cancer, which exemplifies an additive or synergistic effect on antitumor activity in clinical studies and the combination of nivolumab and ipilimumab, which results in significant increases in progression-free and overall survival in patients with advanced melanoma. However, other combinations may lead to unfavorable results, such as bevacizumab with cetuximab or panitumumab in advanced colorectal cancer. These combinations result in shorter progression-free survival and increased toxicity compared to therapy with a single antibody. In summary, the different published studies showed widely varying results, depending on the combination of antibodies, indication and patient population. More preclinical and clinical studies are necessary to unravel the mechanisms behind synergistic or antagonistic effects of combining monoclonal antibodies. Most research on combination therapies is still in an early stage, but it is expected that for several tumor types the use of combination therapy of antibodies will become standard of care in the near future.

  8. 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

  9. Characterization of a monoclonal antibody against CREPT, a novel protein highly expressed in tumors.

    PubMed

    Ren, Fangli; Wang, Ruoke; Zhang, Yanquan; Liu, Chunxiao; Wang, Yinyin; Hu, Jim; Zhang, Linqi; Chang, Zhijie

    2014-12-01

    CREPT (cell-cycle related and expression-elevated protein in tumor), a novel gene also called RPRD1B and C20ORF77, was recently identified to promote tumorigenesis through up-regulation of the expression of genes related to cell cycle. The previous study demonstrated that CREPT is highly expressed in a variety of tumors and enhances the expression of Cyclin D1 by promoting the formation of a chromatin loop. To study the correlation of CREPT expression with clinical factors in different tumors, we generated a monoclonal antibody (3E10) using purified recombinant human GST-CREPT protein as an antigen. In this study, we characterized the specificity of the monoclonal antibody and cloned the gene encoding the antibody for preparation of industrial production. Our results showed that the monoclonal antibody 3E10 was sensitive and specific to recognize human endogenous CREPT protein. We have mapped the epitope of the antibody and cloned the variable region sequence of the gene encoding the antibody. We confirmed that the cloned gene produced an equivalent antibody as that produced by the original hybridoma. This study provided a basis for large-scale production of the CREPT antibody, which will be useful for the study of the role of CREPT in different tumors.

  10. Characterization of a Monoclonal Antibody Against CREPT, a Novel Protein Highly Expressed in Tumors

    PubMed Central

    Ren, Fangli; Wang, Ruoke; Zhang, Yanquan; Liu, Chunxiao; Wang, Yinyin; Hu, Jim; Zhang, Linqi

    2014-01-01

    CREPT (cell-cycle related and expression-elevated protein in tumor), a novel gene also called RPRD1B and C20ORF77, was recently identified to promote tumorigenesis through up-regulation of the expression of genes related to cell cycle. The previous study demonstrated that CREPT is highly expressed in a variety of tumors and enhances the expression of Cyclin D1 by promoting the formation of a chromatin loop. To study the correlation of CREPT expression with clinical factors in different tumors, we generated a monoclonal antibody (3E10) using purified recombinant human GST-CREPT protein as an antigen. In this study, we characterized the specificity of the monoclonal antibody and cloned the gene encoding the antibody for preparation of industrial production. Our results showed that the monoclonal antibody 3E10 was sensitive and specific to recognize human endogenous CREPT protein. We have mapped the epitope of the antibody and cloned the variable region sequence of the gene encoding the antibody. We confirmed that the cloned gene produced an equivalent antibody as that produced by the original hybridoma. This study provided a basis for large-scale production of the CREPT antibody, which will be useful for the study of the role of CREPT in different tumors. PMID:25545209

  11. [Single B cell monoclonal antibody technologies and applications].

    PubMed

    Chi, Xiangyang; Yu, Changming; Chen, Wei

    2012-06-01

    Monoclonal antibodies (mAbs) contribute a lot to the development of numerous fields in life science as a pivotal tool in modern biological research. Development of the PCR methods and maturation of antibody production have made it possible to generate mAbs from single human B cells by single cell RT-PCR with successional cloning and expression in vitro. Compared to traditional monoclonal antibody technologies, single B cell technologies require relatively fewer cells, which are highly efficient in obtaining specific mAbs in a rapid way with preservation of the natural heavy and light chain pairing. With so many advantages, single B cell technologies have been proved to be an attractive approach for retrieval of naive and antigen-experienced antibody repertoires generated in vivo, design of rationale structure-based vaccine, evaluation and development of basic B cell biology concepts in health and autoimmunity, and prevention of infectious diseases by passive immunization and therapy for disorders. Accordingly, this review introduced recent progresses in the single B cell technologies for generating monoclonal antibodies and applications.

  12. Therapeutic monoclonal antibodies and multiple sclerosis: The essentials.

    PubMed

    Heliopoulos, Ioannis; Patousi, Athanasia

    2017-09-06

    Monoclonal antibodies (mAbs) are now established as targeted therapies for malignancies, transplant rejection, autoimmune and infectious diseases. Two monoclonal antibodies are available for treatment and other antibodies are currently being tested in multiple sclerosis (MS) patients. The purpose of the present review paper is to outline the antibody engineering technologies, the immunologic and pharmacologic concepts of mΑbs and the current status of treatment in MS with emphasis on clinical efficacy and safety. We conducted a thorough review of the scientific literature published until 31 December 2014 (print and electronic publications) concerning the production, applications and side effects of the use of Mabs. Sixty five articles were used in total (both original research and review papers). With the introduction of mAbs the treatment of MS has entered a new era, both with respect to efficacy and target specificity. However, administration of mAbs carries the risk of immune reactions such as acute anaphylaxis, serum sickness a, infection and other autoimmune diseases. In addition, unexpected consequences arise from our incomplete knowledge of the immune system. For example, natalizumab, a monoclonal antibody targeting α4-integrin on leukocytes increases the risk of developing progressive multifocal leukoencephalopathy, without causing notable immunosuppression. Further study on the use of mabs is required, both in vitro and in the clinical field, in order to increase our knowledge upon these new revolutionary therapeutic agents. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

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

    SciTech Connect

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

    1986-03-01

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

  14. Adsorption of monoclonal antibodies to glass microparticles.

    PubMed

    Hoehne, Matthew; Samuel, Fauna; Dong, Aichun; Wurth, Christine; Mahler, Hanns-Christian; Carpenter, John F; Randolph, Theodore W

    2011-01-01

    Microparticulate glass represents a potential contamination to protein formulations that may occur as a result of processing conditions or glass types. The effect of added microparticulate glass to formulations of three humanized antibodies was tested. Under the three formulation conditions tested, all three antibodies adsorbed irreversibly at near monolayer surface coverages to the glass microparticles. Analysis of the secondary structure of the adsorbed antibodies by infrared spectroscopy reveal only minor perturbations as a result of adsorption. Likewise, front-face fluorescence quenching measurements reflected minimal tertiary structural changes upon adsorption. In contrast to the minimal effects on protein structure, adsorption of protein to suspensions of glass microparticles induced significant colloidal destabilization and flocculation of the suspension.

  15. N-Glycosylation Design and Control of Therapeutic Monoclonal Antibodies.

    PubMed

    Sha, Sha; Agarabi, Cyrus; Brorson, Kurt; Lee, Dong-Yup; Yoon, Seongkyu

    2016-10-01

    The N-linked glycan profiles on recombinant monoclonal antibody therapeutics significantly affect antibody biological functions and are largely determined by host cell genotypes and culture conditions. A key step in bioprocess development for monoclonal antibodies (mAbs) involves optimization and control of N-glycan profiles. With pressure from pricing and biosimilars looming, more efficient and effective approaches are sought in the field of glycoengineering. Metabolic studies and mathematical modeling are two such approaches that optimize bioprocesses by better understanding and predicting glycosylation. In this review, we summarize a group of strategies currently used for glycan profile modulation and control. Metabolic analysis and mathematical modeling are then explored with an emphasis on how these two techniques can be utilized to advance glycoengineering.

  16. [Production of human monoclonal antibody reactive with gastrointestinal carcinoma].

    PubMed

    Soyama, N; Ohyanagi, H; Saitoh, Y

    1990-12-01

    Lymphocytes obtained from regional lymph nodes and spleen in the patients with gastrointestinal carcinoma were fused with the human B lymphoblastoid cell line GC01 and human hybridomas producing human monoclonal antibody (MoAb) were derived. Human MoAb No. 235 (IgM) derived from spleen cell of a gastric cancer patient reacted with adenocarcinoma of stomach, colon, and pancreas in the new immunohistochemical assay, modified direct immunoperoxidase method, and reacted with KATO III cells in cultured cell lines. The antigenic determinant of this antibody was suspected to be protein moiety after enzyme treatment. The competitive binding inhibition assay indicated that its epitope was different from anti-CEA monoclonal antibodies (KM10, A10, B9, AH3, JA4) and KM01. These findings suggested the possible use of human MoAb No. 235 for clinical application of targeting cancer chemotherapy in the future.

  17. Process economics of industrial monoclonal antibody manufacture.

    PubMed

    Farid, Suzanne S

    2007-03-15

    Pressures for cost-effective manufacture of antibodies are growing given their high doses and increasing market potential that have resulted in significant increases in total site capacities of up to 200,000 L. This paper focuses on the process economic issues associated with manufacturing antibodies and reviews the cost studies published in the literature; many of the issues highlighted are not only specific to antibodies but also apply to recombinant proteins. Data collated at UCL suggest current benchmark investment costs of $660-$1580/ft2 ($7130-$17,000/m2) and $1765-$4220/L for antibody manufacturing facilities with total site capacities in the range of 20,000-200,000 L; the limitations of the data are highlighted. The complications with deriving benchmark cost of goods per gram (COG/g) values are discussed, stressing the importance of stating the annual production rate and either titre or fermentation capacity with the cost so as to allow comparisons. The uses and limitations of the methods for cost analysis and the available software tools for process economics are presented. Specific examples found in the literature of process economic studies related to antibody manufacture for different expression systems are reviewed. The key economic drivers are identified; factors such as fermentation titre and overall yield are critical determinants of economic success. Future trends in antibody manufacture that are driven by economic pressures are discussed, such as the use of alternative expression systems (e.g. transgenics, E. coli and yeast), disposables, and improvements to downstream technology. The hidden costs and the challenges in each case are highlighted.

  18. 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.

  19. 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.

  20. Development of murine monoclonal antibodies to methamphetamine and methamphetamine analogues.

    PubMed

    Danger, Yannic; Gadjou, Caroline; Devys, Anne; Galons, Hervé; Blanchard, Dominique; Folléa, Gilles

    2006-02-20

    Methamphetamine and ecstasy are addictive drugs that cause major health problems in young people. Here we report on the development of high-affinity monoclonal antibodies to methamphetamine and its analogues, which may constitute powerful tools for antibody-based therapy. Six haptens, methamphetamine and ecstasy analogues, were synthesized, linked to a carrier protein and injected into mice. Several specific monoclonal antibodies were subsequently obtained following fusion of splenocytes from the immunized animals, with Sp2/O cells. Antibody specificity was fully investigated by competition ELISA, using a series of analogues, to identify specific amphetamine and/or ecstasy-specific antibodies. Antibody affinity was estimated to be in the range of 10(8) M(-1) with an enantiomeric hapten. Finally, two characteristic hybridoma clones (DAS-M243-6H5 and DAS-M278-4B12), secreting specific and potent mAbs were isolated. The development of drug-specific antibodies as in this study may provide promising therapeutic insight into how to neutralize methamphetamine in vivo during acute intoxication.

  1. Sperm-immobilizing monoclonal antibody to human seminal plasma antigens.

    PubMed Central

    Shigeta, M; Watanabe, T; Maruyama, S; Koyama, K; Isojima, S

    1980-01-01

    Rat spleen cells immunized to human azoospermic semen (a mixture of seminal plasma components) and mouse myeloma cells (P3/X63 Ag8U1; P3U1) (Marguilies et al., 1976) were successfully fused with polyethylene glycol (PEG 1500) and 19 of 89 fused cell cultures were found to produce sperm-immobilizing antibody. The cells that produced antibody indicating the highest sperm-immobilizing activity were distributed into wells for further recloning and 10 clones producing sperm-immobilizing antibody were established. The clone (1C4) producing the highest antibody titre was found to produce a large amount of IgG in culture supernatants and to contain a mixture of rat and mouse chromosomes. It was proved by immunodiffusion test that the monoclonal antibody was produced to the human seminal plasma antigen No. 7 which is common to human milk protein. Using this hybridoma which produced a large amount of monoclonal sperm-immobilizing antibody, a new method could be developed for purifying human seminal plasma antigen by immunoaffinity chromatography with bound antibody from the hybridoma. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:6783353

  2. 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

  3. Idiotypic cascades in cancer patients treated with monoclonal antibody CO17-1A.

    PubMed Central

    Wettendorff, M; Iliopoulos, D; Tempero, M; Kay, D; DeFreitas, E; Koprowski, H; Herlyn, D

    1989-01-01

    We have previously shown that gastrointestinal cancer patients treated with monoclonal antibody CO17-1A (Ab1) developed anti-idiotypic antibodies (Ab2) to the Ab1. We now demonstrate that patients produce anti-anti-idiotypic antibodies (Ab3) to their autologous Ab2. Ab3 were demonstrated in culture supernatants of peripheral blood mononuclear cells from five Ab1-treated patients after stimulation of the cells with heterologous Ab2 that functionally mimicked the tumor antigen (Ag) defined by Ab1 and immunologically cross reacted with the patients' Ab2. Ab3 shared idiotopes with Ab1 and were Ab1-like in their binding specificities to tumor cells, Ag, and Ab2. Such antibodies were also elicited by stimulating cells with Ag. However, they were not produced by stimulating posttreatment mononuclear cells with control proteins or by stimulating pretreatment cells with either Ag or Ab2. Our results demonstrate idiotypic cascades in cancer patients treated with monoclonal antibody. Ag-specific Ab3 responses may underlie delayed clinical responses often observed in cancer patients treated with monoclonal antibodies of various specificities. Images PMID:2786203

  4. The treatment of intraperitoneal malignant disease with monoclonal antibody guided 131I radiotherapy.

    PubMed Central

    Ward, B.; Mather, S.; Shepherd, J.; Crowther, M.; Hawkins, L.; Britton, K.; Slevin, M. L.

    1988-01-01

    Seven patients with small volume ovarian carcinoma, remaining after conventional therapy with surgery and a platinum containing chemotherapy regimen, were treated with intraperitoneal monoclonal antibody guided radiotherapy. 100 mCi131I conjugated to 10 mg of monoclonal antibody were injected i.p. in 2,000 ml peritoneal dialysis fluid. Patients were evaluated 3 months later; 3 had clinical progressive disease while third look laparotomy demonstrated progressive disease in 3 of the remaining 4 patients. The seventh patient did not have a third look laparotomy and is currently inevaluable for response. Five patients with recurrent malignant ascites not controlled by diuretics or repeated paracentesis were similarly treated with 75-170 mCi131I conjugated to 10 mg monoclonal antibody. In three patients the ascites was controlled for a mean of 4 months. One patient died too early to assess the control of his ascites but tumour cells disappeared from the ascitic fluid after therapy. In the patient whose ascites were not controlled, a subpopulation of antigen-negative tumour cells was demonstrated. This study was unable to demonstrate a therapeutic benefit for i.p. injected monoclonal antibody guided radiotherapy for solid intraperitoneal tumour but suggests that it may be capable of controlling the accumulation of antigen positive malignant ascites. Images Figure 1 Figure 3 Figure 4 PMID:3219277

  5. Monoclonal antibodies identify a group of nuclear pore complex glycoproteins

    PubMed Central

    1987-01-01

    Using monoclonal antibodies we identified a group of eight polypeptides of rat liver nuclear envelopes that have common epitopes. Most or all of these proteins are structurally distinct, as shown by tryptic peptide mapping and analysis with polyclonal antibodies. While these polypeptides are relatively tightly bound to nuclear membranes, only one is an integral membrane protein. The eight antigens cofractionate with the nuclear pore complex under various conditions of ionic strength and detergent. It can be seen by immunofluorescence microscopy that the monoclonal antibodies reacting with these antigens stain the nuclear surface of interphase cells in a finely punctate pattern. When the nuclear envelope is disassembled and subsequently reformed during mitosis, the proteins are reversibly dispersed throughout the cytoplasm in the form of minute foci. By EM immunogold localization on isolated nuclear envelopes, the monoclonal antibodies label exclusively the nuclear pore complex, at both its nucleoplasmic and cytoplasmic margins. Considered together, our biochemical and localization data indicate that the eight nuclear envelope polypeptides are pore complex components. As shown in the accompanying paper (Holt, G. D., C. M. Snow, A. Senior, R. S. Haltiwanger, L. Gerace, and G. W. Hart, J. Cell Biol., 104:1157-1164) these eight polypeptides contain a novel form of glycosylation, O-linked N-acetylglucosamine. The relative abundance and disposition of these O-linked glycoproteins in the pore complex are consistent with their having a role in nucleocytoplasmic transport. PMID:2437126

  6. Murine monoclonal antibodies generated against mouse/rat hemokinin-1.

    PubMed

    Jin, Liang; Jin, Bo-quan; Song, Chao-jun; Zhang, Yu

    2009-08-01

    The mouse/rat hemokinin-1 (m/rHK-1) was discovered nearly 9 years ago. This molecule is a peptide comprising 11 amino acids. The m/rHK-1 was found to be mainly expressed in central immune organs like bone marrow, and was proven to have lymphopoietic roles in B and T lymphocyte development. m/rHK-1was also reported to have analgesic roles in rat spinal cord, in addition to other functions such as relaxing activity on coronary artery. Unlike its analogues SP, NKA, and NKB, m/rHK-1 does not express in the nervous system. To further study the distribution and function of m/rHK-1, we carried out conventional immunization and cell fusion procedures to acquire the hybridomas secreting specific monoclonal antibodies to m/rHK-1. In the 17 positive clones obtained, three antibodies named 1B12, 2B4, and 4G5 were shown representative in cross-reactivity against m/rHK-1 and its analogues by indirect ELISA, competitive indirect ELISA, and immunofluorescence assays. Among the three clones, the 2B4 monoclonal antibody appeared to be the high-titered and specific clone to m/rHK-1. Monoclonal antibodies to m/rHK-1 will function as good tools in the physiological study of m/rHK-1 in the near future.

  7. 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.

  8. Stimuli-responsive magnetic nanoparticles for monoclonal antibody purification.

    PubMed

    Borlido, Luís; Moura, Leila; Azevedo, Ana M; Roque, Ana C A; Aires-Barros, Maria R; Farinha, José Paulo S

    2013-06-01

    Monoclonal antibodies (mAbs) are important therapeutic proteins. One of the challenges facing large-scale production of monoclonal antibodies is the capacity bottleneck in downstream processing, which can be circumvented by using magnetic stimuli-responsive polymer nanoparticles. In this work, stimuli-responsive magnetic particles composed of a magnetic poly(methyl methacrylate) core with a poly(N-isopropylacrylamide-co-acrylic acid) (P(NIPAM-co-AA)) shell cross-linked with N, N'-methylenebisacrylamide were prepared by miniemulsion polymerization. The particles were shown to have an average hydrodynamic diameter of 317 nm at 18°C, which decreased to 277 nm at 41°C due to the collapse of the thermo-responsive shell. The particles were superparamagnetic in behavior and exhibited a saturation magnetization of 12.6 emu/g. Subsequently, we evaluated the potential of these negatively charged stimuli-responsive magnetic particles in the purification of a monoclonal antibody from a diafiltered CHO cell culture supernatant by cation exchange. The adsorption of antibodies onto P(NIPAM-co-AA)-coated nanoparticles was highly selective and allowed for the recovery of approximately 94% of the mAb. Different elution strategies were employed providing highly pure mAb fractions with host cell protein (HCP) removal greater than 98%. By exploring the stimuli-responsive properties of the particles, shorter magnetic separation times were possible without significant differences in product yield and purity.

  9. Challenges and opportunities for monoclonal antibody therapy in veterinary oncology.

    PubMed

    Beirão, Breno C B; Raposo, Teresa; Jain, Saurabh; Hupp, Ted; Argyle, David J

    2016-12-01

    Monoclonal antibodies (mAbs) have come to dominate the biologics market in human cancer therapy. Nevertheless, in veterinary medicine, very few clinical trials have been initiated using this form of therapy. Some of the advantages of mAb therapeutics over conventional drugs are high specificity, precise mode of action and long half-life, which favour infrequent dosing of the antibody. Further advancement in the field of biomedical sciences has led to the production of different forms of antibodies, such as single chain antibody fragment, Fab, bi-specific antibodies and drug conjugates for use in diagnostic and therapeutic purposes. This review describes the potential for mAbs in veterinary oncology in supporting both diagnosis and therapy of cancer. The technical and financial hurdles to facilitate clinical acceptance of mAbs are explored and insights into novel technologies and targets that could support more rapid clinical development are offered. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Polyester-Based Nanoparticles for the Encapsulation of Monoclonal Antibodies.

    PubMed

    Sousa, Flávia; Fonte, Pedro; Cruz, Andreia; Kennedy, Patrick J; Pinto, Inês Mendes; Sarmento, Bruno

    2018-01-01

    Aliphatic polyesters have been widely explored for biomedical applications (e.g., drug delivery systems, biomedical devices, and tissue engineering). Recently, polyesters have been used in nanoparticle formulations for the controlled release of monoclonal antibodies (mAbs) for the enhanced efficacy of antibody-based therapy. Polyester-based nanoparticles for mAb delivery provide decreased antibody dosage, increased antibody stability and protection and longer therapeutic action, ultimately translating to an increased therapeutic index. Additionally, nanoencapsulation holds the potential for the selective cellular recognition and internalization of mAbs, in the disease context when intracellular organelles and molecules (e.g., enzymes, transcription factors and oncogenic proteins) are the preferred target. We present here a detailed method to prepare mAb-loaded polyester-based nanoparticles and the various techniques to characterize the resulting nanoparticles and mAb structure. Finally, we highlight different biological approaches to assess the in vitro bioactivity of the antibody upon nanoparticle release.

  11. Characterization of a Novel Neutralizing Monoclonal Antibody Against Ebola Virus GP.

    PubMed

    Reynard, Olivier; Volchkov, Viktor E

    2015-10-01

    Ebola virus is the etiological agent of a severe hemorrhagic fever with a high mortality rate. As the only protein exposed on the surface of viral particles, the spike glycoprotein GP is the unique target for neutralizing monoclonal antibodies. In this study, we demonstrate the strong neutralization capacity of the monoclonal antibody #3327 and characterize its activity. GP residues that are required for recognition and neutralization were found to be located both in the internal fusion loop and in the receptor-binding domain. Analysis of Ebola virus entry in the presence of #3327 allows us to hypothesize that this antibody binds to the virus particle before internalization and endosomal processing of GP and likely prevents the final viral fusion step. Importantly, #3327 is able to block entry of virions bearing GP that contain the Q508 escape mutation common to a number of virus-neutralizing antibodies, and therefore provides future perspectives for treatment strategies against Ebola virus infection.

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

    PubMed Central

    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

  13. In-situ Detection of Squalane in Sedimentary Organic Matter Using Monoclonal Antibodies

    NASA Astrophysics Data System (ADS)

    Bailey, J. V.; Corsetti, F. A.; Moldowan, J. M.; Fago, F.; Caron, D.

    2008-12-01

    Sedimentary geolipids can serve as powerful tools for reconstructing ancient ecosystems, but only if investigators can demonstrate that the hydrocarbons are indigenous to their host rocks. The association of molecules with primary sedimentary fabrics could indicate a syngenetic relationship. However, traditional biomarker analyses require extraction from large quantities of powdered rock, confounding detailed spatial correlations. Biological studies commonly use antibodies as extremely sensitive molecular probes. When coupled with fluorescent labels, antibodies allow for the visual localization of molecules. Here we show that monoclonal antibodies that bind specifically to geolipid compounds can be used for in situ detection and labeling of such compounds in mineral-bound organic macerals. Monoclonal antibodies to squalene, produced for human health studies, also react with the geolipid, squalane. We show that squalene antibodies do not react with other common sedimentary hydrocarbons. We also show that squalane antibodies bind specifically to isolated organic-rich lamina in Eocene-age, squalane-containing rocks. These results suggest that squalane is confined to discrete organo-sedimentary fabrics within those rocks, providing evidence for its syngeneity. The chemical similarity of squalane to other sedimentary hydrocarbons hints at the potential for developing monoclonal antibodies to a variety of biomarkers that could then be localized in rocks, sediments, and extant cells.

  14. 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.

  15. 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. © The Author(s).

  16. Cysteinylation of a monoclonal antibody leads to its inactivation

    PubMed Central

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

    2016-01-01

    ABSTRACT 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

  17. Recent Progress toward Engineering HIV-1-Specific Neutralizing Monoclonal Antibodies

    PubMed Central

    Sun, Ming; Li, Yue; Zheng, Huiwen; Shao, Yiming

    2016-01-01

    The recent discoveries of broadly potent neutralizing human monoclonal antibodies represent a new generation of antiretrovirals for the treatment and prophylaxis. Antibodies are generally considered more effective and safer and have been proved to provide passive protection against mucosal challenge in humanized mice and macaques. Several neutralizing Abs could protect animals against HIV-1 but are not effective when used in an established infected model for therapy. In order to overcome the limitation of antiviral activities, multiple antibody-engineering technologies have been explored to generate “the better” neutralizing antibodies against HIV-1 since bNAbs attack viral entry by various mechanisms. Thus, a promising direction of research is to discover and exploit rational antibody combination or engineered antibodies (eAbs) as potential candidate therapeutics against HIV-1. It has been reported that inclusion of fusion-neutralizing antibodies in a set of bNAbs could improve their overall activities and neutralizing spectrum. Here, we review several routes for engineering bNAbs, such as design and generation of bispecific antibodies, specific glycosylation of antibodies to enhance antiviral activity, and variable region-specific modification guided by structure and computer, as well as reviewing antibody-delivery technologies by non-viral vector, viral vector, and human hematopoietic stem/progenitor cells transduced with a lentiviral construct. We also discuss the optimized antiviral activities and benefits of these strategy and potential mechanisms. PMID:27746780

  18. Anti-CD20 monoclonal antibodies in multiple sclerosis.

    PubMed

    Moreno Torres, Irene; García-Merino, Antonio

    2017-04-01

    The therapeutic utility of the anti-CD20 monoclonal antibodies (mAbs) is currently being evaluated in multiple sclerosis (MS) in line with the better understanding of the role of B lymphocytes in MS pathogenesis. Area covered: We conducted a literature search using Medline/Pub Med database of basic research and available controlled trials about anti-CD20 mAbs in MS. Additionally, ongoing studies were identified in the ClinicalTrials.gov database. B cells exert multiple inflammatory and regulatory functions playing an important role in MS pathogenesis as is demonstrated by the production of autoantibodies, infiltration of B cells in MS lesions and the formation of ectopic B cell follicle-like structures in meninges, among others. B-cell depletion by anti-CD20 mAbs has been shown to have an impact on these pathogenic mechanisms. The efficacy of three of them, rituximab, ocrelizumab and ofatumumab in MS has been confirmed by placebo-controlled clinical trials demonstrating a significant reduction of the annualized relapsing rate (ARR), new gadolinium-enhancing (GdE) and T2 lesions. There have been no significant safety problems so far but the overall benefit to risk profile is still to be determined. Expert commentary: After recent good results of these agents in MS therapy, questions related to maintenance therapy, markers of response and control of B cells values remain unanswered.

  19. Monoclonal antibodies that target pathological assemblies of Abeta.

    PubMed

    Lambert, Mary P; Velasco, Pauline T; Chang, Lei; Viola, Kirsten L; Fernandez, Sara; Lacor, Pascale N; Khuon, Daliya; Gong, Yuesong; Bigio, Eileen H; Shaw, Pamela; De Felice, Fernanda G; Krafft, Grant A; Klein, William L

    2007-01-01

    Amyloid beta (Abeta) immunotherapy for Alzheimer's disease has shown initial success in mouse models of Alzheimer's disease and in human patients. However, because of meningoencephalitis in clinical trials of active vaccination, approaches using therapeutic antibodies may be preferred. As a novel antigen to generate monoclonal antibodies, the current study has used Abeta oligomers (amyloid beta-derived diffusible ligands, ADDLs), pathological assemblies known to accumulate in Alzheimer's disease brain. Clones were selected for the ability to discriminate Alzheimer's disease from control brains in extracts and tissue sections. These antibodies recognized Abeta oligomers and fibrils but not the physiologically prevalent Abeta monomer. Discrimination derived from an epitope found in assemblies of Abeta1-28 and ADDLs but not in other sequences, including Abeta1-40. Immunoneutralization experiments showed that toxicity and attachment of ADDLs to synapses in culture could be prevented. ADDL-induced reactive oxygen species (ROS) generation was also inhibited, establishing this response to be oligomer-dependent. Inhibition occurred whether ADDLs were prepared in vitro or obtained from Alzheimer's disease brain. As conformationally sensitive monoclonal antibodies that selectively immunoneutralize binding and function of pathological Abeta assemblies, these antibodies provide tools by which pathological Abeta assemblies from Alzheimer's disease brain might be isolated and evaluated, as well as offering a valuable prototype for new antibodies useful for Alzheimer's disease therapeutics.

  20. Guinea pig line 10 hepatocarcinoma model: characterization of monoclonal antibody and in vivo effect of unconjugated antibody and antibody conjugated to diphtheria toxin A chain.

    PubMed

    Bernhard, M I; Foon, K A; Oeltmann, T N; Key, M E; Hwang, K M; Clarke, G C; Christensen, W L; Hoyer, L C; Hanna, M G; Oldham, R K

    1983-09-01

    Monoclonal antibodies were raised against the guinea pig line 10 (L10) hepatocarcinoma, and an IgG1-producing hybridoma (D3) was selected for further study. D3 is a true monoclonal antibody as demonstrated by two-dimensional gel electrophoresis. Radioimmunoassays on live cells revealed no cross-reactivity with normal tissues or with the line 1 hepatocarcinoma which was used as a control. Membrane immunofluorescence assays demonstrated similar specificity. Immunoperoxidase staining of cryostat sections of tumor and normal tissues of both adult animals and fetuses showed that the D3 monoclonal antibody reacted primarily with the L10 tumor, but some cross-reactivity with smooth muscle, placenta, fetal skeletal muscle, and fetal liver was also demonstrated. Radioimmunoprecipitation of detergent extracts of iodinated L10 cells showed that the antigen is present on the cell surface as a dimer of Mr 290,000 (unit size, Mr 148,000). Therapy studies with unconjugated D3 antibody demonstrated a minor dose-dependent effect on tumor growth. D3 antibody conjugated to the A chain of diphtheria toxin (10(-7) M) was cytotoxic to 100% of L10 cells in vitro. Animals treated with a single 1-mg i.v. injection of this immunoconjugate on Day 7 following the intradermal injection of 10(5) tumor cells demonstrated a highly significant inhibition of tumor growth compared to control animals and those treated with unconjugated antibody.

  1. Application of Tryptophan Fluorescence Bandwidth-Maximum Plot in Analysis of Monoclonal Antibody Structure.

    PubMed

    Huang, Cheng-Yen; Hsieh, Ming-Ching; Zhou, Qinwei

    2017-04-01

    Monoclonal antibodies have become the fastest growing protein therapeutics in recent years. The stability and heterogeneity pertaining to its physical and chemical structures remain a big challenge. Tryptophan fluorescence has been proven to be a versatile tool to monitor protein tertiary structure. By modeling the tryptophan fluorescence emission envelope with log-normal distribution curves, the quantitative measure can be exercised for the routine characterization of monoclonal antibody overall tertiary structure. Furthermore, the log-normal deconvolution results can be presented as a two-dimensional plot with tryptophan emission bandwidth vs. emission maximum to enhance the resolution when comparing samples or as a function of applied perturbations. We demonstrate this by studying four different monoclonal antibodies, which show the distinction on emission bandwidth-maximum plot despite their similarity in overall amino acid sequences and tertiary structures. This strategy is also used to demonstrate the tertiary structure comparability between different lots manufactured for one of the monoclonal antibodies (mAb2). In addition, in the unfolding transition studies of mAb2 as a function of guanidine hydrochloride concentration, the evolution of the tertiary structure can be clearly traced in the emission bandwidth-maximum plot.

  2. 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

  3. Quantitative SPECT of uptake of monoclonal antibodies

    SciTech Connect

    DeNardo, G.L.; Macey, D.J.; DeNardo, S.J.; Zhang, C.G.; Custer, T.R.

    1989-01-01

    Absolute quantitation of the distribution of radiolabeled antibodies is important to the efficient conduct of research with these agents and their ultimate use for imaging and treatment, but is formidable because of the unrestricted nature of their distribution within the patient. Planar imaging methods have been developed and provide an adequate approximation of the distribution of radionuclide for many purposes, particularly when there is considerable specificity of targeting. This is not currently the case for antibodies and is unlikely in the future. Single photon emission computed tomography (SPECT) provides potential for greater accuracy because it reduces problems caused by superimposition of tissues and non-target contributions to target counts. SPECT measurement of radionuclide content requires: (1) accurate determination of camera sensitivity; (2) accurate determination of the number of counts in a defined region of interest; (3) correction for attenuation; (4) correction for scatter and septal penetration; (5) accurate measurement of the administered dose; (6) adequate statistics; and (7) accurate definition of tissue mass or volume. The major impediment to each of these requirements is scatter of many types. The magnitude of this problem can be diminished by improvements in tomographic camera design, computer algorithms, and methodological approaches. 34 references.

