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Bispecific Antibodies: Molecules That Enable Novel Therapeutic Strategies  

Microsoft Academic Search

Bispecific antibodies are unique in the sense that they can bind simultaneously two different antigens. This property enables the development of therapeutic strategies that are not possible with conventional monoclonal antibodies. The large panel of imaginative bispecific antibody formats that has been developed reflects the strong interest for these molecules. Although in many cases the manufacturing of clinical grade material

Nicolas Fischer; Olivier Léger



Dual targeting strategies with bispecific antibodies  

PubMed Central

Monoclonal antibodies are widely used for the treatment of cancer, inflammatory and infectious diseases and other disorders. Most of the marketed antibodies are monospecific and therefore capable of interacting and interfering with a single target. However, complex diseases are often multifactorial in nature, and involve redundant or synergistic action of disease mediators or upregulation of different receptors, including crosstalk between their signaling networks. Consequently, blockade of multiple, different pathological factors and pathways may result in improved therapeutic efficacy. This result can be achieved by combining different drugs, or use of the dual targeting strategies applying bispecific antibodies that have emerged as an alternative to combination therapy. This review discusses the various dual targeting strategies for which bispecific antibodies have been developed and provides an overview of the established bispecific antibody formats. PMID:22453100



Bispecific Antibodies and Gene Therapy  

Microsoft Academic Search

\\u000a Gene therapy is the transfer of therapeutic genes, via gene transfer vectors, into patients for therapeutic purposes. Different\\u000a gene therapy strategies are being pursued, including long-term gene correction of monogenetic diseases, eradication of tumor\\u000a cells in cancer patients, or genetic vaccination for infectious diseases. Bispecific antibodies and gene therapy are connected\\u000a in two ways. First, bispecific antibodies are tools of

Dirk M. Nettelbeck


Bispecific antibodies for cancer therapy: the light at the end of the Patrick Chames1  

E-print Network

1 Bispecific antibodies for cancer therapy: the light at the end of the tunnel? Patrick Chames1, bispecific, cancer, therapy, clinical trials Abbreviation mAb: monoclonal antibodies, bsAbs: bispecific of producing these molecules,1 they have raised many hopes for the development of novel therapies, particularly

Paris-Sud XI, Université de


Monoclonal Antibodies.  

ERIC Educational Resources Information Center

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

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



A multifunctional bispecific antibody protects against Pseudomonas aeruginosa.  


Widespread drug resistance due to empiric use of broad-spectrum antibiotics has stimulated development of bacteria-specific strategies for prophylaxis and therapy based on modern monoclonal antibody (mAb) technologies. However, single-mechanism mAb approaches have not provided adequate protective activity in the clinic. We constructed multifunctional bispecific antibodies, each conferring three mechanisms of action against the bacterial pathogen Pseudomonas aeruginosa by targeting the serotype-independent type III secretion system (injectisome) virulence factor PcrV and persistence factor Psl exopolysaccharide. A new bispecific antibody platform, BiS4, exhibited superior synergistic protection against P. aeruginosa-induced murine pneumonia compared to parent mAb combinations or other available bispecific antibody structures. BiS4?Pa was protective in several mouse infection models against disparate P. aeruginosa strains and unexpectedly further synergized with multiple antibiotic classes even against drug-resistant clinical isolates. In addition to resulting in a multimechanistic clinical candidate (MEDI3902) for the prevention or treatment of P. aeruginosa infections, these antibody studies suggest that multifunctional antibody approaches may be a promising platform for targeting other antibiotic-resistant bacterial pathogens. PMID:25391481

DiGiandomenico, Antonio; Keller, Ashley E; Gao, Cuihua; Rainey, Godfrey J; Warrener, Paul; Camara, Mareia M; Bonnell, Jessica; Fleming, Ryan; Bezabeh, Binyam; Dimasi, Nazzareno; Sellman, Bret R; Hilliard, Jamese; Guenther, Caitlin M; Datta, Vivekananda; Zhao, Wei; Gao, Changshou; Yu, Xiang-Qing; Suzich, JoAnn A; Stover, C Kendall



World Bispecific Antibody Summit, September 27-28, 2011, Boston, MA  

PubMed Central

With more than 30 therapeutic monoclonal antibodies (mAbs) approved and annual global sales of the products at ?$50 billion in 2010, these products have proven to be successful in many ways. Nevertheless, there is room for improvement in performance, and substantial unmet medical needs remain. As a consequence, numerous organizations are devoting resources to engineering novel mAbs such as bispecific antibodies that have increased functionality compared with unmodified IgG molecules. The World Bispecific Antibody Summit, organized by Hanson Wade, drew over 100 participants to Boston to discuss engineering novel bispecific antibodies, generating lead candidates and clinical study and commercialization of the molecules. Approaches such as the trifunctional antibody (TRION), dual variable domain-Ig (Abbott), two-in-one (Genentech), dual affinity retargeting (MacroGenics), kappa-lambda body (NovImmune), bispecific T-cell engager (Micromet) and chemical generation (CovX/Pfizer) were discussed in detail. In addition, posters describing bispecific Affibody® molecules for targeting of EGFR and HER2 (Affibody), T-cell receptor based bi-specifics that target HLA-peptides (Immunocore), a novel mAb-Fv bispecific antibody format utilizing Fc region (Xencore), generation of a tetravalent bispecific antibody against IL4 and IL13 for the treatment of idiopathic pulmonary fibrosis (Sanofi), Combining Affibody® molecules and the AlbumodTM technology to create long acting multispecific protein therapeutics (Royal Institute of Technology, Affibody) and COVA301 as a highly potent bispecific inhibitor of IL-17A and TNF? (Covagen) were presented. PMID:22327426

Dhimolea, Eugen



Tumor-Specific Inhibition of Membrane-Bound Complement Regulatory Protein Crry with Bispecific Monoclonal Antibodies Prevents Tumor Outgrowth in a Rat Colorectal Cancer Lung Metastases Model  

Microsoft Academic Search

Membrane-bound complement regulatory proteins (mCRP) inhibit complement-mediated tumor cell eradication in vitro and in vivo. Immu- notherapy of cancer with monoclonal antibodies (mAbs) that activate complement might be hampered by expression of mCRP on tumor cells. An important strategy to improve mAb immunotherapy can be blocking or overwhelming mCRP at the tumor cells surface in a tumor-specific manner. In our

Kyra A. Gelderman; Peter J. K. Kuppen; Noriko Okada; Gert Jan Fleuren; Arko Gorter


Monoclonal antibodies and immobilized antibodies  

Microsoft Academic Search

Antibodies in both their free and immobilized state have been the object of considerable industrial and academic interest.\\u000a A variety of methods are used for preparing and immobilizing antibodies. Applications for monoclonal antibodies include the\\u000a preparation of therapeutics, diagnostics, and in affinity fractionation. Recent US patents on monoclonal and immobilized antibodies\\u000a and scientific literature on monoclonal antibodies are surveyed. A

Robert J. Linhardt; C. W. Abell; R. M. Denney; B. W. Altrock; R. Auerbach; S. D. Bernal; R. E. Canfield; P. H. Ehrlich; W. R. Moyle; T. S. Chan; T. W. Chang; N. T. Chang; J. A. Cidlowski; M. D. Viceps; R. J. Cote; D. M. Morrissey; A. N. Houghton; E. J. Beattie; H. F. Oettgen; L. J. Old; C. M. Croce; R. S. Cubicciotti; A. E. Karu; R. M. Krauss; J. S. Cullor; A. Deutsch; H. Brandwein; H. Platt; D. M. Hunter; A. Dubitsky; S. M. Durham; F. A. Dolbeare; J. W. Gray; G. R. Dreesman; C. E. Kendall; J. C. Egrie; A. R. Frackelton; H. N. Eisen; A. H. Ross; S. Gay; G. Geirnaert; J. E. Geltosky; E. H. Goldberg; E. Goldwasser; C. Kavinsky; T. L. Weiss; H. G. Gratzner; B. Hampar; M. Zweig; S. D. Showalter; H. H. Handley; M. C. Glassy; Y. Hagiwara; H. Hagiwara; C. M. Huang; S. N. Cohen; J. V. Hughes; E. M. Scolnick; J. E. Tomassini; R. Jefferis; J. Steensgaard; H. S. Kaplan; N. N. H. Teng; K. S. Earn; R. F. Calvo; L. Kass; J. R. Kettman; M. V. Norgard; M. B. Khazaeli; W. H. Beierwaltes; B. G. England; P. C. Kung; G. Goldstein; L. Lanier; J. Phillips; N. L. Warner; J. W. Larrick; A. R. Raubitschek; K. E. Truitt; H. Lazarus; J. F. Schwaber; J. Lewicki; C. Lewis; J. V. Olander; W. R. Tolbert; E. L. Milford; C. B. Carpenter; J. M. Paradysz; D. F. Mosher; J. L. Mulshine; J. D. Minna; K. A. Murray; D. M. Neville; R. J. Youle; M. Nicolson; I. Pastan; M. C. Willingham; D. J. Fitzgerald; A. Pucci; A. M. Smithyman; M. B. Slade; P. W. French; G. Wijffels; C. S. Pukel; K. O. Lloyd; L. R. Travassos; W. G. Dippold; R. P. Reckel; J. L. Harris; R. Wellerson; S. M. Shaw; P. M. Kaplan; E. L. Reinherz; S. F. Schlossman; S. C. Mener; J. Sakamoto; C. C. Cordon; E. Friedman; C. L. Finstad; W. E. Enker; M. R. Melamed; J. F. Oettgen; P. J. Scannon; L. E. Spitler; H. M. Lee; R. T. Kawahata; R. P. Mischak; J. Schlom; D. Colcher; M. Nuti; P. H. Hand; F. Austin; G. D. Shockman; D. E. Jackson; W. Wong; Z. Steplewski; H. Koprowski; M. Herlyn; M. Strand; I. S. Trowbridge; D. L. Urdal; C. J. March; S. K. Dower; J. R. Wands; V. R. Zurawski; C. A. White; R. Dulbecco; W. R. Allen; E. C. Arnold; M. Flasher; H. H. Freedman; T. D. Heath; P. Shek; D. Papahadjopoulos; M. Ikeda; S. Sakamoto; K. Suzuki; M. Kuboyama; Y. Harada; A. Kawashiri; E. Takahashi; H. S. Lee; S. Margel; R. C. Nowinski; A. S. Hoffman; J. W. Peterson; K. B. Platt; D. E. Reed; F. X. Real; M. J. Mattes; P. O. Livingston; A. Rembaum; R. C. K. Yen; R. Rosenstein; B. Schneider



Bispecific antibody generated with sortase and click chemistry has broad antiinfluenza virus activity.  


Bispecific antibodies have therapeutic potential by expanding the functions of conventional antibodies. Many different formats of bispecific antibodies have meanwhile been developed. Most are genetic modifications of the antibody backbone to facilitate incorporation of two different variable domains into a single molecule. Here, we present a bispecific format where we have fused two full-sized IgG antibodies via their C termini using sortase transpeptidation and click chemistry to create a covalently linked IgG antibody heterodimer. By linking two potent anti-influenza A antibodies together, we have generated a full antibody dimer with bispecific activity that retains the activity and stability of the two fusion partners. PMID:25385586

Wagner, Koen; Kwakkenbos, Mark J; Claassen, Yvonne B; Maijoor, Kelly; Böhne, Martino; van der Sluijs, Koenraad F; Witte, Martin D; van Zoelen, Diana J; Cornelissen, Lisette A; Beaumont, Tim; Bakker, Arjen Q; Ploegh, Hidde L; Spits, Hergen



Therapeutic Recombinant Monoclonal Antibodies  

ERIC Educational Resources Information Center

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…

Bakhtiar, Ray



MAbs . Author manuscript Bispecific antibodies for cancer therapy: the light at the end of the tunnel?  

E-print Network

MAbs . Author manuscript Page /1 10 Bispecific antibodies for cancer therapy: the light at the end ; immunology ; therapy ; United States Author Keywords antibodies, bispecific, cancer, therapy, clinical trials for the development of novel therapies, particularly as cancer treatments.1 However, extensive optimization through

Paris-Sud XI, Université de


Monoclonal antibody "gold rush".  


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

Maggon, Krishan



Bispecific Antibody Pretargeting for Improving Cancer Imaging and Therapy  

SciTech Connect

The main objective of this project was to evaluate pretargeting systems that use a bispecific antibody (bsMAb) to improve the detection and treatment of cancer. A bsMAb has specificity to a tumor antigen, which is used to bind the tumor, while the other specificity is to a peptide that can be radiolabeled. Pretargeting is the process by which the unlabeled bsMAb is given first, and after a sufficient time (1-2 days) is given for it to localize in the tumor and clear from the blood, a small molecular weight radiolabeled peptide is given. According to a dynamic imaging study using a 99mTc-labeled peptide, the radiolabeled peptide localizes in the tumor in less than 1 hour, with > 80% of it clearing from the blood and body within this same time. Tumor/nontumor targeting ratios that are nearly 50 times better than that with a directly radiolabeled Fab fragment have been observed (Sharkey et al., ''Signal amplification in molecular imaging by a multivalent bispecific nanobody'' submitted). The bsMAbs used in this project have been composed of 3 antibodies that will target antigens found in colorectal and pancreatic cancers (CEA, CSAp, and MUC1). For the ''peptide binding moiety'' of the bsMAb, we initially examined an antibody directed to DOTA, but subsequently focused on another antibody directed against a novel compound, HSG (histamine-succinyl-glycine).

Sharkey, Robert M.



Heavy chain-only antibodies and tetravalent bispecific antibody neutralizing Staphylococcus aureus leukotoxins  

PubMed Central

Panton–Valentine leukocidin (PVL) is a pore-forming toxin associated with current outbreaks of community-associated methicillin-resistant strains and implicated directly in the pathophysiology of Staphylococcus aureus-related diseases. Humanized heavy chain-only antibodies (HCAb) were generated against S. aureus PVL from immunized transgenic mice to neutralize toxin activity. The active form of PVL consists of the two components, LukS-PV and LukF-PV, which induce osmotic lysis following pore formation in host defense cells. One anti–LukS-PV HCAb, three anti–LukF-PV HCAbs with affinities in the nanomolar range, and one engineered tetravalent bispecific HCAb were tested in vitro and in vivo, and all prevented toxin binding and pore formation. Anti–LukS-PV HCAb also binds to ?-hemolysin C (HlgC) and inhibits HlgC/HlgB pore formation. Experiments in vivo in a toxin-induced rabbit endophthalmitis model showed that these HCAbs inhibit inflammatory reactions and tissue destruction, with the tetravalent bispecific HCAb performing best. Our findings show the therapeutic potential of HCAbs, and in particular, bispecific antibodies. PMID:21930905

Laventie, Benoit-Joseph; Rademaker, Hendrik Jan; Saleh, Maher; de Boer, Ernie; Janssens, Rick; Bourcier, Tristan; Subilia, Audrey; Marcellin, Luc; van Haperen, Rien; Lebbink, Joyce H. G.; Chen, Tao; Prevost, Gilles; Grosveld, Frank; Drabek, Dubravka



Heavy chain-only antibodies and tetravalent bispecific antibody neutralizing Staphylococcus aureus leukotoxins.  


Panton-Valentine leukocidin (PVL) is a pore-forming toxin associated with current outbreaks of community-associated methicillin-resistant strains and implicated directly in the pathophysiology of Staphylococcus aureus-related diseases. Humanized heavy chain-only antibodies (HCAb) were generated against S. aureus PVL from immunized transgenic mice to neutralize toxin activity. The active form of PVL consists of the two components, LukS-PV and LukF-PV, which induce osmotic lysis following pore formation in host defense cells. One anti-LukS-PV HCAb, three anti-LukF-PV HCAbs with affinities in the nanomolar range, and one engineered tetravalent bispecific HCAb were tested in vitro and in vivo, and all prevented toxin binding and pore formation. Anti-LukS-PV HCAb also binds to ?-hemolysin C (HlgC) and inhibits HlgC/HlgB pore formation. Experiments in vivo in a toxin-induced rabbit endophthalmitis model showed that these HCAbs inhibit inflammatory reactions and tissue destruction, with the tetravalent bispecific HCAb performing best. Our findings show the therapeutic potential of HCAbs, and in particular, bispecific antibodies. PMID:21930905

Laventie, Benoît-Joseph; Rademaker, Hendrik Jan; Saleh, Maher; de Boer, Ernie; Janssens, Rick; Bourcier, Tristan; Subilia, Audrey; Marcellin, Luc; van Haperen, Rien; Lebbink, Joyce H G; Chen, Tao; Prévost, Gilles; Grosveld, Frank; Drabek, Dubravka



The new face of bispecific antibodies: targeting cancer and much more.  


The term magic bullet was first coined by bacteriologist Paul Ehrlich in the late 1800s to describe a chemical with the ability to specifically target microorganisms while sparing normal host cells. His concept was later expanded to include treatments for cancer, but it is only in recent decades, with development and improvements in monoclonal antibody (mAb) technology, that the full therapeutic implications of "magic bullet" strategies have been realized. Expanding on the success of mAb-targeting, linking the specificity of two mAbs into a single agent, called a bispecific antibody (BiAb), allows for targeting of a therapeutic biological agent or cell to specific tissue antigens. Classically, BiAbs have been used for several decades to redirect cytotoxic T cells or other effector cells to kill tumor cells. Here, we review preclinical models and ongoing phase I clinical trials in which arming polyclonally activated T cells with BiAbs may provide anti-tumor activity without dose-limiting toxicities. Additionally, we review findings from this novel strategy that merges magic bullet technology with hematopoietic stem cells to repair injured myocardium. Arming stem cells with BiAbs directed at injury-associated antigens enhances specific homing and engraftment to myocardial infarctions and may significantly improve cardiac function, strongly suggesting new paradigms for BiAb-targeting applications in tissue repair. PMID:16413384

Lum, Lawrence G; Davol, Pamela A; Lee, Randall J



Bispecific antibodies and trispecific immunocytokines for targeting the immune system against cancer: preparing for the future.  


Monoclonal anti-tumor antibodies (mAbs) that are clinically effective usually recruit, via their constant fragment (Fc) domain, Fc receptor (FcR)-positive accessory cells of the immune system and engage these additionally against the tumor. Since T cells are FcR negative, these important cells are not getting involved. In contrast to mAbs, bispecific antibodies (bsAbs) can be designed in such a way that they involve T cells. bsAbs are artificially designed molecules that bind simultaneously to two different antigens, one on the tumor cell, the other one on an immune effector cell such as CD3 on T cells. Such dual antibody constructs can cross-link tumor cells and T cells. Many such bsAb molecules at the surface of tumor cells can thus build a bridge to T cells and aggregate their CD3 molecules, thereby activating them for cytotoxic activity. BsAbs can also contain a third binding site, for instance a Fc domain or a cytokine that would bind to its respective cytokine receptor. The present review discusses the pros and cons for the use of the Fc fragment during the development of bsAbs using either cell-fusion or recombinant DNA technologies. The recombinant antibody technology allows the generation of very efficient bsAbs containing no Fc domain such as the bi-specific T-cell engager (BiTE). The strong antitumor activity of these molecules makes them very interesting new cancer therapeutics. Over the last decade, we have developed another concept, namely to combine bsAbs and multivalent immunocytokines with a tumor cell vaccine. The latter are patient-derived tumor cells modified by infection with a virus. The virus-Newcastle Disease Virus (NDV)-introduces, at the surface of the tumor cells, viral molecules that can serve as general anchors for the bsAbs. Our strategy aims at redirecting, in an Fc-independent fashion, activities of T cells and accessory cells against autologous tumor antigens. It creates very promising perspectives for a new generation of efficient and safe cancer therapeutics that should confer long-lasting anti-tumor immunity. PMID:23329400

Fournier, Philippe; Schirrmacher, Volker



Progress in overcoming the chain association issue in bispecific heterodimeric IgG antibodies  

PubMed Central

The development of bispecific antibodies has attracted substantial interest, and many different formats have been described. Those specifically containing an Fc part are mostly tetravalent, such as stabilized IgG-scFv fusions or dual-variable domain (DVD) IgGs. However, although they exhibit IgG-like properties and technical developability, these formats differ in size and geometry from classical IgG antibodies. Thus, considerable efforts focus on bispecific heterodimeric IgG antibodies that more closely mimic natural IgG molecules. The inherent chain association problem encountered when producing bispecific heterodimeric IgG antibodies can be overcome by several methods. While technologies like knobs-into-holes (KiH) combined with a common light chain or the CrossMab technology enforce the correct chain association, other approaches, e.g., the dual-acting Fab (DAF) IgGs, do not rely on a heterodimeric Fc part. This review discusses the state of the art in bispecific heterodimeric IgG antibodies, with an emphasis on recent progress. PMID:22925968

Klein, Christian; Sustmann, Claudio; Thomas, Markus; Stubenrauch, Kay; Croasdale, Rebecca; Schanzer, Jurgen; Brinkmann, Ulrich; Kettenberger, Hubert; Regula, Jorg T.; Schaefer, Wolfgang



Therapeutic bispecific antibodies cross the blood-brain barrier in nonhuman primates.  


