WorldWideScience

Sample records for monoclonal antibody developed

  1. Recent developments in monoclonal antibody radiolabeling techniques

    Energy Technology Data Exchange (ETDEWEB)

    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. Drug Development of Therapeutic Monoclonal Antibodies.

    Science.gov (United States)

    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.

  3. Development of syngeneic monoclonal anti-idiotype antibodies to mouse monoclonal anti-asialoglycoprotein receptor antibody.

    Directory of Open Access Journals (Sweden)

    Hirai M

    2002-06-01

    Full Text Available Anti-idiotype antibodies (Ab2 play an important role in the homeostasis of immune responses and are related to the development and the disease activity of certain autoimmune diseases. The asialoglycoprotein receptor (ASGPR is considered one of the target antigens in the pathogenesis of autoimmune chronic active hepatitis (AIH. We previously developed a mouse monoclonal antibody (clone 8D7 which recognizes rat and human ASGPR. In this study, to help investigate the anti-ASGPR antibody-anti-idiotype antibody network in patients with AIH, we developed a syngeneic mouse monoclonal Ab2 to the 8D7 anti-ASGPR antibody (Ab1. One clone, designated as 3C8, tested positive for specific reactivity to 8D7-Ab1 and did not bind to other irrelevant immunoglobulins. By competitive inhibition assays, the binding of 8D7-Ab1 to liver membrane extracts, i.e., the crude antigen preparation, was inhibited by 3C8-Ab2 in a dose-dependent manner, and the binding of 8D7-Ab1 to 3C8-Ab2 was inhibited by the liver membrane extracts. In the immunohistochemical analysis, 3C8-Ab2 blocked the specific staining of sinusoidal margins of rat hepatocytes by 8D7-Ab1. These results suggest that 3C8 anti-idiotype antibody recognizes the specific idiotypic determinants within the antigen-binding site of 8D7-Ab1.

  4. Development of monoclonal antibodies that recognize Treponema pallidum.

    OpenAIRE

    Saunders, J M; Folds, J D

    1983-01-01

    We developed a panel of monoclonal antibodies to Treponema pallidum (Nichols) antigens, some of which recognize treponemal antigens on T. pallidum (Nichols), T. pallidum strain 14, and Treponema phagedenis biotype Reiter. The antibodies were detected by either an enzyme-linked immunosorbent assay or a radioimmunoassay.

  5. Monoclonal antibodies in myeloma

    DEFF Research Database (Denmark)

    Sondergeld, P.; van de Donk, N. W. C. J.; Richardson, P. G.;

    2015-01-01

    The development of monoclonal antibodies (mAbs) for the treatment of disease goes back to the vision of Paul Ehrlich in the late 19th century; however, the first successful treatment with a mAb was not until 1982, in a lymphoma patient. In multiple myeloma, mAbs are a very recent and exciting add...

  6. Development of Biodegradable Nanocarriers Loaded with a Monoclonal Antibody

    Directory of Open Access Journals (Sweden)

    Andrew Gdowski

    2015-02-01

    Full Text Available Treatments utilizing monoclonal antibody therapeutics against intracellular protein-protein interactions in cancer cells have been hampered by several factors, including poor intracellular uptake and rapid lysosomal degradation. Our current work examines the feasibility of encapsulating monoclonal antibodies within poly(lactic-co-glycolic acid (PLGA nanoparticles using a water/oil/water double emulsion solvent evaporation technique. This method can be used to prepare protective polymeric nanoparticles for transporting functional antibodies to the cytoplasmic compartment of cancer cells. Nanoparticles were formulated and then characterized using a number of physical and biological parameters. The average nanoparticle size ranged from 221 to 252 nm with a low polydispersity index. Encapsulation efficiency of 16%–22% and antibody loading of 0.3%–1.12% were observed. The antibody molecules were released from the nanoparticles in a sustained manner and upon release maintained functionality. Our studies achieved successful formulation of antibody loaded polymeric nanoparticles, thus indicating that a PLGA-based antibody nanoformulation is a promising intracellular delivery vehicle for a large number of new intracellular antibody targets in cancer cells.

  7. Development and Evaluation of Monoclonal Antibodies for Paxilline

    Directory of Open Access Journals (Sweden)

    Chris M. Maragos

    2015-09-01

    Full Text Available 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.

  8. Therapeutic Recombinant Monoclonal Antibodies

    Science.gov (United States)

    Bakhtiar, Ray

    2012-01-01

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

  9. Therapeutic Recombinant Monoclonal Antibodies

    Science.gov (United States)

    Bakhtiar, Ray

    2012-01-01

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

  10. Monoclonal antibody "gold rush".

    Science.gov (United States)

    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.

  11. Heterogeneity of monoclonal antibodies.

    Science.gov (United States)

    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.

  12. Development and monitoring of a novel monoclonal antibody purification strategy

    OpenAIRE

    Capito, Florian

    2014-01-01

    The studies presented in the cumulative part of this thesis illustrate the different steps to develop a polymer-driven antibody purification process. These peer-reviewed reports show in detail fundamental research, additional method development useful in the development of such a purification process as well as implementation of the final process. A strategy for analyzing copolymers, synthesized by a lab in house, was implemented with particular emphasis on copolymer composition analysis. Thi...

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

    Science.gov (United States)

    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

  14. Advances in monoclonal antibody application in myocarditis

    Institute of Scientific and Technical Information of China (English)

    Li-na HAN; Shuang HE; Yu-tang WANG; Li-ming YANG; Si-yu LIU; Ting ZHANG

    2013-01-01

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

  15. Development of a Highly Protective Combination Monoclonal Antibody Therapy against Chikungunya Virus

    NARCIS (Netherlands)

    Pal, Pankaj; Dowd, Kimberly A.; Brien, James D.; Edeling, Melissa A.; Gorlatov, Sergey; Johnson, Syd; Lee, Iris; Akahata, Wataru; Nabel, Gary J.; Richter, Mareike K. S.; Smit, Jolanda M.; Fremont, Daved H.; Pierson, Theodore C.; Heise, Mark T.; Diamond, Michael S.

    2013-01-01

    Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes global epidemics of a debilitating polyarthritis in humans. As there is a pressing need for the development of therapeutic agents, we screened 230 new mouse anti-CHIKV monoclonal antibodies (MAbs) for their ability to inhibit

  16. Introduction to the application of QbD principles for the development of monoclonal antibodies.

    Science.gov (United States)

    Finkler, Christof; Krummen, Lynne

    2016-09-01

    Quality by Design (QbD) is a global regulatory initiative with the goal of enhancing pharmaceutical development through the proactive design of pharmaceutical manufacturing process and controls to consistently deliver the intended performance of the product. The principles of pharmaceutical development relevant to QbD are described in the ICH guidance documents (ICHQ8-11). An integrated set of risk assessments and their related elements developed at Roche/Genentech were designed to provide an overview of product and process knowledge for the production of a recombinant monoclonal antibody. This chapter introduces a publication series on the application of Quality by Design for biopharmaceuticals, with a focus on the development of recombinant monoclonal antibodies. The development of and overview on the QbD concept applied by Roche and Genentech is described and essential QbD elements are presented.

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

    OpenAIRE

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

    1986-01-01

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

  18. Development and characterization of monoclonal antibodies to Marek's disease tumor-associated surface antigen.

    OpenAIRE

    Liu, X. F.; Lee, L F

    1983-01-01

    Four monoclonal antibodies, A35, B94, EB29, and G152, against Marek's disease tumor-associated surface antigen have been developed and their specificities studied against a panel of Marek's disease and lymphoid leukosis primary tumors; Marek's disease, and lymphoid leukosis, and reticuloendotheliosis lymphoblastoid cell lines; and normal chicken cells. A35 and G152 are of the immunoglobulin M class, and B94 and EB29 are of the immunoglobulin G1 subclass.

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

    Science.gov (United States)

    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. Development of a bispecific monoclonal antibody to pesticide carbofuran and triazophos using hybrid hybridomas.

    Science.gov (United States)

    Jin, R Y; Guo, Y R; Wang, C M; Wu, J X; Zhu, G N

    2009-01-01

    A mouse hybrid hybridoma (tetradoma) was derived from fusing hybridomas producing monoclonal antibody to N-methylcarbamate pesticide carbofuran with hybridomas producing monoclonal antibody to organophosphorus pesticide Triazophos. The prepared tetradoma line (12C1 to 2H12) secreted hybrid immunoglobulin exhibiting parental and bispecific binding characteristics. The effect of relevant physicochemical factors on the immunoassay based on the 12C1 to 2H12 bispecific monoclonal antibody had been studied to optimize the ELISA performance. The developed immunoassay showed that the detection limit (I(20)) were 0.36 and 1.89 ng/mL for triazophos and carbofuran, respectively, without obvious cross-reactivity to other related compounds. Water samples spiked with triazophos at 0.5, 1, and 5 ng/mL or carbofuran at 5, 10, and 20 ng/mL were directly analyzed by the developed ELISA format. The mean recovery of triazophos and carbofuran were 108.1% and 107.5%, with variation coefficient of 15.9% and 17.7%, respectively.

  1. Monoclonal antibodies in chronic lymphocytic leukemia.

    Science.gov (United States)

    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.

  2. Biosimilar monoclonal antibodies: preclinical and clinical development aspects.

    Science.gov (United States)

    Gonçalves, João; Araújo, Filipe; Cutolo, Maurizio; Fonseca, João Eurico

    2016-01-01

    Biological drugs and their originated biosimilars are large, highly complex molecules derived from living cells or organisms. Traditional medicines, by contrast, are usually simple molecules of low molecular weight, synthesised by chemical means. The distinct complexities and methods of manufacture create an important difference between biosimilars and conventional generic drugs: while chemical generics can be fully characterised as identical to the originator product, biosimilars cannot. In addition, biological therapies are inherently variable, creating unavoidable differences between even subsequent batches of the same product. An expiring patent does not necessarily mean that the manufacturing process of the originator product becomes available to the biosimilar developers (for instance, the relevant cell line clone and growth medium). Therefore, it cannot be guaranteed that biosimilar products are identical to their reference product on a molecular level. This difference has important implications for the regulation and licensing of biosimilars. While conventional generic drugs require only a limited comparison and demonstration of identical chemical structure to the reference product, biosimilars require far more rigorous testing. In general, there must be a thorough comparison of structural and functional characteristics between biosimilar and originator drug. Stepwise nonclinical in vitro and in vivo approaches are recommended to evaluate the similarity of both drugs and any identified micro-heterogeneities must then be assessed for their impact on safety and clinical performance. Subsequently, clinical pharmacokinetic (PK) studies need to be performed in order to demonstrate a similar PK profile, prior to conducting clinical efficacy trials.

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

    Directory of Open Access Journals (Sweden)

    Seiji Iwasa

    2012-11-01

    Full Text Available Enzyme-linked immunosorbent assay (ELISA based on a monoclonal antibody (MoAb was developed for the neonicotinoid insecticide clothianidin. A new clothianidin hapten (3-[5-(3-methyl-2-nitroguanidinomethyl-1,3-thiazol-2-ylthio] propionic acid was synthesized and conjugated to keyhole limpet hemocyanin, and was used for monoclonal antibody preparation. The resulting MoAb CTN-16A3-13 was characterized by a direct competitive ELISA (dc-ELISA. The 50% of inhibition concentration value with clothianidin was 4.4 ng/mL, and the working range was 1.5–15 ng/mL. The antibody showed high cross-reactivity (64% to dinotefuran among the structurally related neonicotinoid insecticides. The recovery examinations of clothianidin for cucumber, tomato and apple showed highly agreement with the spiked concentrations; the recovery rate was between 104% and 124% and the coefficient of variation value was between 1.8% and 15%. Although the recovery rate of the dc-ELISA was slightly higher than that of HPLC analysis, the difference was small enough to accept the dc-ELISA as a useful method for residue analysis of clothianidin in garden crops.

  4. Fragmentation of monoclonal antibodies

    Science.gov (United States)

    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

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

    DEFF Research Database (Denmark)

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

    2017-01-01

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

  6. Development, characterization and application of monoclonal antibodies against Brazilian Dengue virus isolates.

    Directory of Open Access Journals (Sweden)

    Camila Zanluca

    Full Text Available Dengue is the most prevalent human arboviral disease. The morbidity related to dengue infection supports the need for an early, quick and effective diagnostic test. Brazil is a hotspot for dengue, but no serological diagnostic test has been produced using Brazilian dengue virus isolates. This study aims to improve the development of immunodiagnostic methods for dengue virus (DENV detection through the production and characterization of 22 monoclonal antibodies (mAbs against Brazilian isolates of DENV-1, -2 and -3. The mAbs include IgG2bκ, IgG2aκ and IgG1κ isotypes, and most were raised against the envelope or the pre-membrane proteins of DENV. When the antibodies were tested against the four DENV serotypes, different reactivity patterns were identified: group-specific, subcomplex specific (DENV-1, -3 and -4 and DENV-2 and -3 and dengue serotype-specific (DENV-2 or -3. Additionally, some mAbs cross-reacted with yellow fever virus (YFV, West Nile virus (WNV and Saint Louis encephalitis virus (SLEV. None of the mAbs recognized the alphavirus Venezuelan equine encephalitis virus (VEEV. Furthermore, mAbs D3 424/8G, D1 606/A12/B9 and D1 695/12C/2H were used to develop a capture enzyme-linked immunosorbent assay (ELISA for anti-dengue IgM detection in sera from patients with acute dengue. To our knowledge, these are the first monoclonal antibodies raised against Brazilian DENV isolates, and they may be of special interest in the development of diagnostic assays, as well as for basic research.

  7. Monoclonal antibodies reactive with hairy cell leukemia

    NARCIS (Netherlands)

    Visser, L; Shaw, A; Slupsky, J; Vos, H; Poppema, S

    Monoclonal antibodies reactive with hairy cell leukemia were developed to aid in the diagnosis of this subtype of B cell chronic lymphocytic leukemia and to gain better insight into the origin of hairy cells. Three antibodies were found to be of value in the diagnosis of hairy cell leukemia.

  8. Monoclonal antibodies reactive with hairy cell leukemia

    NARCIS (Netherlands)

    Visser, L; Shaw, A; Slupsky, J; Vos, H; Poppema, S

    1989-01-01

    Monoclonal antibodies reactive with hairy cell leukemia were developed to aid in the diagnosis of this subtype of B cell chronic lymphocytic leukemia and to gain better insight into the origin of hairy cells. Three antibodies were found to be of value in the diagnosis of hairy cell leukemia. Antibod

  9. OBTAINING OF MONOCLONAL ANTIBODIES AGAINST CHOLERA TOXIN AND HEAT LABILE ENTEROTOXIN OF E. coli FOR DEVELOPMENT OF THE TOXINS DIPLEX ANALYSIS IN ENVIRONMENTAL SPECIMENS

    OpenAIRE

    Eu. V. Grishin; T. I. Valiakina

    2013-01-01

    The present study focuses on development of monoclonal antibodies (MAbs) which specifically interact with cholera toxin or heat labile enterotoxin of E. coli. Such monoclonal antibodies MAbs are possessed of ability to identify cholera toxin or heat labile enterotoxin in different immunochemical assays. We obtained hybridoma clones which produced monoclonal antibodies of IgG isotypes to cholera toxin and heat labile enterotoxin. On application of the method of serial dilutions we selected the...

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

    Directory of Open Access Journals (Sweden)

    Phelps Amanda L

    2009-11-01

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

  11. Biotechnology and genetic engineering in the new drug development. Part II. Monoclonal antibodies, modern vaccines and gene therapy.

    Science.gov (United States)

    Stryjewska, Agnieszka; Kiepura, Katarzyna; Librowski, Tadeusz; Lochyński, Stanisław

    2013-01-01

    Monoclonal antibodies, modern vaccines and gene therapy have become a major field in modern biotechnology, especially in the area of human health and fascinating developments achieved in the past decades are impressive examples of an interdisciplinary interplay between medicine, biology and engineering. Among the classical products from cells one can find viral vaccines, monoclonal antibodies, and interferons, as well as recombinant therapeutic proteins. Gene therapy opens up challenging new areas. In this review, a definitions of these processes are given and fields of application and products, as well as the future prospects, are discussed.

  12. Development and Characterization of a Monoclonal Antibody against Ochratoxin B and Its Application in ELISA

    Directory of Open Access Journals (Sweden)

    Alexandra H. Heussner

    2010-06-01

    Full Text Available A monoclonal antibody specific to ochratoxin B (OTB was employed for the development of an indirect competitive OTB-ELISA. The optimized OTB-ELISA resulted in a limit of detection (LOD for OTB of 3 µg/L (8 nM, a limit of quantification (LOQ of 3.7 µg/L (10 nM, and a 50% inhibitory concentration (IC50 of 150 nM. Due to very low cross-reactivity to OTA (2.7% and structurally related molecules (0%, this OTB-ELISA was found to be suitable to detect OTB with excellent precision in different matrices, i.e., beer, coffee and wine. Therefore, this OTB-ELISA will allow screening of OTB in food and feed products.

  13. Development and characterization of monoclonal antibodies to subgroup A avian leukosis virus.

    Science.gov (United States)

    Qiu, Y; Li, X; Fu, L; Cui, Z; Li, W; Wu, Z; Sun, S

    2014-03-01

    Avian leukosis virus subgroup A (ALV-A) is a retrovirus which infects egg-type chickens and is the main pathogen of lymphoid leukosis (LL) and myeloid leukosis (ML). In order to greatly enhance the diagnosis and treatment of clinical avian leukemia, two monoclonal antibodies (MAbs) to ALV-A were developed by fusion between SP2/0 and spleen cells from mice immunized with expressed ALV-A env-gp85 protein. Using immunofluorescence assay (IFA), two MAbs reacted with ALV-A, but not with subgroups B and J of ALV. Western blot tests showed that molecular weight of ALV-A envelope glycoprotein recognized by MAbs was about 53 kD. Isotyping test revealed that two MAbs (A5C1 and A4C8) were IgG1 isotypes. These MAbs can be used for diagnosis and epidemiology of ALV-A.

  14. Uses of monoclonal antibody 8H9

    Science.gov (United States)

    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.

  15. Detection of Campylobacter species using monoclonal antibodies

    Science.gov (United States)

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

    1999-01-01

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

  16. Development of rabbit monoclonal antibodies for detection of alpha-dystroglycan in normal and dystrophic tissue.

    Directory of Open Access Journals (Sweden)

    Marisa J Fortunato

    Full Text Available Alpha-dystroglycan requires a rare O-mannose glycan modification to form its binding epitope for extracellular matrix proteins such as laminin. This functional glycan is disrupted in a cohort of muscular dystrophies, the secondary dystroglycanopathies, and is abnormal in some metastatic cancers. The most commonly used reagent for detection of alpha-dystroglycan is mouse monoclonal antibody IIH6, but it requires the functional O-mannose structure for recognition. Therefore, the ability to detect alpha-dystroglycan protein in disease states where it lacks the full O-mannose glycan has been limited. To overcome this hurdle, rabbit monoclonal antibodies against the alpha-dystroglycan C-terminus were generated. The new antibodies, named 5-2, 29-5, and 45-3, detect alpha-dystroglycan from mouse, rat and pig skeletal muscle by Western blot and immunofluorescence. In a mouse model of fukutin-deficient dystroglycanopathy, all antibodies detected low molecular weight alpha-dystroglycan in disease samples demonstrating a loss of functional glycosylation. Alternately, in a porcine model of Becker muscular dystrophy, relative abundance of alpha-dystroglycan was decreased, consistent with a reduction in expression of the dystrophin-glycoprotein complex in affected muscle. Therefore, these new rabbit monoclonal antibodies are suitable reagents for alpha-dystroglycan core protein detection and will enhance dystroglycan-related studies.

  17. Development of monoclonal antibody-based sandwich ELISA for detection of dextran.

    Science.gov (United States)

    Wang, Sheng-Yu; Li, Zhe; Wang, Xian-Jiang; Lv, Sha; Yang, Yun; Zeng, Lian-Qiang; Luo, Fang-Hong; Yan, Jiang-Hua; Liang, Da-Feng

    2014-10-01

    Dextran as anti-nutritional factor is usually a result of bacteria activity and has associated serial problems during the process stream in the sugar industry and in medical therapy. A sensitive method is expected to detect dextran quantitatively. Here we generated four monoclonal antibodies (MAbs) against dextran using dextran T40 conjugated with bovine serum albumin (BSA) as immunogen in our lab following hybridoma protocol. Through pairwise, an MAb named D24 was determined to be conjugated with horseradish peroxidase (HRP) and was used in the establishment of a sensitive sandwich enzyme-linked immunosorbent assay (ELISA) method for determination of dextran, in which MAb D9 was chosen as a capture antibody. The detection limit and working scope of the developed sandwich ELISA method were 3.9 ng/mL and 7.8-500 ng/mL with a correlation coefficient of 0.9909. In addition, the cross-reaction assay demonstrated that the method possessed high specificity with no significant cross-reaction with dextran-related substances, and the recovery rate ranged from 96.35 to 102.00%, with coefficient of variation ranging from 1.58 to 6.94%. These results indicated that we developed a detection system of MAb-based sandwich ELISA to measure dextran and this system should be a potential tool to determine dextran levels.

  18. Development of a monoclonal antibody against viral haemorrhagic septicaemia virus (VHSV) genotype IVa

    DEFF Research Database (Denmark)

    Ito, T.; Olesen, Niels Jørgen; Skall, Helle Frank

    2010-01-01

    of the spread of genotypes to new geographical areas. A monoclonal antibody (MAb) against VHSV genotype IVa was produced, with the aim of providing a simple method of discriminating this genotype from the other VHSV genotypes (I, II, III and IVb). Balb/c mice were injected with purified VHSV-JF00Ehil (genotype...... IVa) from diseased farmed Japanese flounder. Ten hybridoma clones secreting monoclonal antibodies (MAbs) against VHSV were established. One of these, MAb VHS-10, reacted only with genotype IVa in indirect fluorescent antibody technique (IFAT) and ELISA. Using cell cultures that were transfected...... with each of the viral protein genes, it was shown that the MAb VHS-10 recognizes a nonlinear genotype IVa-specific epitope on the VHSV N-protein....

  19. Development of monoclonal antibodies and quantitative ELISAs targeting insulin-degrading enzyme

    Directory of Open Access Journals (Sweden)

    Dickson Dennis W

    2009-10-01

    Full Text Available Abstract Background Insulin-degrading enzyme (IDE is a widely studied zinc-metalloprotease implicated in the pathogenesis of type 2 diabetes mellitus, Alzheimer disease (AD and varicella zoster virus infection. Despite more than six decades of research on IDE, progress has been hampered by the lack of well-characterized reagents targeting this biomedically important protease. To address this important need, we generated and characterized new mouse monoclonal antibodies (mAbs targeting natively folded human and rodent IDE. Results Eight monoclonal hybridoma cell lines were derived in house from mice immunized with full-length, natively folded, recombinant human IDE. The mAbs derived from these lines were shown to detect IDE selectively and sensitively by a wide range of methods. Two mAbs in particular—designated 6A1 and 6H9—proved especially selective for IDE in immunocytochemical and immunohistochemical applications. Using a variety of methods, we show that 6A1 selectively detects both human and rodent IDE, while 6H9 selectively detects human, but not rodent, IDE, with both mAbs showing essentially no cross reactivity with other proteins in these applications. Using these novel anti-IDE mAbs, we also developed sensitive and quantitative sandwich ELISAs capable of quantifying IDE levels present in human brain extracts. Conclusion We succeeded in developing novel mAbs that selectively detect rodent and/or human IDE, which we have shown to be suitable for a wide range of applications, including western blotting, immunoprecipitation, immunocytochemistry, immunohistochemistry, and quantitative sandwich ELISAs. These novel anti-IDE mAbs and the assays derived from them constitute important new tools for addressing many unresolved questions about the basic biology of IDE and its role in multiple highly prevalent human diseases.

  20. A monoclonal antibody against leptin.

    Science.gov (United States)

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

    2012-10-01

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

  1. ON THE NOTION OF SYNERGY OF MONOCLONAL ANTIBODIES AS DRUGS

    Directory of Open Access Journals (Sweden)

    Michael Sela

    2013-08-01

    Full Text Available History of developing synergy between monoclonal antibodies, anti-tumor activity of monoclonal antibodies against tyrosine-kinases receptors EGFR/ErbB-1 and HER2/ErbB-2 as well as growth factor VEGF in various combinations are considered in the article. There were proposed hypotheses about potential molecular mechanisms underlay synergy between monoclonal antibodies (for homo- and hetero combinations of antibodies appropriately specific for antigenic determinants on the same or different receptors. Future trends in researches necessary to deeper understanding causes of this phenomenon and perspectives for practical application of monoclonal antibodies acted synergistically as immunotherapeutic drugs for human tumors treatment are reviewed.

  2. Develope Monoclonal Antibody against Foot-and-mouth Disease Virus A Type

    Institute of Scientific and Technical Information of China (English)

    Tong Lin; Jing Li; Jun-jun Shao; Guo-zheng Cong; Jun-zheng Du; Shan-dian Gao; Hui-yun Chang

    2011-01-01

    In order to develop an anti-FMDV A Type monoclonal antibo by (mAb),BABL/c mice were immunized with FMDV A type.Monoclonal antibodies (mAbs) 7B11 and 8H4 against Foot-and-mouth disease virus (FMDV) serotype A were produced by fusing SP2/O myeloma cells with splenocyte from the mouse immunized with A/AV88.The microneutralization titer of the mAbs 7B11 and 8H4 were 1024 and 512,respectively.Both mAbs contain kappa light chains,the mAbs were IgG1.In order to define the mAbs binding epitopes,the reactivity of these mAbs against A Type FMDV,were examined using indirect ELISA,the result showed that both mAbs reacted with A Type FMDV.These mAbs may be used for further vaccine studies,diagnostic methods,prophylaxis,etiological and immunological research on FMDV.Characterization of these ncindicated that prepared anti-FMDV A mAbs had no cross-reactivity with Swine Vesicular Disease (SVD) or FMDV O,Asial and C Type antigens.Their titers in abdomen liquor were 1:5×106 and 1:2×106,respectively.7B11 was found to be of subtype IgG1,8H4 was classified as IgG2b subtype.The mAbs prepared in this study,are specific for detection of FMDV serotype A,and is potentially useful for pen-side diagnosis.

  3. Monoclonal antibody technologies and rapid detection assays

    Science.gov (United States)

    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...

  4. Quality Control System for Beer Developed with Monoclonal Antibodies Specific to Barley Lipid Transfer Protein

    Directory of Open Access Journals (Sweden)

    Yukie Murakami-Yamaguchi

    2012-10-01

    Full Text Available Non-specific lipid transfer protein (LTP in barley grain reacted with the IgE in sera drawn from food allergy patients. A sandwich-type of enzyme-linked immunosorbent assay (ELISA was developed with mouse monoclonal antibodies raised against LTP purified with barley flour. This ELISA showed a practical working range of 0.3–3 ng/mL and no cross-reactivity with wheat, adlay and rye. Using this ELISA, LTP was determined in several types of barley-foods, including fermented foods such as malt vinegar, barley-malt miso and beer. LTP content in beer of the same kind was approximately constant, even if manufacturing factory and production days were different. Not only as a factor of foam formation and stability but also as an allergen, controlling and monitoring of LTP in beer should be considered. Taken together, our LTP-detecting ELISA can be proposed as an appropriate system for the quality control of beer.

  5. Development of an enzyme immunoassay using a monoclonal antibody against the psychoactive diterpenoid salvinorin A.

    Science.gov (United States)

    Paudel, Madan Kumar; Shirota, Osamu; Sasaki-Tabata, Kaori; Tanaka, Hiroyuki; Sekita, Setsuko; Morimoto, Satoshi

    2013-09-27

    Salvinorin A (1), the main active constituent in Salvia divinorum, is a highly selective kappa-opioid receptor agonist with hallucinogenic effects, which is regulated in several countries. In the present study, a monoclonal antibody (mAb) against 1 was prepared, and an indirect competitive enzyme-linked immunosorbent assay (icELISA) system was developed for the detection of salvinorins. To raise mAbs against 1, salvinorin B (2) hemisuccinate was synthesized and used to prepare the immunogen 2-bovine serum albumin conjugate. This technique was used to prepare a hybridoma cell line, 3D5, which secreted a mAb that recognized 1. The mAb was shown to have specificity for 1 and other salvinorins in cross-reactivity tests. The intra-assay calibration range by icELISA using the mAb against 1 was 0.0195-0.625 μg/mL. After validating the icELISA using intra- and interassays, a recovery experiment and analysis of several plants in the family Lamiaceae, including S. divinorum, confirmed that the analytical method based on ELISA is not only simple but also precise, accurate, sensitive, and sufficiently reliable. The results indicate that icELISA is a useful tool in the identification of S. divinorum.

  6. Strategies for developing design spaces for viral clearance by anion exchange chromatography during monoclonal antibody production.

    Science.gov (United States)

    Strauss, Daniel M; Cano, Tony; Cai, Nick; Delucchi, Heather; Plancarte, Magdalena; Coleman, Daniel; Blank, Gregory S; Chen, Qi; Yang, Bin

    2010-01-01

    The quality-by-design (QbD) regulatory initiative promotes the development of process design spaces describing the multidimensional effects and interactions of process variables on critical quality attributes of therapeutic products. However, because of the complex nature of production processes, strategies must be devised to provide for design space development with reasonable allocation of resources while maintaining highly dependable results. Here, we discuss strategies for the determination of design spaces for viral clearance by anion exchange chromatography (AEX) during purification of monoclonal antibodies. We developed a risk assessment for AEX using a formalized method and applying previous knowledge of the effects of certain variables and the mechanism of action for virus removal by this process. We then use design-of-experiments (DOE) concepts to perform a highly fractionated factorial experiment and show that varying many process parameters simultaneously over wide ranges does not affect the ability of the AEX process to remove endogenous retrovirus-like particles from CHO-cell derived feedstocks. Finally, we performed a full factorial design and observed that a high degree of viral clearance was obtained for three different model viruses when the most significant process parameters were varied over ranges relevant to typical manufacturing processes. These experiments indicate the robust nature of viral clearance by the AEX process as well as the design space where removal of viral impurities and contaminants can be assured. In addition, the concepts and methodology presented here provides a general approach for the development of design spaces to assure that quality of biotherapeutic products is maintained.

  7. Development of an ELISA kit using monoclonal antibody to Clostridium difficile toxin A

    Institute of Scientific and Technical Information of China (English)

    Si-Wu Fu; Ya-Li Zhang; Dian-Yuan Zhou

    2004-01-01

    AIM: To establish an ELISA kit using monoclonal antibodiesagainst Clostridium difficile ( C. difficile) toxin A.METHODS: An indirect sandwich ElISA was described using the purified rabbit monospecific antiserum as capturing antibody. After the polystyrene microtitre plates with 96 fiat-bottomed wells were coated with rabbit antiserum, the wells were blocked with 100 g/L BSA in PBS-T. C. difficiletoxin A or culture filtrates were added to each well and then monoclonal antibodies IgG-horseradish peroxidase conjugate was added as detecting antibody, tetramethylbenzidine was used as substrate and A450 of the stopped reacting product was recorded in an automated plate reader. RESULTS: The tested specimens included culture filtrates of 2 strains of toxigenic C. difficile, 2 strains of non-toxigenic C. difficile, 26 strains of E. coli, 2 strains of S. dysenteriae, 1 strain of Bif infantis, 5 strains of V. cholera, 2 strains ofS. typhi, 7 strains of C. botulinum, 1 strain of toxigenic C. sordllii, and 1 strain of C. butyricum. A total of 47 strains of culture filtrates were all negative except for 2 strains of toxigenic C. difficile. The detective limitation of toxin A was 0.1 ng/mL.CONCLUSION: An ELISA kit with high specificity and excellent sensitivity for the rapid detection of C. difficile toxin A was established. It will be a useful tool for diagnostic test of C. difficile toxin A.

  8. Development of a monoclonal antibody against viral haemorrhagic septicaemia virus (VHSV) genotype IVa.

    Science.gov (United States)

    Ito, T; Olesen, N J; Skall, H F; Sano, M; Kurita, J; Nakajima, K; Iida, T

    2010-02-24

    The viral haemorrhagic septicaemia virus (VHSV) comprises 4 major genotypes and a number of subtypes with, in most cases, distinct geographical distribution. A quick and simple detection method that can discriminate the different genotypes is desirable for a quick and more efficient prevention of the spread of genotypes to new geographical areas. A monoclonal antibody (MAb) against VHSV genotype IVa was produced, with the aim of providing a simple method of discriminating this genotype from the other VHSV genotypes (I, II, III and IVb). Balb/c mice were injected with purified VHSV-JF00Ehil (genotype IVa) from diseased farmed Japanese flounder. Ten hybridoma clones secreting monoclonal antibodies (MAbs) against VHSV were established. One of these, MAb VHS-10, reacted only with genotype IVa in indirect fluorescent antibody technique (IFAT) and ELISA. Using cell cultures that were transfected with each of the viral protein genes, it was shown that the MAb VHS-10 recognizes a nonlinear genotype IVa-specific epitope on the VHSV N-protein.

  9. Distinct expression profiles of Notch-1 protein in human solid tumors: Implications for development of targeted therapeutic monoclonal antibodies

    OpenAIRE

    Yuan Li; Burns, Janine A.; Carol A Cheney; et al

    2010-01-01

    Yuan Li1, Janine A Burns1, Carol A Cheney1, Ningyan Zhang1, Salvatore Vitelli1, Fubao Wang1, Andrew Bett2, Michael Chastain2, Laurent P Audoly1, Zhi-Qiang Zhang1,31Department of Biologics Research, 2Department of Vaccine Research, Merck Research Laboratories, West Point, PA, USA; 3Clinical Development Laboratory, Merck Research Laboratories, Rahway, NJ, USAAbstract: Biological therapies, such as monoclonal antibodies (mAbs) that target tumor-associated antigens have been considered an effecti...

  10. Development of a monoclonal antibody-based colony blot immunoassay for detection of thermotolerant Campylobacter species.

    Science.gov (United States)

    Huang, Hongsheng; Phipps-Todd, Beverley; McMahon, Tanis; Elmgren, Catherine L; Lutze-Wallace, Cheryl; Todd, Zoe A; Garcia, Manuel M

    2016-11-01

    Campylobacter species, particularly thermotolerant Campylobacter spp., such as C. jejuni, are major human foodborne pathogens. Culture methods have been routinely used for the detection of this organism in various types of samples. An alternative, simple and rapid confirmation test(s) without further tedious biochemical tests would be useful. Meanwhile, Campylobacter-like colonies can be difficult to identify on agar plates overgrown with competitive bacteria, which can lead to false-negative results. This study was to develop a simple colony blot immunoassay using a new monoclonal antibody (Mab) produced in the present study for rapid screening, confirmation and quantification of campylobacters on culture agar plates. The procedure developed in this study was able to specifically detect thermotolerant Campylobacter spp., but not other non-thermotolerant Campylobacter and non-Campylobacter reference strains tested. This assay could detect 10(5) cells in a single dot. This assay showed 100% correlation with the culture method for the blotted membranes from 21 either chicken meat or vegetable samples experimentally inoculated with thermotolerant campylobacters. Among 101 natural samples of chicken meat (n=44), chicken feces (n=20) and vegetables (n=37), this assay also showed positive for 23 chicken meat and 14 fecal samples that were positive for thermotolerant campylobacters by culture method, and identified four additional suspects that were culture negative. Membranes stored at 4°C for at least 4years could also be used for this assay. The assay developed in this study can be used in quantitative study for immediate or archival usage, and for diagnostic test to preliminarily confirm the presence of thermotolerant Campylobacter on agar plates. Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.

  11. Development of murine monoclonal antibodies for the immunohistochemical diagnosis of systemic bovine aspergillosis

    DEFF Research Database (Denmark)

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

    1996-01-01

    Murine monoclonal antibodies (MAbs) against water-soluble somatic antigens (WSSA) and the wall fraction (WF) from Aspergillus fumigatus were produced by fusion of splenocytes from immunized BALB/c mice with mouse myeloma X63-Ag 8.653 cells. The supernatants of in vitro cultured hybridomas were in.......e., aspergillosis was diagnosed. The characteristics of the 2 MAbs (MAb-WF-AF-1 and-2) raised against the WF of A. fumigatus in ELISA and immunoblotting and immunohistochemical assays justify their application for the in situ diagnosis of systemic aspergillosis of cattle....

  12. Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant.

    Science.gov (United States)

    Zeitlin, Larry; Pettitt, James; Scully, Corinne; Bohorova, Natasha; Kim, Do; Pauly, Michael; Hiatt, Andrew; Ngo, Long; Steinkellner, Herta; Whaley, Kevin J; Olinger, Gene G

    2011-12-20

    No countermeasures currently exist for the prevention or treatment of the severe sequelae of Filovirus (such as Ebola virus; EBOV) infection. To overcome this limitation in our biodefense preparedness, we have designed monoclonal antibodies (mAbs) which could be used in humans as immunoprotectants for EBOV, starting with a murine mAb (13F6) that recognizes the heavily glycosylated mucin-like domain of the virion-attached glycoprotein (GP). Point mutations were introduced into the variable region of the murine mAb to remove predicted human T-cell epitopes, and the variable regions joined to human constant regions to generate a mAb (h-13F6) appropriate for development for human use. We have evaluated the efficacy of three variants of h-13F6 carrying different glycosylation patterns in a lethal mouse EBOV challenge model. The pattern of glycosylation of the various mAbs was found to correlate to level of protection, with aglycosylated h-13F6 providing the least potent efficacy (ED(50) = 33 μg). A version with typical heterogenous mammalian glycoforms (ED(50) = 11 μg) had similar potency to the original murine mAb. However, h-13F6 carrying complex N-glycosylation lacking core fucose exhibited superior potency (ED(50) = 3 μg). Binding studies using Fcγ receptors revealed enhanced binding of nonfucosylated h-13F6 to mouse and human FcγRIII. Together the results indicate the presence of Fc N-glycans enhances the protective efficacy of h-13F6, and that mAbs manufactured with uniform glycosylation and a higher potency glycoform offer promise as biodefense therapeutics.

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

    Science.gov (United States)

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

  14. Waiving in vivo studies for monoclonal antibody biosimilar development: National and global challenges.

    Science.gov (United States)

    Chapman, Kathryn; Adjei, Akosua; Baldrick, Paul; da Silva, Antonio; De Smet, Karen; DiCicco, Richard; Hong, Seung Suh; Jones, David; Leach, Michael W; McBlane, James; Ragan, Ian; Reddy, Praveen; Stewart, Donald I H; Suitters, Amanda; Sims, Jennifer

    2016-01-01

    Biosimilars are biological medicinal products that contain a version of the active substance of an already authorised original biological medicinal product (the innovator or reference product). The first approved biosimilar medicines were small proteins, and more recently biosimilar versions of innovator monoclonal antibody (mAb) drugs have entered development as patents on these more complex proteins expire. In September 2013, the first biosimilar mAb, infliximab, was authorised in Europe. In March 2015, the first biosimilar (Zarxio™, filgrastim-sndz, Sandoz) was approved by the US Food and Drug Administration; however, to date no mAb biosimilars have been approved in the US. There are currently major differences between how biosimilars are regulated in different parts of the world, leading to substantial variability in the amount of in vivo nonclinical toxicity testing required to support clinical development and marketing of biosimilars. There are approximately 30 national and international guidelines on biosimilar development and this number is growing. The European Union's guidance describes an approach that enables biosimilars to enter clinical trials based on robust in vitro data alone; in contrast, the World Health Organization's guidance is interpreted globally to mean in vivo toxicity studies are mandatory. We reviewed our own experience working in the global regulatory environment, surveyed current practice, determined drivers for nonclinical in vivo studies with biosimilar mAbs and shared data on practice and study design for 25 marketed and as yet unmarketed biosimilar mAbs that have been in development in the past 5y. These data showed a variety of nonclinical in vivo approaches, and also demonstrated the practical challenges faced in obtaining regulatory approval for clinical trials based on in vitro data alone. The majority of reasons for carrying out nonclinical in vivo studies were not based on scientific rationale, and therefore the authors

  15. Pharmacokinetics interactions of monoclonal antibodies.

    Science.gov (United States)

    Ferri, Nicola; Bellosta, Stefano; Baldessin, Ludovico; Boccia, Donatella; Racagni, Giorgi; Corsini, Alberto

    2016-09-01

    The clearance of therapeutic monoclonal antibodies (mAbs) typically does not involve cytochrome P450 (CYP450)-mediated metabolism or interaction with cell membrane transporters, therefore the pharmacokinetics interactions of mAbs and small molecule drugs are limited. However, a drug may affect the clearance of mAbs through the modulation of immune response (e.g., methotrexate reduces the clearance of infliximab, adalimumab, and golimumab, possibly due to methotrexate's inhibitory effect on the formation of antibodies against the mAbs). In addition, mAbs that are cytokine modulators may modify the metabolism of drugs through their effects on P450 enzymes expression. For example, cytokine modulators such as tocilizumab (anti-IL-6 receptor antibody) may reverse the "inhibitory" effect of IL-6 on CYP substrates, resulting in a "normalization" of CYP activities. Finally, a drug may alter the clearance of mAbs by either increasing or reducing the levels of expression of targets of mAbs on the cell surface. For instance, statins and fibrates induce PCSK9 expression and therefore increase cellular uptake and clearance of alirocumab and evolocumab, anti-PCSK9 antibodies. In the present review, we will provide an overview on the pharmacokinetics properties of mAbs as related to the most relevant examples of mAbs-small molecule drug interaction.

  16. Production and Screening of Monoclonal Peptide Antibodies.

    Science.gov (United States)

    Trier, Nicole Hartwig; Mortensen, Anne; Schiolborg, Annette; Friis, Tina

    2015-01-01

    Hybridoma technology is a remarkable and indispensable tool for generating high-quality monoclonal antibodies. Hybridoma-derived monoclonal antibodies not only serve as powerful research and diagnostic reagents, but have also emerged as the most rapidly expanding class of therapeutic biologicals. In this chapter, an overview of hybridoma technology and the laboratory procedures used routinely for hybridoma production and antibody screening are presented, including characterization of peptide antibodies.

  17. Development of a mouse monoclonal antibody cocktail for post-exposure rabies prophylaxis in humans.

    Directory of Open Access Journals (Sweden)

    Thomas Müller

    Full Text Available As the demand for rabies post-exposure prophylaxis (PEP treatments has increased exponentially in recent years, the limited supply of human and equine rabies immunoglobulin (HRIG and ERIG has failed to provide the required passive immune component in PEP in countries where canine rabies is endemic. Replacement of HRIG and ERIG with a potentially cheaper and efficacious alternative biological for treatment of rabies in humans, therefore, remains a high priority. In this study, we set out to assess a mouse monoclonal antibody (MoMAb cocktail with the ultimate goal to develop a product at the lowest possible cost that can be used in developing countries as a replacement for RIG in PEP. Five MoMAbs, E559.9.14, 1112-1, 62-71-3, M727-5-1, and M777-16-3, were selected from available panels based on stringent criteria, such as biological activity, neutralizing potency, binding specificity, spectrum of neutralization of lyssaviruses, and history of each hybridoma. Four of these MoMAbs recognize epitopes in antigenic site II and one recognizes an epitope in antigenic site III on the rabies virus (RABV glycoprotein, as determined by nucleotide sequence analysis of the glycoprotein gene of unique MoMAb neutralization-escape mutants. The MoMAbs were produced under Good Laboratory Practice (GLP conditions. Unique combinations (cocktails were prepared, using different concentrations of the MoMAbs that were capable of targeting non-overlapping epitopes of antigenic sites II and III. Blind in vitro efficacy studies showed the MoMab cocktails neutralized a broad spectrum of lyssaviruses except for lyssaviruses belonging to phylogroups II and III. In vivo, MoMAb cocktails resulted in protection as a component of PEP that was comparable to HRIG. In conclusion, all three novel combinations of MoMAbs were shown to have equal efficacy to HRIG and therefore could be considered a potentially less expensive alternative biological agent for use in PEP and prevention of

  18. Characterization of site-specific glycation during process development of a human therapeutic monoclonal antibody.

    Science.gov (United States)

    Miller, Amanda K; Hambly, David M; Kerwin, Bruce A; Treuheit, Michael J; Gadgil, Himanshu S

    2011-07-01

    A therapeutic recombinant monoclonal antibody (mAb1) was found to be highly susceptible to glycation during production. Up to 42% glycation was observed in mAb1, which was significantly greater than the glycation observed in 17 other monoclonal antibodies (mAbs). The majority of the glycation was localized to lysine 98 of a unique sequence in the heavy chain complementarity determining region 3. Upon incubation with 5% glucose at 37 °C for 5 days, the level of glycation rose to 80% of the total protein where the majority of the additional glycation was on the lysine 98 residue. These data suggested that the lysine 98 residue was highly susceptible to glycation. However, three other mAbs with a lysine residue in the same position did not show high rates of glycation in the forced glycation assay, suggesting that primary and perhaps secondary structural constraints could contribute to the rate of glycation at that lysine. Interestingly, a portion of the glycation in mAb1 was found to be reversible and upon incubation in phosphate buffer (pH 7) at 37 °C for 5 days, the glycation dropped from starting levels of 42% to 20%. Variation was observed in the total glycation levels between different lots of mAb1. The variability in glycation introduced charge heterogeneity in the form of an acidic peak on cation exchange chromatography and lead to product inconsistency. Mutation of lysine 98 to arginine reduced the starting level of glycation without any impact on potency. Copyright © 2011 Wiley-Liss, Inc. and the American Pharmacists Association

  19. A Strategy for Screening Monoclonal Antibodies for Arabidopsis Flowers

    Science.gov (United States)

    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

  20. 77 FR 9678 - Prospective Grant of Exclusive License: The Development of Human Anti-CD22 Monoclonal Antibodies...

    Science.gov (United States)

    2012-02-17

    ... and m972 (SMB-002) monoclonal antibodies as therapies for the treatment of B cell cancers and... designated m971 and m972 (SMB-002; applicant designation). CD22 is a cell surface antigen that is...

  1. Development of a double-monoclonal antibody sandwich ELISA: Tool for chicken interferon-γ detection ex vivo

    Science.gov (United States)

    Dai, Hua; Xu, Zheng-zhong; Wang, Meiling; Chen, Jun-hua; Chen, Xiang; Pan, Zhi-ming; Jiao, Xin-an

    2016-01-01

    The aim of the present work was to develop reagents to set up a chicken interferon-γ (ChIFN-γ) assay. Four monoclonal antibodies (mAbs) specific for ChIFN-γ were generated to establish sandwich ELISA based on 2 different mAbs. To improve the detection sensitivity of ChIFN-γ, a double-monoclonal antibody sandwich ELISA was developed using mAb 3E5 as capture antibody and biotinylated mAb 3E3 as a detection reagent. The results revealed that this ELISA has high sensitivity, allowing for the detection of 125 to 500 pg/mL of recombinant ChIFN-γ, and also has an excellent capacity for detecting native ChIFN-γ. This ELISA was then used to detect ChIFN-γ level in chickens immunized with a Newcastle disease virus (NDV) vaccine, the immunized chicken splenocytes were stimulated by NDV F protein as recall antigen. From our results, it appears that the sensitivity range of this sandwich ELISA test is adequate to measure the ex vivo release of ChIFN-γ. PMID:27127340

  2. OBTAINING OF MONOCLONAL ANTIBODIES AGAINST CHOLERA TOXIN AND HEAT LABILE ENTEROTOXIN OF E. coli FOR DEVELOPMENT OF THE TOXINS DIPLEX ANALYSIS IN ENVIRONMENTAL SPECIMENS

    Directory of Open Access Journals (Sweden)

    Eu. V. Grishin

    2013-08-01

    Full Text Available The present study focuses on development of monoclonal antibodies (MAbs which specifically interact with cholera toxin or heat labile enterotoxin of E. coli. Such monoclonal antibodies MAbs are possessed of ability to identify cholera toxin or heat labile enterotoxin in different immunochemical assays. We obtained hybridoma clones which produced monoclonal antibodies of IgG isotypes to cholera toxin and heat labile enterotoxin. On application of the method of serial dilutions we selected the clones which produced monoclonal antibodies with specific activity against only one of the toxins. We found the 16 pairs of monoclonal antibodies to cholera toxin and 28 ones to heat labile enterotoxin. By means of these monoclonal antibodies it was possible to realize the quantitative analysis of theses toxins in sandwich immunoassay ELISA and diplex sandwich xMAP-assay. The limits of detection of cholera toxin and heat labile enterotoxin in ELISA in control buffer were 0.2 and 0.4 ng/ml, respectively, and in xMAP assay — 0.01 and 0.08 ng/ml, respectively. In probes of cow milk, meat soup, pond water and nasopharyngeal washes cholera toxin was detected in the both assays with the same limits of detections, but heat labile enterotoxin limits of detections were above the ones in control buffers.

  3. Development of a monoclonal antibody-based immunoassay for the detection and quantification of Anguillospora longissima colonizing leaf material.

    OpenAIRE

    Bermingham, S; Dewey, F. M.; Maltby, L

    1995-01-01

    Monoclonal antibodies (MAbs) to the aquatic hyphomycete Anguillospora longissima were raised in mice by using a coimmunization program. A cell line was raised that produced a MAb of the immunoglobulin M class that was specific for A. longissima both in an enzyme-linked immunosorbent assay (ELISA) and by immunofluorescence but that did not recognize other members of the aquatic hyphomycetes. This MAb (AL-HH8c) was used to develop a quantitative ELISA in vitro. The antigen recognized by AL-HH8c...

  4. Aggregates in monoclonal antibody manufacturing processes.

    Science.gov (United States)

    Vázquez-Rey, María; Lang, Dietmar A

    2011-07-01

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

  5. Application of GP5 Protein to Develop Monoclonal Antibody against Porcine Reproductive and Respiratory Syndrome Virus

    Institute of Scientific and Technical Information of China (English)

    Hong Tian; Yan Cheng; Jin-yang Wu; Jian-hui He; You-jun Shang; Xiang-tao Liu

    2011-01-01

    In this study,a panel of monoclonal antibodies (mAbs) against Porcine reproductive and respiratory syndrome virus(PRRSV),named as 8C9 and4B4,were produced by fusing SP2/0 myeloma cells and spleen cells of BALB/c mice immunized with the PRRSV (TCID50=5.5),screened by the indirect ELISA and subjected to several limiting dilutions.mAbs were then identified by biological characterization.Among the two fusion cell strains,8C9 belonged to the IgG1 subclass and 4B4 belonged to the IgG2a subclass.The titers in cell culture supernatant and abdomen liquor reached to 1:104and 1:105,respectively.The specificity test indicated that the two cells had specific reactions for the PRRSV and GP5 protein respectively,and no reaction with Classical swine fever virus (CSFV) or Swine vesicular disease virus (SVDV).The molecular weights of the heavy chain and light chain were about 45.0 kDa and 25.0 kDa,respectively.In neutralization activity tests,the results showed that the prepared mAb 4B4 can protect 50% of cells with no CPE in dilution up to 1:512,but mAB 8C9 has no neutralization activities to PRRSV.

  6. Development of novel monoclonal antibodies directed against catechins for investigation of antibacterial mechanism of catechins.

    Science.gov (United States)

    Miyamoto, Takahisa; Zhang, Xiaoguang; Ueyama, Yuuki; Apisada, Kitichalermkiat; Nakayama, Motokazu; Suzuki, Yasuto; Ozawa, Tadahiro; Mitani, Asako; Shigemune, Naofumi; Shimatani, Kanami; Yui, Koji; Honjoh, Ken-Ichi

    2017-03-24

    Catechins are major polyphenolic compounds of green tea. To investigate mechanism for antibacterial action of catechins, 11 monoclonal antibodies (MAbs) were raised against a 3-succinyl-epicatechin (EC)-keyhole limpet hemocyanin (KLH) conjugate. Amino acid sequences of variable regions determined for MAbs b-1058, b-1565, and b-2106 confirmed their innovative character. MAb b-1058 strongly interacted with its target substances in the following order of magnitude: theaflavin-3,3'-di-O-gallate (TFDG)>theaflavin-3-O-gallate (TF3G)≥theaflavin-3'-O-gallate (TF3'G)>gallocatechin gallate (GCg)>penta-O-galloyl-β-d-glucose (PGG)>epigallocatechin gallate (EGCg), as determined using surface plasmon resonance (SPR) on MAb-immobilized sensor chips. The affinity profiles of MAbs b-1058 and b-2106 to the various polyphenols tested suggested that flavan skeletons with both carbonyl oxygen and hydroxyl groups are important for this interaction to take place. S. aureus cells treated with EGCg showed green fluorescence around the cells after incubation with FITC-labeled MAb b-1058. The fluorescence intensity increased with increasing concentrations of EGCg. These MAbs are effective to investigate antibacterial mechanism of catechins and theaflavins.

  7. Accelerated formulation development of monoclonal antibodies (mAbs) and mAb-based modalities: review of methods and tools.

    Science.gov (United States)

    Razinkov, Vladimir I; Treuheit, Michael J; Becker, Gerald W

    2015-04-01

    More therapeutic monoclonal antibodies and antibody-based modalities are in development today than ever before, and a faster and more accurate drug discovery process will ensure that the number of candidates coming to the biopharmaceutical pipeline will increase in the future. The process of drug product development and, specifically, formulation development is a critical bottleneck on the way from candidate selection to fully commercialized medicines. This article reviews the latest advances in methods of formulation screening, which allow not only the high-throughput selection of the most suitable formulation but also the prediction of stability properties under manufacturing and long-term storage conditions. We describe how the combination of automation technologies and high-throughput assays creates the opportunity to streamline the formulation development process starting from early preformulation screening through to commercial formulation development. The application of quality by design (QbD) concepts and modern statistical tools are also shown here to be very effective in accelerated formulation development of both typical antibodies and complex modalities derived from them.