  4. Production and characterization of monoclonal antibodies against aflatoxin B1.

    PubMed

    Soukhtanloo, Mohammad; Talebian, Elham; Golchin, Mehdi; Mohammadi, Mojgan; Amirheidari, Bagher

    2014-01-01

    In this article, we embarked on production of mouse monoclonal antibodies against aflatoxin B1 which is the most commonly occurring fungal toxin in food and feed products. After immunization and fusion with myloma cells, two stable clones (A218 and B319) were selected. Isotyping showed that these monoclonal antibodies (mAbs) were IgG2b with kappa light chains. The affinity of A218 and B319 clons were 5×10(11) M(-1) and 6×10(9) M(-1), respectively. Competitive indirect ELISA results indicated these mAbs had complete (100%) cross-reaction with four major types of aflatoxins: B1, B2, G1, and G2. These mAbs could be used for immunoassay measurement of aflatoxins with high affinity and low detection limits.

  5. 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.

  6. 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

  7. Anaphylaxis: implications of monoclonal antibody use in oncology.

    PubMed

    Gleich, Gerald J; Leiferman, Kristin M

    2009-02-01

    Anaphylaxis is currently classified as an immunologically triggered response with reactions that are IgE-mediated and reactions that are not IgE-mediated. This immunologically mediated phenomenon can result in various clinical manifestations, including decreased blood pressure, generalized skin inflammation, such as hives and pruritus, and respiratory symptoms, such as wheezing or bronchospasm. The severity of anaphylaxis can range from a mild allergic reaction to a potentially fatal anaphylactic shock. Numerous causative agents trigger anaphylactic reactions, and some of the best described include food and bee sting allergens. Monoclonal antibodies, which are increasingly used in the treatment of various malignancies, also can cause anaphylaxis. In this review, the mechanisms governing anaphylaxis along with treatment strategies are reviewed. Diagnostic aids for anaphylaxis are also discussed. Increased awareness of the mechanisms, symptoms, and treatment of anaphylaxis can aid caregivers to make informed decisions when new agents, such as monoclonal antibodies, are introduced into the clinic.

  8. Monoclonal antibodies for the detection of trace chemicals

    SciTech Connect

    Vanderlaan, M.; Van Emon, J.; Watkins, B.; Stanker, L.

    1986-08-15

    Problems in analytical chemistry may limit monitoring for trace organic residues by traditional chromatographic methods. For example, the cost and analysis time per sample may preclude adequate sampling, making the development of alternative technologies desirable. Immunoassays are one such alternative, with the potential for cost reduction by automation and parallel sample processing. A particularly significant advance in the past decade has been the development of monoclonal antibodies, which offer greater selectivity and reproducibility than conventional antisera. Immunoassays can be developed that use simple, field-portable instrumentation, give rapid results, and have detection limits of less than a part-per-billion. This paper reviews the general technology for developing monoclonal antibodies to small organic molecules using the immunoassay of 2,3,7,8-tetrachlorodibenzodioxin (2,3,7,8-TCDD) as an example. 18 refs., 2 figs., 1 tab.

  9. 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

  10. Identification of a Monoclonal Antibody Against Pneumococcal Pilus 1 Ancillary Protein Impairing Bacterial Adhesion to Human Epithelial Cells.

    PubMed

    Amerighi, Fulvia; Valeri, Maria; Donnarumma, Danilo; Maccari, Silvia; Moschioni, Monica; Taddei, Annarita; Lapazio, Lucia; Pansegrau, Werner; Buccato, Scilla; De Angelis, Gabriella; Ruggiero, Paolo; Masignani, Vega; Soriani, Marco; Pezzicoli, Alfredo

    2016-02-15

    The adhesion of Streptococcus pneumoniae is a key step during colonization of human respiratory tract mucosae. Here we demonstrate that pneumococcal type I pilus significantly increases the adhesiveness of poorly adhering highly capsulated strains in vitro. Interestingly, preincubation of bacteria with antibodies against the major pilus backbone subunit (RrgB) or the adhesin component (RrgA) impaired pneumococcal association to human epithelial cells. Screening for anti-RrgA monoclonal antibodies specifically affecting the adhesive capacity of S. pneumoniae led to the identification of the monoclonal 11B9/61 antibody, which greatly reduced pilus-dependent cell contact. Proteomic-based epitope mapping of 11B9/61 monoclonal antibody revealed a well-exposed epitope on the D2 domain of RrgA as the target of this functional antibody. The data presented here confirm the importance of pilus I for S. pneumoniae pathogenesis and the potential use of antipilus antibodies to prevent bacterial colonization.

  11. Rhenium-186-labeled monoclonal antibodies for radioimmunotherapy: preparation and evaluation.

    PubMed

    John, E; Thakur, M L; DeFulvio, J; McDevitt, M R; Damjanov, I

    1993-02-01

    Rhenium-186 has been determined to be a leading radionuclide for radioimmunotherapy. However, the use of 186Re has been limited due to the lack of a convenient and efficient method by which the radionuclide can be bound to monoclonal antibodies. We have developed a simple technique to label IgM, IgG, fragmented antibodies and tumor necrosis factor-alpha with 186Re. This technique uses ascorbic acid (AA) for controlled reduction of antibody disulfide groups to sulfhydryls and SnCl2 in citric acid for the reduction of 186ReO4-. The labeling yields as determined by instant thin-layer chromatography, molecular filtration and gel filtration were greater than 95% and the colloid formation was less than 5%. The labeled antibodies were stable when challenged with 100 and 250 molar excess of DTPA and HSA for 24 hr at 37 degrees C. SDS-PAGE analysis and autoradiography of labeled IgM, IgG and F(ab')2 monoclonal antibodies indicated uniform labeling and that no fragmentation of the monoclonal antibodies had taken place during the labeling procedure. Immunospecificity of 186Re-labeled human neutrophil specific IgM, as determined by in vitro antigen excess assay, was comparable to that of indium-111-labeled c-DTPA-IgM and technetium-99m-labeled-IgM. A nuclear histone specific 186Re-TNT-1-F(ab')2 was evaluated in mice bearing experimental tumors. The tumor/muscle ratios at 4 and 24 hr were 5.9 +/- 0.21 and 13.8 +/- 6.7, respectively compared to that of 2.4 +/- 0.3 at 4 hr p.i. with a nonspecific protein. The labeling technique is simple, reliable and has already been adapted to a single-vial kit preparation.

  12. 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.

  13. 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.

  14. 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

  15. Neutralizing Monoclonal Antibodies against Biological Toxins. Phase 1

    DTIC Science & Technology

    1993-08-14

    the original copy. AD-B’i76 29`8 CONTRACT NO: DAMD17-93-C-3083 TITLE: NEUTRALIZING MONOCLONAL ANTIBODIES AGAINST BIOLOGIC4JL TOXINS PRINCIPAL...Biological ’ Toxins DAMDl7-93-C-308.3 6. AUTHOR($) 65502A 30665502MB02. S4 .274 Mark C. Glassy, Ph.D. WUDA336206 7. PERFORMING ORGANIZATION NAMI(S...NUMBER OF PAGES RA I, SBIR, Antibody, Toxins , BL2, BD 16. PRICE COUE 17. SEC.URIly (LASSIFICATION 18. SECURITY CLASSIMICATION 19. SECURITY

  16. 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

  17. Monoclonal antibodies in the treatment of pancreatic cancer

    PubMed Central

    Huang, Zhi-Qiang; Buchsbaum, Donald J

    2009-01-01

    Human pancreatic cancer is a malignant disease with almost equal incidence and mortality. Effective diagnostic and therapeutic strategies are still urgently needed to improve its survival rate. With advances in structural and functional genomics, recent work has focused on targeted molecular therapy using monoclonal antibodies. This review summarizes the target molecules on the tumor cell surface and normal tissue stroma, which are related to pancreatic cancer oncogenesis, tumor growth or resistance to chemotherapy, as well as molecules involved in regulating inflammation and host immunoresponses. Targeted molecules include cell-surface receptors, such as the EGF receptor, HER2, death receptor 5 and IGF-1 receptor. Effects of monoclonal antibodies against these target molecules alone or in combination with chemotherapy, small-molecule signal transduction inhibitors, or radiation therapy are also discussed. Also discussed are the use of toxin or radioisotope conjugates, and information relating to the use of these targeting agents in pancreatic cancer clinical trials. Although targeted molecular therapy with monoclonal antibodies has made some progress in pancreatic cancer treatment, especially in preclinical studies, its clinical application to improve the survival rate of pancreatic cancer patients requires further investigation. PMID:20046965

  18. Isolation and characterization of monoclonal antibodies specific for chondroitin sulfate E.

    PubMed

    Watanabe, Ippei; Hikita, Tomoya; Mizuno, Haruka; Sekita, Risa; Minami, Akira; Ishii, Ami; Minamisawa, Yuka; Suzuki, Kiyoshi; Maeda, Hiroshi; Hidari, Kazuya I P J; Suzuki, Takashi

    2015-09-01

    Chondroitin sulfate E (CSE) is a polysaccharide containing mainly disaccharide units of D-glucuronic acid (GlcA) and 4,6-O-disulfated N-acetyl-D-galactosamine (GalNAc) residues (E-unit) in the amount of ∼ 60%. CSE is involved in many biological and pathological processes. In this study, we established new monoclonal antibodies, termed E-12C and E-18H, by using CSE that contained more than 70% of E-units as an immunogen. These antibodies recognized CSE but not other CSs isomers or dermatan sulfate (DS). We evaluated the reactivities of the antibodies to 6-O-sulfated CSA (6S-CSA) and DS (6S-DS) that possessed ∼ 60% of GalNAc (4S, 6S) moieties in their structures. Neither of the antibodies reacted with 6S-DS. The antibodies strictly distinguished the structural difference of GlcA and L-iduronic acid in the polysaccharide. Binding affinities of the antibodies were determined by a surface plasmon resonance assay using CSE and 6S-CSA. The binding affinities were strongly associated with the molecular weight of CSE and the E-unit content of 6S-CSA. Moreover, we demonstrated that the antibodies are applicable to histochemical analysis. In conclusion, the new anti-CSE monoclonal antibodies specifically recognize the E-unit of CSE. The antibodies will become useful tools for the investigation of the biological and pathological significance of CSE.

  19. [ICO-166 monoclonal antibodies against the CD45RA antigen].

    PubMed

    Frolova, E A; Baryshnikov, A Iu; Novikov, V V; Syrkin, A B

    1993-07-01

    Monoclonal antibodies (MCA) ICO-166 against CD45RA antigen were generated and characterized. In the indirect IFA, MCA ICO-166 reacted with 54.1 +/- 1.9% lymphocytes of human peripheral blood and 15.2 +/- 2.3% monocytes but not with granulocytes or thrombocytes. The method of double labelling of cells demonstrated that MCA ICO-166 detected all B-lymphocytes, all NK-cells and 31% of mature T-lymphocytes but only 55% of CD8 suppressor cells and only 21% of CDA helper cells carried this antigen on the surface. Experiments were carried out to block binding of FITC-labeled MCA ALB11 against CD45RA antigen with human lymphocytes by pretreatment of cells with different concentrations of MCA ICO-166. Treatment of cells with MCA ALB11 blocked binding of MCA ALB11-FITC by 85% on the average. MCA ICO-166 blocked binding of MCA ALB11-FITC by 66% on the average. When different dilutions of MCA ICO-166 were used, the dose-dependent effect of blocking of MCA ALB11-FITC binding was observed. MCA ICO-166 immunoprecipitated a protein band of molecular weight 220 kDa from lysates of mononuclear cells of the human peripheral blood.

  20. 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

  1. Monoclonal antibody probe for assessing beer foam stabilizing proteins.

    PubMed

    Onishi, A; Proudlove, M O; Dickie, K; Mills, E N; Kauffman, J A; Morgan, M R

    1999-08-01

    A monoclonal antibody (Mab; IFRN 1625) has been produced, which is specific for the most hydrophobic polypeptides responsible for foam stabilization. The binding characteristics of the Mab suggest that it is the conformation of certain hydrophobic polypeptides which is important for foam stabilization. An enzyme-linked immunosorbent assay (ELISA) for assessing the foam-positive form of the foam-stabilizing polypeptides in beer was developed using IFRN 1625. A good correlation was obtained between ELISA determination of foam-stabilizing polypeptides and an empirical means of determining foaming, that is, the Rudin head retention values, for a collection of beers of various foam qualities. Application of the ELISA to different stages of the brewing process showed that the amounts of foam-positive polypeptides increased during barley germination. During the brewing process the proportion of foam-positive polypeptides present after fermentation increased slightly, although a large amount was lost along with other beer proteins during subsequent steps, such as filtering. The present study demonstrates that the amounts of beer polypeptide present in a foam-positive form have a direct relationship with the foaming potential of beer, that their levels are altered by processing, and that there is potential for greater quality control.

  2. Monoclonal anti-thrombopoietin antibodies generated by genetic immunization.

    PubMed

    Lim, Nam-Kyu; Kim, Jung-Hwan; Kim, Se-Yeon; Kang, Hyun-Jung; Kim, Keun-Soo; Lee, Sangyoon; Hong, Hyo Jeong; Inn, Kyung-Soo

    2006-04-01

    Thrombopoietin (TPO) is a megakaryocyte growth and differentiation factor that is currently being investigated as a therapeutic for cancer patients undergoing myelosuppressive chemotherapy. We generated monoclonal antibodies (MAbs) specific for human thrombopoietin (hTPO) by genetic immunization using an hTPO expression plasmid and an adjuvant plasmid that encodes mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). All genetically immunized mice exhibited a high humoral immune response. Splenocytes from these mice were used to generate hybridomas. Two MAbs, designated 2B9A10 and 4C16B15 (of IgG1 and IgG3 isotypes, respectively), were subsequently selected and produced. They specifically recognized and precipitated recombinant hTPO produced by mammalian cells and were effective in sandwich enzyme-linked immunosorbent assays (ELISAs) for hTPO quantitation. Our results demonstrate that these MAbs should be useful for purification and quantitation of hTPO in clinical and laboratory settings.

  3. Therapeutic monoclonal antibodies: strategies and challenges for biosimilars development.

    PubMed

    Calvo, Begoña; Zuñiga, Leyre

    2012-01-01

    Biosimilar medicines already on the market may have a primary structure identical to their reference products (e.g., amino acid sequences should be identical). In the case of monoclonal antibodies (mAbs), and due to their more complex structure, a greater level of demand would be in order and identity at other levels (e.g., post-translational modifications within the Fc region of the molecule) should be proved to establish "similarity". These requirements would lead to a greater development in the process and tighter quality controls during the production of biosimilar mAbs. The following issues should be taken into account in the comparability exercise: - The designs of the studies carried out to obtain approval of the reference product are not always adequate to show that safety and efficacy of the biosimilar mAbs are comparable. A similar efficacy does not necessarily imply a similar safety profile between the innovator and biosimilar products. - The design of clinical tests to demonstrate comparability must be flexible and adaptable throughout the development of the product. - The European Medicines Agency (EMA) will consider suitable goals in the evaluation of biosimilar mAbs for their approval (e.g., to specify whether their goal is to check similarity with the reference product or to show that the treatment is effective at a clinical level).

  4. The interaction between pertussis toxin and 10 monoclonal antibodies.

    PubMed

    Schou, C; Au-Jensen, M; Heron, I

    1987-10-01

    Data on the epitope specificity of 10 monoclonal hybridoma antibodies (Mabs) that showed positive reaction in enzyme-linked immunosorbent assay (ELISA) towards pertussins toxin (Ptx) are presented. The relative functional affinity of the Mabs was determined in a catching ELISA system. The Mabs were tested for their ability to inhibit the biological activities of this toxin in two in vitro systems, viz. haemagglutination (HA) and Chinese Hamster Ovary cell (CHO) test, and in three in vivo assays: histamine sensitization (HS), leucocytosis-promoting activity (LP) and protection against intra-cerebral challenge (i.c.) with virulent B. pertussis organisms. Four Mabs were found inhibiting HA and three inhibited the effect on CHO cells. Two Mabs showed demonstrable protective effect on mice in i.c. test. The same two Mabs were also able to inhibit HS and LP activity of Ptx. Five of the ten Mabs reacted with Ptx subjected to blotting after separation of the toxin subunits in sodium-dodecyl sulphate polyacrylamide gel electrophoresis. The five Mabs all bound to more than one subunit. The epitopes defined by several of the Mabs might be useful in the context of a third-generation whooping cough vaccine.

  5. Ontogeny of Rat Thymic Epithelium Defined by Monoclonal Anticytokeratin Antibodies

    PubMed Central

    Jovanović, Suzana; Vasiljevski, Milijana; Dujić, Aleksandar

    1990-01-01

    Ontogenetic study on the expression of cytokeratin (CK) polypeptides within particular subsets of rat thymic epithelial cells (TEC) has been performed by a large panel of anti-CK monoclonal antibodies (mAbs) using the streptavidin-biotin immunoperoxidase method. Simultaneous presence of two or more CK subunits in the same TEC has been demonstrated by double immunoflouorescence labeling. The obtained results showed that the expression of CK polypeptides in fetal and neonatal thymus differed from the adult patterns. The main difference was observed in expression of CK10, 18, and 19 polypeptides. During fetal ontogeny, CK10 and 18 are markers for most medullary TEC or a subset of medullary TEC, respectively, whereas CK19 is mainly a pan-TEC marker. In the adult animals, they are localized in the cortical and a subset of medullary TEC (CK18), subcapsular/perivascular and some medullary TEC (CK19), or in a subset of medullary TEC and Hasall’s corpuscles (HC) (CK10). The switch in their expression in the cortex was observed during the first two weeks of postnatal life. PMID:1726554

  6. The therapeutic potential of anti-interleukin-20 monoclonal antibody.

    PubMed

    Hsu, Yu-Hsiang; Chang, Ming-Shi

    2014-01-01

    Interleukin (IL)-20, a member of the IL-10 family of cytokines, was discovered in 2001. IL-20 acts on multiple cell types by activating on a heterodimer receptor complex of either IL-20R1-IL-20R2 or IL-22R1-IL-20R2. Recent evidence indicates that IL-20's interaction with its receptors might have proinflammatory effects on chronic inflammatory diseases, particularly rheumatoid arthritis (RA), osteoporosis, and breast cancer. Updated information about IL-20, such as its identification, expression, receptors, signaling, and biological activities, is illustrated in this review based on our research and the data available in the literature. IL-20 is a pleiotropic cytokine, which promotes inflammation, angiogenesis, and chemotaxis. IL-20 also regulates osteoclast differentiation by altering the receptor activator of NF-κB (RANK) and RANK ligand (RANKL) axis. Inflammation, angiogenesis, and osteoclastogenesis are critical for the pathogenesis of RA, osteoporosis, and breast cancer-induced osteolysis. Based on the in vitro and in vivo data and clinical samples, we demonstrated that IL-20 plays pivotal roles in these three diseases. In experimental models, anti-IL-20 monoclonal antibody ameliorates arthritis severity, protects against ovariectomized-induced bone loss, and inhibits breast tumor-induced osteolysis. This review presents the clinical implications of IL-20, which will lead to a better understanding of the biological functions of IL-20 in these diseases and provide new therapeutic options in the future.

  7. Binding and structural diversity among high-affinity monoclonal anti-digoxin antibodies.

    PubMed

    Mudgett-Hunter, M; Anderson, W; Haber, E; Margolies, M N

    1985-04-01

    High-affinity monoclonal antibodies specific for the cardiac glycoside digoxin provide a useful system for the study of structure-function relationships between antibody combining site and specific antigenic determinants. Fifteen high-affinity monoclonal anti-digoxin antibodies were produced when spleen cells from A/J mice immunized with digoxin coupled to human serum albumin (Dig-HSA) were fused with the non-secreting murine myeloma Sp2/0 cell line. Each subcloned hybridoma antibody was analyzed for affinity and specificity for structurally related cardiac glycosides by a radioimmunoassay based on the adsorption of free [3H]digoxin to dextran-coated charcoal. All of the anti-digoxin hybridoma proteins demonstrated high affinity constants ranging from 10(9) to 10(12) M-1. Using seven different analogs of digoxin, binding specificities of the monoclonal antibodies were assessed by inhibition radioimmunoassay. The 15 hybridomas produced from fusions involving five mice could be divided into eight sets on the basis of these binding specificities. Certain antibodies exhibit a preference for the aglycone portion of digoxin, while others are more specific for the tridigitoxose sugar moiety of digoxin. Monoclonal antibody H- and L-chains were subjected to N-terminal amino acid sequence analysis. The antibodies may be divided into several sequence homology sets for both H- and L-chains. In most instances, homologous heavy chains are associated with a set of homologous light chains. Homologous partial sequences, however, do not correlate with similar antigenic specificities and affinities for digoxin. Thus the fine specificity for antigen is not dependent on VH- and VL-encoded sequences alone. These data illustrate the broad diversity of the elicited response to a single hapten, even in inbred mice.

  8. Comparison of avian Chlamydia psittaci isolates by restriction endonuclease analysis and serovar-specific monoclonal antibodies.

    PubMed Central

    Andersen, A A

    1991-01-01

    Avian Chlamydia psittaci isolates were examined by restriction endonuclease analysis and serovar-specific monoclonal antibodies and compared with ovine abortion and polyarthritis isolates. The avian isolates were divided into four serovars (turkey, psittacine, pigeon, and duck) based on their reactivity to the monoclonal antibodies. The DNA digest patterns were similar across the four avian serovars; most bands were identical when the isolates were tested with PstI, BamHI, and EcoRI restriction endonuclease enzymes. The turkey group restriction endonuclease analysis patterns were distinguished from those of the other avian strains by three to four band differences with all enzymes. The duck and pigeon isolates showed only minor DNA pattern differences when compared with the psittacine isolates. Four psittacine isolates from various locations in Texas had an extra band with the EcoRI restriction enzyme, suggesting that they were from a common source; however, they were indistinguishable from the other psittacine isolates when examined with the monoclonal antibodies. The avian isolates were distinctly different from either abortion or polyarthritis isolates by both restriction endonuclease analysis and monoclonal antibody analysis. The data demonstrate that the avian isolates form a distinct group or separate biovar with at least four serovars. Images PMID:1848867

  9. Mitosis-specific monoclonal antibodies block cleavage in amphibian embryos.

    PubMed

    Davis, F M; Wright, D A; Penkala, J E; Rao, P N

    1989-04-01

    By microinjecting monoclonal antibodies that bind specifically to mitotic and meiotic cells of a variety of species, we studied the biological activity of antigens recognized by these antibodies. The antibodies recognize a family of phosphoprotein antigens that are found throughout the cytoplasm of mitotic cells and particularly at microtubule organizing centers, including centrosomes and kinetochores. Their binding is dependent on phosphorylation of the polypeptides. Immunoglobulins were introduced into Xenopus laevis and Rana pipiens oocytes or cleaving embryos using glass micropipettes. The ability of the antibody-injected oocytes to undergo mitosis or meiosis was compared with those injected with control mouse immunoglobulins. The antibodies failed to block chromosome condensation and germinal vesicle breakdown in progesterone-treated oocytes. However, functional mitotic spindles were not assembled in cleavage stage frog embryos injected with antibodies. In vitro, the binding of the antibodies to the antigens inhibited the dephosphorylation of the antigens by alkaline phosphatase. The antibody binding to the activated microtubule organizing centers (MTOC) seems to block not only the nucleation of microtubules and the organization of the mitotic spindle, but also the dephosphorylation of proteins associated with the MTOC that normally occurs at the mitosis-G1 transition.

  10. Intravesical administration of radiolabeled antitumor monoclonal antibody in bladder carcinoma

    SciTech Connect

    Bamias, A.; Keane, P.; Krausz, T.; Williams, G.; Epenetos, A.A. )

    1991-01-15

    Tumor associated AUA1 monoclonal antibody and 11.4.1. nonspecific monoclonal antibody, which does not react with human tissues, were radiolabeled with 111In and administered intravesically to 23 patients undergoing cystoscopy for bladder carcinoma. The antibody solution remained in the bladder for 1 h and then was washed out prior to cystoscopy. Tumor and nontumor samples were obtained during cystoscopy and were counted in a gamma counter. Conventional and immunoperoxidase staining with both antibodies were also performed. AUA1 reacted with all bladder carcinomas while 11.4.1. was negative in all cases. The mean uptake of AUA1 at 2, 24, and 48 h after the instillation (expressed as 10(3) x percentage of injected dose/g of tissue) was: 6.12 +/- 5.50 (SD), 1.70 +/- 2.57, 0.30 +/- 0.17 in the tumors and 0.32 +/- 0.50, 0.22 +/- 0.30, 0 in the nontumor areas, and for 11.4.1. it was: 0.075 +/- 0.075, 0.025 +/- 0.025 in the tumors and 0.30 +/- 0.42, 0.15 +/- 0.26 in the nontumor areas. The uptake of AUA1 by the tumors correlated with the tumor grade. There was no radioactivity in the blood at 2 h, and at 1, 2, and 3 days after the instillation. Our results indicate that intravesical administration of radiolabeled monoclonal antibody AUA1 targets selectively to tumor tissue without any significant normal tissue uptake. This finding might allow the development of a nontoxic and specific therapeutic approach for superficial bladder carcinoma.

  11. 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.

  12. Monoclonal antibody epitope mapping of Plasmodium falciparum rhoptry proteins.

    PubMed

    Sam-Yellowe, T Y; Ndengele, M M

    1993-02-01

    Plasmodium falciparum rhoptry proteins of the 140/130/110-kDa high molecular weight complex (HMWC) are secreted into the erythrocyte membrane during merozoite invasion. Epitopes of membrane-associated HMWC proteins can be detected using rhoptry-specific antibodies by immunofluorescence assays. Phospholipase treatment of ring-infected intact human erythrocytes, membrane ghosts, and inside-out vesicles results in the release of the HMWC as demonstrated by immunoblotting. We characterized the membrane-associating properties of the 110-kDa protein in more detail. PLA2 from three different sources; bee venom, Naja naja venom, and porcine pancreas, were examined and all were equally effective in releasing the 110-kDa protein. Furthermore, PLA2 activity was inhibited by o-phenanthroline, quinacrine, maleic anhydride, and partially by p-bromophenacyl bromide, indicating that the activity of PLA2 is specific. Using sequential protease and phospholipase digestion experiments to map the immunoreactive and functional epitopes of the 110-kDa protein, a 35-kDa protease-resistant protein associated with mouse and human erythrocyte membranes was identified. Limited proteolysis of the 110-kDa protein and analysis by immunoblotting demonstrated several immunoreactive cleavage products, including a highly protease-resistant peptide fragment of approximately 35-kDa which corresponds to the membrane-associated protein. Epitope mapping of the 130-kDa rhoptry protein resulted in a different pattern of cleavage products. Stage-specific metabolic labeling of P. falciparum with [3H] palmitate and [3H] myristate was performed to determine the lipophilic properties of the HMWC. Results showed the incorporation of label into proteins of approximate molecular weight 200 and 45-kDa, predominantly in the late schizont stage. Interestingly, proteins of 140 and 110/100-kDa, corresponding to [35S] methionine-labeled proteins were labeled with [3H]palmitate in ring-infected erythrocyte membranes

  13. Cathepsin B-deficient mice as source of monoclonal anti-cathepsin B antibodies

    PubMed Central

    Weber, Ekkehard; Barbulescu, Elena; Medek, Rita; Reinheckel, Thomas; Sameni, Mansoureh; Anbalagan, Arulselvi; Moin, Kamiar; Sloane, Bonnie F.

    2015-01-01

    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

  14. A histoplasma capsulatum-specific IgG1 isotype monoclonal antibody, H1C, to a 70-kilodalton cell surface protein is not protective in murine histoplasmosis.

    PubMed

    Lopes, Livia Cristina Liporagi; Guimarães, Allan J; de Cerqueira, Mariana Duarte; Gómez, Beatriz L; Nosanchuk, Joshua D

    2010-07-01

    Monoclonal antibodies to Histoplasma capsulatum can modify pathogenesis. We now show that monoclonal antibody H1C to a 70-kDa antigen increases intracellular fungal growth and reduces macrophage nitric oxide release but has no effect on fungal burden or survival in murine infection. This further demonstrates the complexities of host-pathogen interactions.

  15. Antibody immunoprophylaxis and immunotherapy for influenza virus infection: utilization of monoclonal or polyclonal antibodies?

    PubMed

    Berry, Cassandra M

    2017-08-30

    Control programs for emerging influenza are in urgent need of novel therapeutic strategies to mitigate potentially devastating threats from pathogenic strains with pandemic potential. Current vaccines and antivirals have inherent limitations in efficacy, especially with rapid evolutionary changes of influenza viruses. Antibody-based antiviral protection harnesses the natural power of the immune system. Antibodies present prophylactic and therapeutic intervention options for prevention and control of influenza, especially for at-risk populations. Specific monoclonal antibodies are well defined in purity and initial efficacy but polyclonal antibodies are easier to scale-up and cost-effective with long-term efficacy, using batches with broadly neutralizing properties against influenza variants. This review presents the pros and cons of monoclonal versus polyclonal antibody therapy for influenza.

  16. Monoclonal antibodies bind identically to both spores and hyphae of Aspergillus fumigatus.

    PubMed

    Reijula, K E; Kurup, V P; Kumar, A; Fink, J N

    1992-05-01

    Immunoelectron microscopy (IEM) was used to determine the binding of six monoclonal antibodies (MoAbs) produced against Aspergillus fumigatus antigens present on or within the conidia and hyphae of the fungus. Antigen-antibody complexes were demonstrated in EM using labelled colloidal gold particles (15 nm). Three out of 6 MoAbs (C9, F12 and H10) reacted only with the cytoplasmic components of A. fumigatus while the remaining three (B12, F6G5 and D6E6) showed reactivity to both cytoplasm and cell wall of the conidia and hyphae. The results indicate that IEM is of considerable value in determining and selecting monoclonal antibodies having specific reactivity with diverse antigenic components.

  17. Efficient generation of human IgA monoclonal antibodies.

    PubMed

    Lorin, Valérie; Mouquet, Hugo

    2015-07-01

    Immunoglobulin A (IgA) is the most abundant antibody isotype produced in humans. IgA antibodies primarily ensure immune protection of mucosal surfaces against invading pathogens, but also circulate and are present in large quantities in blood. IgAs are heterogeneous at a molecular level, with two IgA subtypes and the capacity to form multimers by interacting with the joining (J) chain. Here, we have developed an efficient strategy to rapidly generate human IgA1 and IgA2 monoclonal antibodies in their monomeric and dimeric forms. Recombinant monomeric and dimeric IgA1/IgA2 counterparts of a prototypical IgG1 monoclonal antibody, 10-1074, targeting the HIV-1 envelope protein, were produced in large amounts after expression cloning and transient transfection of 293-F cells. 10-1074 IgAs were FPLC-purified using a novel affinity-based resin engrafted with anti-IgA chimeric Fabs, followed by a monomers/multimers separation using size exclusion-based FPLC. ELISA binding experiments confirmed that the artificial IgA class switching of 10-1074 did not alter its antigen recognition. In summary, our technical approach allows the very efficient production of various forms of purified recombinant human IgA molecules, which are precious tools in dissecting IgA B-cell responses in physiological and pathophysiological conditions, and studying the biology, function and therapeutic potential of IgAs.

  18. 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

  19. Transformation-Related Antigens Identified by Monoclonal Antibodies

    NASA Astrophysics Data System (ADS)

    Strand, Mette

    1980-06-01

    Tumor-cell proteins that were antigenic in a syngeneic animal were identified by immunoprecipitation with monoclonal antibodies. Spleen cells of BALB/c mice immunized with plasma membranes of Kirsten RNA sarcoma virus-transformed BALB/3T3 cells were fused with NS-l myeloma cells. Antibodies secreted into the culture fluid from these hybridomas were distinguished by their reactivity against proteins of different target cells. A total of 191 cultures were established; 143 produced antibodies that bound to BALB/3T3 cells transformed by the RNA sarcoma virus, of which antibodies from 82 bound to BALB/3T3 transformed with simian virus 40, and antibodies from 56 bound to BALB/3T3 cells. Thus, more than 50% of the cultures produced antibodies that possibly were specific to antigens of the transformed cell. Twenty different hybridomas have been cloned, and antibodies from eight of these were found to immunoprecipitate five different proteins. A protein of approximately 32,000 daltons was precipitated from BALB/3T3 cells transformed by the RNA sarcoma virus, simian virus 40, or methylcholanthrene but not from untransformed BALB/3T3 cells. A protein of about 300,000 daltons was precipitated from all four cell lines; precipitation was enhanced in the viral transformed cells. Proteins of approximately 57,000, 54,000, and 8500 daltons were immunoprecipitated from all four cell lines.