Using therapeutic antibodies that need to cross the blood-brain barrier (BBB) to treat neurological disease is a difficult challenge. We have shown that bispecific antibodies with optimized binding to the transferrin receptor (TfR) that target ?-secretase (BACE1) can cross the BBB and reduce brain amyloid-? (A?) in mice. Can TfR enhance antibody uptake in the primate brain? We describe two humanized TfR/BACE1 bispecific antibody variants. Using a human TfR knock-in mouse, we observed that anti-TfR/BACE1 antibodies could cross the BBB and reduce brain A? in a TfR affinity-dependent fashion. Intravenous dosing of monkeys with anti-TfR/BACE1 antibodies also reduced A? both in cerebral spinal fluid and in brain tissue, and the degree of reduction correlated with the brain concentration of anti-TfR/BACE1 antibody. These results demonstrate that the TfR bispecific antibody platform can robustly and safely deliver therapeutic antibody across the BBB in the primate brain. PMID:25378646

Yu, Y Joy; Atwal, Jasvinder K; Zhang, Yin; Tong, Raymond K; Wildsmith, Kristin R; Tan, Christine; Bien-Ly, Nga; Hersom, Maria; Maloney, Janice A; Meilandt, William J; Bumbaca, Daniela; Gadkar, Kapil; Hoyte, Kwame; Luk, Wilman; Lu, Yanmei; Ernst, James A; Scearce-Levie, Kimberly; Couch, Jessica A; Dennis, Mark S; Watts, Ryan J



Development of a Two-part Strategy to Identify a Therapeutic Human Bispecific Antibody That Inhibits IgE Receptor Signaling*  

PubMed Central

The development of bispecific antibodies as therapeutic agents for human diseases has great clinical potential, but broad application has been hindered by the difficulty of identifying bispecific antibody formats that exhibit favorable pharmacokinetic properties and ease of large-scale manufacturing. Previously, the development of an antibody technology utilizing heavy chain knobs-into-holes mutations and a single common light chain enabled the small-scale generation of human full-length bispecific antibodies. Here we have extended the technology by developing a two-part bispecific antibody discovery strategy that facilitates proof-of-concept studies and clinical candidate antibody generation. Our scheme consists of the efficient small-scale generation of bispecific antibodies lacking a common light chain and the hinge disulfides for proof-of-concept studies coupled with the identification of a common light chain bispecific antibody for large-scale production with high purity and yield. We have applied this technology to generate a bispecific antibody suitable for development as a human therapeutic. This antibody directly inhibits the activation of the high affinity IgE receptor Fc?RI on mast cells and basophils by cross-linking Fc?RI with the inhibitory receptor Fc?RIIb, an approach that has strong therapeutic potential for asthma and other allergic diseases. Our approach for producing human bispecific full-length antibodies enables the clinical application of bispecific antibodies to a validated therapeutic pathway in asthma. PMID:20444694

Jackman, Janet; Chen, Yongmei; Huang, Arthur; Moffat, Barbara; Scheer, Justin M.; Leong, Steven R.; Lee, Wyne P.; Zhang, Juan; Sharma, Navneet; Lu, Yanmei; Iyer, Suhasini; Shields, Robert L.; Chiang, Nancy; Bauer, Michele C.; Wadley, Diana; Roose-Girma, Merone; Vandlen, Richard; Yansura, Daniel G.; Wu, Yan; Wu, Lawren C.



Monoclonal antibody therapy of cancer  

Microsoft Academic Search

The most significant recent advances in the application of monoclonal antibodies (mAbs) to oncology have been the introduction and approval of bevacizumab (Avastin), an anti–vascular endothelial growth factor antibody, and of cetuximab (Erbitux), an anti–epidermal growth factor antibody. In combination with standard chemotherapy regimens, bevacizumab significantly prolongs the survival of patients with metastatic cancers of the colorectum, breast and lung.

Gregory P Adams; Louis M Weiner



Uses of monoclonal antibody 8H9  


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.

Cheung, Nai-Kong V.



Monoclonal antibodies for treating cancer  

SciTech Connect

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

Dillman, R.O. (Hoag Cancer Center, Newport Beach, CA (USA))



Detection of Campylobacter species using monoclonal antibodies  

NASA Astrophysics Data System (ADS)

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.

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



Monoclonal antibodies: application in radiopharmacy.  


In this study was carried on a systematic review of the data was carried out in the topic of monoclonal antibodies in the last 40 years. All the data collected and summarized revealed that this new class of medicine may bring great advance in the field of radiopharmacy, oncology and imaging. PMID:24251731

Ligiero, Thais Braga; de Souza Albernaz, Marta; de Carvalho, Samira Marques; de Oliveira, Silvia Maria Velasques; Santos-Oliveira, Ralph



LC-MS characterization and purity assessment of a prototype bispecific antibody  

PubMed Central

Bispecific IgG asymmetric (heterodimeric) antibodies offer enhanced therapeutic efficacy, but present unique challenges for drug development. These challenges are related to the proper assembly of heavy and light chains. Impurities such as symmetric (homodimeric) antibodies can arise with improper assembly. A new method to assess heterodimer purity of such bispecific antibody products is needed because traditional separation-based purity assays are unable to separate or quantify homodimer impurities. This paper presents a liquid chromatography-mass spectrometry (LC-MS)-based method for evaluating heterodimeric purity of a prototype asymmetric antibody containing two different heavy chains and two identical light chains. The heterodimer and independently expressed homodimeric standards were characterized by two complementary LC-MS techniques: Intact protein mass measurement of deglycosylated antibody and peptide map analyses. Intact protein mass analysis was used to check molecular integrity and composition. LC-MSE peptide mapping of Lys-C digests was used to verify protein sequences and characterize post-translational modifications, including C-terminal truncation species. Guided by the characterization results, a heterodimer purity assay was demonstrated by intact protein mass analysis of pure deglycosylated heterodimer spiked with each deglycosylated homodimeric standard. The assay was capable of detecting low levels (2%) of spiked homodimers in conjunction with co-eluting half antibodies and multiple mass species present in the homodimer standards and providing relative purity differences between samples. Detection of minor homodimer and half-antibody C-terminal truncation species at levels as low as 0.6% demonstrates the sensitivity of the method. This method is suitable for purity assessment of heterodimer samples during process and purification development of bispecific antibodies, e.g., clone selection. PMID:23884083

Woods, R. Jeremy; Xie, Michael Hongwei; Von Kreudenstein, Thomas Spreter; Ng, Gordon Y.; Dixit, Surjit B.



Monoclonal antibody therapeutics with up to five specificities  

PubMed Central

The recognition that few human diseases are thoroughly addressed by mono-specific, monoclonal antibodies (mAbs) continues to drive the development of antibody therapeutics with additional specificities and enhanced activity. Historically, efforts to engineer additional antigen recognition into molecules have relied predominantly on the reformatting of immunoglobulin domains. In this report we describe a series of fully functional mAbs to which additional specificities have been imparted through the recombinant fusion of relatively short polypeptides sequences. The sequences are selected for binding to a particular target from combinatorial libraries that express linear, disulfide-constrained, or domain-based structures. The potential for fusion of peptides to the N- and C- termini of both the heavy and light chains affords the bivalent expression of up to four different peptides. The resulting molecules, called zybodies, can gain up to four additional specificities, while retaining the original functionality and specificity of the scaffold antibody. We explore the use of two clinically significant oncology antibodies, trastuzumab and cetuximab, as zybody scaffolds and demonstrate functional enhancements in each case. The affect of fusion position on both peptide and scaffold function is explored, and penta-specific zybodies are demonstrated to simultaneously engage five targets (ErbB2, EGFR, IGF-1R, Ang2 and integrin ?v?3). Bispecific, trastuzumab-based zybodies targeting ErbB2 and Ang2 are shown to exhibit superior efficacy to trastuzumab in an angiogenesis-dependent xenograft tumor model. A cetuximab-based bispecific zybody that targeting EGFR and ErbB3 simultaneously disrupted multiple intracellular signaling pathways; inhibited tumor cell proliferation; and showed efficacy superior to that of cetuximab in a xenograft tumor model. PMID:23575268

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



Multimodal Cancer Therapy Involving Oncolytic Newcastle Disease Virus, Autologous Immune Cells, and Bi-Specific Antibodies  

PubMed Central

This paper focuses on oncolytic Newcastle disease virus (NDV). This paper summarizes (i) the peculiarities of this virus as an anti-cancer and immune stimulatory agent and (ii) the approaches to further harness this virus as a vector to combat cancer. Special emphasis is given on combining virus therapy with cell therapy and on improving tumor targeting. The review will include some of the authors work on NDV, bi-specific antibodies, and cell therapy as building blocks for a new perspective of multimodal cancer therapy. The broad anti-tumor immune reactivation includes innate and adaptive, tumor antigen (TA) specific and TA independent activities PMID:25309868

Schirrmacher, Volker; Fournier, Philippe



A Monoclonal Antibody Against PMEL.  


PMEL, also known as Pmel17 or gp100, is a melanocyte-specific glycoprotein that is essential for the formation of stage II melanosomes. As it has a highly restricted expression pattern in normal tissues and a transient presence on the cell surface, PMEL is believed to be a potential target for antibody drug conjugate therapy in some pigmentary diseases. The production of a high specificity and high affinity monoclonal antibody against human PMEL was helpful for the antibody drug conjugate therapy study. In the present study, monoclonal antibodies (MAbs) against PMEL were obtained by immunizing BALB/c mice with the recombinant PMEL-GST fusion protein. Three mAbs (A3F, G11B, and J7E) with a titer of 1:6000, 1:10,000, and 1:3000, respectively, were obtained. Immunoglobulin subclass assay revealed that A3F was IgG2b, G11B was IgG1, and J7E was IgG2a. Specificity analysis by Western blotting demonstrated that A3F and J7E cross-reacted with GPNMB or LAMP; however, G11B reacted with PMEL only. Immunohistochemistry experiments showed that G11B could bind human PMEL antigen in normal skin. Flow cytometry assay demonstrated that G11B could bind to the surface of PMEL positive melanoma cells but not PMEL negative cells. Taken together, these results show that this G11B provides a useful tool for the antibody drug conjugate therapy study in some pigmentary diseases. PMID:25118787

Shi, Fangyuan; Xu, Zhenjie; Chen, Hongdong; Wang, Xin; Cui, Jihong; Zhang, Ping; Zhang, Ping; Xie, Xin



A new class of bispecific antibodies to redirect T cells for cancer immunotherapy.  


Various constructs of bispecific antibodies (bsAbs) to redirect effector T cells for the targeted killing of tumor cells have shown considerable promise in both preclinical and clinical studies. The single-chain variable fragment (scFv)-based formats, including bispecific T-cell engager (BiTE) and dual-affinity re-targeting (DART), which provide monovalent binding to both CD3 on T cells and to the target antigen on tumor cells, can exhibit rapid blood clearance and neurological toxicity due to their small size (~55 kDa). Herein, we describe the generation, by the modular DOCK-AND-LOCK™) (DNL™) method, of novel T-cell redirecting bispecific antibodies, each comprising a monovalent anti-CD3 scFv covalently conjugated to a stabilized dimer of different anti-tumor Fabs. The potential advantages of this design include bivalent binding to tumor cells, a larger size (~130 kDa) to preclude renal clearance and penetration of the blood-brain barrier, and potent T-cell mediated cytotoxicity. These prototypes were purified to near homogeneity, and representative constructs were shown to provoke the formation of immunological synapses between T cells and their target tumor cells in vitro, resulting in T-cell activation and proliferation, as well as potent T-cell mediated anti-tumor activity. In addition, in vivo studies in NOD/SCID mice bearing Raji Burkitt lymphoma or Capan-1 pancreatic carcinoma indicated statistically significant inhibition of tumor growth compared with untreated controls. PMID:24492297

Rossi, Diane L; Rossi, Edmund A; Cardillo, Thomas M; Goldenberg, David M; Chang, Chien-Hsing



Uses of monoclonal antibody 8H9  


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

Cheung, Nai-Kong V



The future of monoclonal antibody technology  

PubMed Central

With the rapid growth of monoclonal antibody-based products, new technologies have emerged for creating modified forms of antibodies, including fragments, conjugates and multi-specific antibodies. We created a database of 450 therapeutic antibodies in development to determine which technologies and indications will constitute the “next generation” of antibody products. We conclude that the antibodies of the future will closely resemble the antibodies that have already been approved for commercial sale. PMID:20676053

Zider, Alexander



An alternative chemical redox method for the production of bispecific antibodies: implication in rapid detection of food borne pathogens.  


Bi-functional antibodies with the ability to bind two unrelated epitopes have remarkable potential in diagnostic and bio-sensing applications. In the present study, bispecific antibodies that recognize human red blood cell (RBC) and the food borne pathogen Listeria monocytogenes (L. monocytogenes) were engineered. The procedure involves initial reduction of a mixture of anti-RBC and anti-Listeria antibodies followed by gradual re-oxidation of the reduced disulphides. This facilitates association of the separated antibody chains and formation of hybrid immunoglobulins with affinity for the L. monocytogenes and human RBC. The bispecific antibodies caused the agglutination of the RBCs only in the presence of L. monocytogenes cells. The agglutination process necessitated the specific presence of L. monocytogenes and the red colored clumps formed were readily visible with naked eyes. The RBC agglutination assay described here provides a remarkably simple approach for the rapid and highly specific screening of various pathogens in their biological niches. PMID:24637674

Owais, Mohammad; Kazmi, Shadab; Tufail, Saba; Zubair, Swaleha



Improved monoclonal antibodies to halodeoxyuridine  


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

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



Improving biophysical properties of a bispecific antibody scaffold to aid developability: quality by molecular design.  


While the concept of Quality-by-Design is addressed at the upstream and downstream process development stages, we questioned whether there are advantages to addressing the issues of biologics quality early in the design of the molecule based on fundamental biophysical characterization, and thereby reduce complexities in the product development stages. Although limited number of bispecific therapeutics are in clinic, these developments have been plagued with difficulty in producing materials of sufficient quality and quantity for both preclinical and clinical studies. The engineered heterodimeric Fc is an industry-wide favorite scaffold for the design of bispecific protein therapeutics because of its structural, and potentially pharmacokinetic, similarity to the natural antibody. Development of molecules based on this concept, however, is challenged by the presence of potential homodimer contamination and stability loss relative to the natural Fc. We engineered a heterodimeric Fc with high heterodimeric specificity that also retains natural Fc-like biophysical properties, and demonstrate here that use of engineered Fc domains that mirror the natural system translates into an efficient and robust upstream stable cell line selection process as a first step toward a more developable therapeutic. PMID:23924797

Von Kreudenstein, Thomas Spreter; Escobar-Carbrera, Eric; Lario, Paula I; D'Angelo, Igor; Brault, Karine; Kelly, John; Durocher, Yves; Baardsnes, Jason; Woods, R Jeremy; Xie, Michael Hongwei; Girod, Pierre-Alain; Suits, Michael D L; Boulanger, Martin J; Poon, David K Y; Ng, Gordon Y K; Dixit, Surjit B



Original article Monoclonal antibodies against goldfish  

E-print Network

immunoglobulin and antibody levels by ELISA in carp (Cyprinus carpio) AK Siwicki C Vergnet J Charlemagne M Dunier decreased until day 28. monoclonal antibody / ELISA / ELISPOT / antibody-secreting cells / Cyprinus carpio suite. anticorps monoc/ona//EL/S/Cyprinus carpio

Paris-Sud XI, Université de


Cost modeling for monoclonal antibody manufacturing  

E-print Network

The Novartis BioPharmOps division is responsible for manufacturing large molecule products, including monoclonal antibodies, for late stage clinical trials and commercial sales. The BioPharmOps site in Huningue, France is ...