  8. Polyclonal and monoclonal antibodies in clinic.

    Science.gov (United States)

    Wootla, Bharath; Denic, Aleksandar; Rodriguez, Moses

    2014-01-01

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

  9. Emerging monoclonal antibodies against Clostridium difficile infection.

    Science.gov (United States)

    Péchiné, Séverine; Janoir, Claire; Collignon, Anne

    2017-04-01

    Clostridium difficile infections are characterized by a high recurrence rate despite antibiotic treatments and there is an urgent need to develop new treatments such as fecal transplantation and immonotherapy. Besides active immunotherapy with vaccines, passive immunotherapy has shown promise, especially with monoclonal antibodies. Areas covered: Herein, the authors review the different assays performed with monoclonal antibodies against C. difficile toxins and surface proteins to treat or prevent primary or recurrent episodes of C. difficile infection in animal models and in clinical trials as well. Notably, the authors lay emphasis on the phase III clinical trial (MODIFY II), which allowed bezlotoxumab to be approved by the Food and Drug Administration and the European Medicines Agency. They also review new strategies for producing single domain antibodies and nanobodies against C. difficile and new approaches to deliver them in the digestive tract. Expert opinion: Only two human Mabs against TcdA and TcdB have been tested alone or in combination in clinical trials. However, many animal model studies have provided rationale for the use of Mabs and nanobodies in C. difficile infection and pave the way for further clinical investigation.

  10. Development and evaluation of a competitive ELISA using a monoclonal antibody for antibody detection after goose parvovirus virus-like particles (VLPs) and vaccine immunization in goose sera.

    Science.gov (United States)

    Wang, Qian; Ju, Huanyu; Li, Yanwei; Jing, Zhiqiang; Guo, Lu; Zhao, Yu; Ma, Bo; Gao, Mingchun; Zhang, Wenlong; Wang, Junwei

    2014-12-01

    An assay protocol based on a monoclonal antibody-based competitive enzyme-linked immunosorbent assay (MAb-based C-ELISA) for detecting antibodies against goose parvovirus (GPV) and its virus-like particles (VLPs) is described. The assay was developed using baculovirus-expressed recombinant VP2 virus-like particles (rVP2-VLPs) as antigens and a monoclonal antibody against GPV as the competitive antibody. Of the four anti-GPV MAbs that were screened, MAb 1G3 was selected as it was blocked by the GPV positive serum. Based on the distribution of percent inhibition (PI) of the known negative sera (n=225), a cut-off value was set at 36% inhibition. Using this cut-off value, the sensitivity of the assay was 93.3% and the specificity was 95.8%, as compared with the gold standard (virus neutralization assay). The rVP2-VLPs did not react with anti-sera to other goose pathogens, indicating that it is specific for the recognization of goose parvovirus antibodies. The assay was then validated with serum samples from goslings vaccinated with several VLPs (rVP1-VLPs, rVP2-VLPs, rVP3-VLPs, and rCGV-VLPs) and other vaccines (inactivated and attenuated). The C-ELISA described in this study is a sensitive and specific diagnostic test and should have wide applications for the sero-diagnosis and immunologic surveillance of GPV. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Preparation of Monoclonal Antibody and Development of Enzyme-linked Immunosorbent Assay Specific for Escherichia coli O157 in Foods

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Objective To prepare monoclonal antibodies (Mab) and antisera specific for Escherichia coli (E.coli) O157, and to develop a sandwich enzyme-linked immunosorbent assay (ELISA) to detect E.coli O157 in foods. Methods Spleen cells from BALB/c mice immunized with the somatic antigen of E.coli O157:H7 were fused with murine Sp2/0 myeloma cells. The hybridoma cell line specific for E.coli O157 was established after having been subcloned. Antisera specific for E.coli O157 was prepared by intravenous injection into New Zealand rabbits with a stain of E.coli O157:H7. The sandwich ELISA was developed with the polyclonal antibody as the capture antibody and the Mab 3A5 as the detection antibody. The inoculated ground poultry meat and pasteurized milk were tested to confirm efficiency of the method. Results Mab 3A5 specific for E.coli O157 and O113:H21 belonged to subtype IgM. The ascetic titers of the antibody was 1:1×106. No cross-reactivity of the Mab was observed with strains of Salmonella spp, Yersinia enterocolitica, Shigella dysenteriae, etc. The purified polyclonal antibody had a titer of 1:1×105 with E.coli O157. The detection limit of this sandwich ELISA was 103-104 cfu E.coli O157/mL in pure culture with a high specificity, which was characterized by every non-O157 strain with negative response. With 10h enrichment procedure, E.coli O157:H7 recovered well from inoculated ground poultry meat and pasteurized milk at levels of 0.1 cfu/g and 0.1 cfu/mL. Conclusion Mab 3A5 specific for E.coli O157 and O113:H21 can be produced by immunizing BALB/c mice with a strain of E.coli O157:H7. Then a sandwich ELISA can be developed with the polyclonal antibody as the capture antibody and the Mab 3A5 as the detection antibody. The method is proved to be a sensitive and specific technique to detect low number of E.coli O157 in food.

  12. Development of monoclonal antibodies to human microsomal epoxide hydrolase and analysis of “preneoplastic antigen”-like molecules

    Energy Technology Data Exchange (ETDEWEB)

    Duan, Hongying [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Yoshimura, Kazunori [Department of Physiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Kobayashi, Nobuharu; Sugiyama, Kazuo [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Sawada, Jun-ichi; Saito, Yoshiro [Division of Biochemistry and Immunochemistry, National Institute of Health Sciences, Kamiyoga 1-18-1, Setagaya-ku, Tokyo 158-8501 (Japan); Morisseau, Christophe; Hammock, Bruce D. [Department of Entomology and Cancer Center, University of California, Davis, One Shields Avenue, Davis, CA 95616-8584 (United States); Akatsuka, Toshitaka, E-mail: akatsuka@saitama-med.ac.jp [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan)

    2012-04-01

    Microsomal epoxide hydrolase (mEH) is a drug metabolizing enzyme which resides on the endoplasmic reticulum (ER) membrane and catalyzes the hydration of reactive epoxide intermediates that are formed by cytochrome P450s. mEH is also thought to have a role in bile acid transport on the plasma membrane of hepatocytes. It is speculated that efficient execution of such multiple functions is secured by its orientation and association with cytochrome P450 enzymes on the ER membrane and formation of a multiple transport system on the plasma membrane. In certain disease status, mEH loses its association with the membrane and can be detected as distinct antigens in the cytosol of preneoplastic foci of liver (preneoplastic antigen), in the serum in association with hepatitis C virus infection (AN antigen), or in some brain tumors. To analyze the antigenic structures of mEH in physiological and pathological conditions, we developed monoclonal antibodies against different portions of mEH. Five different kinds of antibodies were obtained: three, anti-N-terminal portions; one anti-C-terminal; and one, anti-conformational epitope. By combining these antibodies, we developed antigen detection methods which are specific to either the membrane-bound form or the linearized form of mEH. These methods detected mEH in the culture medium released from a hepatocellular carcinoma cell line and a glioblastoma cell line, which was found to be a multimolecular complex with a unique antigenic structure different from that of the membrane-bound form of mEH. These antibodies and antigen detection methods may be useful to study pathological changes of mEH in various human diseases. -- Highlights: ► Monoclonal antibodies against different portions of mEH were developed. ► They discriminate between the membrane-bound and the linearized forms of mEH. ► We analyze the antigenic structure of the altered form of mEH in tumor cells. ► Preneoplastic antigen is a multimolecular complex of mEH with

  13. Progress in biosimilar monoclonal antibody development: the infliximab biosimilar CT-P13 in the treatment of rheumatic diseases.

    Science.gov (United States)

    Braun, Jürgen; Kudrin, Alex

    2015-01-01

    Biosimilars are biologic medical products whose active drug substance is made by a living organism or derived from it. The term is used to describe a subsequent version of an innovator biopharmaceutical product aiming at approval following patent expiry on the reference product. Biosimilars of monoclonal need to demonstrate similar but not identical quality of nonclinical and clinical attributes. Not all data of the originator product need to be recapitulated, as large numbers of patient-years of exposure data are already available. Thus, biosimilar development is largely based on the safety profiles of the originator product. The evaluation of biosimilarity includes immunogenicity attributed risks. CT-P13 (Remsima™/Inflectra™, Celltrion/Hospira), a biosimilar of the innovator drug infliximab (INF), was the first approved complex biosimilar monoclonal antibody in the EU, within the framework of WHO, EMA and US FDA biosimilar guidelines. CT-P13 has shown analytical and nonclinical features highly similar to INF including pharmacokinetics, efficacy, safety and immunogenicity profiles in ankylosing spondylitis and rheumatoid arthritis. The objective of this article is to highlight the recent biosimilar development and to review the results from the studies PLANETRA and PLANETAS, which have supported the approval of CT-P13 for several indications.

  14. Monoclonal Antibody Therapy for Advanced Neuroblastoma

    Science.gov (United States)

    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.

  15. Preclinical and clinical evaluation of elotuzumab, a SLAMF7-targeted humanized monoclonal antibody in development for multiple myeloma.

    Science.gov (United States)

    Palumbo, Antonio; Sonneveld, Pieter

    2015-08-01

    Although multiple myeloma has historically been treated with chemotherapy, prolonged survival has only been possible since the introduction of thalidomide, lenalidomide and bortezomib. However, multiple myeloma remains largely incurable, and new treatments are needed to improve long-term outcome. Elotuzumab is a humanized IgG1 monoclonal antibody that targets Signaling Lymphocyte Activation Molecule Family member 7 (SLAMF7) to activate NK cells, enabling selective killing of myeloma cells with minimal effects on normal tissue. The combination of elotuzumab with antimyeloma therapies that stimulate host immunity may be an attractive treatment option for patients with newly diagnosed or relapsed/refractory multiple myeloma. Here, we review the role of SLAMF7 in the pathogenesis of multiple myeloma and the preclinical and clinical development of elotuzumab.

  16. Development and evaluation of a monoclonal antibody-based inhibition ELISA for the quantification of chymosin in solution.

    Science.gov (United States)

    Rolet-Répécaud, O; Arnould, C; Dupont, D; Gavoye, S; Beuvier, E; Achilleos, C

    2015-05-20

    Chymosin is the major enzyme of natural rennet, traditionally used in cheese making for its high milk-clotting activity. For technical reasons, an accurate characterization of rennet should include its total clotting activity and also its enzymatic composition. Monoclonal antibodies specific to chymosin were obtained from mice immunized with purified bovine chymosin, and an inhibition enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of chymosin in solution. No cross-reactivity was observed with other milk-clotting enzymes commonly used in cheese making. The limit of detection and limit of quantification were 125 and 400 ng/mL, respectively. The values of precision within and among runs were 7.23 and 7.39%, respectively, and satisfying recovery, from 92 to 119%, was found for spiked samples. The inhibition ELISA was successfully applied to commercial rennets, and the results were consistent with those obtained using the standard chromatographic method (IDF 110: A, 1987).

  17. Monoclonal antibodies for the control of influenza virus vaccines.

    NARCIS (Netherlands)

    H.J.M. van de Donk; M.F. van Olderen; A.D.M.E. Osterhaus (Albert); J.C. de Jong (Jan)

    1984-01-01

    textabstractHybridomas producing haemagglutination inhibiting monoclonal antibodies against influenza A/Texas/1/77 H3N2 were developed. One hybridoma producing antibodies reacting with Victoria/3/75, Texas/1/77 Bangkok/1/79 and England/496/80 was selected to determine the potency of influenza virusv

  18. Production of Monoclonal Antibody against Human Nestin.

    Science.gov (United States)

    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.

  19. Passive immunization against dental caries and periodontal disease: development of recombinant and human monoclonal antibodies.

    Science.gov (United States)

    Abiko, Y

    2000-01-01

    Indigenous micro-organisms in the oral cavity can cause two major diseases, dental caries and periodontal diseases. There is neither agreement nor consensus as to the actual mechanisms of pathogenesis of the specific virulence factors of these micro-organisms. The complexity of the bacterial community in dental plaque has made it difficult for the single bacterial agent of dental caries to be determined. However, there is considerable evidence that Streptococcus mutans is implicated as the primary causative organism of dental caries, and the cell-surface protein antigen (SA I/II) as well as glucosyltransferases (GTFs) produced by S. mutans appear to be major colonization factors. Various forms of periodontal diseases are closely associated with specific subgingival bacteria. Porphyromonas gingivalis has been implicated as an important etiological agent of adult periodontitis. Adherence of bacteria to host tissues is a prerequisite for colonization and one of the important steps in the disease process. Bacterial coaggregation factors and hemagglutinins likely play major roles in colonization in the subgingival area. Emerging evidence suggests that inhibition of these virulence factors may protect the host against caries and periodontal disease. Active and passive immunization approaches have been developed for immunotherapy of these diseases. Recent advances in mucosal immunology and the introduction of novel strategies for inducing mucosal immune responses now raise the possibility that effective and safe vaccines can be constructed. In this regard, some successful results have been reported in animal experimental models. Nevertheless, since the public at large might be skeptical about the seriousness of oral diseases, immunotherapy must be carried out with absolute safety. For this goal to be achieved, the development of safe antibodies for passive immunization is significant and important. In this review, salient advances in passive immunization against caries

  20. Development and use of a monoclonal antibody to detect semi-digested proteins of the English grain aphid, Sitobion avenae, in the guts of ladybird beetle predators

    NARCIS (Netherlands)

    Gao, S.J.; Zhou, X.R.; Pang, B.P.; Loon, van J.J.A.; Zhao, G.Q.

    2009-01-01

    A monoclonal antibody (McAb), EGA-4A9, was developed to detect the semi-digested proteins of the English grain aphid, Sitobion avenae (Fabricius) (Hemiptera: Aphididae), in predatory ladybird beetles (species of the genera Adonia, Coccinella, Hippodamia, and Propylea) using the gut homogenate of Ado

  1. Monoclonal antibodies to desmin: evidence for stage-dependent intermediate filament immunoreactivity during cardiac and skeletal muscle development.

    Science.gov (United States)

    Fischman, D A; Danto, S I

    1985-01-01

    Monoclonal antibodies reactive with desmin (D3 and D76) have been generated and their specificities validated by immunoblots, RIAs, and immunocytochemistry. No cross-reaction with other IFPs has been observed. The McAbs recognized different epitopes but both reside in the amino-terminal rod domain of desmin. Whereas McAb D3 produces a staining pattern characteristic of desmin throughout the development of cardiac and skeletal muscles, McAb D76 was selectively unreactive with certain regions of early (three days in ovo) embryonic cardiac anlage, with cultured cardiac myocytes derived from 7-day-old embryos, and with skeletal myotubes in early stages of myogenesis in vitro. Positive reactivity of D76 was seen at stages of myofibrillogenesis when the sarcomeres assume lateral alignment. Evidence was presented that differential reactivity of D76 did not result from the biosynthesis of a new desmin isoform or the post-translational modification of an existing protein. We suggest that the appearance of D76 immunoreactivity during striated muscle development represents an unmasking of the epitope by some IF-associated protein. Since this transition during skeletal muscle differentiation occurs during lateral alignment of the myofibrils, this antibody may serve as a useful probe for exploring this reorganization of the contractile apparatus during myogenesis and muscle regeneration.

  2. The Development of a Recombinant scFv Monoclonal Antibody Targeting Canine CD20 for Use in Comparative Medicine.

    Directory of Open Access Journals (Sweden)

    Saurabh Jain

    Full Text Available Monoclonal antibodies are leading agents for therapeutic treatment of human diseases, but are limited in use by the paucity of clinically relevant models for validation. Sporadic canine tumours mimic the features of some human equivalents. Developing canine immunotherapeutics can be an approach for modeling human disease responses. Rituximab is a pioneering agent used to treat human hematological malignancies. Biologic mimics that target canine CD20 are just being developed by the biotechnology industry. Towards a comparative canine-human model system, we have developed a novel anti-CD20 monoclonal antibody (NCD1.2 that binds both human and canine CD20. NCD1.2 has a sub-nanomolar Kd as defined by an octet red binding assay. Using FACS, NCD1.2 binds to clinically derived canine cells including B-cells in peripheral blood and in different histotypes of B-cell lymphoma. Immunohistochemical staining of canine tissues indicates that the NCD1.2 binds to membrane localized cells in Diffuse Large B-cell lymphoma, Marginal Zone Lymphoma, and other canine B-cell lymphomas. We cloned the heavy and light chains of NCD1.2 from hybridomas to determine whether active scaffolds can be acquired as future biologics tools. The VH and VL genes from the hybridomas were cloned using degenerate primers and packaged as single chains (scFv into a phage-display library. Surprisingly, we identified two scFv (scFv-3 and scFv-7 isolated from the hybridoma with bioactivity towards CD20. The two scFv had identical VH genes but different VL genes and identical CDR3s, indicating that at least two light chain mRNAs are encoded by NCD1.2 hybridoma cells. Both scFv-3 and scFv-7 were cloned into mammalian vectors for secretion in CHO cells and the antibodies were bioactive towards recombinant CD20 protein or peptide. The scFv-3 and scFv-7 were cloned into an ADEPT-CPG2 bioconjugate vector where bioactivity was retained when expressed in bacterial systems. These data identify a

  3. The Development of a Recombinant scFv Monoclonal Antibody Targeting Canine CD20 for Use in Comparative Medicine.

    Science.gov (United States)

    Jain, Saurabh; Aresu, Luca; Comazzi, Stefano; Shi, Jianguo; Worrall, Erin; Clayton, John; Humphries, William; Hemmington, Sandra; Davis, Paul; Murray, Euan; Limeneh, Asmare A; Ball, Kathryn; Ruckova, Eva; Muller, Petr; Vojtesek, Borek; Fahraeus, Robin; Argyle, David; Hupp, Ted R

    2016-01-01

    Monoclonal antibodies are leading agents for therapeutic treatment of human diseases, but are limited in use by the paucity of clinically relevant models for validation. Sporadic canine tumours mimic the features of some human equivalents. Developing canine immunotherapeutics can be an approach for modeling human disease responses. Rituximab is a pioneering agent used to treat human hematological malignancies. Biologic mimics that target canine CD20 are just being developed by the biotechnology industry. Towards a comparative canine-human model system, we have developed a novel anti-CD20 monoclonal antibody (NCD1.2) that binds both human and canine CD20. NCD1.2 has a sub-nanomolar Kd as defined by an octet red binding assay. Using FACS, NCD1.2 binds to clinically derived canine cells including B-cells in peripheral blood and in different histotypes of B-cell lymphoma. Immunohistochemical staining of canine tissues indicates that the NCD1.2 binds to membrane localized cells in Diffuse Large B-cell lymphoma, Marginal Zone Lymphoma, and other canine B-cell lymphomas. We cloned the heavy and light chains of NCD1.2 from hybridomas to determine whether active scaffolds can be acquired as future biologics tools. The VH and VL genes from the hybridomas were cloned using degenerate primers and packaged as single chains (scFv) into a phage-display library. Surprisingly, we identified two scFv (scFv-3 and scFv-7) isolated from the hybridoma with bioactivity towards CD20. The two scFv had identical VH genes but different VL genes and identical CDR3s, indicating that at least two light chain mRNAs are encoded by NCD1.2 hybridoma cells. Both scFv-3 and scFv-7 were cloned into mammalian vectors for secretion in CHO cells and the antibodies were bioactive towards recombinant CD20 protein or peptide. The scFv-3 and scFv-7 were cloned into an ADEPT-CPG2 bioconjugate vector where bioactivity was retained when expressed in bacterial systems. These data identify a recombinant anti-CD20

  4. Generation of Recombinant Porcine Parvovirus Virus-Like Particles in Saccharomyces cerevisiae and Development of Virus-Specific Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Paulius Lukas Tamošiūnas

    2014-01-01

    Full Text Available Porcine parvovirus (PPV is a widespread infectious virus that causes serious reproductive diseases of swine and death of piglets. The gene coding for the major capsid protein VP2 of PPV was amplified using viral nucleic acid extract from swine serum and inserted into yeast Saccharomyces cerevisiae expression plasmid. Recombinant PPV VP2 protein was efficiently expressed in yeast and purified using density gradient centrifugation. Electron microscopy analysis of purified PPV VP2 protein revealed the self-assembly of virus-like particles (VLPs. Nine monoclonal antibodies (MAbs against the recombinant PPV VP2 protein were generated. The specificity of the newly generated MAbs was proven by immunofluorescence analysis of PPV-infected cells. Indirect IgG ELISA based on the recombinant VLPs for detection of PPV-specific antibodies in swine sera was developed and evaluated. The sensitivity and specificity of the new assay were found to be 93.4% and 97.4%, respectively. In conclusion, yeast S. cerevisiae represents a promising expression system for generating recombinant PPV VP2 protein VLPs of diagnostic relevance.

  5. Generation of Recombinant Schmallenberg Virus Nucleocapsid Protein in Yeast and Development of Virus-Specific Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Justas Lazutka

    2014-01-01

    Full Text Available Schmallenberg virus (SBV, discovered in continental Europe in late 2011, causes mild clinical signs in adult ruminants, including diarrhoea and reduced milk yield. However, fetal infection can lead to severe malformation in newborn offspring. To develop improved reagents for SBV serology, a high-level yeast expression system was employed to produce recombinant SBV nucleocapsid (N protein. Recombinant SBV N protein was investigated as an antigen in SBV-specific IgG enzyme immunoassay and used for generation of monoclonal antibodies (MAbs. Yeast-expressed SBV N protein was reactive with anti-SBV IgG-positive cow serum specimens collected from different farms of Lithuania. After immunization of mice with recombinant SBV N protein, four MAbs were generated. The MAbs raised against recombinant SBV N protein reacted with native viral nucleocapsids in SBV-infected BHK cells by immunofluorescence assay. The reactivity of recombinant N protein with SBV-positive cow serum specimens and the ability of the MAbs to recognize virus-infected cells confirm the antigenic similarity between yeast-expressed SBV N protein and native viral nucleocapsids. Our study demonstrates that yeast expression system is suitable for high-level production of recombinant SBV N protein and provides the first evidence on the presence of SBV-specific antibodies in cow serum specimens collected in Lithuania.

  6. Development and characterization of monoclonal antibodies against Shiga toxin 2 and their application for toxin detection in milk.

    Science.gov (United States)

    He, Xiaohua; McMahon, Stephanie; Skinner, Craig; Merrill, Paul; Scotcher, Miles C; Stanker, Larry H

    2013-03-29

    Human infection by Shiga toxin producing Escherichia coli (STEC) is one of the most prevalent foodborne diseases. Shiga toxin type 2 (Stx2) is the major contributor to hemolytic-uremic syndrome (HUS) and other systemic complications caused by STEC. Although outbreaks of HUS due to the consumption of dairy products occur frequently, very few reports are available on assays for the detection of Stx2 in milk. In this study, we describe the development of five high-affinity monoclonal antibodies (dissociation constants below nM range) against Stx2 using a recombinant toxoid as an immunogen. These antibodies, designated Stx2-1, Stx2-2, Stx2-3, Stx2-4, and Stx2-5 are IgG1 or IgG2a heavy-chain subclass with kappa light-chains, did not cross-react with Stx1 and showed different preferences to variants of Stx2. Western blot analyses demonstrate that mAbs Stx2-2 and Stx2-5 bind both the A- and B-subunits, whereas the other 3 mAbs bind the A-subunit of Stx2a only. All antibodies bound stronger to the native than to the denatured Stx2a except the mAb Stx2-3, which bound equally well to both forms of the toxin. Of the five mAbs, Stx2-5 was capable of neutralizing Stx2a mediated cytotoxicity in Vero cells. Highly sensitive ELISA and immuno-PCR assays, capable of detecting 1 and 0.01 pg/mL of Stx2a in milk, were developed using mAb pair Stx2-1 and Stx2-2. Such assays are useful for routine diagnosis of Stx2 contamination in milk production process, thus reducing the risk of STEC outbreaks. Published by Elsevier B.V.

  7. Development of monoclonal antibodies and quantitative sandwich enzyme linked immunosorbent assay for the characteristic sialoglycoprotein of edible bird's nest.

    Science.gov (United States)

    Zhang, Shiwei; Lai, Xintian; Liu, Xiaoqing; Li, Yun; Li, Bifang; Huang, Xiuli; Zhang, Qinlei; Chen, Wei; Lin, Lin; Yang, Guowu

    2013-01-01

    The article presents a sandwich enzyme linked immunosorbent assay (ELISA) for identification of edible bird's nest. The characteristic sialoglycoproteins were found by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and purified by liquid-phase isoelectric focusing (LIEF). According to the analysis, the molecular weight was 106-128 kDa and the isoelectric point was ≤pH 3.0. Two anti-characteristic sialoglycoprotein monoclonal antibodies were produced. The monoclonal antibodies were examined by western-blot assay. One of the monoclonal antibody was used as coating and the other as the enzyme-labeled antibody after being coupled to horseradish peroxidase (HRP). Based on the optimized ELISA condition, the method was established with IC(50) of 1.5 ng/mL, and low cross-reactivity with various fake materials (ELISA provided a suitable means for screening of a large number of samples. The coefficients of variation were between 2.9% and 5.8%.

  8. Monoclonal antibodies in animal production; their use in diagnostics and passive immunization.

    NARCIS (Netherlands)

    Booman, P.

    1989-01-01

    One of the landmarks in immunology was the invention and development of monoclonal antibody-secreting hybridomas by Milstein and his coworkers. The enormous promise of monoclonal antibody technology, which became apparent soon after its discovery, may explain the unusual speed with which monoclonal

  9. Development and characterization of human monoclonal antibodies that neutralize multiple TGFβ isoforms.

    Science.gov (United States)

    Bedinger, Daniel; Lao, Llewelyn; Khan, Shireen; Lee, Steve; Takeuchi, Toshihiko; Mirza, Amer M

    2016-01-01

    Transforming growth factor (TGF)β levels are elevated in, and drive the progression of, numerous disease states such as advanced metastatic cancer and systemic and ocular fibrosis. There are 3 main isoforms, TGFβ1, 2, and 3. As multiple TGFβ isoforms are involved in disease processes, maximal therapeutic efficacy may require neutralization of 2 or more of the TGFβ isoforms. Fully human antibody phage display libraries were used to discover a number of antibodies that bind and neutralize various combinations of TGFβ1, 2 or 3. The primary panning did not yield any uniformly potent pan-isoform neutralizing antibodies; therefore, an antibody that displayed potent TGFβ 1, 2 inhibition, but more modest affinity versus TGFβ3, was affinity matured by shuffling with a light chain sub-library and further screening. This process yielded a high affinity pan-isoform neutralizing clone. Antibodies were analyzed and compared by binding affinity, as well as receptor and epitope competition by surface plasmon resonance methods. The antibodies were also shown to neutralize TGFβ effects in vitro in 3 assays: 1) interleukin (IL)-4 induced HT-2 cell proliferation; 2) TGFβ-mediated IL-11 release by A549 cells; and 3) decreasing SMAD2 phosphorylation in Detroit 562 cells. The antibodies' potency in these in vitro assays correlated well with their isoform-specific affinities. Furthermore, the ability of the affinity-matured clone to decrease tumor burden in a Detroit 562 xenograft study was superior to that of the parent clone. This affinity-matured antibody acts as a very potent inhibitor of all 3 main isoforms of TGFβ and may have utility for therapeutic intervention in human disease.

  10. Monoclonal antibodies based on hybridoma technology.

    Science.gov (United States)

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

    2013-03-01

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

  11. A protein-conjugate approach to develop a monoclonal antibody-based antigen detection test for the diagnosis of human brucellosis.

    Science.gov (United States)

    Patra, Kailash P; Saito, Mayuko; Atluri, Vidya L; Rolán, Hortensia G; Young, Briana; Kerrinnes, Tobias; Smits, Henk; Ricaldi, Jessica N; Gotuzzo, Eduardo; Gilman, Robert H; Tsolis, Renee M; Vinetz, Joseph M

    2014-06-01

    Human brucellosis is most commonly diagnosed by serology based on agglutination of fixed Brucella abortus as antigen. Nucleic acid amplification techniques have not proven capable of reproducibly and sensitively demonstrating the presence of Brucella DNA in clinical specimens. We sought to optimize a monoclonal antibody-based assay to detect Brucella melitensis lipopolysaccharide in blood by conjugating B. melitensis LPS to keyhole limpet hemocyanin, an immunogenic protein carrier to maximize IgG affinity of monoclonal antibodies. A panel of specific of monoclonal antibodies was obtained that recognized both B. melitensis and B. abortus lipopolysaccharide epitopes. An antigen capture assay was developed that detected B. melitensis in the blood of experimentally infected mice and, in a pilot study, in naturally infected Peruvian subjects. As a proof of principle, a majority (7/10) of the patients with positive blood cultures had B. melitensis lipopolysaccharide detected in the initial blood specimen obtained. One of 10 patients with relapsed brucellosis and negative blood culture had a positive serum antigen test. No seronegative/blood culture negative patients had a positive serum antigen test. Analysis of the pair of monoclonal antibodies (2D1, 2E8) used in the capture ELISA for potential cross-reactivity in the detection of lipopolysaccharides of E. coli O157:H7 and Yersinia enterocolitica O9 showed specificity for Brucella lipopolysaccharide. This new approach to develop antigen-detection monoclonal antibodies against a T cell-independent polysaccharide antigen based on immunogenic protein conjugation may lead to the production of improved rapid point-of-care-deployable assays for the diagnosis of brucellosis and other infectious diseases.

  12. Development of a rapid RP-UHPLC-MS method for analysis of modifications in therapeutic monoclonal antibodies.

    Science.gov (United States)

    Zhang, Bing; Jeong, Justin; Burgess, Braydon; Jazayri, Mansour; Tang, Yun; Taylor Zhang, Yonghua

    2016-10-01

    Chemical or enzymatic modifications of therapeutic monoclonal antibodies (MAbs) that have high risk to safety and efficacy are defined as critical quality attributes (CQAs). During therapeutic MAbs process development, thorough characterization and quantitative monitoring of CQAs requires a variety of analytical techniques. This paper describes the development of a rapid analytical method to assess modifications in MAbs, based on the analysis of subdomains with molecular weights of ∼25kDa. These subdomains were generated by digestion with a highly specific IdeS protease, followed by disulfide bond reduction. A reversed phase UHPLC-MS method was developed that provides efficient separation and identification of the subdomains (Fc, LC, and Fd) and related variants within 10min. Sample preparation and UHPLC instrument parameters were systematically evaluated. The methodology was applied to MAb stress panel characterization to capture the degradations induced by various stress conditions. Among the CQAs monitored by this method, Fc oxidation levels were compared with the values obtained by the more complicated and time-consuming peptide mapping method. The similar trends observed by the two methods demonstrated that the IdeS-UHPLC method is valuable as a higher throughput alternative to peptide mapping for monitoring modifications. In particular, a high-throughput methodology is preferred for analysis of the many samples associated with process development studies. Overall the method has been demonstrated as a fast, convenient and informative platform approach for analysis of therapeutic MAbs modifications including CQAs. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Distinct expression profiles of Notch-1 protein in human solid tumors: Implications for development of targeted therapeutic monoclonal antibodies

    OpenAIRE

    Li, Yuan; Burns, Janine A.; Carol A Cheney; Zhang, Ningyan; Vitelli, Salvatore; Wang, Fubao; Bett, Andrew; Chastain, Michael; Audoly, Laurent P.; Zhang, Zhi-Qiang

    2010-01-01

    Biological therapies, such as monoclonal antibodies (mAbs) that target tumor-associated antigens have been considered an effective therapeutic approach in oncology. In considering Notch-1 receptor as a potential target, we performed immunohistochemistry on tissue microarrays to determine 1) whether the receptor is overexpressed in tumor cells as compared to their corresponding normal tissues and 2) the clinical significance of its expression levels in human breast, colorectal, lung and prosta...

  14. Development of a mouse monoclonal antibody against the chondroitin sulfate-protein linkage region derived from shark cartilage.

    Science.gov (United States)

    Akatsu, Chizuru; Fongmoon, Duriya; Mizumoto, Shuji; Jacquinet, Jean-Claude; Kongtawelert, Prachya; Yamada, Shuhei; Sugahara, Kazuyuki

    2010-05-01

    Glycosaminoglycans (GAGs) like chondroitin sulfate (CS) and heparan sulfate (HS) are synthesized on the tetrasaccharide linkage region, GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser, of proteoglycans. The Xyl can be modified by 2-O-phosphate in both CS and HS, whereas the Gal residues can be sulfated at C-4 and/or C-6 in CS but not in HS. To study the roles of these modifications, monoclonal antibodies were developed against linkage glycopeptides of shark cartilage CS proteoglycans, and one was characterized in detail. This antibody bound hexa- and pentasaccharide-peptides more strongly than unsaturated tetrasaccharide-peptides with the unnatural fourth sugar residue (unsaturated hexuronic acid), suggesting the importance of the fifth and/or fourth saccharide residue GalNAc-5 and/or GlcA-4. Its reactivity was not affected by treatment with chondro-4-sulfatase or alkaline phosphatase, suggesting that 4-O-sulfate on the Gal residues and 2-O-phosphate on the Xyl residue were not recognized. Treatment with weak alkali to cleave the Xyl-Ser linkage completely abolished the binding activity, suggesting the importance of the peptide moiety of the hexasaccharide-peptide for the binding. Based on the amino acid composition and matrix-assisted laser desorption ionization time-of-flight mass spectrometry analyses, it was revealed that the peptide moiety is composed of four amino acids, Ser, Pro, Gly, and Glu. Furthermore, the antibody stained wild-type CHO cells significantly, but much weakly mutant cells deficient in xylosyl- or galactosyltransferase-I required for the biosynthesis of the linkage region. These results suggest that the antibody recognizes the structure GalNAc(+/-6-O-sulfate)-GlcA-Gal-Gal-Xyl-Ser-(Pro, Gly, Glu). The antibody will be a useful tool for investigating the significance of the linkage region in the biosynthesis and/or intracellular transport of different GAG chains especially since such tools to study the linkage region are lacking.

  15. Monoclonal antibodies AC-43 and AC-29 disrupt Plasmodium vivax development in the Indian malaria vector Anopheles culicifacies (Diptera: culicidae)

    Indian Academy of Sciences (India)

    Manoj Chugh; B R Gulati; S K Gakhar

    2010-03-01

    A repertoire of monoclonal antibodies (mAbs) was generated against the midgut proteins of Anopheles culicifacies mosquitoes. The mAbs AC-43 and AC-29 significantly inhibited Plasmodium vivax development inside the mosquito midgut. The number of oocysts that developed was reduced by 78.6% when mosquitoes ingested a combination of these two mAbs along with the blood meal. AC-43 mAb binds to the epitope common in 97, 80 and 43 kDa polypeptides from the midgut protein extract, as indicated by western blot analysis. Similarly, the mAb AC-29 recognized 52, 44, 40 and 29 kDa polypeptides. These female midgut-specific polypeptides are shared between An. culicifacies and An. stephensi, two major vectors of malaria in India. Deglycosylation assays revealed that -linked carbohydrates are the major components in epitopes corresponding to AC-43 and AC-29. Gold particle labelling revealed that both these mAbs preferentially bind to glycoproteins at the apical microvilli and the microvillus-associated network present inside transverse sections of the gut epithelium. These regions are particularly known to have receptors for ookinetes, which enable them to cross this epithelial barrier and provide them with certain necessary chemicals or components for further development into oocysts. Therefore, these glycoproteins appear to be potential candidates for a vectordirected transmission-blocking vaccine (TBV).

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

    Science.gov (United States)

    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.

  17. Development of novel monoclonal antibodies that define differentiation stages of human stromal (mesenchymal) stem cells

    DEFF Research Database (Denmark)

    Andersen, Ditte Caroline; Kortesidis, Angela; Zannettino, Andrew C W;

    2011-01-01

    fewer differentiated alkaline phosphatase(+) cells compared to STRO-1(+/-)/Collagen VI(+) hMSC, suggesting that Collagen VI on the cell membrane exclusively defines differentiated MSCs. In conclusion, we have generated a panel of high quality antibodies to be used for characterization of MSCs...... of clonogenic hMSC from BMMNCs as single reagents. Using mass-spectrometric analysis, we identified the antigen recognised by DJ3 as CD44, whereas DJ9 and DJ18 recognized HLA-DRB1 and Collagen VI, respectively. The identified proteins were highly expressed throughout in vitro osteogenic- and adipogenic...... mice with hMSC, and by using a panel of subsequent screening methods. Flow cytometry analysis revealed that 83.5, 1.1, and 8.5% of primary cultures of hMSC were double positive for STRO-1 and either of DJ 3, 9, and 18, respectively. However, none of the three DJ antibodies allowed enrichment...

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

    Science.gov (United States)

    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.

  19. Preparation and Identification of Anti-rabies Virus Monoclonal Antibodies

    Institute of Scientific and Technical Information of China (English)

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

    2012-01-01

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

  20. Monoclonal antibodies to Treponema Pallidum.

    NARCIS (Netherlands)

    H.J.M. van de Donk; J.D.A. van Embden; M.F. van Olderen; A.D.M.E. Osterhaus (Albert); J.C. de Jong (Jan)

    1984-01-01

    textabstractThree successive fusions of mouse myeloma cells and spleen lymphocytes of a mouse immunized with Treponema Pallidum resulted in one hybridoma producing anti T. pallidum antibodies for each fusion. The mice were immunized with live pallidum cells respectively 1, 3 and 5 months before fusi

  1. Effects of ICOS+ T cell depletion via afucosylated monoclonal antibody MEDI-570 on pregnant cynomolgus monkeys and the developing offspring.

    Science.gov (United States)

    Nicholson, Simone M; Carlesso, Gianluca; Cheng, Lily I; Cook, Halie; DaCosta, Karma; Leininger, Joel; McKeever, Kathleen; Scott, Stephen Weasel; Taylor, Devon; Streicher, Katie; Eck, Steve; Reed, Molly; Faggioni, Raffaella; Herbst, Ronald; Dixit, Rakesh; Ryan, Patricia C

    2017-09-13

    MEDI-570 is a fully human afucosylated monoclonal antibody (MAb) against Inducible T-cell costimulator (ICOS), highly expressed on CD4+ T follicular helper (TFH) cells. Effects of MEDI-570 were evaluated in an enhanced pre-postnatal development toxicity (ePPND) study in cynomolgus monkeys. Administration to pregnant monkeys did not cause any abortifacient effects. Changes in hematology and peripheral blood T lymphocyte subsets in maternal animals and infants and the attenuated infant IgG immune response to keyhole limpet hemocyanin (KLH) were attributed to MEDI-570 pharmacology. Adverse findings included aggressive fibromatosis in one dam and two infant losses in the high dose group with anatomic pathology findings suggestive of atypical lymphoid hyperplasia. The margin of safety relative to the no observed adverse effect level (NOAEL) for the highest planned clinical dose in the Phase 1a study was 7. This study suggests that women of child bearing potential employ effective methods of contraception while being treated with MEDI-570. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. ERBB oncogene proteins as targets for monoclonal antibodies.

    Science.gov (United States)

    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.

  3. Human Monoclonal Antibodies as a Countermeasure Against Botulinum Toxins

    Science.gov (United States)

    2012-11-30

    REPORT Human monoclonal antibodies as a countermeasure against Botulinum toxins 14. ABSTRACT 16. SECURITY CLASSIFICATION OF: In this report, we...Prescribed by ANSI Std. Z39.18 - 31-Aug-2012 Human monoclonal antibodies as a countermeasure against Botulinum toxins Report Title ABSTRACT In this report...DTRA Final Report: Human monoclonal antibodies as a countermeasure against Botulinum toxins   Page 1 of 22 DTRA Final Report: Human monoclonal

  4. Development of a monoclonal antibody-based enzyme-linked immunosorbent assay to quantify soluble beta-glucans in oats and barley.

    Science.gov (United States)

    Rampitsch, Christof; Ames, Nancy; Storsley, Joanne; Marien, Lindsay

    2003-09-24

    A set of 31 murine monoclonal antibodies was produced against (1-->3,1-->4)beta-d-glucan from oats (Avena sativa L.) chemically cross-linked to keyhole limpet hemocyanin. Monoclonal antibodies were tested for their cross-reactivity to related and unrelated polysaccharides. The antibodies reacted strongly to unmodified beta-glucan from oats and barley (Hordeum vulgare L.) and to lichenan from Icelandic moss, a polysaccharide with a structure similar to that of beta-glucan but which is not encountered in cereals. Cross-reaction to other polysaccharides tested was minimal at physiological levels. An enzyme-linked immunosorbent assay (ELISA) that could routinely detect and quantify nanogram levels of soluble beta-glucan extracted from the flour of oats or barley was designed with one of these monoclonal antibodies. The beta-glucan extraction procedure from ground oat and barley samples and the ELISA were both optimized for reproducibility, accuracy, and throughput, and results were compared to values obtained from an established, commercially available enzyme-based assay. Correlations between the two assays were consistently high (r (2) > 0.9), indicating that the ELISA presented in this paper is a valuable alternative for assaying beta-glucan levels in cereals and cereal products, both routinely and in preparations in which beta-glucans are present in nanogram amounts. Development of the extraction procedure for ELISA is discussed.

  5. Analytical QbD: development of a native gel electrophoresis method for measurement of monoclonal antibody aggregates.

    Science.gov (United States)

    Pathak, Mili; Dutta, Debayon; Rathore, Anurag

    2014-08-01

    This paper presents a quality by design (QbD) based development of a novel native PAGE (N-PAGE) method as a low-cost analytical tool for analysis of aggregates of monoclonal antibodies. Comparability to the present gold standard of SEC has been established. The motivation is the fact that SEC requires relatively expensive equipment and consumables, thus making N-PAGE relevant to those academicians and other small companies involved in early-stage development of biotherapeutics that do not have access to SEC, especially in developing countries. Furthermore, SEC suffers from certain disadvantages including the possibility of secondary interactions between the stationary phase and analyte resulting in higher elution time and therefore underestimation of the analyte size. The proposed N-PAGE method can also serve as an orthogonal analytical method for aggregate analysis. A QbD-based approach has been used for development and optimization of the protocol. First, initial screening studies were carried out with parameters including the running buffer pH, running buffer molarity, gel buffer pH, loading dye, sample concentration, and running voltage. Next, optimization of operating parameters was performed using principles of design of experiments. The final optimized protocol was compared to the traditional SEC method and the results were found to be comparable. While N-PAGE has been in use for protein analysis for several decades, use of N-PAGE for analysis of mAb aggregates with data comparable to SEC such as the case presented here is novel. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Development and characterization of neutralizing monoclonal antibodies against canine distemper virus hemagglutinin protein.

    Science.gov (United States)

    Bi, Zhenwei; Xia, Xingxia; Wang, Yongshan; Mei, Yongjie

    2015-04-01

    Canine distemper virus (CDV) causes a serious multisystemic disease in dogs and other carnivora. Hemagglutinin (H) protein-specific antibodies are mainly responsible for protective immunity against CDV infection. In the present study, six neutralizing MAbs to the H protein of CDV were newly obtained and characterized by immunizing BALB/c mice with a recent Chinese field isolate. Competitive binding inhibition assay revealed that they recognized four distinct antigenic regions of the H protein. Immunofluorescence assay and western blotting showed that all MAbs recognize the conformational rather than the linear epitopes of the H protein. Furthermore, in immunofluorescence and virus neutralization assays, two of the MAbs were found to react only with the recent Chinese field isolate and not with older CDV strains, including vaccine strain Onderstepoort, indicating there are neutralization-related antigenic variations between the recent Chinese field isolate and the older CDV strains examined in this study. The newly established MAbs are useful for differentiating the expanding CDV strains and could be used in immunotherapy and immunodiagnosis against infection with CDV.

  7. Monoclonal Antibodies to Plant Growth Regulators

    Science.gov (United States)

    Eberle, Joachim; Arnscheidt, Angelika; Klix, Dieter; Weiler, Elmar W.

    1986-01-01

    Four high affinity monoclonal antibodies, which recognize two plant growth regulators from the cytokinin group, namely trans-zeatin riboside and dihydrozeatin riboside and their derivatives are reported. Six hybridomas were produced from three independent fusions of Balb/c spleen cells with P3-NS1-Ag 4-1 (abbreviated NS1) or X63-Ag 8.653 (X63) myeloma cells. The mice had been hyperimmunized with zeatin riboside-bovine serum albumin conjugate or dihydrozeatin riboside-bovine serum albumin conjugate for 3 months. The hybridomas secrete antibodies of the IgG 1 or IgG 2b subclass and allow the detection of femtomole amounts of the free cytokinins, their ribosides, and ribotides in plant extracts. The use of these monoclonals in radio- and enzyme-linked immunosorbent assay is also discussed. PMID:16664848

  8. Development and characterization of a panel of cross-reactive monoclonal antibodies generated using H1N1 influenza virus.

    Science.gov (United States)

    Guo, Chun-yan; Tang, Yi-gui; Qi, Zong-li; Liu, Yang; Zhao, Xiang-rong; Huo, Xue-ping; Li, Yan; Feng, Qing; Zhao, Peng-hua; Wang, Xin; Li, Yuan; Wang, Hai-fang; Hu, Jun; Zhang, Xin-jian

    2015-08-01

    To characterize the antigenic epitopes of the hemagglutinin (HA) protein of H1N1 influenza virus, a panel consisting of 84 clones of murine monoclonal antibodies (mAbs) were generated using the HA proteins from the 2009 pandemic H1N1 vaccine lysate and the seasonal influenza H1N1(A1) vaccines. Thirty-three (39%) of the 84 mAbs were found to be strain-specific, and 6 (7%) of the 84 mAbs were subtype-specific. Twenty (24%) of the 84 mAbs recognized the common HA epitopes shared by 2009 pandemic H1N1, seasonal A1 (H1N1), and A3 (H3N2) influenza viruses. Twenty-five of the 84 clones recognized the common HA epitopes shared by the 2009 pandemic H1N1, seasonal A1 (H1N1) and A3 (H3N2) human influenza viruses, and H5N1 and H9N2 avian influenza viruses. We found that of the 16 (19%) clones of the 84 mAbs panel that were cross-reactive with human respiratory pathogens, 15 were made using the HA of the seasonal A1 (H1N1) virus and 1 was made using the HA of the 2009 pandemic H1N1 influenza virus. Immunohistochemical analysis of the tissue microarray (TMA) showed that 4 of the 84 mAb clones cross-reacted with human tissue (brain and pancreas). Our results indicated that the influenza virus HA antigenic epitopes not only induce type-, subtype-, and strain-specific monoclonal antibodies against influenza A virus but also cross-reactive monoclonal antibodies against human tissues. Further investigations of these cross-reactive (heterophilic) epitopes may significantly improve our understanding of viral antigenic variation, epidemics, pathophysiologic mechanisms, and adverse effects of influenza vaccines.

  9. Improved detection of Pneumocystis carinii by an immunofluorescence technique using monoclonal antibodies

    DEFF Research Database (Denmark)

    Orholm, M; Holten-Andersen, W; Lundgren, Jens Dilling

    1990-01-01

    To assess whether a recently developed indirect immunofluorescent stain using monoclonal antibodies was more sensitive in detecting Pneumocystis carinii than the combination of Giemsa and methenamine silver nitrate stains which has routinely been used in the laboratory, 88 lavage fluid specimens...... and 34 induced sputum specimens were examined. All specimens were stained by five techniques: immunofluorescence using a combination of three monoclonal antibodies (from the National Institutes of Health, USA), immunofluorescence using a single monoclonal antibody (from Dakopatts), Giemsa, methenamine...

  10. Monoclonal antibodies for the detection of Puccinia striiformis urediniospores

    DEFF Research Database (Denmark)

    Skottrup, Peter Durand; Frøkiær, Hanne; Hearty, Stephen

    2007-01-01

    The fungal pathogen Pst causes yellow rust disease in wheat plants leading to crop losses. The organism spreads by releasing wind-dispersed urediniospores from infected plants. In this study a library of novel monoclonal antibodies (mAbs) was developed against Pst urediniospores. Nine mAb-produci...

  11. Development of a bead-based Luminex assay using lipopolysaccharide specific monoclonal antibodies to detect biological threats from Brucella species.

    Science.gov (United States)

    Silbereisen, Angelika; Tamborrini, Marco; Wittwer, Matthias; Schürch, Nadia; Pluschke, Gerd

    2015-10-05

    Brucella, a Gram-negative bacterium, is classified as a potential bioterrorism agent mainly due to the low dose needed to cause infection and the ability to transmit the bacteria via aerosols. Goats/sheep, cattle, pigs, dogs, sheep and rodents are infected by B. melitensis, B. abortus, B. suis, B. canis, B. ovis and B. neotomae, respectively, the six classical Brucella species. Most human cases are caused by B. melitensis and B. abortus. Our aim was to specifically detect Brucellae with 'smooth' lipopolysaccharide (LPS) using a highly sensitive monoclonal antibody (mAb) based immunological assay. To complement molecular detection systems for potential bioterror agents, as required by international biodefense regulations, sets of mAbs were generated by B cell hybridoma technology and used to develop immunological assays. The combination of mAbs most suitable for an antigen capture assay format was identified and an immunoassay using the Luminex xMAP technology was developed. MAbs specific for the LPS O-antigen of Brucella spp. were generated by immunising mice with inactivated B. melitensis or B. abortus cells. Most mAbs recognised both B. melitensis and B. abortus and antigen binding was not impeded by inactivation of the bacterial cells by γ irradiation, formalin or heat treatment, a step required to analyse the samples immunologically under biosafety level two conditions. The Luminex assay recognised all tested Brucella species with 'smooth' LPS with detection limits of 2×10(2) to 8×10(4) cells per mL, depending on the species tested. Milk samples spiked with Brucella spp. cells were identified successfully using the Luminex assay. In addition, the bead-based immunoassay was integrated into a multiplex format, allowing for simultaneous, rapid and specific detection of Brucella spp., Bacillus anthracis, Francisella tularensis and Yersinia pestis within a single sample. Overall, the robust Luminex assay should allow detection of Brucella spp. in both natural

  12. Distinct expression profiles of Notch-1 protein in human solid tumors: Implications for development of targeted therapeutic monoclonal antibodies

    Directory of Open Access Journals (Sweden)

    Yuan Li

    2010-06-01

    Full Text Available Yuan Li1, Janine A Burns1, Carol A Cheney1, Ningyan Zhang1, Salvatore Vitelli1, Fubao Wang1, Andrew Bett2, Michael Chastain2, Laurent P Audoly1, Zhi-Qiang Zhang1,31Department of Biologics Research, 2Department of Vaccine Research, Merck Research Laboratories, West Point, PA, USA; 3Clinical Development Laboratory, Merck Research Laboratories, Rahway, NJ, USAAbstract: Biological therapies, such as monoclonal antibodies (mAbs that target tumor-associated antigens have been considered an effective therapeutic approach in oncology. In considering Notch-1 receptor as a potential target, we performed immunohistochemistry on tissue microarrays to determine 1 whether the receptor is overexpressed in tumor cells as compared to their corresponding normal tissues and 2 the clinical significance of its expression levels in human breast, colorectal, lung and prostate cancers. We found that the expression of Notch-1 protein was overexpressed in primary colorectal adenocarcinoma and nonsmall cell lung carcinoma (NSCLC, but not in primary ductal breast carcinoma or prostate adenocarcinoma. Further analysis revealed that higher levels of Notch-1 protein expression were significantly associated with poorer differentiation of breast and prostate tumors. Strikingly, for NSCLC, the expression levels of Notch-1 protein were found to be inversely correlated with tumor differentiation and progression. For colorectal tumors, however, no correlation of Notch-1 protein expression was found with any tumor clinicopathological parameters, in spite of its overexpression in tumor cells. Our data demonstrated the complexity of Notch-1 protein expression in human solid tumors and further supported the notion that the roles of Notch-1 expression in tumorigenesis are highly context-dependent. The findings could provide the basis for development of distinct therapeutic strategies of Notch-1 mAbs for its applications in the treatment of suitable types of human cancers.Keywords: Notch

  13. Assay for the specificity of monoclonal antibodies in crossed immunoelectrophoresis

    DEFF Research Database (Denmark)

    Skjødt, K; Schou, C; Koch, C

    1984-01-01

    A method is described based on crossed immunoelectrophoresis of a complex antigen mixture in agarose gel followed by incubation of the gel with the monoclonal antibody. The bound monoclonal antibody is detected by the use of a secondary enzyme-labelled antibody. Using this technique we have been...... I molecules. In other experiments using the same technique we demonstrated the reaction of a monoclonal antibody specific for chicken Ig light chains. Udgivelsesdato: 1984-Aug-3...

  14. Development and characterization of monoclonal antibody against non-structural protein-2 of Chikungunya virus and its application.

    Science.gov (United States)

    Chattopadhyay, Soma; Kumar, Abhishek; Mamidi, Prabhudutta; Nayak, Tapas Kumar; Das, Indrani; Chhatai, Jagamohan; Basantray, Itishree; Bramha, Umarani; Maiti, Prasanta Kumar; Singh, Sujay; Suryawanshi, Amol Ratnakar; Chattopadhyay, Subhasis

    2014-04-01

    The recent epidemics of Chikungunya viruses (CHIKV) with unprecedented magnitude and unusual clinical severity have raised a great public health concern worldwide, especially due to unavailability of vaccine or specific therapy. This emphasizes the need to understand the biological processes of this virus in details. Although CHIKV associated research has been initiated, the availability of CHIKV specific reagents for in-depth investigation of viral infection and replication are scanty. For Alphavirus replication, non-structural protein 2 (nsP2) is known to play a key regulatory role among all other non-structural proteins. The current study describes the development and characterization of nsP2 specific monoclonal antibody (mAb) against a synthetic peptide of CHIKV. Reactivity and efficacy of this mAb have been demonstrated by ELISA, Western blot, Flow cytometry and Immunofluorescence assay. Time kinetic study confirms that this mAb is highly sensitive to CHIKV-nsP2 as this protein has been detected very early during viral replication in infected cells. Homology analysis of the selected epitope sequence reveals that it is conserved among all the CHIKV strains of different genotypes, while analysis with other Alphavirus sequences shows that none of them are 100% identical to the epitope sequence. Moreover, using the mAb, three isoforms of CHIKV-nsP2 have been detected in 2D blot analysis during infection in mammalian cells. Accordingly, it can be suggested that the mAb reported in this study can be a sensitive and specific tool for experimental investigations of CHIKV replication and infection.