  20. Modulation of desmin intermediate filament assembly by a monoclonal antibody

    PubMed Central

    1988-01-01

    We have used a monoclonal antibody against desmin to examine the assembly of intermediate filaments (IF) from their building blocks, the tetrameric protofilaments. The antibody, designated D76, does not cross react with any other IF proteins (Danto, S.I., and D.A. Fischman. 1984. J. Cell Biol. 98:2179-2191). It binds to a region amino-terminal to cys- 324 of avian desmin that is resistant to chymotrypsin and trypsin digestion, and in the electron microscope appears to bind to the ends of tetrameric protofilaments. In combination, these findings suggest that the epitope of the antibody resides at the amino-terminal end of the alpha-helical rod domain. Preincubation of desmin protofilaments with an excess of D76 antibodies blocks their subsequent assembly into IF. In the presence of sub-stoichiometric amounts of antibodies, IF are assembled from protofilaments but they are morphologically aberrant in that (a) they are capped by IgG molecules at one or both ends; (b) they are unraveled to varying degree, revealing a characteristic right- handed helical arrangement of sub-filamentous strands of different diameters. The antibody binds only to the ends but not along the length of desmin IF. The most straightforward explanation for this is that the epitope resides in a part of the desmin molecule that becomes buried within the core of the filament upon polymerization and is therefore inaccessible to the antibody. PMID:2450097

  1. Model-based prediction of monoclonal antibody retention in ion-exchange chromatography.

    PubMed

    Guélat, Bertrand; Delegrange, Lydia; Valax, Pascal; Morbidelli, Massimo

    2013-07-12

    In order to support a model-based process design in ion-exchange chromatography, an adsorption equilibrium model was adapted to predict the protein retention behavior from the amino acid sequence and from structural information on the resin. It is based on the computation of protein-resin interactions with a colloidal model and accounts for the contribution of each ionizable amino acid to the protein charge. As a verification of the protein charge model, the experimental titration curve of a monoclonal antibody was compared to its predicted net charge. Using this protein charge model in the computation of the protein-resin interactions, it is possible to predict the adsorption equilibrium constant (i.e. retention factor or Henry constant) with an explicit pH and salt dependence. The application of the model-based predictions for an in silico screening of the protein retention on various stationary phases or, alternatively, for the comparison of various monoclonal antibodies on a given cation-exchanger was demonstrated. Furthermore, considering the structural differences between charge variants of a monoclonal antibody, it was possible to predict their individual retention times. The selectivity between the side variants and the main isoform of the monoclonal antibody were computed. The comparison with the experimental data showed that the model was reliable with respect to the identification of the operating conditions maximizing the selectivity, i.e. the most promising conditions for a monoclonal antibody variant separation. Such predictions can be useful in reducing the experimental effort to identify the parameter space.

  2. Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies.

    PubMed

    Leon, Paul E; Wohlbold, Teddy John; He, Wenqian; Bailey, Mark J; Henry, Carole J; Wilson, Patrick C; Krammer, Florian; Tan, Gene S

    2017-08-29

    Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

  3. Improved monoclonal antibody tumor/background ratios with exchange transfusions.

    PubMed

    Henry, C A; Clavo, A C; Wahl, R L

    1991-01-01

    Blood exchange transfusions were performed in nude rats with subcutaneous HTB77 human ovarian carcinoma xenografts in an attempt to improve specific monoclonal antibody (MoAb) tumor/non-tumor uptake ratios. Animals were injected intravenously with both 131I-5G6.4 specific and 125I-UPC-10 non-specific MoAb. Twenty-four hours later 65-80% of the original blood was exchanged with normal heparinized rat blood and then these rodents were sacrificed. Exchange transfusion significantly (P less than 0.05) decreased normal tissue activities of 131I (except for muscle) by 63-85%, while tumor activity decreased only 5%. Tumor to background ratios increased from 0.1-0.8 to 2.3-6.3. Exchange transfusions substantially enhance tumor/normal tissue antibody uptake ratios and, along with plasmapheresis, may be useful in enhancing antibody localization in vivo, particularly for therapy.

  4. Monoclonal antibody aggregation intermediates visualized by atomic force microscopy.

    PubMed

    Lee, Hanjoo; Kirchmeier, Marc; Mach, Henryk

    2011-02-01

    Ubiquitous but highly variable processes of therapeutic protein aggregation remain poorly characterized, especially in the context of common infusion reactions and clinical immunogenicity. Among the numerous challenges is the characterization of intermediate steps that lead to the appearance of precipitates. Although the biophysical methods for elucidation of secondary and tertiary structures as well as overall size distribution are typically well established in the development laboratories, the use of molecular scale imaging techniques is still relatively rare due to low throughput and technical complexity. In this work, we present the use of atomic force microscopy to examine morphology of monoclonal antibody aggregates. Despite varying in primary structure as a result of different complementarity defining regions, most antibodies studied exhibited a similar aggregation intermediate consisting of several monomers. However, the manner of subsequent condensation of these oligomers appeared to differ between the antibodies, suggesting stability-dependent mechanisms.

  5. Daratumumab: monoclonal antibody therapy to treat multiple myeloma.

    PubMed

    Xia, C; Ribeiro, M; Scott, S; Lonial, S

    2016-10-01

    Daratumumab (Darzalex[TM]) is a human monoclonal antibody (MAb) that targets CD38; a surface protein highly expressed across multiple myeloma (MM) cells. Preclinical studies have shown daratumumab induces MM cell death through several mechanisms, including complement-dependent cytotoxicity (CDC) antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), apoptosis upon secondary crosslinking and immunomodulatory effects via a decrease in immune suppressive cells. Daratumumab has a favorable toxicity profile and encouraging clinical activity as a single agent and in combination with lenalidomide in heavily pretreated, relapsed patients in whom other novel agents (such as bortezomib, thalidomide and lenalidomide) and stem cell transplant have already failed. Given the encouraging efficacy and acceptable safety profile, daratumumab has emerged as a novel treatment option for MM both as a monotherapy and in combination with conventional and novel anti-MM agents. This review will focus on preclinical pharmacology, pharmacokinetics, safety and clinical development of daratumumab in MM.

  6. Mycobacterium leprae antigens involved in human immune responses. I. Identification of four antigens by monoclonal antibodies

    SciTech Connect

    Britton, W.J.; Hellqvist, L.; Basten, A.; Raison, R.L.

    1985-12-01

    Four distinct antigens were identified in soluble sonicates of Mycobacterium leprae by using a panel of 11 monoclonal antibodies. Cross-reactivity studies with other mycobacterial species were conducted by using ELISA and immunoblot assays, and demonstrated that determinants on two of the antigens were present in many mycobacteria, whereas the other two were limited in distribution. Competitive inhibition experiments with radiolabeled monoclonal antibodies showed cross-inhibition between antibodies identifying two of the four antigenicbands. These two bands, of M/sub tau/ 4.5 to 6 KD and 30 to 40 KD, were resistant to protease treatment after immunoblotting. In contrast the two other bands of 16 and 70 KD were protease-sensitive. Although all four bands reacted with some human lepromatous leprosy sera in immunoblots, the 4.5 to 6 KD and 30 to 40 KD bands were most prominent. Lepromatous leprosy sera also inhibited the binding of radiolabeled monoclonal antibodies to each of the four antigens, with the mean titer causing 50% inhibition being higher for antibodies reacting with the 4.5 to 6 KD and 30 to 40 KD bands. These findings indicated that all four antigens were involved in the human B cell response to M. leprae.

  7. Characterization of a monoclonal antibody that specifically inhibits triosephosphate isomerase activity of Taenia solium.

    PubMed

    Víctor, Sanabria-Ayala; Yolanda, Medina-Flores; Araceli, Zavala-Carballo; Lucía, Jiménez; Abraham, Landa

    2013-08-01

    In the present study, we obtained and characterized partially a monoclonal antibody (4H11D10B11 mAb) against triosephosphate isomerase from Taenia solium (TTPI). This antibody recognized the enzyme by both ELISA and western blot and was able to inhibit its enzymatic activity in 74%. Moreover, the antigen-binding fragments (Fabs), products of digestion of the monoclonal antibody with papain, retained almost the same inhibitory effect. We determined the binding site by ELISA; synthetic peptides containing sequences from different non-conserved regions of the TTPI were confronted to the 4H11D10B11 mAb. The epitope recognized by the monoclonal antibody was located on peptide TTPI-56 (ATPAQAQEVHKVVRDWIRKHVDAGIADKARI), and an analysis of mimotopes, obtained with the 4H11D10B11 mAb, suggests that the epitope spans the sequence WIRKHVDAGIAD, residues 193-204 of the enzyme. This epitope is located within helix 6, next to loop 6, an essential active loop during catalysis. The antibody did not recognize triosephosphate isomerase from man and pig, definitive and intermediary hosts of T. solium, respectively. Furthermore, it did not bind to the catalytic site, since kinetic analysis demonstrated that inhibition had a non-competitive profile.

  8. Immunohistochemical staining of cyclooxygenases with monoclonal antibodies.

    PubMed

    Saed, Ghassan M

    2008-01-01

    Immunohistochemistry is an important tool that is often used for the diagnosis of several diseases in the pathology laboratory. The quality and sensitivity of immunohistochemical staining is affected by formalin fixation, which results in variable loss of antigenicity, known as a masking effect. While the sensitivity of immunohistochemistry is excellent for certain antigens, other antigens such as COX-1 and COX-2 are difficult to identify, especially in formalin-fixed, paraffin sections. Antigen retrieval is a technique that re-exposes epitopes and allows detection of masked antigens with standard immunohistochemical procedures. One common method involves partial, enzymatic pre-digestion with trypsin or pepsin while other, nonenzymatic procedures or heat-mediated antigen retrieval methods include pressure-cookers, hot plates, or microwave (MW) irradiation of tissue sections in water or a variety of antigen-retrieval solutions. In this chapter, we will describe a technique that provides a more reliable, much simpler approach for the demonstration of cyclooxygenase-1 and cyclooxygenase-2 expression in frozen, vibratome or paraffin sections, and/or cells in cultures.

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

    PubMed

    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.

  10. 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

  11. Monoclonal antibodies to the cell surface and a soluble form of the human nerve growth factor receptor

    SciTech Connect

    Clagett-Dame, M.; Chung, C.; Chao, M.V.; DiStefano, P.S. )

    1990-12-01

    Monoclonal antibodies (designated IIIG5, VIID1, VIIIC8, and XIF1) have been produced that bind to the human nerve growth factor receptor (NGF-R) as well as to a soluble, truncated form of the receptor (NGF-Rt). The antibodies were generated against partially purified NGF-Rt from the conditioned medium of E9b cells, a transfected mouse fibroblast cell line (Ltk-) that expresses large numbers of the low affinity form of the human NGF-R on its cell surface. Hybridomas were screened by radiometric immunosorbent assay (RISA) and by immunoprecipitation of solubilized cell surface receptor covalently cross-linked to {sup 125}I-NGF. Four positive lines were cloned by limiting dilution and were found to secrete monoclonal antibodies of the IgGl,k subclass. All monoclonal antibodies bound to both NGF-R and NGF-Rt. Two monoclonal antibodies (VIID1, XIF1) immunoblotted the NGF-R from E9b cell preparations resolved on non-reducing sodium dodecyl sulfate (SDS)-polyacrylamide gels. The antibodies immunoprecipitated NGF-R from both E9b cells and from SH-SY5Y human neuroblastoma cells. The monoclonal antibodies bound to monkey (rhesis and cynomolgus) NGF-Rt, but did not cross-react with NGF-R from chick or rat. Results of antibody competition studies demonstrated that three antibodies bound to a similar or overlapping epitope on the NGF-Rt and one monoclonal antibody (IIIG5) recognized a distinct receptor epitope. Antibodies that bound to different sites on the receptor were used to develop a sensitive 2-site RISA. The 2-site RISA can be used to rapidly quantitate NGF-R and NGF-Rt in large numbers of biological samples in the absence of added {sup 125}I-labeled NGF.

  12. 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

  13. [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.

  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. 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.

  16. Practical considerations for nonclinical safety evaluation of therapeutic monoclonal antibodies

    PubMed Central

    Lynch, Carmel M; Hart, Bruce W

    2009-01-01

    Monoclonal antibodies (mAbs) are a well established class of therapeutics as evidenced by a large number of FDA approved mAbs for the treatment of cancers and autoimmune diseases. Monoclonal antibodies that are molecularly engineered for enhanced functions and pharmacokinetic properties are routinely being considered for development by many biotechnology companies. Safety evaluation of current generation of mAbs poses new challenges due to the highly complex nature of engineering aspects and variability induced by the diverse recombinant cell systems to generate them. This review provides a basic outline for nonclinical safety evaluation of therapeutic antibodies. Important considerations for planning a preclinical program, the types of nonclinical safety studies, and a general timeline for their conduct in relation to clinical trials are described. A list of relevant regulatory documents issued by government agencies is also provided. Adoption of these principles will greatly enhance the quality and relevance of the nonclinical safety data generated and will facilitate future development of mAb therapeutics. PMID:20046568

  17. Trends in capacity utilization for therapeutic monoclonal antibody production.

    PubMed

    Langer, Eric S

    2009-01-01

    The administration of high doses of therapeutic antibodies requires large-scale, efficient, cost effective manufacturing processes. An understanding of how the industry is using its available production capacity is important for production planning, and facility expansion analysis. Inaccurate production planning for therapeutic antibodies can have serious financial ramifications. In the recent 5(th) Annual Report and Survey of Biopharmaceutical Manufacturing Capacity and Production, 434 qualified respondents from 39 countries were asked to indicate, among other manufacturing issues, their current trends and future predictions with respect to the production capacity utilization of monoclonal antibodies in mammalian cell culture systems. While overall production of monoclonals has expanded dramatically since 2003, the average capacity utilization for mammalian cell culture systems, has decreased each year since 2003. Biomanufacturers aggressively attempt to avoid unanticipated high production demands that can create a capacity crunch. We summarize trends associated with capacity utilization and capacity constraints which indicate that biopharmaceutical manufacturers are doing a better job planning for capacity. The results have been a smoothing of capacity use shifts and an improved ability to forecast capacity and outsourcing needs. Despite these data, today, the instability and financial constraints caused by the current global economic crisis are likely to create unforeseen shifts in our capacity utilization and capacity expansion trends. These shifts will need to be measured in subsequent studies.

  18. Monoclonal antibodies to guinea pig Ia antigens. II. Effect on alloantigen-, antigen-, and mitogen-induced T lymphocyte proliferation in vitro

    PubMed Central

    1980-01-01

    Four xenographic monoclonal antibodies to guinea pig Ia antigens were tested for their inhibitory effects on antigen-, alloantigen-, and mitogen-induced T cell proliferation. All four monoclonal antibodies reacted with strain 2 Ia antigens, and all four were capable of inhibiting the strain 13 against strain 2 mixed leukocyte reaction (MLR) by 50-70%; the two monoclonals that reacted with strain 13 Ia antigens were also capable of inhibiting the strain 2 against strain 13 MLR. In contrast, an analysis of the effects of a single monoclonal antibody on the responses to several antigens demonstrated a selective monoclonal pattern of inhibition in that the responses to some, but not all, antigens were inhibited. These results suggest that monoclonal antibodies react with different parts of Ia molecules that may have different functional roles and that certain parts of an Ia molecule participate in the presentation of certain antigens, whereas other regions of the same molecule present different antigens. PMID:6158543

  19. 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.

  20. Generation of human monoclonal antibodies reactive with cellular antigens.

    PubMed Central

    Cote, R J; Morrissey, D M; Houghton, A N; Beattie, E J; Oettgen, H F; Old, L J

    1983-01-01

    Human lymphocytes from lymph node, peripheral blood, spleen, and tumor specimens have been fused with the LICR-LON-HMy2 (LICR-2) or SKO-007 human cell lines or the NS-1 mouse myeloma line. Over 75 fusions with the three myeloma-lymphoblastoid lines have been performed. Several factors appeared to improve the fusion outcome, including maintenance of the myeloma-lymphoblastoid lines in logarithmic phase growth at greater than or equal to 95% viability, a delay of 24 hr in the introduction of aminopterin to the fused cells, and preselection of the fetal calf serum used in the medium. For a given number of lymphocytes, fusions with NS-1 produced 5-20 times more clones than fusions with LICR-2 or SKO-007, and LICR-2 produced 4 times as many clones as SKO-007. The percentage of clones secreting human immunoglobulin, the range of immunoglobulin production, and the proportion of IgM, IgA, and IgG secretors were comparable for clones derived from the three myeloma-lymphoblastoid lines. Stable Ig-secreting clones were isolated with approximately equal frequency from LICR-2 and NS-1 fusions. A number of stable clones producing human monoclonal antibodies reacting with cell-surface, cytoplasmic, or nuclear antigens have been isolated from tumor-bearing patients and normal individuals. A surface antigenic system present on normal and malignant cells has been defined with a human monoclonal antibody derived from a patient with breast cancer. Techniques for producing human monoclonal antibody now appear to be sufficiently advanced to initiate a serological dissection of the humoral immune response to cancer. Images PMID:6572959

  1. Production and characterization of a monoclonal antibody against enrofloxacin.

    PubMed

    Chusri, Manaspong; Wongphanit, Pitikarn; Palaga, Tanapat; Puthong, Songchan; Sooksai, Sarintip; Komolpis, Kittinan

    2013-01-01

    Enrofloxacin is a fluoroquinolone antibiotic approved for the treatment of infections in animals. Because of the side effects to consumers of animal products, the maximum residue limits (MRLs) of enrofloxacin in animal tissues for consumption are regulated. In this study, a monoclonal antibody (mAb) against enrofloxacin was prepared and characterized for the development of a direct competitive enzyme-linked immunosorbent assay (ELISA). The obtained mAb, Enro44, was highly specific for enrofloxacin and had a 50% inhibition concentration (IC(50)) of 1.99 ng/ml in a competitive ELISA, and the limit of detection (LOD) was 0.50 ng/ml. The cross-reactivity of the mAb with other quinolones and fluoroquinolones was lower than 0.01%. The subclass of the mAb Enro44 was identified as IgG1. The antigen (Ag)-captured direct competitive ELISA using the mAb Enro44 was tested on different spiked samples, including chicken muscle, cattle milk, and cattle urine, and the assay demonstrated recoveries of 82-112%, 80-125%, and 78-124%, respectively. Furthermore, the quantitation of enrofloxacin obtained from the ELISA and from high-performance liquid chromatography (HPLC) was in good agreement, with the linear regression coefficient between 0.933 and 1.056. The cDNAs encoding a heavy-chain Fd fragment (VH and CH1) and a light chain of the mAb Enro44 were cloned and sequenced. Taken together, the results obtained reveal a potential use of this mAb in an ELISA for the detection of enrofloxacin in food samples. The information of amino acid sequence of this mAb will be useful for further modification and production of the mAb in a bioreactor.

  2. Monoclonal Antibodies as Prophylactic and Therapeutic Agents Against Chikungunya Virus.

    PubMed

    Clayton, April M

    2016-12-15

    Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that is responsible for considerable epidemics worldwide and recently emerged in the Americas in 2013. CHIKV may cause long-lasting arthralgia after acute infection. With currently no licensed vaccines or antivirals, the design of effective therapies to prevent or treat CHIKV infection is of utmost importance and will be facilitated by increased understanding of the dynamics of chikungunya. In this article, monoclonal antibodies against CHIKV as viable prophylactic and therapeutic agents will be discussed. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  3. 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.

  4. Monoclonal antibody therapy in the treatment of Reye's syndrome.

    PubMed

    Treon, S P; Broitman, S A

    1992-11-01

    A role for lipopolysaccharides (endotoxins, LPS) in 7 the pathogenesis of Reye's syndrome (RS) has previously been suggested. Impairment of hepatic LPS clearance can lead to systemic endotoxemia as previous studies by this and other laboratories have suggested for several hepatic disorders including RS. Systemic LPS may mediate many of the clinical findings associated with RS by eliciting monokines such as tumor necrosis factor-alpha, interleukin-1, interleukin-6, and interleukin-8. Monoclonal antibody therapy directed at LPS, and monokines may represent a novel approach to the treatment of RS.

  5. Monoclonal antibody against mouse CAR following genetic immunization.

    PubMed

    Carson, Steven D; Switzer, Barbara L; Tracy, Steven M; Chapman, Nora M

    2004-02-01

    To broaden our repertoire of monoclonal antibodies against CAR (coxsackievirus and adenovirus receptor), we inoculated mice with an expression vector containing the cDNA encoding human CAR extracellular and transmembrane sequence, and boosted the response by inoculation with soluble human CAR protein produced in E. coli. Of the hybridomas obtained following this immunization protocol, one secreted IgG with exceptional reactivity against mouse CAR. Since CAR has been shown to form dimers, expression of human CAR in cells that express mouse CAR may have stimulated the host immune system to recognize endogenous CAR in heterodimers.

  6. 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.

  7. Generation of cell lines for monoclonal antibody production.

    PubMed

    Alvin, Krista; Ye, Jianxin

    2014-01-01

    Monoclonal antibodies (mAbs) represent the largest group of therapeutic proteins with 30 products approved in the USA and hundreds of therapies currently undergoing clinical trials. The complex nature of mAbs makes their development as therapeutic agents constrained by numerous criteria such as quality, safety, regulation, and quantity. Identification of a clonal cell line expressing high levels of mAb with adequate quality attributes and generated in compliance with regulatory standards is a necessary step prior to a program moving to large-scale production for clinical material. This chapter outlines the stable transfection technology that generates clonal cell lines for commercial manufacturing processes.

  8. 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.

  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. Humanized Monoclonal Antibody That Passively Protects Mice against Systemic and Intranasal Ricin Toxin Challenge

    PubMed Central

    Sully, Erin K.; Bohorova, Natasha; Bohorov, Ognian; Kim, Do; Pauly, Michael H.; Whaley, Kevin J.

    2016-01-01

    PB10 is a murine monoclonal antibody against an immunodominant epitope on ricin toxin's enzymatic subunit. Here, we characterize a fully humanized version of PB10 IgG1 (hPB10) and demonstrate that it has potent in vitro and in vivo toxin-neutralizing activities. We also report the minimum serum concentrations of hPB10 required to protect mice against 10 times the 50% lethal dose of ricin when delivered by injection and inhalation. PMID:27466351

  11. Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats.

    PubMed

    Byrnes-Blake, Kelly A; Laurenzana, Elizabeth M; Carroll, F Ivy; Abraham, Philip; Gentry, W Brooks; Landes, Reid D; Owens, S Michael

    2003-02-14

    Our studies examined pharmacokinetic mechanisms involved in high-affinity (K(d) approximately 11 nM) monoclonal antibody-based antagonism of (+)-methamphetamine-induced locomotor effects. Male rats received (+)-methamphetamine (0.3, 1, or 3 mg/kg i.v.) followed 30 min later by saline or anti-(+)-methamphetamine monoclonal antibody. All groups received a constant dose of monoclonal antibody that was equimolar in binding sites to the body burden of a 1 mg/kg i.v. (+)-methamphetamine dose 30 min after administration. The monoclonal antibody antagonized locomotor effects due to 0.3 and 1 mg/kg (+)-methamphetamine. In contrast, monoclonal antibody treatment increased locomotor activity due to 3 mg/kg (+)-methamphetamine. We also investigated the serum and brain pharmacokinetics of (+)-methamphetamine without and with the monoclonal antibody. Rats received (+)-methamphetamine (1 mg/kg i.v.) followed by saline or monoclonal antibody treatment at 30 min. The monoclonal antibody significantly increased serum methamphetamine concentrations and significantly decreased brain methamphetamine concentrations. These data indicate that anti-(+)-methamphetamine monoclonal antibody-induced pharmacodynamics are complex, but are related to time-dependent changes in (+)-methamphetamine brain distribution. Copyright 2003 Elsevier Science B.V.

  12. Monoclonal antibodies to pancreatic stone protein. Radioimmunoassay and immunological comparison with trypsin 1.

    PubMed

    Provansal-Cheylan, M; Lusher, M; De Caro, A; Multigner, L; Montalto, G; Sarles, H; Delaage, M

    1986-09-01

    Monoclonal antibodies were prepared against pancreatic stone protein, a protein which inhibits calcium carbonate precipitation. Two monoclonal antibodies designated D4 and 2E7 were characterized. Immunoadsorbant columns, obtained by linkage of these monoclonal antibodies to Affigel 10, have been used to isolate immunoreactive forms of pancreatic stone protein from nonactivated human pancreatic juice. These monoclonal antibodies permitted us to test the possible immunological relationship between pancreatic stone protein and human trypsin 1. No immunological similarity was found, in agreement with our previous results, and it was established that pancreatic stone protein is a novel protein and not a degradation product of human trypsin(ogen) 1.

  13. Binding specificity of a monoclonal anti-DNA antibody.

    PubMed

    Pisetsky, D S; Caster, S A

    1982-05-01

    To investigate the interaction of DNA and anti-DNA antibodies in the immune complex disease of systemic lupus erythematosus, the fine specificity of binding of a monoclonal anti-DNA antibody was determined. This antibody, termed Cll, was derived from the fusion of spleen cells from an autoimmune MRL-lpr/lpr mouse with the myeloma cell line M45. In a solid-phase ELISA assay to measure anti-DNA activity, Cll showed preference for single stranded compared to double stranded DNA of animal origin. The Cll antibody also bound some deoxyribohomopolymers as well as ribohomopolymers, but failed to bind synthetic DNA duplexes. Defined size oligonucleotides with a size range of 2-(12-18) failed to inhibit the binding of Cll to single stranded DNA. This pattern of binding is consistent with the recognition of a unique structural determinant that can be represented by a variety of nucleic acids. The absence of antigenic activity among the oligonucleotides suggests that an extended polynucleotide structure is required for antibody binding, possibly because of a bivalent or 'monogamous' mode of interaction. The binding properties of Cll further suggest that its ability to participate in immune complex formation may be limited by the nature of the available DNA antigen.

  14. Monoclonal Antibody-Based Candidate Therapeutics Against HIV Type 1

    PubMed Central

    Dimitrov, Dimiter S.

    2012-01-01

    Abstract Treatment of HIV-1 infection has been highly successful with small molecule drugs. However, resistance still develops. In addition, long-term use can lead to toxicity with unpredictable effects on health. Finally, current drugs do not lead to HIV-1 eradication. The presence of the virus leads to chronic inflammation, which can result in increased morbidity and mortality after prolonged periods of infection. Monoclonal antibodies (mAbs) have been highly successful during the past two decades for therapy of many diseases, primarily cancers and immune disorders. They are relatively safe, especially human mAbs that have evolved in humans at high concentrations to fight diseases and long-term use may not lead to toxicities. Several broadly neutralizing mAbs (bnmAbs) against HIV-1 can protect animals but are not effective when used for therapy of an established infection. We have hypothesized that HIV-1 has evolved strategies to effectively escape neutralization by full-size antibodies in natural infections but not by smaller antibody fragments. Therefore, a promising direction of research is to discover and exploit antibody fragments as potential candidate therapeutics against HIV-1. Here we review several bnmAbs and engineered antibody domains (eAds), their in vitro and in vivo antiviral efficacy, mechanisms used by HIV-1 to escape them, and strategies that could be effective to develop more powerful mAb-based HIV-1 therapeutics. PMID:21827278

  15. Monoclonal antibodies with specificity for hairy cell leukemia cells.

    PubMed Central

    Posnett, D N; Chiorazzi, N; Kunkel, H G

    1982-01-01

    Hairy cell leukemia is a well described clinical entity, but the cell of origin for this leukemic cell and its function are still unknown. There are no totally specific markers for this cell, although tartrate-resistant acid phosphatase staining has been used extensively as a diagnostic test. This study describes three monoclonal murine antibodies with variable specificity for hairy cells. Antibody 1 was highly specific for hairy cells and was not found to react with normal or leukemic cells in this limited study. It did not react with the cells of all patients. It also did not react with all of the hairy cells of some of the positive cases. Antibodies 2 and 3 reacted with virtually all hairy cells but not with normal peripheral blood cells. However, reactions were obtained with certain leukemic myelomonoblasts and some activated B cells. The most obvious use for these three antibodies is for diagnostic purposes. They should also be helpful reagents to investigate the origin of the leukemic hairy cell. The possibility that antibody 1 detects a tumor-specific antigen is discussed. Images PMID:7047565

  16. Monoclonal antibody-based candidate therapeutics against HIV type 1.

    PubMed

    Chen, Weizao; Dimitrov, Dimiter S

    2012-05-01

    Treatment of HIV-1 infection has been highly successful with small molecule drugs. However, resistance still develops. In addition, long-term use can lead to toxicity with unpredictable effects on health. Finally, current drugs do not lead to HIV-1 eradication. The presence of the virus leads to chronic inflammation, which can result in increased morbidity and mortality after prolonged periods of infection. Monoclonal antibodies (mAbs) have been highly successful during the past two decades for therapy of many diseases, primarily cancers and immune disorders. They are relatively safe, especially human mAbs that have evolved in humans at high concentrations to fight diseases and long-term use may not lead to toxicities. Several broadly neutralizing mAbs (bnmAbs) against HIV-1 can protect animals but are not effective when used for therapy of an established infection. We have hypothesized that HIV-1 has evolved strategies to effectively escape neutralization by full-size antibodies in natural infections but not by smaller antibody fragments. Therefore, a promising direction of research is to discover and exploit antibody fragments as potential candidate therapeutics against HIV-1. Here we review several bnmAbs and engineered antibody domains (eAds), their in vitro and in vivo antiviral efficacy, mechanisms used by HIV-1 to escape them, and strategies that could be effective to develop more powerful mAb-based HIV-1 therapeutics.

  17. Monoclonal antibody therapy directed against human acute myeloid leukemia stem cells.

    PubMed

    Majeti, R

    2011-03-03

    Accumulating evidence indicates that many human cancers are organized as a cellular hierarchy initiated and maintained by self-renewing cancer stem cells. This cancer stem cell model has been most conclusively established for human acute myeloid leukemia (AML), although controversies still exist regarding the identity of human AML stem cells (leukemia stem cell (LSC)). A major implication of this model is that, in order to eradicate the cancer and cure the patient, the cancer stem cells must be eliminated. Monoclonal antibodies have emerged as effective targeted therapies for the treatment of a number of human malignancies and, given their target antigen specificity and generally minimal toxicity, are well positioned as cancer stem cell-targeting therapies. One strategy for the development of monoclonal antibodies targeting human AML stem cells involves first identifying cell surface antigens preferentially expressed on AML LSC compared with normal hematopoietic stem cells. In recent years, a number of such antigens have been identified, including CD123, CD44, CLL-1, CD96, CD47, CD32, and CD25. Moreover, monoclonal antibodies targeting CD44, CD123, and CD47 have demonstrated efficacy against AML LSC in xenotransplantation models. Hopefully, these antibodies will ultimately prove to be effective in the treatment of human AML.

  18. Monoclonal antibodies to human plasma low-density lipoproteins. I. Enhanced binding of 125I-labeled low-density lipoproteins by combined use of two monoclonal antibodies.

    PubMed

    Mao, S J; Patton, J G; Badimon, J J; Kottke, B A; Alley, M C; Cardin, A D

    1983-11-01

    Four monoclonal antibodies (IgG2b) to human plasma low-density lipoproteins (LDL) have been characterized. The binding affinities of each monoclonal antibody to 125I-labeled LDL were moderately high, ranging from 10(8) to 10(10) L/mol at 4 degrees C, but were reduced by at least 50-70% at 37 degrees C. The maximum binding of each monoclonal antibody was unique, ranging from 20 to 95% of total 125I-labeled LDL, suggesting that LDL particles were immunochemically heterogeneous. One antibody, LP-34, had both high and low binding affinities to LDL. Another, LP-47, exhibited high affinity for isolated LDL, yet reacted poorly with native LDL in plasma, indicating that the conformation of isolated LDL differs from that of native LDL in plasma. Unlike polyclonal serum antibodies, a mixture of four monoclonal antibodies failed to precipitate LDL, but did show a drastic increase in binding to LDL. We found that only two of our monoclonal antibodies were necessary for such synergistic enhancement. We propose that one of the monoclonal antibodies may serve as a catalytic reagent, and discuss the clinical significance of this finding.

  19. 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

  20. Enhanced antibody-dependent cellular phagocytosis by chimeric monoclonal antibodies with tandemly repeated Fc domains.

    PubMed

    Nagashima, Hiroaki; Ootsubo, Michiko; Fukazawa, Mizuki; Motoi, Sotaro; Konakahara, Shu; Masuho, Yasuhiko

    2011-04-01

    We previously reported that chimeric monoclonal antibodies (mAbs) with tandemly repeated Fc domains, which were developed by introducing tandem repeats of Fc domains downstream of 2 Fab domains, augmented binding avidities for all Fcγ receptors, resulting in enhanced antibody (Ab)-dependent cellular cytotoxicity. Here we investigated regarding Ab-dependent cellular phagocytosis (ADCP) mediated by these chimeric mAbs, which is considered one of the most important mechanisms that kills tumor cells, using two-color flow cytometric methods. ADCP mediated by T3-Ab, a chimeric mAb with 3 tandemly repeated Fc domains, was 5 times more potent than that by native anti-CD20 M-Ab (M-Ab hereafter). Furthermore, T3-Ab-mediated ADCP was resistant to competitive inhibition by intravenous Ig (IVIG), although M-Ab-mediated ADCP decreased in the presence of IVIG. An Fcγ receptor-blocking study demonstrated that T3-Ab mediated ADCP via both FcγRIA and FcγRIIA, whereas M-Ab mediated ADCP exclusively via FcγRIA. These results suggest that chimeric mAbs with tandemly repeated Fc domains enhance ADCP as well as ADCC, and that Fc multimerization may significantly enhance the efficacy of therapeutic Abs.