Simpson, Christina M. (Christina Margaret)



T cell activation and retargeting using staphylococcal enterotoxin B and bispecific antibody: An effective in vivo antitumor strategy  

Microsoft Academic Search

The aim of this work was to test for cure and immunity in a micrometastatic tumor model using in vivo T cell activation with\\u000a staphylococcal enterotoxin B (SEB) and retargeting with antitumor×anti-CD3 F(ab?)2 bispecific antibodies (bsAb). All studies were performed in C3H\\/HeN mice using syngeneic tumor cell lines. For survival studies,\\u000a mice were injected intravenously on day 0 with CL62

Lewis E. Porterm; Heidi Nelson; I. Ethem Gecim; David C. Rice; Claude Thibault; Andrei I. Chapoval



CD3 × CD28 cross-interacting bispecific antibodies improve tumor cell dependent T-cell activation  

Microsoft Academic Search

Bispecific antibodies (Bs-Abs) containing an anti-CD3 and an anti-TAA specificity can recruit T cells to the tumor for cancer immunotherapy. To be effective, efficient activation at the tumor site is a prerequisite. This can be achieved by triggering both the T-cell receptor and the co-stimulatory molecule CD28. We engineered two recombinant cross-interacting Bs-Abs (CriBs-Abs) by incorporating a peptide tag and

An Willems; Steve Schoonooghe; Dominique Eeckhout; Geert De Jaeger; Johan Grooten; Nico Mertens



Cytosine Arabinoside Promotes Cytotoxic Effect of T Cells on Leukemia Cells Mediated by Bispecific Antibody  

PubMed Central

Abstract Chemotherapeutic drugs can enhance an immune response of the host against the tumor in addition to killing cancer cells by direct cytotoxicity. Therefore, the combination of chemotherapy and immunotherapy is a promising approach for eliminating tumors, particularly in advanced stages. A strategic medication is to use a bispecific antibody format that is capable of recruiting polyclonal T cells around antibody-target-expressing tumor cells. Recently, we have constructed a bispecific antibody, anti-CD3×anti-CD19, in a diabody configuration. In this study, we measured B7 family members B7.1 (CD80) and B7.2 (CD86) expressed on a CD19+ human leukemia cell line, Nalm-6, stimulated by cytosine arabinoside (Ara-C). We found that a low concentration of Ara-C could upregulate CD80 expressed on CD19+ Nalm-6 cells. The cytotoxicity of T lymphocytes against Nalm-6 cells in vitro and in vivo mediated by the anti-CD3×anti-CD19 diabody with or without a low dose of Ara-C was compared. The combination of the anti-CD3×anti-CD19 diabody and Ara-C showed the greatest effectiveness in enhancing the cytotoxicity of T cells against the tumor cells in vitro and in vivo. Activated T cells expressed higher levels of CD25 and CD69 and released more interleukin 2. Both perforin/granzyme B system and Fas/FasL pathway were involved in the diabody-induced T-cell cytotoxicity. Moreover, the activated T cells could upregulate ICAM-3 expression on Nalm-6 cells, and inhibition of LFA-1–ICAM-3 interaction impaired cytotoxicity of T cells. It was noted that Ara-C could upregulate CD80 expressed on two of five specimens of acute B lymphoblastic leukemia patient-derived cells. Cytotoxicity of T cells against these two patient-derived cells was enhanced in the presence of the anti-CD3×anti-CD19 diabody. These findings indicate that treatment strategy using both cytotoxic lymphocyte-based immunotherapy and chemotherapy may have synergistic effects. PMID:23879717

Li, Wei; Yang, Ming; Yan, Yan; Shi, RuiZan; Cheng, JunPing; Li, ZhenZhen; Zhang, MengNan; Wang, JianXiang; Xiong, DongSheng



A novel bispecific EGFR/Met antibody blocks tumor-promoting phenotypic effects induced by resistance to EGFR inhibition and has potent antitumor activity  

PubMed Central

Simultaneous targeting of epidermal growth factor receptor (EGFR) and Met in cancer therapy is under pre-clinical and clinical evaluation. Here, we report the finding that treatment with EGFR inhibitors of various tumor cells, when stimulated with hepatocyte growth factor (HGF) and EGF, results in transient upregulation of phosphorylated AKT. Furthermore, EGFR inhibition in this setting stimulates a pro-invasive phenotype as assessed in Matrigel-based assays. Simultaneous treatment with AKT and EGFR inhibitors abrogates this invasive growth, hence functionally linking signaling and phenotype. This observation implies that during treatment of tumors a balanced ratio of EGFR and Met inhibition is required. To address this, we designed a bispecific antibody targeting EGFR and Met, which has the advantage of a fixed 2:1 stoichiometry. This bispecific antibody inhibits proliferation in tumor cell cultures and co-cultures with fibroblasts in an additive manner compared with treatment with both single agents. In addition, cell migration assays reveal a higher potency of the bispecific antibody in comparison with the antibodies' combination at low doses. We demonstrate that the bispecific antibody inhibits invasive growth, which is specifically observed with cetuximab. Finally, the bispecific antibody potently inhibits tumor growth in a non-small cell lung cancer xenograft model bearing a strong autocrine HGF-loop. Together, our findings strongly support a combination treatment of EGFR and Met inhibitors and further evaluation of resistance mechanisms to EGFR inhibition in the context of active Met signaling. PMID:23812422

Castoldi, R; Ecker, V; Wiehle, L; Majety, M; Busl-Schuller, R; Asmussen, M; Nopora, A; Jucknischke, U; Osl, F; Kobold, S; Scheuer, W; Venturi, M; Klein, C; Niederfellner, G; Sustmann, C



Monoclonal antibodies to rat renal antigens.  

PubMed Central

We have developed hybridoma cell lines which secrete monoclonal antibodies to some rat renal antigens, namely the brush border of proximal tubular epithelium and the cytoplasm of tubular cells. The immunoglobulin class of the hybridoma was found to be IgG1. Specific antibody activity against either glomerular basement membrane (GBM) and tubular basement membrane (TBM) or Bowman's capsule and a part of TBM was observed, although these hybridoma cell lines have not yet been successfully established. In particular, the hybridoma secreting antibodies to TBM did not remain stable during antibody production, and was lost during the culture and cloning procedures. These monoclonal antibodies should be of value in research on the pathogenesis of human glomerulonephritis. Images Figure 1 Figure 2 PMID:6376337

Shimizu, F; Orikasa, M; Sato, K; Oite, T



Production of human monoclonal antibodies to myeloperoxidase.  

PubMed Central

Two mouse-human heterohybridomas secreting human antibodies to myeloperoxidase (MPO) were derived from the peripheral blood of a patient who developed microscopic polyarteritis as the result of long-term treatment with hydralazine. Forty-five immunoglobulin-secreting lines were obtained from the fusion of patient lymphocytes with the CB-F7 heteromyeloma cell line. Of these, two antibodies, one IgG and one IgM, bound to myeloperoxidase in solid phase ELISA and gave a perinuclear staining pattern on ethanol-fixed human neutrophil cytospin preparations. The staining patterns were similar to those seen with serum from the patient. Antigen-inhibition studies revealed that the affinity of the IgG monoclonal antibody was 28 times higher (k = 1.4 x 10(-7)) than the IgM antibody (k = 5 x 10(-5)). Cross-inhibition studies further suggested that the two monoclonal antibodies recognized the same epitope on MPO. Of the other secreting cell lines, none produced antibody which reacted with the panel of autoantigens used for testing. Neither mononuclear antibody reacted with this panel indicating that they were not simply polyreactive natural autoantibodies. These are the first human monoclonal antibodies to native myeloperoxidase to be reported. Images Figure 2 Figure 3 PMID:1337335

Ehrenstein, M R; Leaker, B; Isenberg, D; Cambridge, G



Magic Bullets and Monoclonals: An Antibody Tale  

NSDL National Science Digital Library

FASEB Breakthroughs in Bioscience article. This most recent article describes the century of fundamental immunology research that led to todayÃÂs cutting edge monoclonal antibody therapies, used to treat millions of patients for several types of cancer, autoimmune and inflammatory disorders, and infectious disease.

Margie Patlak (Federation of American Societies for Experimental Biology Office of Public Affairs)



Subcutaneous Administration of Monoclonal Antibodies in Oncology  

PubMed Central

Treatment with monoclonal antibodies (mabs) has become an established component of oncological therapy. The monoclonal antibodies available for this purpose are mainly administered intravenously in individually adapted doses according to body weight over longer treatment times. For other chronic diseases such as, for example, diabetes mellitus, the subcutaneous administration of drugs is an established therapy option. For the subcutaneous administration of larger volumes as needed for mab solutions the extracellular matrix of the subcutaneous tissue represents a problem. The co-formulation with recombinant human hyaluronidase makes the relatively pain-free administration of larger fluid volumes and thus the subcutaneous administration of monoclonal antibodies possible, as illustrated by the development of a subcutaneous formulation of trastuzumab. This constitutes a less invasive, time-optimised and flexible form of administration for patients with HER2-positive breast cancer that, with its fixed dosing possibilities, contributes to therapeutic safety. The example of trastuzumab shows that the subcutaneous administration of monoclonal antibodies can simplify oncological long-term therapy not only for the patients but also for the medical personnel. PMID:25076790

Jackisch, C.; Muller, V.; Maintz, C.; Hell, S.; Ataseven, B.



Innovative monoclonal antibody therapies in multiple sclerosis  

Microsoft Academic Search

The recent years have witnessed great efforts in establishing new therapeutic options for multiple sclerosis (MS), especially for relapsing-remitting disease courses. In particular, the application of monoclonal antibodies provide innovative approaches allowing for blocking or depleting specific molecular targets, which are of interest in the pathogenesis of MS. While natalizumab received approval by the US Food and Drug Administration and

Ralf A. Linker; Bernd C. Kieseier


Human Monoclonal Antibody Against Mesothelin

Mesothelin is a cell surface protein that is naturally expressed at very low levels, but that is significantly increased in aggressive tumors such as mesotheliomas, and pancreatic and ovarian tumors. Therefore, mesothelin is an excellent candidate for tumor-targeted immunotherapeutics. However, the only antibodies against mesothelin that are currently available for clinical trials are of murine origin. The use of these antibodies may be limited by their potential to elicit adverse immune responses in patients with repeated doses.


Functional comparison of single-chain and two-chain anti-CD3-based bispecific antibodies in gene immunotherapy applications  

PubMed Central

Gene therapy to achieve in vivo secretion of recombinant anti-CD3 x anti-tumor bispecific antibodies in cancer patients is being explored as a strategy to counterbalance rapid renal elimination, thereby sustaining levels of bispecific antibodies in the therapeutic range. Here, we performed a comparative analysis between single- and two-chain configurations for anti-CD3 x anti-CEA (carcinoembryonic antigen) bispecific antibodies secreted by genetically-modified human cells. We demonstrate that tandem single-chain variable fragment (scFv) antibodies and two-chain diabodies are expressed as soluble secreted proteins with similar yields. However, we found significant differences in their biological functionality (i.e., antigen binding) and in their ability to induce non-specific T cell activation. Whereas single-chain tandem scFvs induced human T cell activation and proliferation in an antigen-independent manner, secreted two-chain diabodies exerted almost no proliferative stimulus when human T cells were cultured alone or in co-cultures with CEA negative cells. Thus, our data suggest that two-chain diabodies are preferable to single-chain tandem scFvs for immunotherapeutic strategies comprising in vivo secretion of bispecific antibodies aiming to recruit and activate anticancer specific lymphocytic effector T cells. PMID:25057445

Compte, Marta; Alvarez-Cienfuegos, Ana; Nunez-Prado, Natalia; Sainz-Pastor, Noelia; Blanco-Toribio, Ana; Pescador, Nuria; Sanz, Laura; Alvarez-Vallina, Luis



Innovative Monoclonal Antibody Therapies in Multiple Sclerosis  

PubMed Central

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

Kieseier, Bernd C.



Next generation and biosimilar monoclonal antibodies  

PubMed Central

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



Monoclonal antibody targets, kills leukemia cells

Researchers at the University of California, San Diego Moores Cancer Center have identified a humanized monoclonal antibody that targets and directly kills chronic lymphocytic leukemia (CLL) cells. The findings, published in the online Early Edition of the Proceedings of the National Academy of Sciences on March 25, 2013 represent a potential new therapy for treating at least some patients with CLL, the most common type of blood cancer in the United States.


[Therapeutic monoclonal antibodies in onco-hematology].  


Rituximab, a chimeric monoclonal anti-CD20 antibody, was introduced into clinical practice in 1997, and has proven to be highly effective in the treatment of B-lymphoproliferative disorders and autoimmune diseases. Despite such success, in vivo mechanisms of action of anti-CD20 have only recently began to be unraveled, pointing to the crucial role of antibody-dependent cellular cytotoxicity response mediated through Fcg receptor signalling. Better understanding of pharmacokinetics and factors influencing individual exposure mediated through anti-CD20 will allow to engineer these molecules to increase their effector responses. Meanwhile, other formats have also been investigated, such as radiolabeled anti-CD20, or coupling of antibodies to cytotoxic drugs such as anti-CD33 used in myeloid leukemia. However these antibodies are used in combination with standard chemotherapy and cannot substitute for cytotoxic drugs. This review summarizes the knowledge acquired through our clinical use of anti-CD20 and authorized monoclonal antibodies in oncohematology and proposes some news areas that will lead to the development of new and more effective therapeutic strategies. PMID:20035683

Cartron, Guillaume; Rossi, Jean-François



Development of murine monoclonal antibodies to methamphetamine and methamphetamine analogues  

Microsoft Academic Search

Methamphetamine and ecstasy are addictive drugs that cause major health problems in young people. Here we report on the development of high-affinity monoclonal antibodies to methamphetamine and its analogues, which may constitute powerful tools for antibody-based therapy. Six haptens, methamphetamine and ecstasy analogues, were synthesized, linked to a carrier protein and injected into mice. Several specific monoclonal antibodies were subsequently

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



Monoclonal antibodies and method for detecting dioxins and dibenzofurans  


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

Vanderlaan, Martin (San Ramon, CA); Stanker, Larry H. (Livermore, CA); Watkins, Bruce E. (Livermore, CA); Bailey, Nina R. (Berkley, CA)



Monoclonal antibodies specific for sickle cell hemoglobin  

SciTech Connect

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

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



Trial Watch: Monoclonal antibodies in cancer therapy.  


Since the advent of hybridoma technology, dating back to 1975, monoclonal antibodies have become an irreplaceable diagnostic and therapeutic tool for a wide array of human diseases. During the last 15 years, several monoclonal antibodies (mAbs) have been approved by FDA for cancer therapy. These mAbs are designed to (1) activate the immune system against tumor cells, (2) inhibit cancer cell-intrinsic signaling pathways, (3) bring toxins in the close proximity of cancer cells, or (4) interfere with the tumor-stroma interaction. More recently, major efforts have been made for the development of immunostimulatory mAbs that either enhance cancer-directed immune responses or limit tumor- (or therapy-) driven immunosuppression. Some of these antibodies, which are thought to facilitate tumor eradication by initiating or sustaining a tumor-specific immune response, have already entered clinical trials. In this Trial Watch, we will review and discuss the clinical progress of the most important mAbs that are have entered clinical trials after January 2008. PMID:22720209

Galluzzi, Lorenzo; Vacchelli, Erika; Fridman, Wolf Hervé; Galon, Jerome; Sautès-Fridman, Catherine; Tartour, Eric; Zucman-Rossi, Jessica; Zitvogel, Laurence; Kroemer, Guido



Phylogenetic study of transcortin using monoclonal antibodies.  


We produced monoclonal antibodies that recognise three distinct epitopes of human transcortin. These epitopes are present on transcortin of humans with normal and altered transcortin levels, as well as on a variant with lower affinity for cortisol. One epitope is present on transcortin of Old World Monkeys and apes, the others are only present on transcortin of apes. The epitopes are not present on transcortin of other species. These results indicate that human transcortin contains a highly evolved and a more conserved part. PMID:2428359

Faict, D; De Moor, P



Bispecific multivalent antibody studied by real-time interaction analysis for the development of an antigen-inhibition enzyme-linked immunosorbent assay.  


A bispecific antibody with specificities for both 7-hydroxycoumarin (7-OHC) and alkaline phosphatase (AP) was produced by chemically cross-linking two parental polyclonal antibodies. Real-time interaction analysis of the bispecific multivalent antibody (bsMAb) was performed using BIAcore, a surface plasmon resonance (SPR)-based biosensor, in order to confirm its bispecific nature. A 7-OHC-BSA conjugate was covalently immobilized to a dextran matrix to serve as the reaction surface and unconjugated bovine serum albumin (BSA) was immobilized on to a separate dextran matrix as a control surface. Immunoaffinity-purified bsMAb, parental anti-7-OHC antibody and AP were injected over both surfaces. The bsMAb was shown to bind both antigens, 7-OHC and AP, simultaneously. Comparison of the ratio of mass bound for bsMAb and AP (5:1) with the ratio of the molecular masses of bsMAb (approximately 300 kDa) and AP (85 kDa) (3.5:1) suggests that most of the bsMAb species possess both specificities. The bsMAb was employed in a one-step antigen-inhibition ELISA for the detection of 7-OHC. The assay was compared with a conventional ELISA approach employing an AP-labelled secondary antibody. The bispecific antibody approach proved to be faster and more sensitive, with a detection limit of 6 ng ml-1 as compared with approximately 50 ng ml-1 for the conventional approach. The assay was used for the quantification of free and total 7-OHC in urine samples from two healthy volunteers who had been administered coumarin. The accuracy and precision of the assay were assessed. The bispecific antibody-based assay gave similar results, accuracy and precision, but proved to be far more sensitive (limit of determination 6 ng ml-1 for total 7-OHC). It is concluded that real-time interaction analysis using BIAcor provides a rapid method for the evaluation of the bsMAb and it was verified that the bispecific product formed by chemical cross-linking of two parental antibodies offers a simple alternative for the development of a highly sensitive ELISA. PMID:8763206

Reinartz, H W; Quinn, J G; Zänker, K; O'Kennedy, R



Screening individual hybridomas by microengraving to discover monoclonal antibodies  

E-print Network

The demand for monoclonal antibodies (mAbs) in biomedical research is significant, but the current methodologies used to discover them are both lengthy and costly. Consequently, the diversity of antibodies available for ...

Ogunniyi, Adebola Oluwakayode


Monoclonal Antibodies against Cytosolic Thyroid Hormone Binding Protein.  

National Technical Information Service (NTIS)

The present invention related to the preparation of monoclonal antibodies having specific binding affinity for a cytoplasmic thyroid hormone binding protein. Heretofore, not even polyclonal antibodies to the cytosolic thyroid hormone binding protein exist...

S. Y. Cheng



NCI Requests Cancer Targets for Monoclonal Antibody Production and Characterization

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.


NCI Requests Targets for Monoclonal Antibody Production and Characterization

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.


Virotherapy, gene transfer and immunostimulatory monoclonal antibodies  

PubMed Central

Malignant cells are susceptible to viral infection and consequent cell death. Virus-induced cell death is endowed with features that are known to stimulate innate and adaptive immune responses. Thus danger signals emitted by cells succumbing to viral infection as well as viral nucleic acids are detected by specific receptors, and tumor cell antigens can be routed to professional antigen-presenting cells. The anticancer immune response triggered by viral infection is frequently insufficient to eradicate malignancy but may be further amplified. For this purpose, transgenes encoding cytokines as co-stimulatory molecules can be genetically engineered into viral vectors. Alternatively, or in addition, it is possible to use monoclonal antibodies that either block inhibitory receptors of immune effector cells, or act as agonists for co-stimulatory receptors. Combined strategies are based on the ignition of a local immune response at the malignant site plus systemic immune boosting. We have recently reported examples of this approach involving the Vaccinia virus or Semliki Forest virus, interleukin-12 and anti-CD137 monoclonal antibodies. PMID:23243597

Quetglas, Jose I.; John, Liza B.; Kershaw, Michael H.; Alvarez-Vallina, Luis; Melero, Ignacio; Darcy, Phillip K.; Smerdou, Cristian



A new monoclonal antibody to human subcapsular thymic epithelial cells  

Microsoft Academic Search

Summary A monoclonal antibody, termed K-20, was generated against an anaplastic thymic carcinoma cell line, Ty-82. Subcapsular thymic epithelial cells of the thymus and blood vessels in various organs were shown to react with the K-20 monoclonal antibody by immunohistochemical staining. Immunofluorescent study revealed that various haematopoietic fresh cells and cell lines did not show any significant reactivity with K-20,

Tamotsu Takeuchi; Ichiro Kubonishi; Yuji Ohtsuki; Isao Miyoshi



Monoclonal Antibodies Against Xenopus Greatwall Kinase  

PubMed Central

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

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



Monoclonal antibodies against Xenopus greatwall kinase.  


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

Wang, Ling; Fisher, Laura A; Wahl, James K; Peng, Aimin



Monoclonal antibodies based on hybridoma technology.  


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

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



SPECT assay of radiolabeled monoclonal antibodies  

SciTech Connect

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

Jaszczak, R.J.



Production and characterization of profilin monoclonal antibodies.  


Profilins have been identified as a pan-allergen of different plant pollens and foods. In this paper, we describe the generation of monoclonal antibodies (MAbs) by immunizing BALB/c mice with Helianthus annuus purified profilin in order to characterize this important and common allergen. A panel of forty MAbs has been obtained, and twenty of them were used to map antigenic determinants in this molecule. At least two different antigenic determinants were recognized in H. annuus profilin by immunoblotting. Using the purified MAbs produced against sunflower profilin, we have analyzed the common epitope determinants in pollens of different plants: Olea europaea, Cynodon dactylon, Mercurialis annua, Phleum pratense, Parietaria judaica and Betula verrucosa. These experiments showed different cross-reactivity patterns. PMID:9208051

Arilla, M C; Asturias, J A; Gómez-Bayón, N; Martínez, A; Martínez, J; Palacios, R



Simultaneous targeting of TNF and Ang2 with a novel bispecific antibody enhances efficacy in an in vivo model of arthritis.  