  15. Preparation of a specific monoclonal antibody to asiaticoside for the development of an enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Juengwatanatrakul, Thaweesak; Sritularak, Boonchoo; Amornnopparattanakul, Paveena; Tassanawat, Patcharin; Putalun, Waraporn; Tanaka, Hiroyuki; Morimoto, Satoshi

    2011-03-01

    Asiaticoside (AS), the major active component of Centella asiatica (L.) Urban, is used as a memory enhancer and for wound healing. We have successfully prepared monoclonal antibodies (MAbs) against AS, and developed an enzyme-linked immunosorbent assay (ELISA) system for its determination. AS was conjugated to the carrier protein bovine serum albumin (BSA), which acted as an immunogen. In order to confirm its immunogenicity, the ratio of hapten in the AS-BSA conjugate was determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). After immunization, hybridomas secreting MAbs against AS were produced by fusing splenocytes with the mouse myeloma cell line, SP2/0-Ag14. After the screening, anti-asiaticoside MAb 2B4 was obtained. Weak cross-reactivities occurred with madecassoside (7.08%), but no cross-reactivities were observed with other related triterpenoid glycosides (<0.01%). The assay was suitable for quantitating AS in the range of 0.78 to 50 µg mL(-1). A good correlation of AS concentrations in crude extracts of C. asiatica between ELISA and HPLC methods was obtained (r(2) = 0.999). The contents of AS in various cultivated C. asiatica samples were assayed by the newly established ELISA. The recovery rates of AS in the samples were in the range of 95-103% with coefficients of variation of <10%. The intra- and inter-assay variations were 3.9 and 4.5%, respectively. The ELISA method described should prove useful as an analytical tool for quality control and standardization of medicinal plants and pharmaceutical products containing AS.

  16. Development of Monoclonal Antibodies That Target 1-Cys Peroxiredoxin and Differentiate Plasmodium falciparum from P. vivax and P. knowlesi.

    Science.gov (United States)

    Hakimi, Hassan; Nguyen, Thu-Thuy; Suganuma, Keisuke; Masuda-Suganuma, Hirono; Angeles, Jose Ma M; Inoue, Noboru; Kawazu, Shin-Ichiro

    2013-06-01

    Prompt and accurate diagnosis of malarial patients is a crucial factor in controlling the morbidity and mortality of the disease. Effective treatment decisions require a correct diagnosis among mixed-species malarial patients. Differential diagnosis is particularly important in cases of Plasmodium vivax, a species that shares endemicity with P. falciparum in most endemic areas. Moreover, it is difficult to identify P. knowlesi on the basis of morphology alone, and rapid diagnostic tests are still not available for this malaria species. Therefore, the development of diagnostic tests applicable to the field is urgently needed. 1-Cys peroxiredoxin (1-Cys-Prx) in P. falciparum is abundantly expressed in the mature asexual stages, making it a promising candidate as a diagnostic antigen. In this study, we produced five monoclonal antibodies (mAbs) against P. falciparum 1-Cys-Prx (Pf1-Cys-Prx) by immunizing BALB/c mice with recombinant Pf1-Cys-Prx and subsequent hybridoma production. Cross reactivity of established mAbs with the orthologous molecule of Pf1-Cys-Prx in P. vivax (Pv1-Cys-Prx) and P. knowlesi (Pk1-Cys-Prx) was examined. Western blot analyses showed that three mAbs reacted with Pv1-Cys-Prx and Pk1-Cys-Prx but two mAbs did not. These results indicate that the two mAbs were effective in differentiating P. falciparum from P. vivax and P. knowlesi and could be used in differential diagnosis as well as comparative molecular studies of human Plasmodium species.

  17. Therapeutic monoclonal antibody for Sporotrichosis

    Directory of Open Access Journals (Sweden)

    Sandro eAlmeida

    2012-11-01

    Full Text Available Sporotrichosis is a chronic subcutaneous mycosis that affects either humans or animals and occurs worldwide. This subcutaneous mycosis had been attributed to a single etiological agent, Sporothrix schenckii. S. schenckii exhibits a considerable genetic variability, where recently, was suggesting 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 the cutaneous forms but, recently, occurrences of more severe clinical forms of this mycosis were described, especially among immunocompromized individuals. The immunological mechanisms involved in prevention and control of sporotrichosis are still not very well understood. Some works 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 have not been studied in detail. In a previous study, we showed that antigens secreted by S. schenckii induce a specific humoral response in infected animals, mainly against the 70-kDa molecules, indicating a possible participation of specific antibodies to this molecule in infection control. In an other work of the our group, we produced a mAb against a 70-kDa glycoprotein of S. schenckii in order to better understand the effect of passive immunization of mice infected with S. schenckii. Results showed a significant reduction in the number of CFU in organs of mice when the mAb was injected before and during S. schenckii infection. Similar results were observed when T-cell deficient mice were used. Drugs of choice in the treatment of sporothrichosis require long periods and frequently relapses are observed, mainly in immunocompromized patients. The strong protection induced by mAb against a 70-kDa glycoprotein makes it a strong candidate for a

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

    Science.gov (United States)

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

    2014-01-25

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

  19. Production and Characterization of Monoclonal Antibodies of Shrimp White Spot Syndrome Virus Envelope Protein VP28

    Institute of Scientific and Technical Information of China (English)

    Wan-gang GU; Jun-fa YUAN; Ge-lin XU; Li-juan LI; Ni LIU; Cong ZHANG; Jian-hong ZHANG; Zheng-li SHI

    2007-01-01

    BALB/c mice were immunized with purified White spot syndrome virus (WSSV).Six monoclonal antibody cell lines were selected by ELISA with VP28 protein expressed in E.coll in vitro neutralization experiments showed that 4 of them could inhibit the virus infection in crayfish.Westernblot suggested that all these monoclonal antibodies were against the conformational structure of VP28.The monoclonal antibody 7B4 was labeled with colloidal gold particles and used to locate the VP28 on virus envelope by immunogold labeling.These monoclonal antibodies could be used to develop immunological diagnosis methods for WSSV infection.

  20. The value of non-human primates in the development of therapeutic monoclonal antibodies

    NARCIS (Netherlands)

    Van Meer, P.J.K.; Kooijman, M.; Van Der Laan, J.W.; Moors, E.H.M.; Schellekens, H.

    2011-01-01

    The pharmaceutical industry is increasingly focusing on the development of biological therapeutics. These molecules generally cause no off-target toxicity and are highly species specific. Therefore, non-human primates (NHPs) are often the only relevant species in which to conduct regulatory safety

  1. The value of non-human primates in the development of therapeutic monoclonal antibodies

    NARCIS (Netherlands)

    Van Meer, P.J.K.; Kooijman, M.; Van Der Laan, J.W.; Moors, E.H.M.; Schellekens, H.

    2011-01-01

    The pharmaceutical industry is increasingly focusing on the development of biological therapeutics. These molecules generally cause no off-target toxicity and are highly species specific. Therefore, non-human primates (NHPs) are often the only relevant species in which to conduct regulatory safety t

  2. The value of non-human primates in the development of therapeutic monoclonal antibodies

    NARCIS (Netherlands)

    Van Meer, P.J.K.; Kooijman, M.; Van Der Laan, J.W.; Moors, E.H.M.; Schellekens, H.

    2011-01-01

    The pharmaceutical industry is increasingly focusing on the development of biological therapeutics. These molecules generally cause no off-target toxicity and are highly species specific. Therefore, non-human primates (NHPs) are often the only relevant species in which to conduct regulatory safety t

  3. Seizing the strategic opportunities of emerging technologies by building up innovation system: monoclonal antibody development in China.

    Science.gov (United States)

    Zhang, Mao-Yu; Li, Jian; Hu, Hao; Wang, Yi-Tao

    2015-11-04

    Monoclonal antibodies (mAbs), as an emerging technology, have become increasingly important in the development of human therapeutic agents. How developing countries such as China could seize this emerging technological opportunity remains a poorly studied issue in prior literature. Thus, this paper aims to investigate the research and development of mAbs in China based on an innovation system functions approach and probes into the question of how China has been taking advantage of emerging technologies to overcome its challenges of building up a complete innovation system in developing mAbs. Mixed research methods were applied by combining archival data and field interviews. Archival data from the China Food and Drug Administration, Web of Science, the United States Patent and Trademark Office, the Chinese Clinical Trial Registry, and the National Science and Technology Report Service were used to examine the status quo of the technology and research and development (R&D) activities in China, while the opinions of researchers and managers in this field were synthesized from the interviews. From the perspective of innovation system functions, technological development of mAb in China is being driven by incentives such as the subsidies from the State and corporate R&D funding. Knowledge diffusion has been well served over the last 10 years through exchanging information on networks and technology transfer with developed countries. The State has provided clear guidance on search of emerging mAb technologies. Legitimacy of mAb in China has gained momentum owing to the implementation of government policies stipulated in the "The Eleventh Five-year Plan" in 2007, as well as national projects such as the "973 Program" and "863 Program", among others. The potential of market formation stays high because of the rising local demand and government support. Entrepreneurial activities for mAb continue to prosper. In addition, the situation of resource supply has been improved

  4. Monoclonal IgA Antibodies for Aflatoxin Immunoassays

    OpenAIRE

    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 ...

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

    Science.gov (United States)

    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.

  6. Generation and Characterization of Novel Human IRAS Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Bo Wang

    2009-01-01

    Full Text Available Imidazoline receptors were first proposed by Bousquet et al., when they studied antihypertensive effect of clonidine. A strong candidate for I1R, known as imidazoline receptor antisera-selected protein (IRAS, has been cloned from human hippocampus. We reported that IRAS mediated agmatine-induced inhibition of opioid dependence in morphine-dependent cells. To elucidate the functional and structure properties of I1R, we developed the newly monoclonal antibody against the N-terminal hIRAS region including the PX domain (10–120aa through immunization of BALB/c mice with the NusA-IRAS fusion protein containing an IRAS N-terminal (10–120aa. Stable hybridoma cell lines were established and monoclonal antibodies specifically recognized full-length IRAS proteins in their native state by immunoblotting and immunoprecipitation. Monoclonal antibodies stained in a predominantly punctate cytoplasmic pattern when applied to IRAS-transfected HEK293 cells by indirect immunofluorescence assays and demonstrated excellent reactivity in flow immunocytometry. These monoclonal antibodies will provide powerful reagents for the further investigation of hIRAS protein functions.

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

    Science.gov (United States)

    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.

  8. Generation of monoclonal antibodies against highly conserved antigens.

    Directory of Open Access Journals (Sweden)

    Hongzhe Zhou

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

  9. Monoclonal IgA Antibodies for Aflatoxin Immunoassays

    Directory of Open Access Journals (Sweden)

    Özlem Ertekin

    2016-05-01

    Full Text Available 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.

  10. Monoclonal IgA Antibodies for Aflatoxin Immunoassays

    Science.gov (United States)

    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

  11. Monoclonal IgA Antibodies for Aflatoxin Immunoassays.

    Science.gov (United States)

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

    2016-05-12

    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.

  12. Development and application of triple antibody sandwich enzyme-linked immunosorbent assays for begomovirus detection using monoclonal antibodies against Tomato yellow leaf curl Thailand virus.

    Science.gov (United States)

    Seepiban, Channarong; Charoenvilaisiri, Saengsoon; Warin, Nuchnard; Bhunchoth, Anjana; Phironrit, Namthip; Phuangrat, Bencharong; Chatchawankanphanich, Orawan; Attathom, Supat; Gajanandana, Oraprapai

    2017-05-30

    Tomato yellow leaf curl Thailand virus, TYLCTHV, is a begomovirus that causes severe losses of tomato crops in Thailand as well as several countries in Southeast and East Asia. The development of monoclonal antibodies (MAbs) and serological methods for detecting TYLCTHV is essential for epidemiological studies and screening for virus-resistant cultivars. The recombinant coat protein (CP) of TYLCTHV was expressed in Escherichia coli and used to generate MAbs against TYLCTHV through hybridoma technology. The MAbs were characterized and optimized to develop triple antibody sandwich enzyme-linked immunosorbent assays (TAS-ELISAs) for begomovirus detection. The efficiency of TAS-ELISAs for begomovirus detection was evaluated with tomato, pepper, eggplant, okra and cucurbit plants collected from several provinces in Thailand. Molecular identification of begomoviruses in these samples was also performed through PCR and DNA sequence analysis of the CP gene. Two MAbs (M1 and D2) were generated and used to develop TAS-ELISAs for begomovirus detection. The results of begomovirus detection in 147 field samples indicated that MAb M1 reacted with 2 begomovirus species, TYLCTHV and Tobacco leaf curl Yunnan virus (TbLCYnV), whereas MAb D2 reacted with 4 begomovirus species, TYLCTHV, TbLCYnV, Tomato leaf curl New Delhi virus (ToLCNDV) and Squash leaf curl China virus (SLCCNV). Phylogenetic analyses of CP amino acid sequences from these begomoviruses revealed that the CP sequences of begomoviruses recognized by the narrow-spectrum MAb M1 were highly conserved, sharing 93% identity with each other but only 72-81% identity with MAb M1-negative begomoviruses. The CP sequences of begomoviruses recognized by the broad-spectrum MAb D2 demonstrated a wider range of amino acid sequence identity, sharing 78-96% identity with each other and 72-91% identity with those that were not detected by MAb D2. TAS-ELISAs using the narrow-specificity MAb M1 proved highly efficient for the detection of

  13. Development and characterization of mouse monoclonal antibodies against monomeric dengue virus non-structural glycoprotein 1 (NS1).

    Science.gov (United States)

    Gelanew, Tesfaye; Poole-Smith, B Katherine; Hunsperger, Elizabeth

    2015-09-15

    Dengue virus (DENV) nonstructural-1 (NS1) glycoprotein is useful for diagnosis of DENV infections in the first 8 days of illness with any of the four serotypes (DENV-1, DENV-2, DENV-3 and DENV-4). However, NS1 diagnostics are less sensitive for secondary DENV infections so the utility of NS1 diagnostics in dengue endemic countries where there is predominantly secondary infections is being questioned. Heat-mediated immunecomplex dissociation (ICD) prior to testing serum samples can significantly improve NS1 test sensitivity in secondary infections but requires monoclonal antibodies (MAbs) reactive to heat-denatured NS1. In order to incorporate a simple heat-mediated ICD step, a crucial step was to develop new MAbs with high affinity and specificity to heat-denatured DENV NS1 protein. In the present study, six new MAbs were isolated from BALB/c mice immunized with recombinant monomeric NS1 of DENV-1 and DENV-2. Characterization using three different methods: indirect ELISA, fixed cell ELISA and western blot revealed that all six MAbs are serotype-cross-reactive and capable of recognizing dimeric and hexameric isoforms as well as heat-denatured NS1 from all four DENV serotypes. No cross-reactivity to NS1 of West Nile virus and Yellow fever virus was observed on western blot and indirect ELISA. Five of the six MAbs mapped to the DENV NS1 region of 105-119 amino acids. The remaining MAb mapped to DENV NS1 region of 25-39 amino acids. These two NS1 regions were found to be highly conserved among all four DENV serotypes by sequences analysis and database comparison. These MAbs were used to develop an NS1 capture ELISA and tested using a small panel of clinical specimens. The results from the NS1 capture ELISA indicated at least a three-fold increase in NS1 antigen detection in heat-denatured samples compared to untreated specimens. Furthermore, artificial immunecomplexed results also demonstrated the binding efficiency of these MAbs to heat denatured NS1. Taken together

  14. Anaphylaxis to chemotherapy and monoclonal antibodies.

    Science.gov (United States)

    Castells, Mariana C

    2015-05-01

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

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Mohamad Cherry

    2010-10-01

    Full Text Available 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.

  17. Induction and characterization of monoclonal anti-idiotypic antibodies reactive with idiotopes of canine parvovirus neutralizing monoclonal antibodies.

    NARCIS (Netherlands)

    G.F. Rimmelzwaan (Guus); J. van Es (Johan); G.A. Drost; F.G.C.M. Uytdehaag (Fons); A.D.M.E. Osterhaus (Albert)

    1991-01-01

    textabstractMonoclonal anti-idiotypic (anti-Id) antibodies (Ab2) were generated against idiotypes (Id) of canine parvovirus (CPV) specific monoclonal antibodies (MoAbs). The binding of most of these anti-Id antibodies to their corresponding Id could be inhibited by antigen, thus classifying these an

  18. Monoclonal antibody disulfide reduction during manufacturing

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  20. Development of an enzyme-linked immunosorbent assay for the detection of isomiroestrol, an identical marker, in White Kwao Krua using a monoclonal antibody.

    Science.gov (United States)

    Kitisripanya, Tharita; Jutathis, Kamonthip; Inyai, Chadathorn; Komaikul, Jukrapun; Udomsin, Orapin; Tanaka, Hiroyuki; Putalun, Waraporn

    2017-04-15

    Pueraria candollei var. mirifica or White Kwao Krua (WKK) is a phytoestrogen-rich plant widely used among women to improve climacteric symptoms. Additionally, the tuberous roots of this plant have been added as an active ingredient for skin rejuvenation and breast enlargement effects in various functional foods. However, most of the products on the market containing WKK have not been sufficiently standardized with respect to the active compound or identical marker. To control the quality of these plant materials, an enzyme-linked immunosorbent assay (ELISA) using anti-isomiroestrol antibodies was established for the determination of isomiroestrol, an identical marker in WKK. Polyclonal and monoclonal antibodies against isomiroestrol were generated and their specificity characterized in this study. Monoclonal antibody 12C1 showed higher specificity to isomiroestrol and was thus selected to develop the ELISA. Based on the validation analysis and the tested performance of the developed ELISA in variably sourced WKK samples, the assay can provide an alternative approach that is reliable and highly sensitive for the quantitative analysis of isomiroestrol in plant.

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

    Science.gov (United States)

    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.

  2. Generation of monoclonal antibodies to native active human glycosyltransferases

    DEFF Research Database (Denmark)

    Vester-Christensen, Malene Bech; Bennett, Eric Paul; Clausen, Henrik;

    2013-01-01

    using monoclonal antibodies therefore provides an excellent strategy to analyze the glycosylation process in cells. A major drawback has been difficulties in generating antibodies to glycosyltransferases and validating their specificities. Here we describe a simple strategy for generating...

  3. A monoclonal antibody toolkit for C. elegans.

    Directory of Open Access Journals (Sweden)

    Gayla Hadwiger

    Full Text Available 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 beta-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 alpha-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

  4. Development and Characterization of a Novel Anti-idiotypic Monoclonal Antibody to Growth Hormone, Which Can Mimic Physiological Functions of Growth Hormone in Primary Porcine Hepatocytes.

    Science.gov (United States)

    Lan, Hai-Nan; Jiang, Hai-Long; Li, Wei; Wu, Tian-Cheng; Hong, Pan; Li, Yu Meng; Zhang, Hui; Cui, Huan-Zhong; Zheng, Xin

    2015-04-01

    B-32 is one of a panel of monoclonal anti-idiotypic antibodies to growth hormone (GH) that we developed. To characterize and identify its potential role as a novel growth hormone receptor (GHR) agonist, we determined that B-32 behaved as a typical Ab2β based on a series of enzyme-linked immunosorbent assay assays. The results of fluorescence-activated cell sorting, indirect immunofluorescence and competitive receptor binding assays demonstrated that B-32 specifically binds to the GHR expressed on target cells. Next, we examined the resulting signal transduction pathways triggered by this antibody in primary porcine hepatocytes. We found that B-32 can activate the GHR and Janus kinase (2)/signal transducers and activators of transcription (JAK2/STAT5) signalling pathways. The phosphorylation kinetics of JAK2/STAT5 induced by either GH or B-32 were analysed in dose-response and time course experiments. In addition, B32 could also stimulate porcine hepatocytes to secrete insulin-like growth factors-1. Our work indicates that a monoclonal anti-idiotypic antibody to GH (B-32) can serve as a GHR agonist or GH mimic and has application potential in domestic animal (pig) production.

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

    Science.gov (United States)

    Wilkinson, Trevor C I

    2016-06-15

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

  6. Strategies for Treating Autoimmune Disease With Monoclonal Antibodies

    OpenAIRE

    Wofsy, David

    1985-01-01

    There is no safe and reliable therapy for most serious autoimmune diseases, such as systemic lupus erythematosus. Severe cases usually require treatment with corticosteroids or cytotoxic drugs or both, which frequently provide inadequate disease control and can cause serious complications. These therapies are not restricted in their effects to cells of the immune system, but rather have a broad range of toxic effects on cells throughout the body. The development of monoclonal antibodies has l...

  7. Immunochemical Characterization of Anti-Acetylcholinesterase Inhibitory Monoclonal Antibodies

    Science.gov (United States)

    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

  8. Monoclonal Antibodies Specific for Hippurate Hydrolase of Campylobacter jejuni

    OpenAIRE

    Steele, Marina; Gyles, Carlton; Chan, Voon Loong; Odumeru, Joseph

    2002-01-01

    Eleven monoclonal antibodies raised against recombinant Campylobacter jejuni hippurate hydrolase were tested for binding to lysates from 19 C. jejuni strains, 12 other Campylobacter strains, and 21 non-Campylobacter strains. Several monoclonal antibodies bound to C. jejuni but not to other Campylobacter species and may be useful in a species-specific immunoassay.

  9. Monoclonal Antibody-Based Therapeutics for Melioidosis and Glanders

    Directory of Open Access Journals (Sweden)

    Hyung-Yong Kim

    2011-01-01

    Full Text Available Problem statement: Burkholderia Pseudomallei (BP and B. Mallei (BM were two closely related pathogenic gram-negative bacteria. They were the causative agents of melioidosis and glanders, respectively and are recognized by CDC as category B select agents. Significant efforts had been devoted to developing the diagnostic and therapeutic measures against these two pathogens. Monoclonal antibody-based therapeutic was a promising targeted therapy to fight against melioidosis and glanders. Valuable findings have been reported by different groups in their attempt to identify vaccine targets against these two pathogens. Approach: Our group has generated neutralizing Monoclonal Antibodies (MAbs against BP and BM and characterized them by both in vitro and in vivo experiments. We present an overview of the MAb-based therapeutic approaches against BP and BM and demonstrate some of our efforts for developing chimeric and fully human MAbs using antibody engineering. Results: Throughout conventional mouse hybridoma technique and antibody engineering (chimerization and in vitro antibody library techniques, we generated 10 chimeric MAbs (3 stable MAbs and 7 transient MAbs and one fully human MAb against BP and BM. In addition, we present the reactive antigen profiles of these MAbs. Our approaches had potentials to accelerate the development of therapeutics for melioidosis and glanders in humans. Conclusion: Our experience and findings presented here will be valuable for choosing the best antigenic targets and ultimately for the production of effective vaccines for these two pathogens.

  10. Real-time kinetic analysis applied to the production of bispecific monoclonal antibodies for radioimmunodetection of cancer.

    Science.gov (United States)

    Horenstein, A L; Poiesi, C; DeMonte, L; Camagna, M; Mariani, M; Albertini, A; Malavasi, F

    1993-01-01

    An automated biosensor system designed for measuring molecular interactions in real-time and without labelling of the reactants has been used to evaluate the association/dissociation rate and affinity constants of bivalent monoclonal antibodies and a monovalent bispecific monoclonal antibody. Observed differences in affinity between parental and bispecific antibody produced were related to the association rate constants, since the dissociation rate constants were in the same range. Values were also closely related to radioimmunochemical data. These results indicate that the biosensor system, besides presenting several advantages for characterizing antigen-antibody interaction, is valuable for selecting monoclonal antibodies with properties which might be useful in the development of bispecific monoclonal antibodies.

  11. Surface activity of a monoclonal antibody.

    Science.gov (United States)

    Mahler, Hanns-Christian; Senner, Frank; Maeder, Karsten; Mueller, Robert

    2009-12-01

    The development of high concentration antibody formulations presents a major challenge for the formulation scientist, as physical characteristics and stability behavior change compared to low concentration protein formulations. The aim of this study was to investigate the potential correlation between surface activity and shaking stress stability of a model antibody-polysorbate 20 formulation. The surface activities of pure antibody and polysorbate 20 were compared, followed by a study on the influence of a model antibody on the apparent critical micelle concentration (CMC) of polysorbate 20 over a protein concentration range from 10 to 150 mg/mL. In a shaking stress experiment, the stability of 10, 75, and 150 mg/mL antibody formulations was investigated containing different concentrations of polysorbate 20, both below and above the CMC. The antibody increased significantly the apparent CMC of antibody-polysorbate 20 mixtures in comparison to the protein-free buffer. However, the concentration of polysorbate required for stabilization of the model antibody in a shaking stress experiment did not show dependence on the CMC. A polysorbate 20 level of 0.005% was found sufficient to stabilize both at low and high antibody concentration against antibody aggregation and precipitation.

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

    Science.gov (United States)

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

    1993-04-01

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

  13. Monoclonal Antibodies Against Xenopus Greatwall Kinase

    Science.gov (United States)

    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

  14. 89Zr-PET radiochemistry in the development and application of therapeutic monoclonal antibodies and other biologicals.

    Science.gov (United States)

    Vugts, Danielle J; Visser, Gerard W M; van Dongen, Guus A M S

    2013-01-01

    Positron emission tomography with (89)Zr can be used to follow the behaviour of therapeutic monoclonal antibodies (mAbs) and other biologicals in vivo. The favourable radiophysical characteristics of (89)Zr allow multiple days PET scanning after injection. For the coupling of (89)Zr to proteins six desferrioxamine (DFO)-based bifunctional chelators have been described, five of which forming stable complexes in vivo. Of the methods that give stable complexes three are based on random lysine modification of mAbs and two on site-specific engineering. Up to now only two methods, random lysine modification with N-suc-DFO or DFO-Bz-NCS, have been used in clinical studies. In this review firstly aspects of the physicochemical properties and production of (89)Zr are emphasized as well as important items that have to be taken into account for current good manufacturing practice (cGMP) compliant production of (89)Zr-labeled proteins. Next, the different DFO-based conjugation strategies will be discussed with respect to synthesis, and their (pre)clinical evaluation particularly in the field of oncology.

  15. Development and applications of AlphaScreen-based FcRn binding assay to characterize monoclonal antibodies.

    Science.gov (United States)

    Wu, Qiang; Lee, Ho Young; Wong, Pin Yee; Jiang, Guoying; Gazzano-Santoro, Hélène

    2015-05-01

    IgG antibodies are important pharmaceutical molecules that successfully treat a variety of human diseases. The neonatal Fc receptor (FcRn) interacts with IgG Fc in the CH2-CH3 domain and plays a key role in IgG antibody homeostasis and affects its pharmacokinetic properties. An in vitro FcRn binding assay could be a highly valuable complementary tool to assess IgG antibody pharmacokinetics in IgG engineering and screening during the early optimization stage. In addition, it could be useful in biological characterization studies for antibody minor variants, process optimization, and comparability study at later stages of antibody development. Here we developed a homogeneous AlphaScreen-based FcRn assay to assess the binding of FcRn to IgG antibody in vitro. The assay is found to be accurate, precise, specific, and simple: donor beads loaded with FcRn and acceptor beads loaded with IgG1 mAb1 are mixed together with sample IgG at various dilutions and incubated for 1h before acquiring data with a fluorescence reader. This assay can run up to four samples per plate in 2h, which is time and cost effective compared with other FcRn binding methods such as cell-based fluorescent-activated cell scan and surface plasma resonance. Our data demonstrated that this assay is suitable for assessing the FcRn binding in vitro and provides a platform approach that can be readily applied to various antibodies. Copyright © 2015. Published by Elsevier B.V.

  16. Development of an antigen-capture enzyme-linked immunosorbent assay using monoclonal antibodies for detecting H6 avian influenza viruses.

    Science.gov (United States)

    Chen, Yi-Tung; Tsao, Zak; Chang, Shu-Ting; Juang, Ron-Huay; Wang, Lih-Chiann; Chang, Chung-Ming; Wang, Ching-Ho

    2012-06-01

    The H6 subtype of avian influenza virus (AIV) infection occurs frequently in wild and domestic birds. AIV antigen detection is preferred for controlling AIV as birds are infected before they produce antibodies. The purpose of this study was to develop an early diagnostic method for AIV detection. Six monoclonal antibodies (mAbs) developed from a field H6N1 AIV strain were tested for their ability to bind to viruses. The two that showed the greatest binding ability to AIVs were used for antigen detection. An antigen-capture enzyme-linked immunosorbent assay (ELISA) to detect H6 AIVs was developed using these mAbs. One mAb was coated onto an ELISA plate as the capture antibody. The other mAb was used as the detector antibody after labeling with horseradish peroxidase. The antigen-capture ELISA detected H6N1 AIVs but not H5 AIVs, human H1N1, H3N2 influenza or other viruses. This antigen-capture ELISA could be used to specifically detect H6N1 AIV.

  17. A MONOCLONAL-ANTIBODY AGAINST HUMAN BETA-GLUCURONIDASE FOR APPLICATION IN ANTIBODY-DIRECTED ENZYME PRODRUG THERAPY

    NARCIS (Netherlands)

    Haisma, Hidde; VANMUIJEN, M; SCHEFFER, G; SCHEPER, RJ; PINEDO, HM; BOVEN, E

    1995-01-01

    The selectivity of anticancer agents may be improved by antibody-directed enzyme prodrug therapy (ADEPT), The immunogenicity of antibody-enzyme conjugates and the low tumor to normal tissue ratio calls for the use of a human enzyme and the development of a monoclonal antibody (MAb) against that enzy

  18. Treatment with anti-interferon-δ monoclonal antibodies modifies experimental autoimmune encephalomyelitis in interferon-δ receptor knockout mice

    DEFF Research Database (Denmark)

    Espejo, C.; Penkowa, Milena; Saez-Torres, I.

    2001-01-01

    Neuroinflammation, neuronal degeneration, regeneration, monoclonal antibodies, multiple schlerosis......Neuroinflammation, neuronal degeneration, regeneration, monoclonal antibodies, multiple schlerosis...

  19. Monoclonal antibodies against naturally occurring bioactive compounds.

    Science.gov (United States)

    Shoyama, Y; Tanaka, H; Fukuda, N

    1999-09-01

    The ratio of hapten to bovine serum albumin (BSA) in an antigen conjugate was determined by matrix-assisted laser desorption/ionization (MALDI) tof mass spectrometry. A hybridoma secreting monoclonal antibody (MAb) was produced by fusing splenocytes immunized with an antigen-BSA conjugate with HAT-sensitive mouse myeloma cells. The cross-reaction of anti-forskolin antibodies with 7-deacetyl forskolin was 5.6%. A very small cross-reaction appeared with other derivatives. The full measuring range of the assay extends from 5 ng to 5 mug/ml of forskolin. Immunoaffinity column chromatography using anti-forskolin MAbs appears to be far superior to previously published separation methods. The capacity of the immunoaffinity column as determined by ELISA is 9 mug/ml. Forskolin has been isolated directly from the crude extracts of tuberous roots and the callus culture of Coleus forskohlii. A MAb against tetrahydrocannabinolic acid (THCA) was produced. The cross-reaction of anti-THCA antibody against other cannabinoids was very wide. Many cannabinoids and a spiro-compound were reactive, but did not react with other phenolics. It became evident that this ELISA was able to be applied to the biotransformation experiments of cannabinoids in plant tissue culture system. Anti-ginsenoside Rb1 MAbs were produced. New western blotting method of determination for ginsenosides was established. Ginsenosides separated by silica gel TLC were transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was treated with NaIO(4) solution followed by BSA, resulting in a ginsenoside-BSA conjugate. Immunostaining of ginsenosides was more sensitive compared to other staining. Immunostaining of ginsenosides in the fresh ginseng root was succeeded using anti-ginsenoside Rb1 (GRb1) MAb after blotting to PVDF membrane.

  20. Development of an ErbB4 monoclonal antibody that blocks neuregulin-1-induced ErbB4 activation in cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Okazaki, Shogo [Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Nakatani, Fumi [Cell Biology Laboratory, Department of Pharmaceutical Sciences, Faculty of Pharmacy, Kinki University, Higashiosaka, Osaka 577-8502 Japan (Japan); Masuko, Kazue; Tsuchihashi, Kenji [Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Ueda, Shiho; Masuko, Takashi [Cell Biology Laboratory, Department of Pharmaceutical Sciences, Faculty of Pharmacy, Kinki University, Higashiosaka, Osaka 577-8502 Japan (Japan); Saya, Hideyuki [Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Nagano, Osamu, E-mail: osmna@sb3.so-net.ne.jp [Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan)

    2016-01-29

    The use of monoclonal antibodies (mAbs) for cancer therapy is one of the most important strategies for current cancer treatment. The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, which regulates cancer cell proliferation, survival, and migration, is a major molecular target for antibody-based therapy. ErbB4/HER4, which contains a ligand-binding extracellular region, is activated by several ligands, including neuregulins (NRGs), heparin-binding EGF-like growth factor, betacellulin and epiregulin. Although there are clinically approved antibodies for ErbB1 and ErbB2, there are no available therapeutic mAbs for ErbB4, and it is not known whether ErbB4 is a useful target for antibody-based cancer therapy. In this study, we developed an anti-ErbB4 mAb (clone P6-1) that suppresses NRG-dependent activation of ErbB4 and examined its effect on breast cancer cell proliferation in the extracellular matrix. - Highlights: • We newly generated four clones of human ErbB4 specific mAb. • ErbB4 mAb clone P6-1 blocks ErbB4 phosphorylation induced by NRG-1. • ErbB4 mAb clone P6-1 suppresses NRG-1-promoted breast cancer cells proliferation on three dimensional culture condition.

  1. Use of monoclonal antibodies developed against chicken coccidia (Eimeria) to study invasion and development of Eimeria reichenowi in Florida sandhill cranes (Grus canadensis)

    Science.gov (United States)

    Augustine, P.C.; Olsen, G.H.; Danforth, H.D.; Gee, G.F.; Novilla, M.

    2001-01-01

    Eimeria gruis and Eimeria reichenowi are common coccidial parasites of a number of species of cranes. Until recently, little was known about either the site for invasion or the dynamics of early development of the crane coccidia because of the difficulty of identifying sporozoites and early developmental stages of these parasites by conventional staining methods. In the present study, monoclonal antibodies (MAbs) elicited against Eimeria spp. of chickens and turkeys were found to cross-react with sporozoites and developmental stages of E. reichenowi in the tissues of Florida sandhill cranes (Grus canadensis). With these Mabs, E. reichenowi sporozoites were found in specimens taken at 6 hr postinoculation (PI) from just proximal to Meckel's diverticulum in the jejunum to the ileocecal juncture. Fewer were found in the ceca and rectum and none in the duodenal loop. At 24 hr PI, there were markedly fewer sporozoites and their location had shifted to the duodenum. No stages were seen in intestinal cells at 5 days PI (DPI), but trophozoites had developed in the liver and spleen. At 10 DPI, sexual stages were detected in the intestine from the duodenal loop through Meckel's diverticulum but not in other organs. By 14 DPI, numerous developmental stages were detected in the intestine (ceca and jejunum), liver, and lungs but not in the heart, kidney, or brain. The number, location, and maturity of the stages in the ceca differed markedly from those in the jejunum.

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

    Science.gov (United States)

    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.

  3. Monoclonal antibodies and Fc fragments for treating solid tumors

    Directory of Open Access Journals (Sweden)

    Eisenbeis AM

    2012-01-01

    Full Text Available Andrea M Eisenbeis, Stefan J GrauDepartment of Neurosurgery, University Hospital of Cologne, Cologne, GermanyAbstract: Advances in biotechnology, better understanding of pathophysiological processes, as well as the identification of an increasing number of molecular markers have facilitated the use of monoclonal antibodies and Fc fragments in various fields in medicine. In this context, a rapidly growing number of these substances have also emerged in the field of oncology. This review will summarize the currently approved monoclonal antibodies used for the treatment of solid tumors with a focus on their clinical application, biological background, and currently ongoing trials.Keywords: targeted therapy, monoclonal antibodies, cancer, biological therapy

  4. Prediction and Reduction of the Aggregation of Monoclonal Antibodies.

    Science.gov (United States)

    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

    2017-04-21

    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 and their contribution to the thermodynamic stability of the protein. Although aggregation-prone regions 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. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  5. Efficient generation of human IgA monoclonal antibodies.

    Science.gov (United States)

    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.

  6. Muscle precursor cells in the developing limbs of two isopods (Crustacea, Peracarida): an immunohistochemical study using a novel monoclonal antibody against myosin heavy chain.

    Science.gov (United States)

    Kreissl, S; Uber, A; Harzsch, S

    2008-05-01

    In the hot debate on arthropod relationships, Crustaceans and the morphology of their appendages play a pivotal role. To gain new insights into how arthropod appendages evolved, developmental biologists recently have begun to examine the expression and function of Drosophila appendage genes in Crustaceans. However, cellular aspects of Crustacean limb development such as myogenesis are poorly understood in Crustaceans so that the interpretative context in which to analyse gene functions is still fragmentary. The goal of the present project was to analyse muscle development in Crustacean appendages, and to that end, monoclonal antibodies against arthropod muscle proteins were generated. One of these antibodies recognises certain isoforms of myosin heavy chain and strongly binds to muscle precursor cells in malacostracan Crustacea. We used this antibody to study myogenesis in two isopods, Porcellio scaber and Idotea balthica (Crustacea, Malacostraca, Peracarida), by immunohistochemistry. In these animals, muscles in the limbs originate from single muscle precursor cells, which subsequently grow to form multinucleated muscle precursors. The pattern of primordial muscles in the thoracic limbs was mapped, and results compared to muscle development in other Crustaceans and in insects.

  7. Immunolocation of antisperm monoclonal antibody 6B10 and corresponding antigen

    Institute of Scientific and Technical Information of China (English)

    高绍荣; 胡国俊; 段崇文; 刘辉; 韩之明; 宋祥芬; 陈大元

    1999-01-01

    An antisperm monoclonal antibody 6B10 was produced by hybridoma technique of the isotype IgG. The monoclonal antibody was purified by means of ammonium sulfate precipitation and protein A-Sepharose Cl-4B affinity chromatography. SDS polyacrylamide gel electrophoresis was used to evaluate the purity of the antibody. Evaluation of the sperm acrosomal status was determined by chlortetracycline (CTC) staining. It was found that monoclonal antibody 6B10 can inhibit the sperm acrosome reaction induced by progesterone. The corresponding antigen recognized by monoclonal antibody 6B10 was located on the plasma membrane of the sperm acrosome by indirect immunofluorescent microscopy and immunoelectronmicroscopy. Sperm protein was extracted by 1% Triton X-100. The molecular weight of the antigen is 50 ku, detected by Western blot. The antigen is a key protein in the sperm acrosome reaction and may be the receptor of progesterone on the sperm acrosome. It may either be developed as a candidate contraceptive vaccine

  8. Monitoring monoclonal antibody delivery in oncology: the example of bevacizumab.

    Directory of Open Access Journals (Sweden)

    Guillaume Nugue

    Full Text Available Developing therapeutic monoclonal antibodies paves the way for new strategies in oncology using targeted therapy which should improve specificity. However, due to a lack of biomarkers, a personalized therapy scheme cannot always be applied with monoclonal antibodies. As a consequence, the efficacy or side effects associated with this type of treatment often appear to be sporadic. Bevacizumab is a therapeutic monoclonal antibody targeting Vascular Endothelial Growth Factor (VEGF. It is used to limit tumor vascularization. No prognosis or response biomarker is associated with this antibody, we therefore assessed whether the administration protocol could be a possible cause of heterogeneous responses (or variable efficacy. To do this, we developed a bevacizumab assay with a broad sensitivity range to measure blood bevacizumab concentrations. We then analyzed bevacizumab concentrations in 17 patients throughout the first quarter of treatment. In line with previously published data, average blood concentrations were 88+/-27 mg/L following the first dose administered, and 213+/-105 mg/L after the last (6(th dose administered. However, the individual values were scattered, with a mean 4-fold difference between the lowest and the highest concentration for each dose administered. We demonstrated that the bevacizumab administration schedule results in a high inter-individual variability in terms of blood concentrations. Comparison of assay data with clinical data indicates that blood concentrations above the median are associated with side effects, whereas values below the median favor inefficacy. In conclusion, bevacizumab-based therapy could benefit from a personalized administration schedule including follow-up and adjustment of circulating bevacizumab concentrations.

  9. [Development of new approaches to standartization of enzyme immunoassay test systems for detection of antibodies to respiratory syncytial virus using electron microscopy and monoclonal antibodies].

    Science.gov (United States)

    Krivitskaia, V Z; Sirotkin, A K; Samoĭlovich, M P; Sominina, A A

    1999-01-01

    Respiratory syncytial virus (RSV), strain Long, was purified through 20-60% sucrose gradient. The virions from different sucrose density zones were tested by ELISA for reactivity with monoclonal antibodies (MAB) to F- (MAB 9C5) and N- (MAB 8B10) proteins of RSV. Comparative study of the same patterns of RSV by electron microscopy after negative staining showed a close relationship between the virion morphology and MAB binding in ELISA. MAB 9C5 were highly reactive with the surface domains of both mature RSV virions and "empty" virion envelopes without formed inner nucleocapsid structures. MAB 8B10 reacted well only with mature virions with completely assembled nucleocapsids. These MAB failed to reorganize the N-protein epitope of immature and destroyed virions, which indicated a conformation dependence of the 8B10 binding site. For practical purposes, MAB tests can be used to determine the RSV patterns, which can be used in ELISA for serologic diagnosis of RSV infection. Testing with these MAB demonstrate the stability of RSV to extreme exposures (lyophilization, storage, heating), which is important for creation of sensitive ELISA test systems and their standardization.

  10. Production and characterization of monoclonal antibodies against mink leukocytes

    DEFF Research Database (Denmark)

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

    1997-01-01

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

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

    Science.gov (United States)

    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."

  12. Fluorescence polarization immunoassay for salinomycin based on monoclonal antibodies

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    A fluorescence polarization immunoassay(FPIA) for the determination of salinomycin(SAL) was developed by using anti-SAL monoclonal antibodies(mAb).Fluorescein labeled SAL(tracer) was synthesized by the N-hydroxysuccinimide active ester method and purified using thin layer chromatography(TLC).The developed FPIA for SAL had a dynamic range from 0.60 to 2193 ng/mL with an IC50 value of 33.2 ng/mL and a detection limit(LOD) of 0.08 ng/mL.No significant cross-reactivities were observed with other drugs but 67.6% with narasin.

  13. A monoclonal antibody against human MUDENG protein.

    Science.gov (United States)

    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.

  14. Generation and characterization of monoclonal antibodies specific to Coenzyme A

    Directory of Open Access Journals (Sweden)

    Malanchuk O. M.

    2015-06-01

    Full Text Available Aim. Generation of monoclonal antibodies specific to Coenzyme A. Methods. Hybridoma technique. KLH carrier protein conjugated with CoA was used for immunization. Screening of positive clones was performed with BSA conjugated to CoA. Results. Monoclonal antibody that specifically recognizes CoA and CoA derivatives, but not its precursors ATP and cysteine has been generated. Conclusion. In this study, we describe for the first time the production and characterization of monoclonal antibodies against CoA. The monoclonal antibody 1F10 was shown to recognize specifically CoA in Western blotting, ELISA and immunoprecipitation. These properties make this antiboby a particularly valuable reagent for elucidating CoA function in health and disease.

  15. Purification of Murine Monoclonal IgM Antibody

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    This paper presents the purification of a monoclonal IgM antibody against human tumor associated antigen Lewis-Y by ion exchange chromatography and gel filtration.Enzyme-linked immunosorbent assay (ELISA) and SDS-polyacrylamide gel electrophoresis (PAGE) were used to identify purified IgM antibody.In flow cytometry analysis, the purified IgM antibody recognizes human breast tumor cell line MCF-7 which expresses Lewis-Y antigen.This work presents a new way for the purification of murine monoclonal IgM antibody.

  16. Monoclonal Antibody Therapy and Renal Transplantation: Focus on Adverse Effects

    Directory of Open Access Journals (Sweden)

    Gianluigi Zaza

    2014-02-01

    Full Text Available A series of monoclonal antibodies (mAbs are commonly utilized in renal transplantation as induction therapy (a period of intense immunosuppression immediately before and following the implant of the allograft, to treat steroid-resistant acute rejections, to decrease the incidence and mitigate effects of delayed graft function, and to allow immunosuppressive minimization. Additionally, in the last few years, their use has been proposed for the treatment of chronic antibody-mediated rejection, a major cause of late renal allograft loss. Although the exact mechanism of immunosuppression and allograft tolerance with any of the currently used induction agents is not completely defined, the majority of these medications are targeted against specific CD proteins on the T or B cells surface (e.g., CD3, CD25, CD52. Moreover, some of them have different mechanisms of action. In particular, eculizumab, interrupting the complement pathway, is a new promising treatment tool for acute graft complications and for post-transplant hemolytic uremic syndrome. While it is clear their utility in renal transplantation, it is also unquestionable that by using these highly potent immunosuppressive agents, the body loses much of its innate ability to mount an adequate immune response, thereby increasing the risk of severe adverse effects (e.g., infections, malignancies, haematological complications. Therefore, it is extremely important for clinicians involved in renal transplantation to know the potential side effects of monoclonal antibodies in order to plan a correct therapeutic strategy minimizing/avoiding the onset and development of severe clinical complications.

  17. Development of an immunoaffinity column method using broad-specificity monoclonal antibodies for simultaneous extraction and cleanup of quinolone and sulfonamide antibiotics in animal muscle tissues

    Science.gov (United States)

    This paper describes a novel mixed-bed immunoaffinity column (IAC) method. The IAC was produced by coupling anti-fluoroquinolone and anti-sulfonamide broad-specificity monoclonal antibodies (Mabs) to Sepharose 4B for simultaneously isolating 13 fluoroquinolones (FQs) and 6 sulfonamides (SAs) from s...

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

    Science.gov (United States)

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

    2016-04-22

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

  19. TSOL18 Vaccine Antigen of Taenia solium: Development of Monoclonal Antibodies and Field Testing of the Vaccine in Cameroon

    Directory of Open Access Journals (Sweden)

    Assana, E.

    2010-01-01

    lack of knowledge of the taeniasis-cysticercosis complex and the absence of a pig pen in the household were associated with pig cysticercosis. Chapter 3 reports the investigations that were undertaken to characterise whether the principal antibody specificities raised by TSOL18 in pigs were against linear or conformational determinants. TSOL18 was expressed in two truncated forms representing either the amino terminal portion or the carboxy terminal portion, with the two truncations overlapping in sequence by 25 amino acids. The original protein (designated TSOL18N— and the two truncations (TSOL18N—-1 and TSOL18N—-2 were used in inhibition ELISA to determine their ability to inhibit the binding of protective pig antibodies to TSOL18. TSOL18N— was shown to be capable of completely inhibiting the binding of pig anti-TSOL18N— antibodies to TSOL18N— in ELISA. However, neither TSOL18N—-1 nor TSOL18N—-2, either alone or combined, was capable of inhibiting any detectable amount of reactivity of pig anti-TSOL18N— antibodies with TSOL18N—. It is concluded that the dominant antibody specificities, and likely the host-protective specificities, of TSOL18 are conformational epitopes. Chapter 4 describes the development of an antibody detection test for the specific diagnosis of porcine cysticercosis. A fraction with a major band of 14 kDa was obtained from crude cyst fluid (CF of T. solium cysticerci by 2-step chromatography. A first fraction isolated by gel filtration was purified using an anion exchange column on High Performance Liquid Chromatography (HPLC. Evaluation of the analytic sensitivity of this fraction (F3 was carried out in an antibody detection enzyme-linked immunosorbent assay (Ab-ELISA-F3 using serum samples from pigs experimentally infected with different doses of T. solium eggs. The cross-reactivity of F3 was evaluated with serum samples from pigs that were naturally or experimentally infected with Taenia hydatigena, Taenia saginata asiatica

  20. ELISA Detection of Francisella tularensis using Polyclonaland Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Miroslav Pohanka

    2008-09-01

    Full Text Available The mouse monoclonal and polyclonal antibodies were produced for the detection of intracellular pathogenand potential warfare agent Francisella tularensis. Antibody titers obtained were 1:640 for polyclonal antibodiesand 1:320 for monoclonal antibodies. Both antibodies were used in the indirect enzyme-linked immunosorbentassay (ELISA found to detect F. tularensis whole cells. The limit of detection was 5.4×106 CFU/ml for polyclonalantibodies and 6.9×106 CFU/ml for monoclonal antibodies. The value sample could  be distinguished from anyconcentration of another gram-negative bacterium: Escherichia coli.Defence Science Journal, 2008, 58(5, pp.698-702, DOI:http://dx.doi.org/10.14429/dsj.58.1693

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

    Science.gov (United States)

    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.

  2. Development of a serotype colloidal gold strip using monoclonal antibody for rapid detection type Asia1 foot-and-mouth disease

    Directory of Open Access Journals (Sweden)

    Gao Shan-dian

    2011-09-01

    Full Text Available Abstract Background In this study, we developed a rapid, one step colloid gold strip (CGS capable of specifically detecting type Asia1 foot-and-mouth disease virus (FMDV. We have produced two monoclonal antibodies (mAb to type Asia1 FMD (named 1B8 and 5E2. On the test strip, the purified 1B8 labelled with the colloidal gold was used as the detector, and the purified 5E2 and goat anti-mouse antibodies were wrapped onto nitrocellulose (NC membranes as the test and the control line, respectively. The rapid colloidal gold stereotype diagnostic strip was housed in a plastic case. Results In specificity and sensitivity assay, there was no cross-reaction of the antigen with the other type of FMD and SVDV. The detection sensitivity was found to be as high as 10-5 dilution of Asia1/JSL/05 (1 × 107.2TCID50/50 μL. There was excellent agreement between the results obtained by CGS and reverse indirect hemagglutination assay (RIHA, and the agreement can reach to 98.75%. Conclusion We developed colloidal gold strips that have good qualities and does not require specialized equipment or technicians. This method provided a feasible, convenient, rapid, and effective for detecting type Asia1 FMDV in the fields.

  3. Moving through three-dimensional phase diagrams of monoclonal antibodies.

    Science.gov (United States)

    Rakel, Natalie; Baum, Miriam; Hubbuch, Jürgen

    2014-01-01

    Protein phase behavior characterization is a multivariate problem due to the high amount of influencing parameters and the diversity of the proteins. Single influences on the protein are not understood and fundamental knowledge remains to be obtained. For this purpose, a systematic screening method was developed to characterize the influence of fluid phase conditions on the phase behavior of proteins in three-dimensional phase diagrams. This approach was applied to three monoclonal antibodies to investigate influences of pH, protein and salt concentrations, with five different salts being tested. Although differences exist between the antibodies, this extensive study confirmed the general applicability of the Hofmeister series over the broad parameter range analyzed. The influence of the different salts on the aggregation (crystallization and precipitation) probability was described qualitatively using this Hofmeister series, with a differentiation between crystallization and precipitation being impossible, however.

  4. MONOCLONAL ANTIBODIES TO IDENTIFY TOMATO MOSAIC TOBAMOVIRUS (TOMV

    Directory of Open Access Journals (Sweden)

    Duarte Keila M.R.

    2001-01-01

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

  5. Serological comparison of tospovirus isolates using polyclonal and monoclonal antibodies.

    NARCIS (Netherlands)

    Adam, G.; Peters, D.; Goldbach, R.W.

    1996-01-01

    A test was conducted to compare tospovirus isolates using different poly- and monoclonal antibodies. All isolates and antibodies were compared under identical conditions. From 130 tospovirus isolates, which were obtained from all over the world and included well-characterized isolates from all four

  6. Serological comparison of tospovirus isolates using polyclonal and monoclonal antibodies.

    NARCIS (Netherlands)

    Adam, G.; Peters, D.; Goldbach, R.W.

    1996-01-01

    A test was conducted to compare tospovirus isolates using different poly- and monoclonal antibodies. All isolates and antibodies were compared under identical conditions. From 130 tospovirus isolates, which were obtained from all over the world and included well-characterized isolates from all four

  7. Development and characterization of a monoclonal antibody specific for human basophils and the identification of a unique secretory product of basophil activation.

    Science.gov (United States)

    McEuen, A R; Buckley, M G; Compton, S J; Walls, A F

    1999-01-01

    Despite increasing evidence that basophils can infiltrate into inflamed tissues during allergic reactions, determination of the extent of infiltration and elucidation of their role in allergic disease has been frustrated by the lack of reliable means for detecting this cell type in tissues. In the present study, we report on a new monoclonal antibody specific for basophils and on the initial characterization of the antigen it recognizes. Basophils were isolated from peripheral blood by Percoll density gradient centrifugation and a positive-selection immunomagnetic procedure and injected into mice to produce monoclonal antibodies. A hybridoma clone, designated BB1, secreted antibody of the IgG2a isotype; this antibody bound selectively to basophils on immunocytochemistry but did not react with any other cell type or tissue structure, although it did stain a proportion of cells from the basophilic cell line KU812F. In sections of mixed populations of peripheral blood cells, similar numbers of cells stained with Alcian blue dye and BB1 over a wide range of basophil purity. BB1 antibody was effective in identifying basophils in sections of mixed cells or in tissues after fixation with ethanol, Carnoy's solution, or formalin. Staining of basophils with BB1 gave a granular appearance, although flow cytometry indicated that some antigen was also present on the surface of the cell. Activation of these cells with anti-IgE antibody or with the calcium ionophore A23187 provoked release of the antigen in parallel with that of histamine. BB1 antibody did not, by itself, stimulate histamine release. The molecular mass of the antigen was determined on Hedrick-Smith gels to be 124+/-11 kd. This new monoclonal antibody will be a valuable experimental tool in future studies, allowing the reliable detection of basophils in tissues of patients with allergic and chronic inflammatory disease; in addition, the antigen it identifies has potential as a unique marker of basophil activation.

  8. Fingerprinting of Natural Product by Eastern Blotting Using Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Hiroyuki Tanaka

    2012-01-01

    Full Text Available We succeeded in developing the fingerprint of natural product by eastern blotting using monoclonal antibodies. After developing and separating them on a TLC plate, solasodine glycosides are oxidized by NaIO4 and reacted with a protein to give conjugates which are recognized with anti-solamargine monoclonal antibody (MAb. Anti-solamargine MAb having wide cross-reactivity can stain and detect all solasodine glycosides by fingerprint. Different sensitivity between solamargine and solasonine was observed. The detection limit was 1.6 ng of solasonine. The hydrolysed products of solamargine were determined by fingerprint of eastern blotting compared to their Rf values depending on the sugar number. Fingerprint by eastern blotting using anti-ginsenoside Rb1 MAb distinguished the formula containing ginseng prescribed in traditional Chinese medicine. By double-staining of ginsenosides it is possible to suggest that the staining color shows the pharmacological activity, such as the purple bands indicate ginsenosides having stimulation activity, and the blue color indicated compound like ginsenosides possessed the depression affect for the central nervous system (CNS, respectively.