  1. Monoclonal antibodies to the extracellular glucosyltransferases from Streptococcus sobrinus 6715.

    PubMed Central

    McCabe, M M; Alberts, M; Stein, J

    1987-01-01

    Murine monoclonal antibodies (MAbs) were raised against the glucosyltransferases (GTFs) of Streptococcus sobrinus 6715. The antibody panels included MAbs raised against the primer-independent, soluble product enzyme (GTF-Si) which did not cross-react with other GTFs, as well as MAbs raised against the primer-dependent, soluble product enzyme (GTF-Sd) which recognized both GTF-Si and GTF-Sd, thus indicating that these catalytically distinct enzymes share epitopes. MAbs raised against GTF-I recognized several forms of GTF-I and did not cross-react with the GTF-S enzymes. None of the MAbs recognized the major glucan-binding protein of S. sobrinus. Two MAbs inhibited glucan synthesis, one blocking primer synthesis by GTF-Si by 89% and the second inhibiting that by GTF-I by 92%. Images PMID:2956196

  2. Discovery and characterization of hydroxylysine in recombinant monoclonal antibodies

    PubMed Central

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

    2016-01-01

    ABSTRACT 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 MSn 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

  3. The application of monoclonal antibodies in cancer diagnosis.

    PubMed

    Zhang, Xuemei; Soori, Gamini; Dobleman, Thomas J; Xiao, Gary G

    2014-01-01

    Cancer becomes the second leading cause of death in the world. An effective strategy for early diagnosis of the disease is key to reduce the mortality and morbidity. Development of effective monoclonal antibody (mAb)-based assays or diagnostic imaging techniques for detection of antigens and small molecules that are released from cancerous cells will enhance modern diagnostic medicine of cancer significantly. Although mAb technology is still under development, recent advances in preparation of recombinant antigen and antibody engineering techniques have dramatically enhanced the applications of this technology in cancer diagnosis. Compared with other methods, mAb-based assays may provide spatial, temporal, accurate and quantitative measurement for diagnosis of the disease. This review summarizes the progress of the mAb-based assays in the field of molecular diagnosis of cancer.

  4. 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.

  5. Development of a multi-product leached protein A assay for bioprocess samples containing recombinant human monoclonal antibodies.

    PubMed

    Ren, Diya; Darlucio, Maria R; Chou, Judy H

    2011-03-07

    The detection of low level of protein A leached from monoclonal antibody downstream purification process is often interfered by the presence of excess amount of product antibody. In order to prevent this interference, we developed a new multi-product leached protein A assay that used acidification to completely dissociate the IgG-ProteinA complex, followed by neutralization under selected condition to prevent re-formation of the IgG-ProteinA complex. The amount of protein A was then determined by a sandwich immunoassay using Meso Scale Discovery technology. The assay takes approximately 3h to complete for one 96-well plate of samples, and this has been successfully applied to samples containing different monoclonal antibody products examined so far. The data demonstrates that this assay is robust and efficient in determining leached protein A contamination during purification of recombinant monoclonal antibodies. Copyright © 2010 Elsevier B.V. All rights reserved.

  6. Development of a PBPK model for monoclonal antibodies and simulation of human and mice PBPK of a radiolabelled monoclonal antibody.

    PubMed

    Heiskanen, Tomi; Heiskanen, Tomas; Kairemo, Kalevi

    2009-01-01

    Physiology based pharmacokinetic (PBPK) modeling and simulation is a useful method for prediction of biodistribution of both macromolecules and small molecules. It can enhance our understanding of the underlying mechanisms of biodistribution and hence may help in rational design of macromolecules used as diagnostic and therapeutic agents. In this review we discuss PBPK modeling and simulation of a radiolabelled Monoclonal Antibody ((111)In-DOTA-hAFP31 IgG) ("MAB") in mice without tumor and in a human with tumor. This study is part of Xemet Co.'s effort to develop a more accurate and reliable PBPK model and simulation platform, which is applicable both for small molecules and macromolecules. The simulated results were fitted to experimental time series data by varying parameters which were not fixed a priori. It was demonstrated that the PBPK model describes the main features of the pharmacokinetics of the studied systems. It was also shown that simulation can be used for evaluating the parameters of the system and scaling up the pharmacokinetics of MAB from mice to man. We identified several areas of improvement and further development needed to improve the accuracy of PBPK simulation for MAB and other macromolecules. It was concluded that the transvascular permeabilities are the most important parameters and more research is needed to enable prediction of permeabilities from molecular characteristics of macromolecules. It would also be necessary to understand better and describe with a more detailed model the microstructure of the tumor and to measure or predict the antigen concentration in tumor. Non-specific, non-saturable binding in other organs/tissues should be understood better and the kinetic constants of the binding should be measured experimentally. Although the metabolism and clearance were neglected in this study they need to be included in more detailed studies. Also the intracellular trafficking of macromolecules, which was not included in this study

  7. Studying host cell protein interactions with monoclonal antibodies using high throughput protein A chromatography.

    PubMed

    Sisodiya, Vikram N; Lequieu, Joshua; Rodriguez, Maricel; McDonald, Paul; Lazzareschi, Kathlyn P

    2012-10-01

    Protein A chromatography is typically used as the initial capture step in the purification of monoclonal antibodies produced in Chinese hamster ovary (CHO) cells. Although exploiting an affinity interaction for purification, the level of host cell proteins in the protein A eluent varies significantly with different feedstocks. Using a batch binding chromatography method, we performed a controlled study to assess host cell protein clearance across both MabSelect Sure and Prosep vA resins. We individually spiked 21 purified antibodies into null cell culture fluid generated with a non-producing cell line, creating mock cell culture fluids for each antibody with an identical composition of host cell proteins and antibody concentration. We demonstrated that antibody-host cell protein interactions are primarily responsible for the variable levels of host cell proteins in the protein A eluent for both resins when antibody is present. Using the additives guanidine HCl and sodium chloride, we demonstrated that antibody-host cell protein interactions may be disrupted, reducing the level of host cell proteins present after purification on both resins. The reduction in the level of host cell proteins differed between antibodies suggesting that the interaction likely varies between individual antibodies but encompasses both an electrostatic and hydrophobic component.

  8. Biosimilar monoclonal antibodies (mAbs) in oncology.

    PubMed

    Moore, Caroline

    2017-09-07

    Biological medicines are derived from living cells and organisms. Monoclonal antibodies (mAbs) are biological agents that are widely used to treat malignancies including non-Hodgkin's lymphomas and chronic lymphocytic leukaemia. They are effective but expensive. The patents for many mAbs are expiring, so biosimilar medicines, which contain a version of the active ingredient of the original drug, are being developed. Biological medicines cannot be assessed in the same way as standard generic medications because they are difficult to copy and can change over time. A pathway regulates how biosimilars are assessed and compared with the original drug to ensure they are highly similar and have no clinically meaningful differences in terms of structure, function, pharmacodynamics and mechanism of action, pharmacokinetic properties, clinical efficacy and safety. Truxima(® ▾)(rituximab), the first biosimilar monoclonal antibody to be approved for use in the UK in an oncology setting, is biosimilar to intravenous (IV) rituximab; rituximab improves the effectiveness of standard chemotherapy for lymphoma. The two drugs are comparable in efficacy and safety and have the same indications, dosing regimen and storage procedures.

  9. [Obtaining monoclonal antibodies against outer membrane glycoproteins of Entamoeba histolytica].

    PubMed

    Agundis, C; Isibasi, A; Ortíz, V; Reyes, J L; Paniagua, J; Ramírez, A; Kumate, J

    1990-01-01

    The goal of this paper was the production of monoclonal antibodies capable of detecting relevant antigens from the surface of Entamoeba histolytica trophozoites, with the purpose of using them as a diagnostic test. The cellular fusion for obtaining the monoclonal antibodies (mAb) was done with spleen cells from BALB/c mice, previously immunized with glycoproteins from the membrane, as well as Sp2/0 cells. The hybridoma supernatants were tested with ELISA, using glycoproteins and lipopeptide phosphoglycans (LPPG) as antigens. Seven hybridomas producing mAb against the glycoproteins were found. Among these, three recognize LPPG. The ability of reacting with the mAb against two molecules disappeared for all the LPPG positive ones when were treated with meta-periodate, and only three reacted against the glycoproteins. All of the mAb were of the Ig M isotypes. They were characterized by Dot blot and Western blot assays. From the results, one may deduce that some mAb recognize as epitopes the polysaccharide portion, and thus infer that they are directed of against the surface and therefore, in the future, could be used with a diagnostic purpose.

  10. Generation of a rat monoclonal antibody specific for hsp72.

    PubMed

    Tanaka, Masako; Shiota, Masayuki; Okada, Seiji; Harada, Akihito; Odawara, Jun; Mun, Saya; Iwao, Hiroshi; Ohkawa, Yasuyuki

    2011-08-01

    The heat shock protein 70 (Hsp70) family members function as ATP-dependent molecular chaperones that assist in the folding of newly synthesized polypeptides and in the refolding of misfolded/aggregated proteins. These heat shock proteins comprise at least eight sets of molecular groups that share high homology, but differ from each other in their expression level and subcellular localization. Hsp72, which is also known as Hsp70 and Hsp70-1, is localized mainly in the cytoplasm but is also found in the nucleus. Stress-induced Hsp72 functions as a chaperone enabling the cells to cope with harmful aggregations of denatured proteins during and following stress. The difference in the function of Hsp72 from that of other Hsp70 members, however, remains unclear. We report the establishment of a monoclonal antibody specific for Hsp72 using the rat medial iliac lymph node method. Immunoblot analysis revealed that our monoclonal antibody against Hsp72 specifically identified the 65 kDa protein. Immunocytochemical staining also revealed that Hsp72 localized in the cytoplasm and nucleus, and aggregated in the nucleus in response to heat stress. This MAb against Hsp72 will allow for further studies to elucidate the mechanism by which Hsp72 is localized in the cell in response to stress stimuli, and aid in the identification of specific interacting molecules.

  11. Bothropic antivenom based on monoclonal antibodies, is it possible?

    PubMed

    Frauches, Thiago S; Petretski, Jorge H; Arnholdt, Andrea C V; Lasunskaia, Elena B; de Carvalho, Eulógio C Q; Kipnis, Thereza L; da Silva, Wilmar D; Kanashiro, Milton M

    2013-09-01

    Neutralizing monoclonal antibodies against three major toxic components of Bothrops atrox venom were produced and tested. The mAbs against phospholipase A2, hemorrhagic metalloprotease, and thrombin-like enzymes were produced in large amounts and purified with caprylic acid followed by ammonium sulfate precipitation. Purified mAbs were analyzed by SDS-PAGE and their ability to neutralize the respective toxins was tested. Five Swiss mice were injected i.p. with 13.5 mg of pooled mAbs and challenged via s.c. route with venom. Survival rate was recorded for the next 48 h. All mice treated and challenged with venom survived, whereas only one mouse in the control group survived. Bleeding time in mice treated with mAbs was similar to that observed in control mice. Our results show that monoclonal antibodies neutralized the lethal toxicity of Bothrops venom and indicate that there is a reasonable possibility of developing antivenoms based on humanized mAbs to treat victims of venomous animals in the future.

  12. Monoclonal antibodies specific to heat-treated porcine blood.

    PubMed

    Raja Nhari, Raja Mohd Hafidz; Hamid, Muhajir; Rasli, Nurmunirah Mohamad; Omar, Abdul Rahman; El Sheikha, Aly Farag; Mustafa, Shuhaimi

    2016-05-01

    Porcine blood is potentially being utilized in food as a binder, gelling agent, emulsifier or colorant. However, for certain communities, the usage of animal blood in food is strictly prohibited owing to religious concerns and health reasons. This study reports the development of monoclonal antibodies (MAbs) against heat-treated soluble proteins (HSPs) of autoclaved porcine blood; characterization of MAbs against blood, non-blood and plasma from different animal species using qualitative indirect non-competitive enzyme-linked immunosorbent assay (ELISA); and immunoblotting of antigenic components in HSPs of porcine blood. Fifteen MAbs are specific to heat-treated and raw porcine blood and not cross-reacted with other animal blood and non-blood proteins (meat and non-meat). Twelve MAbs are specific to porcine plasma, while three MAbs specific to porcine plasma are cross-reacted with chicken plasma. Immunoblotting revealed antigenic protein bands (∼60, ∼85-100 and ∼250 kDa) in porcine blood and plasma recognized by the MAbs. Selection of MAbs that recognized 60 kDa HSPs of porcine blood and plasma as novel monoclonal antibodies would be useful for detection of porcine plasma in processed food using the immunoassay method. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

  13. 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

  14. Microglial internalization and degradation of pathological tau is enhanced by an anti-tau monoclonal antibody

    PubMed Central

    Luo, Wenjie; Liu, Wencheng; Hu, Xiaoyan; Hanna, Mary; Caravaca, April; Paul, Steven M.

    2015-01-01

    Microglia have been shown to contribute to the clearance of brain amyloid β peptides (Aβ), the major component of amyloid plaques, in Alzheimer’s disease (AD). However, it is not known whether microglia play a similar role in the clearance of tau, the major component of neurofibrillary tangles (NFTs). We now report that murine microglia rapidly internalize and degrade hyperphosphorylated pathological tau isolated from AD brain tissue in a time-dependent manner in vitro. We further demonstrate that microglia readily degrade human tau species released from AD brain sections and eliminate NFTs from brain sections of P301S tauopathy mice. The anti-tau monoclonal antibody MC1 enhances microglia-mediated tau degradation in an Fc-dependent manner. Our data identify a potential role for microglia in the degradation and clearance of pathological tau species in brain and provide a mechanism explaining the potential therapeutic actions of passively administered anti-tau monoclonal antibodies. PMID:26057852

  15. Detection of complement activation using monoclonal antibodies against C3d.

    PubMed

    Thurman, Joshua M; Kulik, Liudmila; Orth, Heather; Wong, Maria; Renner, Brandon; Sargsyan, Siranush A; Mitchell, Lynne M; Hourcade, Dennis E; Hannan, Jonathan P; Kovacs, James M; Coughlin, Beth; Woodell, Alex S; Pickering, Matthew C; Rohrer, Bärbel; Holers, V Michael

    2013-05-01

    During complement activation the C3 protein is cleaved, and C3 activation fragments are covalently fixed to tissues. Tissue-bound C3 fragments are a durable biomarker of tissue inflammation, and these fragments have been exploited as addressable binding ligands for targeted therapeutics and diagnostic agents. We have generated cross-reactive murine monoclonal antibodies against human and mouse C3d, the final C3 degradation fragment generated during complement activation. We developed 3 monoclonal antibodies (3d8b, 3d9a, and 3d29) that preferentially bind to the iC3b, C3dg, and C3d fragments in solution, but do not bind to intact C3 or C3b. The same 3 clones also bind to tissue-bound C3 activation fragments when injected systemically. Using mouse models of renal and ocular disease, we confirmed that, following systemic injection, the antibodies accumulated at sites of C3 fragment deposition within the glomerulus, the renal tubulointerstitium, and the posterior pole of the eye. To detect antibodies bound within the eye, we used optical imaging and observed accumulation of the antibodies within retinal lesions in a model of choroidal neovascularization (CNV). Our results demonstrate that imaging methods that use these antibodies may provide a sensitive means of detecting and monitoring complement activation-associated tissue inflammation.

  16. Detection of complement activation using monoclonal antibodies against C3d

    PubMed Central

    Thurman, Joshua M.; Kulik, Liudmila; Orth, Heather; Wong, Maria; Renner, Brandon; Sargsyan, Siranush A.; Mitchell, Lynne M.; Hourcade, Dennis E.; Hannan, Jonathan P.; Kovacs, James M.; Coughlin, Beth; Woodell, Alex S.; Pickering, Matthew C.; Rohrer, Bärbel; Holers, V. Michael

    2013-01-01

    During complement activation the C3 protein is cleaved, and C3 activation fragments are covalently fixed to tissues. Tissue-bound C3 fragments are a durable biomarker of tissue inflammation, and these fragments have been exploited as addressable binding ligands for targeted therapeutics and diagnostic agents. We have generated cross-reactive murine monoclonal antibodies against human and mouse C3d, the final C3 degradation fragment generated during complement activation. We developed 3 monoclonal antibodies (3d8b, 3d9a, and 3d29) that preferentially bind to the iC3b, C3dg, and C3d fragments in solution, but do not bind to intact C3 or C3b. The same 3 clones also bind to tissue-bound C3 activation fragments when injected systemically. Using mouse models of renal and ocular disease, we confirmed that, following systemic injection, the antibodies accumulated at sites of C3 fragment deposition within the glomerulus, the renal tubulointerstitium, and the posterior pole of the eye. To detect antibodies bound within the eye, we used optical imaging and observed accumulation of the antibodies within retinal lesions in a model of choroidal neovascularization (CNV). Our results demonstrate that imaging methods that use these antibodies may provide a sensitive means of detecting and monitoring complement activation–associated tissue inflammation. PMID:23619360

  17. Production and Characterization of Monoclonal Antibody against Recombinant Virus Coat Protein CP42.

    PubMed

    Shibaei, Naeimeh; Majidi, Jafar; Razavi, Khadijeh; Karkhane, Ali Asghar; Sokhandan-Bashir, Nemat; Aghebati-Maleki, Leili

    2017-02-01

    There are many studies related to the production of a ELISA kit for diagnosing virus infections. However, production of most kits depends on purification of whole virus particles, which involves the use of costly equipment and reagents. The purpose of this study was to check out if the anti-CP42 antibodies could be used as a diagnostic assay for detection of Grapevine fanleaf Virus (GFLV). In this study, recombinant GFLV coat protein gene related to selected antigenic determinants was inserted into pET-28a bacterial expression vector and the construct (pET-28a CP42) was cloned into E. coli strain (DE3). Expressed protein was verified with western blotting assay by the use of commercially available anti-GFLV antibody. The recombinant protein was purified using nickel-nitrilotriacetic acid (Ni-NTA) resin. Balb/c mice were immunized with purified protein and splenocytes of hyperimmunized mice were fused with murine myeloma Sp2/0 cells. Positive hybridomas were selected by ELISA using CP42 as coating antigen. The results showed that monoclonal antibody (MAb) specific to CP42 has been successfully generated. Efficiency of produced antibody was analyzed by ELISA and western blotting assay using some confirmed grapevine samples. The infection was confirmed previously based on morphological features and ELISA assay, performed using commercial anti-GFLV antibody. The monoclonal antibody reacted with antigen in ELISA and immunoblot method. Our results demonstrated that anti recombinant CP42 monoclonal antibodies are able to diagnose whole virus in infected grapevine sample using ELISA test.

  18. 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

  19. Elicitation of distinct populations of monoclonal antibodies specific for the variable domains of monoclonal anti-fluorescein antibody 4-4-20.

    PubMed

    Weidner, K M; Denzin, L K; Kim, M L; Mallender, W D; Miklasz, S D; Voss, E W

    1993-08-01

    Armenian hamsters were immunized with non-liganded, partially liganded or affinity-labeled anti-fluorescein Mab 4-4-20. Seventeen hybridoma producing monoclonal anti-4-4-20 antibodies were characterized from chemically-mediated fusions of immune hamster lymphocytes with murine Sp2/O-Ag14 myeloma cells. Distinct populations of anti-4-4-20 monoclonal antibodies were isolated from hamsters receiving immunizations with partially liganded Mab 4-4-20 relative to those receiving affinity-labeled 4-4-20. Two of the three monoclonal antibodies produced in response to partially liganded 4-4-20 were inhibited in their interaction with 4-4-20 by fluorescyl ligand. These two clones, 1F4 and 1B7, recognized unique epitopes on the 4-4-20 molecules, as demonstrated by non-reactivity with members of the 4-4-20 idiotype family. Additionally, 1F4 and 1B7 demonstrated the ability to delay the association of fluorescein with Mab 4-4-20. The 14 characterized non-ligand-inhibitable Mabs elicited to affinity-labeled 4-4-20 were classified into four separate groups based on various binding properties with members of the 4-4-20 idiotype family and binding to resolved H- and L-chains in a western blot. Members of three of the four groups showed strong reactivity with both 04-01 Ig and 04-01 SCA, which utilizes the same L-chain as Mab 4-4-20. Six non-ligand-inhibitable Mabs, 4A6, P1E11, 3A5-1, 2C3, 2C4, and 1A4, delayed the dissociation rate of ligand from Mab 4-4-20 and mutant 4-4-20 SCA L32phe.

  20. 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

  1. Rapid Isolation of Monoclonal Antibodies. Monitoring Enzymes in the Phytochelatin Synthesis Pathway

    PubMed Central

    Li, Y.; Kandasamy, M. K.; Meagher, R. B.

    2001-01-01

    Abstract Genomics projects have identified thousands of interesting new genes whose protein products need to be examined at the tissue, subcellular, and molecular levels. Furthermore, modern metabolic engineering requires accurate control of expression levels of multiple enzymes in complex pathways. The lack of specific immune reagents for characterization and monitoring of these numerous proteins limits all proteomic and metabolic engineering projects. We describe a rapid method of isolating monoclonal antibodies that required only sequence information from GenBank. We show that large synthetic peptides were highly immunogenic in mice and crude protein extracts were effective sources of antigen, thus eliminating the time-consuming step of purifying the target proteins for antibody production. A case study was made of the three-enzyme pathway for the synthesis of phytochelatins. Enzyme-linked immunosorbent assays and western blots with the recombinant proteins in crude extracts demonstrated that the monoclonal antibodies produced to synthetic peptides were highly specific for the different target proteins, gamma-glutamyl cysteine synthetase, glutathione synthetase, and phytochelatin synthase. Moreover, immunofluorescence localization studies with antibacterial -glutamyl cysteine synthetase and antiglutathione synthetase antibodies demonstrated that these immune reagents reacted strongly with their respective target proteins in chemically fixed cells from transgenic plants. This approach enables research to progress rapidly from the genomic sequence of poorly characterized target genes, to protein-specific antibodies, to functional studies. PMID:11706154

  2. 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. Submissions will be accepted through July 9, 2012.

  3. NCI Requests Cancer 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. Submissions will be accepted through July 11, 2014.

  4. 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.

  5. 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

  6. Intracavitary use of two radiolabeled tumor-associated monoclonal antibodies

    SciTech Connect

    Malamitsi, J.; Skarlos, D.; Fotiou, S.; Papakostas, P.; Aravantinos, G.; Vassilarou, D.; Taylor-Papadimitriou, J.; Koutoulidis, K.; Hooker, G.; Snook, D.

    1988-12-01

    Six patients with metastatic breast cancer and malignant pleural effusions and 13 patients with known or suspected ovarian cancer, underwent immunoscintigraphy after intracavitary (intrapleural or intraperitoneal) administration of iodine-131-(131I) or indium-111-(111In) labeled tumor associated monoclonal antibodies HMFG2 and H17E2. This method proved to be sensitive and specific with a true-positive result in 13 out of 14 patients with tumor and a true-negative result in five out of five patients without tumor. At any one time, 65%-80% of the whole-body radioactivity was closely associated with the cavity into which the radiolabeled antibody was administered while the radioactivity in the blood was always low, (approximately 4 X 10(-3) of administered dose/ml of blood). Concentrations of radiolabeled antibody (per gram of tumor tissue) ranged from 0.02%-0.1% of the injected dose in intracavitary tumors, but only 0.002% in a retroperitoneal metastasis. The specificity of this approach was documented in four control patients with benign ovarian cysts and in two patients who were imaged using both specific and nonspecific radiolabeled antibody. We conclude that the intracavitary administration of 131I- or 111In-labeled HMFG2 and H17E2 is a favorable route of administration and offers significant advantages over previously reported intravenous administration for the localization of breast or ovarian metastases confined to the pleural or peritoneal cavities.

  7. [Monoclonal antibodies against PCSK9: from bench to clinic].

    PubMed

    Guijarro Herraiz, Carlos

    2016-05-01

    Antibodies are glycoproteins with high specificity binding to multiple antigens due to the large number of structural conformations of the variable chains. Hybridoma technology (fusion of myeloma cells with immunoglobulin-producing lymphocytes) has allowed the synthesis of large quantities of unique antibodies (monoclonal [mAb]). mAbs were initially murine. Subsequently, chimeric mAbs were developed, followed by humanized mAbs and finally human mAbs. The high selectivity and good tolerance of human mAbs allows their therapeutic administration to block specific exogenous or endogenous molecules. Selective human mAbs to the catalytic domain of PCSK9 have recently been developed. These antibodies block PCSK9, favour low-density lipoprotein receptor recycling and markedly reduce circulating cholesterol. Preliminary studies indicate that lowering cholesterol through anti-PCSK9 antibodies may significantly reduce the cardiovascular complications of arteriosclerosis. Copyright © 2016 Elsevier España, S.L.U. y Sociedad Española de Arteriosclerosis. All rights reserved.

  8. Generation and characterization of monoclonal antibodies against FABP4.

    PubMed

    Gorbenko, Olena; Filonenko, Valeriy; Gout, Ivan

    2006-04-01

    Fatty acid binding protein 4 (FABP4) is a key mediator of intracellular transport and metabolism of fatty acids in adipose tissues. FABP4 binds fatty acids with high affinity and transports them to various compartments in the cell. When in complex with fatty acids, FABP4 interacts with and modulates the activity of two important regulators of metabolism: hormone-sensitive lipase and peroxisome proliferator-activated receptor gamma. Genetic studies in mice clearly indicated that deregulation of FABP4 function may lead to the development of severe diseases such as diabetes II type and atherosclerosis. In this study, we report the production and detailed characterization of monoclonal antibodies (MAbs) against FABP4. Recombinant glutathione S-transferase (GST)-FABP4 or His-FABP4 was expressed in bacteria, affinity purified, and used for immunization of mice, enzyme-linked immunosorbent assay (ELISA) screening, and characterization of selected clones. We have isolated two hybridoma clones that produced antibodies specific for recombinant and native FABP4, as shown by Western blotting and immunoprecipitation. The specificity of generated antibodies was further tested in a cell-based model of adipogenesis. In this analysis, the accumulation of FABP4 during NIH 3T3-L1 differentiation into adipocytes was detected by generated antibodies, which correlates well with previously published data. Taken together, we produced MAbs that will be useful for the scientific community working on fatty acid-binding proteins and lipid metabolism.

  9. Advantage of dose fractionation in monoclonal antibody-targeted radioimmunotherapy

    SciTech Connect

    Schlom, J.; Molinolo, A.; Simpson, J.F.; Siler, K.; Roselli, M.; Hinkle, G.; Houchens, D.P.; Colcher, D. )

    1990-05-02

    Monoclonal antibody (MAb) B72.3 IgG was radiolabeled with 131I and administered to female athymic NCr-nu mice bearing the LS-174T human colon adenocarcinoma xenograft to determine if fractionation of MAb dose had any advantage in tumor therapy. In the LS-174T xenograft, only approximately 30%-60% of tumor cells express the B72.3-reactive TAG-72 antigen. The LS-174T xenograft was used to reflect the heterogeneity of the TAG-72 antigen often seen in biopsy specimens from patients. In contrast to a single 600-muCi dose of 131I-B72.3 IgG where 60% of the animals died from toxic effects, two 300-muCi doses of 131I-B72.3 IgG reduced or eliminated tumor growth in 90% of mice, with only 10% of the animals dying from toxic effects. Dose fractionation even permitted escalation of the dose to three doses of 300 muCi of 131I-B72.3 IgG, resulting in even more extensive tumor reduction or elimination and minimal toxic effects. The use of an isotype-matched control MAb revealed a nonspecific component to tumor growth retardation, but the use of the specific B72.3 IgG demonstrated a much greater therapeutic effect. Tumors that had escaped MAb therapy were analyzed for expression of the B72.3-reactive TAG-72 antigen with the use of the immunoperoxidase method; they were shown to have the same antigenic phenotype as the untreated tumors. We verified tumor elimination by killing the test animals after a 7-week observation period and performing histologic examination of tumor sites. We also monitored toxic effects by histologic examination of numerous organs. These studies thus demonstrate the advantage of dose fractionation of a radiolabeled MAb for tumor therapy. We anticipate that the concept of dose fractionation can be practically applied in radioimmunotherapeutic clinical trials with the development and use of recombinant-chimeric MAbs and modified constructs.

  10. 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

    2016-01-05

    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.

  11. From the discovery of monoclonal antibodies to their therapeutic application: an historical reappraisal.

    PubMed

    Ribatti, Domenico

    2014-09-01

    Vertebrate make billions of different antibodies, each with a binding site that recognizes a specific region of a macromolecule. The hybridoma technique allows monoclonal antibodies, highly specific antibodies produced in the laboratory by a variety of methods. In the last 35 years since the first process for creating monoclonal antibodies was introduced, their application have improved the growing biotechnology industry, but the most important application concerns the therapy of human malignancies.

  12. 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).

  13. 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

  14. [Study of the antigenic structure of human immunoglobulin lambda-chain using monoclonal antibodies].

    PubMed

    Arsen'eva, E L; Bogacheva, G T; Solomon, A; Weiss, D; Ibragimov, A R; Rokhlin, O V

    1990-01-01

    Nine monoclonals against human Ig lambda chains were produced, 4 antibodies react with C-domain, 5--with V-domain of the lambda chain. Anti-C lambda domain antibodies recognize not less than 3 epitopes and one of them is expressed only on the isolated chain. Anti-V lambda antibodies bind both isolated lambda chain and intact IgG, IgM, IgA. Four epitopes are expressed by few lambda Bence Jones proteins of the III subgroup, the immunogen possessing the same isotype. The 4 mentioned epitopes represent private idiotypic determinants. The epitope 3E10 is characteristic of 50% Bence Jones proteins of the II and III V lambda-subgroups thus representing a common idiotypic determinant. Using anti-V lambda antibodies germ line variability of V lambda III proteins was analysed and the similarity of antigenic structure of normal and myeloma human Ig lambda chains was demonstrated.

  15. Cell type specific targeted intracellular delivery into muscle of a monoclonal antibody that binds myosin IIb.

    PubMed

    Weisbart, Richard H; Yang, Fusheng; Chan, Grace; Wakelin, Rika; Ferreri, Kevin; Zack, Debra J; Harrison, Brooke; Leinwand, Leslie A; Cole, Greg M

    2003-03-01

    Methods for cell type specific targeted intracellular delivery of proteins in vivo remain limited. A murine monoclonal anti-dsDNA antibody, mAb 3E10, was selectively transported into skeletal muscle cells in vivo. The antibody bound a 200 kDa protein only found in lysates of skeletal muscle by Western blotting. The 200 kDa protein was purified from muscle lysate by antibody affinity chromatography and identified as the skeletal muscle specific heavy chain of myosin IIb by electrospray mass spectrometry. Antibody binding specificity for myosin IIb was demonstrated in Western blots by binding myosin in skeletal muscle lysates from mice null for myosin IId but not in mice null for myosin IIb. Myosin IIb is implicated in the specific targeting of mAb 3E10 to skeletal muscle.

  16. Using monoclonal antibodies as an international standard for the measurement of anti-adalimumab antibodies.

    PubMed

    van Schouwenburg, Pauline A; Kruithof, Simone; Wolbink, Gertjan; Wouters, Diana; Rispens, Theo

    2016-02-20

    Comparing studies investigating anti-drug antibody (ADA) formation is hampered by the lack of comparability between study protocols, assay formats, and standardized reference materials. In this respect, the use of an international standard would mean a major step forward. Here we compared 11 fully human monoclonal antibodies against adalimumab in two assays commonly used for ADA measurement; the bridging ELISA and the antigen binding test (ABT). Our results show non-parallel titration of the monoclonal antibodies in both assays, which we also find for polyclonal ADA sources. Moreover, we observed that the output of the bridging ELISA depends to a large degree on the affinity of the monoclonal antibody. For the ABT, results reflect a combination of affinity and avidity. This suggests that rather than reporting ADA values in nanogram per milliliter, arbitrary units may be more appropriate. Together our data highlight the difficulty of ADA standardization by identifying several pitfalls that should be taken into account when selecting a standard for ADA testing.

  17. 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.

  18. 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

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

  20. 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.

  1. 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.

  2. Adsorption and recovery issues of recombinant monoclonal antibodies in reversed-phase liquid chromatography.

    PubMed

    Fekete, Szabolcs; Beck, Alain; Wagner, Elsa; Vuignier, Karine; Guillarme, Davy

    2015-01-01

    The poor recovery of large biomolecules is a well-known issue in reversed-phase liquid chromatography. Several papers have reported this problem, but the reasons behind this behavior are not yet fully understood. In the present study, state-of-the-art reversed-phase wide-pore stationary phases were used to evaluate the adsorption of therapeutic monoclonal antibodies. These biomolecules possess molar mass of approximately 150,000 g/mol and isoelectric points between 6.6 and 9.3. Two types of stationary phases were tested, the Phenomenex Aeris Widepore (silica based), with 3.6 μm superficially porous particles, and the Waters Acquity BEH300 (ethylene-bridged hybrid), with 1.7 μm fully porous particles. A systematic investigation was carried out using 11 immunoglobulin G1, G2, and G4 antibodies, namely, panitumumab, natalizumab, cetuximab, bevacizumab, trastuzumab, rituximab, palivizumab, belimumab, adalimumab, denosumab, and ofatumumab. All are approved by the Food and Drug Administration and the European Medicines Agency in various therapeutic indications and are considered as reference antibodies. Several test proteins, such as human serum albumin, transferrin, apoferritin, ovalbumin, and others, possessing a molar mass between 42,000 and 443,000 g/mol were also evaluated to draw reliable conclusions. The purpose of this study was to find a correlation between the adsorption of monoclonal antibodies and their physicochemical properties. Therefore, the impact of isoelectric point, molar mass, protein glycosylation, and hydrophobicity was investigated. The adsorption of intact antibodies on the stationary phase was significantly higher than that of proteins of similar size, isoelectric point, or hydrophobicity. The present study also demonstrates the unique behavior of monoclonal antibodies, contributing some unwanted and unpredictable strong secondary interactions.