Despite the clinical success of anti-tumor necrosis factor (TNF) therapies in the treatment of inflammatory conditions such as rheumatoid arthritis, Crohn disease and psoriasis, full control of the diseases only occurs in a subset of patients and there is a need for new therapeutics with improved efficacy against broader patient populations. One possible approach is to combine biological therapeutics, but both the cost of the therapeutics and the potential for additional toxicities needs to be considered. In addition to the various mediators of immune and inflammatory pathways, angiogenesis is reported to contribute substantially to the overall pathogenesis of inflammatory diseases. The combination of an anti-angiogenic agent with anti-TNF into one molecule could be more efficacious without the risk of severe immunosuppression. To evaluate this approach with our Zybody technology, we generated bispecific antibodies that contain an Ang2 targeting peptide genetically fused to the anti-TNF antibody adalimumab (Humira®). The bispecific molecules retain the binding and functional characteristics of the anti-TNF antibody, but with additional activity that neutralizes Ang2. In a TNF transgenic mouse model of arthritis, the bispecific anti-TNF-Ang2 molecules showed a dose-dependent reduction in both clinical symptoms and histological scores that were significantly better than that achieved by adalimumab alone. PMID:22864384

Kanakaraj, Palanisamy; Puffer, Bridget A; Yao, Xiao-Tao; Kankanala, Spandana; Boyd, Ernest; Shah, Rutul R; Wang, Geping; Patel, Dimki; Krishnamurthy, Rajesh; Kaithamana, Shashi; Smith, Rodger G; LaFleur, David W; Barbas, Carlos F; Hilbert, David M; Kiener, Peter A; Roschke, Viktor V



Autoantibody potential of cancer therapeutic monoclonal antibodies.  


We and others have reported that multiple autoantibodies are unmasked in human polyclonal antibody preparations after exposure to physiological oxidizing agents (hemin) or electromotive force. We now have asked if oxidation unmasks autoantibody reactivities in monoclonal antibodies (mAb). To do this, we have studied 9 FDA approved mAb used therapeutically, including 4 chimeric, 4 humanized and 1 chemically modified chimeric Fab that were exposed to the physiological oxidizing agent hemin at 36 degrees C for 20 hr. These mAb were studied for autoantibody activity to phospholipids and DNA before and after oxidation with hemin and found to develop autoantibody activities after oxidation, while retaining their original specificity as measured by mAb anti-glycophorin A binding of erythrocytes, CD 19 binding to B lymphocytes and anti-HLA-A29 binding to A29-positive lymphocytes. The finding that certain mAb have the potential to unmask autoantibody activities as a consequence of exposure to physiological redox reactions in vitro gives pause to our present understanding of the immunological basis of tolerance and concern for potential autoimmune side effects in patients receiving mAb for diagnosis or treatment. PMID:19904753

McIntyre, John A; Faulk, And W Page



Retargeting T Cells for HER2-Positive Tumor Killing by a Bispecific Fv-Fc Antibody  

PubMed Central

To exploit the biological and pharmacological properties of immunoglobulin constant domain Fc fragment and increase the killing efficacy of T cells, a single chain variable fragment specific to CD3 was fused with Fcab (Fc antigen binding), a mutant Fc fragment with specificity against Human epidermal growth factor receptor 2 (HER2) developed by F-star. The bispecific fusion named as FcabCD3 was expressed by transient transfection in HEK-293T cells and purified by affinity chromatography. Specific cytolytic activity of retargeted T cells to kill HER2 positive SKBR3 cell line was evaluated in vitro. FcabCD3 was able to retarget T cells to kill both Herceptin insensitive Colo205-luc cell line and HER2 low expression MDA-MB-231-luc cell line. Furthermore, FcabCD3 was effective in eliminating the Colo205 tumor established on BALB/c nu/nu mice. PMID:24086580

Wang, Lei; He, Yanran; Zhang, Ge; Ma, Juan; Liu, Changzhen; He, Wen; Wang, Wei; Han, Huamin; Boruah, Bhargavi M.; Gao, Bin



Monoclonal antibody specific for a pigmentation associated antigen  

SciTech Connect

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

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



Structural design of disialoganglioside GD2 and CD3-bispecific antibodies to redirect T cells for tumor therapy.  


Antibody-based immunotherapy has proven efficacy for patients with high-risk neuroblastoma. However, despite being the most efficient tumoricidal effectors, T cells are underutilized because they lack Fc receptors. Using a monovalent single-chain fragment (ScFv) platform, we engineered tandem scFv bispecific antibodies (BsAbs) that specifically target disialoganglioside (GD2) on tumor cells and CD3 on T cells. Structural variants of BsAbs were constructed and ranked based on binding to GD2, and on competency in inducing T-cell-mediated tumor cytotoxicity. In vitro thermal stability and binding measurements were used to characterize each of the constructs, and in silico molecular modeling was used to show how the orientation of the variable region heavy (VH) and light (VL) chains of the anti-GD2 ScFv could alter the conformations of key residues responsible for high affinity binding. We showed that the VH-VL orientation, the (GGGGS)3 linker, disulfide bond stabilization of scFv, when combined with an affinity matured mutation provided the most efficient BsAb to direct T cells to lyse GD2-positive tumor cells. In vivo, the optimized BsAb could efficiently inhibit melanoma and neuroblastoma xenograft growth. These findings provide preclinical validation of a structure-based method to assist in designing BsAb for T-cell-mediated therapy. PMID:24895182

Cheng, Ming; Ahmed, Mahiuddin; Xu, Hong; Cheung, Nai-Kong V



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

Microsoft Academic Search

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 antibodies have been applied to biological and medical sciences.In animal production monoclonal antibodies are increasingly finding application in

P. Booman



Human and Improved Murine Monoclonal Antibodies Against CD22

The National Cancer Institute's Nanobiology Program is seeking statements of capability or interest from parties interested in collaborative research to further develop, evaluate, or commercialize human monoclonal antibodies expressed in types of lymphoma.



E-print Network

agglutina- tion of rat red blood cells ( criticisms and his encouragements. J.F. Vautherot, J. Laporte Abstract Hybrid cells secreting monoclonal antibodies against Bovine Enteric Coronavirus (BECV strain G110) were obtained by fusion between SP2

Paris-Sud XI, Université de



EPA Science Inventory

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


Anti-Mesothelin Monoclonal Antibodies for the Treatment of Cancer

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


Monoclonal Antibody-Mediated Tumor Regression by Induction of Apoptosis  

Microsoft Academic Search

To characterize cell surface molecules involved in control of growth of malignant lymphocytes, monoclonal antibodies were raised against the human B lymphoblast cell line SKW6.4. One monoclonal antibody, anti-APO-1, reacted with a 52-kilodalton antigen (APO-1) on a set of activated human lymphocytes, on malignant human lymphocyte lines, and on some patient-derived leukemic cells. Nanogram quantities of anti-APO-1 completely blocked proliferation

Bernhard C. Trauth; Christiane Klas; Anke M. J. Peters; Siegfried Matzku; Peter Moller; Werner Falk; Klaus-Michael Debatin; Peter H. Krammer



Monoclonal Antibodies for Systemic Lupus Erythematosus (SLE) †  

PubMed Central

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

Ponticelli, Claudio; Moroni, Gabriella



A Monoclonal Antibody Against Human MUDENG Protein  

PubMed Central

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. PMID:23909422

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



Radioiodacao de anticorpo monoclonal anti-CEA intacto. (Radioiodination of monoclonal antibody intact anti-CEA).  

National Technical Information Service (NTIS)

The purpose of this study is to examine a convenient system that can be used to iodinate monoclonal antibodies which is rapid, simple, efficient and reproducible, and which can be accomplished in radiopharmaceutical laboratories. It is important to rememb...

H. Okada, I. T. T. Souza, C. P. G. Silva



Characterization and utilization of a monoclonal antibody against pancreatic carcinoma  

SciTech Connect

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

Kurtzman, S.H.; Sindelar, W.F.; Atcher, R.W.; Mitchell, J.B.; DeGraff, W.G.; Gamson, J.; Russo, A. [National Cancer Institute, Bethesda, MD (United States); Friedman, A.M.; Hines, J.J. [Argonne National Lab., IL (United States)



Rescue of Impaired NK Cell Activity in Hodgkin Lymphoma With Bispecific Antibodies In Vitro and in Patients  

PubMed Central

Natural killer (NK) cells represent a key component of the innate immune system against cancer. Nevertheless, malignant diseases arise in immunocompetent individuals despite tumor immunosurveillance. Hodgkin lymphoma (HL) is characterized by CD30+ tumor cells and a massive infiltration of immune effector cells in affected lymph nodes. The latter obviously fail to eliminate the malignant cell population. Here, we tested for functional NK cell defects in HL and suggest an improvement of NK function by therapeutic means. We demonstrate that peripheral NK cells (pNK) from patients with HL fail to eliminate HL cell lines in ex vivo killing assays. Impaired NK cell function correlated with elevated serum levels of soluble ligands for NK cell receptors NKp30 (BAG6/BAT3) and NKG2D (MICA), factors known to constrict NK cell function. In vitro, NK cell cytotoxicity could be restored by an NKG2D/NKp30-independent bispecific antibody construct (CD30xCD16A). It artificially links the tumor receptor CD30 with the cytotoxicity NK cell receptor CD16A. Moreover, we observed that NK cells from patients treated with this construct were generally activated and displayed a restored cytotoxicity against HL target cells. These data suggest that reversible suppression of NK cell activity contributes to immune evasion in HL and can be antagonized therapeutically. PMID:23459515

Reiners, Katrin S.; Kessler, Jorg; Sauer, Maike; Rothe, Achim; Hansen, Hinrich P.; Reusch, Uwe; Hucke, Christian; Kohl, Ulrike; Durkop, Horst; Engert, Andreas; von Strandmann, Elke Pogge



Rescue of impaired NK cell activity in hodgkin lymphoma with bispecific antibodies in vitro and in patients.  


Natural killer (NK) cells represent a key component of the innate immune system against cancer. Nevertheless, malignant diseases arise in immunocompetent individuals despite tumor immunosurveillance. Hodgkin lymphoma (HL) is characterized by CD30(+) tumor cells and a massive infiltration of immune effector cells in affected lymph nodes. The latter obviously fail to eliminate the malignant cell population. Here, we tested for functional NK cell defects in HL and suggest an improvement of NK function by therapeutic means. We demonstrate that peripheral NK cells (pNK) from patients with HL fail to eliminate HL cell lines in ex vivo killing assays. Impaired NK cell function correlated with elevated serum levels of soluble ligands for NK cell receptors NKp30 (BAG6/BAT3) and NKG2D (MICA), factors known to constrict NK cell function. In vitro, NK cell cytotoxicity could be restored by an NKG2D/NKp30-independent bispecific antibody construct (CD30xCD16A). It artificially links the tumor receptor CD30 with the cytotoxicity NK cell receptor CD16A. Moreover, we observed that NK cells from patients treated with this construct were generally activated and displayed a restored cytotoxicity against HL target cells. These data suggest that reversible suppression of NK cell activity contributes to immune evasion in HL and can be antagonized therapeutically. PMID:23459515

Reiners, Katrin S; Kessler, Jörg; Sauer, Maike; Rothe, Achim; Hansen, Hinrich P; Reusch, Uwe; Hucke, Christian; Köhl, Ulrike; Dürkop, Horst; Engert, Andreas; von Strandmann, Elke Pogge



Cancer immunotherapeutics: evolution of monoclonal antibodies to peptide immunogens.  


The Wistar Institute under Hilary Koprowski's direction played a preeminent role in bringing the fruits of basic science to clinic through biotechnology and life science enterprises. Koprowski's early view on the utility of monoclonal antibodies has been validated to some extent, because monoclonal therapeutics form one of the fastest growing and most successful and lucrative segments within the biopharmaceutical sector. Over 30 monoclonal antibody drugs have now been approved in the United States and Europe. However, monoclonal antibodies might be viewed in the context of a bridge to the future for vaccines. The Wistar Institute's reputation, while built on vaccines for infectious diseases, can be translated in years to come into vaccines for cancer. PMID:24892996

Murali, Ramachadran; Kieber-Emmons, Thomas



Characterization of BIS20x3, a bi-specific antibody activating and retargeting T-cells to CD20-positive B-cells  

PubMed Central

This paper describes a bi-specific antibody, which was called BIS20x3. It retargets CD3?-positive cells (T-cells) to CD20-positive cells and was obtained by hybrid–hybridoma fusion. BIS20x3 could be isolated readily from quadroma culture supernatant and retained all the signalling characteristics associated with both of its chains. Cross-linking of BIS20x3 on Ramos cells leads to DNA fragmentation percentages similar to those obtained after Rituximab-cross-linking. Cross-linking of BIS20x3 on T-cells using cross-linking F(ab?)2-fragments induced T-cell activation. Indirect cross-linking of T-cell-bound BIS20x3 via Ramos cells hyper-activated the T-cells. Furthermore, it was demonstrated that BIS20x3 effectively re-targets T-cells to B-cells, leading to high B-cell cytotoxicity. The results presented in this paper show that BIS20x3 is fully functional in retargeting T-cells to B-cells and suggest that B-cell lymphomas may represent ideal targets for T-cell retargeting bi-specific antibodies, because the retargeted T-cell is maximally stimulated in the presence of B-cells. Additionally, since B-cells may up-regulate CD95/ Fas expression upon binding of CD20-directed antibodies, B-cells will become even more sensitive for T-cell mediated killing via CD95L/ Fas L, and therefore supports the intention to use T-cell retargeting bi-specific antibodies recognizing CD20 on B-cell malignancies as a treatment modality for these diseases. © 2001 Cancer Research Campaign PMID:11308263

Withoff, S; Bijman, M N A; Stel, A J; Delahaye, L; Calogero, A; Jonge, M W A de; Kroesen, B J; Leij, L de



Monoclonal antibody therapy in multiple sclerosis  

PubMed Central

Therapeutic approaches to multiple sclerosis (MS) are based on altering the functions of the immune system, either by using broad immunosuppressive drugs used for transplantation rejection and rheumatology, or by modulating them more discreetly with beta interferon and synthetic amino-acid copolymers. These strategies are only partially successful, have important safety and tolerability limitations, and have shown to be mostly effective in earlier stages of the disease, in which acute relapses dominate the clinical picture. For progressive phenotypes of MS there are currently no effective therapeutic options. As very specific and potent immunosuppressive agents, monoclonal antibodies (mAbs) may offer considerable advantages over other therapies for MS. During the last decade, anti-a4 integrin natalizumab became the first approved mAb for treatment of relapsing MS, after convincingly demonstrating clinically significant effects on two large Phase 3 trials. Moreover, the concept of disease remission was introduced for the first time to describe patients who show no signs of clinical or imaging markers of disease activity during therapy with natalizumab. Of the mAbs under development for MS, alemtuzumab and rituximab have also shown promising evidence of effectiveness and potentially expanded the therapeutic horizon to reversal of disease progression in early relapsing patients and progressive patients who previously had not been studied. However, the appearance of progressive multifocal leukoencephalopathy (PML) in natalizumab-treated MS patients, as well as in patients with lymphoma, lupus and rheumatoid arthritis, treated with rituximab and autoimmune-type complications in alemtuzumab-treated MS patients underlines the fact that extended efficacy comes with significant clinical risks. The challenge is then how best to utilize therapies that have evidently superior efficacy in a chronic disease of young adults to obtain the best benefit-risk ratio and how to monitor and prevent emergent safety concerns. PMID:21124072



The growth and potential of human antiviral monoclonal antibody therapeutics  

Microsoft Academic Search

Monoclonal antibodies (mAbs) have long provided powerful research tools for virologists to understand the mechanisms of virus entry into host cells and of antiviral immunity. Even so, commercial development of human (or humanized) mAbs for the prophylaxis, preemptive and acute treatment of viral infections has been slow. This is surprising, as new antibody discovery tools have increased the speed and

Wayne A Marasco; Jianhua Sui



Monoclonal Antibody Therapy for Relapsed or Treatment-Resistant Neuroblastoma

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 high-risk neuroblastoma.


A Monoclonal Antibody, Py, Distinguishes Different Classes of Hippocampal Neurons  

Microsoft Academic Search

A monoclonal antibody, Py, was produced by immunizing mice with a glycoprotein fraction isolated from 3-week-old rat hippocampus. Py antibodies gave strong immunocyto- chemical staining of the perikarya and dendrites of large neurons in many areas of the rat brain, including the cerebral cortex, hippocampus, cerebellum, brain stem, and olfactory bulb. lmmunoelectron microscopy showed the antigen to be predominantly intracellular,

Peter L. Woodhams; Michael Webb; D. John Atkinson; P. John Seeleyl



Antigenic heterogeneity of Bacteroides intermedius as recognized by monoclonal antibodies.  

PubMed Central

Four hybrid cell lines secreting monoclonal antibodies against antigens of Bacteroides intermedius were generated by fusing murine NSI cells with splenocytes from a rat immunized with B. intermedius strain OMZ248. An enzyme-linked immunosorbent assay was used to analyze the distribution of the recognized antigens on 39 strains from various Bacteroides species and on 5 strains from other genera. Only Bacteroides species B. intermedius, B. loescheii, B. melaninogenicus, and B. corporis were found to express at least one of the recognized antigens. Strains of the two asaccharolytic black-pigmenting Bacteroides species were negative. Among the strains capable of binding to one or more of the monoclonal antibodies, five groups with different reactivity patterns could be distinguished. Two of the monoclonal antibodies were specific for B. intermedius. The B. intermedius strains were metabolically almost identical, expressed at least three of the recognized antigens, and fell into three distinct antibody reactivity groups, suggesting a tentative separation of this species into three new serogroups. Oral and nonoral isolates of B. intermedius were, however, not distinguished by the monoclonal antibodies. One monoclonal antibody was directed against an antigen strongly expressed on all saccharolytic black-pigmenting Bacteroides strains tested so far, thus confirming the previously noted antigenic relationship between the species which had emerged from the former B. melaninogenicus subsp. intermedius and B. melaninogenicus subsp. melaninogenicus groups. PMID:6196291

Gmur, R; Guggenheim, B



Bispecific anti?CD3 x anti?HER2 antibody mediates T cell cytolytic activity to HER2?positive colorectal cancer in vitro and in vivo.  


Targeting HER2 overexpressed breast cancer cells with anti?HER2 monoclonal antibodies inhibits tumor growth. Here we investigated whether HER2 can serve as a target for T cell?mediated immunotherapy of human colorectal carcinoma. Specific cytolytic activity of activated T cells (ATCs) armed with anti?CD3 x anti?HER2 bispecific antibody (HER2Bi?Ab) against HER2+ tumor cells was evaluated by bioluminescent signal generated by luciferase reporter on tumor cells in vitro and in vivo. In contrast to unarmed ATCs, increased cytotoxic activity of HER2Bi?armed ATCs against HER2+ tumor cells was observed. Moreover, HER2Bi?armed ATCs expressed higher level of activation marker CD69 and secreted significantly higher levels of IFN?? than the unarmed ATC counterpart. In addition, compared with anti?HER2 mAb (Herceptin®) or unarmed ATC, HER2Bi?armed ATCs showed significant suppression against colorectal carcinoma cells. In colorectal tumor cell xenograft mice, infusion of HER2Bi?armed ATCs successfully inhibited the growth of Colo205?luc cells. The HER2Bi?armed ATCs with anti?tumor effects may provide a promising immunotherapy for colorectal carcinoma in the future. PMID:25242665

Han, Huamin; Ma, Juan; Zhang, Keming; Li, Wei; Liu, Changzhen; Zhang, Yu; Zhang, Ganlin; Ma, Pan; Wang, Lei; Zhang, Ge; Tao, Hua; Gao, Bin



Monoclonal antibodies in the treatment of multiple sclerosis.  