  9. High throughput production of mouse monoclonal antibodies using antigen microarrays

    DEFF Research Database (Denmark)

    De Masi, Federico; Chiarella, P.; Wilhelm, H.;

    2005-01-01

    Recent advances in proteomics research underscore the increasing need for high-affinity monoclonal antibodies, which are still generated with lengthy, low-throughput antibody production techniques. Here we present a semi-automated, high-throughput method of hybridoma generation and identification....... Monoclonal antibodies were raised to different targets in single batch runs of 6-10 wk using multiplexed immunisations, automated fusion and cell-culture, and a novel antigen-coated microarray-screening assay. In a large-scale experiment, where eight mice were immunized with ten antigens each, we generated...

  10. Development and application of a monoclonal-antibody technique for counting Aureococcus anophagefferens, an alga causing recurrent brown tides in the Mid-Atlantic United States.

    Science.gov (United States)

    Caron, David A; Dennett, Mark R; Moran, Dawn M; Schaffner, Rebecca A; Lonsdale, Darcy J; Gobler, Christopher J; Nuzzi, Robert; McLean, Tim I

    2003-09-01

    A method was developed for the rapid detection and enumeration of Aureococcus anophagefferens, the cause of harmful algal blooms called "brown tides" in estuaries of the Mid-Atlantic United States. The method employs a monoclonal antibody (MAb) and a colorimetric, enzyme-linked immunosorbent assay format. The MAb obtained exhibits high reactivity with A. anophagefferens and very low cross-reactivities with a phylogenetically diverse array of other protists and bacteria. Standard curves are constructed for each 96-well microtiter plate by using known amounts of a preserved culture of A. anophagefferens. This approach allows estimation of the abundance of the alga in natural samples. The MAb method was compared to an existing method that employs polyclonal antibodies and epifluorescence microscopy and to direct microscopic counts of A. anophagefferens in samples with high abundances of the alga. The MAb method provided increased quantitative accuracy and greatly reduced sample processing time. A spatial survey of several Long Island estuaries in May 2000 using this new approach documented a range of abundances of A. anophagefferens in these bays spanning nearly 3 orders of magnitude.

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

    Science.gov (United States)

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

    2016-01-01

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

  12. Development of capillary size exclusion chromatography for the analysis of monoclonal antibody fragments extracted from human vitreous humor.

    Science.gov (United States)

    Rea, Jennifer C; Lou, Yun; Cuzzi, Joel; Hu, Yuhua; de Jong, Isabella; Wang, Yajun Jennifer; Farnan, Dell

    2012-12-28

    Recombinant antigen-binding fragments (Fabs) are currently on the market and in development for the treatment of ophthalmologic indications. Recently, Quality by Design (QbD) initiatives have been implemented that emphasize understanding the relationship between quality attributes of the product and their impact on safety and efficacy. In particular, changes in product quality once the protein is administered to the patient are of particular interest. Knowledge of protein aggregation in vivo is of importance due to the possibility of antibody aggregates eliciting an immunogenic response in the patient. Presently, there are few analytical methods with adequate sensitivity to analyze Fab aggregates in human vitreous humor (HVH) because the Fab amount available for analysis is often quite low. Here, we report the development of a highly sensitive capillary size exclusion chromatography (SEC) methodology for Fab aggregate analysis in HVH. We demonstrate a process to perform capillary SEC to analyze Fabs with picogram sensitivity and an RSD of less than 8% for the relative peak area of high molecular weight species (HMWS). In addition, we have developed a Protein G affinity chromatography method to capture Fabs from HVH for capillary SEC analysis. Recovery efficiencies ranging from 86 to 99% were achieved using this recovery method with 300 μL HVH samples containing Fab1. Finally, we demonstrate the applicability of the methodology by quantifying Fab aggregates in HVH, which can potentially be used for aggregate analysis of clinically relevant samples. Copyright © 2012 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    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.

  14. Development and evaluation of a DAS-ELISA for rapid detection of Tembusu virus using monoclonal antibodies against the envelope protein.

    Directory of Open Access Journals (Sweden)

    Hao Chen

    Full Text Available Since April 2010, Tembusu virus (TMUV which is a contagious pathogen of waterfowls, causing symptoms of high fever, loss of appetite and fall in egg production, has been reported in east of China. A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA which detects for TMUV was developed, using two monoclonal antibodies (mAbs against the TMUV envelope (E protein. BALB/c mice were immunized with purified recombinant E protein expressed in E. coli. Three hybridoma cell lines designated as 12B1, 10C6 and 2D2, were screened by cell fusion and indirect ELISA for their ability to recognize different linear epitopes on the E protein, and were characterized subsequently. High-affinity mAbs 12B1 and 2D2 were used as capture and detection antibodies, respectively. The reaction conditions for the DAS-ELISA were optimized for TMUV detection. The cross-reactivity of the DAS-ELISA was determined using TMUV, duck plague virus, avian influenza virus subtype H9, Newcastle disease virus, duck hepatitis A virus type 1 and duck reovirus samples. A total of 191 homogenized tissues of field samples were simultaneously detected by DAS-ELISA and by RT-PCR. The former was found to have a high specificity of 99.1% and a sensitivity of 93.1%. These results reveal a positive coincidence between DAS-ELISA and RT-PCR at a coincidence rate of 95.8%. The method developed in this study can be used for the diagnosis of TMUV infection of duck origin.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  18. Development, characterization, and epitope mapping of a panel of twenty-four monoclonal antibodies specific for human inducible nitric oxide synthase.

    Science.gov (United States)

    Webber, Robert J; Rodriguez, John G; Webber, Douglas S; Dunnebacke, Thelma H

    2005-02-01

    A panel of monoclonal antibodies (MAbs) to human inducible nitric oxide synthase (hiNOS) has been developed. By isotype analysis of the MAbs cloned from the 24 different positive hybridomas, 13 were determined to be mouse IgG1, two were mouse IgG2a, two were mouse IgG2b, and the seven others were mouse IgM antibodies: all contained kappa light chains. The anti-hiNOS MAbs were initially characterized by ELISA, RIA, Western blot, and immunocytochemistry, and then they were epitope mapped using synthetic peptides and a three-step mapping procedure. In the first step, each of the 24 MAbs was tested by indirect ELISA for binding to 96 overlapping 18-amino acid-long peptides that span the entire 1153-amino acid length of hiNOS. Eight IgG class anti-hiNOS MAbs were found to bind to one of five different peptides. In the second step, a series of amino terminal and carboxyl terminal truncated peptides were synthesized for each of the five peptides to which one or more of the MAbs bound. Each of the eight anti-hiNOS MAbs was found to bind to the truncated peptides with a unique specificity that identified the amino acid segment involved in binding. The third step in the epitope mapping process utilized three series of overlapping 5-, 6-, 7-, 8-, and 9-amino acid-long peptides for each of these segments and identified the exact amino acids of hiNOS involved in antibody binding. Anti-hiNOS MAbs 2A1-F8, 2D2-B2, 21C10-1D10, and 24B10-2C7 were found to be especially useful in different immunoassays.

  19. Rationale for the development of IMC-3G3, a fully human immunoglobulin G subclass 1 monoclonal antibody targeting the platelet-derived growth factor receptor alpha.

    Science.gov (United States)

    Shah, Gaurav D; Loizos, Nick; Youssoufian, Hagop; Schwartz, Jonathan D; Rowinsky, Eric K

    2010-02-15

    A large body of evidence suggests that the platelet-derived growth factor (PDGF) family and associated receptors are potential targets in oncology therapeutic development because of their critical roles in the proliferation and survival of various cancers and in the regulation and growth of the tumor stroma and blood vessels. Several small molecules that nonspecifically target the PDGF signaling axis are in current use or development as anticancer therapies. However, for the majority of these agents, PDGF and its receptors are neither the primary targets nor the principal mediators of anticancer activity. IMC-3G3, a fully human monoclonal antibody of the immunoglobulin G subclass 1, specifically binds to the human PDGF receptor alpha (PDGFRalpha) with high affinity and blocks PDGF ligand binding and PDGFRalpha activation. The results of preclinical studies and the frequent expression of PDGFRalpha in many types of cancer and in cancer-associated stroma support a rationale for the clinical development of IMC-3G3. Currently, IMC-3G3 is being evaluated in early clinical development for patients with several types of solid malignancies.

  20. Development of a Model Immunoassay Utilizing Monoclonal Antibodies of Different Specificities for Quantitative Determination of Dieldrin and Heptachlors in Their Mixtures.

    Science.gov (United States)

    Kataoka, Chiwa; Nihei, Masumi; Nimata, Masaomi; Sawadaishi, Kazuyuki

    2016-11-23

    The presence of dieldrin and heptachlor residues in cucurbitaceous crops at concentrations exceeding the limits set by the Japanese Food Sanitation Law constitutes a serious problem. To prevent accumulation of these residues in cucurbitaceous crops, development of high-throughput analysis methods for the detection of contaminants in the soil before cultivation is required. This study aimed to develop a model immunoassay using new monoclonal antibodies (MAbs) to quantitatively determine dieldrin and heptachlor contents in their mixtures. Three distinctive MAbs were obtained from mice immunized with the respective immunogens. MAb DrA-04 showed high reactivity toward dieldrin with ca. 20% cross-reactivity toward heptachlors. MAb DrC-02 displayed a similar reactivity toward dieldrin and heptachlors. The specificity and sensitivity of MAbs DrA-04 and DrC-02 were largely unaffected by the composition ratio of heptachlors in a mixture. Six standard mixtures with different dieldrin and heptachlor contents were prepared. Concentrations of dieldrin and heptachlors in standard mixtures, calculated on the basis of an immunoassay with MAbs DrA-04 and DrC-02, were 88.1-125 and 96.2-115% of the theoretical values, respectively, revealing excellent sensitivity and specificity of this assay. The developed method paves the way for a facile and rapid quantitative determination of chlorinated cyclodiene pesticides in soil.

  1. From gene to harvest: insights into upstream process development for the GMP production of a monoclonal antibody in transgenic tobacco plants.

    Science.gov (United States)

    Sack, Markus; Rademacher, Thomas; Spiegel, Holger; Boes, Alexander; Hellwig, Stephan; Drossard, Juergen; Stoger, Eva; Fischer, Rainer

    2015-10-01

    The EU Sixth Framework Programme Integrated Project 'Pharma-Planta' developed an approved manufacturing process for recombinant plant-made pharmaceutical proteins (PMPs) using the human HIV-neutralizing monoclonal antibody 2G12 as a case study. In contrast to the well-established Chinese hamster ovary platform, which has been used for the production of therapeutic antibodies for nearly 30 years, only draft regulations were initially available covering the production of recombinant proteins in transgenic tobacco plants. Whereas recombinant proteins produced in animal cells are secreted into the culture medium during fermentation in bioreactors, intact plants grown under nonsterile conditions in a glasshouse environment provide various 'plant-specific' regulatory and technical challenges for the development of a process suitable for the acquisition of a manufacturing licence for clinical phase I trials. During upstream process development, several generic steps were addressed (e.g. plant transformation and screening, seed bank generation, genetic stability, host plant uniformity) as well as product-specific aspects (e.g. product quantity). This report summarizes the efforts undertaken to analyse and define the procedures for the GMP/GACP-compliant upstream production of 2G12 in transgenic tobacco plants from gene to harvest, including the design of expression constructs, plant transformation, the generation of production lines, master and working seed banks and the detailed investigation of cultivation and harvesting parameters and their impact on biomass, product yield and intra/interbatch variability. The resulting procedures were successfully translated into a prototypic manufacturing process that has been approved by the German competent authority.

  2. Development of monoclonal antibodies (MAbs) to feline interferon (fIFN)-γ as tools to evaluate cellular immune responses to feline infectious peritonitis virus (FIPV).

    Science.gov (United States)

    Satoh, Ryoichi; Kaku, Ayumi; Satomura, Megumi; Kohori, Michiyo; Noura, Kanako; Furukawa, Tomoko; Kotake, Masako; Takano, Tomomi; Hohdatsu, Tsutomu

    2011-06-01

    Feline infectious peritonitis virus (FIPV) can cause a lethal disease in cats, feline infectious peritonitis (FIP). The antibody-dependent enhancement (ADE) of FIPV infection has been recognised in experimentally infected cats, and cellular immunity is considered to play an important role in preventing the onset of FIP. To evaluate the importance of cellular immunity for FIPV infection, monoclonal antibodies (MAbs) against feline interferon (fIFN)-γ were first created to establish fIFN-γ detection systems using the MAbs. Six anti-fIFN-γ MAbs were created. Then, the difference in epitope which those MAbs recognise was demonstrated by competitive enzyme-linked immunosorbent assay (ELISA) and IFN-γ neutralisation tests. Detection systems for fIFN-γ (sandwich ELISA, ELISpot assay, and two-colour flow cytometry) were established using anti-fIFN-γ MAbs that recognise different epitopes. In all tests, fIFN-γ production from peripheral blood mononuclear cells (PBMCs) obtained from cats experimentally infected with an FIPV isolate that did not develop the disease was significantly increased by heat-inactivated FIPV stimulation in comparison with medium alone. Especially, CD8(+)fIFN-γ(+) cells, but not CD4(+)fIFN-γ(+) cells, were increased. In contrast, fIFN-γ production from PBMCs isolated from cats that had developed FIP and specific pathogen-free (SPF) cats was not increased by heat-inactivated FIPV stimulation. These results suggest that cellular immunity plays an important role in preventing the development of FIP. Measurement of fIFN-γ production with the anti-fIFN-γ MAbs created in this study appeared to be useful in evaluating cellular immunity in cats.

  3. Antibodies in infectious diseases: polyclonals, monoclonals and niche biotechnology.

    Science.gov (United States)

    Berry, Jody D; Gaudet, Ryan G

    2011-09-01

    Antibody preparations have a long history of providing protection from infectious diseases. Although antibodies remain the only natural host-derived defense mechanism capable of completely preventing infection, as products, they compete against inexpensive therapeutics such as antibiotics, small molecule inhibitors and active vaccines. The continued discovery in the monoclonal antibody (mAb) field of leads with broadened cross neutralization of viruses and demonstrable synergy of antibody with antibiotics for bacterial diseases, clearly show that innovation remains. The commercial success of mAbs in chronic disease has not been paralleled in infectious diseases for several reasons. Infectious disease immunotherapeutics are limited in scope as endemic diseases necessitate active vaccine development. Also, the complexity of these small markets draws the interest of niche companies rather than big pharmaceutical corporations. Lastly, the cost of goods for mAb therapeutics is inherently high for infectious agents due to the need for antibody cocktails, which better mimic polyclonal immunoglobulin preparations and prevent antigenic escape. In cases where vaccine or convalescent populations are available, current polyclonal hyperimmune immunoglobulin preparations (pIgG), with modern and highly efficient purification technology and standardized assays for potency, can make economic sense. Recent innovations to broaden the potency of mAb therapies, while reducing cost of production, are discussed herein. On the basis of centuries of effective use of Ab treatments, and with growing immunocompromised populations, the question is not whether antibodies have a bright future for infectious agents, but rather what formats are cost effective and generate safe and efficacious treatments to satisfy regulatory approval. Copyright © 2011 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    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.

  5. Establishment of highly specific and quantitative immunoassay systems for staphylococcal enterotoxin A, B, and C using newly-developed monoclonal antibodies.

    Science.gov (United States)

    Sasaki, Takanori; Terano, Yoshitake; Shibata, Tadayoshi; Kawamoto, Hiroyoshi; Kuzuguchi, Tsuyoshi; Kohyama, Erina; Watanabe, Toshihiro; Ohyama, Tohru; Gemba, Munekazu

    2005-01-01

    Staphylococcal enterotoxin (SE) activities remain after boiling or treating with proteases. The main symptoms such as vomiting and diarrhea, are caused by the ingestion of SEs. Among SEs, SEA has been reported to be the major and most toxic protein. A highly specific and simple assay system is required to diagnose staphylococcal food poisoning. Therefore, the development of a suitable assay system is strongly anticipated. In this study, we have established a highly specific and sensitive avidin-biotin sandwich ELISA (ABS-ELISA) system for SEA, SEB, and SEC1 using newly-developed monoclonal antibodies. The linearity of these systems obtained was in the range of 0.78-25 ng/ml for each SE, and furthermore, the lower concentrations of SEs could also be detected. The recoveries of SEs from murine serum, skim milk solution, and raw milk were found to be over 90%, suggesting that our systems could detect SEs without any interventions, such as these from milk or serum proteins. We were also able to quantify SEs in 22 specimens of culture supernatants of S. aureus isolated in past occurrences. Our established system should be very useful not only in the clinical field but also in various fields of investigation because of its quantifi-cation and simplicity in detecting SEs.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  8. Development and application of pathovar-specific monoclonal antibodies that recognize the lipopolysaccharide O antigen and the type IV fimbriae of Xanthomonas hyacinthi

    Energy Technology Data Exchange (ETDEWEB)

    Doorn, J. van; Ojanen-Reuhs, T.; Hollinger, T.C.; Reuhs, B.L.; Schots, A.; Boonekamp, P.M.; Oudega, B.

    1999-09-01

    The objective of this study was to develop a specific immunological diagnostic assay for yellow disease in hyacinths, using monoclonal antibodies (MAbs). Mice were immunized with a crude cell wall preparation (shear fraction) from Xanthomonas hyacinthi and with purified type IV fimbriae. Hybridomas were screened for a positive reaction with X. hyacinthi cells or fimbriae and for a negative reaction with X. translucens pv. graminis or Erwinia carotovora subsp. carotovora. Nine MAbs recognized fimbrial epitopes, as shown by immunoblotting, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy; however, three of these MAbs had weak cross-reactions with two X. translucens pathovars in immunoblotting experiments. Seven MAbs reacted with lipopolysaccharides and yielded a low-mobility ladder pattern on immunoblots. Subsequent analysis of MAb 2E5 showed that it specifically recognized an epitope on the O antigen, which was found to consist of rhamnose and fucose in a 2:1 molar ratio. The cross-reaction of MAb 2E5 with all X. hyacinthi strains tested showed that this O antigen is highly conserved within this species. MAb 1B10 also reacted with lipopolysaccharides. MAbs 2E5 and 1B10 were further tested in ELISA and immunoblotting experiments with cells and extracts from other pathogens. No cross-reaction was found with 27 other Xanthomonas pathovars tested or with 14 other bacterial species from other genera, such as Erwinia and Pseudomonas, indicating the high specificity of these antibodies. MAbs 2E5 and 1B10 were shown to be useful in ELISA for the detection of X. hyacinthi in infected hyacinths.

  9. Taenia taeniaeformis: early inflammatory response around developing metacestodes in the liver of resistant and susceptible mice I. Identification of leukocyte response with monoclonal antibodies.

    Science.gov (United States)

    Letonja, T; Hammerberg, C

    1987-10-01

    Female BALB/cJ (resistant), C3H/HeJ (intermediate resistant), and C3H/HeDub (susceptible) inbred mice, 4-5 wk old, were infected with Taenia taeniaeformis. Liver sections were stained by an immunoperoxidase technique (avidin-biotin complex, ABC) for the differentiation antigens Lyt-1, Lyt-2, Mac-1, Mac-2, Mac-3, and B220. Binding of ABC to the cytoplasm of hepatocytes around the developing parasite was observed at 4 days postinfection (PI) in all 3 strains of mice, persisting in BALB/cJ and C3H/HeJ liver sections at 5 and 6 days PI, suggesting the presence of high concentrations of biotin, a fatty acid synthesis mediator. Two cell populations were labeled with B220 monoclonal antibodies: lymphocytes and polymorphonuclear (PMN) cells. At 4 days PI the number of labeled PMN cells peaked in infected C3H/HeJ and BALB/cJ mice; however a low number of PMN cells were labeled in infected C3H/HeDub mice. Few lymphocytes bound the B220 antibody in either BALB/cJ, C3H/HeJ, or C3H/HeDub infected mice. The number of Mac-1+ cells detected in infected C3H/HeJ and BALB/cJ liver sections were similar whereas fewer Mac-1+ cells were present in infected C3H/HeDub mice. Mac-2+ cells appeared in high numbers around the growing parasite at 5 and 6 days PI in the liver of C3H/HeDub mice, but not in the liver of BALB/cJ mice. Mac-3+ cells followed a similar pattern to that of the cell population defined by Mac-2. Few Lyt-1+ and Lyt-2+ cells were detected around the parasite site in the 3 strains of mice.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. Development of EMab-51, a Sensitive and Specific Anti-Epidermal Growth Factor Receptor Monoclonal Antibody in Flow Cytometry, Western Blot, and Immunohistochemistry.

    Science.gov (United States)

    Itai, Shunsuke; Kaneko, Mika K; Fujii, Yuki; Yamada, Shinji; Nakamura, Takuro; Yanaka, Miyuki; Saidoh, Noriko; Handa, Saori; Chang, Yao-Wen; Suzuki, Hiroyoshi; Harada, Hiroyuki; Kato, Yukinari

    2017-09-11

    The epidermal growth factor receptor (EGFR) is a member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases and is involved in cell growth and differentiation. EGFR homodimers or heterodimers with other HER members, such as HER2 and HER3, activate downstream signaling cascades in many cancers. In this study, we developed novel anti-EGFR monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. First, we expressed the full-length or ectodomain of EGFR in LN229 glioblastoma cells and then immunized mice with LN229/EGFR or ectodomain of EGFR, and performed the first screening using enzyme-linked immunosorbent assays. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening), Western blot (third screening), and immunohistochemical (fourth screening) analyses. Among 100 mAbs, only one clone EMab-51 (IgG1, kappa) reacted with EGFR in Western blot analysis. Finally, immunohistochemical analyses with EMab-51 showed sensitive and specific reactions against oral cancer cells, warranting the use of EMab-51 to detect EGFR in pathological analyses of EGFR-expressing cancers.

  11. Parallel development of chromatographic and mass-spectrometric methods for quantitative analysis of glycation on an IgG1 monoclonal antibody.

    Science.gov (United States)

    Viski, Kornél; Gengeliczki, Zsolt; Lenkey, Krisztián; Baranyáné Ganzler, Katalin

    2016-10-01

    Monitoring post-translational modifications (PTMs) in biotherapeutics is of paramount importance. In pharmaceutical industry, chromatography with optical detection is the standard choice of quantitation of product related impurities; and mass spectrometry is used only for characterization. Parallel development of a boronate affinity chromatographic (BAC) and a mass spectrometric methods for quantitative measurement of glycation on a monoclonal antibody (mAb) shed light on the importance of certain characteristics of the individual methods. Non-specific interactions in BAC has to be suppressed with the so-called shielding reagent. We have found that excessive amount of shielding reagents in the chromatographic solvents may cause significant underestimation of glycation. Although contamination of the retained peak with the non-glycated isoforms in BAC is unavoidable, our work shows that it can be characterized and quantitated by mass spectrometry. It has been demonstrated that glycation can be measured by mass spectrometry at the intact protein level with an LOQ value of 3.0% and error bar of ±0.5%. The BAC and MS methods have been found to provide equivalent results. These methods have not been compared from these points of view before.

  12. Comparison of Laboratory-Developed and Commercial Monoclonal Antibody-Based Sandwich Enzyme-Linked Immunosorbent Assays for Almond (Prunus dulcis) Detection and Quantification.

    Science.gov (United States)

    Liu, Changqi; Chhabra, Guneet S; Zhao, Jing; Zaffran, Valerie D; Gupta, Sahil; Roux, Kenneth H; Gradziel, Thomas M; Sathe, Shridhar K

    2017-09-04

    A commercially available monoclonal antibody (mAb)-based direct sandwich enzyme-linked immunosorbent assay (ELISA) kit (BioFront Technologies, Tallahassee, Fla., U.S.A.) was compared with an in-house developed mAb 4C10-based ELISA for almond detection. The assays were comparable in sensitivity (limit of detection < 1 ppm full fat almond, limit of quantification < 5 ppm full fat almond), specificity (no cross-reactivity with 156 tested foods at a concentration of 100000 ppm whole sample), and reproducibility (intra- and interassay variability < 15% CV). The target antigens were stable and detectable in whole almond seeds subjected to autoclaving, blanching, frying, microwaving, and dry roasting. The almond recovery ranges for spiked food matrices were 84.3% to 124.6% for 4C10 ELISA and 81.2% to 127.4% for MonoTrace ELISA. The almond recovery ranges for commercial and laboratory prepared foods with declared/known almond amount were 30.9% to 161.2% for 4C10 ELISA and 38.1% to 207.6% for MonoTrace ELISA. Neither assay registered any false-positive or negative results among the tested commercial and laboratory prepared samples. © 2017 Institute of Food Technologists®.

  13. ANTITUMOR EFFECTS OF MONOCLONAL ANTIBODY FAB′ FRAGMENT CONTAINING IMMUNOCONJUGATES

    Institute of Scientific and Technical Information of China (English)

    刘小云; 甄永苏

    2002-01-01

    Objective.Using monoclonal antibody (mAb) Fab′ fragment to develop mAb immunoconjugates for cancer. Methods.Fab′ fragment of mAb 3A5 was prepared by digestion of the antibody with pepsin and then reduced by dithiothreitol (DTT),while Fab′ fragment of mAb 3D6 was obtained by digestion of the antibody with ficin and subsequently reduced by β mercaptoethanol.The conjugation between Fab′ fragment and pingyangmycin (PYM),an antitumor antibiotic,was mediated by dextran T 40.Immunoreactivity of Fab′ PYM conjugates with cancer cells was determined by ELISA,and the cytotoxicity of those conjugates to cancer cells was determined by clonogenic assay.Antitumor effects of the Fab′ PYM conjugates were evaluated by subcutaneously transplanted tumors in mice. Results.The molecular weight of Fab′ fragment was approximately 53 kD,while the average molecular weight of Fab′ PYM conjugate was 170 kD.The Fab′ PYM conjugates showed immunoreactivity with antigen relevant cancer cells and selective cytotoxicity against target cells.Administered intravenously,Fab′ PYM conjugates were more effective against the growth of tumors in mice than free PYM and PYM conjugated with intact mAb. Conclusion.Fab′ PYM conjugate may be capable of targeting cancer cells and effectively inhibiting tumor growth,suggesting its therapeutic potential in cancer treatment.

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

    Science.gov (United States)

    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.

  15. Generation and characterization of monoclonal antibodies against the transcription factor Nkx6.1.

    Science.gov (United States)

    Pedersen, Inger L; Klinck, Rasmus; Hecksher-Sorensen, Jacob; Zahn, Stefan; Madsen, Ole D; Serup, Palle; Jorgensen, Mette C

    2006-05-01

    We present the generation of a panel of monoclonal antibodies (F55A10, F55A12, F64A6B4, and F65A2) against the homeodomain transcription factor Nkx6.1, one of the essential transcription factors that regulates the multistep differentiation process of precursor cells into endocrine beta-cells in the pancreas. Expression of Nkx6.1 can be detected in developing pancreatic epithelium and in adult insulin-producing beta-cells, making this transcription factor a unique beta-cell marker. For production of monoclonal antibodies, RBF mice were immunized with a GST-Nkx6.1 fusion protein containing a 66-amino acid C-terminal fragment of rat Nkx6.1. Four clones were established as stable hybridoma cell lines and the produced antibodies were of the mouse IgG1/kappa subtype. When applied for immunohistochemistry on frozen sections of adult mouse pancreas, monoclonal antibodies stain specifically the beta-cells in the endocrine islets of Langerhans with patterns comparable to that of a previously produced polyclonal rabbit serum. Monoclonal antibodies can be divided into two groups that appear to recognize different epitopes, as determined by competition ELISA. The presented antibodies are useful tools for the further characterization of the role and function of Nkx6.1 in pancreatic development, especially for use in double-labeling experiments with existing polyclonal rabbit antibodies.

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

    Science.gov (United States)

    Strohl, William R

    2014-03-01

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

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

    Science.gov (United States)

    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.

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

    OpenAIRE

    Ming Sun; Yue Li; Huiwen Zheng; Yiming Shao

    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 ...

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  1. Characterization of Binding Epitopes of CA125 Monoclonal Antibodies

    DEFF Research Database (Denmark)

    Marcos-Silva, Lara; Narimatsu, Yoshiki; Halim, Adnan

    2014-01-01

    The most used cancer serum biomarker is the CA125 immunoassay for ovarian cancer that detects the mucin glycoprotein MUC16. Several monoclonal antibodies (mAbs) including OC125 and M11 are used in CA125 assays. However, despite considerable efforts, our knowledge of the molecular characteristics...

  2. Production and potential use of monoclonal antibodies against polio viruses.

    NARCIS (Netherlands)

    A.D.M.E. Osterhaus (Albert); A.L. van Wezel; G. van Steenis (Bert); A.G. Hazendonk

    1982-01-01

    textabstractLymphocyte hybridomas secreting monoclonal antibodies against different strains of polio virus type 1, 2, or 3 have been produced. For this purpose Balb/C mice were immunized with purified and inactivated virus suspensions and their splenocytes were fused with P3X63Ag8 mouse myeloma cell

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

    DEFF Research Database (Denmark)

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

    1996-01-01

    Murine monoclonal antibodies (Mabs) against water-soluble somatic antigens (WSSA) and the wall fraction (WF) from Rhizopus arrhizus (Rhizopus oryzae) were produced in vitro by fusion of splenocytes from immunized BALB/c mice with mouse myeloma X63-Ag 8.653 cells. Supernatants reacting only with h...... for the in situ diagnosis of systemic bovine zygomycosis....

  4. A mouse monoclonal antibody against Alexa Fluor 647.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    2007-07-01

    Mycotoxins as Biological Weapons PRINCIPAL INVESTIGATOR: Marta Feldmesser, M.D. CONTRACTING ORGANIZATION: Albert Einstein College of...Monoclonal Antibodies to Prevent Use of Mycotoxins as Biological Weapons 5b. GRANT NUMBER W81XWH-06-1-0085 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR

  6. Monoclonal antibodies specific for the organophosphate pesticide azinphos-methyl

    NARCIS (Netherlands)

    Jones, WT; Harvey, D; Jones, SD; Ryan, GB; Wynberg, H; TenHoeve, W; Reynolds, PHS

    1995-01-01

    2-(2-Mercapto-5-methyl-1,3,2-dioxaphosphorinan-5-yl,2-sulphide) methoxyacetic acid has been synthesized and used to prepare an azinphos hapten and protein conjugates. Monoclonal antibodies of high affinity against the pesticide azinphos-methyl were prepared from mice immunized with the

  7. Monoclonal antibodies specific for the organophosphate pesticide azinphos-methyl

    NARCIS (Netherlands)

    Jones, WT; Harvey, D; Jones, SD; Ryan, GB; Wynberg, H; TenHoeve, W; Reynolds, PHS

    1995-01-01

    2-(2-Mercapto-5-methyl-1,3,2-dioxaphosphorinan-5-yl,2-sulphide) methoxyacetic acid has been synthesized and used to prepare an azinphos hapten and protein conjugates. Monoclonal antibodies of high affinity against the pesticide azinphos-methyl were prepared from mice immunized with the hapten-ovalbu

  8. Anti-bacterial monoclonal antibodies: back to the future?

    Science.gov (United States)

    Oleksiewicz, Martin B; Nagy, Gábor; Nagy, Eszter

    2012-10-15

    Today's medicine has to deal with the emergence of multi-drug resistant bacteria, and is beginning to be confronted with pan-resistant microbes. This worsening inadequacy of the antibiotics concept, which has ruled infectious medicine in the last six decades creates an increasing unmet medical need that can be addressed by passive immunization. While past experience from the pre-antibiotic era with serum therapy was in many cases encouraging, antibacterial monoclonal antibodies have so far suffered high attrition rates in the clinic, generally from lack of efficacy. Yet, we believe that recent developments in a number of areas such as infectious disease pathogenesis research, translational medicine, mAb engineering, mAb manufacturing and rapid bedside diagnostics are converging to make the medium-term future permissive for antibacterial mAb development. Here, we review antibacterial mAb-based approaches that are or were in clinical development, and may potentially act as paradigms with regards to molecular targets, antibody formats and mode-of-action, pre-clinical validation and selection of most relevant patient populations, in order to increase the likelihood of successful product development in this field.

  9. Structure and specificity of lamprey monoclonal antibodies

    OpenAIRE

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

    2008-01-01

    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...

  10. Development of a monoclonal antibody against the left wing of ciguatoxin CTX1B: thiol strategy and detection using a sandwich ELISA.

    Science.gov (United States)

    Tsumuraya, Takeshi; Takeuchi, Katsutoshi; Yamashita, Shuji; Fujii, Ikuo; Hirama, Masahiro

    2012-09-01

    Ciguatera fish poisoning (CFP) is a form of food poisoning caused by the ingestion of a variety of reef fish that have accumulated trace amounts of ciguatoxins produced by dinoflagellates of the genus Gambierdiscus through the food chain. CFP affects more than 50,000 people each year. The extremely low level of the causative neurotoxins, ciguatoxins, in fish has hampered the preparation of antibodies for detecting the toxins. In this paper, we describe a thiol strategy for synthesizing a keyhole limpet hemocyanin (KLH)-conjugate (20) of the ABCDE-ring fragment of the Pacific ciguatoxins, CTX1B (1) and 54-deoxyCTX1B (4). We succeeded in producing a monoclonal antibody (3G8) against the left wings of these ciguatoxins by immunizing mice with the hapten-KLH conjugate (20) as the synthetic antigen. The most promising mAb, 3G8, does not cross-react with other related marine toxins. Sandwich enzyme-linked immunosorbent assay (ELISA) utilizing 3G8 and the previously prepared monoclonal antibody (8H4) enabled us to detect 1 specifically at less than 0.28 ng/mL.

  11. 治疗性单克隆抗体药物的现状及发展趋势%The Status Quo and Development Trend of the Therapeutic Monoclonal Antibody Drugs

    Institute of Scientific and Technical Information of China (English)

    王志明; 高健; 李耿

    2013-01-01

    After more than 30 years of development,monoclonal antibodies have become an important class of clinical drugs.Currently,these antibody drugs are used to treat many kinds of diseases,such as cancer,autoimmune diseases,infectious diseases and transplant rejection reactions.Ended in 2012,there are 29 kinds of therapeutic monoclonal antibody drugs through the FDA approval and on sale.And in 2011,there were more than 1000 kinds of antibody drugs undergoing clinical trials in the European Union or the United States,of which about 164 kinds of antibodies in clinical phase Ⅲ trials.The safety and efficacy of therapeutic monoclonal antibody drug is largely decided by the target.Some monoclonal antibody drugs in research have the same targets with the antibodies on sale,and some have the unique targets.At the same time,more and more new targets are being found.There are four kinds of the most popular targets of research:tumor necrosis factor-α,cluster of differentiation 20,epidermal growth factor receptor and vascular endothelial growth factor.Detailed review which focuses on the different targets of the monoclonal antibody drugs,whether in research or on sale,is expected to provide valuable references for the monoclonal antibody drug development trend.The status quo of monoclonal antibody drug in our country was discussed,and the development strategies in the future were proposed.%治疗性单克隆抗体药物经历了三十多年的发展,已经成为生物医药的最重要组成部分之一.在疾病治疗上具有广阔的应用前景,成功用于治疗肿瘤、自身免疫性疾病、感染性疾病和移植排斥反应等多种疾病.截至2012年已有29种治疗性单克隆抗体药物通过FDA审批并上市销售.治疗性单抗的安全性和有效性很大程度上由其作用的靶点决定,上市和在研的单抗药物有些靶向相同的靶点,有些有自己独特的作用靶点,新的作用靶点也在不断出现.以治疗性单抗的作用靶点

  12. Development of a Monoclonal Antibody-Based icELISA for the Detection of Ustiloxin B in Rice False Smut Balls and Rice Grains

    Directory of Open Access Journals (Sweden)

    Xiaoxiang Fu

    2015-08-01

    Full Text Available Rice false smut is an emerging and economically-important rice disease caused by infection by the fungal pathogen Villosiclava virens. Ustiloxin B is an antimitotic cyclopeptide mycotoxin isolated from the rice false smut balls that formed in the pathogen-infected rice spikelets. A monoclonal antibody (mAb designated as mAb 1B5A10 was generated with ustiloxin B—ovalbumin conjugate. A highly-sensitive and specific indirect competitive enzyme-linked immunosorbent assay (icELISA was then developed. The median inhibitory concentration (IC50 of the icELISA was 18.0 ng/mL for the detection of ustiloxin B; the limit of detection was 0.6 ng/mL, and the calibration range was from 2.5 to 107.4 ng/mL. The LOD/LOQ values of the developed ELISA used for the determination of ustiloxin B in rice false smut balls and rice grains were 12/50 μg/g and 30/125 ng/g, respectively. The mAb 1B5A10 cross-reacted with ustiloxin A at 13.9% relative to ustiloxin B. Average recoveries of ustiloxin B ranged from 91.3% to 105.1% for rice false smut balls at spiking levels of 0.2 to 3.2 mg/g and from 92.6% to 103.5% for rice grains at spiking levels of 100 to 5000 ng/g. Comparison of ustiloxin B content in rice false smut balls and rice grains detected by both icELISA and high performance liquid chromatography (HPLC demonstrated that the developed icELISA can be employed as an effective and accurate method for the detection of ustiloxin B in rice false smut balls, as well as rice food and feed samples.

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

    Science.gov (United States)

    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.

  14. The Preparation and Identification of a Monoclonal Antibody against Citrinin and the Development of Detection via Indirect Competitive ELISA

    Directory of Open Access Journals (Sweden)

    Shimuye Kalayu Yirga

    2017-03-01

    Full Text Available Citrinin (CTN is a hepato-nephrotoxic mycotoxin produced by fungi genera of Aspergillus, Monauscus, and Penicillium. CTN contaminates grains, fruits, juices and vegetables, and causes various toxic effects to humans and animals. It has small molecular weight, which is non-immunogenic to animals. Thus, CTN was conjugated to bovine serum albumin (BSA and ovalbumin (OVA, respectively, by amide bonds using 1-ethyl-3-(3-dimethylaminopropyl carbodiimide hydrochloride (EDC and N-hydroxysuccinimide (NHS. Mice were immunized with CTN-BSA conjugates, and spleen cells of the immunized mice were fused with Sp2/0 myeloma cells to obtain 21H27 hybriodoma cell. Ascitic fluid of hybridoma cell was produced in mice abdomen, and purified using caprylic/ammonium sulfate precipitation method. The 21H27 anti-CTN mcAb was the IgG2a antibody subclass, and cross-reactivity results indicated that anti-CTN mcAb is specific to CTN with high affinity (2.0 × 108 L/mol. Indirect competitive ELISA (ic-ELISA results showed that the linear range of detection was 0.01–5.96 ng/mL and the IC50 was 0.28 ng/mL with a lower detection limit (LOD of 0.01 ng/mL. The average recovery was 93.8% ± 1.6% with a coefficient variation of 1.0%–4.3%. Hence, anti-CTN mcAb secreted by 21H27 hybridoma cell was successfully produced and can be used to detect CTN contaminated feed and foodstuffs.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  16. Adsorption of monoclonal antibodies to glass microparticles.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  18. Development of a H2 O2 -sensitive quantum dots-based fluorescent sandwich ELISA for sensitive detection of bovine β-lactoglobulin by monoclonal antibody.

    Science.gov (United States)

    He, Shengfa; Li, Xin; Gao, Jinyan; Tong, Ping; Chen, Hongbing

    2017-06-16

    Bovine β-lactoglobulin (BLG) is the major allergen in cows' milk, and the specific epitope plays a key role in food allergy. Developing a method specifically bind to the IgE epitope is necessary for testing BLG and its allergenic residues. The monoclonal antibody (1G9) specific to the IgE linear epitope for BLG was identified as high affinity and specificity. Based on 1G9, a sensitive fluorescent sandwich enzyme-linked immunosorbent assay (sELISA) was successfully developed using catalase-mediated fluorescence quenching of thiolated CdTe quantum dots in the presence of hydrogen peroxide as fluorescent signal output. The fluorescent sELISA showed high sensitivity and specificity, the limit of detection was 0.49 ng mL(-1) , which was 16-fold lower than horseradish peroxidase (HRP)-based sELISA. The linear range for BLG detection were 125-4000 ng mL(-1) (r = 0.9939) and 0.48-62.5 ng mL(-1) (r = 0.9919). The recoveries and coefficients of variation were 94.25-109.83% and 4.38-20.29%, respectively. Allergenic residues were also detected in hydrolysed infant formulas. The results of fluorescent sELISA showed good performance as HRP-based sELISA and commercial sELISA kit. This proposed fluorescent sELISA could be employed to detect BLG and its allergenic residues in food with highly sensitivity, reliability, and recovery. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

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

    Science.gov (United States)

    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 (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.

  20. Development, evaluation, and in-vivo validation of two non-invasive methods for quantitation of activity and dosimetry of monoclonal antibodies in humans

    Energy Technology Data Exchange (ETDEWEB)

    Hammond, N.D.; Moldofsky, P.J.; Exten, R.E.; Gatenby, R.A.; Broder, G.J.

    1985-05-01

    The authors have applied both a conjugate view imaging method and a first pass study for quantitation of absolute I-131 activity in lesions and normal tissue of patients with colon carcinoma in order to study biological clearance of the I-131 F(ab)'/sub 2/ fragments of mouse monoclonal antibody and the resultant dosimetry. Both methods require a transmission scan for determining patient attenuation and measurement of patient lesion or organ size in the region of interest. The conjugate view method is analyzed for both SPECT and planar imaging. The percent error of both methods relates to lesion size and absolute activity when compared to actual well-counter assayed samples of malignant and normal tissue obtained from CT-guided needle biopsies or surgical specimens. Dosimetric evaluation was based on determination of activity, clearance from computer-generated time-activity curves and lesion or organ volumes from volumetric CT scan data. The dose to the thyroid gland was calculated for one population receiving Lugol's solution 3 days prior and for the other who received Lugol's at the time of administration. Data showed no significant difference in absorbed thyroid dose. Lastly, the absolute uptake of I-131, lesion to background ratios, and the dosimetry data were compared for three different monoclonal antibody fragments.

  1. The Use of Monoclonal Antibodies in Human Prion Disease

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Sindy Liao-Chan

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

  3. Development of porcine ficolin-alpha monoclonal and polyclonal antibodies for determining the binding capacity of multiple GlcNAc-binding proteins to bacterial danger components.

    Science.gov (United States)

    Nahid, M Abu; Ross, Steven J; Umiker, Benjamin R; Li, Huapeng; Sugii, Sunji; Bari, Latiful

    2016-02-01

    Ficolins are a group of oligomeric defense proteins assembled from collagen-like stalks and fibrinogen-like domains that have common biochemical specificity for N-acetyl-d-glucose amine (GlcNAc) and can function as opsonins. In this report, GlcNAc-binding protein (GBP) purified from porcine nonimmune serum was biochemically characterized as ficolin-α. Ficolin-α was used as an immunogen to generate both rabbit polyclonal and murine monoclonal anti-ficolin-α antibodies, which are not yet commercially available. GBPs have been shown to be present in many animals, including humans; however, their functions are largely unknown. GBPs from chicken, dog, horse, bovine, and human sera were isolated using various chromatography methods. Interestingly, anti-ficolin-α antibody showed cross-reaction with those animal sera GBPs. Furthermore, anti-ficolin-α antibody was reactive with the GlcNAc eluate of Escherichia coli O26-bound and Salmonella-bound porcine serum proteins. Functionally, GBPs and bacteria-reactive pig serum proteins were able to bind with pathogen-associated molecular patterns such as lipopolysaccharides and lipoteichoic acids. Our studies demonstrate that ficolin-α specific antibody was reactive with GBPs from many species as well as bacteria-reactive serum proteins. These proteins may play important roles in innate immunity by sensing danger components that can lead to antibacterial activity.

  4. A recombinant, fully human monoclonal antibody with antitumor activity constructed from phage-displayed antibody fragments

    NARCIS (Netherlands)

    Huls, GA; Heijnen, IAFM; Cuomo, ME; Koningsberger, JC; Boel, E; de Vries, ARV; Loyson, SAJ; Helfrich, W; Henegouwen, GPV; van Meijer, M; de Kruif, J; Logtenberg, T

    1999-01-01

    A single-chain Fv antibody fragment specific for the tumor-associated Ep-CAM molecule was isolated from a semisynthetic phage display library and converted into an intact, fully human IgG1 monoclonal antibody (huMab), The purified huMab had an affinity of 5 nM and effectively mediated tumor cell kil

  5. Generation and applications of monoclonal antibodies for livestock production.

    Science.gov (United States)

    Van Der Lende, T

    1994-01-01

    Monoclonal antibodies (MCAs) have found widespread applications in livestock production. Although the generation of murine MCAs is at present a routine, the production of homologous MCAs, especially important for in vivo applications, is still hampered by the lack of efficient homologous fusion partners for immortalization of antibody producing lymphocytes of livestock species. At present, MCAs are used in immunodiagnostic tests e.g. to monitor livestock reproduction and quality of livestock products. In the future MCAs will also be used in immunosensors for real-time and on-site applications in the same areas. The commercial application of MCAs for the immunomodulation of (pharmacologically induced) physiological processes underlying important (re)production traits is at present limited to the use of anti-PMSG MCAs in PMSG-induced superovulation. However, many potentially interesting applications are under investigation (e.g. immunopotentiation of growth hormone to enhance growth; immunocytolysis of adipocytes to increase lean meat production; immunoneutralization of GnRH for immunocastration; immunoimitation of hormone activity with anti-idiotype antibodies). Attempts to use specific MCAs for the sexing of embryos have been disappointing, mainly because of the relatively low accuracy. In the future, MCAs against membrane proteins which are specific for X- or Y-chromosome bearing spermatozoa might be used for bulk separation of livestock sperm. In general, it is expected that engineered (homologous) recombinant MCAs will largely contribute to the development of a new generation of rapid immunodiagnostic tests and effective immunomodulation applications. They will further increase the use of MCAs in livestock production.

  6. Three monoclonal antibodies against the serpin protease nexin-1 prevent protease translocation

    DEFF Research Database (Denmark)

    Kousted, Tina Mostrup; Skjoedt, K; Petersen, S V

    2013-01-01

    , conversion to an inactive conformation or induction of serpin substrate behaviour. Until now, no inhibitory antibodies against PN-1 have been thoroughly characterised. Here we report the development of three monoclonal antibodies binding specifically and with high affinity to human PN-1. The antibodies all...... the loop connecting α-helix F with β-strand 3A and the loop connecting α-helix A with β-strand 1B. We conclude that antibody binding causes a direct blockage of the final critical step of protease translocation, resulting in abortive inhibition and premature release of reactive centre cleaved PN-1...

  7. Production of monoclonal antibodies to human glomerular basement membrane.

    Directory of Open Access Journals (Sweden)

    Mino,Yasuaki

    1984-10-01

    Full Text Available Using the technique of somatic cell fusion, we produced monoclonal antibodies to collagenase-digested human glomerular basement membrane (GBM. Fourteen monoclonal antibodies which reacted with normal human kidney in indirect immunofluorescence (IIF studies were produced. An analysis of the binding patterns indicated that the antigens recognized could be divided into six broad groups. Monoclonal antibody B3-H10 (Group 1 reacted with only GBM in a fine granular pattern. A5-B12 and B5-C2 (Group 2 reacted with GBM and peritubular capillary in a linear pattern. B2-A12 (Group 3 reacted with only epithelial cells. Al-C9 and A4-E2 (Group 4 showed a mesangial pattern in glomerulus and a lineal pattern in tubular basement membrane (TBM, Bowman's capsule and peritubular capillary. A1-E1, A1-E11, A2-E6, A3-B6, A4-F8 and B5-H2 (Group 5 recognized determinants common to GBM, TBM, Bowman's capsule and/or peritubular capillary. A3-F1 and B5-E10 (Group 6 reacted with TBM and Bowman's capsule. The staining pattern of B3-H10 (Group 1 was characteristic because it was not linear, but finely granular along the GBM. The staining pattern of B2-A12 (Group 3 was also characteristic because only epithelial cells were stained, and processes of epithelial cells were observed as fine fibrils. To the best of our knowledge, these two types of monoclonal antibodies have not been reported previously.

  8. Quantification of Moraxella bovis haemagglutinating adhesins with monoclonal antibodies.

    Science.gov (United States)

    Gil-Turnes, C; Aleixo, J A

    1991-08-01

    Six monoclonal antibodies (MAbs) against Moraxella bovis GF 9 were used to quantify haemagglutinating adhesins of 16 strains of this organism. The amount of each MAb necessary to inhibit one haemagglutinating unit of each strain varied between 4 and 0.007 times that required by strain GF 9. Five strains reacted with six MAbs, one with five, two with four, one with three, two with two and three with none. The procedures used enabled to detect dominant strains candidates for vaccines.

  9. Monoclonal antibody to native P39 protein from Borrelia burgdorferi.

    OpenAIRE

    Sullivan, T J; Hechemy, K E; Harris, H L; Rudofsky, U H; Samsonoff, W A; Peterson, A J; Evans, B. D.; Balaban, S L

    1994-01-01

    We have produced, by using a sonicate of Borrelia burgdorferi, a monoclonal antibody (MAb), NYSP39H, that is specific for the P39 protein band. This MAb reacted with 13 isolates of B. burgdorferi but not with eight different spirochetes (four borrelias, two leptospiras, and two treponemas). Surface labeling of B. burgdorferi with biotin and subsequent treatment with Nonidet P-40 showed that P39 was not biotinylated but was extracted with Nonidet P-40, indicating that it is present within the ...

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  12. Molecular imaging of rheumatoid arthritis by radiolabelled monoclonal antibodies: new imaging strategies to guide molecular therapies

    Energy Technology Data Exchange (ETDEWEB)

    Malviya, G.; Dierckx, R.A. [Department of Nuclear Medicine and Molecular Imaging, University Medical Centre Groningen, University of Groningen (Netherlands); Conti, F. [Rheumatology Unit, I Faculty of Medicine and Surgery, Sapienza University of Rome (Italy); Chianelli, M. [Department of Nuclear Medicine and Molecular Imaging, University Medical Centre Groningen, University of Groningen (Netherlands); Unit of Nuclear Medicine, Regina apostolorum Hospital, Albano, Rome (Italy); Scopinaro, F. [Nuclear Medicine Department, Sapienza University of Rome, St. Andrea Hospital, Rome (Italy); Signore, A. [Department of Nuclear Medicine and Molecular Imaging, University Medical Centre Groningen, University of Groningen (Netherlands); Nuclear Medicine Department, Sapienza University of Rome, St. Andrea Hospital, Rome (Italy)

    2010-02-15

    The closing of the last century opened a wide variety of approaches for inflammation imaging and treatment of patients with rheumatoid arthritis (RA). The introduction of biological therapies for the management of RA started a revolution in the therapeutic armamentarium with the development of several novel monoclonal antibodies (mAbs), which can be murine, chimeric, humanised and fully human antibodies. Monoclonal antibodies specifically bind to their target, which could be adhesion molecules, activation markers, antigens or receptors, to interfere with specific inflammation pathways at the molecular level, leading to immune-modulation of the underlying pathogenic process. These new generation of mAbs can also be radiolabelled by using direct or indirect method, with a variety of nuclides, depending upon the specific diagnostic application. For studying rheumatoid arthritis patients, several monoclonal antibodies and their fragments, including anti-TNF-{alpha}, anti-CD20, anti-CD3, anti-CD4 and anti-E-selectin antibody, have been radiolabelled mainly with {sup 99m}Tc or {sup 111}In. Scintigraphy with these radiolabelled antibodies may offer an exciting possibility for the study of RA patients and holds two types of information: (1) it allows better staging of the disease and diagnosis of the state of activity by early detection of inflamed joints that might be difficult to assess; (2) it might provide a possibility to perform 'evidence-based biological therapy' of arthritis with a view to assessing whether an antibody will localise in an inflamed joint before using the same unlabelled antibody therapeutically. This might prove particularly important for the selection of patients to be treated since biological therapies can be associated with severe side-effects and are considerably expensive. This article reviews the use of radiolabelled mAbs in the study of RA with particular emphasis on the use of different radiolabelled monoclonal antibodies for

  13. Human Monoclonal Antibodies Broadly Neutralizing against Influenza B Virus

    Science.gov (United States)

    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

  14. PREPARATION AND CHARACTERIZATION OF MONOCLONAL ANTIBODY AGAINST HUMAN TELOMERASE REVERSE TRANSCRIPTASE

    Institute of Scientific and Technical Information of China (English)

    王俊梅; 张波; 杨邵敏; 韩继生; 李冰思; 侯琳

    2003-01-01

    Objective. To develop monoclonal antibodies against the catalytic subunit of human telomerase reverse transcriptase (hTERT) for its expression detection of human tumors. Methods. A dominant epitope in hTERT (peptide hTERT7)was automatically synthesized based on Fmoc method, and was used to immunize Balb/c mice. Hybridomas were generated and screened by ELISA for specific monoclonal antibodies, and the characterization was performed by Western blotting and immunohistochemical staining. The heavy chain variable region of antibody was cloned by RT-PCR and sequenced. Results. Antigenic peptide hTERT7 was synthesized and confirmed by MALDI-TOF-MS and HPLC analysis. One hybridoma cell line secreting anti-hTERT7 antibodies designated as M2 was established after primary screening and consequent 3 rounds of limited dilution. M2 was IgG1 in isotyping. The competi tive assay showed that the M2 antibody was hTERT7 -specific, and the affinity constant was about 1×106 mol-1. The antibody reacted with cell extracts from HeLa cancer cells but not with those from normal 2BS cells in ELISA assay. For in situ staining of immunohistochemistry, the positive staining presented in the nuclear compartment of HeLa, while 2BS was negative. The heavy chain variable region from M2 re vealed that the monoclonal antibody was mouse origin. Conclusions. The developed mouse monoclonal antibody is hTERT-specific and able to recognize native cellular hTERT in ELISA and immunohistochemistry, which makes the immuno-detection of telom erase hTERT expression in cancer cells or tissues possible.

  15. Development and characterization of a specific IgG monoclonal antibody toward the Lewis x antigen using splenocytes of Schistosoma mansoni-infected mice.