  3. Monoclonal Antibodies Detect a Spectrin-Like Protein in Normal and Dystrophic Human Skeletal Muscle

    NASA Astrophysics Data System (ADS)

    Appleyard, S. T.; Dunn, M. J.; Dubowitz, V.; Scott, M. L.; Pittman, S. J.; Shotton, D. M.

    1984-02-01

    Spectrin is the major protein of the erythrocyte membrane skeleton, which is bound to the cytoplasmic surface of the membrane's lipid bilayer and is responsible for cell shape and membrane elasticity. Inability to identify spectrin in other cell types led to the assumption that this protein was unique to erythrocytes. However, spectrin-like proteins have been demonstrated recently in a variety of cell types, including skeletal and cardiac muscle, in several species. We used monoclonal antibodies against human erythrocyte spectrin subunits in an immunocytochemical study to detect related proteins in normal and diseased human skeletal muscle. Six of seven monoclonal antibodies against β -spectrin determinants were bound at the cytoplasmic surface of muscle fiber plasma membranes, whereas none of six monoclonal antibodies against α -spectrin determinants was bound. Muscle fibers of patients with neuromuscular diseases showed similar distribution and specificity of antibody binding to those of normal subjects, but the intensity of binding was increased. In contrast, probable regenerating fibers in muscle of patients with muscular dystrophies showed reduced binding of antibodies, but reduced binding was not seen in fetal muscle fibers nor in those of a patient with a myotubular myopathy. We conclude that human skeletal muscle fibers possess a spectrin-related protein associated with their plasma membrane that shows extensive β -chain similarities to erythrocyte spectrin but differs significantly with respect to the α -subunit. Its function may be associated with the maintenance of membrane and myofibril integrity during contraction, and the increased antibody binding in diseased muscle may reflect a structural rearrangement of spectrin or a compensatory increase in spectrin abundance in response to increased stress on these systems.

  4. Myosin at the apical pole of ciliated epithelial cells as revealed by a monoclonal antibody

    PubMed Central

    1986-01-01

    A monoclonal antibody (CC-212), obtained in a fusion experiment in which basal bodies from quail oviduct were used as immunogen, has been shown to label the apical pole of ciliated cells and to react with a 200-kD protein. This monoclonal antibody was demonstrated to be an anti- myosin from smooth muscle or from nonmuscular cells using the following criteria: On Western blots it reacted with the myosin heavy chains from gizzard and platelet extracts and from cultured cell line extracts, but did not react with striated muscle myosin heavy chains. By immunofluorescence it decorated the stress fibers of well-spread cells with a characteristic striated pattern, while it did not react with myotubes containing organized myofibrils. On native ciliated cells as well as on Triton-extracted ciliated cortices from quail oviduct, this monoclonal antibody decorated the apical pole with a stronger labeling of the periphery of the apical area. Ultrastructural localization was attempted using the immunogold technique on the same preparation. Myosin was associated with a filamentous material present between striated rootlets and the proximal extremities of the basal bodies. No labeling of the basal body itself or of axoneme was observed. PMID:3525577

  5. Neurological Complications of Therapeutic Monoclonal Antibodies: Trends from Oncology to Rheumatology.

    PubMed

    Gill, Chandler; Rouse, Stasia; Jacobson, Ryan D

    2017-08-17

    This review is to describe the scope of neurological complications associated with monoclonal antibody-based therapies, applied across medical specialties, to demonstrate the common and rare neurological syndromes that may be encountered in clinical practice according to the therapeutic agent being receive, and to explain appropriate work-up, diagnosis, and management of drug complications, as supported by the literature. The number of commercially available, evidence-based therapeutic monoclonal antibodies continues to expand. In oncology, immune checkpoint inhibitors are particularly important, as a wide range of central and peripheral nervous system complications are described. In rheumatology, anti-TNF alpha drugs remain associated with demyelinating syndromes. The number of therapeutic monoclonal antibodies encountered in practice continues to grow, as does the number of described neurological complications. Recognition of a possible drug complication is key, as these are typically complex patients at risk of other causes of neurological injury. Identification of a complication of therapy often leads to intervention and a change in management.

  6. Rapid high-resolution characterization of functionally important monoclonal antibody N-glycans by capillary electrophoresis.

    PubMed

    Szabo, Zoltan; Guttman, András; Bones, Jonathan; Karger, Barry L

    2011-07-01

    Characterization of the N-glycosylation present in the Fc region of therapeutic monoclonal antibodies requires rapid, high-resolution separation methods to guarantee product safety and efficacy during all stages of process development. Determination of fucosylated oligosaccharides is particularly important during clone selection, product characterization, and lot release as fucose has been shown to adversely affect the ability of mAbs to induce antibody dependent cellular cytotoxicity (ADCC). Here, we apply a general capillary electrophoresis optimization strategy to separate functionally relevant fucosylated and afucosylated glycans on mononclonal antibody products in the presence of several high mannose oligosaccharides. The N-glycans chosen represent those most commonly reported on CHO cell derived therapeutic antibodies. A rapid (<7 min) high-resolution separation of 12 commonly reported and functionally important IgG glycans was developed by systematically evaluating the effects of selectivity (boric acid) and efficiency (linear polyacrylamide) enhancing additives. The approach can be used to rapidly optimize capillary electrophoresis separation of other glycan mixtures. Following optimization, the method was applied to overnight sample processing for automated 96 well plate-based glycosylation analyses of two nonproprietary therapeutic monoclonal antibodies, demonstrating ruggedness and suitability for high-throughput process and product monitoring applications.

  7. 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.

  8. A monoclonal antibody that blocks poliovirus attachment recognizes the lymphocyte homing receptor CD44.

    PubMed Central

    Shepley, M P; Racaniello, V R

    1994-01-01

    A monoclonal antibody, AF3, was previously shown to specifically inhibit poliovirus binding to HeLa cells and to detect a 100-kDa glycoprotein only in cell lines and tissues permissive for poliovirus infection. These results suggested that the 100-kDa protein may be involved in the pathogenesis of poliomyelitis and the cellular function of the poliovirus receptor site. To study further the role of the 100-kDa protein in poliovirus attachment, immunoaffinity purification, amino acid sequencing, and cDNA cloning were undertaken. The results demonstrate that antibody AF3 reacts with the lymphocyte homing receptor CD44, a multifunctional cell surface glycoprotein involved in the homing of circulating lymphocytes to lymph nodes and the modulation of lymphocyte adhesion and activation. Antibody AF3 reacts with a subset of CD44 molecules (AF3CD44H), which appears to be a small fraction of the heterogeneously glycosylated CD44 molecules expressed on hematopoietic and nonhematopoietic cells. Anti-CD44 monoclonal antibodies, previously reported to induce CD44-mediated modulation of lymphocyte activation and adhesion, compete with 125I-AF3 in binding assays, demonstrating functional overlap among the epitopes. The anti-CD44 monoclonal antibody A3D8, which binds to a greater molecular weight range of CD44 than does AF3, inhibits poliovirus binding to a similar degree. CD44 does not act as a poliovirus receptor, since CD44-expressing mouse L-cell transformants did not bind poliovirus. The poliovirus receptor and AF3CD44H may be noncovalently associated, or they may interact through the cytoskeleton or signal transduction pathways. Images PMID:7508992

  9. [ICO-10 monoclonal antibodies to the Thy-1 antigen].

    PubMed

    Korotkova, O V; Baryshnikov, A Iu; Tupitsyn, N N; Chimishkian, K L; Kostrykina, V N

    1989-01-01

    Mouse monoclonal antibodies (MAB) ICO-10 to Thy-1 antigen were obtained. MAB ICO-10 reacted in indirect immunofluorescence test with 5.7 +/- 0.8% human thymocytes. Antibodies did not react with granulocytes, monocytes, T- and non-T cells from peripheral blood, and with marrow cells of healthy donors. MAB ICO-10 reacted with blast cells from 25 of 53 patients with T-cell acute lymphoblastic leukemia (ALL), from 2 of 5 patients with B-cell ALL. This antigen was absent on blood and marrow cells from some patients with ALL, 80 patients with chronic lymphoid leukemia, 54 patients with chronic granulocytic leukemia at the stage of blastic crisis, 128 patients with acute nonlymphoblastic leukemia. Antibodies are specifically bound to thymocytes and spleen cells of Thy 1.1 and Thy 1.2 mice. MAB ICO-10 detect Thy-1 antigen expressed on human hematopoietic cells. MAB ICO-10 may be applied for human leukemia and lymphoma immune diagnosis.

  10. [Identification and production of monoclonal antibody of Siberian tiger's immunoglobulin].

    PubMed

    Zhang, Yaonglong; Zhang, Duanling; Zhou, Ming; Xue, Yuan; Hua, Yuping; Ma, Jianzhang

    2010-03-01

    To purify immunoglobulin (Ig) of Siberian Tiger and prepare monoclonal antibody (mAb) against the Ig,which can be used to develop immunological diagnostic kits for diagnosing infectious disease in Siberian Tiger. The Ig of Siberian tigers was purified with saturated ammonium sulfate combined with recombinant Protein G. The C57BL/6 mice were immunized with the purified Ig. Spleno-cytes of the mice immunized were collected and fused with the mouse myeloma cell line (Sp2/0-Ag14). The positive hybridoma clones were selected by ELISA and were identified by western blot. The sandwich ELISA was used to detect immunocompetence of the purified Ig and the mAb. We obtained three mouse hybridoma clones that produced mAbs against Ig of Siberian Tiger. The derived McAbs could recognize Ig heavy chain of Siberian Tiger specifically. The biological activity of the Ig and obtained McAbs also could be identified by detecting the antibody induced by panleukopenia virus (FPV-HLJ) vaccine in Siberian Tiger. The antibody also would be useful for assess the vaccine efficacy against the infectious disease on the Siberian Tiger. Protein G can be used in Ig purification of Siberian Tiger. The obtained McAbs from the hybridoma ADT11 in this study owned strong ability to bind Ig of Siberian Tiger and have a stable immunocompetence. They can be used to develop diagnostic methods for detecting infectious disease in Siberian Tiger and vaccine research.

  11. Molecular mechanisms of resistance to the EGFR monoclonal antibody cetuximab.

    PubMed

    Brand, Toni M; Iida, Mari; Wheeler, Deric L

    2011-05-01

    The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase belonging to the HER family of receptor tyrosine kinases. Receptor activation upon ligand binding leads to down stream activation of the PI3K/AKT, RAS/RAF/MEK/ERK and PLCγ/PKC pathways that influence cell proliferation, survival and the metastatic potential of tumor cells. Increased activation by gene amplification, protein overexpression or mutations of the EGFR has been identified as an etiological factor in a number of human epithelial cancers (e.g., NSCLC, CRC, glioblastoma and breast cancer). Therefore, targeting the EGFR has been intensely pursued as a cancer treatment strategy over the last two decades. To date, five EGFR inhibitors, including three small molecule tyrosine kinase inhibitors (TKIs) and two monoclonal antibodies have gained FDA approval for use in oncology. Both approaches to targeting the EGFR have shown clinical promise and the anti-EGFR antibody cetuximab is used to treat HNSCC and CRC. Despite clinical gains arising from use of cetuximab, both intrinsic resistance and the development of acquired resistance are now well recognized. In this review we focus on the biology of the EGFR, the role of EGFR in human cancer, the development of antibody-based anti-EGFR therapies and a summary of their clinical successes. Further, we provide an in depth discussion of described molecular mechanisms of resistance to cetuximab and potential strategies to circumvent this resistance.

  12. A monoclonal antibody recognizing a differentiation marker on rat gonocytes.

    PubMed

    van Dissel-Emiliani, F M; van Kooten, P J; de Boer-Brouwer, M; de Rooij, D G; van der Donk, J A

    1993-01-01

    Monoclonal antibodies (MAb) were raised against a testicular membrane fraction from 18-day post coitum (p.c.) rat testes. One antibody, designated 4B6.3E10 (mu, kappa), was obtained which specifically reacted with gonocytes in the fetal testis. No significant cross-reactivity with other tissues from the 18-day p.c. embryo was found. MAb 4B6.3E10 was reactive with rat gonocytes from 17-day p.c. until the day of birth. Germ cells at later stages of testis development did not show any labelling. The epitope recognized by 4B6.3E10 is a carbohydrate as periodate treatment leads to a loss of reactivity of the antibody. By SDS-PAGE and Western blotting of proteins extracted from a testicular membrane fraction from 18-day p.c. testes, MAb 4B6.3E10 was found to recognize at least 3 protein moieties with apparent molecular weights in the ranges of 80-100, 120, 160-180 kDa (either under reducing- or non-reducing conditions). The results suggest that MAb 4B6.3E10 recognizes a specific differentiation marker for fetal rat gonocytes.

  13. 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.

  14. Properties of technetium-99m labeled monoclonal antibodies

    SciTech Connect

    Rhodes, B.A.; Zamora, P.O.; Newell, K.D.; Reed, K.A.

    1984-01-01

    This study was designed to determine the chemical and immunochemical properties of monoclonal antibodies or fragments which have been labeled with Tc-99m using the pretinning method. The labeled proteins were evaluated using: Sephadex G-25 gel column scanning to determine percentage radiolabeled protein; HPLC to determine the molecular weight and purity of the proteins; reactivity with solid phase antigens to determine immunoreactivity under a variety of storage conditions; and the Tc-99m transchelation method of a previous study to determine the strength of the bonding. Percentage labeled protein ranges from 65 to 95%. Under certain labeling conditions small fractions of the F(ab')2 protein can be converted to aggregates of Fab fragments. Immunoreactivity depends on the purity and immunoreactivity of the original protein and is not changed by the labeling procedure. Transchelation is minimal (less than 5% at 4000 molar excess of EDTA). It is concluded that the pretinning method can be used to produce an extremely stable, immunoreactive, Tc-99m labeled antibody or antibody fragments. The labeled proteins retain their biologic activity during storage or during incubation with human plasma.

  15. Molecular mechanisms of resistance to the EGFR monoclonal antibody cetuximab

    PubMed Central

    Brand, Toni M; Iida, Mari

    2011-01-01

    The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase belonging to the HER family of receptor tyrosine kinases. Receptor activation upon ligand binding leads to down stream activation of the PI3K/AKT, RAS/RAF/MEK/ERK and PLCγ/PKC pathways that influence cell proliferation, survival and the metastatic potential of tumor cells. Increased activation by gene amplification, protein overexpression or mutations of the EGFR has been identified as an etiological factor in a number of human epithelial cancers (e.g., NSCLC, CRC, glioblastoma and breast cancer). Therefore, targeting the EGFR has been intensely pursued as a cancer treatment strategy over the last two decades. To date, five EGFR inhibitors, including three small molecule tyrosine kinase inhibitors (TKIs) and two monoclonal antibodies have gained FDA approval for use in oncology. Both approaches to targeting the EGFR have shown clinical promise and the anti-EGFR antibody cetuximab is used to treat HNSCC and CRC. Despite clinical gains arising from use of cetuximab, both intrinsic resistance and the development of acquired resistance are now well recognized. In this review we focus on the biology of the EGFR, the role of EGFR in human cancer, the development of antibody-based anti-EGFR therapies and a summary of their clinical successes. Further, we provide an in depth discussion of described molecular mechanisms of resistance to cetuximab and potential strategies to circumvent this resistance. PMID:21293176

  16. Human liver catalase: cloning, expression and characterization of monoclonal antibodies.

    PubMed

    Jin, Li Hua; Kim, Dae Won; Eum, Won Sik; Yoon, Chang Sik; Jang, Sang Ho; Choi, Hee Soon; Choi, Soo Hyun; Kim, Young Hoon; Kim, So Young; Jung, Mi Ryoung; Kang, Tae-Cheon; Won, Moo Ho; Lee, Hyeon Yong; Kang, Jung Hoon; Kwon, Oh-Shin; Cho, Sung-Woo; Lee, Kil Soo; Park, Jinseu; Choi, Soo Young

    2003-06-30

    We isolated a cDNA encoding liver catalase from a human liver cDNA library. The cDNA had a high degree of sequence similarity to the corresponding enzyme from other sources. It was expressed in E. coli using the pET15b vector. The protein produced was enzymatically active after purification, and its kinetic parameters closely resembled those of other mammalian catalases. Monoclonal antibodies were generated against the purified catalase; six antibodies recognizing different epitopes were obtained, one of which inhibited the enzyme. The cross reactions of the antibodies with brain catalases from human and other mammalian tissues were investigated, and all the immunoreactive bands obtained on Western blots had molecular masses of about 58 kDa. Similarly fractionated extracts of several mammalian cell lines all gave a single band of molecular mass 58 kDa. These results indicate that mammalian livers and human cell lines contain only one major type of immunologically reactive catalase, even though some of catalases have been previously reported to differ in certain properties.

  17. Potent monoclonal antibodies against Clostridium difficile toxin A elicited by DNA immunization.

    PubMed

    Zhang, Chunhua; Jin, Ke; Xiao, Yanling; Cheng, Ying; Huang, Zuhu; Wang, Shixia; Lu, Shan

    2013-10-01

    Recent studies have demonstrated that DNA immunization is effective in eliciting antigen-specific antibody responses against a wide range of infectious disease targets. The polyclonal antibodies elicited by DNA vaccination exhibit high sensitivity to conformational epitopes and high avidity. However, there have been limited reports in literature on the production of monoclonal antibodies (mAb) by DNA immunization. Here, by using Clostridium difficile (C. diff) toxin A as a model antigen, we demonstrated that DNA immunization was effective in producing a panel of mAb that are protective against toxin A challenge and can also be used as sensitive reagents to detect toxin A from various testing samples. The immunoglobulin (Ig) gene usage for such mAb was also investigated. Further studies should be conducted to fully establish DNA immunization as a unique platform to produce mAb in various hosts.

  18. Purification of chimeric heavy chain monoclonal antibody EG2-hFc using hydrophobic interaction membrane chromatography: an alternative to protein-A affinity chromatography.

    PubMed

    Sadavarte, Rahul; Spearman, Maureen; Okun, Natalie; Butler, Michael; Ghosh, Raja

    2014-06-01

    Heavy chain monoclonal antibodies are being considered as alternative to whole-IgG monoclonal antibodies for certain niche applications. Protein-A chromatography which is widely used for purifying IgG monoclonal antibodies is also used for purifying heavy chain monoclonal antibodies as these molecules possess fully functional Fc regions. However, the acidic conditions used to elute bound antibody may sometimes also leach protein-A, which is immunotoxic. Low pH conditions also tend to make the mAb molecules unstable and prone to aggregation. Moreover, protein-A affinity chromatography does not remove aggregates already present in the feed. Hydrophobic interaction membrane chromatography (or HIMC) has already been studied as an alternative to protein-A chromatography for purifying whole-IgG monoclonal antibodies. This paper describes the use of HIMC for capturing a humanized chimeric heavy chain monoclonal antibody (EG2-hFC). Binding and eluting conditions were suitably optimized using pure EG2-hFC. Based on this, an HIMC method was developed for capture of EG2-hFC directly from cell culture supernatant. The EG2-hFc purity obtained in this single-step process was high. The glycan profiles of protein-A and HIMC purified monoclonal antibody samples were similar, clearly demonstrating that both techniques captured similarly glycosylated population of EG2-hFc. Moreover, this technique was able to resolve aggregates from monomeric form of the EG2-hFc.

  19. Monoclonal antibodies to mouse cell-surface antigens.

    PubMed

    Alaverdi, Noosheen

    2002-05-01

    The cluster of differentiation (CD) nomenclature, originally devised by the International Workshop on Human Leukocyte Differentiation Antigens (HLDA) to classify leukocyte surface antigens, has become more accepted by researchers in nonhuman animal models. The CD homologues from other species are usually identified by multiple studies, including biochemical, functional analysis, cloning, and immunological assays. This unit compiles a complete (as of August, 2001) list of monoclonal antibodies (mAb) to the mouse CD antigens and several non-CD surface antigens that are commonly used as phenotypic and functional markers in the mouse system. There are many reagents recognizing polymorphic epitopes of the mouse MHC and different subunits of TCR; for simplicity, only a few mAbs to the framework determinants of these molecules are listed here.

  20. Monoclonal antibodies and the transformation of blood typing.

    PubMed

    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.

  1. A Monoclonal Antibody Specific to Surface Antigen on Candida krusei

    PubMed Central

    Robert, Raymond; Faure, Odile; Carloti, Arnaud; Lebeau, Bernadette; Bernard, Christian; Marot-Leblond, Agnès; Grillot, Renée; Senet, Jean-Marcel

    1998-01-01

    A monoclonal antibody (MAb; MAb 6B3) which reacts specifically with a cell wall antigen found in all strains or isolates of Candida krusei was developed. MAb 6B3 was extensively tested by immunofluorescence assay for cross-reaction with many Candida, Cryptococcus, Saccharomyces, Trichosporon, and Rhodotorula species and was found to react only with the species C. krusei. The specific epitope is expressed on the surface of fungal cells and appears to reside on a protein moiety. Taking into account the increasing importance of fluconazole-resistant strains in nosocomial fungal infections, the very high degree of specificity of this MAb for C. krusei could be useful for the routine detection of C. krusei in culture or in tissue samples. PMID:9455893

  2. High-level iodination of monoclonal antibody fragments for radiotherapy

    SciTech Connect

    Ferens, J.M.; Krohn, K.A.; Beaumier, P.L.; Brown, J.P.; Hellstroem, I.; Hellstroem, K.E.; Carrasquillo, J.A.; Larson, S.M.

    1984-03-01

    Two different murine monoclonal antibody Fab fragments specific for p97, a melanoma-associated antigen, were labeled with I-131 at high activity levels without excessive chemical damage. Up to 20 mg of Fab were labeled with up to 300 mCi of I-131 using the chloramine-T method and large working volumes at room temperature. As much as 90% of the initial activity was recovered as labeled product. The labeled Fabs varied in their sensitivity to radioiodination damage, as measured by an in vitro cell-binding assay. Radioiodination was performed safely using a remote iodination apparatus. The final product was of radiopharmaceutical quality suitable for clinical diagnosis and experimental radiotherapy in humans.

  3. Positron emission tomographic imaging of tumors using monoclonal antibodies

    SciTech Connect

    Zalutsky, M.R.

    1990-12-01

    The overall objective for this research project is to develop methods for utilizing Positron Emission Tomography (PET) to increase the clinical potential of radiolabelled monoclonal antibodies (MAbs). 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 to investigate the following: normal tissue toxicity; radiation dose to the tumor; and early tumor imaging. The research plans of this proposal include the following specific aims: optimize labeling of MAbs with fluorine 18, bromine 76 and bromine 75; label MAb Mel-14 (reactive against human gliomas and melanomas) and its Fab and F(ab{prime}){sub 2} fragments while retaining immunoreactivity; determine the distribution of Mel-14 in athymic mice bearing human gliomas; determine pharmacokinetics of Mel-14 in nonhuman primates. Experiments with another MAb, TP-1, and iodine 124 and 131 are also planned. 8 figs.

  4. Pharmacokinetic, pharmacodynamic and immunogenicity comparability assessment strategies for monoclonal antibodies.

    PubMed

    Putnam, Wendy S; Prabhu, Saileta; Zheng, Yanan; Subramanyam, Meena; Wang, Yow-Ming C

    2010-10-01

    Regulatory guidance stipulates that comparability assessment is required to support manufacturing process changes during the development of a biological product or post-approval. However, strategies for assessing the comparability of pre- and post-change materials are still evolving. A hierarchical risk-based approach is recommended, starting with analytical testing to ensure quality, followed by biological characterization and, if needed, in vivo pharmacokinetic (PK), PK-pharmacodynamic (PD), safety and/or efficacy studies. The need for an in vivo study and the type of study required depend on the magnitude and the potential impact of the changes and the timing in the development process. This review discusses factors affecting the PK, PD and immunogenicity of monoclonal antibodies, and provides guidance for determining non-clinical and clinical comparability assessment strategies. Copyright © 2010 Elsevier Ltd. All rights reserved.

  5. 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. PMID:27433507

  6. Effect of polyol sugars on the stabilization of monoclonal antibodies.

    PubMed

    Nicoud, Lucrèce; Cohrs, Nicholas; Arosio, Paolo; Norrant, Edith; Morbidelli, Massimo

    2015-02-01

    We investigate the impact of sugars and polyols on the heat-induced aggregation of a model monoclonal antibody whose monomer depletion is rate-limited by protein unfolding. We follow the kinetics of monomer consumption by size exclusion chromatography, and we interpret the results in the frame of two mechanistic schemes describing the enhanced protein stability in the presence of polyols. It is found that the stabilization effect increases with increasing polyol concentration with a comparable trend for all of the tested polyols. However, the stabilization effect at a given polyol concentration is polyol specific. In particular, the stabilization effect increases as a function of polyol size until a plateau is reached above a critical polyol size corresponding to six carbon atoms. Our results show that the stabilization by polyols does not depend solely on the volume fraction filled by the polyol molecules, but is also affected by the polyol chemistry.

  7. Identification of Eimeria acervulina conoid antigen using chicken monoclonal antibody.

    PubMed

    Matsubayashi, Makoto; Minoura, Chisa; Kimura, Shintaro; Tani, Hiroyuki; Furuya, Masaru; Lillehoj, Hyun S; Matsuda, Haruo; Takenaka, Shigeo; Hatta, Takeshi; Tsuji, Naotoshi; Sasai, Kazumi

    2016-11-01

    In the poultry industry, Eimeria spp. is one of the important pathogens which cause significant economic losses. We have previously generated a chicken monoclonal antibody (mAb), 6D-12-G10, with specificity for an antigen located in the apical cytoskeleton of Eimeria acervulina and with cross-reactive among Apicomplexan parasites, including other Eimeria spp., Toxoplasma, Neospora, and Cryptosporidium spp. Furthermore, the protein of Cryptosporidium parvum recognized by the 6D-12-G10 has been identified as elongation factor-1α (EF-1α). In the present study, to identify the target molecule of E. acervulina by the mAb, we performed two-dimensional Western blotting analysis. Finally, we found two positive molecules which are identified as EF-1α and a related protein. Our previous finding using C. parvum and the results in this study suggest that EF-1α could be associated with the invasion facilitated by the cytoskeleton at the apical region of zoites.

  8. [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. Copyright © 2014 Elsevier España, S.L.U. All rights reserved.

  9. 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

  10. Molecular Characterization of the Monoclonal Antibodies Composing ZMAb: A Protective Cocktail Against Ebola Virus

    PubMed Central

    Audet, Jonathan; Wong, Gary; Wang, Han; Lu, Guangwen; Gao, George F.; Kobinger, Gary; Qiu, Xiangguo

    2014-01-01

    Ebola virus (EBOV) causes severe viral hemorrhagic fever in humans and non-human primates, with a case fatality rate of up to 88% in human outbreaks. Over the past 3 years, monoclonal antibody (mAb) cocktails have demonstrated high efficacy as treatments against EBOV infection. One such cocktail is ZMAb, which consists of three mouse antibodies, 1H3, 2G4, and 4G7. Here, we present the epitope binding properties of mAbs 1H3, 2G4, and 4G7. We showed that these antibodies have different variable region sequences, suggesting that the individual mAbs are not clonally related. All three antibodies were found to neutralize EBOV variant Mayinga. Additionally, 2G4 and 4G7 were shown to cross-inhibit each other in vitro and select for an escape mutation at the same position on the EBOV glycoprotein (GP), at amino acid 508. 1H3 selects an escape mutant at amino acid 273 on EBOV GP. Surface plasmon resonance studies showed that all three antibodies have dissociation constants on the order of 10−7. In combination with previous studies evaluating the binding sites of other protective antibodies, our results suggest that antibodies targeting the GP1-GP2 interface and the glycan cap are often selected as efficacious antibodies for post-exposure interventions against EBOV. PMID:25375093

  11. Molecular characterization of the monoclonal antibodies composing ZMAb: a protective cocktail against Ebola virus.

    PubMed

    Audet, Jonathan; Wong, Gary; Wang, Han; Lu, Guangwen; Gao, George F; Kobinger, Gary; Qiu, Xiangguo

    2014-11-06

    Ebola virus (EBOV) causes severe viral hemorrhagic fever in humans and non-human primates, with a case fatality rate of up to 88% in human outbreaks. Over the past 3 years, monoclonal antibody (mAb) cocktails have demonstrated high efficacy as treatments against EBOV infection. One such cocktail is ZMAb, which consists of three mouse antibodies, 1H3, 2G4, and 4G7. Here, we present the epitope binding properties of mAbs 1H3, 2G4, and 4G7. We showed that these antibodies have different variable region sequences, suggesting that the individual mAbs are not clonally related. All three antibodies were found to neutralize EBOV variant Mayinga. Additionally, 2G4 and 4G7 were shown to cross-inhibit each other in vitro and select for an escape mutation at the same position on the EBOV glycoprotein (GP), at amino acid 508. 1H3 selects an escape mutant at amino acid 273 on EBOV GP. Surface plasmon resonance studies showed that all three antibodies have dissociation constants on the order of 10(-7). In combination with previous studies evaluating the binding sites of other protective antibodies, our results suggest that antibodies targeting the GP1-GP2 interface and the glycan cap are often selected as efficacious antibodies for post-exposure interventions against EBOV.

  12. PCSK9 Inhibition With Monoclonal Antibodies: Modern Management of Hypercholesterolemia

    PubMed Central

    Santos, Raul D.

    2016-01-01

    Abstract Current guidelines for hypercholesterolemia treatment emphasize lifestyle modification and lipid‐modifying therapy to reduce the risk for cardiovascular disease. Statins are the primary class of agents used for the treatment of hypercholesterolemia. Although statins are effective for many patients, they fail to achieve optimal reduction in lipids for some patients, including those who have or are at high risk for cardiovascular disease. The PCSK9 gene was identified in the past decade as a potential therapeutic target for the management of patients with hypercholesterolemia. Pharmacologic interventions to decrease PCSK9 levels are in development, with the most promising approach using monoclonal antibodies that bind to PCSK9 in the plasma. Two monoclonal antibodies, alirocumab and evolocumab, have recently been approved for the treatment of hypercholesterolemia, and a third one, bococizumab, is in phase 3 clinical development. All 3 agents achieve significant reductions in levels of low‐density lipoprotein cholesterol, as well as reductions in non‐high‐density lipoprotein cholesterol, apolipoprotein B, and lipoprotein(a). Long‐term outcome trials are under way to determine the sustained efficacy, safety, and tolerability of PCSK9 inhibitors and whether this novel class of agents decreases the risk for major cardiovascular events in patients on lipid‐modifying therapy. Available data suggest that PCSK9 inhibitors provide a robust reduction in atherogenic cholesterol levels with a good safety profile, especially for patients who fail to obtain an optimal clinical response to statin therapy, those who are statin intolerant or have contraindications to statin therapy, and those with familial hypercholesterolemia. PMID:27195910

  13. PCSK9 Inhibition With Monoclonal Antibodies: Modern Management of Hypercholesterolemia.

    PubMed

    Ito, Matthew K; Santos, Raul D

    2017-01-01

    Current guidelines for hypercholesterolemia treatment emphasize lifestyle modification and lipid-modifying therapy to reduce the risk for cardiovascular disease. Statins are the primary class of agents used for the treatment of hypercholesterolemia. Although statins are effective for many patients, they fail to achieve optimal reduction in lipids for some patients, including those who have or are at high risk for cardiovascular disease. The PCSK9 gene was identified in the past decade as a potential therapeutic target for the management of patients with hypercholesterolemia. Pharmacologic interventions to decrease PCSK9 levels are in development, with the most promising approach using monoclonal antibodies that bind to PCSK9 in the plasma. Two monoclonal antibodies, alirocumab and evolocumab, have recently been approved for the treatment of hypercholesterolemia, and a third one, bococizumab, is in phase 3 clinical development. All 3 agents achieve significant reductions in levels of low-density lipoprotein cholesterol, as well as reductions in non-high-density lipoprotein cholesterol, apolipoprotein B, and lipoprotein(a). Long-term outcome trials are under way to determine the sustained efficacy, safety, and tolerability of PCSK9 inhibitors and whether this novel class of agents decreases the risk for major cardiovascular events in patients on lipid-modifying therapy. Available data suggest that PCSK9 inhibitors provide a robust reduction in atherogenic cholesterol levels with a good safety profile, especially for patients who fail to obtain an optimal clinical response to statin therapy, those who are statin intolerant or have contraindications to statin therapy, and those with familial hypercholesterolemia. © 2016, The Authors. The Journal of Clinical Pharmacology Published by Wiley Periodicals, Inc. on behalf of American College of Clinical Pharmacology.

  14. 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.

  15. Production and characterization of a monoclonal antibody against the beta-adrenergic agonist ractopamine.

    PubMed

    Shelver, W L; Smith, D J; Berry, E S

    2000-09-01

    A monoclonal antibody was generated toward the beta-adrenergic agonist ractopamine hydrochloride ¿(1R,3R),(1R, 3S)-4-hydroxy-alpha-[[[3-(4-hydroxyphenyl)-1-methylpropyl]amino]methy l]benzenemethanol hydrochloride¿. Ractopamine-glutarate-keyhole limpet hemocyanin (KLH) was used as the antigen for antibody generation in mice. Clone 5G10, secreted antibody with isotype IgG1kappa, was used for the development of an immunoassay. The selected antibody was specific for racemic ractopamine with an IC(50) of 2.69 +/- 0.36 ng/mL (n = 15). Antibody binding toward ractopamine was stereoselective with (1R,3R)-ractopamine having an IC(50) of 0.55 +/- 0.09 ng/mL (n = 3). IC(50) values for the (1S, 3R)-, (1S,3S)-, and (1R,3S)-ractopamine stereoisomers were 2.00 +/- 0.37, 140 +/- 23, and 291+/- 32 ng/mL (n = 3), respectively. Phenethanolamine beta-agonists showed low cross-reactivity. Studies using a series of ractopamine metabolites and ractopamine analogues demonstrated structural requirements for the antibody binding. A free phenolic group on the N-butylphenol moiety was required for high-affinity binding because methoxylated analogues and metabolites glucuronidated at this phenol generally had IC(50) values greater than 200 ng/mL. Ractopamine analogues methoxylated or glucuronidated at the ethanolamine phenol had IC(50) values of 0.7-2.6 ng/mL. Lack of a benzylic hydroxyl group was of less importance to antibody binding than was the correct stereochemical orientation (3R) of ractopamine's N-phenylalkyl group. In conclusion, a highly specific monoclonal antibody to ractopamine hydrochloride was developed that could be of potential utility in screening assays.