Multiple sclerosis (MS), a chronic demyelinating disorder, is characterized by recurrent neurological deficits or progressive impairment with a high risk of permanent disability. Since the exact pathophysiology and etiology remains still unclear, no curing therapy is currently available. However, several treatments with beneficial effect on relapse rate such as the first line therapies interferon-beta and glatiramer acetate were approved for relapsing-remitting MS. One new important tool in the therapy of MS is the use of monoclonal antibodies. Natalizumab is the first and so far only monoclonal antibody that is approved for MS treatment by the US Food and Drug Administration and the European Medicines Agency. In addition to natalizumab other monoclonal antibodies previously used in cancer and other autoimmune disorders or even newly developed for MS are now being tested in clinical trials. With their high target specificity and efficacy monoclonal antibodies are a promising treatment approach in MS. This review summarizes the present knowledge on the use, effectiveness and safety of monoclonal antibodies in MS treatment. PMID:19929782

Di Pauli, F; Berger, T; Reindl, M



The effect of dexamethasone on polyclonal T cell activation and redirected target cell lysis as induced by a CD19\\/CD3-bispecific single-chain antibody construct  

Microsoft Academic Search

BiTE molecules comprise a new class of bispecific single-chain antibodies redirecting previously unstimulated CD8+ and CD4+ T cells for the elimination of target cells. One example is MT103 (MEDI-538; bscCD19xCD3), a CD19-specific BiTE that can\\u000a induce lysis of normal and malignant B cells at low picomolar concentrations, which is accompanied by T cell activation. Here,\\u000a we explored in cell culture

Christian Brandl; Cornelia Haas; Sandrine d’Argouges; Tanja Fisch; Peter Kufer; Klaus Brischwein; Nadja Prang; Ralf Bargou; JoAnn Suzich; Patrick A. Baeuerle; Robert Hofmeister



Serogrouping and subtyping of Legionella pneumophila with monoclonal antibodies.  

PubMed Central

Monoclonal antibodies directed against Legionella pneumophila serogroups 1 to 6 were produced by fusing splenocytes of BALB/c mice with the Sp 2/0-Ag14 or the NSO mouse myeloma cell lines. Specificity of these antibodies was determined by indirect fluorescent-antibody staining: 8 reacted with L. pneumophila serogroup 1 and, respectively, 13, 6, 6, 5, and 10 reacted with serogroups 2, 3, 4, 5, and 6; all except 5 were serogroup specific, and none presented cross-reactions with six other species of Legionellaceae. Serogroup determination of 35 isolates of L. pneumophila with seven selected monoclonal antibodies resulted in correct serogrouping in all instances; a pool of the same seven monoclonal antibodies stained intensely all strains of L. pneumophila without any staining of the other species of Legionellaceae. When 24 serogroup 1 isolates of L. pneumophila were stained with eight serogroup 1-specific monoclonal antibodies, the staining patterns could be clustered in five distinct groups. These hybridomas thus represent an unlimited source of standard reagent that could be used in the detection and serogrouping of L. pneumophila; differences in staining patterns could be used as epidemiological markers for these bacteria. PMID:6358248

Joly, J R; Chen, Y Y; Ramsay, D



Delivery of the ribosome-inactivating protein, gelonin, to lymphoma cells via CD22 and CD38 using bispecific antibodies.  

PubMed Central

It is well established that bispecific antibodies (BsAbs) can be used effectively in targeting the ribosome-inactivating protein (RIP), saporin, against neoplastic B cells. We have now extended this delivery system for use with gelonin. By measuring antigen-binding characteristics and epitope mapping a panel of anti-gelonin MAbs using the IAsys resonant mirror bisensor, we were able to rapidly select the most suitable for making BaAbs. The Fab' fragments from these MAbs were chemically conjugated with Fab' from either anti-CD22 or anti-CD38. Cytotoxicity assays showed that BsAbs were highly efficient at delivering gelonin to cultured Daudi cells and achieved levels of toxicity which correlated closely with the affinity of the BsAbs. Using pairs of anti-CD22 BsAbs we were able to generate bivalent BsAb-gelonin complexes which achieved IC50 values of 2 x 10(-11) M gelonin, a potency which is equivalent to that reached by saporin in this targeting system. However, because gelonin is 5-10 times less toxic than saporin, the therapeutic ratio for gelonin is superior, making it potentially a more useful agent for human treatment. Cytotoxicity assays and kinetic analysis showed that targeting gelonin via CD38 was 2-5 times less effective than delivery through CD22. However, with a pair of BsAbs designed to co-target gelonin via CD22 and CD38, the cytotoxicity achieved equalled that obtained with a pair of anti-CD22 BsAbs (IC50 = 1 x 10(-11) M). This important result suggests that the anti-CD38 helps bind the gelonin to the cell and is then 'dragged' or 'piggy-backed' into the cell by the anti-CD22 BsAb. The implication of these findings for cancer therapy is discussed. PMID:7734325

French, R. R.; Penney, C. A.; Browning, A. C.; Stirpe, F.; George, A. J.; Glennie, M. J.



Targeted siRNA Delivery and mRNA Knockdown Mediated by Bispecific Digoxigenin-binding Antibodies  

PubMed Central

Bispecific antibodies (bsAbs) that bind to cell surface antigens and to digoxigenin (Dig) were used for targeted small interfering RNA (siRNA) delivery. They are derivatives of immunoglobulins G (IgGs) that bind tumor antigens, such as Her2, IGF1-R, CD22, and LeY, with stabilized Dig-binding variable domains fused to the C-terminal ends of the heavy chains. siRNA that was digoxigeninylated at its 3?end was bound in a 2:1 ratio to the bsAbs. These bsAb–siRNA complexes delivered siRNAs specifically to cells that express the corresponding antigen as demonstrated by flow cytometry and confocal microscopy. The complexes internalized into endosomes and Dig-siRNAs separated from bsAbs, but Dig-siRNA was not released into the cytoplasm; bsAb-targeting alone was thus not sufficient for effective mRNA knockdown. This limitation was overcome by formulating the Dig-siRNA into nanoparticles consisting of dynamic polyconjugates (DPCs) or into lipid-based nanoparticles (LNPs). The resulting complexes enabled bsAb-targeted siRNA-specific messenger RNA (mRNA) knockdown with IC50 siRNA values in the low nanomolar range for a variety of bsAbs, siRNAs, and target cells. Furthermore, pilot studies in mice bearing tumor xenografts indicated mRNA knockdown in endothelial cells following systemic co-administration of bsAbs and siRNA formulated in LNPs that were targeted to the tumor vasculature. PMID:23344238

Schneider, Britta; Grote, Michael; John, Matthias; Haas, Alexander; Bramlage, Birgit; lckenstein, Ludger M; Jahn-Hofmann, Kerstin; Bauss, Frieder; Cheng, Weijun; Croasdale, Rebecca; Daub, Karin; Dill, Simone; Hoffmann, Eike; Lau, Wilma; Burtscher, Helmut; Ludtke, James L; Metz, Silke; Mundigl, Olaf; Neal, Zane C; Scheuer, Werner; Stracke, Jan; Herweijer, Hans; Brinkmann, Ulrjch



Use of monoclonal antibodies in a radioimmunoassay for human transcortin.  


We describe the production of monoclonal antibodies to human transcortin and their use in a radioimmunoassay (RIA). A high-affinity antibody (Ka = 4 X 10(10) L/mol) made possible a sensitive RIA for transcortin (detection limit = 0.23 ng per tube), whereas use of an antibody of moderate affinity (Ka = 5 X 10(8) L/mol) was more suitable for the routine measurement of transcortin in serum, only a 25-fold dilution of the sample being required instead of 1500-fold. The correlation was good between both RIAs (r = 0.959) and between each of the RIAs and radial immunodiffusion (r = 0.955 and 0.976 for the methods with high- and low-affinity antibody, respectively). Although monoclonal antibodies were used in the RIAs and polyclonal ones in the radial immunodiffusion procedure, similar values were obtained by all techniques. PMID:6421509

Faict, D; De Moor, P



Quantitative analysis of monoclonal antibodies by cation-exchange chromatofocusing.  


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. PMID:19560777

Rozhkova, Anna



Three rat monoclonal antibodies to human C3.  

PubMed Central

Three monoclonal antibodies to human C3 have been obtained from a fusion of the rat myeloma line Y3 Ag 1.2.3. with spleen cells from rats immunized against C3. One, from clone 4, reacts with an antigenic determinant in C3c showing the expected reactivity of the 'C' antigen of C3. The specificity of the other two monoclonal antibodies correspond less clearly with known C3 antigens. By agglutination analysis of complement coated cells the determinant reacting with clone 3 is present in C3d while that for clone 9 appears as a neoantigen on C3bi. In both cases the co-precipitation results are anomalous and more direct studies are needed to define the exact specificity. The possibility that internal sequence duplications in C3 may explain some anomalies is discussed. None of the monoclonal antibodies significantly inhibit C3 functions. The monoclonal antibodies have been found to have unusual properties in co-precipitation assays being able to diffuse through a precipitation line with which they react to react with a further line. One antibody is also able to react strongly with the anodal half of what appears as a single line with a polyclonal antiserum. Images Figure 3 Figure 7 PMID:6161872

Lachmann, P J; Oldroyd, R G; Milstein, C; Wright, B W



Characterization of monoclonal antibodies against human apolipoprotein E.  

PubMed Central

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

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



Therapeutic efficacy of potent neutralizing HIV-1-specific monoclonal antibodies in SHIV-infected rhesus monkeys  

E-print Network

Human immunodeficiency virus type 1 (HIV-1)-specific monoclonal antibodies with extraordinary potency and breadth have recently been described. In humanized mice, combinations of monoclonal antibodies have been shown to ...

Barouch, Dan H.


Antibody discovery: sourcing of monoclonal antibody variable domains.  


Historically, antibody variable domains for therapeutic antibodies have been sourced primarily from the mouse IgG repertoire, and typically either chimerized or humanized. More recently, human antibodies from transgenic mice producing human IgG, phage display libraries, and directly from human B lymphocytes have been used more broadly as sources of antibody variable domains for therapeutic antibodies. Of the total 36 antibodies approved by major maket regulatory agencies, the variable domain sequences of 26 originate from the mouse. Of these, four are marketed as murine antibodies (of which one is a mouse-rat hybrid IgG antibody), six are mouse-human chimeric antibodies, and 16 are humanized. Ten marketed antibodies have originated from human antibody genes, three isolated from phage libraries of human antibody genes and seven from transgenic mice producing human antibodies. Five antibodies currently in clinical trials have been sourced from camelids, as well as two from non-human primates, one from rat, and one from rabbit. Additional sources of antibody variable domains that may soon find their way into the clinic are potential antibodies from sharks and chickens. Finally, the various methods for retrieval of antibodies from humans, mouse and other sources, including various display technologies and amplification directly from B cells, are described. PMID:24168292

Strohl, William R



Phase 1 trial of the novel bispecific molecule H22xKi-4 in patients with refractory Hodgkin lymphoma  

Microsoft Academic Search

CD30 is an excellent target for immuno- therapy of Hodgkin lymphoma (HL) be- cause it is overexpressed on Hodgkin and Reed-Sternberg cells. We developed a novel bispecific molecule (BSM) consist- ing of F(ab) fragments derived from the murine anti-CD30 monoclonal antibody (MoAb) Ki-4 and the humanized CD64- specific MoAb H22. In vitro experiments of H22xKi-4 demonstrated specific phago- cytosis of

Peter Borchmann; Roland Schnell; Irene Fuss; Oliver Manzke; Thomas Davis; Lionel D. Lewis; Detlev Behnke; Claudia Wickenhauser; Petra Schiller; Volker Diehl; Andreas Engert



Mechanisms of monoclonal antibody stabilization and release from silk biomaterials  

PubMed Central

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

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



The safety and side effects of monoclonal antibodies  

Microsoft Academic Search

Monoclonal antibodies (mAbs) are now established as targeted therapies for malignancies, transplant rejection, autoimmune and infectious diseases, as well as a range of new indications. However, administration of mAbs carries the risk of immune reactions such as acute anaphylaxis, serum sickness and the generation of antibodies. In addition, there are numerous adverse effects of mAbs that are related to their

Harald Kropshofer; Thomas Singer; Jane A. Mitchell; Andrew J. T. George; Trevor T. Hansel



Monoclonal antibody identification of infiltrating mononuclear leukocytes in lupus nephritis  

Microsoft Academic Search

Monoclonal antibody identification of infiltrating mononuclear leukocytes in lupus nephritis (LN). Populations of mononuclear inflammatory cells infiltrating the renal interstitium in LN were studied by means of an avidin–biotin immunoperoxidase technique applied to cryostat sections of 26 renal biopsies (3 WHO class IIb; 4 class III; 8 class IV; 4 class V; 4 class III and V; and 3 class

Vivette D D'Agati; Gerald B Appel; Dorothy Estes; Daniel M Knowles; Conrad L Pirani



[Single B cell monoclonal antibody technologies and applications].  


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. PMID:23016302

Chi, Xiangyang; Yu, Changming; Chen, Wei



Mechanisms of Allergen-Antibody Interaction of Cockroach Allergen Bla g 2 with Monoclonal Antibodies  

E-print Network

Mechanisms of Allergen-Antibody Interaction of Cockroach Allergen Bla g 2 with Monoclonal is strongly associated with asthma, and involves the production of IgE antibodies against inhaled allergens. Reports of conformational epitopes on inhaled allergens are limited. The conformational epitopes for two


The Use of Monoclonal Antibodies in Human Prion Disease  

NASA Astrophysics Data System (ADS)

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.

Bodemer, Walter


Affinity of monoclonal antibodies for Globo-series glycans  

PubMed Central

Globo-series glycans are human cell-surface carbohydrates that include stem-cell marker SSEA-4 and cancer-cell antigen Globo H. These two hexasaccharides differ only in their terminal saccharide: N-acetylneuraminic acid in SSEA-4 and l-fucose in Globo H. Herein, we evaluated the affinity of the monoclonal antibodies ?-SSEA-4 and ?-GH for the glycans SSEA-4 and Globo H. Using fluorescence polarization, we find that the two monoclonal antibodies have affinity for their cognate glycanin the low nanomolar range, and have negligible affinity for the non-cognate glycan. Using surface plasmon resonance, we find that each cognate affinity is ~20-fold greater if the glycanis immobilized on a surface rather than free in solution. We conclude that the terminal saccharide plays a dominant role in the ability of monoclonal antibodies to recognize these Globo-series glycans and that the extraordinary specificity of these antibodies supports their use for identifying and sorting stem-cells (?-SSEA-4) and as an agent in cancer immunotherapy (?-GH). PMID:25163606

Eller, Chelcie H.; Yang, Guangbin; Ouerfelli, Ouathek; Raines, Ronald T.



[Batch release of immunoglobulin and monoclonal antibody products].  


The Paul-Ehrlich Institute (PEI) is an independent institution of the Federal Republic of Germany responsible for performing official experimental batch testing of sera. The institute decides about the release of each batch and performs experimental research in the field. The experimental quality control ensures the potency of the product and also the absence of harmful impurities. For release of an immunoglobulin batch the marketing authorization holder has to submit the documentation of the manufacture and the results of quality control measures together with samples of the batch to the PEI. Experimental testing is performed according to the approved specifications regarding the efficacy and safety. Since implementation of the 15th German drug law amendment, the source of antibody is not defined anymore. According to § 32 German drug law, all batches of sera need to be released by an official control laboratory. Sera are medicinal products, which contain antibodies, antibody fragments or fusion proteins with a functional antibody portion. Therefore, all batches of monoclonal antibodies and derivatives must also be released by the PEI and the marketing authorization holder has to submit a batch release application. Under certain circumstances a waiver for certain products can be issued with regard to batch release. The conditions for such a waiver apply to the majority of monoclonal antibodies. PMID:25200488

Gross, S



Secretory expression of a bispecific antibody targeting tumor necrosis factor and ED-B fibronectin in Pichia pastoris and its functional analysis.  


Specific targeting of tumor necrosis factor (TNF)-? antagonist to the inflamed site could increase its efficacy and reduce side-effects. Here, we constructed a bispecific diabody (BsDb) that targets TNF-? and ED-B-containing fibronectin, a fibronectin isoform specifically expressed in the pannus of the inflamed synovium in rheumatoid arthritis. BsDb was secreted from Pichia pastoris as functional protein and was purified to homogeneity. BsDb could simultaneously bind to human TNF-? and B-FN and neutralize TNF-? action. Additionally, BsDb showed a significant gain both in the antigen-binding affinity and in TNF-?-neutralizing ability as compared to its original antibodies, L19 and anti-TNF-? scFv, which were produced in E. coli. BsDb was constructed and was endowed with enhanced bioactivities and improved production processing. Therefore, it holds great potential for in vivo applications. PMID:25129049

Liu, Meng-Yuan; Hu, Xue-Ping; Xie, Mian; Jiang, Si-Jing; Li, Lu-Jun; Liu, Dong-Xu; Yang, Xiao-Song



Improved Iodine Radiolabels for Monoclonal Antibody Therapy1  

Microsoft Academic Search

A major disadvantage of 131iodine (I)-labeled monoclonal antibodies (MAbs) for radioimmunotherapy has been the rapid diffusion of iodoty- rosine from target cells after internalization and catabolism of the radio- iodinated MAbs. We recently reported that a radioiodinated, diethylene- triaminepentaacetic acid-appended peptide, designated immunomedics' residualizing peptide 1 (IMP-R1), was a residualizing iodine label that overcame many of the limitations that had

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


Trypanosoma cruzi: amastigote polymorphism defined by monoclonal antibodies  

Microsoft Academic Search

We have raised monoclonal antibodies (mAbs) directed towards amastigote forms of Trypanosoma cruzi, and shown that mAbs 1D9 and 4B9 are carbohydrate while mAb 4B5 activity is resistant to periodate oxidation of the antigen. Here we used an ELISA to quanti- tate and compare the expression of surface epitopes on fixed parasites among different parasite isolates. The expression of markers

N. V. Verbisck; S. Da-Silva; R. A. Mortara



Monoclonal antibodies defining distinctive human T cell surface antigens  

Microsoft Academic Search

Three novel monoclonal antibodies (designated OKT1, OKT3, and OKT4) were generated against surface determinants of human peripheral T cells. Both OKT1 and OKT3 reacted with all human peripheral T cells and 5 to 10 percent of thymocytes but differed in their reactivities with T cell lines. By contrast, OKT4 reacted with 55 percent of human peripheral T cells and 80

P. C. Kung; G. Goldstein; E. L. Reinherz; S. F. Schlossman



Iodine 123 radiolabeling of monoclonal antibodies for in vivo procedures  

Microsoft Academic Search

When labeled to monoclonal antibodies (MAbs) or their fragments, ¹²³I can be used for imaging or for predicting the treatment potential and radiation dosimetry of ¹³¹I labeled to the same molecular species. Because ¹²³I (p,5n) from the Crocker Nuclear Laboratory is in dilute solution, when compared with commercial ¹²⁵I of labeling grade, we have evaluated labeling parameters using Chloramine-T as

S. L. Mills; S. J. Denardo; G. L. Denardo; A. L. Epstein; J. S. Peng; D. Colcher



Arthritogenic Monoclonal Antibodies from K\\/BxN Mice  

Microsoft Academic Search

Arthritis in the K\\/BxN mouse model is provoked by pathogenic antibodies (Abs) directed against a ubiquitously expressed protein, glucose-6-phosphate isomerase (GPI). To begin dis- secting the repertoire of arthritogenic immunoglobulins (Igs) in the K\\/BxN model, and to pro- vide a basis for comparison with RA patientswe have generated anti-GPI monoclonal Abs (mAbs) from spontaneously activated B cells in the lymphoid

Mariana Maccioni; Gabrielle Zeder-Lutz; Haochu Huang; Claudine Ebel; Philippe Gerber; Josiane Hergueux; Patricia Marchal; Veronique Duchatelle; Claude Degott; Marc van Regenmortel; Christophe Benoist; Diane Mathis



Monoclonal antibodies in the therapy of multiple sclerosis  

Microsoft Academic Search

\\u000a Abstract\\u000a   With the generation of monoclonal antibodies (mAbs), a new therapeutical concept has gained importance. MAbs aim against selective\\u000a antigens and so have changed our treatment strategies from non-specific to specific. Four therapeuticals have gained importance\\u000a in the therapy of multiple sclerosis (MS): One has already been approved for therapy (natalizumab), whereas the other three\\u000a are either in clinical trials

P. S. Rommer; O. Stüve; R. Goertsches; E. Mix; U. K. Zettl



Monoclonal antibodies to human growth hormone modulate its biological properties  

Microsoft Academic Search

Previous results indicated that monoclonal antibodies (mAbs) termed mAb AE5, mAb AC8 and mAb F11, recognizing the human growth hormone (hGH) region left exposed after binding to lactogenic, somatogenic and hGH-specific receptors, produce allosteric changes in the hormone which modify its binding properties. To study whether these mAbs could also influence hGH biological activity, experiments were carried out with Nb2

Leonor P. Roguin; Rubén C. Aguilar; Lilia A. Retegui



Tumor-specific novel taxoid-monoclonal antibody conjugates.  