    Science.gov (United States)

    Mandalasi, Msano; Dorabawila, Nelum; Smith, David F; Heimburg-Molinaro, Jamie; Cummings, Richard D; Nyame, A Kwame

    2013-07-01

    The parasitic blood fluke Schistosoma mansoni synthesizes immunogenic glycans containing the human Lewis x antigen (Le(x); Galactose-β1-4(Fucα1-3)N-acetylglucosamine-β-R, also called CD15), but the biological role(s) of this antigen in the parasites and in humans is poorly understood. To develop IgG-based monoclonal antibodies (mAbs) specific for Le(x), we harvested splenocytes from S. mansoni-infected Swiss Webster mice at Week 10 postinfection, when peak IgG responses to glycan antigens occur, and generated a panel of hybridomas secreting anti-glycan IgG that recognize periodate-sensitive epitopes in soluble egg antigens of the parasites, and also recognizes a neoglycoprotein containing a pentasaccharide with the Le(x) sequence. One murine mAb, an IgG3 designated F8A1.1, bound to glycoproteins and glycolipids from schistosome adults and human promyelocytic leukemic HL-60 cells that express Le(x) antigens, as assessed by a wide variety of approaches including immunofluorescence staining, confocal microscopy, flow cytometry and western blotting, as well as overlay assays of glycolipids after thin-layer chromatography. In contrast, F8A1.1 bound weakly to cercariae, 3-h schistosomula and human Jurkat cells. We also directly compared the glycan specificity of F8A1.1 with commercially available anti-CD15 IgG1 (clone W6D3) using a defined glycan microarray. The results demonstrated that F8A1.1 recognized glycans expressing Le(x) epitopes in a terminal nonreducing position, whereas anti-CD15 bound to glycans with multiple repeats of Le(x) epitopes, but not to glycans with a single, terminal Le(x) epitope. Our results show that F8A1.1 recognizes terminal Le(x) epitopes and can be used for identification, immunolocalization, immunoprecipitation and purification of Le(x)-containing glycoconjugates from schistosomes and mammalian cells.

  16. Ofatumumab: a novel monoclonal anti-CD20 antibody

    Directory of Open Access Journals (Sweden)

    Thomas S Lin

    2010-05-01

    Full Text Available Thomas S LinGlaxoSmithKline Oncology R&D, Collegeville, PA, USAAbstract: Ofatumumab, a novel humanized monoclonal anti-CD20 antibody, was recently approved by the FDA for the treatment of fludarabine and alemtuzumab refractory chronic lymphocytic leukemia (CLL. Ofatumumab effectively induces complement-dependent cytotoxicity (CDC in vitro, and recent studies demonstrated that ofatumumab also effectively mediates antibody-dependent cellular cytotoxicity (ADCC. Pharmacokinetic studies indicated that increased exposure to the antibody correlated with improved clinical outcome in CLL. Thus, pharmacogenomics may be important in identifying which patients are more likely to respond to ofatumumab therapy, although such studies have not yet been performed. Patients with the high-affinity FCGR3a 158 V/V polymorphism may be more likely to respond to therapy, if ADCC is the primary in vivo mechanism of action of ofatumumab. Patients with increased expression of the complement defense proteins CD55 and CD59 may be less likely to respond if ofatumumab works in vivo primarily via CDC. Patients with increased metabolism and clearance of ofatumumab may have lower exposure and be less likely to respond clinically. Thus, pharmacogenomics may determine the responsiveness of patients to ofatumumab therapy.Keywords: monoclonal antibody, CD20, CLL, NHL, lymphoma

  17. Monoclonal Antibody Production against Human Spermatozoal Surface Antigens

    Directory of Open Access Journals (Sweden)

    M Jedi-Tehrani

    2005-10-01

    Full Text Available Introduction: As monoclonal antibodies are potential tools for characterization of soluble or cellular surface antigens, use of these proteins has always been considered in infertility and reproduction research. Therefore, in this study, monoclonal antibodies against human sperm surface antigens were produced. Material and Methods: To produce specific clones against human sperm surface antigens, proteins were extracted using solubilization methods. Balb/c mice were immunized intraperitoneally with the proteins using complete Freund’s adjuvant in the first injection and incomplete Adjuvant in the following booster injections. Hybridoma cells producing ASA were cloned by limiting dilution. Results: Five stable ASA producing hybridoma clones were achieved and their antibody isotypes were determined by ELISA. All the isotypes were of IgG class. Their cross reactivity with rat and mice spermatozoa was examined but they did not have any cross reactivity. Conclusion: The produced antibodies can be used in further studies to characterize and evaluate each of the antigens present on human sperm surface and determining their role in fertilization.

  18. Radiolabelled peptides and monoclonal antibodies for therapy decision making in inflammatory diseases

    NARCIS (Netherlands)

    Malviya, G.; Signore, A.; Lagana, B.; Dierckx, R. A.

    2008-01-01

    Radiolabelled peptides and monoclonal antibodies are an emerging class of radiopharmaceuticals for imaging inflammation with clinical implications for several chronic inflammatory disorders for diagnosis, therapy decision making and follow up. In the last decades, a number of novel monoclonal antibo

  19. Characterization of monoclonal antibodies against waterfowl parvoviruses VP3 protein

    Directory of Open Access Journals (Sweden)

    Yin Xiuchen

    2012-11-01

    Full Text Available Abstract Background The VP3 protein of goose parvovirus (GPV or Muscovy duck parvovirus (MDPV, a major structural protein, can induce neutralizing antibodies in geese and ducks, but monoclonal antibodies (MAbs against VP3 protein has never been characterized. Results Three hybridoma cell lines secreting anti-GPV VP3 MAbs were obtained and designated 4A8, 4E2, and 2D5. Immunoglobulin subclass tests differentiated them as IgG2b (4A8 and 4E2 and IgG2a (2D5. Dot blotting assays showed that three MAbs reacted with His-VP3 protein in a conformation-independent manner. A competitive binding assay indicated that the MAbs delineated two epitopes, A and B of VP3. Immunofluorescence assay showed that MAbs 4A8, 4E2, and 2D5 could specifically bind to goose embryo fibroblast cells (GEF or duck fibroblast cells (DEF infected with GPV and MDPV. Dot blotting also showed that the MAbs recognized both nature GPV and MDPV antigen. Western blotting confirmed that the MAbs recognized VP3 proteins derived from purified GPV and MDPV particles. The MAbs 4A8 and 2D5 had universal reactivity to heterologous GPV and MDPV tested in an antigen-capture enzyme-linked immunosorbent assay. Conclusions Preparation and characterization of these the MAbs suggests that they may be useful for the development of a MAb-capture ELISA for rapid detection of both GPV and MDPV. Virus isolation and PCR are reliable for detecting GPV and MDPV infection, but these procedures are laborious, time-consuming, and requiring instruments. These diagnosis problems highlight the ongoing demand for rapid, reproducible, and automatic methods for the sensitive detection of both GPV and MDPV infection.

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

    Science.gov (United States)

    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.

  1. Immunomodulatory Monoclonal Antibodies in Combined Immunotherapy Trials for Cutaneous Melanoma

    Directory of Open Access Journals (Sweden)

    Mariana Aris

    2017-08-01

    Full Text Available In the last few years, there has been a twist in cancer treatment toward immunotherapy thanks to the impressive results seen in advanced patients from several tumor pathologies. Cutaneous melanoma is a highly mutated and immunogenic tumor that has been a test field for the development of immunotherapy. However, there is still a way on the road to achieving complete and long-lasting responses in most patients. It is desirable that immunotherapeutic strategies induce diverse immune reactivity specific to tumor antigens, including the so-called neoantigens, as well as the blockade of immunosuppressive mechanisms. In this review, we will go through the role of promising monoclonal antibodies in cancer immunotherapy with immunomodulatory function, especially blocking of the inhibitory immune checkpoints CTLA-4 and PD-1, in combination with different immunotherapeutic strategies such as vaccines. We will discuss the rational basis for these combinatorial approaches as well as different schemes currently under study for cutaneous melanoma in the clinical trials arena. In this way, the combination of “push and release” immunomodulatory therapies can contribute to achieving a more robust and durable antitumor immune response in patients.

  2. An update on newer monoclonal antibodies in lymphoma therapy

    Directory of Open Access Journals (Sweden)

    Subhashini Archana Kadavakolan

    2016-01-01

    Full Text Available In 2014, an estimated 9.4% of all new cancers in the US were accounted to hematological cancers. Most of these cancers have a B-cell origin and on the cell surface express antigen CD20-known to restrict B-cells. Considering the intrinsic immune status of the patients receiving chemotherapy, monoclonal antibodies (mAbs are designed to provide active or passive immunotherapy. Clinical success of rituximab-anti-CD20 mAb in the treatment of lymphoma has led to the development of newer generations of mAb to increase the anti-tumor activity. Hence, recent advances in lymphoma therapy are being built on the conventional prototype of anti-CD20 mAb-rituximab. Our review is an update on the advances in lymphoma therapy using mAb against CD20 including the second generation-ofatumumab, veltuzumab, ocrelizumab, and the third-generation mAbs-ocaratuzumab and obinutuzumab.

  3. Production and Characterization of Monoclonal Antibody Against Recombinant Human Erythropoietin

    Institute of Scientific and Technical Information of China (English)

    JIE-BO MI; JIN YAN; XIAO-JIE DING; ZHEN-QUAN GUO; MEI-PING ZHAO; WEN-BAO CHANG

    2007-01-01

    Objective To produce specific monoclonal antibody(mAb)against recombinant human erythropoietin(rHuEPO)for development of higmy efficient methods for erythropoietin detection in biological fluids.Methods rHuEPO was covalently coupled with bovine serum albumin(BSA)and the conjugate was used to immunize mice to produce specific mAb against rHuEPO based on hybridoma technology.The obtained F3-mAb was characterized by enzyme-linked immunosorbent assay (ELISA),SDS-PAGE and Western blot.Results The isotype of F3-mAb Was found to be IgM with an affinity constant of 2.1x108 L/mol.The competitive ELISA using the obtained IgM showed a broader linear range and lower detection limit compared with previous work.Conclusions The modification of rHuEPO was proved to be successful in generating required specific mAb with high avidity to rHuEPO.

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

    Science.gov (United States)

    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.

  5. Studies on Purification of Methamidophos Monoclonal Antibodies and Comoarative Immunoactivity of Purified Antibodies

    Institute of Scientific and Technical Information of China (English)

    SU-QING ZHAO; YUAN-MING SUN; CHUN-YAN ZHANG; XIAO-YU HUANG; HOU-RUI ZHANG; ZHEN-YU ZHU

    2003-01-01

    Objective To purify Methamidophos (Met) monoclonal antibodies with two methods andcompare immune activity of purified antibodies. Method Caprylic acid ammonium sulphateprecipition (CAASP) method and Sepharose protein-A (SPA) affinity chromatography method wereused to purify Met monoclonal antibodies, UV spectrum scanning was used to determine proteincontent and recovery of purified antibodies, sodium dodecylsulphate polyacrylamide gelelectrophoresis (SDS-PAGE) was used to analyze the purity of purified antibodies, and enzyme-linkedimmunosorbent assay (ELISA) was used to determine immune activity of purified antibodies.Results Antibody protein content and recovery rate with CAASP method were 7.62 mg/mL and8.05% respectively, antibody protein content and recovery rate with SPA method were 6.45 mg/mLand 5.52% respectively. Purity of antibodies purified by SPA method was higher than that by CAASPmethod. The half-maximal inhibition concentration (IC50) of antibodies purified by SPA to Met was181.26 μg/mL, and the linear working range and the limit of quantification (LOD) were 2.43-3896.01μg/mL and 1.03 μg/mL, respectively. The IC50 of antibodies purified by CAASP to Met was 352.82μg/mL, and the linear working range and LOD were 10.91-11412.29 ug/mL and 3.42 μg/mL,respectively. Conclusion Antibodies purified by SPA method are better than those by CAASPmethod, and Met monoclonal antibodies purified by SPA method can be used to prepare gold-labelledtesting paper for analyzing Met residue in vegetable and drink water.

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

    Science.gov (United States)

    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

  7. Development and characterization of murine monoclonal antibody specific for the P1.4 PorA proteins from strain B:4:P1.(7b.4. of Neisseria meningitidis

    Directory of Open Access Journals (Sweden)

    María Elena Pérez

    2011-08-01

    Full Text Available Neisseria meningitidis isolates are conventionally classified by serosubtyping that characterizes the reactivities of the PorA outer membrane protein variable-region epitopes with monoclonal antibodies. Porins are outer membrane proteins (OMPs of N. meningitidis serogroup B and have attracted study principally for two reasons: their use in the classification of meningococcal isolates into serotype and subtype and as potential components of vaccines against this important pathogen. New murine hybridomas, secreting specific monoclonal antibodies against PorA serotype P1.4 of N. meningitidis serogroup B, were generated using conventional hybridoma procedures. The monoclonal antibodies obtained were characterized by Western blot and whole cell ELISA, using reference strains from different N. meningitidis serotypes and subtypes. All monoclonal antibodies belong to isotype IgG1. Others hybridomas producing MAbs against PorB and FrpB were also obtained.

  8. Monoclonal antibodies:Principles and applications of immmunodiagnosis and immunotherapy for hepatitis C virus

    Institute of Scientific and Technical Information of China (English)

    Ashraf; Tabll; Aymn; T; Abbas; Sherif; El-Kafrawy; Ahmed; Wahid

    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(m Ab) 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 mA b 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 mA b 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 mA b. Finally,we will discuss available and new trends to produce antibodies,such as egg yolk-based antibodies(Ig Y),production in transgenic plants,and the synthetic antibody mimics approach.

  9. Cell culture processes for monoclonal antibody production.

    Science.gov (United States)

    Li, Feng; Vijayasankaran, Natarajan; Shen, Amy Yijuan; Kiss, Robert; Amanullah, Ashraf

    2010-01-01

    Animal cell culture technology has advanced significantly over the last few decades and is now generally considered a reliable, robust and relatively mature technology. A range of biotherapeutics are currently synthesized using cell culture methods in large scale manufacturing facilities that produce products for both commercial use and clinical studies. The robust implementation of this technology requires optimization of a number of variables, including 1) cell lines capable of synthesizing the required molecules at high productivities that ensure low operating cost; 2) culture media and bioreactor culture conditions that achieve both the requisite productivity and meet product quality specifications; 3) appropriate on-line and off-line sensors capable of providing information that enhances process knowledge; and 4) good understanding of culture performance at different scales to ensure smooth scale-up. Successful implementation also requires appropriate strategies for process development, scale-up and process characterization and validation that enable robust operation that is compliant with current regulations. This review provides an overview of the state-of-the art technology in key aspects of cell culture, e.g., engineering of highly productive cell lines and optimization of cell culture process conditions. We also summarize the current thinking on appropriate process development strategies and process advances that might affect process development.

  10. Cell culture processes for monoclonal antibody production

    Science.gov (United States)

    Li, Feng; Vijayasankaran, Natarajan; Shen, Amy (Yijuan); Kiss, Robert

    2010-01-01

    Animal cell culture technology has advanced significantly over the last few decades and is now generally considered a reliable, robust and relatively mature technology. A range of biotherapeutics are currently synthesized using cell culture methods in large scale manufacturing facilities that produce products for both commercial use and clinical studies. The robust implementation of this technology requires optimization of a number of variables, including (1) cell lines capable of synthesizing the required molecules at high productivities that ensure low operating cost; (2) culture media and bioreactor culture conditions that achieve both the requisite productivity and meet product quality specifications; (3) appropriate on-line and off-line sensors capable of providing information that enhances process control; and (4) good understanding of culture performance at different scales to ensure smooth scale-up. Successful implementation also requires appropriate strategies for process development, scale-up and process characterization and validation that enable robust operation and ensure compliance with current regulations. This review provides an overview of the state-of-the art technology in key aspects of cell culture, e.g., generation of highly productive cell lines and optimization of cell culture process conditions. We also summarize the current thinking on appropriate process development strategies and process advances that might affect process development. PMID:20622510

  11. Monoclonal antibody production and indirect competitive enzyme-linked immunosorbent assay development of 3-methyl-quinoxaline-2-carboxylic acid based on novel haptens.

    Science.gov (United States)

    Li, Guopeng; Zhao, Liang; Zhou, Feng; Li, Jiaying; Xing, Yuan; Wang, Tiangang; Zhou, Xilong; Ji, Baoping; Ren, Wanpeng

    2016-10-15

    Two novel immunizing haptens of 3-methyl-quinoxaline-2-carboxylic acid (MQCA) were synthesized and conjugated with cationized bovine serum albumin. Female BALB/c mice were immunized with above conjugates, splenocytes were fused with Sp2/0 cells to produce monoclonal antibody. Compared with previous studies, antibodies raised in this work showed higher sensitivity. Meantime, a novel heterologous coating hapten was also prepared. The indirect competitive enzyme-linked immunosorbent assay (icELISA) based on the optimum condition showed an IC50 of 3.1μg/kg (ppb), and the linear range of 0.46-10.5ppb for MQCA. The limit of detect (LOD) of MQCA in swine muscle, swine liver and chicken was 0.32, 0.54, and 0.28ppb, respectively. The LOD of this assay can satisfy the minimum required performance levels (4ppb) for MQCA. These results indicated that the proposed ELISA, with high sensitivity and specificity, as well as good reproducibility and accuracy, is suitable for determination of MQCA residues in food samples.

  12. CHO cell line specific prediction and control of recombinant monoclonal antibody N-glycosylation.

    Science.gov (United States)

    Grainger, Rhian K; James, David C

    2013-11-01

    Here we demonstrate that it is possible to predict and control N-glycan processing of a secreted recombinant monoclonal antibody during manufacturing process development using a combination of statistical modelling and comparative measurement of cell surface glycans using fluorescent lectins. Using design of experiments--response surface modelling (DoE-RSM) methodology to adjust the relative media concentrations of known metabolic effectors of galactosylation (manganese, galactose, and uridine) we have shown that β1,4-galactosylation of the same recombinant IgG4 monoclonal antibody produced by different CHO cell lines can be precisely controlled in a cell line specific manner. For two cell lines, monoclonal antibody galactosylation could be increased by over 100% compared to control, non-supplemented cultures without a reduction in product titre and with minimal effect on cell growth. Analysis of galactosylation effector interactions by DoE-RSM indicated that Mn²⁺ alone was necessary but not sufficient to improve galactosylation, and that synergistic combinations of Gal and Urd were necessary to maximize galactosylation, whilst minimizing the deleterious effect of Urd on cell growth. To facilitate rapid cell culture process development we also tested the hypothesis that substrate-level control of cellular galactosylation would similarly affect both cell surface and secreted monoclonal antibody glycans, enabling facile indirect prediction of product glycan processing. To support this hypothesis, comparative quantitation of CHO cell surface β1,4-galactosylation by flow cytometry using fluorescent derivatives of RCA and ConA lectins revealed that substrate-controlled variation in monoclonal antibody galactosylation and cell surface galactosylation were significantly correlated. Taken together, these data show that precision control of a complex, dynamic cellular process essential for the definition of protein product molecular heterogeneity and bioactivity is

  13. Immunohistochemical diagnosis of fusariosis with monoclonal antibodies

    DEFF Research Database (Denmark)

    Jensen, H.E.; Aalbæk, B.; Jungersen, Gregers

    for fusariosis. A panel of newly developed Mabs for immunohistochemical diagnosis of fusariosis was screened for specificity on experimentally infected laboratory animal tissue and on skin tissue biopsies from two neutropenic patients with Fusarium sepsis. Methods: Somatic antigens were made from F. solani (CBS......Objectives: Fusariosis is an emerging fungal infection, especially in neutropenic patients. Proper identification of Fusarium spp. is important because the choice of antifungal treatment of fusariosis differs from that of aspergillosis, candidosis or scedosporidiosis (pseudalleceriosis). Cultural...... isolation attempts from fusarium lesions often fail, and because the tissue forms of Fusarium spp. are histologically indistinguishable from fungal elements of aspergillosis, true hyphae containing candidosis and scedosporidiosis (pseudalleceriosis), alternative diagnostic techniques are often necessary...

  14. Metrics for antibody therapeutics development.

    Science.gov (United States)

    Reichert, Janice M

    2010-01-01

    A wide variety of full-size monoclonal antibodies (mAbs) and therapeutics derived from alternative antibody formats can be produced through genetic and biological engineering techniques. These molecules are now filling the preclinical and clinical pipelines of every major pharmaceutical company and many biotechnology firms. Metrics for the development of antibody therapeutics, including averages for the number of candidates entering clinical study and development phase lengths for mAbs approved in the United States, were derived from analysis of a dataset of over 600 therapeutic mAbs that entered clinical study sponsored, at least in part, by commercial firms. The results presented provide an overview of the field and context for the evaluation of on-going and prospective mAb development programs. The expansion of therapeutic antibody use through supplemental marketing approvals and the increase in the study of therapeutics derived from alternative antibody formats are discussed.

  15. Studies towards the improvement of an anti-cocaine monoclonal antibody for treatment of acute overdose.

    Science.gov (United States)

    Zhou, Bin; Eubanks, Lisa M; Jacob, Nicholas T; Ellis, Beverly; Roberts, Amanda J; Janda, Kim D

    2016-10-15

    There is currently no clinically-approved antidote for cocaine overdose. Efforts to develop a therapy via passive immunization have resulted in a human monoclonal antibody, GNCgzk, with a high affinity for cocaine (Kd=0.18nM). Efforts to improve the production of antibody manifolds based on this antibody are disclosed. The engineering of an HRV 3C protease cleavage site into the GNCgzk IgG has allowed for increased production of a F(ab')2 with a 20% superior capacity to reduce mortality for cocaine overdose in mice. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Membrane adsorbers as purification tools for monoclonal antibody purification.

    Science.gov (United States)

    Boi, Cristiana

    2007-03-15

    Downstream purification processes for monoclonal antibody production typically involve multiple steps; some of them are conventionally performed by bead-based column chromatography. Affinity chromatography with Protein A is the most selective method for protein purification and is conventionally used for the initial capturing step to facilitate rapid volume reduction as well as separation of the antibody. However, conventional affinity chromatography has some limitations that are inherent with the method, it exhibits slow intraparticle diffusion and high pressure drop within the column. Membrane-based separation processes can be used in order to overcome these mass transfer limitations. The ligand is immobilized in the membrane pores and the convective flow brings the solute molecules very close to the ligand and hence minimizes the diffusional limitations associated with the beads. Nonetheless, the adoption of this technology has been slow because membrane chromatography has been limited by a lower binding capacity than that of conventional columns, even though the high flux advantages provided by membrane adsorbers would lead to higher productivity. This review considers the use of membrane adsorbers as an alternative technology for capture and polishing steps for the purification of monoclonal antibodies. Promising industrial applications as well as new trends in research will be addressed.

  17. Monoclonal anti-elastin antibody labelled with technetium-99m

    Energy Technology Data Exchange (ETDEWEB)

    Oliveira, Marcia B.N. de; Silva, Claudia R. da; Araujo, Adriano C. de; Bernardo Filho, Mario [Universidade do Estado, Rio de Janeiro, RJ (Brazil). Instituto de Biologia Roberto Alcantara Gomes. Lab. de Radiofarmacia; Porto, Luis Cristovao M.S.; Gutfilen, Bianca [Universidade do Estado, Rio de Janeiro, RJ (Brazil). Instituto de Biologia Roberto Alcantara Gomes; Souza, J.E.Q. [Instituto Nacional do Cancer, Rio de Janeiro, RJ (Brazil). Centro de Pesquisa Basica; Frier, Malcolm [University Hospital, Nottingham (United Kingdom). Dept. of Medical Physics

    1999-11-01

    Technetium-99m ({sup 99m} Tc) is widely employed in nuclear medicine due to its desirable physical, chemical and biological properties. Moreover, it is easily available and normally is inexpensive. A reducing agent is necessary to label cells and molecules with {sup 99m} Tc and stannous chloride (Sn C L{sub 2}) is usually employed. Elastin is the functional protein component of the elastic fiber and it is related with some diseases such as arteriosclerosis, pulmonary emphysema and others. The present study refers to the preparation of the {sup 99m} Tc labeled monoclonal anti-elastin antibody. The monoclonal antibody was incubated with an excess of 2-iminothiolane. The free thiol groups created, were capable of binding with the reduced technetium. Labeling was an exchange reaction with {sup 99m} Tc-glucoheptonate. The labeled preparation was left at 4 deg C for one hour. Then, it was passed through a Sephadex G50 column. Various fractions were collected and counted. A peak corresponding to the radiolabeled antibody was obtained. Stability studies of the labelled anti-elastin were performed at 0,3 6, 24 hours, at both 4 deg C or room temperature. The biodistribution pattern of the {sup 99m} Tc-anti-elastin was studied in healthy male Swiss mice. The immunoreactivity was also determined. An useful labeled-anti-elastin was obtained to future immunoscintigraphic investigations. (author) 4 refs., 7 figs., 6 tabs.

  18. Characterization of oxidative carbonylation on recombinant monoclonal antibodies.

    Science.gov (United States)

    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.

  19. Discovery and characterization of antibody variants using mass spectrometry-based comparative analysis for biosimilar candidates of monoclonal antibody drugs.

    Science.gov (United States)

    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. High-efficiency screening of monoclonal antibodies for membrane protein crystallography.

    Directory of Open Access Journals (Sweden)

    Hyun-Ho Lim

    Full Text Available Determination of crystal structures of membrane proteins is often limited by difficulties obtaining crystals diffracting to high resolution. Co-crystallization with Fab fragments of monoclonal antibodies has been reported to improve diffraction of membrane proteins crystals. However, it is not simple to generate useful monoclonal antibodies for membrane protein crystallography. In this report, we present an optimized process for efficient screening from immunization to final validation of monoclonal antibody for membrane protein crystallography.

  1. Alemtuzumab and Natalizumab: The Monoclonal Antibody Story Continues

    Directory of Open Access Journals (Sweden)

    BL Johnston

    2006-01-01

    Full Text Available In the July/August 2006 issue of this journal, the infectious complications associated with the use of infliximab, etanercept and adalimumab were reviewed (1. These represent only three of the many monoclonal antibodies either licensed or in clinical trials for therapeutic use in cancer and autoimmune disease or to prevent rejection in both solid organ and hematopoietic stem cell transplantation. While most of these agents have not been associated with increased infection rates, alemtuzumab and natalizumab have gained particular attention related to either the frequency or type of infection seen in some individuals who have received them.

  2. Rapid analysis of small samples containing forskolin using monoclonal antibodies.

    Science.gov (United States)

    Yanagihara, H; Sakata, R; Shoyama, Y; Murakami, H

    1996-04-01

    The effective range of the competitive ELISA test for detection of forskolin content in clonally propagated plant organs of Coleus forskohlii using monoclonal antibodies extends from 5ng to 5 micrograms. A correlation between the forskolin accumulation and the growth rate was investigated using the clonally propagated shoots. An increase of forskolin content was noted, beginning at week 6. Flowers, rachises, leaves, stems, tuberous roots, and roots were analyzed. Tuberous roots and the stem base contained higher amounts of forskolin than other organs. The forskolin content in the stem decreased gradually towards the top of the shoot.

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

    Science.gov (United States)

    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.

  4. Large-scale production of monoclonal antibodies in suspension culture.

    Science.gov (United States)

    Backer, M P; Metzger, L S; Slaber, P L; Nevitt, K L; Boder, G B

    1988-10-01

    Monoclonal antibodies are being manufactured for clinical trials in suspension culture at the 1300-L scale. Suspension culture offers some advantages relative to high-density mammalian cell culture methods; in particular, the ability to closely monitor the behavior of cells in a homogeneous environment. Computer control and on-line mass spectrography of exit gases provide instantaneous information about the culture metabolic activity. Air sparging and agitation by marine impeller provide aeration sufficient to maintain a constant dissolved oxygen tension at cell concentrations up to 5.0 x 10(6) cells/mL without causing apparent cell damage.

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

    Science.gov (United States)

    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.

  6. Indium-111-monoclonal antimyosin antibody studies after the first year of heart transplantation. Identification of risk groups for developing rejection during long-term follow-up and clinical implications

    Energy Technology Data Exchange (ETDEWEB)

    Ballester, M.; Obrador, D.; Carrio, I.; Auge, J.M.; Moya, C.; Pons-Llado, G.; Caralps-Riera, J.M. (Hospital de la Santa Creu i Sant Pau, Barcelona (Spain))

    1990-12-01

    The long-term clinical course and results of biopsies in 21 patients studied with monoclonal antimyosin antibodies more than 12 months after heart transplantation according to the presence and degree of antimyosin-antibody uptake is described. Eighteen men and three women aged 20-52 years (39 +/- 9 years) were studied with antimyosin antibodies 12-40 months (mean, 22 +/- 9 months) after heart transplantation, and followed for a mean of 18 months (10-28 months). The number of biopsies performed during follow-up was 102. Results showed normal antimyosin-antibody studies in nine patients and abnormal studies in 12 patients. Myocyte damage was identified in 18 of the 102 biopsies (17.6%), one in the normal antimyosin-antibody group of patients and 17 in those patients with myocardial antimyosin-antibody uptake. Patients who developed rejection comprised 11% and 67% of each respective group; the mean number of rejection episodes per patient was 0.11 +/- 0.33 and 1.41 +/- 1.41, respectively (p less than 0.01). A trend was noted by which higher heart-to-lung ratios were associated with greater probability of rejection. Conclusively, (1) antimyosin-antibody studies performed after more than 1 year after heart transplantation indicate the presence and level of rejection activity, (2) groups of patients at risk for developing rejection at biopsy during long-term follow-up may be detected by antimyosin-antibody study, and (3) surveillance for rejection and the degree of immunosuppression should be tailored to meet individual patient needs.

  7. Monoclonal antibodies to intermediate filament proteins of human cells: unique and cross-reacting antibodies.

    Science.gov (United States)

    Gown, A M; Vogel, A M

    1982-11-01

    Monoclonal antibodies were generated against the intermediate filament proteins of different human cells. The reactivity of these antibodies with the different classes of intermediate filament proteins was determined by indirect immunofluorescence on cultured cells, immunologic indentification on SDS polyacrylamide gels ("wester blot" experiments), and immunoperoxidase assays on intact tissues. The following four antibodies are described: (a) an antivimentin antibody generated against human fibroblast cytoskeleton; (b), (c) two antibodies that recognize a 54-kdalton protein in human hepatocellular carcinoma cells; and (d) an antikeratin antibody made to stratum corneum that recognizes proteins of molecular weight 66 kdaltons and 57 kdaltons. The antivimentin antibody reacts with vimentin (58 kdaltons), glial fibrillary acidic protein (GFAP), and keratins from stratum corneum, but does not recognize hepatoma intermediate filaments. In immunofluorescence assays, the antibody reacts with mesenchymal cells and cultured epithelial cells that express vimentin. This antibody decorates the media of blood vessels in tissue sections. One antihepatoma filament antibody reacts only with the 54 kdalton protein of these cells and, in immunofluorescence and immunoperoxidase assays, only recognizes epithelial cells. It reacts with almost all nonsquamous epithelium. The other antihepatoma filament antibody is much less selective, reacting with vimentin, GFAP, and keratin from stratum corneum. This antibody decorates intermediate filaments of both mesenchymal and epithelial cells. The antikeratin antibody recognizes 66-kdalton and 57-kdalton proteins in extracts of stratum corneum and also identifies proteins of similar molecular weights in all cells tested. However, by immunofluorescence, this antibody decorates only the intermediate filaments of epidermoid carcinoma cells. When assayed on tissue sections, the antibody reacts with squamous epithelium and some, but not all

  8. Novel neutralizing monoclonal antibodies protect rodents against lethal filovirus challenges

    Directory of Open Access Journals (Sweden)

    Caleb D. Marceau

    2014-01-01

    Full Text Available Filoviruses are the causative agents of lethal hemorrhagic fever in human and non-human primates (NHP. The family of Filoviridae is composed of three genera, Ebolavirus, Marburgvirus and Cuevavirus. There are currently no approved vaccines or antiviral therapeutics for the treatment of filovirus infections in humans. Passive transfer of neutralizing antibodies targeting the Ebola virus (EBOV glycoprotein (GP has proven effective in protecting mice, guinea pigs and NHP from lethal challenges with EBOV. In this study, we generated two neutralizing monoclonal antibodies (MAbs, termed S9 and M4 that recognize the GP of EBOV or multiple strains of Marburg virus (MARV, respectively. We characterized the putative binding site of S9 as a linear epitope on the glycan cap of the GP1 subunit of the EBOV-GP. The M4 antibody recognizes an unknown conformational epitope on MARV-GP. Additionally, we demonstrated the post-exposure protection potential of these antibodies in both the mouse and guinea pig models of filovirus infection. These data indicate that MAbs S9 and M4 would be good candidates for inclusion in an antibody cocktail for the treatment of filovirus infections.

  9. Preliminary Studies on the Development of Monoclonal Antibodies Against Mycelia of Ganoderma boninense, the Causal Pathogen of Basal Stem Rot of Oil Palm

    Directory of Open Access Journals (Sweden)

    Shamala, S.

    2006-01-01

    Full Text Available This study aimed to raise specific MAbs against G. boninense, the causal pathogen of basal stem rot (BSR of oil palm. Crude mycelium extract of G. boninense was used as immunogen to generate MAbs. Mycelium was harvested from liquid culture and freeze-dried followed by re-suspension in phosphate buffer saline (PBS. Two 10-week old BALB-C mice were immunized with the mycelial extract. The mice were boosted once before harvesting their spleens for fusion. The MAbs were fused with myeloma cells from BALB-C mice. Initial screening was carried out using plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA with mycelial immunogen of G. boninense. The MAbs with positive signals were verified via secondary screening and cloned for cross-reactivity test. Cross-reactivity testing was carried out with 2 other fungi namely; Trichoderma and Botrytis along with 2 different species of Ganoderma commonly found in oil palm plantations namely; G. zonatum, and G. miniatocinctum. This study found that the MAbs raised against G. boninense were not specific as the MAbs gave positive signals through the cross-reactivity test with all fungi tested in the cross-reactivity. Future work would be using these MAbs in a co-immunization program whereby the generated Ganoderma sp generic monoclonal antibody will be pre-mixed with the G. boninense mycelium immunogen to allow reduction in the potential cross-reactivity of newly generated antibodies with Ganoderma sp. Our efforts are also currently directed at optimizing the immunogen preparation for the production of MAbs specific to G. boninense.

  10. Belimumab: anti-BLyS monoclonal antibody; Benlysta; BmAb; LymphoStat-B.

    Science.gov (United States)

    2010-01-01

    Belimumab is a fully human monoclonal antibody for the treatment of autoimmune disorders that is being developed by Human Genome Sciences and GlaxoSmithKline. Two pivotal phase III trials in systemic lupus erythematosus have been concluded with the primary endpoints being met in both studies. A phase II trial in rheumatoid arthritis has also been completed, with positive results. Marketing authorization submissions are being prepared in the major markets worldwide. This review discusses the development history and scientific profile of belimumab.

  11. [Generation and characterization of monoclonal antibodies against chicken interleukin 4].

    Science.gov (United States)

    Guan, Xiaoyu; Xu, Zhichao; Wang, Yongqiang; Li, Xiaoqi; Cao, Hong; Zheng, Shijun

    2017-01-25

    To develop monoclonal antibodies (McAbs) against chicken interleukin 4 (chIL-4), we subcloned the mature chIL-4 gene into prokaryotic expression vectors pET-28a and pGEX-6P-1, then expressed and purified the recombinant proteins. We immunized BALB/c mice with the purified His-chIL-4 protein and fused the murine splenocytes with SP2/0 after 4 times of immunization. We used the GST-chIL-4 protein as a coating antigen to establish an indirect ELISA to screen positive clones. After screening and 3 rounds of cloning process, we obtained 3 hybridomas that stably secreted McAbs against chIL-4, and named 1G11-3B, 2E5-3D, and 1G11-5H. The isotypes of these McAbs were all IgG1 and the dissociation constant (Kd) of these McAbs were 1.79×10⁻⁹, 1.61×10⁻⁹, and 2.36×10⁻⁹, respectively. These McAbs specifically bound to chIL-4 expressed by either prokaryotic or eukaryotic system as determined by Western blotting and indirect immunofluorescence assay. The binding domains of chIL-4 recognized by 1G11-3B, 2E5-3D, and 1G11-5H were located between aa 1-40, 80-112, and 40-80, respectively, as determined by Western blotting. These McAbs would help to detect chIL-4 and to elucidate the biological roles of chIL-4 in immune responses.

  12. Capillary ion-exchange chromatography with nanogram sensitivity for the analysis of monoclonal antibodies.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Thomas A Torode

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  16. Immunotherapy of hepatoma with a monoclonal antibody against murine endoglin

    Institute of Scientific and Technical Information of China (English)

    Guang-Hong Tan; Feng-Ying Huang; Hua Wang; Yong-Hao Huang; Ying-Ying Lin; Yue-Nan Li

    2007-01-01

    AIM: To explore the capability of a monoclonal antibody(mAb) against murine endoglin to inhibit tumor angiogenesis and suppression of hepatoma growth in murine models.METHODS: A monoclonal antibody against murine endoglin was purified by affinity chromatography and passively transfused through tail veins in two murine hepatoma models. Tumor volume and survival time were observed at three-day intervals for 48 d. Microvessels in tumor tissues were detected by immunohistochemistry against CD31, and angiogenesis in vivo was determined by alginate encapsulated assay. In addition, tumor cell apoptosis was detected by TUNEL assay.RESULTS: Passive immunotherapy with anti-endoglin mAb could effectively suppress tumor growth, and prolonged the survival time of hepatoma-bearing mice.Angiogenesis was apparently inhibited within the tumor tissues, and the vascularization of alginate beads was also reduced in the mice passively transfused with antiendoglin mAb. In addition, increased apoptotic cells were observed within the tumor tissues from the mice passively transfused with anti-endoglin mAb.CONCLUSION: Passive immunotherapy with antiendoglin mAb effectively inhibits tumor growth via inhibiting tumor angiogenesis and increasing tumor cell apoptosis, which may be highly correlated with the blockage of endoglin-related signal pathway induced by anti-endoglin mAb.

  17. Monoclonal antibody proteomics: use of antibody mimotope displaying phages and the relevant synthetic peptides for mAb scouting.

    Science.gov (United States)

    Hajdú, István; Flachner, Beáta; Bognár, Melinda; Végh, Barbara M; Dobi, Krisztina; Lőrincz, Zsolt; Lázár, József; Cseh, Sándor; Takács, László; Kurucz, István

    2014-08-01

    Monoclonal antibody proteomics uses nascent libraries or cloned (Plasmascan™, QuantiPlasma™) libraries of mAbs that react with individual epitopes of proteins in the human plasma. At the initial phase of library creation, cognate protein antigen and the epitope interacting with the antibodies are not known. Scouting for monoclonal antibodies (mAbs) with the best binding characteristics is of high importance for mAb based biomarker assay development. However, in the absence of the identity of the cognate antigen the task represents a challenge. We combined phage display, and surface plasmon resonance (Biacore) experiments to test whether specific phages and the respective mimotope peptides obtained from large scale studies are applicable to determine key features of antibodies for scouting. We show here that mAb captured phage-mimotope heterogeneity that is the diversity of the selected peptide sequences, is inversely correlated with an important binding descriptor; the off-rate of the antibodies and that represents clues for driving the selection of useful mAbs for biomarker assay development. Carefully chosen synthetic mimotope peptides are suitable for specificity testing in competitive assays using the target proteome, in our case the human plasma.

  18. Therapeutic monoclonal antibodies for respiratory diseases: Current challenges and perspectives, March 31 - April 1, 2016, Tours, France.

    Science.gov (United States)

    Desoubeaux, Guillaume; Reichert, Janice M; Sleeman, Matthew; Reckamp, Karen L; Ryffel, Bernhard; Adamczewski, Jörg P; Sweeney, Theresa D; Vanbever, Rita; Diot, Patrice; Owen, Caroline A; Page, Clive; Lerondel, Stéphanie; Le Pape, Alain; Heuze-Vourc'h, Nathalie

    2016-01-01

    Monoclonal antibody (mAb) therapeutics have tremendous potential to benefit patients with lung diseases, for which there remains substantial unmet medical need. To capture the current state of mAb research and development in the area of respiratory diseases, the Research Center of Respiratory Diseases (CEPR-INSERM U1100), the Laboratory of Excellence "MAbImprove," the GDR 3260 "Antibodies and therapeutic targeting," and the Grant Research program ARD2020 "Biotherapeutics" invited speakers from industry, academic and government organizations to present their recent research results at the Therapeutic Monoclonal Antibodies for Respiratory Diseases: Current challenges and perspectives congress held March 31 - April 1, 2016 in Tours, France.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  1. New monoclonal antibodies directed against human renin. Powerful tools for the investigation of the renin system.

    OpenAIRE

    Galen, F X; Devaux, C.; Atlas, S; Guyenne, T; Menard, J; Corvol, P; Simon, D.; Cazaubon, C; Richer, P; Badouaille, G

    1984-01-01

    Monoclonal antibodies directed against human renin were obtained by the fusing of myeloma cells with spleen cells from Balb/c or high-responder Biozzi mice injected with pure tumoral or highly purified renal renin. These procedures resulted in the production of seven stable monoclonal antibodies to human renin. Antibodies in the hybridoma culture medium were screened by binding to pure iodinated renin or insolubilized renin in a solid phase assay. The concentration of purified antibodies that...

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

    Directory of Open Access Journals (Sweden)

    Rong-Hong Hua

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

  3. The Efficacy of an anti-CD4 Monoclonal Antibody for HIV-1 Treatment

    OpenAIRE

    Fessel, W. Jeffrey; Anderson, Brooke; Follansbee, Stephen E.; Winters, Mark A.; Lewis, Stanley; Weinheimer, Steven; Christos J Petropoulos; Shafer, Robert W.

    2011-01-01

    The availability of 24 antiretroviral (ARV) drugs within six distinct drug classes has transformed HIV-1 infection (AIDS) into a treatable chronic disease. However, the ability of HIV-1 to develop resistance to multiple classes continues to present challenges to the treatment of many ARV treatment-experienced patients. In this case report, we describe the response to ibalizumab, an investigational CD4-binding monoclonal antibody (mAb), in a patient with advanced immunodeficiency and high-leve...

  4. Discovery and Characterization of Phage Display-Derived Human Monoclonal Antibodies against RSV F Glycoprotein.

    Directory of Open Access Journals (Sweden)

    Zhifeng Chen

    Full Text Available Respiratory syncytial virus (RSV is a leading cause of lower respiratory tract infection in infants, the elderly and in immunosuppressed populations. The vast majority of neutralizing antibodies isolated from human subjects target the RSV fusion (F glycoprotein, making it an attractive target for the development of vaccines and therapeutic antibodies. Currently, Synagis® (palivizumab is the only FDA approved antibody drug for the prevention of RSV infection, and there is a great need for more effective vaccines and therapeutics. Phage display is a powerful tool in antibody discovery with the advantage that it does not require samples from immunized subjects. In this study, Morphosys HuCAL GOLD® phage libraries were used for panning against RSV prefusion and postfusion F proteins. Panels of human monoclonal antibodies (mAbs against RSV F protein were discovered following phage library panning and characterized. Antibodies binding specifically to prefusion or postfusion F proteins and those binding both conformations were identified. 3B1 is a prototypic postfusion F specific antibody while 2E1 is a prototypic prefusion F specific antibody. 2E1 is a potent broadly neutralizing antibody against both RSV A and B strains. Epitope mapping experiments identified a conformational epitope spanning across three discontinuous sections of the RSV F protein, as well as critical residues for antibody interaction.

  5. Monoclonal antibodies against DNA-binding tips of DNABII proteins disrupt biofilms in vitro and induce bacterial clearance in vivo

    Directory of Open Access Journals (Sweden)

    Laura A. Novotny

    2016-08-01

    Full Text Available The vast majority of chronic and recurrent bacterial diseases are attributed to the presence of a recalcitrant biofilm that contributes significantly to pathogenesis. As such, these diseases will require an innovative therapeutic approach. We targeted DNABII proteins, an integral component of extracellular DNA (eDNA which is universally found as part of the pathogenic biofilm matrix to develop a biofilm disrupting therapeutic. We show that a cocktail of monoclonal antibodies directed against specific epitopes of a DNABII protein is highly effective to disrupt diverse biofilms in vitro as well as resolve experimental infection in vivo, in both a chinchilla and murine model. Combining this monoclonal antibody cocktail with a traditional antibiotic to kill bacteria newly released from the biofilm due to the action of the antibody cocktail was highly effective. Our results strongly support these monoclonal antibodies as attractive candidates for lead optimization as a therapeutic for resolution of bacterial biofilm diseases.

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

    Directory of Open Access Journals (Sweden)

    Fedosov Sergey N

    2006-01-01

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

  7. Preparation of monoclonal antibody to P53 and its clinical application

    Institute of Scientific and Technical Information of China (English)

    Wenqing Wei; Junhua Wu; Jing Liu; Yuxia Wang

    2013-01-01

    Objective:The aim of this study was to prepare monoclonal antibody against P53, a kind of tumor suppressor protein,and use the antibody initial y in clinical immunoassay. Methods:Monoclonal antibody was prepared and identified via the classic protocol of monoclonal antibody preparation. Identified monoclonal antibodies were purified by af inity chro-matography. Antibody titer was determined by enzyme linked immunosorbent assay (ELISA). The specific binding activity of antibody was detected by Western blotting and immunohistochemistry. Results:Three strains of monoclonal antibodies named 1P15, 2P37 and 3P40 were obtained and purified by af inity chromatography. The purity of antibodies was higher than 90%. The titers of antibodies were more than 1:6000. Western blot and immunohistochemistry assay showed that the specific antibody can combine with endogenous P53 protein in the tumor celllines and determine the expression of P53 in tumor tis-sue. Conclusion:Three strains of monoclonal antibodies with high af inity to P53 were successful y established, which can be used for detecting the expression of P53 in tumor cells or tissue.

  8. Monoclonal antibodies against a synthetic peptide from human immunodeficiency virus type 1 Nef protein

    DEFF Research Database (Denmark)

    Steinaa, L; Wulff, A M; Saermark, T

    1994-01-01

    Monoclonal antibodies against a synthetic peptide (aa 138-152) from HIV-1 Nef protein were produced and characterized. Three hybridoma lines producing monoclonal antibodies (MAbs) against the synthetic peptide were generated by fusion between P3-X63 Ag8.653 myeloma cells and BALB/c splenocytes fr...

  9. Preparation of Europium Induced Conformation—specific anti—calmodulin Monoclonal Antibody

    Institute of Scientific and Technical Information of China (English)

    WeiGuoLI; ChaoQI; 等

    2002-01-01

    Monoclonal antibody technique was employed to detect the conformational difference of CaM induced by metal ions. A trivalent europium ion induced conformation-specific anti-calmodulin monoclonal antibody was successfully prepared with europium-saturated calmodulin as antigen.

  10. Preparation of Europium Induced Conformation-specific anti-calmodulin Monoclonal Antibody

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Monoclonal antibody technique was employed to detect the conformational difference of CaM induced by metal ions. A trivalent europium ion induced conformation-specific anti-calmodulin monoclonal antibody was successfully prepared with europium-saturated calmodulin as antigen.

  11. Development and Characterization of Monoclonal Antibodies to Yellow Fever Virus and Application in Antigen Detection and IgM Capture Enzyme-Linked Immunosorbent Assay.

    Science.gov (United States)

    Adungo, Ferdinard; Yu, Fuxun; Kamau, David; Inoue, Shingo; Hayasaka, Daisuke; Posadas-Herrera, Guillermo; Sang, Rosemary; Mwau, Matilu; Morita, Kouichi

    2016-08-01

    Yellow fever (YF) is an acute hemorrhagic viral infection transmitted by mosquitoes in Africa and South America. The major challenge in YF disease detection and confirmation of outbreaks in Africa is the limited availability of reference laboratories and the persistent lack of access to diagnostic tests. We used wild-type YF virus sequences to generate recombinant envelope protein in an Escherichia coli expression system. Both the recombinant protein and sucrose gradient-purified YF vaccine virus 17D (YF-17D) were used to immunize BALB/c mice to generate monoclonal antibodies (MAbs). Eight MAbs were established and systematically characterized by indirect enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and immunofluorescence assay (IFA). The established MAbs showed strong reactivity with wild-type YF virus and recombinant protein with no detectable cross-reactivity to dengue virus or Japanese encephalitis virus. Epitope mapping showed strong binding of three MAbs to amino acid positions 1 to 51, while two MAbs mapped to amino acid positions 52 to 135 of the envelope protein. The remaining three MAbs did not show reactivity to envelope fragments. The established MAbs exert no neutralization against wild-type YF and 17D viruses (titer of <10 for both strains). The applicability of MAbs 8H3 and 3F4 was further evaluated using IgM capture ELISA. A total of 49 serum samples were analyzed, among which 12 positive patient and vaccinee samples were correctly identified. Using serum samples that were 2-fold serially diluted, the IgM capture ELISA was able to detect all YF-positive samples. Furthermore, MAb-based antigen detection ELISA enabled the detection of virus in culture supernatants containing titers of about 1,000 focus-forming units.

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

    DEFF Research Database (Denmark)

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

    1988-01-01

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

  13. The use of monoclonal antibodies for the characterization and production of Mycobacterium leprae antigens

    Directory of Open Access Journals (Sweden)

    J. Ivanyi

    1987-01-01

    Full Text Available Similar immunizations of mice and hybridoma technology were used by several investigators to raise monoclonal antibodies which identified a limited range of epitopes and antigenic molecules. Further studies would have the scope for revealing yet more novel structures. The existing MABs are agreed standard reagents, avaiable to investigators and valuable for several applications. At least six epitopes specific for M. leprae were defined in molecular terms. Monoclonal antibody based immunoassays proved to be invaluable for the screening of recombinant DNA clones and for the topographic study of individual epitopes. Purification of antigens using affinity chromatography requires further development of techniques whilst serology of leprosy is open for clinical and epidemiological evaluation.

  14. Detection of Penicillinase in Milk by Sandwich ELISA Based Polyclonal and Monoclonal Antibody.

    Science.gov (United States)

    Zhao, Yinli; Li, Guoxi

    2016-01-01

    A sandwich ELISA has been developed using polyclonal and monoclonal antibody for the determination of penicillinase in milk. For this purpose, specific polyclonal and monoclonal antibodies against penicillinase were generated and characterized. Using penicillinase standards prepared from 1-128 ng/mL, the method indicated that the detection limit of the sandwich ELISA, as measured in an ELISA plate reader, was as low as 0.86 ng/mL of penicillinase. For determine the accuracy, raw milk containing 2, 8, 32, and 64 ng/mL of penicillinase were tested by sandwich ELISA. Recoveries were from 93-97.5%, and the coefficient of variation [CV (%)] were from 5.55-8.38%. For interassay reproducibility, recoveries were from 89.5-95.1%, the coefficient of variation [CV (%)] were from 5.26-9.58%. This sandwich ELISA provides a useful screening method for quantitative detection of penicillinase in milk.

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

    DEFF Research Database (Denmark)

    Bzorek, M.; Stamp, I.M.; Frederiksen, L.

    2008-01-01

    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...

  16. Monoclonal antibodies against human granulocytes and myeloid differentiation antigens.

    Science.gov (United States)

    Mannoni, P; Janowska-Wieczorek, A; Turner, A R; McGann, L; Turc, J M

    1982-12-01

    Monoclonal antibodies (MCA) were obtained by immunizing BALB/c mice with 99% pure granulocytes from normal donors or with a whole leukocyte suspension obtained from a chronic myelogenous leukemia (CML) patient, and then fusing the mouse spleen cells with a 315-43 myeloma cell clone. Four MCA were selected and studied using ELISA, immunofluorescence, cytotoxicity assays, and FACS analysis. Antibodies 80H.1, 80H.3, and 80H.5 (from normals) and 81H.1 (from CML) detected antigens expressed on neutrophils. Antibodies 80H.1 and 80H.3 (IgG) also reacted with monocytes but not with other blood cell subsets. Antibodies 80H.5 and 81H.1 (IgM) were cytotoxic and reacted strongly with most of the cells of the neutrophil maturation sequence, i.e., myeloblasts, promyelocytes, myelocytes, and mature granulocytes. Antibodies 80H.5 and 81H.1 also inhibited CFU-GM growth stimulated by leukocyte feeder layers or placental conditioned media, but did not inhibit BFU-E and CFU-E. Antigens recognized by 80H.3, 80H.5, and 81H.1 were expressed both on a proportion of cells from HL.60, KG.1, ML.1, and K562 myeloid cell lines, and on a proportion of blast cells isolated from patients with acute myelogenous leukemia. They were not found on lymphoid cell lines or lymphoid leukemia cells. These MCA recognize either late differentiation antigens expressed on mature neutrophils and monocytes (80H.1 and 80H.3) or early differentiation antigens (80H.5 and 81H.1) specific to the granulocytic lineage. They may be useful for a better definition of those antigens specific to hematopoietic stem cells and their relationship with normal or neoplastic hematopoiesis.

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

    Science.gov (United States)

    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.

  18. Simulation model for overloaded monoclonal antibody variants separations in ion-exchange chromatography.

    Science.gov (United States)

    Guélat, Bertrand; Ströhlein, Guido; Lattuada, Marco; Delegrange, Lydia; Valax, Pascal; Morbidelli, Massimo

    2012-08-31

    A model was developed for the design of a monoclonal antibody charge variants separation process based on ion-exchange chromatography. In order to account for a broad range of operating conditions in the simulations, an explicit pH and salt concentration dependence has been included in the Langmuir adsorption isotherm. The reliability of this model was tested using experimental chromatographic retention times as well as information about the structural characteristics of the different charge variants, e.g. C-terminal lysine groups and deamidated groups. Next, overloaded isocratic elutions at various pH and salt concentrations have been performed to determine the saturation capacity of the ion-exchanger. Furthermore, the column simulation model was applied for the prediction of monoclonal antibody variants separations with both pH and salt gradient elutions. A good prediction of the elution times and peak shapes was observed, even though none of the model parameters was adjusted to fit the experimental data. The trends in the separation performance obtained through the simulations were generally sufficient to identify the most promising operating conditions. The predictive column simulation model thus developed in this work, including a set of parameters determined through specific independent experiments, was experimentally validated and offers a useful basis for a rational optimization of monoclonal antibody variants separation processes on ion-exchange chromatography.

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

    Science.gov (United States)

    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.