  16. Comprehensive characterization and optimization of anti-LRRK2 (leucine-rich repeat kinase 2) monoclonal antibodies.

    PubMed

    Davies, Paul; Hinkle, Kelly M; Sukar, Nour N; Sepulveda, Bryan; Mesias, Roxana; Serrano, Geidy; Alessi, Dario R; Beach, Thomas G; Benson, Deanna L; White, Charles L; Cowell, Rita M; Das, Sonal S; West, Andrew B; Melrose, Heather L

    2013-07-01

    Missense mutations in LRRK2 (leucine-rich repeat kinase 2) are a major cause of PD (Parkinson's disease). Several antibodies against LRRK2 have been developed, but results using these polyclonal antibodies have varied widely leading to conflicting conclusions. To address this challenge, the Michael J. Fox Foundation for Parkinson's Research generated a number of monoclonal antibodies targeting epitopes across the LRRK2 protein. In the present paper, we report optimized protocols and results for ten monoclonal antibodies for immunoblotting, immunohistochemistry, immunoprecipitation and kinase activity assays, in rat, mouse and human brain tissue. Several efficacious antibodies were identified, but results demonstrate that the mouse monoclonal N241A/34 is suitable for most applications, with the best overall rabbit monoclonal antibody being c41-2. These antibodies produced a dominant band of the expected size via immunoblotting and a lack of labelling in tissue derived from LRRK2-knockout animals under optimized conditions. A significant proportion of LRRK2 protein localizes to insoluble fractions and no evidence of truncated LRRK2 protein was detected in any fraction from rodent or human tissues. An assay was developed for the robust detection of LRRK2 kinase activity directly from frozen mouse and human brain tissue, but precipitous declines in activity were observed that corresponded to increasing post-mortem intervals and processing times. Finally, we demonstrate the highest levels of brain-localized LRRK2 in the striatum, but note differential expression patterns between rat and mouse in both striatum and cortex. Anti-LRRK2 monoclonal antibodies that are unlimited in availability together with the proposed standardized protocols should aid in the definition of LRRK2 function in both health and disease.

  17. 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

  18. 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

  19. The role of isotype and epitope specificity of monoclonal antibody mixtures in immunodiffusion reactions.

    PubMed Central

    Griffiths, B W; Godard, A; Caron, J A; Tostowaryk, W

    1987-01-01

    Twenty monoclonal antibodies to human alpha-fetoprotein have been characterized in terms of IgG subclass, epitope specificity and immunodiffusion properties. The Ig molecules consisted of six G1, ten G2a and four G2b isotypes. The epitope analysis was conducted by a solid-phase RIA employing 125I-labelled AFP. The RIA analysis resulted in the identification of seven determinants, two of which were specific for one antibody each, while others were specific for two or more antibodies. In Ouchterlony tests with the antibodies, none formed bands of immunoprecipitate when diffused individually against the antigen. Tests with all possible mixtures of pairs of the antibodies (190) resulted in positive immunodiffusion responses with only two mixtures. These each contained antibodies of the 2b isotype and demonstrated distinct epitope specificities. The immunodiffusion of mixtures of three antibodies from a group of 12 that were selected to represent equal numbers of isotypes resulted in 84 positive responses and 136 negative responses, i.e. from a total of 220 mixtures. A striking correlation was noted between positive immunodiffusion tests and composition of antibody isotypes and of epitope specificities of the mixtures. Each mixture was found to contain antibodies of at least one 2b isotype and of different epitope specificities. None of the mixtures that lacked a 2b isotype (56) responded in immunodiffusion tests. Similarly, in instances (46) in which the epitope specificities of the antibodies in the mixture were the same, i.e. duplicated or triplicated, the results were again negative despite the presence of a 2b isotype. The comparison of these studies with similar studies of a smaller group of antibodies to pregnancy-specific beta 1-glycoprotein strongly suggests that the G3 antibody isotype may have an immune precipitation enhancing effect similar to the G2b isotype. PMID:2438204

  20. Generation of monoclonal antibody targeting fibroblast growth factor receptor 3.

    PubMed

    Gorbenko, Olena; Ovcharenko, Galyna; Klymenko, Tetyana; Zhyvoloup, Olexandr; Gaman, Nadia; Volkova, Darija; Gout, Ivan; Filonenko, Valeriy

    2009-08-01

    Fibroblast growth factor receptor 3 (FGFR3) is a member of the FGFR family of receptor tyrosine kinases, whose function has been implicated in diverse biological processes, including cell proliferation, differentiation, survival, and tumorigenesis. Deregulation of FGFR3 signaling has been implicated with human pathologies, including cancer. Activating mutations in FGFR3 gene are frequently detected in bladder cancer, multiple myeloma, and noninvasive papillary urothelial cell carcinomas, while the overexpression of the receptor is observed in thyroid lymphoma and bladder cancer. The main aim of this study was to generate hybridoma clones producing antibody that could specifically recognize FGFR3/S249C mutant, but not the wild-type FGFR. To achieve this, we used for immunization bacterially expressed fragment of FGFR3 corresponding to loops II-III of the extracellular domain (GST-His/FGFR3/S249C-LII-III), which possesses oncogenic mutation at Ser249 detected in at least 50% of bladder cancers. Primary ELISA screening allowed us to isolate several hybridoma clones that showed specificity towards FGFR3/S249C, but not FGFR3wt protein. Unfortunately, these clones were not stable during single-cell cloning and expansion and lost the ability to recognize specifically FGFR3/S249C. However, this study allowed us to generate several monoclonal antibodies specific towards both FGFR3wt and FGFR3/S249C recombinant proteins. Produced hybridomas secreted MAbs that were specific in Western blotting towards bacterially expressed FGFR3wt and FGFR3/S249C, as well as the full-length receptors ectopically expressed in Sf21 and HEK293 cells. Moreover, transiently expressed wild-type and oncogenic forms of FGFR were efficiently immunoprecipitated with selected antibodies from the lysates of infected Sf21 and transiently transfected HEK293. In summary, generated antibodies should be useful as tools for examining the expression pattern and biological functions of FGFR3 in normal and

  1. Feasibility studies of using the Catfish Immune System to produce monoclonal antibodies

    SciTech Connect

    Poston, T.M.

    1987-03-01

    The objective of these studies was to determine the feasibility of using a teleost cell line to produce monoclonal antibodies. Studies were undertaken to demonstrate the production of a polyclonal response of channel catfish (Icatalurus punctatus) challenged with mycotoxins coupled to a protein carrier. Companion studies were also performed to induce a permanent cell line with catfish lymphocytes. Attempts to demonstrate a polyclonal response to haptenized mycotoxins were inconclusive. Tests to induce an immortal, permanent cell line with benzene and x-ray irradiated cells were also inconclusive. 3 refs., 13 tabs.

  2. Production and Characterization of Monoclonal Antibodies Against the Protective Antigen Component of Bacillus anthracis Toxin

    DTIC Science & Technology

    1988-01-21

    F. Jaquet, P. Luethy, R. Huetter, and D. G. Braun. 1986. Characterization of mcnoclonal antibodies to a crystal protein of Bacillus thuringiensis ...AD-A192 855 UT FILE COPY Production and Characterization of Monoclonal Antibodies Against the Protective Antigen Component of Bacillus anthracis...Author Tel. No. 301-663-7341 1--ac",- 88 3 14 05 6 Krhirty-six monoclonal antibodies to the protective antigen protein of Bacillus anthracis exotoxin

  3. Regional distribution of prostaglandin endoperoxide synthase studied by enzyme-linked immunoassay using monoclonal antibodies.

    PubMed

    Yoshimoto, T; Magata, K; Ehara, H; Mizuno, K; Yamamoto, S

    1986-06-11

    Prostaglandin endoperoxide synthase transforms arachidonic acid to prostaglandin H2 via prostaglandin G2. The enzyme purified from bovine vesicular gland was given to mice as antigen, and monoclonal antibodies were raised by the hybridoma technique. Two species of the monoclonal antibody recognizing different sites of the enzyme were utilized to establish a peroxidase-linked immunoassay of prostaglandin endoperoxide synthase. Fab' fragment of one of the antibodies was prepared and conjugated to horseradish peroxidase. The conjugate was then bound to prostaglandin endoperoxide synthase, and the labeled enzyme was precipitated by the addition of the other antibody. The peroxidase activity of the immunoprecipitate correlated linearly with the amount of prostaglandin endoperoxide synthase. This sensitive and convenient method to determine the enzyme amount rather than the enzyme activity was utilized to extensively screen the amount of prostaglandin endoperoxide synthase in various bovine tissues. In addition to vesicular gland, platelets and kidney medulla previously known as rich enzyme sources, the immunoenzymometric assay demonstrated a high content of the enzyme in various parts of alimentary tract and a low but significant amount of enzyme in some parts of brain.

  4. Preparation and characterization of new anti-PSMA monoclonal antibodies with potential clinical use.

    PubMed

    Moffett, Serge; Mélançon, Dominic; DeCrescenzo, Gregory; St-Pierre, Caroline; Deschénes, François; Saragovi, H Uri; Gold, Phil; Cuello, A Claudio

    2007-12-01

    Monoclonal antibodies with high specificity for prostate cancer tissue are of interest for diagnostic and therapeutic applications employing targeted therapy. The prostate-specific membrane antigen (PSMA) is a protein predominantly found in epithelial cells of prostate tissue origin and its expression correlates with tumor aggressiveness. Here, we report the development and characterization of new antibodies against PSMA. Murine monoclonal antibodies (MAb) were obtained by immunizing mice with a peptide corresponding to PSMA extracellular residues 490-500 -- GKSLYESWTKK (PSMA(490-500)). The MAbs react specifically to PSMA and to the prostate cancer cell line LNCaP with an affinity for PSMA in the low nanomolar range. This study also demonstrates the potential use of these antibodies for targeted drug delivery to prostate cancer cells. Nanomolar concentrations of PSMA-specific MAb in association with a molecule with cytotoxic potential were sufficient to allow for binding and uptake by LNCaP cells within minutes, leading to complete cell death within 3 days. These MAbs have potential clinical value in the development of diagnostic and therapeutic applications for prostate cancer.

  5. 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.

  6. Monoclonal antibodies: pharmacokinetics as a basis for new dosage regimens?

    PubMed

    Azanza, J-R; Sádaba, B; Gómez-Guiu, A

    2015-10-01

    Complete monoclonal IgG antibodies which are in use in clinical practice share some pharmacological properties resulting in high concentrations in plasma. This fact is reflected in their low volumes of distribution, which can also be correlated with a high molecular weight and water solubility. This feature allows a novel approach to be applied to the dosing schedule for this group of drugs with fixed doses being used instead of the initially developed weight- or body surface-adjusted dosing schedules. In addition, the development of a new formulation containing hyaluronidase allows a subcutaneous route of administration to be used, because hyaluronidase creates a space in the subcutaneous tissue that helps antibody absorption. This method requires higher doses, but has allowed testing the feasibility of administering a fixed dose, with no individual dose adjustments based on weight or body surface. Moreover, loading doses are not needed, because the first dose results, within 3 weeks, in minimum concentrations that are higher than effective concentrations.

  7. 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.

  8. Generation and characterization of monoclonal antibodies to equine CD16.

    PubMed

    Noronha, Leela E; Harman, Rebecca M; Wagner, Bettina; Antczak, Douglas F

    2012-04-15

    The low-affinity Fc receptor CD16 plays a central role in the inflammatory and innate immune responses of many species, but has not yet been investigated in the horse. Using the predicted extracellular region of equine CD16 expressed as a recombinant fusion protein with equine IL-4 (rIL-4/CD16), we generated a panel of mouse monoclonal antibodies (mAbs) that recognize equine CD16. Nine mAbs were chosen for characterization based upon recognition of CD16, but not IL-4, in ELISA. All nine mAbs recognized full-length, cell-surface CD16 expressed as a GFP fusion protein by CHO cells, but not the closely related Fc receptor CD32 expressed in the same system. In flow cytometric analysis with equine peripheral leukocytes, the mAbs labeled cells in the granulocyte, monocyte, and lymphocyte populations in a pattern consistent with other species. Monocytes that were strongly labeled with CD16 mAb 9G5 were also positive for the LPS receptor CD14. Cytospins made with peripheral leukocytes were immunohistochemically labeled and showed mAb recognition of primarily mononuclear cells. ELISA revealed that the nine mAbs can be grouped into three patterns of epitope recognition. These new antibodies will serve as useful tools in the investigation of equine immune responses and inflammatory processes.

  9. Production of human anti-HLA monoclonal antibodies

    SciTech Connect

    Walker, M.C.; Mercier, F.; Roger, J.; Varin, M.

    1986-03-01

    Only 40% of the several hundred anti-HLA murine monoclonal antibodies (MAbs) that have been made detect HLA-A,B,C or DR specificities previously defined by human alloantisera, the range of recognized specificities is very narrow, and few of the MAbs have proven useful as tissue typing reagents. In hopes of obtaining HLA typing reagents, the authors are developing a protocol for the production of human anti-HLA MAbs from HLA-antigen (Ag) immunized peripheral blood B cells of volunteering renal patients, immunized to one or more HLA Ags through therapeutic blood transfusions. A simple enrichment of the donor B cells has not been sufficient for anti-HLA MAb production, the authors are currently delineating the conditions necessary for increasing the number of HLA-specific donor B cells by in vitro stimulation with cells expressing the HLA Ag to which the B cell donor is immunized. For the production of MAbs, the stimulated B cells are transformed with Epstein-Barr virus and subsequently fused with KR-4 lymphoblastoid cells. Hybridomas are selected by HAT and Ouabain. Supernatants are screened for anti-HLA activity against lymphocyte targets expressing the original immunizing HLA Ag by complement mediated /sup 51/Cr release assay. Antibody specificity is determined by the complement-dependent microcytotoxicity test used for HLA typing.

  10. 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.

  11. Direct detection of idiotypic determinants on blotted monoclonal antibodies.

    PubMed

    Petit, C; Sauron, M E; Gilbert, M; Thèze, J

    1982-01-01

    The protein-blotting technique has been tested as a mean to study the expression of idiotypic determinants. A monoclonal BALB/c antipoly (Glu60-Ala30-Tyr10) GAT antibody (G5) was caused to migrate on SDS gel and transferred to a nitrocellulose filter. To facilitate the renaturation of the idiotypic determinants, the blotted proteins were incubated in NP40 buffer, immediately after the transfer. The ability of two anti-idiotypic sera to detect two defined idiotypic specificities of the blotted G5 molecules was investigated. When G5 was electrophoresed on SDS gel under non-reducing conditions, a specific detection of two idiotypic specificities of the G5-blotted molecules was obtained. On the other hand, when G5 was migrated under reducing conditions, none of the two antiidiotypic sera gave a staining of the heavy and the light chains. This result indicates that molecules expressing conformational idiotypic determinants can be detected by protein-blotting technique after migration on SDS gel. Moreover, this suggests the possible interest of this technique to analyse non-antibody molecules bearing idiotypic determinants.

  12. Monoclonal antibody-based therapies for microbial diseases

    PubMed Central

    Saylor, Carolyn; Dadachova, Ekaterina; Casadevall, Arturo

    2009-01-01

    The monoclonal antibody (mAb) revolution that currently provides many new options for the treatment of neoplastic and inflammatory diseases has largely bypassed the field of infectious diseases. Only one mAb is licensed for use against an infectious disease, although there are many in various stages of development. This situation is peculiar given that serum therapy was one of the first effective treatments for microbial diseases and that specific antibodies have numerous antimicrobial properties. The underdevelopment and underutilization of mAb therapies for microbial diseases has various complex explanations that include the current availability of antimicrobial drugs, small markets, high costs and microbial antigenic variation. However, there are signs that the climate for mAb therapeutics in infectious diseases is changing given increasing antibiotic drug resistance, the emergence of new pathogenic microbes for which no therapy is available, and development of mAb cocktail formulations. Currently, the major hurdle for the widespread introduction of mAb therapies for microbial diseases is economic, given the high costs of immunoglobulin preparations and relatively small markets. Despite these obstacles there are numerous opportunities for mAb development against microbial diseases and the development of radioimmunotherapy provides new options for enhancing the magic bullet. Hence, there is cautious optimism that the years ahead will see more mAbs in clinical use against microbial diseases. PMID:20006139

  13. Assessing immunogenicity of biosimilar therapeutic monoclonal antibodies: regulatory and bioanalytical considerations.

    PubMed

    Chamberlain, Paul

    2013-03-01

    This article reflects on methodological limitations for interpretation of relative immunogenicity of biosimilar and reference therapeutic monoclonal antibodies, in emphasizing the relevance of correlation of bioanalytical signals with appropriate clinical end points, and the possible need for post-marketing observational studies to indicate the impact of detected differences in anti-drug antibody incidence and magnitude on sustainability of treatment benefit. Given the current uncertainty regarding the longer term clinical impact of undesirable immunogenicity for reference products, there can be no predefined margin for an acceptable difference based on incidence and magnitude of detected anti-drug antibodies. Any detected differences should be assessed in relation to clinical parameters; and the designation of biosimilarity made with reference to the similarity demonstrated in the directly comparative quality, nonclinical and clinical evaluations. Application of this 'totality of evidence' approach is illustrated for infliximab and adalimumab.

  14. Common epitope on urinary antigen derived from different Legionella pneumophila serogroup 1 strains recognized by a monoclonal antibody.

    PubMed

    Helbig, J H; Lück, P C; Pilz, C; Witzleb, W

    1990-09-01

    Guinea pigs were infected intraperitoneally with 4 subgroup reference strains (Knoxville 1, Philadelphia 1, Bellingham 1, OLDA) and 2 clinical isolates of Legionella pneumophila serogroup 1. Antigenuria was demonstrated by the enzyme-linked immunosorbent assay using polyclonal antibodies and monoclonal antibody (mab) F8/5. Mab F8/5 recognizes a hitherto undetected common epitope on urinary antigen of the investigated strains.

  15. Natalizumab: AN 100226, anti-4alpha integrin monoclonal antibody.

    PubMed

    2004-01-01

    Natalizumab [AN 100226, anti-alpha4 integrin monoclonal antibody, Antegren] is a humanised monoclonal antibody that blocks alpha4beta1 integrin-mediated leukocyte migration. Natalizumab is in phase III trials for the treatment of multiple sclerosis in North America and the UK, and for the treatment of Crohn's disease also in the UK. It may have potential in the treatment of other immune-related inflammatory disease. Elan Corporation intends to examine the potential of natalizumab in rheumatoid arthritis and ulcerative colitis. 4beta1 integrin on circulating leukocytes binds to vascular cell adhesion molecule-1, which is expressed at high levels in the blood vessels in the CNS during exacerbations of multiple sclerosis. This allows leukocytes expressing alpha4beta1 integrin (very late antigen-4) to move from the peripheral blood into the CNS. Inflammatory proteins and other factors released from lymphocytes in the brain lead to the progression of symptoms. A limitation of natalizumab is that it must be injected and cannot be administered orally. Scientists have transformed the large anti-alpha4 monoclonal antibody into much smaller, drug-like molecules suitable for oral administration. Protein Design Labs has granted a worldwide nonexclusive licence under its antibody humanisation patents to Elan Pharmaceuticals for natalizumab. Biogen Inc. has entered into an agreement with Elan for a worldwide exclusive collaboration to develop, manufacture and commercialise natalizumab for multiple sclerosis and Crohn's disease and rheumatoid arthritis. Development of natalizumab is also being funded, in part, by Axogen (acquired by Elan in 1999). In November 2003, Biogen and IDEC Pharmaceuticals merged to form Biogen Idec. Elan repurchased royalty rights on a package of products, including natalizumab, from Autoimmune Disease Research Company. Elan and Genzyme Transgenics Corporation signed an agreement to produce natalizumab in GTC's genetically engineered goats, which will

  16. Monoclonal antibodies that inhibit ADP-ribosyltransferase but not NAD-glycohydrolase activity of pertussis toxin.

    PubMed Central

    Kaslow, H R; Schlotterbeck, J D; Kenimer, J G

    1990-01-01

    Kenimer et al. (J. G. Kenimer, J. Kim, P. G. Probst, C. R. Manclark, D. G. Burstyn, and J. L. Lowell, Hybridoma 8:37-51, 1989) identified three classes of monoclonal antibodies, termed A, B, and C, that recognize the S1 subunit of pertussis toxin. This report presents data demonstrating that class A monoclonal antibodies (3CX4, 6D11C, and 3C4D), which block the ADP-ribosyltransferase activity and recognize the predominant neutralizing epitope on the S1 subunit of the toxin, do not inhibit the NAD-glycohydrolase activity of the toxin. In addition, alkylation of cysteine 41 of the S1 subunit, which may interact with NAD, inactivates the toxin but does not prevent binding by class A antibodies. Taken together, these results support the conclusion that proper alterations of amino acids that interact with NAD should allow for inactivation of the toxin without destruction of the predominant neutralizing epitope. The class A antibodies recognized control but not heat-treated pertussis toxin spotted onto nitrocellulose, indicating that class A antibodies do not recognize denatured S1 subunit. In contrast, a nonneutralizing class C antibody (X2X5) failed to bind to control toxin or S1 subunit in solution and recognized heat-treated pertussis toxin better than control toxin when spotted onto nitrocellulose. Thus, this type of analysis presents a heterogeneous mixture of fully or partially denatured and native S1 proteins and fails to distinguish between neutralizing and nonneutralizing antibodies. Images PMID:2155182

  17. Development and characterization of a monoclonal antibody to human embryonal carcinoma

    SciTech Connect

    Khazaeli, M.B.; Beierwaltes, W.H.; Pitt, G.S.; Kabza, G.A.; Rogers, K.J.; LoBuglio, A.F.

    1987-06-01

    A monoclonal anti-testicular carcinoma antibody was obtained via the somatic cell fusion technique by immunization of BALB/c mice with freshly prepared single cell suspension from a patient with testicular embryonal carcinoma with choriocarcinoma components. The hybridoma supernates were screened against the testicular carcinoma cells used in the immunization as well as normal mononuclear white blood cells isolated from the same patient. An antibody (5F9) was selected which bound to fresh tumor cells from two patients with embryonal testicular carcinoma and failed to bind to fresh tumor cells from 24 patients (2 seminoma, 2 melanoma, 3 neck, 2 esophageal, 1 ovarian, 3 colon, 1 prostate, 2 breast, 1 liposarcoma, 3 endometrial, 1 kidney, 1 adrenal, 1 larynx and 1 bladder tumors) or cell suspensions prepared from normal liver, lung, spleen, ovary, testes, kidney, red blood cells or white blood cells. The antibody was tested for its binding to several well established cancer cell lines, and was found to bind to the BeWo human choriocarcinoma and two human embryonal carcinoma cell lines. The antibody did not react with 22 other cell lines or with hCG. The antibody was labeled with /sup 131/I and injected into nude mice bearing BeWo tumors and evaluated for tumor localization by performing whole body scans with a gamma camera 5 days later. Six mice injected with the antibody showed positive tumor localization without the need for background subtraction while six mice injected with MOPC-21, a murine myeloma immunoglobulin, demonstrated much less tumor localization. Tissue distribution studies performed after scanning showed specific tumor localization (8:1 tumor: muscle) for the monoclonal antibody and no specific localization for MOPC-21.

  18. Novel Monoclonal Antibodies against the Proximal (Carboxy-Terminal) Portions of MUC16

    PubMed Central

    Rao, Thapi Dharma; Park, Kay J.; Smith-Jones, Peter; Iasonos, Alexia; Linkov, Irina; Soslow, Robert A.; Spriggs, David R.

    2013-01-01

    The CA125 antigen, recognized by the OC125 antibody, is a tissue-specific, circulating antigen expressed in ovarian cancer. The CA125 antigen is encoded by the MUC16 gene, cloned by Lloyd and Yin. The full-length gene describes a complex tethered mucin protein present primarily in a variety of gynecologic tissues, especially neoplasms. OC125 and other related antibodies react with glycosylation-dependent antigens present exclusively in the cleaved portion of the molecule. These antibodies are not useful as screening tools, nor can they detect the proximal residual MUC16 protein fragment after cleavage. This has limited its diagnostic and therapeutic applications. Using synthetic peptides we raised novel-specific antibodies to the carboxy-terminal portion of MUC16, retained by the cell, proximal to the putative cleavage site. These antibodies were characterized using fluorescence-activated cell-sorting analysis, enzyme-linked immunoassay, Western blot analysis, and immunohistochemistry. Each of the selected monoclonal antibodies was reactive against recombinant GST-ΔMUC16c114 protein and the MUC16 transfected SKOV3 cell line. Three antibodies, 4H11, 9C9, and 4A5 antibodies demonstrated high affinities by Western blot analysis and saturation-binding studies of transfected SKOV3 cells, and displayed antibody internalization. Immunohistochemical positivity with novel antibody 4H11 was similar to OC125, but with important differences, including diffuse positivity in lobular breast cancer and a small percentage of OC125-negative ovarian carcinomas that showed intense and diffuse 4H11. Development of such antibodies may be useful for the characterization of MUC16 biology and allow for future studies in targeted therapy and diagnostics. PMID:20453816

  19. Monoclonal antibodies against rabbit mammary prolactin receptors. Specific antibodies to the hormone binding domain

    SciTech Connect

    Katoh, M.; Djiane, J.; Kelly, P.A.

    1985-09-25

    Three monoclonal antibodies (M110, A82, and A917) were obtained by fusing myeloma cells and spleen cells from mice immunized with partially purified rabbit mammary gland prolactin (PRL) receptors. All 3 antibodies were capable of complete inhibition of SVI-ovine prolactin (oPRL) binding to rabbit mammary PRL receptors in either particulate or soluble form. M110 showed slightly greater potency than oPRL in competing for SVI-oPRL binding. These antibodies also inhibited PRL binding to microsomal fractions from rabbit liver, kidney, adrenal, ovary, and pig mammary gland, although A82 showed poor inhibition in pig mammary gland. There was no cross-reaction of any of the 3 monoclonal antibodies (mAbs) for the other species tested: human (T-47D breast cancer cells) and rat (liver, ovary). In order to confirm that these antibodies are specific to the binding domain, antibodies were purified, iodinated, and binding characteristics were investigated. SVI-M110 and SVI-A82 binding was completely inhibited by lactogenic hormones, whereas nonlactogenic hormones did not cross-react. Competition of 125I-M110 by oPRL was comparable to that of SVI-oPRL by unlabeled oPRL, while SVI-A917 binding was only partially competed (30-60%) by lactogenic hormones. Tissue and species specificity of labeled antibody binding paralleled results of binding inhibition experiments using 125I-oPRL. In addition, A82 and A917 completely inhibited 125I-M110 binding. In contrast, 125I-A82 binding was stimulated by A917 and 125I-A917 binding was stimulated by A82.

  20. Selectivity verification of cardiac troponin monoclonal antibodies for cardiac troponin detection by using conventional ELISA

    NASA Astrophysics Data System (ADS)

    Fathil, M. F. M.; Arshad, M. K. Md; Gopinath, Subash C. B.; Adzhri, R.; Ruslinda, A. R.; Hashim, U.

    2017-03-01

    This paper presents preparation and characterization of conventional enzyme-linked immunosorbent assay (ELISA) for cardiac troponin detection to determine the selectivity of the cardiac troponin monoclonal antibodies. Monoclonal antibodies, used to capture and bind the targets in this experiment, are cTnI monoclonal antibody (MAb-cTnI) and cTnT monoclonal antibody (MAb-cTnT), while both cardiac troponin I (cTnI) and T (cTnT) are used as targets. ELISA is performed inside two microtiter plates for MAb-cTnI and MAb-cTnT. For each plate, monoclonal antibodies are tested by various concentrations of cTnI and cTnT ranging from 0-6400 µg/l. The binding selectivity and level of detection between monoclonal antibodies and antigen are determined through visual observation based on the color change inside each well on the plate. ELISA reader is further used to quantitatively measured the optical density of the color changes, thus produced more accurate reading. The results from this experiment are utilized to justify the use of these monoclonal antibodies as bio-receptors for cardiac troponin detection by using field-effect transistor (FET)-based biosensors coupled with substrate-gate in the future.

  1. The history of monoclonal antibody development – Progress, remaining challenges and future innovations

    PubMed Central

    Liu, Justin K.H.

    2014-01-01

    As medicine progresses into a new era of personalised therapy, the use of monoclonal antibodies to treat a wide range of diseases lies at the heart of this new forefront. Since the licencing of the first monoclonal antibody for clinical use 30 years ago, the monoclonal antibody industry has expanded exponentially and is now valued at billions of dollars. With major advances in genetic sequencing and biomedical research, much research into monoclonal antibodies now focuses on identifying new targets for development and maximising their efficacy for use in clinical practice. However, a balance has to be struck with regards to reducing numbers of side-effects and overall economic cost, which arguably somewhat blighted their early clinical and commercial successes. Nowadays, there are approximately 30 monoclonal antibodies that have been approved for use in clinical practice with many more currently being tested in clinical trials. Some of the current major limitations include: the use of inefficient models for generation, a lack of efficacy and issues of cost-effectiveness. Some of the current research focuses on ways to improve the efficacy of existing monoclonal antibodies through optimising their effects and the addition of beneficial modifications. This review will focus on the history of monoclonal antibody development – how it has increasingly moved away from using laborious animal models to a more effective phage display system, some of the major drawbacks from a clinical and economical point of view and future innovations that are currently being researched to maximise their effectiveness for future clinical use. PMID:25568796

  2. Monoclonal antibodies for the rapid diagnosis of influenza-B virus infections by ELISA: production and characterization.

    PubMed

    Glikmann, G; Chen, S N; Mordhorst, C H; Koch, C

    1995-07-01

    Monoclonal antibodies directed against conserved epitopes of viral proteins have substantially improved the accuracy of several immunochemical methods in diagnostic virology. To characterize mouse monoclonal antibodies directed against structural protein antigens of influenza-B virus and evaluate their use as diagnostic reagents for the direct detection of such antigens in clinical specimens from patients with respiratory infections of unknown aetiology. (a) Production and characterization of monoclonal antibodies against influenza-B viral antigens, and (b) their use in two different ELISA systems for detecting influenza-B antigen either directly in clinical specimens or after confirmation by rapid culture in MDCK cells. Four monoclonal antibodies were selected for their specificity for the nucleoprotein antigen as demonstrated by Western blot analysis. The specificity of these antibodies for different epitopes of the nucleoprotein was demonstrated by competition experiments, using unlabelled and biotin-labelled purified antibodies in a sandwich assay. All four antibodies belong to the mouse IgG(2a) isotype, lack haemagglutination inhibition and neutralization properties and exhibit titres as high as 10(-6) in ELISA with as little as 30 ng purified influenza-B virus. ELISA methods using these antibodies detected only influenza-B viral antigens in direct testing of clinical specimens from patients with known influenza-B or influenza-A infections, or after reisolating virus from such specimens in tissue culture of MDCK cells. The antibodies were suitable for the direct detection and typing of influenza-B virus in clinical specimens or for use in rapid confirmation cultures.

  3. Kinetic analysis of the multistep aggregation mechanism of monoclonal antibodies.

    PubMed

    Nicoud, Lucrèce; Arosio, Paolo; Sozo, Margaux; Yates, Andrew; Norrant, Edith; Morbidelli, Massimo

    2014-09-11

    We investigate by kinetic analysis the aggregation mechanism of two monoclonal antibodies belonging to the IgG1 and IgG2 subclass under thermal stress. For each IgG, we apply a combination of size exclusion chromatography and light scattering techniques to resolve the time evolution of the monomer, dimer, and trimer concentrations, as well as the average molecular weight and the average hydrodynamic radius of the aggregate distribution. By combining the detailed experimental characterization with a theoretical kinetic model based on population balance equations, we extract relevant information on the contribution of the individual elementary steps on the global aggregation process. The analysis shows that the two molecules follow different aggregation pathways under the same operating conditions. In particular, while the monomer depletion of the IgG1 is found to be rate-limited by monomeric conformational changes, bimolecular collision is identified as the rate-limiting step in the IgG2 aggregation process. The measurement of the microscopic rate constants by kinetic analysis allows the quantification of the protein-protein interaction potentials expressed in terms of the Fuchs stability ratio (W). It is found that the antibody solutions exhibit large W values, which are several orders of magnitude larger than the values computed in the frame of the DLVO theory. This indicates that, besides net electrostatic repulsion, additional effects delay the aggregation kinetics of the antibody solutions with respect to diffusion-limited conditions. These effects likely include the limited efficiency of the collision events due to the presence of a limited number of specific aggregation-prone patches on the heterogeneous protein surface, and the contribution of additional repulsive non-DLVO forces to the protein-protein interaction potential, such as hydration forces.