Taxoids bearing methyldisulfanyl(alkanoyl) groups for taxoid-antibody immunoconjugates were designed, synthesized and their activities evaluated. A highly cytotoxic C-10 methyldisulfanylpropanoyl taxoid was conjugated to monoclonal antibodies recognizing the epidermal growth factor receptor (EGFR) expressed in human squamous cancers. These conjugates were shown to possess remarkable target-specific antitumor activity in vivo against EGFR-expressing A431 tumor xenografts in severe combined immune deficiency mice, resulting in complete inhibition of tumor growth in all the treated mice. PMID:12477344

Ojima, Iwao; Geng, Xudong; Wu, Xinyuan; Qu, Chuanxing; Borella, Christopher P; Xie, Hongsheng; Wilhelm, Sharon D; Leece, Barbara A; Bartle, Laura M; Goldmacher, Victor S; Chari, Ravi V J



Tumor specific novel taxoid-monoclonal antibody conjugates.  


The basic principle of the targeted delivery approach is that the conjugation of a drug to a tumor-specific molecule renders the drug inactive until it reaches the target site. Monoclonal antibodies (mAbs), which have shown high binding specificity for tumor-specific antigens, could be used as targeting agents. Paclitaxel has brought significant impact on the current cancer chemotherapy, but seriously suffers from the lack of tumor specificity. A series of paclitaxel-monoclonal antibody conjugates via C-2' ester linkage were reported. Taking into account the fact that the cytotoxicity of paclitaxel is not good enough and thus not applicable to this target delivery prodrug approach, new taxoids bearing methyldisulfanyl(alkanoyl) groups were designed, synthesized, and their activities evaluated. A highly cytotoxic C-10 methyldisulfanyl-propanoyl taxoid, was conjugated to monoclonal antibodies recognizing the epidermal growth factor receptor (EGFR). These conjugates were shown to possess remarkable target-specific antitumor activity in vivo against EGFR-expressing A431 tumor xenografts in SCID mice, resulting in complete inhibition of tumor growth in all the treated mice without any noticeable toxicity to the animals. PMID:14965224

Wu, X; Ojima, I



Monoclonal antibodies in the treatment of pancreatic cancer  

PubMed Central

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

Huang, Zhi-Qiang; Buchsbaum, Donald J



Monoclonal antibody against a Burkitt lymphoma-associated antigen.  


A monoclonal antibody, referred to as 38.13, was obtained by fusing murine myeloma cells with Lewis rat splenocytes sensitized with Daudi cells (human Burkitt lymphoma containing Epstein--Barr virus genome but lacking HLA-A, -B, and -C and beta 2-microglobulin molecules at the cell surface). 38.13 antibody was demonstrated to be a rat IgM. By complement-dependent microcytotoxicity and indirect immunofluorescence assays, 38.13 antibody was shown to react specifically with cells derived from Burkitt tumors, including both Epstein--Barr virus genome-carrying and Epstein--Barr virus-negative Burkitt lymphoma. By contrast, Epstein--Barr virus-containing lymphoblastoid cell lines derived from normal B lymphocytes were not recognized by 38.13 antibody. Fresh malignant cells from patients affected with various lymphoproliferative disorders were negative, except 4/8 having abdominal Burkitt-like lymphomas. Normal lymphocytes from peripheral blood, spleen, lymph node, tonsil, and bone marrow and mitogen (phytohemagglutinin, pokeweed mitogen, and concanavalin A)-activated blasts were also negative. Thus, 38.13 antibody apparently recognized a Burkitt-associated antigen that is not related to Epstein--Barr virus. The pattern of reactivity of 38.13 antibody with various Burkitt lymphoma cells appeared quite heterogenous and some Burkitt cells were consistently negative. 38.13 antibody thus defines a subset of Burkitt lymphomas. PMID:7031655

Wiels, J; Fellous, M; Tursz, T



Monkey-derived monoclonal antibodies against Plasmodium falciparum  

SciTech Connect

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

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



Monoclonal antibodies to a phosphoprotein from chromatin of rat liver.  

PubMed Central

Three monoclonal antibody subclasses (IgG1, IgG2a, and IgM) were raised to the phosphoprotein B2 (Mr 68000, pI6.5-8.2) which has been shown previously to be associated with the nucleosomes of rat liver nuclei. These antibodies do not show any significant cross reactivity with CM-cellulose 'unbound' non-histone chromosomal proteins, bovine serum albumin or histones. Further verification of the specificity of these antibodies to this phosphoprotein was carried out using both 'dot' blot and immunological transfer analysis ('Western blot'). The monoclonal antibodies (IgG1 and IgG2a) could also be used to semi-quantify the phosphoprotein B2 in rat liver nuclei. The high specificity and unlimited availability of this type of probe provides a means to study the role(s) of this phosphoprotein in the overall scheme of actively transcribed chromatin. Images Fig. 1. Fig. 3. Fig. 4. PMID:3919706

Halikowski, M J; Liew, C C



Removal of digoxin from the circulation using immobilized monoclonal antibodies.  


High-affinity (Ka = 3.0 X 10(8) M-1) monoclonal antidigoxin antibodies were covalently coupled to Sepharose CL-6B, Bio Gel A5m, Affi-gel 15, and agarose:polyacrolein microsphere (APAM) beads. Antibody immobilized to these supports was characterized in its ability to remove digoxin in vitro from biological fluids. Equilibrium binding studies of digoxin in plasma revealed that antibody coupled to Sepharose, Bio Gel, and Affi-gel 15 retained 38-42% of theoretical binding capacity, whereas antibody coupled to APAM beads retained only 13%. Increasing flow rates from 0.5 to 5.0 mL/min across antibody immobilized to APAM beads decreased the removal of digoxin from plasma from 90 to 55%. In comparison, no decreases were observed in the amount of digoxin removed from plasma as the flow rates were increased across antibody immobilized to the other agarose supports. Antibody coupled to Affi-gel 15 was used for in vivo extracorporeal hemoperfusion studies. These studies indicated that although 5-20% of the total dose was removed from three guinea pigs, plasma digoxin levels were not lowered when compared with preperfusion concentrations. In addition, antibody columns which were reused following regeneration with glycine:HCl, pH 2.5 revealed that 80% of the original antibody activity was lost after two hemoperfusions. Hematological parameters were unchanged with the exception that platelets were significantly decreased following the 3-h perfusion study. These data indicate that immobilized antidigoxin antibodies were able to remove digoxin from the circulation, and immunoaffinity hemoperfusion may be a viable means of removing substances from blood. PMID:2746478

Brizgys, M V; Pincus, S; Siebert, C J; Rollins, D E



Monoclonal antibody to simian virus 40 small t.  

PubMed Central

A monoclonal antibody, PAb280, was produced that recognizes simian virus 40 (SV40) small t but does not react with SV40 large T. The specificity of the antibody was analyzed by immunoprecipitation of labeled cell extracts, Western blotting, and immunocytochemistry. Small t was found to accumulate late in the SV40 lytic cycle and was localized in both the cytoplasm and the nucleus of cells infected with wild-type SV40. Importantly, antibodies against determinants common to SV40 large T and small t did not appear to be able to recognize the cytoplasmic form of SV40 small t at the immunocytochemical level. The localization of small t within the nucleus appeared to be distinct from that of large T. Images PMID:6088798

Montano, X; Lane, D P



Bispecific antibodies directed to CD4 domain 2 and HIV envelope exhibit exceptional breadth and picomolar potency against HIV-1  

PubMed Central

In the absence of an effective HIV-1 vaccine, passive immunization using broadly neutralizing Abs or Ab-like molecules could provide an alternative to the daily administration of oral antiretroviral agents that has recently shown promise as preexposure prophylaxis. Currently, no single broadly neutralizing Ab (bNAb) or combination of bNAbs neutralizes all HIV-1 strains at practically achievable concentrations in vivo. To address this problem, we created bispecific Abs that combine the HIV-1 inhibitory activity of ibalizumab (iMab), a humanized mAb directed to domain 2 of human CD4, with that of anti-gp120 bNAbs. These bispecific bNAbs (BibNAbs) exploit iMab’s potent anti–HIV-1 activity and demonstrated clinical efficacy and safety to anchor and thereby concentrate a second broadly neutralizing agent at the site of viral entry. Two BibNabs, PG9-iMab and PG16-iMab, exhibit exceptional breadth and potency, neutralizing 100% of the 118 viruses tested at low picomolar concentrations, including viruses resistant to both parental mAbs. The enhanced potency of these BibNAbs was entirely dependent on CD4 anchoring, not on membrane anchoring per se, and required optimal Ab geometry and linker length. We propose that iMab-based BibNAbs, such as PG9-iMab and PG16-iMab, are promising candidates for passive immunization to prevent HIV-1 infection. PMID:23878231

Pace, Craig S.; Song, Ruijiang; Ochsenbauer, Christina; Andrews, Chasity D.; Franco, David; Yu, Jian; Oren, Deena A.; Seaman, Michael S.; Ho, David D.



Blood-brain barrier protein recognized by monoclonal antibody.  

PubMed Central

An IgG1 mouse monoclonal antibody produced in response to immunization with rat brain homogenate reacted with endothelial cells in the central and peripheral nervous system. Because antibody reactivity was associated with endothelia that have a selective permeability barrier, the antibody was called anti-endothelial-barrier antigen (anti-EBA). Paraffin sections of Bouins'-fixed rat tissue were used for initial screening and subsequent characterization of antibody reactivity. The antibody was generally unreactive with endothelial cells in other organs and with nonendothelial cells in or outside of the nervous system. Antibody binding was greatly reduced or absent in endothelia of the area postrema and choroid plexus, sites known to possess fenestrated blood vessels. In developing rat brain, anti-EBA binding to some microvessels was seen at 3 days postnatally. Anti-EBA reactivity outside the nervous system occurred in spleen and skin. Patchy reaction with portions of some spleen blood vessels and binding to some cells in the spleen were observed. In the skin, small cells, tentatively identified as Langerhans cells, which participate in Ia presentation, were stained. On immunoblots of rat brain microvessel preparations electrophoresed in Na-DodSO4/polyacrylamide gels, anti-EBA reacted with a protein triplet of Mr 30,000, 25,000, and 23,500 components. Images PMID:3500474

Sternberger, N H; Sternberger, L A



Production of recombinant woodchuck IFNalpha and development of monoclonal antibodies.  


Interferon alpha (IFNalpha) is the first line treatment for chronic hepatitis B and C. In order to test new IFNalpha delivery systems and investigate the function of this cytokine in the woodchuck model, the best animal model of chronic hepatitis B, we produced and purified recombinant woodchuck IFNalpha and used it to produce monoclonal antibodies. wIFNalpha5 was cloned in a prokaryotic expression system, expressed as His-tagged protein and then purified. The rwIFNalpha5 protein was found to induce STAT-3 phosphorylation, to enhance 2',5'-oligoadenylate synthetase mRNA levels and to possess a potent antiviral activity. Two monoclonal antibodies were obtained through immunization of rats with rwIFNalpha5. Both recognized rwIFNalpha5 in western blot analysis and one was able to neutralize the antiviral activity of the rwIFNalpha5 and lymphoblastoid IFNalpha preparations. Finally, a capture rwIFNalpha5 ELISA was developed using both antibodies. In summary, the tools generated in this study will allow different forms of IFNalpha delivery as well as different combination therapies in woodchuck hepatitis virus infection to be tested, thus providing useful information for the design of new strategies to treat chronic hepatitis B in humans. PMID:19014334

Berraondo, Pedro; Crettaz, Julien; Ochoa, Laura; Vales, Africa; Ruiz, Juan; Prieto, Jesús; Martinez-Ansó, Eduardo; González-Aseguinolaza, Gloria



Labeling of cerebral amyloid in vivo with a monoclonal antibody.  


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

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



Practical considerations for nonclinical safety evaluation of therapeutic monoclonal antibodies  

PubMed Central

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

Lynch, Carmel M; Hart, Bruce W



A human fetal monoclonal DNA-binding antibody shares idiotypes with fetal and adult murine monoclonal DNA-binding antibodies.  

PubMed Central

A human DNA-binding monoclonal antibody was produced by fusing the hepatocytes from a 12-week-old human fetus with the lymphoblastoid cell line GM 4672 using polyethylene glycol. This antibody, designated BEG 2, binds to single-stranded (ss) DNA but also binds to double-stranded (ds) DNA, poly(dT), polyI and poly(ADP-ribose), but not to RNA, cardiolipin or K-30. The binding of BEG 2 to these polynucleotides can be inhibited by incubation with polynucleotides in the fluid phase. A rabbit polyclonal anti-idiotype was raised, and using this reagent it was shown that the BEG 2 idiotype is present in normal human serum (7%), systemic lupus erythematosus (SLE) sera (8%) and rheumatoid arthritis sera (23%). The extent of idiotypic sharing between BEG 2 and murine monoclonal DNA-binding antibodies, in particular monoclonal antibody (mAb) 423 (derived from a 15-day-old fetal MRL/Mp-lpr/lpr mouse) and mAb 402 (derived from an adult MRL/lpr mouse), was also investigated. Using a competition ELISA, it was shown that preincubation of BEG 2 with rabbit anti-423 and rabbit anti-402 inhibits the binding of BEG 2 to DNA, and the binding of 402 to DNA by anti-BEG 2 and anti-423. These data suggest that mAb BEG 2, 423 and 402 share common idiotypes, that autoreactivity is present in early fetal life, and that autoantibodies may be encoded for by germline genes, which have been conserved through evolution. Images Figure 2 Figure 3 Figure 7 PMID:2312159

Watts, R A; Ravirajan, C T; Staines, N A; Isenberg, D A



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

PubMed Central

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

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



The production and characterization of protective monoclonal antibodies against Fasciola hepatica and Schistosoma mansoni  

E-print Network

F 1 h~*t 2C. Binding of monoclonal antibody Ell culture supernatant to a cryostat section of adult ~F* o1 h~t 2D. Binding of monoclonal antibody H 8 culture supernatant to a cryostat section of adult F 1 h~lt 2E. Binding of Sp2 supernatant to a... cryostat t f d 1t ~F'o1 h~*t' 3A. Binding of monoclonal antibody Ell culture supernatant to 1 week juvenile Fasciola ~ht ' 3B. Binding of Sp2 supernatant to 1 week juvenile F 1 h~t' Western blot analysis of monoclonal antibody D4 binding on a 104 SDS...

Hicks, Carolyn Sue



Immunosuppression associated with novel chemotherapy agents and monoclonal antibodies.  


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

Morrison, Vicki A



Anti-CD20 monoclonal antibodies: historical and future perspectives  

PubMed Central

Antibodies to CD20 have confirmed the hypothesis that monoclonal reagents can be given in vivo to alleviate human diseases. The targeting of CD20 on normal, malignant and auto-immune B-lymphocytes by rituximab has demonstrated substantial benefits for patients with a variety of B-cell lymphomas, as well as some with autoimmune disorders. There has been a notable increase in the survival rates from B-cell lymphoma in the decade since anti-CD20 therapy was introduced. PMID:19773256

Lim, Sean H.; Beers, Stephen A.; French, Ruth R.; Johnson, Peter W.M.; Glennie, Martin J.; Cragg, Mark S.



Monoclonal antibodies directed against surface molecules of multicell spheroids  

NASA Technical Reports Server (NTRS)

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

Martinez, Andrew O.



Antiepidermal Growth Factor Receptor Monoclonal Antibodies: Applications in Colorectal Cancer  

PubMed Central

Patients with metastatic colorectal cancer have a poor prognosis and present a challenge to clinicians. The role of the antiepidermal growth factor receptor (EGFR) pathway in tumorogenesis and tumor progression has been well defined. This paper will review the use of anti-EGFR monoclonal antibodies in the treatment of operable, as well as metastatic colorectal cancer both in the setting of KRAS mutation unselected patients and later in KRAS wild-type patients. Active investigations designed to further identify predictive biomarkers that may be potentially druggable are reviewed as well. PMID:23091721

Azizi, Efat; Kittai, Adam; Kozuch, Peter



Generation and characterization of monoclonal antibodies to TDRD7 protein.  


TDRD7 is a scaffold protein whose specific function is unknown. It has been identified in complexes with proteins that regulate cytoskeleton dynamics and centrosomal movements, mRNA transport, and protein translation apparatus. Here we report the generation and characterization of monoclonal antibodies against TDRD7 protein. Bacterially expressed His-tagged fragments of TDRD7 were used as antigens. Spleen cells from immunized mice were collected and fused with SP2/0 myeloma cells using PEG 2000. High titer anti-TDRD7 antibody-producing hybridoma cell lines were identified by enzyme-linked immunosorbent assay (ELISA) and then subcloned by limiting dilution. Antibodies produced by E6 clone were further tested for their reactivity with the TDRD7 recombinant proteins. The results obtained clearly indicate that E6 anti-TDRD7 antibodies recognize specifically recombinant 6His-tagged TDRD7 proteins and endogenous TDRD7 in Western blotting, immunoprecipitation, and immunocytochemistry. In summary, these antibodies will be useful for researchers investigating TDRD7 and molecular complexes involving this protein. PMID:18582216

Skorokhod, Oleksandr; Nemazanyy, Ivan; Breus, Oksana; Filonenko, Valeriy; Panasyuk, Ganna



Internal radiation dosimetry for clinical testing of radiolabeled monoclonal antibodies  

SciTech Connect

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

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



Anti-factor IXa/X bispecific antibody ACE910 prevents joint bleeds in a long-term primate model of acquired hemophilia A.  