  20. Characteristics of Monoclonal Antibody Against Infectious Bursal Disease Virus

    Institute of Scientific and Technical Information of China (English)

    LiYan-Fei; WangWei; 等

    1999-01-01

    Thirteen strains of monoclonal antibodies(McAbs) against infections bursal disease virus(IBDV) were obtained by using hydridoma technique and their characteristics were studied by double immunodiffusion,enzyme-linked immunosorbent assay(ELISA),virus neutralization test(VNT) and Western-blotting assay (WBA).The result showed that nine of the thirteen McAbs belonged to IgG class and four of them belonged to IgM class.No crossreactions were detected betwween the McAbs and Newscastle disease virus (NDV),infectious bronchitis virus(IBV) and Marek's disease virus(MDV).All of McAbs were positively specific reactive with IBDV and five of them can neutralize viral infectivity.Their recognized epitopes of the neutralizing McAbs were all presented on VP2 of the IBDV.

  1. Characteristics of Monoclonal Antibody Against Infectious Bursal Disease Virus

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    Thirteen strains of monoclonal antibodies (McAbs) against infectious bursal disease virus (IBDV) were obtained by using hybridoma technique and their characteristics were studied by double immunodiffusion,en- zyme- linked immunosorbent assay (ELISA), virus neutralization test (VNT) and Western- blotting assay (WBA). The result showed that nine of the thirteen McAbs belonged to IgG class and four of them belonged to IgM class. No crossreactions were detected betwween the McAbs and Newscastle disease virus (NDV) ,in- fectious bronchitis virus(IBV) and Marek's disease virus(MDV). All of McAbs were positively specific reac- tive with IBDV and five of them can neutralize viral infectivity. Their recognized epitopes of the neutralizing McAbs were all presented on VP2 of the IBDV.

  2. Preparation and Identification of Monoclonal Antibodies Against Vibrio anguillarum

    Institute of Scientific and Technical Information of China (English)

    Chen Shiyong(陈师勇); Zhang Peijun; Mo Zhaolan; Zhang Zhendong; Zou Yuxia; Xu Yongli

    2004-01-01

    Monoclonal antibodies (Mabs) against V.anguillarum strain M3 are prepared, and their isotypes are also characterized. Among them, C1C5 is the only Mab which does not crossreact with other eleven non-V.anguillarum strains. The proteinase K digestion test shows that the epitopes recognized by C1C5, C6C3 and C6C32 Mabs contained protein. The periodate oxidation test showed that the epitopes recognized by Mabs except C1C5 are glycosylated. In addition, results of additivity test indicate that the epitopes recognized by C6C3 and C6C32 Mabs are similar, and quite different from those recognized by Mab C1C5.

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

    Science.gov (United States)

    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.

  4. Automated pipeline for rapid production and screening of HIV-specific monoclonal antibodies using pichia pastoris.

    Science.gov (United States)

    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).

  5. Identification of novel proteins in Neospora caninum using an organelle purification and monoclonal antibody approach.

    Science.gov (United States)

    Sohn, Catherine S; Cheng, Tim T; Drummond, Michael L; Peng, Eric D; Vermont, Sarah J; Xia, Dong; Cheng, Stephen J; Wastling, Jonathan M; Bradley, Peter J

    2011-04-04

    Neospora caninum is an important veterinary pathogen that causes abortion in cattle and neuromuscular disease in dogs. Neospora has also generated substantial interest because it is an extremely close relative of the human pathogen Toxoplasma gondii, yet does not appear to infect humans. While for Toxoplasma there are a wide array of molecular tools and reagents available for experimental investigation, relatively few reagents exist for Neospora. To investigate the unique biological features of this parasite and exploit the recent sequencing of its genome, we have used an organelle isolation and monoclonal antibody approach to identify novel organellar proteins and develop a wide array of probes for subcellular localization. We raised a panel of forty-six monoclonal antibodies that detect proteins from the rhoptries, micronemes, dense granules, inner membrane complex, apicoplast, mitochondrion and parasite surface. A subset of the proteins was identified by immunoprecipitation and mass spectrometry and reveal that we have identified and localized many of the key proteins involved in invasion and host interaction in Neospora. In addition, we identified novel secretory proteins not previously studied in any apicomplexan parasite. Thus, this organellar monoclonal antibody approach not only greatly enhances the tools available for Neospora cell biology, but also identifies novel components of the unique biological characteristics of this important veterinary pathogen.

  6. Identification of novel proteins in Neospora caninum using an organelle purification and monoclonal antibody approach.

    Directory of Open Access Journals (Sweden)

    Catherine S Sohn

    Full Text Available Neospora caninum is an important veterinary pathogen that causes abortion in cattle and neuromuscular disease in dogs. Neospora has also generated substantial interest because it is an extremely close relative of the human pathogen Toxoplasma gondii, yet does not appear to infect humans. While for Toxoplasma there are a wide array of molecular tools and reagents available for experimental investigation, relatively few reagents exist for Neospora. To investigate the unique biological features of this parasite and exploit the recent sequencing of its genome, we have used an organelle isolation and monoclonal antibody approach to identify novel organellar proteins and develop a wide array of probes for subcellular localization. We raised a panel of forty-six monoclonal antibodies that detect proteins from the rhoptries, micronemes, dense granules, inner membrane complex, apicoplast, mitochondrion and parasite surface. A subset of the proteins was identified by immunoprecipitation and mass spectrometry and reveal that we have identified and localized many of the key proteins involved in invasion and host interaction in Neospora. In addition, we identified novel secretory proteins not previously studied in any apicomplexan parasite. Thus, this organellar monoclonal antibody approach not only greatly enhances the tools available for Neospora cell biology, but also identifies novel components of the unique biological characteristics of this important veterinary pathogen.

  7. Antibodies to poliovirus detected by immunoradiometric assay with a monoclonal antibody

    Energy Technology Data Exchange (ETDEWEB)

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

    1982-10-01

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

  8. Human Cell Line-Derived Monoclonal IgA Antibodies for Cancer Immunotherapy

    Science.gov (United States)

    Hart, Felix; Danielczyk, Antje; Goletz, Steffen

    2017-01-01

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

  9. Human Cell Line-Derived Monoclonal IgA Antibodies for Cancer Immunotherapy

    Directory of Open Access Journals (Sweden)

    Felix Hart

    2017-05-01

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

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

    Science.gov (United States)

    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.

  11. Preparation and Biological Evaluation of 188Re Labeled Monoclonal Antibody TGLA

    Institute of Scientific and Technical Information of China (English)

    WEN; Kai; ZHANG; Jun-li; CHEN; Bao-jun; CUI; Hai-ping

    2012-01-01

    <正>Monoclonal antibody TGLA is a specific targeting CD20 chimeric antibody. It can kill tumor cells and inhibit tumor cells’ growth effectively, which has been applied to clinical therapy of lymphoma cell B. 188 Re is easy to get, and emits both β and γ rays. 188Re labeled monoclonal antibody TGLA can be used for the study of lymphoma therapy and imaging. This work got the product 188Re-TGLA by direct labeling

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2013-11-01

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

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

    Science.gov (United States)

    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.

  18. Immunogenicity assessment of monoclonal antibody products: A simulated case study correlating antibody induction with clinical outcomes.

    Science.gov (United States)

    Knezevic, Ivana; Kang, Hye-Na; Thorpe, Robin

    2015-09-01

    Monoclonal antibodies are large molecules with complex structure and functions. They have a wide application for treatment of a broad range of chronic diseases and represent the largest class of biotherapeutic products. Given that biotherapeutic products may induce unwanted humoral and/or cellular immune responses in recipients, it is essential to investigate the immunogenicity of a product prior to licensure. The immune response is influenced by many factors and data generated in the pre-licensure studies are usually somewhat difficult for regulatory review. The knowledge and expertise required for this requires a thorough understanding of animal and human immunology as well as specific product characteristics, including mechanism of action, antibody assays and assessment of results in a given clinical context. The appropriate interpretation of immunogenicity data is of critical importance for defining the safety profile of a monoclonal antibody. Two case studies described in this paper were prepared to mimic a real situation in which regulators need to evaluate immunogenicity studies conducted by manufacturers of monoclonal antibody products. The specific objective of the case studies was to illustrate assessment of unwanted immunogenicity and the important factors that need to be considered in this context. Regulators and manufacturers who attended the World Health Organization (WHO) implementation workshop on Evaluation of Biotherapeutic Products, held in Seoul, Republic of Korea, in May 2014, participated in the case studies and provided valuable input. This article outlines the main aspects of immunogenicity discussed in these case studies and a summary of the lessons learned at this occasion. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  19. Monoclonal antibodies neutralizing the haemolytic activity of box jellyfish (Chironex fleckeri) tentacle extracts.

    Science.gov (United States)

    Collins, S P; Comis, A; Marshall, M; Hartwick, R F; Howden, M E

    1993-09-01

    1. Three monoclonal antibodies have been produced which neutralize in vitro the haemolytic activity present in tentacle extracts of the box jellyfish (Chironex fleckeri). 2. Two of these monoclonal antibodies bound specifically to a component of relative molecular mass 50,000 in tentacle extract on Western blots. 3. This binding only occurred when the extracts were electrophoresed under non-reducing conditions. 4. The third monoclonal antibody did not display binding to Western blots of tentacle extract under any of our experimental conditions.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.''

  1. Monoclonal antibodies against proteins of the IBR virus nucleocapside and their assessing by ELISA

    Directory of Open Access Journals (Sweden)

    Jeannette Navarrete 0.

    2011-12-01

    Full Text Available Monoclonal antibodies were produced against the nucleocapside of a field IBR virus strain. The virus was growth in MOBK cell line. Viral components were purified and concentrated in a continuous 30% sacarose gradient followed by chemical precipitation. Nucleocapsides were run in a 10% SDS-PAGE gel. Positive hybridomes were tested using a direct ELISA developed in our laboratory. As a result eigth ELISA positive clones were obtained, from these five were also positive to seronetralization and immunodot. The clones recognize a 39.8Kda protein. Aditionally, a capture-ELISA was developed using the monoclonal antobodies from this research. This ELISA is useful to detect a reference as a field nm virus strain.

  2. Application of {sup 99m}Tc-labeled chimeric Fab fragments of monoclonal antibody A7 for radioimmunoscintigraphy of pancreatic cancer

    Energy Technology Data Exchange (ETDEWEB)

    Matsumura, Hiroomi [Kyoto Prefectural Univ. of Medicine (Japan)

    1999-06-01

    Pancreatic cancer is one of the most lethal diseases and its prognosis is still poor. To improve the survival rate, it is essential to develop new technologies for early and definitive diagnosis. In this study, chimeric Fab fragments of monoclonal antibody A7 were successfully radio-labeled with {sup 99m}Tc, preventing depression of the antigen-binding activity. {sup 99m}Tc-labeled monoclonal antibody A7, {sup 99m}Tc-labeled chimeric Fab fragments of monoclonal antibody A7, {sup 99m}Tc-labeled normal mouse IgG and {sup 99m}Tc-labeled Fab fragments of normal mouse IgG were injected intravenously into nude mice bearing human pancreatic cancer xenografts and the radioactivity was subsequently measured. The tumor accumulation was significantly higher with labeled monoclonal antibody A7 than with normal mouse IgG, and higher with chimeric Fab fragments of monoclonal antibody A7 than with Fab fragments of normal mouse IgG. The tumor/blood ratio of radioactivity increased rapidly over time with chimeric Fab fragments of monoclonal antibody A7. These results suggest that chimeric Fab fragments of monoclonal antibody A7 may be useful for diagnosing pancreatic cancer by means of radioimmunoscintigraphy. (author)

  3. Paper-PEG-based membranes for hydrophobic interaction chromatography: purification of monoclonal antibody.

    Science.gov (United States)

    Yu, Deqiang; Chen, Xiaonong; Pelton, Robert; Ghosh, Raja

    2008-04-15

    This article discusses the preparation of novel Paper-PEG interpenetrating polymer network-based membranes as inexpensive alternative to currently available adsorptive membranes. The Paper-PEG membranes were developed for carrying out hydrophobic interaction membrane chromatography (HIMC). PEG is normally very hydrophilic but can undergo phase separation and become hydrophobic in the presence of high antichaotropic salt concentrations. Two variants of the Paper-PEG membranes, Paper-PEG 1 and Paper-PEG 2 were prepared by grafting different amounts of the polymer on filter paper and these were tested for their hydraulic properties and antibody binding capacity. The better of the two membranes (Paper-PEG 1) was then used for purifying the monoclonal antibody hIgG1-CD4 from simulated mammalian cell culture supernatant. The processing conditions required for purification were systematically optimized. The dynamic antibody binding capacity of the Paper-PEG 1 membrane was about 9 mg/mL of bed volume. A single step membrane chromatographic process using Paper-PEG 1 membrane gave high monoclonal antibody purity and recovery. The hydraulic permeability of the paper-based membrane was high and was maintained even after many runs, indicating that membrane fouling was negligible and the membrane was largely incompressible.

  4. {sup 90}Nb: potential radionuclide for application in immuno-PET. Development of appropriate production strategy and first in vivo evaluation of {sup 90}Nb-labeled monoclonal antibody

    Energy Technology Data Exchange (ETDEWEB)

    Radchenko, Valery

    2013-07-01

    labeling of monoclonal antibody (Rituximab) (iv) provides high in vitro stability. An alternative rapid separation strategy was developed, which provided a source of {sup 90}Nb within one hour, with appropriated radiochemical and radionuclidic purity for subsequent labeling of biomolecules. Finally, for the first time {sup 90}Nb labeled biomolecules were investigated in vivo. Further, experiments on searching of optimal bifunctional chelating (BFC) agents were conducted. Several common BFC were examined on complex formation with NbV. Desferrioxamine (Df) was found to be the most appropriate chelator for {sup 90}Nb. The monoclonal antibody bevacizumab (Avastin {sup registered}) was labeled with {sup 90}Nb and biodistribution study and PET imaging was conducted. All these results proved that {sup 90}Nb is a potential radionuclide for immuno-PET, and may even be further commercially applied for clinical usage.

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

    Science.gov (United States)

    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

  6. Generation of monoclonal antibodies against peptidylarginine deiminase 2 (PAD2) and development of a PAD2-specific enzyme-linked immunosorbent assay

    DEFF Research Database (Denmark)

    Damgaard, Dres; Palarasah, Yaseelan; Skjødt, Karsten

    2014-01-01

    Abs) against rabbit PAD2 and evaluated their cross-reactivity with human PAD2 by indirect enzyme-linked immunosorbent assay (ELISA), western blotting and immunohistological staining of inflamed synovial tissue. Moreover, we established a sandwich ELISA detecting human PAD2, based on two different monoclonal...

  7. Efficient Methods To Isolate Human Monoclonal Antibodies from Memory B Cells and Plasma Cells.

    Science.gov (United States)

    Corti, Davide; Lanzavecchia, Antonio

    2014-10-01

    In this article, we highlight the advantages of isolating human monoclonal antibodies from the human memory B cells and plasma cell repertoires by using high-throughput cellular screens. Memory B cells are immortalized with high efficiency using Epstein-Barr virus (EBV) in the presence of a toll-like receptor (TLR) agonist, while plasma cells are maintained in single-cell cultures by using interleukin 6 (IL-6) or stromal cells. In both cases, multiple parallel assays, including functional assays, can be used to identify rare cells that produce antibodies with unique properties. Using these methods, we have isolated potent and broadly neutralizing antibodies against a variety of viruses, in particular, a pan-influenza-A-neutralizing antibody and an antibody that neutralizes four different paramyxoviruses. Given the high throughput and the possibility of directly screening for function (rather than just binding), these methods are instrumental to implement a target-agnostic approach to identify the most effective antibodies and, consequently, the most promising targets for vaccine design. This approach is exemplified by the identification of unusually potent cytomegalovirus-neutralizing antibodies that led to the identification of the target, a pentameric complex that we are developing as a candidate vaccine.

  8. A human-mouse hybridoma producing monoclonal antibody against human sperm coating antigen.

    Science.gov (United States)

    Kyurkchiev, S D; Shigeta, M; Koyama, K; Isojima, S

    1986-01-01

    Since anti-sperm antibodies were first discovered in the sera of women, the relationship of these antibodies to sterility has been studied by many investigators. In order to determine the antigens of spermatozoa responsible for raising antibodies to spermatozoa in humans, many studies have been carried out by purifying human spermatozoa cell membrane and seminal plasma components. Since it was found that the purification was difficult by physiochemical procedures, the immunoaffinity chromatography bound monoclonal antibody (Mab) to spermatozoa antigens was attempted for this purpose. The establishment of hybridomas producing Mabs to human seminal plasma and human spermatozoa was reported by Shigeta et al. (1980), Isojima, Koyoma & Fujiwara (1982), Lee et al. (1982) and Isahakia & Alexander (1984). The ordinary approaches to obtain the Mabs consisted of xenogenic immunization with human semen and cell fusion of immunized spleen cells with mouse myeloma cells. However, the antigenic epitopes of human spermatozoa, which induced antibody production, are xenogenic for the mouse, and therefore there is a possibility that there is a difference in recognized antigenic epitopes in humans as isotypic and in mice as xenogenic. In order to study these antigenic epitopes, which correspond to antibodies against spermatozoa in women, the establishment of human-mouse hybridomas, which produced anti-semen antibodies as produced in sterile women, became essential. In these studies, we used recently developed cell fusion techniques to fuse immunized human peripheral lymphocytes with mouse myeloma cells. PMID:3456978

  9. Generation and characterization of human B lymphocyte stimulator blocking monoclonal antibody.

    Science.gov (United States)

    Zhuang, Weiliang; Zhang, Jianjun; Pei, Lili; Fang, Shuping; Liu, Honghao; Wang, Ruixue; Su, Yunpeng

    2016-09-01

    The cytokine, B lymphocyte stimulator (Blys) is essential for activation and proliferation of B cells and is involved in the pathogenesis of B-cell mediated autoimmune diseases. Based on its essential activity, Blys may be a potential therapeutic target for human autoimmune diseases. In this article, we have described the development of a novel humanized anti-Blys antibody, NMB04, that binds with high affinity and specificity to both soluble and membrane bound Blys. This monoclonal antibody has the potential to block Blys binding to all its three receptors, TACI, BCMA and BR-3. Further in vivo studies revealed that NMB04 possessed more potent inhibitory activity against human Blys as compared to an existing antibody, Belimumab. Therefore, NMB04 may have potential as a therapeutic candidate targeting autoimmune diseases.

  10. A monoclonal antibody that recognizes an antigenic determinant shared by HLA A2 and B17.

    Science.gov (United States)

    McMichael, A J; Parham, P; Rust, N; Brodsky, F

    1980-09-01

    A hybridoma monoclonal anti-HLA antibody has been produced by the technique of Kohler and Milstein [1]. This antibody recognizes a new specificity common to HLA A2 and B17. It was shown to be a single antibody by isoelectric focusing and absorption experiments.

  11. EFFECT OF POLYCLONAL AND MONOCLONAL-ANTIBODIES ON SURFACE-PROPERTIES OF STREPTOCOCCUS-SOBRINUS

    NARCIS (Netherlands)

    VANRAAMSDONK, M; VANDERMEI, HC; DESOET, JJ; BUSSCHER, HJ; DEGRAAFF, J

    1995-01-01

    In this study, the effect of antibody adsorption on physicochemical properties of Streptococcus sobrinus was studied. Bacteria were preincubated with polyclonal antibodies or with OMVU10, a monoclonal antibody (MAb) reactive with S. sobrinus. The zeta potentials and the hydrophobicity as determined

  12. Development of a Blocking ELISA Using a Monoclonal Antibody to a Dominant Epitope in Non-Structural Protein 3A of Foot-and-Mouth Disease Virus, as a Matching Test for a Negative-Marker Vaccine

    Science.gov (United States)

    Fu, Yuanfang; Li, Pinghua; Cao, Yimei; Wang, Na; Sun, Pu; Shi, Qian; Ji, Xincheng; Bao, Huifang; Li, Dong; Chen, Yingli; Bai, Xingwen; Ma, Xueqing; Zhang, Jing; Lu, Zengjun; Liu, Zaixin

    2017-01-01

    Foot-and-mouth disease (FMD) is a devastating animal disease. Strategies for differentiation of infected from vaccinated animals (DIVA) remain very important for controlling disease. Development of an epitope-deleted marker vaccine and accompanying diagnostic method will improve the efficiency of DIVA. Here, a monoclonal antibody (Mab) was found to recognize a conserved “AEKNPLE” epitope spanning amino acids 109–115 of non-structural protein (NSP) 3A of foot-and-mouth disease virus (FMDV; O/Tibet/CHA/99 strain), which could be deleted by a reverse-genetic procedure. In addition, a blocking ELISA was developed based on this Mab against NSP 3A, which could serve as a matching test for a negative-marker vaccine. The criterion of this blocking ELISA was determined by detecting panels of sera from different origins. The serum samples with a percentage inhibition (PI) equal or greater than 50% were considered to be from infected animals, and those with <50% PI were considered to be from non-infected animals. This test showed similar performance when compared with other 2 blocking ELISAs based on an anti-NSP 3B Mab. This is the first report of the DIVA test for an NSP antibody based on an Mab against the conserved and predominant “AEKNPLE” epitope in NSP 3A of FMDV. PMID:28107470

  13. Use of radiolabeled monoclonal antibodies for diagnostic imaging

    Energy Technology Data Exchange (ETDEWEB)

    Endo, Keigo (Kyoto Univ. (Japan). Faculty of Medicine)

    1990-08-01

    Monoclonal antibodies (MoAbs) are expected to carry radionuclides selectively to target tissues and to offer antigen-specific diagnosis. Indium (In)-111 has many favorable nuclear properties and is efficiently labeled with MoAbs using DAPA as a bifunctional chelating agent. In-111 labeled MoAbs are clinically employed for the diagnosis of malignant melanoma, colorectal cancer and acute myocardial infarction in Japan. Although non-specific deposit of In-111 was seen in liver and bone-marrow, scintigraphy using In-111 labeled MoAbs was encouraging, since it detected about 80% of tumors, tumors missed by conventional diagnostic methods such as CT, and tumors in patients with normal serum CEA values, and acute myocarditis as well as acute myocardial infarction was positive with In-111 labeled Fab fraction of anti-myosin Ab. Acute or subacute toxicity was not observed. Human anti-murine antibody (HAMA) was detected in 53 of 64 (82.8%) patients who were intravenously administered with 20 to 42 mg of anti-melanoma or anti-CEA MoAbs (whole IgG). In contrast, only 5 of 406 (1.2%) patients had detectable levels of HAMA in their serum after receiving 0.5 mg of Fab fraction of MoAb. Recently mouse-human chimeric Ab has been produced by recombinant DNA techniques, which localized well in xenografted tumors and seems to be promising for clinical use. Investigations are under way to increase the tumor to non-tumor ratio by modifying chelating agents for coupling MoAbs with radionuclides. (author).

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  16. Immunotherapy of melanoma with the immune costimulatory monoclonal antibodies targeting CD137

    Directory of Open Access Journals (Sweden)

    Li SY

    2013-09-01

    Full Text Available Shi-Yan Li, Yizhen Liu Cancer Research Institute, Scott and White Healthcare, Temple, TX, USA Abstract: Knowledge of how the immune system recognizes and attempts to control cancer growth and development has improved dramatically. The advent of immunotherapies for cancer has resulted in robust clinical responses and confirmed that the immune system can significantly inhibit tumor progression. Until recently, metastatic melanoma was a disease with limited treatment options and a poor prognosis. CD137 (also known as 4-1BB a member of the tumor necrosis factor (TNF receptor superfamily, is an activation-induced T cell costimulator molecule. Growing evidence indicates that anti-CD137 monoclonal antibodies possess strong antitumor properties, the result of their powerful capability to activate CD8+ T cells, to produce interferon (IFN-γ, and to induce cytolytic markers. Combination therapy of anti-CD137 with other anticancer agents, such as radiation, has robust tumor-regressing abilities against nonimmunogenic or poorly immunogenic tumors. Of importance, targeting CD137 eliminates established tumors, and the fact that anti-CD137 therapy acts in concert with other anticancer agents and/or radiation therapy to eradicate nonimmunogenic and weakly immunogenic tumors is an additional benefit. Currently, BMS-663513, a humanized anti-CD137 antibody, is in clinical trials in patients with solid tumors, including melanoma, renal carcinoma, ovarian cancer, and B-cell malignancies. In this review, we discuss the basis of the therapeutic potential of targeting CD137 in cancer treatment, focusing in particular, on BMS-663513 as an immune costimulatory monoclonal antibody for melanoma immunotherapy. Keywords: anti-CD137 monoclonal antibodies, immune costimulator molecule, BMS-663513

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

    Science.gov (United States)

    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.

  18. Role of cosolutes in the aggregation kinetics of monoclonal antibodies.

    Science.gov (United States)

    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.

  19. Kinetics of Monoclonal Antibody Aggregation from Dilute toward Concentrated Conditions.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  1. Characterization of Endotrypanum Parasites Using Specific Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Ramos Franco Antonia Maria

    1997-01-01

    Full Text Available A large number of Endotrypanum stocks (representing an heterogeneous population of strains have been screened against a panel of monoclonal antibodies (MAbs derived for selected species of Endotrypanum or Leishmania, to see whether this approach could be used to group/differentiate further among these parasites. Using different immunological assay systems, MAbs considered specific for the genus Endotrypanum (E-24, CXXX-3G5-F12 or strain M6159 of E. schaudinni (E-2, CXIV-3C7-F5 reacted variably according to the test used but in the ELISA or immunofluorescence assay both reacted with all the strains tested. Analyses using these MAbs showed antigenic diversity occurring among the Endotrypanum strains, but no qualitative or quantitative reactivity pattern could be consistently related to parasite origin (i.e., host species involved or geographic area of isolation. Western blot analyses of the parasites showed that these MAbs recognized multiple components. Differences existed either in the epitope density or molecular forms associated with the antigenic determinants and therefore allowed the assignment of the strains to specific antigenic groups. Using immunofluorescence or ELISA assay, clone E-24 produced reaction with L. equatorensis (which is a parasite of sloth and rodent, but not with other trypanosomatids examined. Interestingly, the latter parasite and the Endotrypanum strains cross-reacted with a number of MAbs that were produced against members of the L. major-L. tropica complex

  2. Monoclonal antibodies against NS1 protein of Goose parvovirus.

    Science.gov (United States)

    Qiu, Zheng; Tian, Wei; Yu, Tianfei; Li, Li; Ma, Bo; Wang, Junwei

    2012-04-01

    In the present study, monoclonal antibodies (MAbs) against NS1 protein of Goose parvovirus (GPV) were generated. The secreted MAbs were obtained by fusing mouse myeloma cells and spleen cells of BALB/c mice, which were immunized with the plasmid pcDNA3.1-GPV-NS1 and recombinant protein of GPV-NS1. With indirect ELISA, six hybridoma cell lines against GPV-NS1 were screened. The subtypes of the two MAbs were IgG2a; the others were IgM. The light chain was κ. Western blot analysis showed that six MAbs reacted with recombinant protein GPV-NS1. GPV-NS1 was dissected into 15 overlapping epitopes, which were used to react with MAbs in Western blot. Results showed that six MAbs recognized NS1 protein linear B-cell epitopes located at the C-terminus 453-514 aa, 485-542 aa, and 533-598 aa.

  3. Establishment of a novel monoclonal antibody against LGR5.

    Science.gov (United States)

    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.

  4. THE MECHANISM OF ANTI-IMPLANTATION EFFECT OF PROGESTERONE MONOCLONAL ANTIBODIES IN MICE

    Institute of Scientific and Technical Information of China (English)

    WANGMin-Yi; HEZhi-Ying; WANGHan-Zheng

    1989-01-01

    The purpose of this study is to investigate the mechanism by which antiprogcsterone monoclonal antibodies block early pregnancy in mice. The mechanism of passive immunization is a complex issue as indicated below:

  5. Purification of a Mycoplasma pneumoniae adhesin by monoclonal antibody affinity chromatography.

    OpenAIRE

    Leith, D K; Baseman, J B

    1984-01-01

    A 165,000-dalton surface protein of Mycoplasma pneumoniae, designated protein P1, appears to be the major attachment ligand of the pathogen. We employed monoclonal antibody affinity chromatography to obtain purified protein P1.

  6. The role of radiolabeled anti-TNFα monoclonal antibodies for diagnostic purposes and therapy evaluation

    NARCIS (Netherlands)

    Glaudemans, A. W.J.M.; Dierckx, R. A.J.O.; Kallenberg, C. G.M.; Anzola Fuentes, K. L.

    2010-01-01

    Radiolabelled cytokines and monoclonal antibodies are an emerging class of radiopharmaceuticals for imaging inflammation. These radiopharmaceuticals bind to their targets with high affinity and specificity and therefore have excellent diagnostic potential for imaging of patients with chronic inflamm

  7. CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST BOTH PIG AND RABBIT ZONA PELLUCIDA

    Institute of Scientific and Technical Information of China (English)

    OURU-QIANG

    1989-01-01

    Three monoclonal antibodies (MAbs) were raised against both pig and rabbit zona pellucida with a dual immunization protocol employing heat soluble pig zona (HSPZ) and heat soluble rabbit zona (HSRZ), Of the 140 wells screencd, 12 wells were positive to

  8. Production of polyclonal and monoclonal antibodies against the Bacillus thuringiensis vegetative insecticidal protein Vip3Aa16.

    Science.gov (United States)

    Ben Hamadou-Charfi, Dorra; Sauer, Annette Juliane; Abdelkafi-Mesrati, Lobna; Jaoua, Samir; Stephan, Dietrich

    2015-03-01

    The aim of this study is to establish a quantitative determination of the vegetative insecticidal protein Vip3A from the culture supernatant of Bacillus thuringiensis either by ELISA or by the conventional quantification method of the Western blot band. The Vip3A protein was produced by fermentation of the B. thuringiensis reference strain BUPM95 in 3 L. By Western blot, the Vip3Aa16 toxin was detected in the culture supernatant during the exponential growth phase of B. thuringiensis BUPM95. However, the detection of Vip3Aa16 on Western blot showed in addition to the toxin two other strips (62 and 180 kDa) recognized by the anti-Vip3Aa16 polyclonal antibodies prepared at the Centre of Biotechnology of Sfax Tunisia. For that reason and in order to develop a technique for reliable quantification of the toxin, we have considered the production of polyclonal antibodies at the Julius Kühn Institute, Germany. These antibodies were the basis for the production of monoclonal antibodies directed against the protein produced by the Vip3Aa16 recombinant strain Escherichia coli BL21 (DE3). These monoclonal antibodies were tested by plate-trapped antigen (PTA) and triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA). The selection of hybridoma supernatants gave us four positive clones producing monoclonal antibodies.

  9. Use of Monoclonal Antibodies in the Sensitive Detection and Neutralization of Botulinum Neurotoxin Serotype B

    Directory of Open Access Journals (Sweden)

    Luisa W. Cheng

    2015-11-01

    Full Text Available Botulinum neurotoxins (BoNT are some of nature’s most potent toxins. Due to potential food contamination, and bioterrorism concerns, the development of detection reagents, therapeutics and countermeasures are of urgent interest. Recently, we have developed a sensitive electrochemiluminescent (ECL immunoassay for BoNT/B, using monoclonal antibodies (mAbs MCS6-27 and anti-BoNT/B rabbit polyclonal antibodies as the capture and detector. The ECL assay detected as little as 1 pg/mL BoNT/B in the buffer matrix, surpassing the detection sensitivities of the gold standard mouse bioassays. The ECL assay also allowed detection of BoNT/B in sera matrices of up to 100% sera with negligible matrix effects. This highly-sensitive assay allowed the determination of the biological half-lives of BoNT/B holotoxin in vivo. We further tested the toxin neutralization potential of our monoclonal antibodies using the mouse systemic and oral intoxication models. A combination of mAbs protected mice in both pre- and post-exposure models to lethal doses of BoNT/B. MAbs were capable of increasing survival of animals when administered even 10 h post-intoxication in an oral model, suggesting a likely time for BoNT/B complexes to reach the blood stream. More sensitive detection assays and treatments against BoNT intoxication will greatly enhance efforts to combat botulism.

  10. Production and immunoanalytical application of 32 monoclonal antibodies against metacestode somatic antigens of Echinococcus multilocularis.

    Science.gov (United States)

    Wang, Xin; Lu, Rui; Liu, Qiao-Feng; Chen, Jian-Ping; Deng, Qiang; Zhang, Ya-Lou; Zhang, Bing-Hua; Xu, Jia-Nan; Sun, Lei; Niu, Qin-Wang; Liang, Quan-Zeng

    2010-06-01

    Alveolar echinococcosis is a rare but potentially fatal disease. Immunodiagnosis based on antibodies or antigens plays an important role in its diagnosis. In this study, metacestode somatic antigens of Echinococcus multilocularis were used to immunize BALB/c mice, and hybridomas were formed by cell fusion. Making use of the inherent effect of monoclonal antibody techniques to isolate different epitopes, we obtained a repertoire of 32 monoclonal antibodies against the metacestode somatic antigens. These monoclonal antibodies were used to investigate the specificity and localization of the metacestode antigens by enzyme-linked immunosorbent assay and immunohistochemistry, respectively. Nine antibodies specifically reacted with E. multilocularis, while 14 and ten cross-reacted with Echinococcus granulosus and Taenia saginata, respectively. Twenty-five antibodies stained the laminated layer. Eight reacted with the tegument of the protoscolex. Fourteen antibodies recognized the germinal layer. Most of the monoclonal antibodies can react with the antigen Em2. One antibody can react with antigen Em2 and Em10. One antibody that cross-reacted with T. saginata stained the germinal layer and protoscolex, especially its hooklets and suckers, but could not react with Em2 and Em10 antigens. It detected protein bands at 26 and 52 kDa. Two E. multilocularis-specific monoclonal antibodies stained both the germinal and laminated layers and could be used not only to purify specific antigens but also for immunohistochemical studies of E. multilocularis. In summary, these 32 monoclonal antibodies could have potential applications as useful tools in further studies of E. multilocularis antigen profiles.

  11. Identification of Haemophilus influenzae type b by a monoclonal antibody coagglutination assay.

    OpenAIRE

    Hamel, J.; Brodeur, B R; Belmaaza, A; Montplaisir, S; Musser, J M; Selander, R K

    1987-01-01

    A coagglutination assay using monoclonal antibody is described for the identification of Haemophilus influenzae type b. An immunoglobulin G2a monoclonal antibody, Hb-2, directed against a serotype-specific outer membrane protein of H. influenzae type b was adsorbed to Staphylococcus aureus Cowan 1 cells. In a dot enzyme immunoassay, Hb-2 reacted with 453 of 455 H. influenzae type b isolates and did not react with H. influenzae of other serotypes, untypeable H. influenzae strains, or other bac...

  12. Inhibition of lipoxygenase activity in lentil protoplasts by monoclonal antibodies introduced into the cells via electroporation

    OpenAIRE

    J. F. G. Vliegenthart; Maccarrone, M.; Veldink, G.A.

    1992-01-01

    The isolation of lentil protoplasts and the transfer of anti-lipoxygenase monoclonal antibodies into plant protoplasts by electroporation is reported. The dependence of the efficiency of monoclonal antibody incorporation on the field strength is shown as well. The transferred immunoglobulins retained their functional and structural integrity and were able to inhibit the intracellular target enzyme, with a linear relationship between inhibition of lipoxygenase activity and amount of incorporat...

  13. Murine monoclonal anti-avidin antibodies enhance the sensitivity of avidin-biotin immunoassays and immunohistologic staining.

    Science.gov (United States)

    Cassano, W F

    1989-02-24

    To improve the sensitivity of conventional immunoassay methods using avidin-biotin-enzyme complex reagents, we have developed several murine monoclonal antibodies to the egg white avidin glycoprotein. These anti-avidin antibodies enhance the sensitivity of avidin-biotin immunoassays by selectively enlarging the avidin-biotin-enzyme complex through bridging avidin to a second layer of avidin-biotin-enzyme complex, thereby increasing the signal from substrate conversion. Six hybridomas producing murine monoclonal antibodies which bind avidin-biotin-enzyme complexes were isolated and cloned. Two of these anti-avidin antibodies, WC19.10 and WC19.7, have been shown to produce a four-fold enhancement of signal obtained in avidin-biotin-enzyme ELISAs and qualitative enhancement of immunohistologic staining procedures.

  14. Characterization of a monoclonal antibody which detects luteinizing hormone from diverse mammalian species.

    Science.gov (United States)

    Matteri, R L; Roser, J F; Baldwin, D M; Lipovetsky, V; Papkoff, H

    1987-07-01

    The present study describes the development and characterization of a monoclonal antibody (518B7) generated against bovine LH (bLH). Although 518B7 was extremely specific for LH, very low species specificity was observed. A RIA using this antibody and radioiodinated equine LH (eLH) showed good sensitivity for all mammalian LH preparations tested, with the exception of human LH (15%, relative to the eLH reference standard). Activities of most mammalian LH's ranged between approximately 50-200%. Much less activity was detected with reptilian LH (less than 1.5%). Amphibian and avian LH fractions were essentially inactive. The reactivities of LH alpha and beta subunits from a variety of mammals clearly showed that the antibody reacts with the beta subunit. Sensitive RIAs were also developed utilizing 125I-bovine and 125I-rat LH. Interestingly, all hormone preparations which showed sufficient reactivity for statistical analysis within the dose ranges used in the present study (0.01-1000 ng/tube) produced a displacement curve parallel to the reference standard. We have also validated the use of 518B7 in detecting LH in serum. Parallel dilution curves relative to purified LH reference standards were observed with equine and bovine serum samples and equine pituitary extract. High (average 94%) recoveries were also seen with bovine serum with known amounts of exogenously added bLH. Similar patterns of LH secretion were detected with a RIA based upon 125I-bLH and 518B7 and a previously described polyclonal antibody-based RIA in bovine serum samples during estrus. Thus, a monoclonal antibody for LH has been produced which can be used to develop sensitive and specific RIAs in many different mammalian species.(ABSTRACT TRUNCATED AT 250 WORDS)

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2004-12-01

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

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

    Science.gov (United States)

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

    2004-12-01

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

  17. Specific Monoclonal Antibody Overcomes the Salmonella enterica Serovar Typhimurium's Adaptive Mechanisms of Intramacrophage Survival and Replication.

    Directory of Open Access Journals (Sweden)

    Swarmistha Devi Aribam

    Full Text Available Salmonella-specific antibodies play an important role in host immunity; however, the mechanisms of Salmonella clearance by pathogen-specific antibodies remain to be completely elucidated since previous studies on antibody-mediated protection have yielded inconsistent results. These inconsistencies are at least partially attributable to the use of polyclonal antibodies against Salmonella antigens. Here, we developed a new monoclonal antibody (mAb-449 and identified its related immunogen that protected BALB/c mice from infection with Salmonella enterica serovar Typhimurium. In addition, these data indicate that the mAb-449 immunogen is likely a major protective antigen. Using in vitro infection studies, we also analyzed the mechanism by which mAb-449 conferred host protection. Notably, macrophages infected with mAb-449-treated S. Typhimurium showed enhanced pathogen uptake compared to counterparts infected with control IgG-treated bacteria. Moreover, these macrophages produced elevated levels of pro-inflammatory cytokine TNFα and nitric oxide, indicating that mAb-449 enhanced macrophage activation. Finally, the number of intracellular bacteria in mAb-449-activated macrophages decreased considerably, while the opposite was found in IgG-treated controls. Based on these findings, we suggest that, although S. Typhimurium has the potential to survive and replicate within macrophages, host production of a specific antibody can effectively mediate macrophage activation for clearance of intracellular bacteria.

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

    Institute of Scientific and Technical Information of China (English)

    XIA Zhi-nan; CAI Xue-ting; CAO Peng

    2012-01-01

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

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

    NARCIS (Netherlands)

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

    1992-01-01

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

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

    NARCIS (Netherlands)

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

    1992-01-01

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

  1. Preparation of anti-ciguatoxin monoclonal antibodies using synthetic haptens: sandwich ELISA detection of ciguatoxins.

    Science.gov (United States)

    Tsumuraya, Takeshi; Fujii, Ikuo; Hirama, Masahiro

    2014-01-01

    Ciguatera fish poisoning (CFP) is a form of food poisoning caused by the consumption of fish that have accumulated a type of sodium channel activator toxin called ciguatoxins (CTXs), which are produced by dinoflagellates of the genus Gambierdiscus through the food chain. CFP affects more than 50000 people each year. The extremely low level of CTXs in tainted fish has hampered the development of antibodies for the detection of these toxins. Monoclonal antibodies (mAbs) specific against major congeners of CTX3C, 51-hydroxyCTX3C, CTX1B, and 54-deoxyCTX1B were prepared by immunization of mice with protein conjugates of rationally designed synthetic haptens in place of the natural toxins. We found that haptenic groups possessing a surface area larger than 400 angstroms2 were required to produce mAbs that can bind strongly to CTXs. Direct sandwich ELISA utilizing two different monoclonal antibodies that bind specifically to one of the two wings of a CTX were established to detect CTXs. No cross-reactivity was observed against the other marine toxins tested, including brevetoxin A, brevetoxin B, okadaic acid, and maitotoxin.

  2. Large Scale Production and Characterization of Anti-Human IgG Monoclonal Antibody in Peritoneum of Balb/c MICE

    Directory of Open Access Journals (Sweden)

    B. Baradaran

    2005-01-01

    Full Text Available Monoclonal antibodies are key reagents that are used in biomedical researches, diagnosis of immunodeficiency diseases such as IgG subclasses deficiency and treatment of diseases like infections and cancers .For large scale production of monoclonal antibody, hybridoma cells that produce monoclonal antibody against human IgG were injected into the peritoneum of the Balb/c mice which have previously been primed with 0.5 ml Pristane. After ten days, approximately 3 ml ascitic fluid was harvested from the peritoneum of each mouse. Ascitic fluid was assayed for the titer of monoclonal antibody in reaction with human IgG and its cross reactivity in reaction with IgM & IgA. The titer of mAb was 100,000 and didn't show cross reactivity with IgM & IgA. Immunobloting was done for confirming the ELISA method. In immunobloting, only one sharp band in the heavy chain position of IgG was developed. The subclass of antibody was IgG1 and its light chain was kappa. Ascitic fluid was purified by ion exchange chromatography and the purified monoclonal antibody was conjugated with HRP. The conjugated monoclonal antibody could have application in diagnosis of infectious diseases like Toxoplasmosis, Rubella and IgG class of all other infectious diseases.

  3. Antigen-specific monoclonal antibodies isolated from B cells expressing constitutively active STAT5.

    Directory of Open Access Journals (Sweden)

    Ferenc A Scheeren

    Full Text Available BACKGROUND: Fully human monoclonal antibodies directed against specific pathogens have a high therapeutic potential, but are difficult to generate. METHODOLOGY/PRINCIPAL FINDINGS: Memory B cells were immortalized by expressing an inducible active mutant of the transcription factor Signal Transducer and Activator of Transcription 5 (STAT5. Active STAT5 inhibits the differentiation of B cells while increasing their replicative life span. We obtained cloned B cell lines, which produced antibodies in the presence of interleukin 21 after turning off STAT5. We used this method to obtain monoclonal antibodies against the model antigen tetanus toxin. CONCLUSIONS/SIGNIFICANCE: Here we describe a novel and relatively simple method of immortalizing antigen-specific human B cells for isolation of human monoclonal antibodies. These results show that STAT5 overexpression can be employed to isolate antigen specific antibodies from human memory B cells.

  4. Immunoscintigraphy and radioimmunotherapy in Cuba: experiences with labeled monoclonal antibodies for cancer diagnosis and treatment (1993-2013).

    Science.gov (United States)

    Peña, Yamilé; Perera, Alejandro; Batista, Juan F

    2014-01-01

    INTRODUCTION The availability of monoclonal antibodies in Cuba has facilitated development and application of innovative techniques (immunoscintigraphy and radioimmunotherapy) for cancer diagnosis and treatment. Objective Review immunoscintigraphy and radioimmunotherapy techniques and analyze their use in Cuba, based on the published literature. In this context, we describe the experience of Havana's Clinical Research Center with labeled monoclonal antibodies for cancer diagnosis and treatment during the period 1993-2013. EVIDENCE ACQUISITION Basic concepts concerning cancer and monoclonal antibodies were reviewed, as well as relevant international and Cuban data. Forty-nine documents were reviewed, among them 2 textbooks, 34 articles by Cuban authors and 13 by international authors. All works published by the Clinical Research Center from 1993 through 2013 were included. Bibliography was obtained from the library of the Clinical Research Center and Infomed, Cuba's national health telematics network, using the following keywords: monoclonal antibodies, immunoscintigraphy and radioimmunotherapy. RESULTS Labeling the antibodies (ior t3, ior t1, ior cea 1, ior egf/r3, ior c5, h-R3, 14F7 and rituximab) with radioactive isotopes was a basic line of research in Cuba and has fostered their use as diagnostic and therapeutic tools. The studies conducted demonstrated the good sensitivity and diagnostic precision of immunoscintigraphy for detecting various types of tumors (head and neck, ovarian, colon, breast, lymphoma, brain). Obtaining different radioimmune conjugates with radioactive isotopes such as 99mTc and 188Re made it possible to administer radioimmunotherapy to patients with several types of cancer (brain, lymphoma, breast). The objective of 60% of the clinical trials was to determine pharmacokinetics, internal dosimetry and adverse effects of monoclonal antibodies, as well as tumor response; there were few adverse effects, no damage to vital organs, and a positive

  5. Determination of critical quality attributes for monoclonal antibodies using quality by design principles.

    Science.gov (United States)

    Alt, Nadja; Zhang, Taylor Y; Motchnik, Paul; Taticek, Ron; Quarmby, Valerie; Schlothauer, Tilman; Beck, Hermann; Emrich, Thomas; Harris, Reed J

    2016-09-01

    Quality by design (QbD) is a global regulatory initiative with the goal of enhancing pharmaceutical development through the proactive design of pharmaceutical manufacturing process and controls to consistently deliver the intended performance of the product. The principles of pharmaceutical development relevant to QbD are described in the ICH guidance documents (ICHQ8-11). An integrated set of risk assessments and their related elements developed at Roche/Genentech were designed to provide an overview of product and process knowledge for the production of a recombinant monoclonal antibody. This chapter describes the identification of critical quality attributes (CQAs) as an important first step for QbD development of biopharmaceuticals. A systematic scientific based risk ranking and filtering approach allows a thorough understanding of quality attributes and an assignment of criticality for their impact on drug safety and efficacy. To illustrate the application of the approach and tools, a few examples from monoclonal antibodies are shown. The identification of CQAs is a continuous process and will further drive the structure and function characterization of therapeutic proteins. Copyright © 2016 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

  6. Development of Enzyme Linked Immunosorbent Assay for Quantita-tive Determination of Agonistic DR5 Monoclonal Antibody%抗 DR5单克隆抗体 ELISA 检测新方法的建立

    Institute of Scientific and Technical Information of China (English)

    郭聪; 陈知航; 许先兴; 刘运龙; 车津晶; 董俊兴; 程远国

    2013-01-01

    Objective: To develop a specific, sensitive and rapid ELISA method for the quantification of agonistic death receptor 5(DR5) monoclonal antibody. Methods: An quantitative sandwich enzyme immunoassay was devel⁃oped in using goat anti-human IgG for capturing as well as detecting. Following that, color was developed by the substrate solution and the reaction was stopped by stop solution. Finally the plate was read at a wavelength of 450 nm using a microplate reader. Results: An ELISA assay was developed with a wide dynamic range of con⁃centrations from 12.5 to 800 ng/mL. The lowest quantification of this assay was 12.5 ng/mL, both accuracy of the intra- and inter-assay were less than 15%. Conclusion: The assay is highly sensitive, accurate, specific, and re⁃producibility over a wide dynamic range of concentrations, which was proven to be a feasible quantitative method for agonistic DR5 monoclonal antibody analysis.%  目的:建立一种灵敏、特异、快速的 ELISA 方法,用猕猴血清中抗死亡受体5(DR5)单克隆抗体的检测.方法:采用双抗夹心 ELISA 法,用猴血清吸附的羊抗人 IgG 包被96孔酶标板,加入待测样品,用 HRP 标记的猴血清吸附的羊抗人 IgG 进行检测,加底物显色,读取 D450nm值.结果:建立了检测抗 DR5单克隆抗体的 ELISA 方法并进行了确证,方法的线性范围为12.5~800 ng/mL,定量下限为12.5 ng/mL,板内和板间精密度均小15%,准确度为±15%,冻融稳定性和稀释稳定性良好.结论:方法学确证结果表明,本研究建立的抗 DR5单克隆抗体检测方法符合新生物制品临床前药代动力学研究指导则要求,可用抗 DR5单克隆抗体的检测.

  7. Two new monoclonal antibodies for biochemical and flow cytometric analyses of human interferon regulatory factor-3 activation, turnover, and depletion.

    Science.gov (United States)

    Rustagi, Arjun; Doehle, Brian P; McElrath, M Juliana; Gale, Michael

    2013-02-01

    Interferon regulatory factor-3 (IRF-3) is a master transcription factor that drives the host intracellular innate immune response to virus infection. The importance of IRF-3 in innate immune responses is highlighted by the fact that pathogenic viruses have developed strategies for antagonism of IRF-3. Several tools exist for evaluation of viral regulation of IRF-3 activation and function, but high-quality monoclonal antibodies that mark the differential activation states of human IRF-3 are lacking. To study IRF-3 activation, turnover, and depletion in a high-throughput manner in the context of virus infection, we have developed two new monoclonal antibodies to human IRF-3. These antibodies detect IRF-3 in virus-infected cells in a wide variety of assays and provide a new tool to study virus-host interactions and innate immune signaling.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1987-11-01

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

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

    Science.gov (United States)

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

    1987-11-01

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

  10. Dye Labelled Monoclonal Antibody Assay for Detection of Toxic Shock Syndrome Toxin -1 from Staphylococcus Aureus

    Directory of Open Access Journals (Sweden)

    V Javid Khojasteh

    2011-12-01

    Full Text Available Objective: The aim of study was to develop a rapid assay, dye labelled monoclonal antibody assay (DLMAA, using non-radioactive organic synthetic dyes for identification of Toxic Shock Syndrome Toxin-1 (TSST-1 producing strains of Staphylococcus aureus.Materials and Methods: The assay protocol required only two simple steps; addition of TSST-1 antigen to a nitrocellulose membrane and then adding a colloidal dye labelled antibody (D/A suspension detection reagent.Results: The sensitivity and specificity of the assay was determined relative to positive and negative strains compared to an ELISA assay. Overall 100% agreement was found between both assays. The sensitivity for detection of TSST-1 was 30 ng.Conclusion: The DLMAA did not require handling and disposal of radioactive materials. It is a rapid qualitative technique for detection of TSST-1 toxin at room temperature within a short time.

  11. High-throughput epitope binning of therapeutic monoclonal antibodies: why you need to bin the fridge.

    Science.gov (United States)

    Brooks, Benjamin D; Miles, Adam R; Abdiche, Yasmina N

    2014-08-01

    Analytical tools are evolving to meet the need for the higher-throughput characterization of therapeutic monoclonal antibodies. An antibody's epitope is arguably its most important property because it underpins its functional activity but, because epitope selection is innate, it remains an empirical process. Here, we focus on the emergence of label-free biosensors with throughput capabilities orders of magnitude higher than the previous state-of-the-art, which can facilitate large assays such as epitope binning so that they can be incorporated alongside functional activity screens, enabling the rapid identification of leads that exhibit unique and functional epitopes. In addition to streamlining the drug development process by saving time and cost, the information from epitope binning assays could provide the basis for intellectual property protection. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodies.

    Science.gov (United States)

    Du, Yi; Walsh, Alison; Ehrick, Robin; Xu, Wei; May, Kimberly; Liu, Hongcheng

    2012-01-01

    The existence of multiple variants with differences in either charge, molecular weight or other properties is a common feature of monoclonal antibodies. These charge variants are generally referred to as acidic or basic compared with the main species. The chemical nature of the main species is usually well-understood, but understanding the chemical nature of acidic and basic species, and the differences between all three species, is critical for process development and formulation design. Complete understanding of acidic and basic species, however, is challenging because both species are known to contain multiple modifications, and it is likely that more modifications may be discovered. This review focuses on the current understanding of the modifications that can result in the generation of acidic and basic species and their affect on antibody structure, stability and biological functions. Chromatography elution profiles and several critical aspects regarding fraction collection and sample preparations necessary for detailed characterization are also discussed.

  13. Targeting the replisome with transduced monoclonal antibodies triggers lethal DNA replication stress in cancer cells.

    Science.gov (United States)

    Desplancq, Dominique; Freund, Guillaume; Conic, Sascha; Sibler, Annie-Paule; Didier, Pascal; Stoessel, Audrey; Oulad-Abdelghani, Mustapha; Vigneron, Marc; Wagner, Jérôme; Mély, Yves; Chatton, Bruno; Tora, Laszlo; Weiss, Etienne

    2016-03-15

    Although chemical inhibition of the DNA damage response (DDR) in cancer cells triggers cell death, it is not clear if the fork blockade achieved with inhibitors that neutralise proteins of the replisome is sufficient on its own to overcome the DDR. Monoclonal antibodies to PCNA, which block the DNA elongation process in vitro, have been developed. When these antibodies were transduced into cancer cells, they are able to inhibit the incorporation of nucleoside analogues. When co-delivered with anti-PCNA siRNA, the cells were flattened and the size of their nuclei increased by up to 3-fold, prior to cell death. Analysis of these nuclei by super-resolution microscopy revealed the presence of large numbers of phosphorylated histone H2AX foci. A senescence-like phenotype of the transduced cells was also observed upon delivery of the corresponding Fab molecules or following PCNA gene disruption or when the Fab fragment of an antibody that neutralises DNA polymerase alpha was used. Primary melanoma cells and leukaemia cells that are resistant to chemical inhibitors were similarly affected by these antibody treatments. These results demonstrate that transduced antibodies can trigger a lethal DNA replication stress, which kills cancer cells by abolishing the biological activity of several constituents of the replisome.

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

    Science.gov (United States)

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

    1981-05-01

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

  15. Partial analysis of the flagellar antigenic determinant recognized by a monoclonal antibody to Clostridium tyrobutyricum.