  4. Development of new staining technology "eastern blotting" using monoclonal antibody.

    PubMed

    Morinaga, Osamu; Shoyama, Yukihiro

    2011-03-01

    Ginsenosides contained in Panax species were separated by silica gel TLC blotted to a polyvinylidene difluoride (PVDF) membrane which was dipped in a sodium periodide (NaIO(4)) solution and reacted with protein, preparing a ginsenoside-protein conjugate for binding a ginsenoside on a PVDF membrane. The blotted spots were stained by anti-ginsenoside-Rb1 monoclonal antibody (MAb) and anti-ginsenoside-Rg1MAb, respectively. The newly established immunostaining method, eastern blotting was applied for the determination of ginsenosides possessing protopanaxadiol and/or protopanaxatriol. Double staining of eastern blotting for ginsenosides using anti-ginsenoside-Rb1 MAb and anti-ginsenoside-Rg1 MAb promoted complete identification of ginsenosides in Panax species. This technique has been devised for the chromatographic separation and identification of ginsenosides using polyethersulfone (PES) membrane. It caused an acceptable separation of ginsenoside-Rb1, -Rc and -Rd in various ginseng extracts. Newly developed technique is quite simple and applies for immunoassay system. Ginsenosides separated using a PES membrane were directly treated with a NaIO(4) solution and then reacted with bovine serum albumin (BSA) for making a ginsenoside-protein conjugate. After the blocking, anti-ginsenoside-Rb1 MAb recognized a ginsenoside on a PES membrane and then a sec-ond antibody labeled with enzyme reacted to the first antibody. Finally a substrate was oxidized with the enzyme and de-veloped the staining of ginsenosides. The staining spots of ginsenosides on membrane were quantitatively evaluated by NIH Image indicating at least 62.5 ng of each ginsenoside-Rb1, -Rc and -Rd were detected with clarity. The determination range of three ginsenosides was from 0.125 to 2.0 µg of direct amount on PES membrane.

  5. Genetic characterization of mouse immunoglobulin allotypic determinants (allotopes) defined by monoclonal antibodies.

    PubMed

    Huang, C M; Parsons, M; Oi, V T; Huang, H J; Herzenberg, L A

    1983-01-01

    We have generated a new series of monoclonal antibodies recognizing allotypic determinants on mouse IgG1, IgG2a, and IgG2b. In this communication we describe their reactivities with immunoglobulins of the inbred mouse strains. Comparison with serology charts indicates that many of these monoclonal antibodies detect allotypic specificities previously defined by conventional antisera; others define previously undescribed specificities. Strain and isotype distribution allows us to assign five new allotypic specificities to Igh-1 and three new specificities to Igh-3. In addition, on the basis of reactivity with the monoclonal antibodies, we have defined a new Igh haplotype in SWR/J mice, Ighp.

  6. 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

  7. A broadly reactive monoclonal antibody detects multiple genotypes of hepatitis B virus X protein.

    PubMed

    Wei, Lili; Shen, Zhongliang; Zhao, Xue; Wu, Yanxin; Liu, Wei; Zhang, Junqi; Xie, Youhua; Liu, Jing

    2014-10-01

    A highly specific and broadly reactive monoclonal antibody against hepatitis B virus X (HBx) protein was developed that detected, in both immunoblotting and immunofluorescence, HBx proteins of seven of the eight currently known genotypes of HBV, which were overexpressed in cultured cells. Evaluation of HBx expression levels in cultured hepatocytes using this monoclonal antibody showed that cells transiently and stably transfected with HBV genomes expressed far less HBx protein than cells transiently transfected with an HBx overexpression plasmid routinely used for studying HBx functions. The availability of such sensitive and broadly reactive monoclonal antibodies against HBx will enable more-quantitative studies of HBx functions.

  8. Immunodiagnosis of tumors in vivo using radiolabeled monoclonal antibody A2B5.

    PubMed

    Reintgen, D S; Shimizu, K; Coleman, E; Briner, W; Kitzmiller, J; Eisenbarth, G; Seigler, H F

    1983-07-01

    Recently a murine monoclonal antibody (A2B5) has been described that reacts with a membrane associated GQ ganglioside common to peptide secreting normal cells and tumors. In vitro binding data demonstrated the presence of this ganglioside on neurons, adrenal medulla, and pancreatic islets, along with neuroendocrine tumors such as insulinomas, pheochromocytomas, melanomas and neuroblastomas. Negative binding has previously been shown for tissue sections from liver, kidney, colon, lung, stomach, and tumors not derived from the neural crest. Because of the specificity at A2B5 in vitro, this monoclonal antibody was labeled with 131I for in vivo tumor localization studies. Daily radionuclear scans were obtained in 5 KX rats bearing the radiation induced rat insulinoma with disappearance of the label from the blood pool and concentration in the tumor so that by the fourth day, the only activity present by scan was in the insulinoma. Tissue-counting data showed tumor/blood ratios (av +/- SE, 1.29 +/- 0.25) of A2B5 activity two to ten times the average activity found in other organs (0.28 +/- 0.05). No tumor concentration of the control nonspecific monoclonal antibody P3X63 was evident (0.27 +/- 0.04). In addition A2B5 also localized to five different human melanoma cells lines grown in nude mice with high tumor/blood levels (1.04 +/- 0.27) compared to normal tissues (0.32 +/- 0.05) (P = .0005), while no localization is seen in nudes carrying osteosarcomas, colon, bladder, and renal cell carcinomas. In addition antibody A2B5 did not concentrate in any normal tissue though the antigen is present on several. The finding that A2B5 reacts across species lines (mouse, rat, man) lends itself to obvious diagnostic and therapeutic possibilities.

  9. 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.''

  10. 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.

  11. Tumor localization by combinations of monoclonal antibodies in a new human colon carcinoma cell line (LIM1899)

    SciTech Connect

    Andrew, S.M.; Teh, J.G.; Johnstone, R.W.; Russell, S.M.; Whitehead, R.H.; McKenzie, I.F.; Pietersz, G.A. )

    1990-09-01

    One of the problems of in vivo diagnosis and therapy of tumors with monoclonal antibodies is their heterogeneity with respect to antigen expression, with some cells expressing no antigen and others being weakly or strongly positive. Selected mixtures of antibodies to different antigens are therefore likely to react with more cells than single antibodies and be more effective for imaging and therapy. With this in mind, we have examined a new human colon cancer cell line (LIM1899) which has a heterogeneous expression of several cell surface molecules: by flow cytometry 38% were carcinoembryonic antigen positive; 64%, human milk fat globule positive, and 73%, CD46 positive; 87% of tumor cells bound a mixture of all three antibodies in vitro. Some blocking of the binding of anti-human milk fat globule antibody by the anti-CD46 antibody was noted. LIM1899 was established as a xenograft in nude mice and in vivo biodistribution studies performed using antibodies alone or in combination. Mixtures of antibodies clearly showed a higher percentage of injected dose of antibody in the tumor than did single antibodies: one antibody gave 10%; two together, 17 to 21%; and all three together gave 29% of the injected dose in the tumor. Tumor:blood ratios were also superior for combinations of antibodies, provided that low doses of the antibodies were used; at higher doses the effect was lost. The study demonstrates that combinations of antibodies are better than single antibodies for localization, provided that the dose used is carefully selected.

  12. Tumor localization by combinations of monoclonal antibodies in a new human colon carcinoma cell line (LIM1899).

    PubMed

    Andrew, S M; Teh, J G; Johnstone, R W; Russell, S M; Whitehead, R H; McKenzie, I F; Pietersz, G A

    1990-09-01

    One of the problems of in vivo diagnosis and therapy of tumors with monoclonal antibodies is their heterogeneity with respect to antigen expression, with some cells expressing no antigen and others being weakly or strongly positive. Selected mixtures of antibodies to different antigens are therefore likely to react with more cells than single antibodies and be more effective for imaging and therapy. With this in mind, we have examined a new human colon cancer cell line (LIM1899) which has a heterogeneous expression of several cell surface molecules: by flow cytometry 38% were carcinoembryonic antigen positive; 64%, human milk fat globule positive, and 73%, CD46 positive; 87% of tumor cells bound a mixture of all three antibodies in vitro. Some blocking of the binding of anti-human milk fat globule antibody by the anti-CD46 antibody was noted. LIM1899 was established as a xenograft in nude mice and in vivo biodistribution studies performed using antibodies alone or in combination. Mixtures of antibodies clearly showed a higher percentage of injected dose of antibody in the tumor than did single antibodies: one antibody gave 10%; two together, 17 to 21%; and all three together gave 29% of the injected dose in the tumor. Tumor:blood ratios were also superior for combinations of antibodies, provided that low doses of the antibodies were used; at higher doses the effect was lost. The study demonstrates that combinations of antibodies are better than single antibodies for localization, provided that the dose used is carefully selected.

  13. Monoclonal antibodies to NF-Y define its function in MHC class II and albumin gene transcription.

    PubMed Central

    Mantovani, R; Pessara, U; Tronche, F; Li, X Y; Knapp, A M; Pasquali, J L; Benoist, C; Mathis, D

    1992-01-01

    NF-Y is a sequence-specific DNA-binding protein which, as a heterodimer, recognizes CCAAT motifs in a variety of transcriptional promoters. We have generated a panel of monoclonal and affinity-purified polyclonal antibodies directed against various epitopes of NF-Y. These reagents are highly specific for either of the A or B subunits; we have mapped the epitopes recognized by the monoclonal antibodies to the glutamine-rich activation domain of NF-YA. The antibodies inhibit in vitro transcription from the promoters of the albumin gene and of Ea, a class II gene of the major histocompatibility complex. These data definitively demonstrate the role of NF-Y in regulating the transcription of two tissue-specific genes whose expression patterns do not overlap. Interestingly, the antibodies cannot inhibit a formed pre-initiation complex, but do block reinitiation of subsequent rounds of transcription from the same templates. Images PMID:1380453

  14. Perspectives of monoclonal antibodies for detection and treatment of head and neck tumours.

    PubMed

    Quak, J; Gerretsen, M; De Bree, R; Brakenhof, R; Van Dongen, G; Snow, G

    1993-01-01

    Monoclonal antibodies (mabs) are potentially powerful tools for the detection and treatment of cancer. To date, only a limited number of mabs are available to head and neck cancer. We produced 5 different groups of mabs to head and neck cancer. These mabs were characterized for their reactivity tumour and non-tumour tissues. Furthermore, biochemical elucidation of recognized antigens was provided. In animal studies the effectiveness of mabs for diagnoses and therapy of cancer is clearly demonstrated. The first results of a clinical study for the detection of head and neck cancer with mabs are shown. Finally, the future of mabs in clinical oncology is discussed.

  15. Targeting and killing glioblastoma with monoclonal antibody to O-acetyl GD2 ganglioside

    PubMed Central

    Fougeray, Sophie; Oliver, Lisa; Geraldo, Fanny; Terme, Mickaël; Dorvillius, Mylène; Loussouarn, Delphine; Vallette, François; Paris, François; Birklé, Stéphane

    2016-01-01

    There are still unmet medical needs in the treatment of glioblastoma, the most common and the most aggressive glioma of all brain tumors. Here, we found that O-acetyl GD2 is expressed in surgically resected human glioblastoma tissue. In addition, we demonstrated that 8B6 monoclonal antibody specific for O-acetylat GD2 could effectively inhibit glioblastoma cell proliferation in vitro and in vivo. Taken together, these results indicate that O-acetylated GD2 represents a novel antigen for immunotherapeutic-based treatment of high-grade gliomas. PMID:27172791

  16. Rheumatoid factor interference in immunogenicity assays for human monoclonal antibody therapeutics.

    PubMed

    Tatarewicz, Suzanna; Miller, Jill M; Swanson, Steven J; Moxness, Michael S

    2010-05-31

    Rheumatoid factors (RFs) are endogenous human antibodies that bind to human gamma globulins. RFs demonstrate preferential binding to aggregated gamma globulins and are involved in the clearing mechanism of immune complexes. Immunoassays designed to measure human anti-human antibodies (HAHA) after administration of monoclonal antibody therapeutics are thus vulnerable to interference from RFs. When using a sensitive electrochemiluminescent (ECL) bridging immunoassay, samples from subjects with rheumatoid arthritis demonstrated much higher baseline reactivity than healthy subjects. Interference was found to be dependent on the aggregation state of the therapeutic antibody that had been conjugated with the detection reagent (ruthenium). Size exclusion high performance liquid chromatography (SE-HPLC) demonstrated that of the total integrated peaks, as little as 0.55% high molecular weight aggregates (>600kDa) were sufficient to cause increased reactivity. Stability studies of the ruthenium and biotin conjugated therapeutic antibody indicated that storage time, temperature and buffer formulation were critical in maintaining the integrity of the reagents. Through careful SE-HPLC monitoring we were able to choose appropriate storage and buffer conditions which led to a reduction in the false reactivity rate in therapeutic-naïve serum from a rheumatoid arthritis population.

  17. Protection of mice against Clostridium chauvoei infection by anti-idiotype antibody to a monoclonal antibody to flagella.

    PubMed

    Kijima-Tanaka, M; Nakamura, M; Nagamine, N; Takahashi, T; Aoki, A; Tamura, Y

    1994-03-01

    Polyclonal rabbit anti-idiotypic antibody (anti-Id) against the protective monoclonal antibody specific to the flagella of Clostridium chauvoei was produced, purified, and characterized. Anti-Id inhibited the binding of its related monoclonal antibody to the flagellar antigen, suggesting that the anti-Id bore an internal image of the flagellar antigen. When mice were immunized with anti-Id intraperitoneally, the survival rate increased significantly, compared with mice immunized with normal rabbit IgG (P < 0.01), and specific anti-flagellar antibodies were induced.

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

    PubMed

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

    2002-09-06

    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.

  19. Antibody specificity and antigen characterization of rat monoclonal antibodies against Streptococcus mutans cell wall-associated protein antigens.

    PubMed Central

    Ackermans, F; Klein, J P; Cormont, F; Bazin, H; Ogier, J A; Frank, R M; Vreven, J

    1985-01-01

    Monoclonal antibodies to Streptococcus mutans OMZ175 (serotype f) cell wall-associated antigens (wall-extracted antigens [WEA]) were derived from the fusion of Lou C plasmocytoma rat cells (IR 983 F) and spleen cells from Wistar R inbred rats immunized with WEA. Four cell lines producing monoclonal antibodies directed against a component of S. mutans WEA have been established. All four monoclonal antibodies reacted only with two antigens of WEA from S. mutans OMZ175 by Western blotting and immunoprecipitation techniques, enzyme-linked immunosorbent assay (ELISA), and competitive ELISA. Western blot analysis of WEA showed that the four monoclonal antibodies recognized two related cell wall-associated proteins with apparent molecular weights of 125,000 and 76,000. Immunoprecipitation of whole cells with the monoclonal antibodies confirmed the surface localization of the two antigens. The ELISA and competitive ELISA were used to analyze the distribution of the epitopes on seven S. mutans serotypes. All S. mutans serotypes were found to express the recognized epitopes; however, different reactivity patterns could be distinguished among the various strains tested, and the four monoclonal antibodies reacted only weakly with S. mutans serotypes d and g. Images PMID:2410364

  20. 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

  1. Pan-ebolavirus and Pan-filovirus Mouse Monoclonal Antibodies: Protection against Ebola and Sudan Viruses

    PubMed Central

    Holtsberg, Frederick W.; Shulenin, Sergey; Vu, Hong; Howell, Katie A.; Patel, Sonal J.; Gunn, Bronwyn; Karim, Marcus; Lai, Jonathan R.; Frei, Julia C.; Nyakatura, Elisabeth K.; Zeitlin, Larry; Douglas, Robin; Fusco, Marnie L.; Froude, Jeffrey W.; Saphire, Erica Ollmann; Herbert, Andrew S.; Wirchnianski, Ariel S.; Lear-Rooney, Calli M.; Alter, Galit; Dye, John M.; Glass, Pamela J.; Warfield, Kelly L.

    2015-01-01

    ABSTRACT The unprecedented 2014-2015 Ebola virus disease (EVD) outbreak in West Africa has highlighted the need for effective therapeutics against filoviruses. Monoclonal antibody (MAb) cocktails have shown great potential as EVD therapeutics; however, the existing protective MAbs are virus species specific. Here we report the development of pan-ebolavirus and pan-filovirus antibodies generated by repeated immunization of mice with filovirus glycoproteins engineered to drive the B cell responses toward conserved epitopes. Multiple pan-ebolavirus antibodies were identified that react to the Ebola, Sudan, Bundibugyo, and Reston viruses. A pan-filovirus antibody that was reactive to the receptor binding regions of all filovirus glycoproteins was also identified. Significant postexposure efficacy of several MAbs, including a novel antibody cocktail, was demonstrated. For the first time, we report cross-neutralization and in vivo protection against two highly divergent filovirus species, i.e., Ebola virus and Sudan virus, with a single antibody. Competition studies indicate that this antibody targets a previously unrecognized conserved neutralizing epitope that involves the glycan cap. Mechanistic studies indicated that, besides neutralization, innate immune cell effector functions may play a role in the antiviral activity of the antibodies. Our findings further suggest critical novel epitopes that can be utilized to design effective cocktails for broad protection against multiple filovirus species. IMPORTANCE Filoviruses represent a major public health threat in Africa and an emerging global concern. Largely driven by the U.S. biodefense funding programs and reinforced by the 2014 outbreaks, current immunotherapeutics are primarily focused on a single filovirus species called Ebola virus (EBOV) (formerly Zaire Ebola virus). However, other filoviruses including Sudan, Bundibugyo, and Marburg viruses have caused human outbreaks with mortality rates as high as 90%. Thus

  2. Pan-ebolavirus and Pan-filovirus Mouse Monoclonal Antibodies: Protection against Ebola and Sudan Viruses.

    PubMed

    Holtsberg, Frederick W; Shulenin, Sergey; Vu, Hong; Howell, Katie A; Patel, Sonal J; Gunn, Bronwyn; Karim, Marcus; Lai, Jonathan R; Frei, Julia C; Nyakatura, Elisabeth K; Zeitlin, Larry; Douglas, Robin; Fusco, Marnie L; Froude, Jeffrey W; Saphire, Erica Ollmann; Herbert, Andrew S; Wirchnianski, Ariel S; Lear-Rooney, Calli M; Alter, Galit; Dye, John M; Glass, Pamela J; Warfield, Kelly L; Aman, M Javad

    2015-10-14

    The unprecedented 2014-2015 Ebola virus disease (EVD) outbreak in West Africa has highlighted the need for effective therapeutics against filoviruses. Monoclonal antibody (MAb) cocktails have shown great potential as EVD therapeutics; however, the existing protective MAbs are virus species specific. Here we report the development of pan-ebolavirus and pan-filovirus antibodies generated by repeated immunization of mice with filovirus glycoproteins engineered to drive the B cell responses toward conserved epitopes. Multiple pan-ebolavirus antibodies were identified that react to the Ebola, Sudan, Bundibugyo, and Reston viruses. A pan-filovirus antibody that was reactive to the receptor binding regions of all filovirus glycoproteins was also identified. Significant postexposure efficacy of several MAbs, including a novel antibody cocktail, was demonstrated. For the first time, we report cross-neutralization and in vivo protection against two highly divergent filovirus species, i.e., Ebola virus and Sudan virus, with a single antibody. Competition studies indicate that this antibody targets a previously unrecognized conserved neutralizing epitope that involves the glycan cap. Mechanistic studies indicated that, besides neutralization, innate immune cell effector functions may play a role in the antiviral activity of the antibodies. Our findings further suggest critical novel epitopes that can be utilized to design effective cocktails for broad protection against multiple filovirus species. Filoviruses represent a major public health threat in Africa and an emerging global concern. Largely driven by the U.S. biodefense funding programs and reinforced by the 2014 outbreaks, current immunotherapeutics are primarily focused on a single filovirus species called Ebola virus (EBOV) (formerly Zaire Ebola virus). However, other filoviruses including Sudan, Bundibugyo, and Marburg viruses have caused human outbreaks with mortality rates as high as 90%. Thus, cross

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

    PubMed

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

    2016-04-22

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

  4. Monoclonal antibodies against the native urease of Helicobacter pylori: synergistic inhibition of urease activity by monoclonal antibody combinations.

    PubMed Central

    Nagata, K; Mizuta, T; Tonokatu, Y; Fukuda, Y; Okamura, H; Hayashi, T; Shimoyama, T; Tamura, T

    1992-01-01

    Monoclonal antibodies (MAbs) against the native urease of Helicobacter pylori NCTC 11637 were found to clearly inhibit the urease activity. Interestingly, synergistic inhibition by two MAbs recognizing different subunits was also observed. Ten MAbs were produced and classified as two isotypes of the immunoglobulin G (IgG) subclass, IgG1, and IgG2a. Western blot (immunoblot) analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that five MAbs recognized the large subunit and the other five recognized the small subunit of the urease. Among the MAbs, L2 and S2, which recognized the large and the small subunits, respectively, were also able to inhibit the urease activity of clinical isolates from H. pylori-infected patients. The combination of L2 and S2 led to augmented synergistic inhibition. L2, but not S2, could also inhibit the urease activity from Helicobacter mustelae; enzyme-linked immunosorbent assay and Western blot analysis showed that L2 cross-reacted with this urease. These results suggested that the epitope recognized by L2 had a structure common to both Helicobacter species and may be involved in the active site of the urease. In contrast to the MAbs, a polyclonal antibody in sera from mice immunized with H. pylori urease did not have the ability to inhibit H. pylori urease activity. However, the polyclonal antibody retained the ability to abolish the inhibitory action of these MAbs. Moreover, other MAbs which could not inhibit H. pylori urease activity also abolished the inhibitory action. Images PMID:1383158

  5. Reagent Target Request for Monoclonal Antibody Production and Characterization | Office of Cancer Clinical Proteomics Research

    Cancer.gov

    NCI's Antibody Characterization Program provides reagents and other critical resources to support protein/peptide measurements and analysis. In an effort to produce and distribute well-characterized monoclonal antibodies to the scientific community, the program is seeking cancer related protein targets for antibody production and characterization for distribution to the research community. Submission Period: May 20, 2011 - July 1, 2011.

  6. Human anti-murine antibody responses in ovarian cancer patients undergoing radioimmunotherapy with the murine monoclonal antibody OC-125

    SciTech Connect

    Muto, M.G.; Finkler, N.J.; Kassis, A.I.; Lepisto, E.M.; Knapp, R.C. )

    1990-08-01

    Human anti-murine antibody (HAMA) responses were monitored in 23 patients with recurrent or persistent epithelial ovarian carcinoma undergoing single-dose intraperitoneal radioimmunotherapy (RIT) with the murine monoclonal antibody OC-125. Sera of patients receiving escalating doses of OC-125 F(ab')2 (10-70 mg) radiolabeled with 18 to 141 mCi of iodine-131 were assayed for HAMA by a protein A-based radioimmunoassay. Overall, 70% of patients (16/23) developed HAMA within 10 to 46 days (median = 29) postinfusion, with peak values (23 +/- 6 to 325 +/- 10 micrograms/ml) at 32 to 102 days (median = 38). HAMA was undetectable prior to infusion in all cases and persisted up to 76 weeks. Of patients receiving a dose of 123 mCi or less, 80% (16/20) developed HAMA, whereas in the 140-mCi group, none of the three patients had detectable levels. Two patients in the 140-mCi group demonstrated dose-limiting bone marrow toxicity (severe thrombocytopenia and neutropenia). It is concluded that a single intraperitoneal dose of monoclonal antibody leads to a high incidence of HAMA production. The results also suggest that the likelihood of HAMA formation in patients who either had undergone recent chemotherapy or had received the highest dose of the radioimmunoconjugate is reduced. These observations may be of significance in designing multiple-dose therapy trials as HAMA has been demonstrated to decrease antibody-to-tumor binding and may potentially increase renal, hepatic, and hematologic toxicity associated with radioimmunotherapy.

  7. 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.

  8. Characterization of monoclonal antibodies that strongly inhibit Electrophorus electricus acetylcholinesterase.

    PubMed

    Remy, M H; Frobert, Y; Grassi, J

    1995-08-01

    In this study, we describe three different monoclonal antibodies (mAbs Elec-403, Elec-408, and Elec-410) directed against Electrophorus electricus acetylcholinesterase (AChE) which were selected as inhibitors for this enzyme. Two of these antibodies (Elec-403 and Elec-410), recognized overlapping but different epitopes, competed with snake venom toxin fasciculin for binding to the enzyme, and thus apparently recognized the peripheral site of AChE. In addition, the binding of Elec-403 was antagonized by 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide (BW284C51) and propidium, indicating that the corresponding epitope encompassed the anionic site involved in the binding of these low-molecular-mass inhibitors. The third mAb (Elec-408), was clearly bound to another site on the AChE molecule, and its inhibitory effect was cumulative with those of Elec-403, Elec-410, and fasciculin. All mAbs bound AChE with high affinity and were as strong inhibitors with an apparent Ki values less than 0.1 nM. Elec-403 was particularly efficient with an inhibitory activity similar to that of fasciculin. Inhibition was observed with both charged (acetylthiocholine) and neutral substrates (o-nitrophenyl acetate) and had the characteristics of a non-competitive process. Elec-403 and Elec-410 probably exert their effect by triggering allosteric transitions from the peripheral site to the active site. The epitope recognized by mAb Elec-408 has not been localized, but it may correspond to a new regulatory site on AChE.

  9. Human Monoclonal Antibodies Broadly Neutralizing against Influenza B Virus

    PubMed Central

    Yasugi, Mayo; Kubota-Koketsu, Ritsuko; Yamashita, Akifumi; Kawashita, Norihito; Du, Anariwa; Sasaki, Tadahiro; Nishimura, Mitsuhiro; Misaki, Ryo; Kuhara, Motoki; Boonsathorn, Naphatsawan; Fujiyama, Kazuhito; Okuno, Yoshinobu; Nakaya, Takaaki; Ikuta, Kazuyoshi

    2013-01-01

    Influenza virus has the ability to evade host immune surveillance through rapid viral genetic drift and reassortment; therefore, it remains a continuous public health threat. The development of vaccines producing broadly reactive antibodies, as well as therapeutic strategies using human neutralizing monoclonal antibodies (HuMAbs) with global reactivity, has been gathering great interest recently. Here, three hybridoma clones producing HuMAbs against influenza B virus, designated 5A7, 3A2 and 10C4, were prepared using peripheral lymphocytes from vaccinated volunteers, and were investigated for broad cross-reactive neutralizing activity. Of these HuMAbs, 3A2 and 10C4, which recognize the readily mutable 190-helix region near the receptor binding site in the hemagglutinin (HA) protein, react only with the Yamagata lineage of influenza B virus. By contrast, HuMAb 5A7 broadly neutralizes influenza B strains that were isolated from 1985 to 2006, belonging to both Yamagata and Victoria lineages. Epitope mapping revealed that 5A7 recognizes 316G, 318C and 321W near the C terminal of HA1, a highly conserved region in influenza B virus. Indeed, no mutations in the amino acid residues of the epitope region were induced, even after the virus was passaged ten times in the presence of HuMAb 5A7. Moreover, 5A7 showed significant therapeutic efficacy in mice, even when it was administered 72 hours post-infection. These results indicate that 5A7 is a promising candidate for developing therapeutics, and provide insight for the development of a universal vaccine against influenza B virus. PMID:23408886

  10. Characterization of monoclonal antibodies to avian Escherichia coli Iss.

    PubMed

    Lynne, Aaron M; Foley, Steven L; Nolan, Lisa K

    2006-09-01

    Colibacillosis accounts for annual multimillion dollar losses in the poultry industry, and control of this disease is hampered by limited understanding of the virulence mechanisms used by avian pathogenic Escherichia coli (APEC). Previous work in our laboratory has found that the presence of the increased serum survival gene (iss) is strongly associated with APEC but not commensal E. coli, making iss and the protein it encodes (Iss) candidate targets of colibacillosis-control procedures. Previously, we produced monoclonal antibodies (MAbs) against Iss to be used as a reagent in studies of APEC virulence and colibacillosis pathogenesis. Unfortunately, the utility of these MAbs was limited because these MAbs exhibited nonspecific binding. It was thought that the lack of specificity might be related to the fact that these MAbs were of the immunoglobulin M (IgM) isotype. In the present study, new MAbs were produced using a different immunization strategy in an effort to generate MAbs of a different isotype. Also, because Iss bears strong similarity to Bor, a lambda-derived protein that occurs commonly among E. coli, MAbs were assessed for their ability to distinguish Iss and Bor. For these studies, the bor gene from an APEC isolate was cloned into an expression vector. The fusion protein expressed from this construct was used to assess the potential of the anti-Iss MAbs produced in the past and present studies to distinguish Bor and Iss. The MAbs produced in this study were of the IgG1 isotype, which appeared to bind more specifically to Iss than previously generated antibodies in certain immunologic procedures. These results suggested that the MAbs generated in this study might prove superior to the previous MAbs as a reagent for study of APEC. However, both MAbs recognized recombinant Iss and Bor, suggesting that any results obtained using anti-Iss MAbs would need to be interpreted with this cross-reactivity in mind.

  11. Monoclonal antibodies recognizing single amino acid substitutions in hemoglobin

    SciTech Connect

    Stanker, L.H.; Branscomb, E.; Vanderlaan, M.; Jensen, R.H.

    1986-06-01

    Four monoclonal antibodies (mAb) to non-human primate hemoglobin referred to as Cap-4, Cap-5, Rh-2, and Rh-4, and two mAb to human hemoglobin, referred to as H-1 and H-3 were isolated and were partially characterized. Binding studies with these mAb on a panel of hemoglobins and isolated ..cap alpha.. and ..beta.. globin chains revealed a unique reactivity pattern for each mAb. Amino acid sequence analysis of the antigens used to generate the binding data suggests that the specific recognition of certain hemoglobin antigens by each mAb is controlled by the presence of a particular amino acid at a specific position within the epitope. The use of synthetic peptides as antigens confirmed this observation for five of the mAb. No synthetic peptides were tested with the sixth mAb, Rh-2. The amino acids required for binding of mAb Cap-4, Cap-5, Rh-4, and Rh-2 to hemoglobin are alanine at ..beta..5, threonine at ..beta..13, glutamine at ..beta..125, and leucine at ..cap alpha..68. The non-human primate hemoglobin antibodies require a specific amino acid that is not present in human hemoglobin. The amino acid required for binding of Cap-4, Cap-5, and Rh-4 could arise by a single base change in the ..beta.. globin gene, whereas the amino acid required for Rh-2 binding could only occur if two base changes occurred. Thus these mAb are candidate probes for a somatic cell mutation assay on the basis of the detection of peripheral blood red cells that possess single amino acid substituted hemoglobin as a result of single base substitutions in the globin genes of precursor cells.

  12. Monoclonal antibodies to human apolipoproteins: application to the study of high density lipoprotein subpopulations.

    PubMed

    Bustos, P; Ulloa, N; Calvo, C; Muller, D; Durán, D; Martínez, J; Salazar, L; Quiroga, A

    2000-09-01

    We produced, selected and cloned hybridomas that secrete monoclonal antibodies against human apolipoprotein (apo) A-I. All of the antibodies corresponded to the IgG(1) subclass and were named 1C11, 2B4, 2C10, 7C5, 8A4 and 8A5. The antibodies were characterized by their reactivity with whole lipoproteins, apolipoproteins, synthetic peptides and fragments generated by cleavage of the apo A-I. Three of the monoclonal antibodies studied (2B4, 2C10 and 7C5) were similarly inhibited by an amino-terminal peptide (amino acid sequence 1-20) of apo A-I, whereas antibodies 1C11, 8A4 and 8A5 had no reaction. Other results show that monoclonal antibody 1C11 recognizes an epitope located between amino acids 135-148. We evaluated the monoclonal antibody 8A4 against different HDL subpopulations by competitive displacement analysis and it showed a similar reactivity with the HDL particles: LpA-I and LpA-I:A-II. This antibody was used to standardize a sandwich ELISA to quantitate LpA-I in plasma. We conclude that these monoclonal antibodies are relevant for the study of apo A-I epitope expression and for quantitating apo A-I containing lipoparticles.

  13. Localization of a tumor cell adhesion domain of laminin by a monoclonal antibody

    SciTech Connect

    Skubitz, A.P.N.; Charonis, A.S.; Tsilibary, E.C.; Furcht, L.T. )

    1987-12-01

    Monoclonal antibodies were prepared to localize the domain(s) of laminin to which tumor cells adhere. Rat Y3-Ag 1.2.3 myeloma cells were fused with spleen cells from a rat immunized with a purified 440-kDa fragment of chymotrypsin-digested laminin. Three monoclonal antibodies (AL-1 to AL-3) that bound to intact laminin in a solid-phase radioimmunoassay were chosen for further analysis. The epitopes recognized by these antibodies were characterized by radioimmunoassays, immunoblotting, radioimmunoprecipitation, and immunoaffinity chromatography. In cell adhesion assays, monoclonal antibody AL-2 inhibited the highly metastatic melanoma cell line, K-1735-M4, to both intact laminin and the 440-kDa fragment of laminin. Electron microscopic examination of laminin-monoclonal antibody interactions showed that monoclonal antibody AL-2 reacted with the long arm of laminin directly below the cross-region. Two monoclonal antibodies that failed to inhibit tumor cell adhesion to laminin reacted with epitopes on the lateral short arms or cross-region of laminin as seen by electron microscopy. These results suggest that a new tumor cell binding domain of laminin may be located close to the cross-region on the long arm of laminin.