ACE910 is a humanized anti-factor IXa/X bispecific antibody mimicking the function of factor VIII (FVIII). We previously demonstrated in nonhuman primates that a single IV dose of ACE910 exerted hemostatic activity against hemophilic bleeds artificially induced in muscles and subcutis, and that a subcutaneous (SC) dose of ACE910 showed a 3-week half-life and nearly 100% bioavailability, offering support for effective prophylaxis for hemophilia A by user-friendly SC dosing. However, there was no direct evidence that such SC dosing of ACE910 would prevent spontaneous bleeds occurring in daily life. In this study, we newly established a long-term primate model of acquired hemophilia A by multiple IV injections of an anti-primate FVIII neutralizing antibody engineered in mouse-monkey chimeric form to reduce its antigenicity. The monkeys in the control group exhibited various spontaneous bleeding symptoms as well as continuous prolongation of activated partial thromboplastin time; notably, all exhibited joint bleeds, which are a hallmark of hemophilia. Weekly SC doses of ACE910 (initial 3.97 mg/kg followed by 1 mg/kg) significantly prevented these bleeding symptoms; notably, no joint bleeding symptoms were observed. ACE910 is expected to prevent spontaneous bleeds and joint damage in hemophilia A patients even with weekly SC dosing, although appropriate clinical investigation is required. PMID:25274508

Muto, Atsushi; Yoshihashi, Kazutaka; Takeda, Minako; Kitazawa, Takehisa; Soeda, Tetsuhiro; Igawa, Tomoyuki; Sampei, Zenjiro; Kuramochi, Taichi; Sakamoto, Akihisa; Haraya, Kenta; Adachi, Kenji; Kawabe, Yoshiki; Nogami, Keiji; Shima, Midori; Hattori, Kunihiro



Monoclonal Antibodies against HLA-DR Antigens Replace T Helper Cells in Activation of B Lymphocytes  

Microsoft Academic Search

In the presence but not in the absence of pokeweed mitogen (PWM), monoclonal antibodies against HLA-DR antigens 147 and 164 helped highly purified B lymphocytes to proliferate and mature to Ig-secreting cells. In contrast, neither anti-DR antibody 231 nor the UCHT1 monoclonal anti-human T cell antibody (both of the same isotype as the 147 and 164 anti-DR antibodies) exhibited any

Ronald Palacios; Otoniel Martinez-Maza; Keith Guy



Computational design of a CNT carrier for a high affinity bispecific anti-HER2 antibody based on trastuzumab and pertuzumab Fabs.  


This is a preliminary cross multidisciplinary theoretical-computational approach for the design of a drug delivery system based on immunoconjugated carbon nanotube against HER2- overexpressing cancer cells. This drug delivery system allows the release of an encapsulated cytotoxic cocktail in a controlled manner under pulsed radio frequency (RF) irradiation. Our effort is focused on the computational aided design of a high affinity bispecific anti-HER2 antibody and an opening mechanism of the carbon nanotube (CNT) based cytotoxic carrier for controlling multiple drug release. We study the main interactions between the antibody and the antigen by a computational scanning mutagenesis approach of trastuzumab and pertuzumab fragment antigen binding (Fab) structures in order to enhance their binding affinity. Then, each Fab fragments is joined by a polypeptide linker which should be stable enough to avoid the "open form" of antibody. On the other hand, we also conjugate the engineered antibody to functionalized CNTs (f-CNTs), which encapsulate the inhibitors of the HER2/PI3K/Akt/mTOR signaling pathway. We take advantage of the fact that f-CNT converts the RF radiation absorption into heat release. A pulsed laser at 13.45 MHz increments the temperature around 40 °C for triggering the nano-caps destabilization, which allows the switching of the opening mechanism of the drug carrier. Nano-caps will be a dual pH/temperature responsive in order to take advantage of lysosome characteristic (acidic pH) and heat release from the carrier. Nano-caps are functionalized with organic amide moieties, which hydrolyze quickly at an acidic pH into primary amines, and protonated amines generate repulsion interactions with other charged species, which trigger the cytotoxics release. PMID:23143677

Salazar-Salinas, Karim; Kubli-Garfias, Carlos; Seminario, Jorge M



Screening individual hybridomas by microengraving to discover monoclonal antibodies  

PubMed Central

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

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



Engineering fully human monoclonal antibodies from murine variable regions.  


Fully human monoclonal antibodies (mAbs) derived from transgenic mice or human antibody libraries are the current state of the art for reducing the immunogenicity risk of antibody drugs. Here, we describe a novel method for generating fully human mAbs from nonhuman variable regions using information from the human germline repertoire. Central to our strategy is the rational engineering of residues within and proximal to CDRs and the V(H)/V(L) interface by iteratively exploring substitutions to the closest human germline sequences using semi-automated computational methods. Starting from the parent murine variable regions of three currently marketed mAbs targeting CD25, vascular endothelial growth factor, and tumor necrosis factor alpha, we have generated fully human antibodies with 59, 46, and 45 substitutions, respectively, compared to the parent murine sequences. A large number of these substitutions were in the CDRs, which are typically avoided in humanization methods. Antigen affinities of the fully human variants were comparable to the chimeric mAbs in each case. Furthermore, in vitro functional characterization indicated that all retain potency of the chimeric mAbs and have comparable activity to their respective marketed drugs daclizumab, bevacizumab, and infliximab. Based on local and global sequence identity, the sequences of our engineered mAbs are indistinguishable from those of fully human mAbs isolated from transgenic mice or human antibody libraries. This work establishes a simple rational engineering methodology for generating fully human antibody therapeutics from murine mAbs produced from standard hybridoma technology. PMID:20045416

Bernett, Matthew J; Karki, Sher; Moore, Gregory L; Leung, Irene W L; Chen, Hsing; Pong, Erik; Nguyen, Duc-Hanh T; Jacinto, Jonathan; Zalevsky, Jonathan; Muchhal, Umesh S; Desjarlais, John R; Lazar, Greg A



Murine monoclonal antibodies reactive with human heart and group A streptococcal membrane antigens.  

PubMed Central

Ten selected murine hybridoma cell lines that produce monoclonal antibodies against M type 5 Streptococcus pyogenes and human heart antigen were isolated. All of the monoclonal antibodies studied were determined to be the immunoglobulin M isotype. The antibodies were characterized on the basis of their reactions with Triton X-100-extracted whole human heart antigens, sodium dodecyl sulfate-extracted sarcolemmal antigens, and whole streptococci or their membranes. Enzyme-linked immunosorbent assays and Western immunoblotting techniques were used to compare the reactivity of the monoclonal antibodies. All 10 of the antibodies were first selected for their reactivity with Triton X-100-extracted heart antigens and whole group A, M type 5 streptococci. These antibodies were then divided into two categories: strong reactors or weak reactors with human sarcolemmal and streptococcal membranes. Among the strong reactors, two different types of monoclonal antibodies were observed on the basis of their immunobanding patterns with sarcolemmal and streptococcal membranes on Western blots. Monoclonal antibodies that were strong reactors with sarcolemmal and group A streptococcal membrane antigen were directed against a determinant on a family of proteins. The major reactants of sarcolemmal extracts were high-molecular-weight proteins near 200,000. Some monoclonal antibodies demonstrated more specificity for the heart than did others when reacted with separated Triton X-100-extracted tissue antigens from the heart, kidney, and skeletal muscle. One of the monoclonal antibodies that reacted with group A streptococci reacted with a Triton X-100-extracted heart antigen ca. 40,000 daltons in size. None of these monoclonal antibodies opsonized type 5 Streptococcus pyogenes, and in enzyme-linked immunosorbent assays most of the antibodies were found to react to a lesser degree with other groups of streptococci. Monoclonal antibody was used to probe normal and rheumatic sarcolemma for differences in reactivity. Although the rheumatic heart reacted more intensely, no major differences between the immunobanding patterns of normal and rheumatic hearts were observed. Images PMID:6384047

Cunningham, M W; Krisher, K; Graves, D C



Nature's best weapons to fight cancer. Revival of human monoclonal IgM antibodies  

Microsoft Academic Search

The unique features of monoclonal antibodies (specificity, effectiveness, purity and unlimited reproducibility) make them ideal tools for the specific treatment of all kind of diseases. The third generation of monoclonal antibodies for the treatment of human diseases will be, after murine and \\

H. Peter Vollmers; Stephanie Brandlein


Affinity Precipitation of a Monoclonal Antibody From an Industrial Harvest Feedstock Using an  

E-print Network

Affinity Precipitation of a Monoclonal Antibody From an Industrial Harvest Feedstock Using an ELP affinity precipi- tation process is developed for monoclonal antibody (mAb) purification from industrial. While the overall mAb yield and HCP clearance for the affinity precipitation process was comparable

Chen, Wilfred


Antigenic Analysis of Punta Toro Virus and Identification of Protective Determinants with Monoclonal Antibodies,  

National Technical Information Service (NTIS)

Hybridomas producing monoclonal antibodies to the three major structural proteins of Punta Toro virus (PTV) were established by fusion of spleen cells with Sp2/0-Ag-14 mouse plasmacytoma cells. Thirty-six independently derived monoclonal antibodies were e...

D. Y. Pifat, J. F. Smith, M. C. Osterling



Characterization of monoclonal antibodies against Muscovy duck reovirus ?B protein  

PubMed Central

Background The ?B protein of Muscovy duck reovirus (DRV), one of the major structural proteins, is able to induce neutralizing antibody in ducks, but the monoclonal antibody (MAb) against ?B protein has never been characterized. Results Four hybridoma cell lines secreting anti-DRV ?B MAbs were obtained, designated 1E5, 2F7, 4E3 and 5D8. Immunoglobulin subclass tests differentiated them as IgG2b (1E5 and 4E3) and IgM (2F7 and 5D8). Dot blot and western blotting assays showed that MAbs reacted with His-?B protein in a conformation-independent manner. Competitive binding assay indicated that the MAbs delineated two epitopes, A and B of ?B. Immunofluorescence assay indicated that the four MAbs could specifically bind to Vero cells infected with DRV and ?B was distributed diffusely in the cytoplasma of infected cells. MAbs had universal reactivity to all DRVs tested in an antigen-capture enzyme-linked immunosorbent assay. Conclusion Results of this research provide important information about the four monoclonal antibodies and therefore the MAbs may be useful candidate for the development of a MAb capture ELISA for rapid detection of DRVs. In addition, it showed that the ?B protein was located in the cytoplasma of infected cells by immunofluorescence assay with MAbs. Virus isolation and RT-PCR are reliable way for detection of DRV infection, but these procedures are laborious, time consuming, and requiring instruments. These obvious diagnosis problems highlight the ongoing demand of rapid, reproducible, and automatic methods for the sensitive detection of DRV. PMID:20569474



Immunologic characterization and specificity of three monoclonal antibodies against the 58-kilodalton protein of Legionella pneumophila.  

PubMed Central

Three monoclonal antibodies against the Legionella pneumophila 58-kDa protein were produced. By using immunoblot analysis, the percentages of reactivity against 47 serogroups of Legionella representing 29 species were determined to be 80.9, 87.2, and 95.6 for monoclonal antibodies GB5BE8, GB5AF6, and CA4AF5, respectively. Specificities obtained from testing 63 heterologous organisms representing 22 genera and 46 species were 90.7, 92.2, and 95.3% for monoclonal antibodies GB5BE8, GB5AF6, and CA4AF5, respectively. No single heterologous strain was reactive with all three monoclonal antibodies. These monoclonal antibodies successfully identified all 10 clinical isolates of Legionella examined in a dot blot assay and should be excellent reagents for use in genuswide diagnostic immunoassays. Images PMID:1890189

Sampson, J S; Plikaytis, B B; Aloisio, C H; Carlone, G M; Pau, C P; Stinson, A R



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

PubMed Central

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

Monzo, Cesar; Urbaneja, Alberto; Ximenez-Embun, Miguel; Garcia-Fernandez, Julia; Garcia, Jose Luis; Castanera, Pedro



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


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

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



Microheterogeneity of the collecting duct system in rabbit kidney as revealed by monoclonal antibodies  

Microsoft Academic Search

This report describes the immunolocalization of three monoclonal antibodies along the collecting duct system in rabbit kidney. The antibodies were raised against antigens derived from a membrane fraction of homogenized papillary tissue. Western Blot analysis demonstrated that each of the antibodies recognized a single band of about 190000 (PCD1), 210000 (PCD2) and 50000 (PCD3) daltons. In renal tissue, the antibodies

P. Gilbert; W. W. Minuth; S. Bachmann



A monoclonal antibody to brush border and passive Heymann nephritis.  

PubMed Central

An IgG2a monoclonal antibody (MoAb) reacting with the brush border of the renal proximal tubule and glomerular capillary wall was produced by fusion of NS1 myeloma cells with spleen cells from BALB/c mice immunized with renal brush border preparations from rat kidney cortex. This antibody reacts with a 90,000 mol. wt protein which can be isolated by immunoprecipitation of radiolabelled brush border or glomerular preparations and localized on these structures by immunoperoxidase electron microscopy, thus demonstrating the presence of common antigenic determinants. Survey of various organs showed that the MoAb reacted with the brush border of the gut, but also with antigens associated with the distal vascular system. In the liver antigenic determinants were located along the sinusoid walls but mainly on bile canaliculi. Specific glomerular binding could be demonstrated in vivo by immunofluorescence after an intravenous injection of 2 mg of antibody or by paired label methodology using tracer amounts. Kinetics however were dramatically different from those observed in classical passive Heymann nephritis since glomerular binding was transient during the first hours after injection. Binding was also found in tubular structures, as well as in lung, liver, spleen and heart. These results identify a well defined antigen-antibody system responsible for the formation of transient extramembranous glomerular deposits and may be relevant to some human cases of glomerulonephritis. They may also provide new models to study glomerular and tubular transfer of membrane bound antibodies. Images Fig. 3 Fig. 4 Fig. 1 Fig. 2 Fig. 5 Fig. 6 Fig. 8 PMID:6365376

Ronco, P; Allegri, L; Melcion, C; Pirotsky, E; Appay, M D; Bariety, J; Pontillon, F; Verroust, P



Modulation of catalysis and inhibition of fetal bovine serum acetylcholinesterase by monoclonal antibodies  

SciTech Connect

Monoclonal antibodies have been raised against acetyicholinesterase isolated from a variety of sources and species. Although none of these antibodies bind to the esteratic site, some of them appear to interact with the region of the catalytic subunit referred to as the peripheral anionic site. We describe here the production and characterization of six inhibitory monoclonal antibodies against fetal bovine serum acetylcholinesterase. Results show that changes in the conformation of acetyicholinesterase caused by interaction with monoclonal antibodies at a site remote from the catalytic site result in the modulation of catalytic activity of acetylcholinesterase.

Doctor, B.P.; Gentry, M.K.; Saxena, A.; Ashani, Y.



Molecular mechanisms of resistance to the EGFR monoclonal antibody cetuximab  

PubMed Central

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

Brand, Toni M; Iida, Mari



Generation and characterization of monoclonal antibodies to prostate secretory protein.  


Monoclonal antibodies (MAbs) were produced against a highly purified preparation of prostate secretory protein (PSP) isolated from normal seminal plasma. Fifteen antibodies were selected for further evaluation based on their strong reactivity and specificity for PSP. All the MAbs had a specificity for prostate epithelial cells and none reacted to any of a variety of normal tissues as determined by immunoperoxidase staining. Six of the MAbs were selected for further immunohistochemical evaluation based on their ability to recognize different antigenic determinants. Using competitive binding immunoassays, a variety of overlapping specificities were observed with at least 2 distinct epitopes identified. Although some staining variability was noted, the 6 antibodies, in general, gave the same pattern of tissue reactivity. Both the normal prostate and the benign prostate hyperplastic ductal epithelial cells stained intensely, with 78 to 100% and 50-100% of the cells staining, respectively. The number and often the staining intensity of the tumor cells decreased as the tumor became more undifferentiated. Approximately 40 to 100% and 15 to 70% of the tumor cells stained in the moderately-differentiated and well-differentiated carcinoma tissues, respectively, whereas either no staining was observed or less than 20% of the tumor cells stained in the poorly-differentiated and undifferentiated tumors. Most of the metastatic prostate tumors showed either no staining or scattered staining in a few cells (i.e., less than 20%). PMID:2194982

Wright, G L; Huang, C L; Lipford, G; Beckett, M L; Liang, H M; Haley, C; Newhall, K; Morningstar, M



Targeting human C-type lectin-like molecule-1 (CLL1) with a bispecific antibody for immunotherapy of acute myeloid leukemia.  


Acute myeloid leukemia (AML), which is the most common acute adult leukemia and the second most common pediatric leukemia, still has a poor prognosis. Human C-type lectin-like molecule-1 (CLL1) is a recently identified myeloid lineage restricted cell surface marker, which is overexpressed in over 90% of AML patient myeloid blasts and in leukemic stem cells. Here, we describe the synthesis of a novel bispecific antibody, ?CLL1-?CD3, using the genetically encoded unnatural amino acid, p-acetylphenylalanine. The resulting ?CLL1-?CD3 recruits cytotoxic T?cells to CLL1 positive cells, and demonstrates potent and selective cytotoxicity against several human AML cell lines and primary AML patient derived cells in?vitro. Moreover, ?CLL1-?CD3 treatment completely eliminates established tumors in an U937 AML cell line xenograft model. These results validate the clinical potential of CLL1 as an AML-specific antigen for the generation of a novel immunotherapeutic for AML. PMID:25056598

Lu, Hua; Zhou, Quan; Deshmukh, Vishal; Phull, Hardeep; Ma, Jennifer; Tardif, Virginie; Naik, Rahul R; Bouvard, Claire; Zhang, Yong; Choi, Seihyun; Lawson, Brian R; Zhu, Shoutian; Kim, Chan Hyuk; Schultz, Peter G



Characterization of a Novel Single-Chain Bispecific Antibody for Retargeting of T Cells to Tumor Cells via the TCR Co-Receptor CD8  

PubMed Central

There is currently growing interest in retargeting of effector T cells to tumor cells via bispecific antibodies (bsAbs). Usually, bsAbs are directed on the one hand to the CD3 complex of T cells and on the other hand to a molecule expressed on the surface of the target cell. A bsAb-mediated cross-linkage via CD3 leads to an activation of CD8+ T cells and consequently to killing of the target cells. In parallel, CD4+ T cells including TH1, TH2, TH17 cells and even regulatory T cells (Tregs) will be activated as well. Cytokines produced by CD4+ T cells can contribute to severe side effects e. g. life-threatening cytokine storms and, thinking of the immunosupressive function of Tregs, can even be counterproductive. Therefore, we asked whether or not it is feasible to limit retargeting to CD8+ T cells e. g. via targeting of the co-receptor CD8 instead of CD3. In order to test for proof of concept, a novel bsAb with specificity for CD8 and a tumor-associated surface antigen was constructed. Interestingly, we found that pre-activated (but not freshly isolated) CD8+ T cells can be retargeted via CD8-engaging bsAbs leading to an efficient lysis of target cells. PMID:24751697

Koristka, Stefanie; Arndt, Claudia; Cartellieri, Marc; Ehninger, Armin; Ehninger, Gerhard; Bachmann, Michael P.



A Th1 cytokine-enriched microenvironment enhances tumor killing by activated T cells armed with bispecific antibodies and inhibits the development of myeloid-derived suppressor cells  

PubMed Central

In this study, we investigated whether activated T cells (ATC) armed with bispecific antibodies (aATC) can inhibits tumor growth and MDSC development in a Th1 cytokine–enriched (IL-2 and IFN-?) microenvironment. Cytotoxicity mediated by aATC was significantly higher (P <0.001) against breast cancer cell lines in the presence of Th1 cytokines as compared with control co-cultures. In the presence of aATC, CD33+/CD11b+/CD14?/HLA-DR?MDSC population was reduced significantly under both control (P <0.03) and Th1-enriched (P <0.036) culture conditions. Cytokine analysis in the culture supernatants showed high levels of MDSC suppressive chemokines CXCL9 and CXCL10 in Th1-enriched culture supernatants with highly significant increase (P <0.001) in the presence of aATC. Interestingly, MDSC recovered from co-cultures without aATC showed potent ability to suppress activated T-cell-mediated cytotoxicity (P <0.001), IFN-? production (P <0.01) and T-cell proliferation (P <0.05) compared to those recovered from aATC-containing co-cultures. These data suggest that aATC can mediate enhanced killing of tumor cells and may suppress MDSC and Treg differentiation, and presence of Th1 cytokines potentiates aATC-induced suppression of MDSC, sug-gesting that Th1-enriching immunotherapy may be benefi-cial in cancer treatment. PMID:21971587

Schalk, Dana; Sarkar, Sanila H.; Al-Khadimi, Zaid; Sarkar, Fazlul H.; Lum, Lawrence G.