    Science.gov (United States)

    Bédouet, L; Arnold, F; Robreau, G; Batina, P; Talbot, F; Malcoste, R

    1998-01-01

    In order to count Clostridium tyrobutyricum spores in milk after membrane filtration, murine 21E7-B12 monoclonal antibody was produced. Elution of the monoclonal antibody from this antigen, the flagellar filament protein, by carbohydrate ligands was used to study the epitope structure. A competitive elution of an anti-dextran monoclonal antibody by carbohydrate ligands served as a control in order to validate the immunological tool applied to flagellin epitope study. The carbohydrate moiety of flagellin contained D-glucose and N-acetyl-glucosamine in a molar ration of 11:1 as determined by gas-liquid chromatography and 2 low-abundancy unidentified compounds. In ELISA, D-glucose and N-acetyl-glucosamine did not dissociate the antibody-flagellin complex contrary to maltose, maltotriose, maltotetraose and maltopentaose. The efficiency of elution increased from the dimer to the pentamer and became nil for maltohexaose and maltoheptaose. The fact that the hexamer and heptamer could not react with the 21E7-B12 monoclonal antibody could be explained by a drastic conformational change. The over-all stretched maltopentaose switch to a helical-shaped maltoheptaose which could not fit the 21E7-B12 monoclonal antibody antigen-combining site. Thus, flagellin epitope may contain alpha (1-->4) linked glucose residues plus either N-actyl-glucosamine or an unidentified compound that maintain it in an extended shape.

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

    Science.gov (United States)

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

  17. Human monoclonal HLA antibodies reveal interspecies crossreactive swine MHC class I epitopes relevant for xenotransplantation.

    NARCIS (Netherlands)

    Mulder, A.; Kardol, M.J.; Arn, J.S.; Eijsink, C.; Franke, M.E.; Schreuder, G.M.; Haasnoot, G.W.; Doxiadis, I.I.; Sachs, D.H.; Smith, D.M.; Claas, F.H.

    2010-01-01

    Crossreactivity of anti-HLA antibodies with SLA alleles may limit the use of pig xenografts in some highly sensitized patients. An understanding of the molecular basis for this crossreactivity may allow better selection of xenograft donors. We have tested 68 human monoclonal HLA class I antibodies (

  18. The Synthesis of N-Morphine Hapten and Production of Monoclonal Antibody

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Those antibodies elicited by different tether site for attachment to carrier protein have different specificity. Herein we reported that a monoclonal antibody against morphine with high specificity and affinity was successfully produced by using different linkers to couple to different carrier proteins.

  19. Synthetic methyl hexagalacturonate hapten inhibitors of antihomogalacturonan monoclonal antibodies LM7, JIM5 and JIM7

    DEFF Research Database (Denmark)

    Clausen, Mads Hartvig; Willats, William George Tycho; Knox, J. Paul

    2003-01-01

    A range of synthetic methyl hexagalacturonates were used as potential hapten inhibitors in competitive-inhibition enzyme-linked immunosorbent assays (ELISAs) with anti-homogalacturonan monoclonal antibodies LM7, JIM5 and JIM7. The selective inhibition of these antibodies by different haptens prov...

  20. B lymphocyte depletion with the monoclonal antibody rituximab in Graves' disease: a controlled pilot study

    DEFF Research Database (Denmark)

    El Fassi, Daniel; Nielsen, Claus H; Bonnema, Steen J

    2007-01-01

    Graves' disease (GD) is a common TSH receptor autoantibody (TRAb)-mediated disorder. Because B lymphocytes are important self-antigen presenting cells and precursors for antibody-secreting plasma cells, temporary B-lymphocyte depletion with the monoclonal antibody rituximab (RTX) might be of bene...

  1. In-column ATR-FTIR spectroscopy to monitor affinity chromatography purification of monoclonal antibodies.

    Science.gov (United States)

    Boulet-Audet, Maxime; Kazarian, Sergei G; Byrne, Bernadette

    2016-07-29

    In recent years many monoclonal antibodies (mAb) have entered the biotherapeutics market, offering new treatments for chronic and life-threatening diseases. Protein A resin captures monoclonal antibody (mAb) effectively, but the binding capacity decays over repeated purification cycles. On an industrial scale, replacing fouled Protein A affinity chromatography resin accounts for a large proportion of the raw material cost. Cleaning-in-place (CIP) procedures were developed to extend Protein A resin lifespan, but chromatograms cannot reliably quantify any remaining contaminants over repeated cycles. To study resin fouling in situ, we coupled affinity chromatography and Fourier transform infrared (FTIR) spectroscopy for the first time, by embedding an attenuated total reflection (ATR) sensor inside a micro-scale column while measuring the UV 280 nm and conductivity. Our approach quantified the in-column protein concentration in the resin bed and determined protein conformation. Our results show that Protein A ligand leached during CIP. We also found that host cell proteins bound to the Protein A resin even more strongly than mAbs and that typical CIP conditions do not remove all fouling contaminants. The insights derived from in-column ATR-FTIR spectroscopic monitoring could contribute to mAb purification quality assurance as well as guide the development of more effective CIP conditions to optimise resin lifespan.

  2. In-column ATR-FTIR spectroscopy to monitor affinity chromatography purification of monoclonal antibodies

    Science.gov (United States)

    Boulet-Audet, Maxime; Kazarian, Sergei G.; Byrne, Bernadette

    2016-07-01

    In recent years many monoclonal antibodies (mAb) have entered the biotherapeutics market, offering new treatments for chronic and life-threatening diseases. Protein A resin captures monoclonal antibody (mAb) effectively, but the binding capacity decays over repeated purification cycles. On an industrial scale, replacing fouled Protein A affinity chromatography resin accounts for a large proportion of the raw material cost. Cleaning-in-place (CIP) procedures were developed to extend Protein A resin lifespan, but chromatograms cannot reliably quantify any remaining contaminants over repeated cycles. To study resin fouling in situ, we coupled affinity chromatography and Fourier transform infrared (FTIR) spectroscopy for the first time, by embedding an attenuated total reflection (ATR) sensor inside a micro-scale column while measuring the UV 280 nm and conductivity. Our approach quantified the in-column protein concentration in the resin bed and determined protein conformation. Our results show that Protein A ligand leached during CIP. We also found that host cell proteins bound to the Protein A resin even more strongly than mAbs and that typical CIP conditions do not remove all fouling contaminants. The insights derived from in-column ATR-FTIR spectroscopic monitoring could contribute to mAb purification quality assurance as well as guide the development of more effective CIP conditions to optimise resin lifespan.

  3. Biosimilar Monoclonal Antibodies for Inflammatory Bowel Disease: Current Comfort and Future Prospects.

    Science.gov (United States)

    Gecse, Krisztina B; Lakatos, Péter L

    2016-10-01

    Biosimilars are biologic medicines that enter the market after a patent for an original reference product expires. The European Medicines Agency (EMA) developed a stringent legislation process for biosimilar monoclonal antibodies, whereby similarity to the reference medicinal product in terms of quality characteristics, biological activity, clinical safety and efficacy must be demonstrated. Biosimilar infliximab CT-P13 was the first biosimilar monoclonal antibody to receive EMA marketing authorization, and further biosimilar molecules are being developed. The phase I and III clinical trials were conducted in ankylosing spondylitis and rheumatoid arthritis, and the use of CT-P13 in inflammatory bowel disease (IBD) was extrapolated on the results of these trials. Medical professionals were initially concerned about the reversed engineering process, the novel legal framework and the lack of clinical data in IBD. Emerging real-world data have confirmed the similarities between CT-P13 and the reference product in terms of efficacy, safety and immunogenicity in IBD. The cost reduction represented by biosimilars promotes industry competition and improves treatment access with sustained quality of care. This article reviews the existing and emerging clinical data for CT-P13 and a future perspective on biosimilar use in IBD.

  4. Mammalian Cell Culture Clarification: A Case Study Using Chimeric Anti-CEA Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Mohamed Ali Abol Hassan

    2011-12-01

    Full Text Available The extracellular expression of monoclonal antibodies (mAbs in mammalian cell culture provides both opportunities and restrictions for the design of robust harvest and clarification operations. With advances in cell culture media and cell lines, it is now possible to achieve high titers of over 5 g/l for mAbs. However, Mammalian cells are sensitive to breakage due to shear stress that can result in release of proteases and other host cell proteins (HCPs which eventually affects product stability and purity. There is larger number of mAbs undergoing clinical development and it has placed significant importance on platform technologies of process development. Generally, Centrifugation and microfiltration are the primary harvest techniques used in the industry and depth filtration is also used as a step operation on clarification. This study compares the unit operations; centrifugation, microfiltration and depth filtration for maximum recovery of monoclonal antibodies. The results have shown that the depth filtration as more suitable operation for mammalian cell culture clarification since it gives 96% recovery of mAbs in comparison to centrifugation and microfiltration. ABSTRAK: Pengungkapan luar sel dari antibodi monoklon (monoclonal antibodies ((mAbs dalam kultur sel mamalia memberi ruang dan batasan terhadap reka bentuk penuaian yang cekap dan penerangan operasi. Dengan kemajuan dalam media sel kultur dan cell lines (produk yang berupa sel kekal yang digunakan untuk tujuan kajian biologi, kini adalah berkemungkinan untuk memperolehi titer tinggi melebihi 5g/l untuk mAbs [2]. Walaupun begitu, sel mamalia sensitif terhadap retakan disebabkan tegasan ricih yang menyebabkan pengeluaran protease dan hos sel protein yang lain, (host cell proteins (HCPs akhirnya mempengaruhi kestabilan dan keaslian produk. Terdapat mAbs dalam jumlah besar yang masih menjalani pembangunan klinikal dan sesungguhnya ini penting sebagai satu landasan teknologi dalam

  5. Secretory leukocyte protease inhibitor (SLPI) might contaminate murine monoclonal antibodies after purification on protein G.

    Science.gov (United States)

    Schenk, Jörg A; Fettke, Joerg; Lenz, Christine; Albers, Katharina; Mallwitz, Frank; Gajovic-Eichelmann, Nenad; Ehrentreich-Förster, Eva; Kusch, Emely; Sellrie, Frank

    2012-03-31

    The large scale production of a monoclonal anti-progesterone antibody in serum free medium followed by affinity chromatography on protein G lead to a contamination of the antibody sample with a protein of about 14 kDa. This protein was identified by mass spectrometry as secretory leukocyte protease inhibitor (SLPI). This SLPI contamination lead to a failure of the fiber-optic based competitive fluorescence assay to detect progesterone in milk. Purification of the monoclonal antibody using protein A columns circumvented this problem.

  6. Rescue and expression of human immunoglobulin genes to generate functional human monoclonal antibodies.

    Science.gov (United States)

    Lewis, A P; Parry, N; Peakman, T C; Crowe, J S

    1992-07-01

    Human monoclonal antibody production has been hampered for many years by the instability of cell lines and low levels of expression of the antibodies. We describe here the rescue of human immunoglobulin genes utilizing micro-mRNA preparation from a small number of human hybridoma cells and conventional cDNA cloning. This allows cloning and immediate high-level expression from full-length human heavy and light chain cDNA molecules and provides a mechanism to rescue whole human monoclonal antibodies of proven efficacy.

  7. Mammalian tissue distribution of a large heparan sulfate proteoglycan detected by monoclonal antibodies

    DEFF Research Database (Denmark)

    Couchman, J R; Ljubimov, A V

    1989-01-01

    A panel of nine monoclonal antibodies has been characterized, all of which have reactivity with the core protein of a large heparan sulfate proteoglycan derived from the murine EHS tumor matrix. These rat monoclonal antibodies stained mouse basement membranes intensely, including those of all...... muscle, endothelia, peripheral nerve fibers and epithelia so far examined. In addition, two of the monoclonal antibodies show cross-species reactivity, staining bovine and human basement membranes, and immunoprecipitating proteoglycans from human endothelial cell cultures. These antibodies do not......, however, cross-react with avian tissues. These results show the ubiquitous distribution of a heparan sulfate proteoglycan in mammalian tissues, which will be useful in vitro and in vivo for studies on the biology of basement membrane proteoglycans and investigations of possible roles of these molecules...

  8. Epidemiologic investigation by macrorestriction analysis and by using monoclonal antibodies of nosocomial pneumonia caused by Legionella pneumophila serogroup 10.

    OpenAIRE

    Lück, P. C.; Helbig, J H; Günter, U; Assmann, M.; Blau, R; Koch, H.; Klepp, M.

    1994-01-01

    A 67-year-old woman was hospitalized with an acute pneumonia of the left lower lobe. Legionella pneumophila serogroup 10 was cultured from two sputum specimens taken on days 18 and 20 and was also detected by direct immunofluorescence assay by using a commercially available species-specific monoclonal antibody as well as serogroup 10-specific monoclonal antibodies. Antigenuria was detected in enzyme-linked immunosorbent assays by using serogroup 10-specific polyclonal and monoclonal antibodie...

  9. Characteristics of 26 kDa antigen of H. Pylori by Monoclonal Antibody.

    Directory of Open Access Journals (Sweden)

    Hossein Ghahremani

    2015-02-01

    Full Text Available Alkylhydroperoxide reductase (AhpC, the 26 kDa antigen is one of the abundant antioxidant enzymes in Helicobacter pylori and seems to have a good potential for use in development of immunoassays to detect H. pylori infection in clinical specimens. This study aimed to investigate some properties of this antigen by the produced monoclonal antibodies. Five established hybridoma cell lines secreting monoclonal antibodies (MAbs against 26 kDa antigen of H. pylori were cultivated and MAbs were purified by affinity chromatography. Subsequently, MAbs were conjugated with biotin, and different combinations of capture and tracer antibodies used in sandwich ELISA. Immunoblotting of bacterial extracts were performed to estimate aggregation status of the antigen. Release of antigen from the cultivated bacteria on solid media was examined by sandwich ELISA, and also, existence of interference in fecal extract was investigated by immunoblotting and sandwich ELISA. Our findings showed that the MAbs against 26 kDa antigen of H. pylori could recognize three bands of nearly 25 kDa, 50 kDa, and 75 kDa in immunoblotting. This study also indicated presence of more antigens in the culture medium around the bacteria than the bacterial extract itself. The results of sandwich ELISA and immunoblotting on fecal extracts suggest the presence of interfering agents that prevent detection of antigen by antibody in ELISA but not in immunoblotting. In this study the oligomerization of the 26 kDa antigen, presence of interfering agents in stool matrix, and release of antigen to outside of bacteria, were demonstrated.

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

    Directory of Open Access Journals (Sweden)

    Kiefer Hans

    2009-06-01

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

  11. Monoclonal antibody-based sandwich ELISA for the detection of staphylococcal enterotoxin A.

    Science.gov (United States)

    Kuang, Hua; Wang, Wenbing; Xu, Liguang; Ma, Wei; Liu, Liqiang; Wang, Libing; Xu, Chuanlai

    2013-04-19

    A sensitive and specific monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (ELISA) was established and validated for the detection of staphylococcal enterotoxin A (SEA). After routine fusion and selection, 10 monoclonal antibodies showed high affinity for SEA. An optimal pair for sandwich ELISA was selected by pairwise interaction analysis. After optimization, the limit of detection (LOD) and linear dynamic range of the method were established, and were found to be 0.0282 ng/mL and 0.06-2 ng/mL, respectively. The recovery in pure milk ranged from 82.67% to 111.95% and the intra- and inter-assay coefficients of variation ranged from 3.16% to 6.05% and from 5.16% to 10.79%, respectively. Cross-reactivity with staphylococcal enterotoxin B (SEB), staphylococcal enterotoxin C (SEC), staphylococcal enterotoxin D (SED), and staphylococcal enterotoxin E (SEE) in this method were insignificant. These results indicate that the sandwich ELISA method developed in our study is effective for routine identification of SEA in food samples.

  12. Stage-specific Monoclonal Antibodies to the Potato Cyst Nematode Globodera pallida Stone (Behrens).

    Science.gov (United States)

    Backett, K D; Atkinson, H J; Forrest, J M

    1993-09-01

    Using standard hybridoma technology and hierarchical screening, monoclonal antibodies (MAbs) were obtained with specific reactivity against two developmental stages of Globodera pallida. The procedure was based on enzyme-linked immunosorbent assay (ELISA) with homogenates prepared from second-stage juveniles, young adult females, and potato roots. Hybridomas were formed by fusing myelomas with splenocytes derived from mice immunized with either infective juveniles or females of G. pallida. About 600 hybridoma lines were screened from the fusion involving the mouse immunized with juveniles. Two MAbs (LJMAbl &2) were identified with high reactivity toward second-stage juveniles but no reactivity with either potato roots or females of G. pallida. A total of 630 cell lines was screened from the corresponding fusion involving the spleen of a mouse receiving immunogens from adult female nematodes. One MAb (LFMAbl) was obtained with the required specificity against only adult female G. pallida. This work extends the application of monoclonal antibodies in nematology from valuable probes for research and species identification to recognition of developmental stages. These specific MAbs have potential value in plant breeding programs for screening for resistant lines unable to support nematode development.

  13. Monoclonal antibodies to the major Lolium perenne (rye grass) pollen allergen Lol p I (Rye I).

    Science.gov (United States)

    Kahn, C R; Marsh, D G

    1986-12-01

    Thirteen monoclonal antibodies (MAbs) were produced against Lol p I (Rye I), the major Lolium perenne (rye grass) pollen allergen. Spleen cells from A/J and SJL mice immunized with highly purified Lol p I (Lol I) were allowed to fuse with cells from the non-secreting Sp2/0-Ag14 myeloma cell line. Each MAb was analyzed for antigenic specificity by radioimmunoassay (RIA) using 125I-Lol I. The epitope specificities of seven of the MAbs were examined by competitive binding against a labelled standard MAb for the Lol I antigen (Ag). The dissociation constant, Kd, of one MAb (No. 3.2) that was studied most extensively was determined by double Ab RIA to be 3.5 X 10(-6) L/M. This MAb recognized the related 27,000-30,000 Group I glycoproteins found in the pollens of nine other species of grass pollens tested, including weak binding to Bermuda grass Group I (Cyn d I), which by conventional analysis using polyclonal anti-Lol I serum shows no detectable binding. Monoclonal antibody No. 3.2 was coupled covalently to Sepharose 4B and used to prepare highly purified Lol I from a partially purified rye pollen extract. Finally, an RIA was developed which permitted the analysis of the Group I components in rye grass and nine other grass pollen species. The latter assay is likely to prove useful in the standardization of grass pollen extracts according to their Group I contents.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1985-09-01

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

  15. Epitope Mapping of Dengue-Virus-Enhancing Monoclonal-Antibody Using Phage Display Peptide Library

    OpenAIRE

    Chung-I Rai; Huan-Yao Lei; Yee-Shin Lin; Hsiao-Sheng Liu; Shun-Hua Chen; Lien-Cheng Chen; Trai-Ming Yeh

    2008-01-01

    The Antibody-Dependent Enhancement (ADE) hypothesis has been proposed to explain why more severe manifestations of Dengue Hemorrhagic Fever and Dengue Shock Syndrome (DHF/DSS) occur predominantly during secondary infections of Dengue Virus (DV) with different serotypes. However, the epitopes recognized by these enhancing antibodies are unclear. Recently, anti-pre-M monoclonal antibody (mAb 70-21), which recognized all DV serotypes without neutralizing activity, were generated and demonstrated...

  16. Purification and identification of Haemophilus ducreyi cytotoxin by use of a neutralizing monoclonal antibody.

    OpenAIRE

    Purvén, M; Frisk, A.; Lönnroth, I.; Lagergard, T

    1997-01-01

    Haemophilus ducreyi produces a cytotoxin responsible for the killing of cultured human epithelial cells. Cytotoxin-neutralizing antibodies were detected in the majority of sera from patients with culture-proven chancroid, and a significantly higher level of such antibodies in patients than in blood donors was noted both in areas where the disease is endemic and those where it is not. We produced neutralizing monoclonal antibodies (MAbs) in mice with a crude osmotic preparation of the cytotoxi...

  17. Overloading ion-exchange membranes as a purification step for monoclonal antibodies

    OpenAIRE

    Brown, Arick; Bill, Jerome; Tully, Timothy; Radhamohan, Asha; Dowd, Chris

    2010-01-01

    The present study examined the overloading of ion-exchange membrane adsorbers, a form of frontal chromatography, as the final purification step in the production of mAbs (monoclonal antibodies) produced from CHO (Chinese-hamster ovary) cells. Preferential binding of impurities over antibody product was exploited using commercially available cation- and anion-exchange membranes. Three different antibody feedstreams previously purified over Protein A and ion-exchange column chromatography were ...

  18. Prevention of herpes simplex virus induced stromal keratitis by a glycoprotein B-specific monoclonal antibody.

    Directory of Open Access Journals (Sweden)

    Adalbert Krawczyk

    Full Text Available The increasing incidence of acyclovir (ACV and multidrug-resistant strains in patients with corneal HSV-1 infections leading to Herpetic Stromal Keratitis (HSK is a major health problem in industrialized countries and often results in blindness. To overcome this obstacle, we have previously developed an HSV-gB-specific monoclonal antibody (mAb 2c that proved to be highly protective in immunodeficient NOD/SCID-mice towards genital infections. In the present study, we examined the effectivity of mAb 2c in preventing the immunopathological disease HSK in the HSK BALB/c mouse model. Therefore, mice were inoculated with HSV-1 strain KOS on the scarified cornea to induce HSK and subsequently either systemically or topically treated with mAb 2c. Systemic treatment was performed by intravenous administration of mAb 2c 24 h prior to infection (pre-exposure prophylaxis or 24, 40, and 56 hours after infection (post-exposure immunotherapy. Topical treatment was performed by periodical inoculations (5 times per day of antibody-containing eye drops as control, starting at 24 h post infection. Systemic antibody treatment markedly reduced viral loads at the site of infection and completely protected mice from developing HSK. The administration of the antiviral antibody prior or post infection was equally effective. Topical treatment had no improving effect on the severity of HSK. In conclusion, our data demonstrate that mAb 2c proved to be an excellent drug for the treatment of corneal HSV-infections and for prevention of HSK and blindness. Moreover, the humanized counterpart (mAb hu2c was equally effective in protecting mice from HSV-induced HSK when compared to the parental mouse antibody. These results warrant the future development of this antibody as a novel approach for the treatment of corneal HSV-infections in humans.

  19. Antibody Positron Emission Tomography Imaging in Anticancer Drug Development

    NARCIS (Netherlands)

    Lamberts, Laetitia E.; Williams, Simon P.; Terwisscha Van Scheltinga, Anton; Lub-de Hooge, Marjolijn N.; Schroeder, Carolien P.; Gietema, Jourik A.; Brouwers, Adrienne H.; de Vries, Elisabeth G. E.

    2015-01-01

    More than 50 monoclonal antibodies (mAbs), including several antibody-drug conjugates, are in advanced clinical development, forming an important part of the many molecularly targeted anticancer therapeutics currently in development. Drug development is a relatively slow and expensive process,

  20. Modified cytokeratins expressed on the surface of carcinoma cells undergo endocytosis upon binding of human monoclonal antibody and its recombinant Fab fragment

    DEFF Research Database (Denmark)

    Ditzel, H J; Garrigues, U; Andersen, C B

    1997-01-01

    Previously, we have reported on successful imaging of colon, rectal, and pancreatic carcinomas in patients by using a radiolabeled all-human monoclonal antibody, COU-1, directed against modified cytokeratin. To further develop this antibody for use as an immunoconjugate, COU-1 was cloned by phage...

  1. Generation of monoclonal antibodies specific for cell surface molecules expressed on early mouse endoderm.

    Science.gov (United States)

    Gadue, Paul; Gouon-Evans, Valerie; Cheng, Xin; Wandzioch, Ewa; Zaret, Kenneth S; Grompe, Markus; Streeter, Philip R; Keller, Gordon M

    2009-09-01

    The development of functional cell populations such as hepatocytes and pancreatic beta cells from embryonic stem cell (ESC) is dependent on the efficient induction of definitive endoderm early in the differentiation process. To monitor definitive endoderm formation in mouse ESC differentiation cultures in a quantitative fashion, we generated a reporter cell line that expresses human CD25 from the Foxa3 locus and human CD4 from the Foxa2 locus. Induction of these reporter ESCs with high concentrations of activin A led to the development of a CD25-Foxa3+CD4-Foxa2+ population within 4-5 days of culture. Isolation and characterization of this population showed that it consists predominantly of definitive endoderm that is able to undergo hepatic specification under the appropriate conditions. To develop reagents that can be used for studies on endoderm development from unmanipulated ESCs, from induced pluripotent stem cells, and from the mouse embryo, we generated monoclonal antibodies against the CD25-Foxa3+CD4-Foxa2+ population. With this approach, we identified two antibodies that react specifically with endoderm from ESC cultures and from the early embryo. The specificity of these antibodies enables one to quantitatively monitor endoderm development in ESC differentiation cultures, to study endoderm formation in the embryo, and to isolate pure populations of culture- or embryo-derived endodermal cells.

  2. Radioimmunoassay of bovine, ovine and porcine luteinizing hormone with a monoclonal antibody and a human tracer

    Energy Technology Data Exchange (ETDEWEB)

    Fosberg, M.; Tagle, R.; Madej, A.; Molina, J.R.; Carlsson, M.-A.

    1993-01-01

    A radioimmunoassay for bovine (bLH), ovine (oLH) and porcine (pLH) luteinizing hormone was developed using a human [sup 125]ILH tracer from a commercial kit and a monoclonal antibody (518B7) specific for LH but with low species specificity. Standard curves demonstrated similar binding kinetics when bLH, oLH and pLH were incubated with tracer and antibody for 2 h at room temperature. A 30-min delay in the addition of the tracer gave sufficient sensitivity when analysing pLH. Separation of antibody-bound LH from free hormone was achieved by using second antibody-coated micro Sepharose beads. The assay was validated and the performance compared with that of an RIA currently in use for determination of bLH (coefficient of correlation: 0.99 and 0.98). Regardless of the standards used, intra-assay coefficients of variation were <10% for LH concentrations exceeding 1 [mu]g/L. The inter-assay coefficients of variation were <15%. The assay was used for clinical evaluation demonstrating the pre-ovulatory LH surge in two cyclic cows, LH pulsatility in an oophorectomized ewe and LH response to GnRH injection in a boar. (au) (7 refs.).

  3. Treatment with FGFR2-IIIc monoclonal antibody suppresses weight gain and adiposity in KKAy mice

    Science.gov (United States)

    Nonogaki, K; Kaji, T; Yamazaki, T; Murakami, Mari

    2016-01-01

    Expression of β-Kotho, fibroblast growth factor receptor (FGFR)-1c and 2c, which bind FGF21, is decreased in the white adipose tissue of obese mice. The aim of the present study was to determine the role of FGFR2c in the development of obesity and diabetes in KKAy mice. Treatment with mouse monoclonal FGFR2-IIIc antibody (0.5 mg kg−1) significantly suppressed body weight gain and epididymal white adipose tissue weight in individually housed KKAy mice while having no effect on daily food intake. In addition, treatment with FGFR2-IIIc antibody significantly increased plasma-free fatty acid levels while having no effect on blood glucose or plasma FGF21 levels. Moreover, treatment with FGFR2-IIIc antibody had no significant effect on the expression of uncoupling protein-1, uncoupling protein-2 or peroxisome proliferator-activated receptor-γ coactivator 1α in the epididymal white adipose tissue. The treatment with FGFR2-IIIc antibody had no significant effects on daily food intake and body weight gain in individually housed KK mice. These findings suggest that FGFR2-IIIc upregulates the adiposity induced by social isolation in KKAy mice, and that decreased expression and/or function of FGFR2c might be a compensatory response to enhanced adiposity. Inhibition of FGFR2-IIIc function might be a novel therapeutic approach for obesity. PMID:27892934

  4. Pathogenic and Nonpathogenic Strains of Entamoeba histolytica can be Differentiated by Monoclonal Antibodies to the Galactose-Specific Adherence Lectin

    Science.gov (United States)

    1991-04-01

    AD- A235 913 DEVELOPMENT Ei ENGINEERING CENTER CRDEC-TR-268 PATHOGENIC AND NONPATHOGENIC STRAINS OF ENTAMOEBA HISTOLYTICA CAN BE DIFFERENTIATED BY...Pathogenic and Nonpathogenic Strains of Entamoeba Histolytica can be Differentiated by Monoclonal PR-IFJlX2XXRPEW Antibodies to the Galactose-Specific...galactose lectin produced by Entamoeba histolytica provide the basis for development of a model system for the environmental detection of adherence and

  5. Development of β-lactoglobulin-specific chimeric human IgEκ monoclonal antibodies for in vitro safety assessment of whey hydrolysates.

    Directory of Open Access Journals (Sweden)

    Karen Knipping

    Full Text Available Cow's milk-derived whey hydrolysates are nutritional substitutes for allergic infants. Safety or residual allergenicity assessment of these whey hydrolysates is crucial. Currently, rat basophilic leukemia RBL-2H3 cells expressing the human IgE receptor α-chain (huFcεRIα-RBL-2H3, sensitized with serum IgE from cow's milk allergic children, are being employed to assess in vitro residual allergenicity of these whey hydrolysates. However, limited availability and inter-lot variation of these allergic sera impede standardization of whey hydrolysate safety testing in degranulation assays.An oligoclonal pool of chimeric human (chuIgE antibodies against bovine β-lactoglobulin (a major allergen in whey was generated to increase sensitivity, specificity, and reproducibility of existing degranulation assays.Mice were immunized with bovine β-lactoglobulin, and subsequently the variable domains of dissimilar anti-β-lactoglobulin mouse IgG antibodies were cloned and sequenced. Six chimeric antibodies were generated comprising mouse variable domains and human constant IgE/κ domains.After sensitization with this pool of anti-β-lactoglobulin chuIgEs, huFcεRIα-expressing RBL-2H3 cells demonstrated degranulation upon cross-linking with whey, native 18 kDa β-lactoglobulin, and 5-10 kDa whey hydrolysates, whereas a 3 kDa whey hydrolysate and cow's milk powder (mainly casein showed no degranulation. In parallel, allergic serum IgEs were less sensitive. In addition, our pool anti-β-lactoglobulin chuIgEs recognized multiple allergenic immunodominant regions on β-lactoglobulin, which were also recognized by serum IgEs from cow's milk allergic children.Usage of our 'unlimited' source and well-defined pool of β-lactoglobulin-specific recombinant chuIgEs to sensitize huFcεRIα on RBL-2H3 cells showed to be a relevant and sensitive alternative for serum IgEs from cow's milk allergic patients to assess safety of whey-based non-allergic hydrolyzed formula.

  6. Characterising the association of latency with α1-antitrypsin polymerisation using a novel monoclonal antibody

    Science.gov (United States)

    Tan, Lu; Perez, Juan; Mela, Marianna; Miranda, Elena; Burling, Keith A; Rouhani, Farshid N; DeMeo, Dawn L; Haq, Imran; Irving, James A; Ordóñez, Adriana; Dickens, Jennifer A; Brantly, Mark; Marciniak, Stefan J; Alexander, Graeme J M; Gooptu, Bibek; Lomas, David A

    2015-01-01

    α1-Antitrypsin is primarily synthesised in the liver, circulates to the lung and protects pulmonary tissues from proteolytic damage. The Z mutant (Glu342Lys) undergoes inactivating conformational change and polymerises. Polymers are retained within the hepatocyte endoplasmic reticulum (ER) in homozygous (PiZZ) individuals, predisposing the individuals to hepatic cirrhosis and emphysema. Latency is an analogous process of inactivating, intra-molecular conformational change and may co-occur with polymerisation. However, the relationship between latency and polymerisation remained unexplored in the absence of a suitable probe. We have developed a novel monoclonal antibody specific for latent α1-antitrypsin and used it in combination with a polymer-specific antibody, to assess the association of both conformers in vitro, in disease and during augmentation therapy. In vitro kinetics analysis showed polymerisation dominated the pathway but latency could be promoted by stabilising monomeric α1-antitrypsin. Polymers were extensively produced in hepatocytes and a cell line expressing Z α1-antitrypsin but the latent protein was not detected despite manipulation of the secretory pathway. However, α1-antitrypsin augmentation therapy contains latent α1-antitrypsin, as did the plasma of 63/274 PiZZ individuals treated with augmentation therapy but 0/264 who were not receiving this medication (p < 10−14). We conclude that latent α1-antitrypsin is a by-product of the polymerisation pathway, that the intracellular folding environment is resistant to formation of the latent conformer but that augmentation therapy introduces latent α1-antitrypsin into the circulation. A suite of monoclonal antibodies and methodologies developed in this study can characterise α1-antitrypsin folding and conformational transitions, and screen methods to improve augmentation therapy. PMID:25462157

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

    Directory of Open Access Journals (Sweden)

    Noda M

    2012-11-01

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

  8. Boronated monoclonal antibody 225. 28S for potential use in neutron capture therapy of malignant melanoma

    Energy Technology Data Exchange (ETDEWEB)

    Tamat, S.R.; Moore, D.E.; Patwardhan, A.; Hersey, P. (Univ. of Sydney (Australia))

    1989-07-01

    The concept of conjugating boron cluster compounds to monoclonal antibodies has been examined by several groups of research workers in boron neutron capture therapy (BNCT). The procedures reported to date for boronation of monoclonal antibodies resulted in either an inadequate level of boron incorporation, the precipitation of the conjugates, or a loss of immunological activity. The present report describes the conjugation of dicesium-mercapto-undecahydrododecaborate (Cs2B12H11SH) to 225.28S monoclonal antibody directed against high molecular weight melanoma-associated antigens (HMW-MAA), using poly-L-ornithine as a bridge to increase the carrying capacity of the antibody and to minimize change in the conformational structure of antibody. The method produces a boron content of 1,300 to 1,700 B atoms per molecule 225.28S while retaining the immunoreactivity. Characterization in terms of the homogeneity of the conjugation of the boron-monoclonal antibody conjugates has been studied by gel electrophoresis and ion-exchange HPLC.

  9. Production and Purification of Monoclonal Antibody Against Tumor Marker of TPA

    Directory of Open Access Journals (Sweden)

    Seyyed Amir Abbas Ghodrat

    2016-05-01

    Full Text Available Considering the invasive nature of cancer cells, one of the most important and best indicator of them is the markers inside them. One of the most important markers that observed in some types of cancer cells in various parts of the body is the Cytokeratin. Tissue plasminogen activator antigen (TPA is a Cytokeratin composed of molecules with various molecular weights. The level of TPA serum as associated with cellular growth level and tumorization of cells. In this research, the hybrid of spleen cells in BALB/c female mouse with myeloma cells was conducted with a ratio of 10:1. The resulting monoclonal antibodies were confirmed by SDS-PAGE and western blot. Protein G chromatography was utilized to purify monoclonal antibodies. The results for determining isotypes showed IgM and IgG classes. The titer of the antibody obtained from various clones was capable of identifying Cytokeratin antigen with a dilution of 1/10000. The resulting antibodies were finally confirmed by western blot and all the 5 resulting monoclonal antibodies were capable of identifying a 48 kDa protein. The results indicate that with the help of TPA marker and the monoclonal antibodies produced against them, this marker can be recognized quickly with great accuracy in suspicious cases of cancer. Thus, appropriate measures will be taken to prevent and fight off its probable side effects. This factor can be further used to build a diagonal kit with high sensitivity.

  10. Production and characterization of monoclonal antibodies to the edta extract of Leptospira interrogans, serovar icterohaemorrhagiae

    Directory of Open Access Journals (Sweden)

    Lilian Terezinha de Queiroz Leite

    1996-10-01

    Full Text Available Monoclonal antibodies (MABs ivere produced against an etbylenediaminetetraacetate (EDTA extract of Leptospira interrogans serovar icterohaemorrhagiae being characterized by gel precipitation as IgM and IgG (IgGl and IgG2b. The EDTA extract was detected as several bands by silver staining in SDS-PAGE. In the Western blot the bands around 20 KDa reacted with a monoclonal antibody, 47B4D6, and was oxidized by periodate and was not digested by pronase, suggesting that the determinant is of carbohydrate nature, lmmunocytochemistry, using colloidal gold labeling, showed that an EDTA extract determinant recognized by monoclonal antibody 47B4D6, is localized under the outer envelope of serovar icterohaemorrhagiae. Hoe AIAB raised against the EDTA extract was not able to protect hamsters from lethal challenge with virulent homologous leptospires.

  11. Simultaneous Raising of Rabbit Monoclonal Antibodies to Fluoroquinolones with Diverse Recognition Functionalities via Single Mixture Immunization.

    Science.gov (United States)

    Liu, Na; Zhao, Zhiyong; Tan, Yanglan; Lu, Lei; Wang, Lin; Liao, Yucai; Beloglazova, Natalia; De Saeger, Sarah; Zheng, Xiaodong; Wu, Aibo

    2016-01-19

    Highly specific monoclonal and polyclonal antibodies are the key components in a diverse set of immunoassay applications, from research work to routine monitoring and analysis. In the current manuscript, combinatorial strategies for a single mixture immunization, screening and rabbit hybridoma cell technology were described. Fluoroquinolones (FQs) drugs were chosen as representative analytes. Six FQs were conjugated with bovine serum albumin and used as immunogens for subsequent immunization, while a mixture of all was injected for coimmunization. The hybridomas obtained against the individual and multiple FQs were used for the production of diverse varieties of rabbit monoclonal antibodies (RabMAbs) against the target analytes. As was proven by indirect competitive ELISA and quantitative lateral flow immunoassay, this approach opens a new way for simultaneously obtaining functional monoclonal antibodies which are capable of recognizing both individual and multiple analytes in a single preparation circle. This addresses various needs of different monitoring regulations as analytical methodology advances.

  12. Reactivity of alveolar epithelial cells in primary culture with type I cell monoclonal antibodies.

    Science.gov (United States)

    Danto, S I; Zabski, S M; Crandall, E D

    1992-03-01

    An understanding of the process of alveolar epithelial cell growth and differentiation requires the ability to trace and analyze the phenotypic transitions that the cells undergo. This analysis demands specific phenotypic probes to type II and, especially, type I pneumocytes. To this end, monoclonal antibodies have been generated to type I alveolar epithelial cells using an approach designed to enhance production of lung-specific clones from a crude lung membrane preparation. The monoclonal antibodies were screened by a combination of enzyme-linked immunosorbent assay and immunohistochemical techniques, with the determination of type I cell specificity resting primarily on immunoelectron microscopic localization. Two of these new markers of the type I pneumocyte phenotype (II F1 and VIII B2) were used to analyze primary cultures of type II cells growing on standard tissue culture plastic and on a variety of substrata reported to affect the morphology of these cells in culture. On tissue culture plastic, the antibodies fail to react with early (days 1 to 3) type II cell cultures. The cells become progressively more reactive with time in culture to a plateau of approximately 6 times background by day 8, with a maximum rate of increase between days 3 and 5. This finding is consistent with the hypothesis that type II cells in primary culture undergo at least partial differentiation into type I cells. Type II cells grown on laminin, which reportedly delays the loss of type II cell appearance, and on fibronectin, which has been reported to facilitate cell spreading and loss of type II cell features, develop the type I cell markers during cultivation in vitro with kinetics similar to those on uncoated tissue culture plastic. Cells on type I collagen and on tissue culture-treated Nuclepore filters, which have been reported to support monolayers with type I cell-like morphology, also increase their expression of the II F1 and VIII B2 epitopes around days 3 to 5. Taken

  13. Serological Differentiation of Plant-parasitic Nematode Species with Polyclonal and Monoclonal Antibodies.

    Science.gov (United States)

    Schots, A; Gommers, F J; Bakker, J; Egberts, E

    1990-01-01

    Although several attempts have been made to differentiate nematode species with polyclonal antisera, these efforts thus far have met with limited success because of extensive crossreactivities of the sera. Since the hybridoma technique offers the opportunity to develop more specific serological reagents, some research groups have recently started to apply this technology to the problem of species identification in nematology. Monoclonal antibodies (MA) that differentiate the potato-cyst nematodes Globodera rostochiensis and G. pallida, as well as MA specific for Meloidogyne species, have been developed. The possibilities of developing serodiagnostic tools for identification of nematodes recovered from soil samples and the implications of such monitoring of nematode infestations in view of integrated control of plant-parasitic nematodes are discussed.

  14. Precipitating and non-precipitating monoclonal antibodies against chicken avidin. Significance of epitope density.

    Science.gov (United States)

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

    1987-01-01

    1. Monoclonal antibodies, generated against chicken avidin, were characterized in Ouchterlony's immunodiffusion. 2. Of the nine antibodies three were non-precipitable but six could form clear visible precipitation lines with egg-white avidin in agarose gel. 3. The latter six antibodies could be divided into two groups according to their reactive pattern in immunodiffusion. 4. Antibodies belonging to the first group precipitated both dimeric as well as tetrameric avidin molecules, while those of the second group precipitated only the tetrameric avidin molecules. 5. The relevance of these results to the structure of avidin as well as possibilities to use monoclonal antibodies and the immunodiffusion technique to compare the structure of avidin induced by different factors are discussed.

  15. Epitope Mapping of Dengue-Virus-Enhancing Monoclonal-Antibody Using Phage Display Peptide Library

    Directory of Open Access Journals (Sweden)

    Chung-I Rai

    2008-01-01

    Full Text Available The Antibody-Dependent Enhancement (ADE hypothesis has been proposed to explain why more severe manifestations of Dengue Hemorrhagic Fever and Dengue Shock Syndrome (DHF/DSS occur predominantly during secondary infections of Dengue Virus (DV with different serotypes. However, the epitopes recognized by these enhancing antibodies are unclear. Recently, anti-pre-M monoclonal antibody (mAb 70-21, which recognized all DV serotypes without neutralizing activity, were generated and demonstrated as an enhancing antibody for DV infection. In the present study, the epitope recognized by mAb 70-21 was identified using a phage-displayed random-peptide library. After three rounds of biopanning, ELISA showed that immunopositive phage clones specifically bound to mAb 70-21 but not to serum or purified IgG from naive mice. DNA sequencing of these phage clones showed a consensus sequence, QNNLGPR. Like mAb70-21, these phage-induced antisera also enhanced the DV infection of cells. In addition, indirect fluorescent assays showed phage-induced antisera bound to human rhabdomyosarcoma or Vero cells. Western blotting and immunoprecipitation analysis showed that phage-induced antisera recognized hsp 60 in BHK cell lysate. Moreover, the sera levels of antibodies against the synthetic peptide QNNLGPR correlated with the disease severity of dengue patients. Taken together, these results suggest that antibodies which recognized epitopes shared by pre-M of DV and hsp 60 of host cells may enhance DV infection and be involved in the development of DHF or DSS.

  16. Screening a hybridoma producing a specific monoclonal antibody to HLA-A24+Bw4 antigen by cytotoxicity inhibition assay.

    Science.gov (United States)

    Hiroishi, S; Kaneko, T; Arita, J

    1987-02-01

    A hybridoma secreting a monoclonal antibody (Tsa-1, IgG3) reacting specifically to HLA-A24+Bw4 was screened by cytotoxicity inhibition assay and micrototoxicity test. The R value of the antibody was 0.843.

  17. Comparison of SEC and CE-SDS methods for monitoring hinge fragmentation in IgG1 monoclonal antibodies.

    Science.gov (United States)

    Dada, Oluwatosin O; Rao, Romesh; Jones, Natalie; Jaya, Nomalie; Salas-Solano, Oscar

    2017-10-25

    Fragmentation of monoclonal antibodies is a critical quality attribute routinely monitored to assess the purity and integrity of the product from development to commercialization. Cleavage in the upper hinge region of IgG1 monoclonal antibodies is a common fragmentation pattern widely studied by size exclusion chromatography (SEC). Capillary electrophoresis with sodium dodecylsulfate (CE-SDS) is a well-established technique commonly used for monitoring antibody fragments as well, but its comparability to SEC in monitoring hinge fragments has not been established until now. We report a characterization strategy that establishes the correlation between hinge region fragments analyzed by SEC and CE-SDS. Monoclonal antibodies with elevated hinge fragments were generated under low pH stress conditions and analyzed by SEC and CE-SDS. The masses of the fragments generated were determined by LC-MS. Electrophoretic migration of the hinge fragmentation products in CE-SDS were determined based on their mass values. Comparative assessment of fragments by SEC, and CE-SDS showed similar correlation with incubation time. This study demonstrates that CE-SDS can be employed as a surrogate technique to SEC for monitoring hinge region fragments. Most importantly, combination of these techniques can be used to obtain comprehensive understanding of fragment related characteristics of therapeutic protein products. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. A novel universal neutralizing monoclonal antibody against enterovirus 71 that targets the highly conserved "knob" region of VP3 protein.

    Directory of Open Access Journals (Sweden)

    Tanja K Kiener

    Full Text Available Hand, foot and mouth disease caused by enterovirus 71(EV71 leads to the majority of neurological complications and death in young children. While putative inactivated vaccines are only now undergoing clinical trials, no specific treatment options exist yet. Ideally, EV71 specific intravenous immunoglobulins could be developed for targeted treatment of severe cases. To date, only a single universally neutralizing monoclonal antibody against a conserved linear epitope of VP1 has been identified. Other enteroviruses have been shown to possess major conformational neutralizing epitopes on both the VP2 and VP3 capsid proteins. Hence, we attempted to isolate such neutralizing antibodies against conformational epitopes for their potential in the treatment of infection as well as differential diagnosis and vaccine optimization. Here we describe a universal neutralizing monoclonal antibody that recognizes a conserved conformational epitope of EV71 which was mapped using escape mutants. Eight escape mutants from different subgenogroups (A, B2, B4, C2, C4 were rescued; they harbored three essential mutations either at amino acid positions 59, 62 or 67 of the VP3 protein which are all situated in the "knob" region. The escape mutant phenotype could be mimicked by incorporating these mutations into reverse genetically engineered viruses showing that P59L, A62D, A62P and E67D abolish both monoclonal antibody binding and neutralization activity. This is the first conformational neutralization epitope mapped on VP3 for EV71.

  19. Combined use of anti-ErbB monoclonal antibodies and erlotinib enhances antibody-dependent cellular cytotoxicity of wild-type erlotinib-sensitive NSCLC cell lines

    Directory of Open Access Journals (Sweden)

    Cavazzoni Andrea

    2012-12-01

    Full Text Available Abstract Background The epidermal growth factor receptor (EGFR is an established target for anti-cancer treatment in different tumour types. Two different strategies have been explored to inhibit this pivotal molecule in epithelial cancer development: small molecules TKIs and monoclonal antibodies. ErbB/HER-targeting by monoclonal antibodies such as cetuximab and trastuzumab or tyrosine-kinase inhibitors as gefitinib or erlotinib has been proven effective in the treatment of advanced NSCLC. Results In this study we explored the potential of combining either erlotinib with cetuximab or trastuzumab to improve the efficacy of EGFR targeted therapy in EGFR wild-type NSCLC cell lines. Erlotinib treatment was observed to increase EGFR and/or HER2 expression at the plasma membrane level only in NSCLC cell lines sensitive to the drug inducing protein stabilization. The combined treatment had marginal effect on cell proliferation but markedly increased antibody-dependent, NK mediated, cytotoxicity in vitro. Moreover, in the Calu-3 xenograft model, the combination significantly inhibited tumour growth when compared with erlotinib and cetuximab alone. Conclusion Our results indicate that erlotinib increases surface expression of EGFR and/or HER2 only in EGFR-TKI sensitive NSCLC cell lines and, in turns, leads to increased susceptibility to ADCC both in vitro and in a xenograft models. The combination of erlotinib with monoclonal antibodies represents a potential strategy to improve the treatment of wild-type EGFR NSCLC patients sensitive to erlotinib.

  20. PURIFICATION OF MONOCLONAL ANTIBODY 3H11 AGAINST GASTRIC CANCER FOR IN VIVO USE

    Institute of Scientific and Technical Information of China (English)

    LI Zhen-fu; ZHANG Hong; NIU Yong-ge

    1999-01-01

    Monoclonal antibody (McAb) 3H11 against gastric cancer was grown in the mouse ascites system. To acquire a clinical grade product for cancer radioimmuno-imaging was purified by two step high performance liquid chromatography (HPLC) protocol using protein A and high-performance hydroxylapatite (HPHT). An analysis of data reported shows the two step HPLC method to be the best purification procedure. This protocol satisfies purity and immunoreactivity requirement, and provides an sample sterility,free-pyrogens, free-mycoplasma and non-specific IgG contamination. This procedure described was capable of generating large amounts of clinical grade monoclonal antibody.

  1. Ramucirumab (IMC-1121B): Monoclonal antibody inhibition of vascular endothelial growth factor receptor-2.

    Science.gov (United States)

    Spratlin, Jennifer

    2011-04-01

    Angiogenesis, a well-recognized characteristic of malignancy, has been exploited more than any other pathway targeted by biologic anti-neoplastic therapies. Vascular endothelial growth factor receptor-2 (VEGFR-2) is the critical receptor involved in malignant angiogenesis with its activation inducing a number of other cellular modifications resulting in tumor growth and metastases. Ramucirumab (IMC-1121B; ImClone Systems Corporation, Branchburg, NJ) is a fully human monoclonal antibody developed to specifically inhibit VEGFR-2. Ramucirumab is currently being investigated in multiple clinical trials across a variety of tumor types. Herein, angiogenesis inhibition in cancer is reviewed and up-to-date information on the clinical development of ramucirumab is presented.

  2. Modeling on-column reduction of trisulfide bonds in monoclonal antibodies during protein A chromatography.

    Science.gov (United States)

    Ghose, Sanchayita; Rajshekaran, Rupshika; Labanca, Marisa; Conley, Lynn

    2017-01-06

    Trisulfides can be a common post-translational modification in many recombinant monoclonal antibodies. These are a source of product heterogeneity that add to the complexity of product characterization and hence, need to be reduced for consistent product quality. Trisulfide bonds can be converted to the regular disulfide bonds by incorporating a novel cysteine wash step during Protein A affinity chromatography. An empirical model is developed for this on-column reduction reaction to compare the reaction rates as a function of typical operating parameters such as temperature, cysteine concentration, reaction time and starting level of trisulfides. The model presented here is anticipated to assist in the development of optimal wash conditions for the Protein A step to effectively reduce trisulfides to desired levels. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Main Quality Attributes of Monoclonal Antibodies and Effect of Cell Culture Components

    Science.gov (United States)

    Torkashvand, Fatemeh; Vaziri, Behrouz

    2017-05-01

    The culture media optimization is an inevitable part of upstream process development in therapeutic monoclonal antibodies (mAbs) production. The quality by design (QbD) approach defines the assured quality of the final product through the development stage. An important step in QbD is determination of the main quality attributes. During the media optimization, some of the main quality attributes such as glycosylation pattern, charge variants, aggregates, and low-molecular-weight species, could be significantly altered. Here, we provide an overview of how cell culture medium components affects the main quality attributes of the mAbs. Knowing the relationship between the culture media components and the main quality attributes could be successfully utilized for a rational optimization of mammalian cell culture media for industrial mAbs production.

  4. Characterization of epitopes recognized by monoclonal antibodies: experimental approaches supported by freely accessible bioinformatic tools.

    Science.gov (United States)

    Clementi, Nicola; Mancini, Nicasio; Castelli, Matteo; Clementi, Massimo; Burioni, Roberto

    2013-05-01

    Monoclonal antibodies (mAbs) have been used successfully both in research and for clinical purposes. The possible use of protective mAbs directed against different microbial pathogens is currently being considered. The fine definition of the epitope recognized by a protective mAb is an important aspect to be considered for possible development in epitope-based vaccinology. The most accurate approach to this is the X-ray resolution of mAb/antigen crystal complex. Unfortunately, this approach is not always feasible. Under this perspective, several surrogate epitope mapping strategies based on the use of bioinformatics have been developed. In this article, we review the most common, freely accessible, bioinformatic tools used for epitope characterization and provide some basic examples of molecular visualization, editing and computational analysis.

  5. Broad neutralizing human monoclonal antibodies against influenza virus from vaccinated healthy donors

    Energy Technology Data Exchange (ETDEWEB)

    Kubota-Koketsu, Ritsuko; Mizuta, Hiroyuki [Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871 (Japan); Oshita, Masatoshi; Ideno, Shoji [Osaka Research Laboratory, Benesis Corporation, Yodogawa-ku, Osaka 532-6505 (Japan); Yunoki, Mikihiro [Osaka Research Laboratory, Benesis Corporation, Yodogawa-ku, Osaka 532-6505 (Japan); Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871 (Japan); Kuhara, Motoki [Ina Laboratory, Medical and Biological Laboratories Corporation, Ltd., Ina, Nagano 396-0002 (Japan); Yamamoto, Naomasa [Department of Biochemistry, School of Pharmaceutical Sciences, Ohu University, Koriyama, Fukushima 963-8611 (Japan); Okuno, Yoshinobu [Kanonji Institute, The Research Foundation for Microbial Diseases of Osaka University, Kanonji, Kagawa 768-0061 (Japan); Ikuta, Kazuyoshi, E-mail: ikuta@biken.osaka-u.ac.jp [Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871 (Japan)

    2009-09-11

    Human monoclonal antibodies (HuMAbs) prepared from patients with viral infections could provide information on human epitopes important for the development of vaccines as well as potential therapeutic applications. Through the fusion of peripheral blood mononuclear cells from a total of five influenza-vaccinated volunteers, with newly developed murine-human chimera fusion partner cells, named SPYMEG, we obtained 10 hybridoma clones stably producing anti-influenza virus antibodies: one for influenza A H1N1, four for influenza A H3N2 and five for influenza B. Surprisingly, most of the HuMAbs showed broad reactivity within subtype and four (two for H3N2 and two for B) showed broad neutralizing ability. Importantly, epitope mapping revealed that the two broad neutralizing antibodies to H3N2 derived from different donors recognized the same epitope located underneath the receptor-binding site of the hemagglutinin globular region that is highly conserved among H3N2 strains.

  6. Application of monoclonal antibody panels in the virological and epidemiological review of poliomyelitis in Poland, 1981-1990.

    OpenAIRE

    Jarzabek, Z.; Zabicka, J.; John, A.; Howlett, J.; Dunn, G.; Wood, D. J.

    1992-01-01

    Monoclonal antibody panels developed to differentiate vaccine-derived and wild-type strains of polio-viruses were applied to isolates from cases of paralytic poliomyelitis, non-paralytic poliomyelitis, and healthy excreters of poliovirus from Poland. All isolates from poliomyelitis cases were shown to be vaccine-derived, as were most other strains. However, two strains associated with meningitis had wild-type antigenic phenotypes and, as shown by partial genomic sequencing, wild-type genotype...