  14. An immunogen synthesis strategy for the development of specific anti-deoxynivalenol monoclonal antibodies.

    PubMed

    Sanders, Melanie; Guo, Yirong; Iyer, Abhishek; García, Yara Ruiz; Galvita, Anastasia; Heyerick, Arne; Deforce, Dieter; Risseeuw, Martijn D P; Van Calenbergh, Serge; Bracke, Marc; Eremin, Sergei; Madder, Annemieke; De Saeger, Sarah

    2014-01-01

    An immunogen synthesis strategy was designed to develop anti-deoxynivalenol (DON) monoclonal antibodies with low cross-reactivity against structurally similar trichothecenes. A total of eight different DON immunogens were synthesised, differing in the type and position of the linker on the DON molecule. After immunisation, antisera from mice immunised with different DON immunogens were checked for the presence of relevant antibodies. Then, both homologous and heterologous enzyme-linked immunosorbent assays (ELISAs) were performed for hybridoma screening. Finally, three monoclonal antibodies against DON and its analogues were generated. In addition, monoclonal antibody 13H1 could recognise DON and its analogues in the order of HT-2 toxin > 15-acetyldeoxynivalenol (15-ADON) > DON, with IC₅₀ ranging from 1.14 to 2.13 µg ml⁻¹. Another monoclonal antibody 10H10 manifested relatively close sensitivities to DON, 3-acetyldeoxynivalenol (3-ADON) and 15-ADON, with IC₅₀ values of 22, 15 and 34 ng ml⁻¹, respectively. Using an indirect ELISA format decreases the 10H10 sensitivity to 15-ADON with 92%. A third monoclonal antibody 2A9 showed to be very specific and sensitive to 3-ADON, with IC₅₀ of 0.38 ng ml⁻¹. Using both 2A9 and 10H10 monoclonal antibodies allows determining sole DON contamination.

  15. Solubility Challenges in High Concentration Monoclonal Antibody Formulations: Relationship with Amino Acid Sequence and Intermolecular Interactions.

    PubMed

    Pindrus, Mariya; Shire, Steven J; Kelley, Robert F; Demeule, Barthélemy; Wong, Rita; Xu, Yiren; Yadav, Sandeep

    2015-11-02

    The purpose of this work was to elucidate the molecular interactions leading to monoclonal antibody self-association and precipitation and utilize biophysical measurements to predict solubility behavior at high protein concentration. Two monoclonal antibodies (mAb-G and mAb-R) binding to overlapping epitopes were investigated. Precipitation of mAb-G solutions was most prominent at high ionic strength conditions and demonstrated strong dependence on ionic strength, as well as slight dependence on solution pH. At similar conditions no precipitation was observed for mAb-R solutions. Intermolecular interactions (interaction parameter, kD) related well with high concentration solubility behavior of both antibodies. Upon increasing buffer ionic strength, interactions of mAb-R tended to weaken, while those of mAb-G became more attractive. To investigate the role of amino acid sequence on precipitation behavior, mutants were designed by substituting the CDR of mAb-R into the mAb-G framework (GM-1) or deleting two hydrophobic residues in the CDR of mAb-G (GM-2). No precipitation was observed at high ionic strength for either mutant. The molecular interactions of mutants were similar in magnitude to those of mAb-R. The results suggest that presence of hydrophobic groups in the CDR of mAb-G may be responsible for compromising its solubility at high ionic strength conditions since deleting these residues mitigated the solubility issue.

  16. Rapid quantitative analysis of monoclonal antibody heavy and light chain charge heterogeneity

    PubMed Central

    Vanam, Ram P; Schneider, Michael A; Marlow, Michael S

    2015-01-01

    An alternative method to traditional 2-dimensional gel electrophoresis (2D-PAGE) and its application in characterizing the inherent charge heterogeneity of chromatographically isolated monoclonal antibody heavy and light chains is described. This method, referred to as ChromiCE, utilizes analytical size-exclusion chromatography (SEC), performed under reducing and denaturing conditions, followed by imaged capillary isoelectric focusing (icIEF) of the chromatographically separated heavy and light chains. Under conditions suitable for the subsequent icIEF analysis, the absolute and relative SEC elution volumes of the heavy and light chains were found to be highly pH dependent, a phenomenon that can be exploited in optimizing chromatographic separation. Compared to 2D-PAGE, the ChromiCE method substantially decreases the time and labor needed to complete the analysis, improves reproducibility, and provides fully quantitative assessment of charge heterogeneity. The ChromiCE methodology was applied to a set of diverse monoclonal antibodies to demonstrate suitability for quantitative charge variant analysis of heavy and light chains. A typical application of ChromiCE in extended characterization and stability studies of a purified antibody is shown. PMID:26305772

  17. Protective effect of polyclonal and monoclonal antibodies against abortion in mice infected by Chlamydia psittaci.

    PubMed

    Buzoni-Gatel, D; Bernard, F; Andersen, A; Rodolakis, A

    1990-08-01

    The role of antibody in preventing placental and fetal infection by Chlamydia psittaci was studied in mice. Pregnant mice were passively immunized with polyclonal sera or monoclonal antibodies (mAbs) at day 11 of gestation. The mice were intravenously challenged the following day with the virulent AB7 ovine abortion strain of C. psittaci. Mice were either killed on day 16 of gestation to determine placental and fetal chlamydial infection levels or were permitted to have and raise their young until 8 days old for comparison of survival rates. Immune sera produced a decrease in both placental and fetal infection and reduced the number of young dying in utero or shortly after birth. Polyclonal sera to the highly invasive AB7 and AB4 strains or to the invasive 1B strain were more effective than serum to the invasive AB13 strain. The B577/F3 and B577/A11 monoclonal antibodies gave almost complete protection, with only low levels of placental infection and no detectable fetal infection or decrease in survival rate. The study demonstrates that immune sera and type-specific mAbs can passively transfer resistance to placental and fetal colonization and to abortion and fetal loss in mice intravenously challenged with P. psittaci.

  18. Immunodiagnosis of tumors in vivo using radiolabeled monoclonal antibody A2B5

    SciTech Connect

    Reintgen, D.S.; Shimizu, K.; Coleman, E.; Briner, W.; Kitzmiller, J.; Eisenbarth, G.; Seigler, H.F.

    1983-07-01

    Recently a murine monoclonal antibody (A2B5) has been described that reacts with a membrane associated GQ ganglioside common to peptide secreting normal cells and tumors. In vitro binding data demonstrated the presence of this ganglioside on neurons, adrenal medulla, and pancreatic islets, along with neuroendocrine tumors such as insulinomas, pheochromocytomas, melanomas and neuroblastomas. Negative binding has previously been shown for tissue sections from liver, kidney, colon, lung, stomach, and tumors not derived from the neural crest. Because of the specificity at A2B5 in vitro, this monoclonal antibody was labeled with /sup 131/I for in vivo tumor localization studies. Daily radionuclear scans were obtained in 5 KX rats bearing the radiation induced rat insulinoma with disappearance of the label from the blood pool and concentration in the tumor so that by the fourth day, the only activity present by scan was in the insulinoma. In addition A2B5 also localized to five different human melanoma cells lines grown in nude mice with high tumor/blood levels compared to normal tissues, while no localization is seen in nudes carrying osteosarcomas, colon, bladder, and renal cell carcinomas. In addition antibody A2B5 did not concentrate in any normal tissue though the antigen is present on several. The finding that A2B5 reacts across species lines (mouse, rat, man) lends itself to obvious diagnostic and therapeutic possibilities.

  19. Monoclonal antibodies against soman: Characterization of soman stereoisomers. (Reannouncement with new availability information)

    SciTech Connect

    Lenz, D.E.; Yourick, J.J.; Dawson, J.S.; Scott, J.

    1992-12-31

    Hybridomas were produced which expressed monoclonal anti-soman antibodies as determined by microtiter enzyme-linked-antibody immunoassay (EIA). Each of these antibodies was titrated using a competitive inhibition enzyme immunoassay (CIEIA) with a variety of test ligands. The ligands used included soman (a racemic mixture), sarin, tabun, and each of the four stereoisomers of soman(C+P+, C+P-, C-P+ and C-P-). In all cases the antibodies tested exhibited IC50 values of 10 - 4 - 5 X 10 - 6 M for soman. When sarin or tabun was used as a ligand, the antibodies exhibited no cross reactivity. All of the antibodies cross reacted with the four soman stereoisomers. A second group of hybridomas were produced which expressed monoclonal antibodies against CsPs-soman. These antibodies were used to make preliminary absolute chiral assignments to the four soman stereoisomers. Soman; Antibodies; Stereoisomers; Absolute configuration.

  20. Trial Watch: Tumor-targeting monoclonal antibodies in cancer therapy.

    PubMed

    Vacchelli, Erika; Aranda, Fernando; Eggermont, Alexander; Galon, Jérôme; Sautès-Fridman, Catherine; Zitvogel, Laurence; Kroemer, Guido; Galluzzi, Lorenzo

    2014-01-01

    In 1997, for the first time in history, a monoclonal antibody (mAb), i.e., 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 mAbs that are approved by international regulatory agencies for the treatment of hematopoietic and solid malignancies has not stopped to expand, nowadays encompassing a stunning amount of 15 distinct molecules. This therapeutic armamentarium includes mAbs that target tumor-associated antigens, as well as molecules that interfere with tumor-stroma interactions or exert direct immunostimulatory effects. These three classes of mAbs exert antineoplastic activity via distinct mechanisms, which may or may not involve immune effectors other than the mAbs themselves. In previous issues of OncoImmunology, we provided a brief scientific background to the use of mAbs, all types confounded, in cancer therapy, and discussed the results of recent clinical trials investigating the safety and efficacy of this approach. Here, we focus on mAbs that primarily target malignant cells or their interactions with stromal components, as opposed to mAbs that mediate antineoplastic effects by activating the immune system. In particular, we discuss relevant clinical findings that have been published during the last 13 months as well as clinical trials that have been launched in the same period to investigate the therapeutic profile of hitherto investigational tumor-targeting mAbs.

  1. 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.

  2. Monoclonal Antibodies for Systemic Lupus Erythematosus (SLE) †

    PubMed Central

    Ponticelli, Claudio; Moroni, Gabriella

    2010-01-01

    A number of monoclonal antibodies (mAb) are now under investigation in clinical trials to assess their potential role in Systemic Lupus Erythematosus (SLE). The most frequently used mAb is rituximab, which is directed against CD20, a membrane protein expressed on B lymphocytes. Uncontrolled trials reported an improvement of SLE activity in non-renal patients and other studies even reported an improvement of severe lupus nephritis unresponsive to conventional treatments. However two randomized trials failed to show the superiority of rituximab over conventional treatment in non renal SLE and in lupus nephritis. Preliminary trials reported promising results with epratuzumab, a humanized mAb directed against CD22, and with belimumab, a human mAb that specifically recognizes and inhibits the biological activity of BLyS a cytokine of the tumor-necrosis-factor (TNF) ligand superfamily. Other clinical trials with mAb directed against TNF-alpha, interleukin-10 (Il-10), Il-6, CD154, CD40 ligand, IL-18 or complement component C5 are under way. At present, however, in spite of good results reported by some studies, no firm conclusion on the risk-benefit profile of these mAbs in patients with SLE can be drawn from the available studies. PMID:27713252

  3. Pharmacokinetics of biotech drugs: peptides, proteins and monoclonal antibodies.

    PubMed

    Lin, Jiunn H

    2009-09-01

    With the advances in recombinant DNA biotechnology, molecular biology and immunology, the number of biotech drugs, including peptides, proteins and monoclonal antibodies, available for clinical use has dramatically increased in recent years. Although pharmacokinetic principles are equally applicable to the large molecule drugs and conventional small molecule drugs, the underlying mechanisms for the processes of absorption, distribution, metabolism and excretion (ADME) of large molecule drugs are often very different from that of small molecule drugs. Therefore, a good understanding of the ADME processes of large molecule drugs is essential in support of the development of therapeutic biologics. The purpose of this article is to review the current knowledge of the ADME processes that govern the pharmacokinetics of biotech drugs. The challenges encountered by orally administered peptide and protein drugs, and the nature of lymphatic absorption after subcutaneous administration will be discussed. In addition, molecular mechanisms of biodistribution, metabolism and renal excretion of biotech drugs will also be discussed. Finally, approaches used for prediction of human pharmacokinetics of protein drugs will be briefly discussed.

  4. Guidelines to cell engineering for monoclonal antibody production.

    PubMed

    Rita Costa, A; Elisa Rodrigues, M; Henriques, Mariana; Azeredo, Joana; Oliveira, Rosário

    2010-02-01

    Monoclonal antibodies (mAbs) are currently used for many diagnostic and therapeutic applications. The high demand for these biopharmaceuticals has led to the development of large-scale manufacturing processes, with productivity improvements being mainly achieved by optimization of bioreactor systems. However, more recently, the early steps of production, previous to bioreactor culture, have been presented as alternative areas where productivity enhancements can be achieved. Thus, this review describes the progress made for the improvement of productivity in mammalian expression systems for the high production of mAbs. Advances in the development of mAb-producing cell lines are being made, particularly regarding expression vector design and methods used for transfection, with the intent to create a reproducible methodology. Selection of the most suitable clones is also a critical step that can be improved, by including variables other than the expression level, which is still the common practice. Furthermore, strategies of cell engineering, although still mostly based on trial-and-error experimentation and not in standard protocols, hold great interest to improve cell growth and productivity, as well as product quality in the future. Improvements of the initial steps of the production process would not only result in cells with higher expression ability, but would also speed-up the process development.

  5. 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.

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

    PubMed

    Liu, Shuying; Wang, Shixia; Lu, Shan

    2016-04-06

    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.

  7. Establishment of a novel monoclonal antibody against LGR5.

    PubMed

    Sasaki, Yuka; Kosaka, Hiromichi; Usami, Katsuaki; Toki, Hiroe; Kawai, Hironori; Shiraishi, Norihiko; Ota, Toshio; Nakamura, Kazuyasu; Furuya, Akiko; Satoh, Mitsuo; Hasegawa, Kazumasa; Masuda, Kazuhiro

    2010-04-09

    LGR5 is an orphan G-protein-coupled receptor (GPCR) that is expressed on the cell surface membrane. LGR5 is reported to be overexpressed in colon, liver, and ovary tumor compared to normal tissue. However, a specific ligand for LGR5 has not yet been determined, and the function is still not clear. An LGR5-specific monoclonal antibody (mAb) is needed as a tool for detection and analysis of LGR5 biological function and cancer therapy. To date, no mAb against LGR5 that retains high affinity and specificity has been reported. Here, we report successful establishment and characterization of a mAb (KM4056) that specifically recognizes the extracellular N-terminal domain of human LGR5, but not LGR4 or LGR6. This mAb has potent complement-dependent cytotoxicity (CDC) activity in vitro and shows strong anti-tumor activity in vivo against xenograft model by transplanting LGR5 expressing CHO transfectants into SCID mice. Thus, KM4056 can be a useful tool for detection of LGR5 positive cells and analysis of LGR5 biological function.

  8. Downstream processing of monoclonal antibodies--application of platform approaches.

    PubMed

    Shukla, Abhinav A; Hubbard, Brian; Tressel, Tim; Guhan, Sam; Low, Duncan

    2007-03-15

    This paper presents an overview of large-scale downstream processing of monoclonal antibodies and Fc fusion proteins (mAbs). This therapeutic modality has become increasingly important with the recent approval of several drugs from this product class for a range of critical illnesses. Taking advantage of the biochemical similarities in this product class, several templated purification schemes have emerged in the literature. In our experience, significant biochemical differences and the variety of challenges to downstream purification make the use of a completely generic downstream process impractical. Here, we describe the key elements of a flexible, generic downstream process platform for mAbs that we have adopted at Amgen. This platform consists of a well-defined sequence of unit operations with most operating parameters being pre-defined and a small subset of parameters requiring development effort. The platform hinges on the successful use of Protein A chromatography as a highly selective capture step for the process. Key elements of each type of unit operation are discussed along with data from 14 mAbs that have undergone process development. Aspects that can be readily templated as well as those that require focused development effort are identified for each unit operation. A brief description of process characterization and validation activities for these molecules is also provided. Finally, future directions in mAb processing are summarized.

  9. 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

  10. 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.

  11. 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

  12. [Study of plant lectins from Viscum album using monoclonal antibodies].

    PubMed

    Tonevitskiĭ, A G; Rakhmanova, V A; Shamshiev, A T; Usachaeva, E A; Agapov, I I; Prokov'ev, S A; Denisenko, O N; Pfuller, U; Eifler, R

    1995-01-01

    Monoclonal antibodies (monAT) against both native (TA5, TB12) and denatured (TB33, TB35) plant toxin ML1 from Viscum album have been obtained. The interaction of monAT against native toxin with its isoforms ML2 and ML3 was investigated. It was shown that monAT TA5 to A-chain of ML1 toxin cross-reacted with ML2 and ML3 isoforms. TA5 did not inhibit enzyme activity of A-chain in cell-free rabbit reticulocyte system. It was shown that monAT TB12 reacted with galactose-binding site of B-subunit. Both monAT had no cross-reactions with plant toxin ricin. The binding constants for TA5 with ML1, ML2, ML3 respectively were 4.3.10(7) M-1, 1.2.10(7) M-1, and 0.3.10(7) M-1. The binding constants for TB12 were 2.10(7) M-1 with ML1 toxin, and more than 10(6) M-1 with ML2 and ML3. The nature of heterogeneity in ML toxin family is discussed. Test-systems for ML1 determination in different V. album extracts are suggested.

  13. 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

  14. Anti-ICOS Monoclonal Antibody Treatment of Canine Chronic GVHD.

    PubMed

    Graves, Scott S; Parker, Maura H; Stone, Diane; Sale, George E; Pillai, Smitha P S; Johnson, Melissa M; Storb, Rainer

    2017-09-25

    In murine model systems, inducible costimulator (ICOS) signaling has been implicated in the formation of chronic graft-versus-host disease (GVHD). Previously, we showed that chronic GVHD can be reproducibly produced in the dog hematopoietic cell transplantation (HCT) model and that ICOS expression is upregulated on T cells in dogs with chronic GVHD. The goal of the present study was to determine whether administration of a short course of anti-canine ICOS monoclonal antibody (mAb) could alter the rapid and progressive course of chronic GVHD. Five dogs underwent HCT from dog leukocyte antigen mismatched unrelated donors following total body irradiation. Post-grafting immunosuppression consisted of methotrexate (days 1, 3, 6, and 11) and cyclosporine (days -1 through 78). Anti-ICOS mAb (3 injections, 72 hours apart) was administered upon diagnosis of GVHD. One dog failed to respond to anti-ICOS mAb therapy and succumbed to chronic GVHD in a time course similar to control untreated dogs. Overall, anti-ICOS-treated dogs experienced a significant prolongation in survival from the time of diagnosis of chronic GVHD compared to control dogs. Within the limitations of the number of study dogs, we suggest that a short course of anti-ICOS mAb may be useful in the treatment of chronic canine GVHD. Copyright © 2017. Published by Elsevier Inc.

  15. Kinetics of Monoclonal Antibody Aggregation from Dilute toward Concentrated Conditions.

    PubMed

    Nicoud, Lucrèce; Jagielski, Jakub; Pfister, David; Lazzari, Stefano; Massant, Jan; Lattuada, Marco; Morbidelli, Massimo

    2016-04-07

    Gaining understanding on the aggregation behavior of proteins under concentrated conditions is of both fundamental and industrial relevance. Here, we study the aggregation kinetics of a model monoclonal antibody (mAb) under thermal stress over a wide range of protein concentrations in various buffer solutions. We follow experimentally the monomer depletion and the aggregate growth by size exclusion chromatography with inline light scattering. We describe the experimental results in the frame of a kinetic model based on population balance equations, which allows one to discriminate the contributions of the conformational and of the colloidal stabilities to the global aggregation rate. Finally, we propose an expression for the aggregation rate constant, which accounts for solution viscosity, protein-protein interactions, as well as aggregate compactness. All these effects can be quantified by light scattering techniques. It is found that the model describes well the experimental data under dilute conditions. Under concentrated conditions, good model predictions are obtained when the solution pH is far below the isoelectric point (pI) of the mAb. However, peculiar effects arise when the solution pH is increased toward the mAb pI, and possible explanations are discussed.

  16. Role of cosolutes in the aggregation kinetics of monoclonal antibodies.

    PubMed

    Nicoud, Lucrèce; Sozo, Margaux; Arosio, Paolo; Yates, Andrew; Norrant, Edith; Morbidelli, Massimo

    2014-10-16

    We propose a general strategy based on kinetic analysis to investigate how cosolutes affect the aggregation behavior of therapeutic proteins. We apply this approach to study the impact of NaCl and sorbitol on the aggregation kinetics of two monoclonal antibodies, an IgG1 and an IgG2. By using a combination of size exclusion chromatography and light scattering techniques, we study the impact of the cosolutes on the monomer depletion, as well as on the formation of dimers, trimers, and larger aggregates. We analyze these macroscopic effects in the frame of a kinetic model based on Smoluchowski's population balance equations modified to account for nucleation events. By comparing experimental data with model simulations, we discriminate the effect of cosolutes on the elementary steps which contribute to the global aggregation process. In the case of the IgG1, it is found that NaCl accelerates the kinetics of aggregation by promoting specifically aggregation events, while sorbitol delays the kinetics of aggregation by specifically inhibiting protein unfolding. In the case of the IgG2, whose monomer depletion kinetics is limited by dimer formation, NaCl and sorbitol are found respectively to accelerate and inhibit conformational changes and aggregation events to the same extent.

  17. 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

  18. Anti-CD20 monoclonal antibodies: beyond B-cells.

    PubMed

    Avivi, Irit; Stroopinsky, Dina; Katz, Tamar

    2013-09-01

    Anti-CD20 monoclonal antibodies (MoAbs), employed in treating CD20⁺ lymphomas and autoimmune diseases, appear to have broader functions than just eradicating malignant B-cells and decreasing autoantibody production. Rituximab-induced T-cell inactivation, reported both in-vitro and in-vivo, may contribute to the increased risk of T-cell-dependent infections, observed in patients receiving this therapy. T-cell polarization into a suppressive phenotype, often observed in patients receiving rituximab for autoimmune disorders, was reported to be associated with prolonged remissions. Elimination of B-cells serving as antigen-presenting cells, thereby causing impaired T-cell activation, could play a significant role in induction of these changes. Direct binding of rituximab to a CD20dim T-cell population, inducing its depletion, may contribute to the decreased T-cell activation following rituximab therapy. Further investigation of the complex network through which rituximab and new anti-CD20 MoAbs act, would advance the employment of these agents in different clinical settings.

  19. 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

  20. Production and characterization of a monoclonal antibody to Campylobacter jejuni.

    PubMed

    Heo, S A; Nannapaneni, R; Johnson, M G; Park, J S; Seo, K H

    2009-04-01

    Campylobacter species are a group of spiral-shaped bacteria that can cause disease in humans and animals. We developed a high-affinity monoclonal antibody (MAb) probe that recognizes Campylobacter jejuni cells. Cell suspensions grown under microaerobic conditions at 42 degrees C for 20 h on Bolton agar plates with lysed horse blood were used as live and heat-killed preparations, centrifuged at 8,000 x g for 20 min, and resuspended in carbonate buffer (pH 9.6) for coating on the enzyme-linked immunosorbent assay plates. BALB/c mice were immunized with C. jejuni sonicated cells at 10(7) CFU/ml to generate MAb-producing hybridoma clones. Of about 500 initial hybridoma clones, MAb 33D2, which reacted with C. jejuni and Campylobacter coli, was selected for further evaluation. MAb 33D2 is in the immunoglobulin subclass G2a and had relatively weaker reactivity with the C. coli strains tested. MAb 33D2 did not show any cross-reactions with the nine non-Campylobacter bacteria tested in the enzyme-linked immunosorbent assay and had a stronger affinity for C. jejuni as live versus heat-killed cells. In Western blot assays, MAb 33D2 recognized two major antigens of 62 and 43 kDa in extracts from C. jejuni cells but only one antigen of 62 kDa in extracts from C. coli cells.

  1. 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

  2. Kinase inhibitors and monoclonal antibodies in oncology: clinical implications.

    PubMed

    Gharwan, Helen; Groninger, Hunter

    2016-04-01

    Molecularly targeted cancer therapies, such as small-molecule kinase inhibitors and monoclonal antibodies, constitute a rapidly growing and an important part of the oncology armamentarium. Unlike conventional (cytotoxic) chemotherapeutics, targeted therapies were designed to disrupt cancer cell pathogenesis at specific biological points essential for the development and progression of the tumour. These agents were developed to disrupt specific targets with the aim of minimizing treatment burden compared with conventional chemotherapy. Nevertheless the increasingly common use of targeted therapies has revealed some unanticipated, often clinically significant toxic effects, as well as compromising effective palliative and end-of-life management approaches. Although patients and clinicians welcome improvements in cancer prognosis, these changes can also impact patient quality-of-life. Therefore, as demand for oncology expertise increases, physicians need to apprise themselves of targeted therapies and their clinical implications, including drug-specific side effects, impact on quality of life, and cost issues, especially in relation to end-of-life care. This Review provides a useful summary and guide for professionals treating patients with malignant diseases.

  3. Development of a monoclonal antibody specific to cooked mammalian meats.

    PubMed

    Hsieh, Y H; Sheu, S C; Bridgman, R C

    1998-04-01

    Detection of species adulteration in ground meat products is important for consumer protection and food-labeling law enforcement. This study was conducted to develop monoclonal antibodies (MAbs) that can be used in an enzyme-linked immunosorbent assay (ELISA) for rapid detection of any cooked mammalian meats in cooked poultry products. Soluble muscle proteins extracted from cooked pork (heated at 100 degrees C for 15 min) were used as the antigen to immunized mice for developing the MAb. One that was developed, MAb 2F8 (IgG2b class), strongly reacted with cooked meat of five mammalian species (beef cattle, hogs, sheep, horse, and deer) but did not react with any cooked poultry (chicken, turkey, and duck) or raw meats. At least 0.5% by weight of pork, beef, lamb, and horse meats in a chicken-based mixture could not detect using the indirect ELISA with MAb 2F8. The MAb 2F8 is useful in a single initial screening test to detect the presence of five nonpoultry meat adulterants in cooked poultry products.

  4. 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

  5. Monoclonal Antibodies for the Treatment of Myeloma: Targeting SLAMF7 and CD38.

    PubMed

    Lonial, Sagar

    2016-01-01

    The recent explosion of immune-based treatments for cancer has significantly impacted remission durations and overall survival for many diseases. Multiple myeloma is no exception to this trend, with several immune-based treatments including checkpoint blockade, cellular therapy, and most advanced now antibody-based treatment coming to fruition. While the use of monoclonal antibodies has been a significant interest in myeloma for some time, identifying the ideal target has been an issue. Given the dependence of plasma cells on interleukin 6 signaling for survival and proliferation, there were several trials testing both single agent and combination therapy effects of anti-interleukin 6 antibodies, which did not demonstrate significant clinical activity; however, more recent antibodies targeting receptors such as CD38 and SLAMF7 (previously known as CS1) are demonstrating significant clinical benefit. In this article, we briefly review the preclinical and clinical data surrounding these 2 important targets and the antibodies that clinically will be used as therapeutic agents in the context of multiple myeloma.

  6. HybriFree: a robust and rapid method for the development of monoclonal antibodies from different host species.

    PubMed

    Kivi, Gaily; Teesalu, Kaupo; Parik, Jüri; Kontkar, Elen; Ustav, Mart; Noodla, Liis; Ustav, Mart; Männik, Andres

    2016-01-08

    The production of recombinant monoclonal antibodies in mammalian cell culture is of high priority in research and medical fields. A critical step in this process is the isolation of the antigen-binding domain sequences of antibodies possessing the desired properties. Many different techniques have been described to achieve this goal, but all have shortcomings; most techniques have problems with robustness, are time-consuming and costly, or have complications in the transfer from isolation to production phase. Here, we report a novel HybriFree technology for the development of monoclonal antibodies from different species that is robust, rapid, inexpensive and flexible and can be used for the subsequent production of antibodies in mammalian cell factories. HybriFree technology is illustrated herein via detailed examples of isolating mouse, rabbit and chicken monoclonal antibody sequences from immunized animals. Starting from crude spleen samples, antigen capturing of specific B-cells is performed initially. cDNA of antibody variable domains is amplified from the captured cells and used a source material for simple and rapid restriction/ligation free cloning of expression vector library in order to produce scFv-Fc or intact IgG antibodies. The vectors can be directly used for screening purposes as well as for the subsequent production of the developed monoclonal antibodies in mammalian cell culture. The antibodies isolated by the method have been shown to be functional in different immunoassays, including ELISA, immunofluorescence and Western blot. In addition, we demonstrate that by using a modified method including a negative selection step, we can isolate specific antibodies targeting the desired epitope and eliminate antibodies directed to undesired off-targets. HybriFree can be used for the reliable development of monoclonal antibodies and their subsequent production in mammalian cells. This simple protocol requires neither the culturing of B-cells nor single

  7. 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.

  8. Comparative testing of monoclonal antibodies against Plasmodium falciparum sporozoites for ELISA development*

    PubMed Central

    Wirtz, R. A.; Zavala, F.; Charoenvit, Y.; Campbell, G. H.; Burkot, T. R.; Schneider, I.; Esser, K. M.; Beaudoin, R. L.; Andre, R. G.

    1987-01-01

    Ten monoclonal antibodies developed against Plasmodium falciparum sporozoites at four institutions were evaluated for use in an enzyme-linked immunosorbent assay (ELISA). Four of the antibodies were eliminated because of their low sensitivity or requirement for high concentrations of capture antibody, while an additional four were rejected because they exhibited cross-reactivity with P. berghei sporozoites. Of the two remaining monoclonal antibodies, that designated 2A10 had the highest sensitivity, a requirement for lower concentrations of capture antibody, and had been tested successfully against sporozoites from a wider range of geographical areas than the others. Use of this monoclonal antibody in a standardized ELISA method gave a test ten times more sensitive than previously reported for P. falciparum sporozoites and its detection limit was less than 100 sporozoites per mosquito. PMID:3555879

  9. 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.

  10. High-dose monoclonal antibodies via the subcutaneous route: challenges and technical solutions, an industry perspective.

    PubMed

    Narasimhan, Chakravarthy; Mach, Henryk; Shameem, Mohammed

    2012-07-01

    This review summarizes the various challenges in product development involved in subcutaneous administration of high-dose monoclonal antibodies and attempts to provide an industry perspective of some of the available technologies and potential avenues to overcome these challenges.

  11. Antigenic characterization of human hepatocellular carcinoma. Development of in vitro and in vivo immunoassays that use monoclonal antibodies.

    PubMed Central

    Carlson, R I; Ben-Porath, E; Shouval, D; Strauss, W; Isselbacher, K J; Wands, J R

    1985-01-01

    Several libraries of monoclonal antibodies have been produced by immunization of Balb/c mice with single cell suspensions of nontrypsin-treated human hepatocellular carcinoma cell (HCC) lines in order to study the antigenic properties of transformed hepatocytes. The antibodies were characterized with regards to specificity for hepatoma-associated antigens and their capability for use as reagents in radioimmunoassays (RIAs) and tumor localization in vivo. Three such antibodies namely, P215457, PM4E9917, P232524 of the IgG2a, IgG2a, and IgG1 isotypes, respectively, not only recognized separate and distinct antigenic determinants on four human hepatoma cell lines but also reacted with epitopes present on chemically induced rat hepatoma cell lines. In contrast, only 1 of 38 other human malignant and transformed cell lines demonstrated reactivity with the three antibodies; normal human tissues were also found to be unreactive. Monoclonal antibody P215457 densely stained the plasma membrane by indirect immunofluorescence, showed rapid binding activity to HCC cells in suspension, and precipitated a 50,000-mol wt cell surface protein; antibody PM4E9917 also stained the plasma membrane and precipitated a 65,000-mol wt protein, whereas P232534 recognized cytoplasmic antigenic determinants. With these antibodies "simultaneous sandwich" RIAs were established that detect soluble hepatoma-associated antigens in culture supernatants. Finally, the Fab fragment of P215457 was found to be useful in tumor localization in vivo. This antibody fragment when labeled with 131I was shown to localize by radionuclide-imaging studies in human hepatoma grown in nude mice. Thus, these investigations demonstrate that monoclonal antibodies may be produced against epitopes that reside almost exclusively on transformed hepatocytes and such antibodies may be successfully employed in the development of in vitro and in vivo immunoassays. Images PMID:2991342

  12. In vitro inhibition of Cryptosporidium parvum infection by human monoclonal antibodies.

    PubMed Central

    Elliot, B C; Wisnewski, A V; Johnson, J; Fenwick-Smith, D; Wiest, P; Hamer, D; Kresina, T; Flanigan, T P

    1997-01-01

    Cryptosporidium parvum infection of the small epithelial intestine causes unremitting diarrhea and malabsorption that can lead to chronic and sometimes fatal illness in patients with AIDS. The illness may be ameliorated by passive oral immunoglobulin therapy. The objective of this study was to produce anti-Cryptosporidium human monoclonal antibodies for evaluation as potential therapy. All human monoclonal cell lines that produced C. parvum antibodies were originally generated from the peripheral blood lymphocytes of a human immunodeficiency virus-seronegative woman. She had recovered from C. parvum infection and had a high specific antibody titer. Hybridization of these lymphocytes with a tumor cell line was accomplished by hypo-osmolar electrofusion. Twelve clones were identified by enzyme-