  7. Reduction-mediated technetium-99m labeling of monoclonal antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Mather, S.J.; Ellison, D. (St. Bartholomews Hospital, London (England))

    1990-05-01

    A simple and generally applicable method for labeling antibodies with technetium-99m ({sup 99m}Tc) is described. Following reduction of intrinsic disulphide bonds, the antibody is labeled with {sup 99m}Tc in the presence of a weak competing ligand methylene diphosphonate. High labeling efficiencies (greater than 97%), in a final labeling step taking only a few minutes, can be routinely obtained with high in-vitro stability over 24 hr. No effect upon antibody reactivity is seen.

  8. A high affinity monoclonal antibody recognizing the light chain of human coagulating factor VII.

    Science.gov (United States)

    Sarial, Sheila; Asadi, Farzad; Jeddi-Tehrani, Mahmood; Hadavi, Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Taghizadeh-Jahed, Masoud; Shokri, Fazel; Rabbani, Hodjattallah

    2012-12-01

    Factor VII (FVII) is a serine protease-coagulating element responsible for the initiation of an extrinsic pathway of clot formation. Here we generated and characterized a high affinity monoclonal antibody that specifically recognizes human FVII. Recombinant human FVII (rh-FVII) was used for the production of a monoclonal antibody using BALB/c mice. The specificity of the antibody was determined by Western blot using plasma samples from human, mouse, sheep, goat, bovine, rabbit, and rat. Furthermore, the antibody was used to detect transiently expressed rh-FVII in BHK21 cell line using Western blot and sandwich ELISA. A mouse IgG1 (kappa chain) monoclonal antibody clone 1F1-B11 was produced against rh-FVII. The affinity constant (K(aff)) of the antibody was calculated to be 6.4×10(10) M(-1). The antibody could specifically recognize an epitope on the light chain of hFVII, with no reactivity with factor VII from several other animals. In addition, transiently expressed rh-FVII in BHK21 cells was recognized by 1F1-B11. The high affinity as well as the specificity of 1F1-B11 for hFVII will facilitate the affinity purification of hFVII and also production of FVII deficient plasma and minimizes the risk of bovine FVII contamination when fetal bovine serum-supplemented media are used for production and subsequent purification of rh-FVII.

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

    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.

  11. Target-selective homologous recombination cloning for high-throughput generation of monoclonal antibodies from single plasma cells

    Directory of Open Access Journals (Sweden)

    Isobe Masaharu

    2011-04-01

    Full Text Available Abstract Background Molecular cloning of functional immunoglobulin genes from single plasma cells is one of the most promising technologies for the rapid development of monoclonal antibody drugs. However, the proper insertion of PCR-amplified immunoglobulin genes into expression vectors remains an obstacle to the high-throughput production of recombinant monoclonal antibodies. Results We developed a single-step cloning method, target-selective homologous recombination (TS-HR, in which PCR-amplified immunoglobulin variable genes were selectively inserted into vectors, even in the presence of nonspecifically amplified DNA. TS-HR utilizes Red/ET-mediated homologous recombination with a target-selective vector (TS-vector with unique homology arms on its termini. Using TS-HR, immunoglobulin variable genes were cloned directly into expression vectors by co-transforming unpurified PCR products and the TS-vector into E. coli. Furthermore, the high cloning specificity of TS-HR allowed plasmids to be extracted from pools of transformed bacteria without screening single colonies for correct clones. We present a one-week protocol for the production of recombinant mouse monoclonal antibodies from large numbers of single plasma cells. Conclusion The time requirements and limitations of traditional cloning procedures for the production of recombinant immunoglobulins have been significantly reduced with the development of the TS-HR cloning technique.

  12. Characterization of Two Human Monoclonal Antibodies Neutralizing Influenza A H7N9 Viruses

    Science.gov (United States)

    Wang, Jianmin; Chen, Zhe; Bao, Linlin; Zhang, Weijia; Xue, Ying; Pang, XingHuo; Zhang, Xi

    2015-01-01

    H7N9 was a cause of significant global health concern due to its severe infection and approximately 35% mortality in humans. By screening a Fab antibody phage library derived from patients who recovered from H7N9 infections, we characterized two human monoclonal antibodies (HuMAbs), HNIgGD5 and HNIgGH8. The epitope of these two antibodies was dependent on two residues in the receptor binding site at positions V186 and L226 of the hemagglutinin glycoprotein. Both antibodies possessed high neutralizing activity. PMID:26063436

  13. Anti-TNP monoclonal antibodies as reagents for enzyme immunoassay (ELISA)

    OpenAIRE

    Leo, P; P. Ucelli; Augusto, EFP; Oliveira,MS; Tamashiro, WMSC

    2000-01-01

    The aim of this study was to produce anti-TNP monoclonal antibodies (MAbs) that could be conjugated and used for the detection of antigen-antibody reactions, in which the antigen specific-antibody had been previously bound to trinitrophenyl (TNP). For hybridoma production, SP2/0-Ag14 cells were fused with spleen cells from mice previously immunized with TNP-ovalbumin (TNP-OVA). After 10 days, enzyme-linked immunoadsorbent assay (ELISA) was used to detect anti-TNP antibodies in the supernatant...

  14. T10B9 monoclonal antibody: A short-acting nonstimulating monoclonal antibody that spares γδ T-cells and treats and prevents cellular rejection

    Directory of Open Access Journals (Sweden)

    Thomas H Waid

    2009-06-01

    Full Text Available Thomas H Waid1, John S Thompson1, Maria Siemionow2, Stephen A Brown1 1Department of Internal Medicine, University of Kentucky, Lexington, Kentucky, USA; 2Cleveland Clinic, Cleveland, Ohio, USAAbstract: T10B9.1A-31/MEDI-500 is a nonmitogenic immunoglobulin M kappa murine monoclonal antibody (mAb directed against the alpha-beta (αβ heterodimer of the T-lymphocyte receptor complex. The hybridoma was first produced by fusing spleen cells from BALB/C mice immunized with human peripheral blood T-lymphocytes with SP2/O-Ag14 mutant myeloma cells. The mAb is produced and purified using multistep ion exchange and molecular sieve chromatography protocols. T10B9 has been used successfully to treat acute cellular rejection in renal transplantation and as an immunosuppression induction agent in heart and simultaneous kidney-pancreas transplantation. Because T10B9 is nonmitogenic and causes minimal cytokine release, both treatment of rejection and induction of immunosuppression were accomplished with significantly fewer and milder untoward effects (cytokine release syndrome than its comparator OKT3. Since T10B9 is directed against the αβ heterodimer of the CD3 epitope, it spares the gamma delta (γδ region. These gamma delta (γδ T cells have a unique role in the immune response controlling many serious human diseases and perhaps facilitating the development of immunologic tolerance. T10B9 has a relatively short duration of action, depleting T cells for only 10 to 14 days, unlike the protracted depletion seen with thymoglobulin and Campath-1H. There is no B-lymphocyte depletion with T10B9 as there is with both of the aforementioned reagents. The lack of prolonged lymphocyte depletion may account for less infection observed with T10B9 treatment.Keywords: T10B9.1A-31, γδ T-cell, monoclonal antibody, Campath-1H, thymoglobulin, OKT3

  15. Computational prediction of neutralization epitopes targeted by human anti-V3 HIV monoclonal antibodies.

    Directory of Open Access Journals (Sweden)

    Evgeny Shmelkov

    Full Text Available The extreme diversity of HIV-1 strains presents a formidable challenge for HIV-1 vaccine design. Although antibodies (Abs can neutralize HIV-1 and potentially protect against infection, antibodies that target the immunogenic viral surface protein gp120 have widely variable and poorly predictable cross-strain reactivity. Here, we developed a novel computational approach, the Method of Dynamic Epitopes, for identification of neutralization epitopes targeted by anti-HIV-1 monoclonal antibodies (mAbs. Our data demonstrate that this approach, based purely on calculated energetics and 3D structural information, accurately predicts the presence of neutralization epitopes targeted by V3-specific mAbs 2219 and 447-52D in any HIV-1 strain. The method was used to calculate the range of conservation of these specific epitopes across all circulating HIV-1 viruses. Accurately identifying an Ab-targeted neutralization epitope in a virus by computational means enables easy prediction of the breadth of reactivity of specific mAbs across the diversity of thousands of different circulating HIV-1 variants and facilitates rational design and selection of immunogens mimicking specific mAb-targeted epitopes in a multivalent HIV-1 vaccine. The defined epitopes can also be used for the purpose of epitope-specific analyses of breakthrough sequences recorded in vaccine clinical trials. Thus, our study is a prototype for a valuable tool for rational HIV-1 vaccine design.

  16. A Rapid and Simple Approach to Preparation of Monoclonal Antibody Based on DNA Immunization

    Institute of Scientific and Technical Information of China (English)

    YiNi; KeMa; JingNi; XiujuanZheng; YingWang; SidongXiong

    2004-01-01

    Inoculation with purified specific protein is usually the first step for preparation of monoclonal antibody (mAb). But it is quite difficult to obtain pure proteins especially with natural structures. Here we attempt to replace the protein inoculation with DNA immunization in the preparation of mAb. The eukaryotic expression vectors pcDNA3-PreS2/S and pVAX-PreS2/S encoding the HBV M protein were constructed and prepared for DNA immunization. Female BALB/c mice developed a well antibody response to the target antigen after muscle injection with corresponding plasmids. The mice with effective antibodies induced were used for preparation of mAb. We found the mice immunized with three administrations of pcDNA3-PreS2/S and boosted by intrasplenic injection with the same plasmid could be exploited for preparation of mAb. And positive hybridoma cell 2D3 that can secrete specific mAb was cloned and analyzed. Our studies demonstrate that gene immunization may provide a convenient and efficient way to prepare mAbs. Cellular & Molecular Immunology.

  17. A Rapid and Simple Approach to Preparation of Monoclonal Antibody Based on DNA Immunization

    Institute of Scientific and Technical Information of China (English)

    Yi Ni; Ke Ma; Jing Ni; Xiujuan Zheng; Ying Wang; Sidong Xiong

    2004-01-01

    Inoculation with purified specific protein is usually the first step for preparation of monoclonal antibody (mAb).But it is quite difficult to obtain pure proteins especially with natural structures. Here we attempt to replace the protein inoculation with DNA immunization in the preparation of mAb. The eukaryotic expression vectors pcDNA3-PreS2/S and pVAX-PreS2/S encoding the HBV M protein were constructed and prepared for DNA immunization. Female BALB/c mice developed a well antibody response to the target antigen after muscle injection with corresponding plasmids. The mice with effective antibodies induced were used for preparation of mAb. We found the mice immunized with three administrations of pcDNA3-PreS2/S and boosted by intrasplenic injection with the same plasmid could be exploited for preparation of mAb. And positive hybridoma cell 2D3 that can secrete specific mAb was cloned and analyzed. Our studies demonstrate that gene immunization may provide a convenient and efficient way to prepare mAbs.

  18. Clarification technologies for monoclonal antibody manufacturing processes: Current state and future perspectives.

    Science.gov (United States)

    Singh, Nripen; Arunkumar, Abhiram; Chollangi, Srinivas; Tan, Zhijun George; Borys, Michael; Li, Zheng Jian

    2016-04-01

    Considerable progress has been made increasing productivity of cell cultures to meet the rapidly growing demand for antibody biopharmaceuticals through increased cell densities and longer culture times. This in turn has dramatically increased the burden of process and product related impurities on the purification processes. In addition, current trends in the biopharmaceutical industry point toward both increased productivity and targeting smaller patient populations for new indications. Taken together, these developments are driving the industry to explore alternative separation technologies as a future manufacturing strategy. Clarification technologies well established in other industries, such as flocculation and precipitation are increasingly considered as a viable solution to address this bottleneck in antibody processes. However, several technical issues need to be fully addressed including suitability as a platform application, robustness, process cost, toxicity, and clearance. This review will focus on recent efforts to incorporate new generation clarification technologies for mammalian cell cultures producing monoclonal antibodies as well as challenges to their implementation supported by a case study. © 2015 Wiley Periodicals, Inc.

  19. beta-Adrenergic agonist activity of a monoclonal anti-idiotypic antibody.

    Science.gov (United States)

    Guillet, J G; Kaveri, S V; Durieu, O; Delavier, C; Hoebeke, J; Strosberg, A D

    1985-03-01

    Hybridoma cells bearing monoclonal antibody against the beta-adrenergic ligand alprenolol were used as an immunogen to raise monoclonal anti-idiotypic antibodies. Of six anti-idiotypic antibodies, which inhibit ligand binding, three were able to recognize beta-adrenergic receptors. One of them, mAb2B4, an IgM that could be amplified into ascites, binds to the beta-adrenergic catecholamine receptors of intact epidermoid A431 cells and precipitates receptors solubilized from plasma membranes by digitonin. This antibody identifies the beta 2-adrenergic receptor of A431 cells as a single 55-kDa protein and stimulates adenylate cyclase activity. This stimulation is inhibited by the beta-adrenergic antagonist propranolol.

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

    Science.gov (United States)

    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.

  1. Chromatographic separation of three monoclonal antibody variants using multicolumn countercurrent solvent gradient purification (MCSGP).

    Science.gov (United States)

    Müller-Späth, Thomas; Aumann, Lars; Melter, Lena; Ströhlein, Guido; Morbidelli, Massimo

    2008-08-15

    Multicolumn countercurrent solvent gradient purification (MCSGP) is a continuous chromatographic process developed in recent years (Aumann and Morbidelli, 2007a; Aumann et al., 2007) that is particularly suited for applications in the field of bioseparations. Like batch chromatography, MCSGP is suitable for three-fraction chromatographic separations and able to perform solvent gradients but it is superior in terms of solvent consumption, yield, purity, and productivity due to the countercurrent movement of the liquid and the solid phases. In this work, the MCSGP process is applied to the separation of three monoclonal antibody variants on a conventional preparative cation exchange resin. The experimental process performance was compared to simulations based on a lumped kinetic model. Yield and purity values of the target variant of 93%, respectively were obtained experimentally. The batch reference process was clearly outperformed by the MCSGP process.

  2. Use of anti tumor necrosis factor-alpha monoclonal antibody for ulcerative jejunoileitis

    Institute of Scientific and Technical Information of China (English)

    Gulseren Seven; Adel Assaad; Thomas Biehl; Richard A Kozarek

    2012-01-01

    Ulcerative jejunoileitis is an uncommon clinical syndrome consisting of abdominal pain,weight loss associated with diarrhea,and multiple inflammatory ulcerations and strictures of the small bowel.Ulcerative jejunoileitis can complicate established celiac disease or develop in patients de novo.Increased levels of tumor necrosis factor-alpha (TNF-α) in the small intestine of patients with untreated celiac disease are associated with a role in the immune pathogenesis of this disorder.No specific therapy has been shown to change the course of ulcerative jejunoileitis.We report a case of severe ulcerative jejunoileitis previously unresponsive to traditional therapies,including high dose corticosteroids and cyclosporine.The patient had a dramatic resolution of symptoms and a complete normalization of endoscopic findings after anti-TNF-α monoclonal antibody,infliximab (Remicade(R)).

  3. Monoclonal antibody targeting chikungunya virus envelope 1 protein inhibits virus release.

    Science.gov (United States)

    Masrinoul, Promsin; Puiprom, Orapim; Tanaka, Atsushi; Kuwahara, Miwa; Chaichana, Panjaporn; Ikuta, Kazuyoshi; Ramasoota, Pongrama; Okabayashi, Tamaki

    2014-09-01

    Chikungunya virus (CHIKV) causes an acute clinical illness characterized by sudden high fever, intense joint pain, and skin rash. Recent outbreaks of chikungunya disease in Africa and Asia are a major public health concern; however, there is currently no effective licensed vaccine or specific treatment. This study reported the development of a mouse monoclonal antibody (MAb), CK47, which recognizes domain III within the viral envelope 1 protein and inhibited the viral release process, thereby preventing the production of progeny virus. The MAb had no effect on virus entry and replication processes. Thus, CK47 may be a useful tool for studying the mechanisms underlying CHIKV release and may show potential as a therapeutic agent.

  4. New frontiers in oncology: biosimilar monoclonal antibodies for the treatment of breast cancer.

    Science.gov (United States)

    Thill, Marc

    2015-03-01

    Trastuzumab is a highly successful monoclonal antibody (mAb) that has been used primarily for the treatment of HER2-positive breast cancer. Because of its success and its impending patent expiry in Europe in 2014, a number of copy versions of trastuzumab have been developed and are currently undergoing a comparability exercise for marketing authorization. Although biosimilar products have been approved in Europe since 2006, including two biosimilar mAbs of infliximab approved in 2013, the use of mAbs such as trastuzumab in the cancer setting has raised a number of new concerns. The requirements for the approval of biosimilar mAbs published by the EMA will be discussed and examined in the context of trastuzumab biosimilars to highlight potential controversies.

  5. Generation of a murine monoclonal antibody to capsular polysaccharide Vi from Salmonella Typhi

    Directory of Open Access Journals (Sweden)

    Fátima Reyes-López

    2015-11-01

    Full Text Available The conventional hybridoma technology has enabled the development of monoclonal antibodies (Mabs against many antigens. Mabs have several applications in the field of basic research, diagnosis, immunotherapy and vaccine manufacturing processes. Mabs-producing hybridomas against the capsular polysaccharide from Salmonella Typhi were obtained, after intraperitoneal immunization of BALB/c mice with 10 µg of capsular polysaccharide Vi conjugated to diphtheria toxoid, and subsequent fusion of lymphocytes isolated of the spleen and myeloma cells SP2/O. A Mab was selected, partially characterized, and named as 4G3E11. The isotype of this Mab was IgG1. It was proved by means of a sandwich ELISA that the 4G3E11 Mab reacts with different concentrations of polysaccharide in samples of the vax-TyVi® vaccine. The Mab obtained in this research could be useful as reagent for the detection and quantitation of polysaccharide Vi in typhoid vaccines.

  6. Development of a highly-sensitive multi-plex assay using monoclonal antibodies for the simultaneous measurement of kappa and lambda immunoglobulin free light chains in serum and urine.

    Science.gov (United States)

    Campbell, John P; Cobbold, Mark; Wang, Yanyun; Goodall, Margaret; Bonney, Sarah L; Chamba, Anita; Birtwistle, Jane; Plant, Timothy; Afzal, Zaheer; Jefferis, Roy; Drayson, Mark T

    2013-05-31

    Monoclonal κ and λ immunoglobulin free light chain (FLC) paraproteins in serum and urine are important markers in the diagnosis and monitoring of B cell dyscrasias. Current nephelometric and turbidimetric methods that use sheep polyclonal antisera to quantify serum FLC have a number of well-observed limitations. In this report, we describe an improved method using specific mouse anti-human FLC monoclonal antibodies (mAbs). Anti-κ and anti-λ FLC mAbs were, separately, covalently coupled to polystyrene Xmap® beads and assayed, simultaneously, in a multi-plex format by Luminex® (mAb assay). The mAbs displayed no cross-reactivity to bound LC, the alternate LC type, or other human proteins and had improved sensitivity and specificity over immunofixation electrophoresis (IFE) and Freelite™. The assay gives good linearity and sensitivity (<1 mg/L), and the competitive inhibition format gave a broad calibration curve up to 437.5 mg/L and prevented anomalous results for samples in antigen excess i.e. high FLC levels. The mAbs displayed good concordance with Freelite™ for the quantitation of normal polyclonal FLC in plasma from healthy donors (n=249). The mAb assay identified all monoclonal FLC in serum from consecutive patient samples (n=1000; 50.1% with monoclonal paraprotein by serum IFE), and all FLC in a large cohort of urine samples tested for Bence Jones proteins (n=13090; 22.8% with monoclonal κ, 9.0% with monoclonal λ, and 0.8% with poly LC detected by urine IFE). Importantly this shows that the mAbs are at least close to the ideal of detecting FLC from all patients and neoplastic plasma cell clones. Given the overall effectiveness of the anti-FLC mAbs, further clinical validation is now warranted on serial samples from a range of patients with B cell disorders. Use of these mAbs on other assay platforms should also be investigated.

  7. Anti-TNP monoclonal antibodies as reagents for enzyme immunoassay (ELISA).

    Science.gov (United States)

    Léo, P; Ucelli, P; Augusto, E F; Oliveira, M S; Tamashiro, W M

    2000-12-01

    The aim of this study was to produce anti-TNP monoclonal antibodies (MAbs) that could be conjugated and used for the detection of antigen-antibody reactions, in which the antigen specific-antibody had been previously bound to trinitrophenyl (TNP). For hybridoma production, SP2/0-Ag14 cells were fused with spleen cells from mice previously immunized with TNP-ovalbumin (TNP-OVA). After 10 days, enzyme-linked immunoadsorbent assay (ELISA) was used to detect anti-TNP antibodies in the supernatants, and five cultures were found to be strictly positive for TNP. Three of these were subsequently cloned by limiting dilution, and 15 clones were chosen for expansion based on the criterion of high reactivity against TNP. Anti-TNP MAbs produced by those clones were isotyped as IgG1, and purified by Sepharose-protein G affinity cromatography from ascites developed in BALB/c mice. Two purified MAbs (1B2.1B6 and 1B2.1E12) were coupled to horseradish peroxidase (HRPO). The resulting conjugates were evaluated in ELISA tests for interferon-gamma and interleukin-4 detection, in which the secondary anti-cytokine antibodies were coupled either to TNP or biotin. The performance of anti-TNP conjugates in these assays were compared with a biotin-streptavidin/peroxidase system. Both types of conjugates were similarly able to detect cytokines with r2 (linear correlation coefficient) close to unity value. Growth studies of one of those hybridomas (1B2.1B6) yielded a specific growth rate of 0.042 h(-1) and a doubling time of 16.5 h. Data discussed here show that at least two MAbs against TNP raised in this work can be used as a reagent for enzyme immunoassays.

  8. The Effects of Anti-Hcg Monoclonal Antibodies on Human Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Mirshahi M

    2011-12-01

    Full Text Available Background: Human cancer cell lines express human choriogonadotropin (hCG, its subunits and derivatives, regardless of their origin and type. It appears that hCG is a common phenotype in human cancer cell lines. In this research, the effects of hCG targeting monoclonal antibodies (7D9, T18H7 and T8B12 on human cancer cell lines were evaluated. Methods: Monoclonal antibody secreting hybridomas were proliferated and injected intraperitoneally to Balb/C mice after treatment with pristine. Two weeks later, ascites fluid was collected. Purification of aforementioned antibodies from ascites fluid was performed using G-protein affinity followed by ion exchange chromatography. SDS-PAGE and ELISA confirmed the structure and functional integrity of the purified antibodies, respectively. Two human cancer cell lines "Hela" and "MDA" were treated by the purified antibodies. Three days later, different wells were imaged and the cells counted. Results: SDS-PAGE gel (None-reducing indicated consistency of band migration patterns with control antibodies. ELISA test using hCG antigens indicated that the produced antibodies could detect hCG antigens. Cell lines were cultured and treated with different concentrations of each antibody. Counting and imaging different wells of treated plates, indicated that 7D9 antibody had a more significant (P<0.01 cytotoxic effect on cancer cell lines than the control cells. Conclusion: HCG targeting monoclonal antibodies can be used for targeted cancer therapy, as human cancer cells express hCG gene. 7D9 antibody that exhibits protease activity is a proper candidate for this purpose, as it possesses both antagonistic and enzymatic properties.

  9. A generic sample preparation approach for LC–MS/MS bioanalysis of therapeutic monoclonal antibodies in serum applied to Infliximab

    Directory of Open Access Journals (Sweden)

    Anne J. Kleinnijenhuis

    2015-01-01

    Full Text Available In this study, we developed a generic bioanalytical workflow providing sensitive, specific, and accurate absolute quantification of therapeutic monoclonal antibodies in serum. The workflow involves magnetic beads coated with protein A to pull-down therapeutic monoclonal antibodies with affinity for protein A from the biological matrix, followed by tryptic digestion and LC-MS/MS quantification of a unique signature peptide, considering of course the matrix of interest and other present mAbs, if applicable. The feasibility of this approach was demonstrated for Infliximab (trade name Remicade in rat serum. The assigned signature peptide was monitored in the selected reaction monitoring (SRM mode. Assay variability was determined to be below 20%, except at the QC low level, which was provided through optimization of the sample preparation and monitoring of the LC-MS/MS using a stable isotope labeled signature peptide as internal standard. The 100 ng/ml lower limit of quantification using only 25 μl sample volume, is generally considered as sufficient for pharmaceutical development purposes for monoclonal antibodies.

  10. Isolation of highly active monoclonal antibodies against multiresistant gram-positive bacteria.

    Directory of Open Access Journals (Sweden)

    Friederike S Rossmann

    Full Text Available Multiresistant nosocomial pathogens often cause life-threatening infections that are sometimes untreatable with currently available antibiotics. Staphylococci and enterococci are the predominant Gram-positive species associated with hospital-acquired infections. These infections often lead to extended hospital stay and excess mortality. In this study, a panel of fully human monoclonal antibodies was isolated from a healthy individual by selection of B-cells producing antibodies with high opsonic killing against E. faecalis 12030. Variable domains (VH and VL of these immunoglobulin genes were amplified by PCR and cloned into an eukaryotic expression vector containing the constant domains of a human IgG1 molecule and the human lambda constant domain. These constructs were transfected into CHO cells and culture supernatants were collected and tested by opsonophagocytic assay against E. faecalis and S. aureus strains (including MRSA. At concentrations of 600 pg/ml, opsonic killing was between 40% and 70% against all strains tested. Monoclonal antibodies were also evaluated in a mouse sepsis model (using S. aureus LAC and E. faecium, a mouse peritonitis model (using S. aureus Newman and LAC and a rat endocarditis model (using E. faecalis 12030 and were shown to provide protection in all models at a concentration of 4 μg/kg per animal. Here we present a method to produce fully human IgG1 monoclonal antibodies that are opsonic in vitro and protective in vivo against several multiresistant Gram-positive bacteria. The monoclonal antibodies presented in this study are significantly more effective compared to another monoclonal antibody currently in clinical trials.

  11. Specificities of monoclonal antibodies to domain I of alpha-gliadins.

    Science.gov (United States)

    Ellis, H J; Doyle, A P; Wieser, H; Sturgess, R P; Ciclitira, P J

    1993-03-01

    Eight monoclonal antibodies were raised against a sequenced 54-amino-acid peptide of alpha-gliadin, which is thought to exacerbate coeliac disease. Five of the antibodies cross-reacted with coeliac non-toxic cereals. Two of eight of the antibodies bound specifically to coeliac toxic prolamins. These two antibodies cross-reacted with high molecular weight gliadins, which are closely related to alpha-gliadins and whose toxicity to patients with coeliac disease is unclear. The antibodies were screened by enzyme-linked immunosorbent assay against three amino-acid-sequenced peptides of alpha-gliadin with single amino-acid differences. Differential binding of antibody WC2 suggested that this antibody binds in the region of amino-acid residue 36, a proline residue, where there may be an antigenic beta-reverse turn. This proline residue forms part of a tetrapeptide motif, QQQP, which is thought to be present in all coeliac-active peptides.

  12. Preliminary characterisation of Toxoplasma gondii isolates from Zimbabwe, with stage-specific monoclonal antibodies

    DEFF Research Database (Denmark)

    Hove, T.; Lind, Peter; Mukaratirwa, S.

    2005-01-01

    Cell-culture-derived clones of eight Toxoplasma gondii isolates from Zimbabwe were characterised in IFAT with a panel of five monoclonal antibodies (mAb). Each clone had been established from a single murine brain cyst. The antibodies were bradyzoite-specific (4.3), tachyzoite-specific (4.25, 5.1...... in the IFAT in a similar way to the Danish reference strain of T. gondii, SSI-119....

  13. Monoclonal antibody to human endothelial cell surface internalization and liposome delivery in cell culture.

    Science.gov (United States)

    Trubetskaya, O V; Trubetskoy, V S; Domogatsky, S P; Rudin, A V; Popov, N V; Danilov, S M; Nikolayeva, M N; Klibanov, A L; Torchilin, V P

    1988-02-01

    A monoclonal antibody (mAb), E25, is described that binds to the surface of cultured human endothelial cells. Upon binding E25 is rapidly internalized and digested intracellularly. Selective liposome targeting to the surface of the cells is performed using a biotinylated E25 antibody and an avidin-biotin system. Up to 30% of the cell-adherent liposomal lipid is internalized.

  14. Drug desensitization in the management of hypersensitivity reactions to monoclonal antibodies and chemotherapy.

    Science.gov (United States)

    Mezzano, Veronica; Giavina-Bianchi, Pedro; Picard, Matthieu; Caiado, Joana; Castells, Mariana

    2014-04-01

    Hypersensitivity reactions to monoclonal antibodies and chemotherapy, which may vary in severity from mild to life-threatening, can lead to their discontinuation and replacement by alternative agents that are often less effective, more toxic, and/or more expensive. Drug desensitization has emerged as the best treatment modality capable of allowing re-introduction of the hypersensitivity reaction-inducing medication in highly sensitized patients in need of first line therapies. In recent years, the availability of new anti-neoplastic drugs and therapeutic monoclonal antibodies has increased, as has the potential for hypersensitivity reactions. Development of desensitization protocols for these new medications requires a careful assessment of the potential risks and benefits. The purposes of this review are to provide an overview of the presentation of hypersensitivity reactions amenable to desensitization and to increase awareness of the indications for and outcomes of desensitization protocols. Rapid drug desensitization has proven to be a safe and effective way of administering first line therapy to patients with hypersensitivity reactions, providing an extremely powerful treatment modality for patients for whom alternative drugs are deemed unacceptable. Rapid drug desensitization protocols should be administered only by highly trained allergists and nurses who have experience in determining which reactions are amenable to desensitization, and can identify high risk patients and provide them with appropriate care. Efforts should be made to increase awareness of the remarkable safety and efficacy of rapid drug desensitization among non-allergists, especially in the fields of oncology and rheumatology, so as to favor its universal application. Development of desensitization units to provide state-of-the-art care is possible only through coordinated teamwork.

  15. Production and Identification of High Affinity Monoclonal Antibodies Against Pesticide Carbofuran

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    To produce high-affinity monoclonal antibodies against pesticide carbofuran, and the develop immunochemical assays for people's health and environmental protection, the hapten 4-[[(2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy) carbonyl]-amino]-butanoic acid (BFNB) of carbofuran was synthesized and Balb/c mice were immunized by the hapten-carrier (BFNB-bovine serum albumin, BFNB-BSA) conjugates. The splenocytes of immunized mice were fused with Sp2/0 cells and the cultural supernatants of hybridoma cells were screened by the indirect enzyme-linked immunoabsorbent assay (ELISA), based on BFNB-ovoalbumin conjugates (BFNB-OVA). Purified monoclonal antibody (McAb) was obtained from fluids of ascites, deposited by octanoic acid and ammonium sulfate. The affinity and the specificity of McAb were characterized by ELISA or indirect competitive ELISA. A hybridoma cell line (5D3) secreting anti-carbofuran McAb had been established. The titer of culture medium and ascites was up to 1:2.048 × 103 and 1:1.024 × 106, respectively, and the subtype of the McAb was IgG1. The affinity constant of the McAb was about 2.54 × 109 L mol-1, with an IC50 value of 1.18 ng mL-1 and a detection limit of 0.01 ng mL-1. Cross-reactivity studies showed that the McAb was quiet specific for carbofuran, as among the four analogous compounds, they were all hardly recognized (4.59 × 10-4% for 2,3-dihydro-2,2-dimethyl-7-benzofuranol and less than 3.0 × 10-4% for others). The prepared McAb had a very high affinity and specificity,and it could be used to develop ELISA for rapid determination of carbofuran.

  16. Anti-MrkA Monoclonal Antibodies Reveal Distinct Structural and Antigenic Features of MrkA

    Science.gov (United States)

    Wang, Qun; Chen, Yan; Cvitkovic, Romana; Pennini, Meghan E.; Chang, Chew shun; Pelletier, Mark; Bonnell, Jessica; Wu, Herren; Dall’Acqua, William F.; Stover, C. Kendall; Xiao, Xiaodong

    2017-01-01

    Antibody therapy against antibiotics resistant Klebsiella pneumoniae infections represents a promising strategy, the success of which depends critically on the ability to identify appropriate antibody targets. Using a target-agnostic strategy, we recently discovered MrkA as a potential antibody target and vaccine antigen. Interestingly, the anti-MrkA monoclonal antibodies isolated through phage display and hybridoma platforms all recognize an overlapping epitope, which opens up important questions including whether monoclonal antibodies targeting different MrkA epitopes can be generated and if they possess different protective profiles. In this study we generated four anti-MrkA antibodies targeting different epitopes through phage library panning against recombinant MrkA protein. These anti-MrkA antibodies elicited strong in vitro and in vivo protections against a multi-drug resistant Klebsiella pneumoniae strain. Furthermore, mutational and epitope analysis suggest that the two cysteine residues may play essential roles in maintaining a MrkA structure that is highly compacted and exposes limited antibody binding/neutralizing epitopes. These results suggest the need for further in-depth understandings of the structure of MrkA, the role of MrkA in the pathogenesis of Klebsiella pneumoniae and the protective mechanism adopted by anti-MrkA antibodies to fully explore the potential of MrkA as an efficient therapeutic target and vaccine antigen. PMID:28107434

  17. Development of a monoclonal antibody-based flow-through immunoassay (FTA) for detection of white spot syndrome virus (WSSV) in black tiger shrimp Penaeus monodon.

    Science.gov (United States)

    Patil, R; Shankar, K M; Kumar, B T N; Kulkarni, A; Patil, P; Moger, N

    2013-09-01

    A flow-through immunoassay (FTA), an improved version of immunodot, was developed using a nitrocellulose membrane baked onto adsorbent pads enclosed in a plastic cassette to detect white spot syndrome virus (WSSV) in shrimp. Sharp purple dots developed with WSSV against the white background of the nitrocellulose membrane. The detection limits of WSSV by the FTA and immunodot were 0.312 and 1.2 μg mL(-1) crude WSSV protein, respectively. The FTA could be completed in 8-10 min compared with 90 min for immunodot. The FTA was 100 times more sensitive than 1-step polymerase chain reaction (PCR) and in between that of the 1- and 2-step PCR protocol recommended by the Office of International Epizootics (OIE). In experimental, orally infected shrimp post-larvae, WSSV was first detected 14, 16 and 18 h post-infection (hpi) by FTA, immunodot and one-step PCR, respectively. The FTA detected WSSV 2 and 4 h earlier than immunodot and one-step PCR, respectively. The FTA was more sensitive (25/27) than one-step PCR (23/27) and immunodot (23/27) for the detection of WSSV from white spot disease outbreak ponds. The reagent components of the FTA were stable giving expected results for 6 m at 4-8 °C. The FTA is available as a rapid test kit called 'RapiDot' for the early detection of WSSV under field conditions.

  18. Quantitative impurity analysis of monoclonal antibody size heterogeneity by CE-LIF: example of development and validation through a quality-by-design framework.

    Science.gov (United States)

    Michels, David A; Parker, Monica; Salas-Solano, Oscar

    2012-03-01

    This paper describes the framework of quality by design applied to the development, optimization and validation of a sensitive capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) assay for monitoring impurities that potentially impact drug efficacy or patient safety produced in the manufacture of therapeutic MAb products. Drug substance or drug product samples are derivatized with fluorogenic 3-(2-furoyl)quinoline-2-carboxaldehyde and nucleophilic cyanide before separation by CE-SDS coupled to LIF detection. Three design-of-experiments enabled critical labeling parameters to meet method requirements for detecting minor impurities while building precision and robustness into the assay during development. The screening design predicted optimal conditions to control labeling artifacts while two full factorial designs demonstrated method robustness through control of temperature and cyanide parameters within the normal operating range. Subsequent validation according to the guidelines of the International Committee of Harmonization showed the CE-SDS/LIF assay was specific, accurate, and precise (RSD ≤ 0.8%) for relative peak distribution and linear (R > 0.997) between the range of 0.5-1.5 mg/mL with LOD and LOQ of 10 ng/mL and 35 ng/mL, respectively. Validation confirmed the system suitability criteria used as a level of control to ensure reliable method performance.

  19. Monoclonal antibodies to proliferating cell nuclear antigen (PCNA)/cyclin as probes for proliferating cells by immunofluorescence microscopy and flow cytometry.

    Science.gov (United States)

    Kurki, P; Ogata, K; Tan, E M

    1988-04-22

    Proliferating cell nuclear antigen (PCNA)/cyclin is an intranuclear polypeptide antigen that is found in both normal and transformed proliferating cells. We have recently described two mouse monoclonal antibodies reacting with PCNA. In this report we describe the application of these antibodies to the study of proliferating human cells by indirect immunofluorescence microscopy and by flow cytometry. A fixation/permeation procedure was developed in order to obtain satisfactory binding of monoclonal PCNA-specific antibodies to proliferating cells. This method involved fixation with 1% paraformaldehyde followed by methanol treatment. For the staining of cells in suspension with the IgM type monoclonal antibodies lysolecithin was added to the paraformaldehyde solution to achieve a better permeation by the antibody molecules. This procedure gave a good ratio of specific staining relative to the background staining. It also preserved the shape and normal architecture of the cells as judged by visual microscopic observation and by light scatter measurements using a flow cytometer. Furthermore, this fixation technique permits simultaneous labeling of DNA by propidium iodide and PCNA by monoclonal antibodies. PCNA was detected in various types of normal and transformed proliferating cells by indirect immunofluorescence. Quiescent peripheral blood mononuclear cells were PCNA-negative whereas a fraction of lectin-stimulated lymphocytes became PCNA-positive. Similarly, early passages of fetal skin fibroblasts were PCNA-positive but non-proliferating senescent fibroblasts of later passages were PCNA-negative. The association of PCNA-staining by monoclonal antibodies with cell proliferation was confirmed by flow cytometry. Simultaneous labeling of PCNA and DNA showed that the PCNA signal increased during the G1 phase of the cell cycle, reached its maximum in the S-phase, and declined during the G2/M phase. Using cell sorting we demonstrated that mitotic cells had a very low PCNA

  20. Rapid production of antigen-specific monoclonal antibodies from a variety of animals

    Directory of Open Access Journals (Sweden)

    Kurosawa Nobuyuki

    2012-09-01

    Full Text Available Abstract Background Although a variety of animals have been used to produce polyclonal antibodies against antigens, the production of antigen-specific monoclonal antibodies from animals remains challenging. Results We propose a simple and rapid strategy to produce monoclonal antibodies from a variety of animals. By staining lymph node cells with an antibody against immunoglobulin and a fluorescent dye specific for the endoplasmic reticulum, plasma/plasmablast cells were identified without using a series of antibodies against lineage markers. By using a fluorescently labeled antigen as a tag for a complementary cell surface immunoglobulin, antigen-specific plasma/plasmablast cells were sorted from the rest of the cell population by fluorescence-activated cell sorting. Amplification of cognate pairs of immunoglobulin heavy and light chain genes followed by DNA transfection into 293FT cells resulted in the highly efficient production of antigen-specific monoclonal antibodies from a variety of immunized animals. Conclusions Our technology eliminates the need for both cell propagation and screening processes, offering a significant advantage over hybridoma and display strategies.

  1. Inhibition of iodothyronine transport into rat liver cells by a monoclonal antibody

    Energy Technology Data Exchange (ETDEWEB)

    Mol, J.A.; Krenning, E.P.; Docter, R.; Rozing, J.; Hennemann, G.

    1986-06-15

    The role of the rat liver plasma membrane in the regulation of uptake and subsequent deiodination of thyroxine (T4) or the biologically active thyroid hormone 3,3',5-triiodothyronine (T3) was investigated. Here we report on the production of monoclonal antibodies raised against rat hepatocytes. Two antibodies were selected. Antibody ER-22 did bind to a Mr 52,000 membrane protein and inhibited the 1- and 5-min uptake of both T4 and T3 by primary cultured rat hepatocytes in a dose-dependent fashion. As the uptake of T4 and T3 depends on the presence of a sodium gradient over the plasma membrane, the inhibitory potency of ER-22 on the Na+,K+-ATPase activity was investigated. No inhibition of the uptake of 86Rb+ could be determined, indicating that antibody ER-22 is not directed against the Na+,K+-ATPase but probably the carrier protein itself. Clearance of T3 from the medium and concomitant iodide production by cultured rat hepatocytes during a 20-h incubation in the presence of ER-22 were both inhibited by 50% with respect to a control incubation in the absence of monoclonal antibody, pointing to the importance of carrier-mediated transport in cellular uptake and metabolism of T3. A second monoclonal antibody did bind to two other plasma membrane proteins but did not inhibit transport of thyroid hormone.

  2. Anti-interleukin-17 monoclonal antibody ixekizumab in chronic plaque psoriasis

    DEFF Research Database (Denmark)

    Leonardi, Craig; Matheson, Robert; Zachariae, Claus;

    2012-01-01

    Type 17 helper T cells have been suggested to play a pathological role in psoriasis. They secrete several proinflammatory cytokines, including interleukin-17A (also known as interleukin-17). We evaluated the safety and efficacy of ixekizumab (LY2439821), a humanized anti-interleukin-17 monoclonal...... antibody, for psoriasis treatment....

  3. Survey of citrus tristeza virus populations in Central California that react with MCA13 monoclonal antibody

    Science.gov (United States)

    The Citrus Pest Detection Program (CPDP) of the Central California Tristeza Eradication Agency monitors Citrus tristeza virus (CTV) in Central California. MCA13 is a severe strain discriminating monoclonal antibody used to screen for potentially virulent CTV isolates. MCA13-reactive CTV isolates are...

  4. Harnessing the immune system's arsenal: producing human monoclonal antibodies for therapeutics and investigating immune responses

    Science.gov (United States)

    Sullivan, Meghan; Kaur, Kaval; Pauli, Noel

    2011-01-01

    Monoclonal antibody technology has undergone rapid and innovative reinvention over the last 30 years. Application of these technologies to human samples revealed valuable therapeutic and experimental insights. These technologies, each with their own benefits and flaws, have proven indispensable for immunological research and in our fight to provide new treatments and improved vaccines for infectious disease. PMID:21876728

  5. Two courses of rituximab (anti-CD20 monoclonal antibody) for recalcitrant pemphigus vulgaris

    DEFF Research Database (Denmark)

    Faurschou, A.; Gniadecki, R.

    2008-01-01

    Background Pemphigus vulgaris (PV) is a severe autoimmune blistering disease involving the skin and mucous membranes. The response to therapy varies greatly amongst patients and treatment may be challenging. Rituximab is a chimeric monoclonal antibody that selectively targets cell surface antigen...

  6. Inhibition of middle east respiratory syndrome coronavirus infection by anti-CD26 monoclonal antibody

    NARCIS (Netherlands)

    K. Ohnuma (Kei); B.L. Haagmans (Bart); R. Hatano (Ryo); V.S. Raj (Stalin); H. Mou (Huihui); S. Iwata (Satoshi); R.L. Dang (Rong); B.J. Bosch (Berend Jan); C. Morimoto (Chikao)

    2013-01-01

    textabstractWe identified the domains of CD26 involved in the binding of Middle East respiratory syndrome coronavirus (MERS-CoV) using distinct clones of anti-CD26 monoclonal antibodies (MAbs). One clone, named 2F9, almost completely inhibited viral entry. The humanized anti-CD26 MAb YS110 also sign

  7. A phase II trial of chimeric monoclonal antibody G250 for advanced renal cell carcinoma patients.

    NARCIS (Netherlands)

    Bleumer, I.; Knuth, A.; Oosterwijk, E.; Hofmann, R.; Varga, Z.; Lamers, C.B.H.W.; Kruit, W.; Melchior, S.; Mala, C.; Ullrich, S.; Mulder, P.; Mulders, P.F.A.; Beck, J.L.M.

    2004-01-01

    Chimeric monoclonal antibody G250 (WX-G250) binds to a cell surface antigen found on >90% of renal cell carcinoma (RCC). A multicentre phase II study was performed to evaluate the safety and efficacy of WX-G250 in metastatic RCC (mRCC) patients. In all, 36 patients with mRCC were included. WX-G250 w

  8. Purification of infectious canine parvovirus from cell culture by affinity chromatography with monoclonal antibodies.

    NARCIS (Netherlands)

    J. Groen (Jan); N. Juntti; J.S. Teppema; F.G.C.M. Uytdehaag (Fons); A.D.M.E. Osterhaus (Albert); G.F. Rimmelzwaan (Guus)

    1987-01-01

    textabstractImmuno affinity chromatography with virus neutralizing monoclonal antibodies, directed to the haemagglutinating protein of canine parvovirus (CPV) was used to purify and concentrate CPV from infected cell culture. The procedure was monitored by testing the respective fractions in an infe

  9. Competitive adsorption of monoclonal antibodies and nonionic surfactants at solid hydrophobic surfaces

    DEFF Research Database (Denmark)

    Kapp, Sebastian J; Larsson, Iben; van de Weert, Marco

    2015-01-01

    Two monoclonal antibodies from the IgG subclasses one and two were compared in their adsorption behavior with hydrophobic surfaces upon dilution to 10 mg/mL with 0.9% NaCl. These conditions simulate handling of the compounds at hospital pharmacies and surfaces encountered after preparation, such ...

  10. Comprehensive analysis of varicella-zoster virus proteins using a new monoclonal antibody collection

    NARCIS (Netherlands)

    T.L. Roviš (Tihana Lenac); S.M. Bailer (Susanne); V.R. Pothineni (Venkata R); W.J.D. Ouwendijk (Werner ); H. Šimić (Hrvoje); M. Babić (Marina); K. Miklić (Karmela); S. Malić (Suzana); M.C. Verweij; M. Baiker (Martin); O. Gonzalez (Orland); A. Brunn (Albrecht von); R. Zimmer; K. Früh (Klaus); G.M.G.M. Verjans (George); S. Jonjic (Stipan); J. Haasb (Jürgeni)

    2013-01-01

    textabstractVaricella-zoster virus (VZV) is the etiological agent of chickenpox and shingles. Due to the virus's restricted host and cell typetropism and the lack of tools for VZV proteomics, it is one of the least-characterized human herpesviruses. We generated 251monoclonal antibodies (MAbs) again

  11. Detection of tomato spotted wilt virus using monoclonal antibodies and riboprobes.

    NARCIS (Netherlands)

    C. Huguenot; G.J.P.M. van den Dobbelsteen (Germie); P. de Haan (Jurre); C.A.M. Wagemakers; G.A. Drost; A.D.M.E. Osterhaus (Albert); D. Peters

    1990-01-01

    textabstractThe immunoreactivity of a panel of monoclonal antibodies raised to tomato spotted wilt virus (TSWV) was examined in enzyme-linked immunosorbent assays (ELISA) and dot immunobinding assays (DIBA) procedures. MAbs 6.12.15 and 2.9 were specific for the nucleocapsid protein of TSWV. The

  12. Clinical efficacy and management of monoclonal antibodies targeting CD38 and SLAMF7 in multiple myeloma

    DEFF Research Database (Denmark)

    van de Donk, Niels W C J; Moreau, Philippe; Plesner, Torben;

    2016-01-01

    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...

  13. HIV monoclonal antibodies: a new opportunity to further reduce mother-to-child HIV transmission.

    Directory of Open Access Journals (Sweden)

    Yegor Voronin

    2014-04-01

    Full Text Available Yegor Voronin and colleagues explore how monoclonal antibodies against HIV could provide a new opportunity to further reduce mother-to-child transmission of HIV and propose that new interventions should consider issues related to implementation, feasibility, and access. Please see later in the article for the Editors' Summary.

  14. Prolonged skin graft survival by administration of anti-CD80 monoclonal antibody with cyclosporin A

    NARCIS (Netherlands)

    Ossevoort, MA; Lorre, K; Boon, L; van den Hout, Y; de Boer, M; De Waele, P; Jonker, M; VandeVoorde, A

    1999-01-01

    Costimulation via the B7/CD28 pathway is an important signal for the activation of T cells. Maximal inhibition of T-cell activation and the induction of alloantigen-specific nonresponsiveness in vitro was achieved using anti-CD80 monoclonal antibody (mAb) in combination with cyclosporin A (CsA). Bas

  15. Intravenous cidofovir for resistant cutaneous warts in a patient with psoriasis treated with monoclonal antibodies.

    LENUS (Irish Health Repository)

    McAleer, M A

    2012-02-01

    Human papilloma virus is a common and often distressing cutaneous disease. It can be therapeutically challenging, especially in immunocompromised patients. We report a case of recalcitrant cutaneous warts that resolved with intravenous cidofovir treatment. The patient was immunocompromised secondary to monoclonal antibody therapy for psoriasis.

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

    DEFF Research Database (Denmark)

    Koch, C; Behrendt, N

    1986-01-01

    A mouse monoclonal antibody, HAV 4-1, obtained after immunization of a BALB/c mouse with purified C3F, detected a novel genetic polymorphism of human complement component C3 in a simple immunoblotting system. The frequency of HAV 4-1-positive genes was 20.1%. Reactivity of HAV 4-1 was closely rel...

  17. New Insights into the Functional Behavior of Antibodies as Revealed by Binding Studies on an Anti-Uranium Monoclonal Antibody

    Energy Technology Data Exchange (ETDEWEB)

    Blake, Diane A.; Xia Li; Haini Yu; Blake, Robert C.

    2004-03-17

    As part of an ongoing effort to develop immunoassays for chelated uranium(VI) on a hand-held flow fluorimeter, an anti-uranium monoclonal antibody designated as 8A11 was fluorescently labeled using two different strategies. When 8A11 was coupled via reactive lysines to either ALEXATM 488 or Cy5TM, the resulting fluorescent antibody conjugate exhibited positive cooperativity in the presence of its antigen, U(VI) chelated with 2,9-dicarboxy-1,10-phenanthroline (U(VI)-DCP). That is, when one of the two binding sites on the covalently modified 8A11 was occupied with bound antigen, the affinity of the remaining site on the antibody for U(VI)-DCP appeared to increase. Unmodified 8A11 bound U(VI)-DCP with the expected hyperbolic dependence on the concentration of antigen, consistent with independent and equal binding of ligand at both sites. Proteolytic cleavage of the fluorescently conjugated 8A11 to produce the fluorescent monovalent Fab fragment yielded an active preparation that now bound U(VI)-DCP with no evidence of positive cooperativity. Although, in principle, any divalent antibody has the potential to exhibit positive cooperativity in its binding interactions with its antigen, very little literature precedent for this type of behavior exists. Native 8A11 was also noncovalently labeled with highly fluorescent ZENONTM reagents. These reagents are fluorescently-labeled Fab fragments of goat anti-mouse antibodies that bind to the Fc portion of 8A11. These high-affinity, monovalent fluorescent reagents permitted the intact 8A11 mouse antibody to be labeled in situ with no covalent modifications. Incubation of the 8A11 with ZENON 647 produced a fluorescent protein complex that showed an 8-fold higher affinity for U(VI)-DCP than did the free 8A11 alone. Again, very few literature precedents exist for this phenomenon, where agents that bind to the Fc portion of an intact antibody change the affinity of the antibody for the antigen at the structurally distant Fab portion

  18. Data on the characterization of follicle-stimulating hormone monoclonal antibodies and localization in Japanese eel pituitary

    Directory of Open Access Journals (Sweden)

    Dae-Jung Kim

    2016-09-01

    In support of our recent publication, "Production and characterization of monoclonal antibodies against recombinant tethered follicle-stimulating hormone from Japanese eel Anguilla japonica" [1], it was important to characterize the specificity of eel follicle-stimulating hormone antibodies. Here, the production and ELISA system of these monoclonal antibodies are presented. The affinity-purified monoclonal antibodies specifically detected eel rec-FSH in ELISA and on western blots of rec-FSH produced from CHO cells. Immunohistochemical analysis revealed that FSH staining was specifically localized in the eel pituitary.

  19. Antibody networks and imaging: elicitation of anti-fluorescein antibodies in response to the metatypic state of fluorescein-specific monoclonal antibodies.

    Science.gov (United States)

    Cedergren, A M; Miklasz, S D; Voss, E W

    1996-01-01

    Studies are described regarding generation of anti-hapten antibodies starting with a monoclonal Ig immunogen in the ligand-induced conformation or metatypic state. Liganded monoclonal Ab1 antibodies represent the unique feature of the study since previous reports investigating internal imaging in the original Idiotype Network Hypothesis [Jerne, 1974 (Ann. Immun. 125C, 373-389)] were based on the non-liganded or idiotypic state [as reviewed in: Rodkey, 1980 (Microbiol. Rev. 44, 631-659); Kohler et al., 1979 (In: Methods in Enzymology: Antibodies, Antigens and Molecular Mimicry, pp. 3-35); Greenspan and Bona, 1993 (FASEB J. 7,437-444)]. Affinity-labeled liganded murine monoclonal anti-fluorescein antibodies served as immunogens administered both in the syngenic and xenogenic modes to determine if the metatypic state elicited anti-hapten antibodies through imaging-like mechanisms. Polyclonal and monoclonal anti-Ab1 reagents in various hosts were assayed for anti-fluorescein and/or anti-metatype specificity. Significant anti-fluorescein responses were measured indicating that the metatypic state directly or indirectly stimulates an anti-hapten antibody population.

  20. Using Monoclonal Antibody to Determine Lead Ions with a Localized Surface Plasmon Resonance Fiber-optic Biosensor

    Directory of Open Access Journals (Sweden)

    Mon-Fu Chung

    2008-01-01

    Full Text Available A novel reflection-based localized surface plasmon resonance (LSPR fiber-optic probe has been developed to determine the heavy metal lead ion concentration. Monoclonal antibody as the detecting probe containing massive amino groups to capture Pb(II-chelate complexes was immobilized onto gold nanoparticle-modified optical fiber (NMAuOF. The optimal immobilizing conditions of monoclonal antibody on to the NMAuOF are 189 μg/mL in pH7.4 PBS for 2 h at 25°C. The absorbability of the functionalized NMAuOF sensor increases to 12.2 % upon changing the Pb(II-EDTA level from 10 to 100 ppb with a detection limit of 0.27 ppb. The sensor retains 92.7 % of its original activity and gives reproducible results after storage in 5% D-( -Trehalose dehydrate solution at 4°C for 35 days. In conclusion, the monoclonal antibody-functionalized NMAuOF sensor shows a promising result for determining the concentration of Pb(II with high sensitivity.