One-step mixing with humanized anti-mPEG bispecific antibody enhances tumor accumulation and therapeutic efficacy of mPEGylated nanoparticles.  


Methoxy PEGylated nanoparticles (mPEG-NPs) are increasingly used for cancer imaging and therapy. Here we describe a general and simple approach to confer tumor tropism to any mPEG-NP. We demonstrate this approach with humanized bispecific antibodies (BsAbs) that can bind to both mPEG molecules on mPEG-NPs and to EGFR or HER2 molecules overexpressed on the surface of cancer cells. Simple mixing of BsAbs with mPEG-NPs can mediate preferential binding of diverse mPEG-NPs to cancer cells that overexpress EGFR or HER2 under physiological conditions and significantly increase cancer cell killing by liposomal doxorubicin to EGFR(+) and HER2(+) cancer cells. BsAbs modification also enhanced accumulation of fluorescence-labeled NPs and significantly increased the anticancer activity of drug-loaded NPs to antigen-positive human tumors in a mouse model. Anti-mPEG BsAbs offer a simple one-step method to confer tumor specificity to mPEG-NPs for enhanced tumor accumulation and improved therapeutic efficacy. PMID:25212525

Kao, Chien-Han; Wang, Jaw-Yuan; Chuang, Kuo-Hsiang; Chuang, Chih-Hung; Cheng, Ta-Chun; Hsieh, Yuan-Chin; Tseng, Yun-Long; Chen, Bing-Mae; Roffler, Steve R; Cheng, Tian-Lu



Monoclonal Antibodies to the S1 Spike and Membrane Proteins of Avian Infectious Bronchitis Coronavirus Strain Massachusetts M41  

Microsoft Academic Search

SUMMARY We have established four murine hybridoma cell lines which secrete specific antibody to avian infectious bronchitis virus (IBV) strain Massachusetts M41. Two monoclonal antibodies reacted with the spike protein and two reacted with the membrane protein. The specificity of the monoclonal antibodies for the external structural proteins was detected by immunoprecipitations using radiolabelled virus. The reactions of the monoclonal




Pretargeted ImmunoPET Imaging of CEA-expressing Tumors with a Bispecific Antibody and a 68Ga- and 18F-labeled Hapten-peptide in Mice with Human Tumor Xenografts  

PubMed Central

18F-Fluorodeoxyglucose (18F-FDG) is the most common molecular imaging agent in oncology, with a high sensitivity and specificity for detecting a number of cancers. Antibodies could enhance specificity; therefore, procedures were developed for radiolabeling a small (?1.5 kD) hapten-peptide with 68Ga or 18F to compare their specificity to 18F-FDG for detecting tumors using a pretargeting procedure. Mice were implanted with carcinoembryonic (CEA; CEACAM5)-expressing LS174T human colonic tumors, a CEA-negative tumor, or an inflammation was induced in thigh muscle. A bispecific monoclonal (bsMAb) anti-CEA × anti-hapten antibody was given to mice, and 16 h later, 5 MBq of 68Ga- or 18F-labeled hapten-peptides were administered intravenously. Within 1 h, tissues showed high and specific targeting of the 68Ga-IMP-288, with 10.7 ± 3.6% ID/g uptake in the tumor and very low uptake in normal tissues (e.g., tumor/blood 69.9 ± 32.3), in a CEA-negative tumor (0.35 ± 0.35% ID/g), and inflamed muscle (0.72 ± 0.20% ID/g). 18F-FDG localized efficiently in the tumor (7.42 ± 0.20% ID/g), but also in the inflamed muscle (4.07 ± 1.13% ID/g) and in a number of normal tissues; thus, pretargeted 68Ga-IMP-288 provided better specificity and sensitivity. PET/CT images reinforced the improved specificity of the pretargeting method. 18F-labeled IMP-449 distributed similarly in the tumor and normal tissues as the 68Ga-labeled IMP-288, indicating that either radiolabeled hapten-peptide could be used. Thus, pretargeted immunoPET performs exceptionally well with short-lived radionuclides, and is a highly sensitive procedure that is more specific than 18F-FDG-PET. PMID:20354120

Schoffelen, Rafke; Sharkey, Robert M.; Goldenberg, David M.; Franssen, Gerben; McBride, William J.; Rossi, Edmund A.; Chang, Chien-Hsing; Laverman, Peter; Disselhorst, Jonathan A.; Eek, Annemarie; van der Graaf, Winette T.A.; Oyen, Wim J.G.; Boerman, Otto C.



Infectious Complications Associated with Monoclonal Antibodies and Related Small Molecules  

PubMed Central

Summary: Biologics are increasingly becoming part of routine disease management. As more agents are developed, the challenge of keeping track of indications and side effects is growing. While biologics represent a milestone in targeted and specific therapy, they are not without drawbacks, and the judicious use of these “magic bullets” is essential if their full potential is to be realized. Infectious complications in particular are not an uncommon side effect of therapy, whether as a direct consequence of the agent or because of the underlying disease process. With this in mind, we have reviewed and summarized the risks of infection and the infectious disease-related complications for all FDA-approved monoclonal antibodies and some related small molecules, and we discuss the probable mechanisms involved in immunosuppression as well as recommendations for prophylaxis and treatment of specific disease entities. PMID:19366915

Salvana, Edsel Maurice T.; Salata, Robert A.



Freezing-induced perturbation of tertiary structure of monoclonal antibody  

E-print Network

and methods Model protein: Monoclonal antibody (IgG) 0.5mg/ml Buffers: 10 mM Potassium Phosphate, pH 3,8 10 mM Sodium Acetate, pH 4 Salt: 0, 150mM Potassium Chloride 1-anilino-8-naphthalene sulfonate (ANS): 75?M Instruments: Quanta...-thawed 20C Peak shift due to freezing pH8 338.2 ? 0.1 nm 338.5 ? 0.1 nm 337.9 ? 0.2 nm 0.3 nm pH3 340.0 ? 0.2 nm 333.9 ? 0.1 nm 340.8 ? 0.2 nm 6.1 nm pH4 339.1 ? 0.1 nm 333.2 ? 0.3 nm 339.2 ? 0.2 nm 5.9 nm Figure 1 IgG intrinsic...

Liu, Lu; Kueltzo, L. A.; Jones, L. S.; Carpenter, J. F.



Monoclonal Antibody-Directed Effector Cells Selectively Lyse Human Melanoma Cells in vitro and in vivo  

NASA Astrophysics Data System (ADS)

Monoclonal antibody 9.2.27 (mAb 9.2.27) directed to a chondroitin sulfate proteoglycan on human melanoma cells was able to suppress tumor growth in athymic (nu/nu) mice more effectively when bound with polyethylene glycol to murine effector cells than when injected alone. These ``armed'' effector cells also proved more effective than the monoclonal antibody in eliciting antibody-dependent cellular cytotoxicity against human melanoma target cells in vitro.

Schulz, Gregor; Bumol, Thomas F.; Reisfeld, Ralph A.



Neutralization of Yellow Fever Virus Studied Using Monoclonal and Polyclonal Antibodies  

Microsoft Academic Search

SUMMARY Monoclonal and polyclonal antibodies with known specificity for either the 54K envelope glycoprotein or the 48K non-structural glycoprotein of yellow fever (YF) virus-infected cells were studied in plaque reduction neutralization tests. Viruses employed in the tests comprised wild-type and vaccine strains of YF and a selection of other flaviviruses. Of 17 monoclonal antibodies examined, six of the 54K-specific antibodies

A. Buckley; E. A. Gould



Characteristics and performance of a bispecific F (ab'gamma)2 antibody for delivering saporin to a CD7+ human acute T-cell leukaemia cell line.  

PubMed Central

We have investigated the efficacy of a F(ab'gamma)2 bispecific antibody (BsAb) with dual specificity for the CD7 molecule in one Fab arm and for the ribosome inactivating protein (rip) saporin in the other arm, for delivering saporin to the acute T-cell leukaemia cell line HSB-2. Saporin titration experiments revealed that BsAb increased the toxicity of saporin 435-fold for HSB-2 cells, reducing the IC50 for saporin alone from 0.1 mumol to 0.23 nmol when BsAb was included. The rate of protein synthesis inactivation brought about by BsAb-mediated toxin delivery to HSB-2 cells was very similar to that described for conventional immunotoxins (IT's) with a t10 (time taken for a one log inhibition of protein synthesis compared with controls) of 46 h obtained at a saporin concentration of 1 nmol and 226 h at 0.1 nmol. BsAb titration studies demonstrated a clear dose response effect of BsAb concentration on target cell protein synthesis inhibition and cell proliferation. The absolute specificity of toxin delivery was unequivocally demonstrated by a failure of BsAb to deliver an effective dose of saporin to the CD7- cell line HL60 and by the blocking of BsAb-mediated delivery of saporin to HSB-2 cells with an excess of F(ab)2 fragments of the anti-CD7 antibody, HB2. These studies have clearly demonstrated the effectiveness of this BsAb for delivering saporin to a T-ALL cell line utilising CD7 as the target molecule on the cell surface. BsAb's would therefore appear to offer a realistic alternative to IT's for toxin delivery to tumour cells and may even offer certain advantages over conventional IT's for clinical use. PMID:1716453

Flavell, D. J.; Cooper, S.; Morland, B.; Flavell, S. U.



Antibody-guided irradiation of advanced ovarian cancer with intraperitoneal monoclonal antibodies  

SciTech Connect

Twenty-four patients with persistent epithelial ovarian cancer after chemotherapy with or without external beam irradiation, were treated with intraperitoneally administered /sup 131/I-labeled monoclonal antibodies HMFG1, HMFG2, AUA1, H17E2, directed against tumor-associated antigens. Acute side effects were mild abdominal pain, pyrexia, diarrhea, and moderate reversible pancytopenia. One patient developed a subphrenic abscess requiring surgical drainage. Eight patients with large volume disease, ie, greater than 2 cm tumor diameter, did not respond to antibody-guided irradiation and died of progressive disease within 9 months of treatment. Sixteen patients had small-volume (less than 2 cm) disease at the time of treatment with radiolabeled antibody. Seven patients failed to respond, and of nine initial responders, four patients remain alive and free from disease 6 months to 3 years from treatment. Analysis of the data on relapse indicated that doses greater than 140 mCi were more effective than lower doses. We conclude that the intraperitoneal administration of 140 mCi or more of /sup 131/I-labeled tumor-associated monoclonal antibodies represents a new and potentially effective form of therapy for patients with small-volume stage III ovarian cancer.

Epenetos, A.A.; Munro, A.J.; Stewart, S.; Rampling, R.; Lambert, H.E.; McKenzie, C.G.; Soutter, P.; Rahemtulla, A.; Hooker, G.; Sivolapenko, G.B.



Production of human anti-HLA monoclonal antibodies  

SciTech Connect

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

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



Macrophages eliminate circulating tumor cells after monoclonal antibody therapy  

PubMed Central

The use of monoclonal antibodies (mAbs) as therapeutic tools has increased dramatically in the last decade and is now one of the mainstream strategies to treat cancer. Nonetheless, it is still not completely understood how mAbs mediate tumor cell elimination or the effector cells that are involved. Using intravital microscopy, we found that antibody-dependent phagocytosis (ADPh) by macrophages is a prominent mechanism for removal of tumor cells from the circulation in a murine tumor cell opsonization model. Tumor cells were rapidly recognized and arrested by liver macrophages (Kupffer cells). In the absence of mAbs, Kupffer cells sampled tumor cells; however, this sampling was not sufficient for elimination. By contrast, antitumor mAb treatment resulted in rapid phagocytosis of tumor cells by Kupffer cells that was dependent on the high-affinity IgG-binding Fc receptor (Fc?RI) and the low-affinity IgG-binding Fc receptor (Fc?RIV). Uptake and intracellular degradation were independent of reactive oxygen or nitrogen species production. Importantly, ADPh prevented the development of liver metastases. Tumor cell capture and therapeutic efficacy were lost after Kupffer cell depletion. Our data indicate that macrophages play a prominent role in mAb-mediated eradication of tumor cells. These findings may help to optimize mAb therapeutic strategies for patients with cancer by helping us to aim to enhance macrophage recruitment and activity. PMID:24430180

Gul, Nuray; Babes, Liane; Siegmund, Kerstin; Korthouwer, Rianne; Bogels, Marijn; Braster, Rens; Vidarsson, Gestur; ten Hagen, Timo L.M.; Kubes, Paul; van Egmond, Marjolein



Production and characterization of monoclonal antibodies to a grouper iridovirus.  


A panel of six monoclonal antibodies (mAbs) against a grouper iridovirus (SGIV) were produced by immunization of Balb/c mice with purified virus preparations. Isotyping test revealed all the mAbs were IgG1. None of the mAbs possessed the ability to neutralize SGIV in cell cultures. Western blot showed that 4 mAbs reacted with 2 SGIV proteins at molecular mass of approximately 100 and 117 kDa in gradient-purified virus. Immunofluorescent studies showed that the two specific viral proteins VP100 and VP117 were localized within virus assembly sites in the cytoplasm of SGIV-infected grouper cells (GP). Fractionations of the iridovirus in a 20-60% sucrose gradient were detected successfully by all the six mAbs using immunodot blot. An antigen-capture enzyme-linked immunosorbent assay (ELISA) system, based on the use of mAb 7E11 for capture and a rabbit polyclonal antibody to SGIV for detection was developed. SGIV antigen was detected in gradient-purified virus and virus-infected grouper blood. These novel mAbs will facilitate the development of more specific and standardized diagnostic techniques for marine fish iridovirus. PMID:12505628

Shi, Chengyin; Wei, Qi; Gin, Karina Yew Hoong; Lam, Toong Jin



A monoclonal antibody against human thrombospondin inhibits platelet aggregation.  

PubMed Central

A monoclonal antibody (C6.7) has been generated against the calcium-replete form of human platelet thrombospondin (TSP). C6.7 is specific for TSP as determined by both competitive radioimmunoassay and immunoprecipitation. This antibody inhibits both thrombin- and A23187-induced aggregation of gel-filtered platelets in a concentration-dependent manner without affecting the secretion of serotonin. The epitope on TSP recognized by C6.7 has been localized to an 18-kDa fragment that is present in mild chymotryptic digests of TSP. This fragment is disulfide-linked to a 120- to 140-kDa fragment in unreduced digests, and both reduction and denaturation are required to separate the 18-kDa peptide from the larger fragments. A 25-kDa heparin binding domain is also present in the chymotryptic digest. However, the 18-kDa peptide is distinct from the heparin binding domain. The amino acid sequence at the NH2 terminus of the 18-kDa fragment is Asp-Thr-Asn-Pro-Thr-Arg-Ala-Gln-Gly-Tyr-. Images PMID:2582413

Dixit, V M; Haverstick, D M; O'Rourke, K M; Hennessy, S W; Grant, G A; Santoro, S A; Frazier, W A



Monoclonal antibody-based therapies for microbial diseases  

PubMed Central

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

Saylor, Carolyn; Dadachova, Ekaterina; Casadevall, Arturo



Isolation of Monoclonal Antibodies with Predetermined Conformational Epitope Specificity  

PubMed Central

Existing technologies allow isolating antigen-specific monoclonal antibodies (mAbs) from B cells. We devised a direct approach to isolate mAbs with predetermined conformational epitope specificity, using epitope mimetics (mimotopes) that reflect the three-dimensional structure of given antigen subdomains. We performed differential biopanning using bacteriophages encoding random peptide libraries and polyclonal antibodies (Abs) that had been affinity-purified with either native or denatured antigen. This strategy yielded conformational mimotopes. We then generated mimotope-fluorescent protein fusions, which were used as baits to isolate single memory B cells from rhesus monkeys (RMs). To amplify RM immunoglobulin variable regions, we developed RM-specific PCR primers and generated chimeric simian-human mAbs with predicted epitope specificity. We established proof-of-concept of our strategy by isolating mAbs targeting the conformational V3 loop crown of HIV Env; the new mAbs cross-neutralized viruses of different clades. The novel technology allows isolating mAbs from RMs or other hosts given experimental immunogens or infectious agents. PMID:22737224

Humbert, Michael; Essono, Sosthene S.; Watkins, Jennifer D.; Vyas, Hemant K.; Shanmuganathan, Vivekanandan; Hemashettar, Girish; Kahn, Maria; Hu, Shiu-Lok; Montefiori, David C.; Polonis, Victoria R.; Schur, Peter H.; Ruprecht, Ruth M.



Characterization of Zaire ebolavirus glycoprotein-specific monoclonal antibodies.  


Zaire ebolavirus (ZEBOV) can be transmitted by human-to-human contact and causes acute haemorrhagic fever with case fatality rates up to 90%. There are no effective therapeutic or prophylactic treatments available. The sole transmembrane glycoprotein (GP) is the key target for developing neutralizing antibodies. In this study, recombinant VSV?G/ZEBOVGP was used to generate monoclonal antibodies (MAbs) against the ZEBOV GP. A total of 8 MAbs were produced using traditional hybridoma cell fusion technology, and then characterized by ELISA using ZEBOV VLPs, Western blotting, an immunofluorescence assay, and immunoprecipitation. All 8 MAbs worked in IFA and IP, suggesting that they are all conformational MAbs, however six of them recognized linearized epitopes by WB. ELISA results demonstrated that one MAb bound to a secreted GP (sGP 1-295aa); three bind to a part of the mucin domain (333-458aa); three MAbs recognized epitopes on the C-terminal domain of GP1 (296-501aa); and one bound to full length GP (VLPs/GP1,2 ?Tm). Using a mouse model these MAbs were evaluated for their therapeutic capacity during a lethal infection. All 8 MAb improved survival rates by 33%-100% against a high dose lethal challenge with mouse-adapted ZEBOV. This work has important implications for further development of vaccines and immunotherapies for ZEBOV infection. PMID:21925951

Qiu, Xiangguo; Alimonti, Judie B; Melito, P Leno; Fernando, Lisa; Ströher, Ute; Jones, Steven M



Monoclonal antibodies against a 97-kilodalton antigen from Aspergillus flavus.  

PubMed Central

We prepared a panel of five monoclonal antibodies (MAbs) directed against Aspergillus flavus that all reacted against one 97-kDa antigen by western blot (immunoblot). Flow cytometry demonstrated that these antibodies bound (in increasing degrees) to all morphologic stages of A. flavus growth: conidia, swollen conidia, and hyphae. Cross-reactivity among species was examined by enzyme-linked immunosorbent assay of fungal culture filtrates. Four MAbs reacted with 10 of 11 A. flavus isolates, and the fifth one reacted with 9 of them. One MAb also reacted with A. fumigatus, two reacted with A. niger, A. wentii, and A. nidulans, and all five reacted with A. ochraceus. None reacted with A. terreus, A. glaucus, A. versicolor, or a Penicillium species. Each MAb bound to A. flavus hyphae in formalin-fixed paraffin sections of a muscle biopsy from a confirmed human case of invasive aspergillosis. In summary, these MAbs identified a 97-kDa antigen found on A. flavus that is both surface bound and an exoantigen. Either the same or a cross-reacting antigen is present in A. fumigatus and other Aspergillus species. Images PMID:7496924

Hetherington, S V; Henwick, S; Parham, D M; Patrick, C C



WHO collaborative study on the use of monoclonal antibodies for the intratypic differentiation of poliovirus strains*  

PubMed Central

An international collaborative study was carried out to identify monoclonal antibodies that could reliably discriminate between wild polioviruses and strains derived from Sabin vaccine viruses. For poliovirus types 2 and 3, monoclonal antibodies were identified that reacted specifically with type 2 or type 3 strains which gave T1-oligonucleotide maps similar to or indistinguishable from that of Sabin vaccine virus, thus indicating their vaccine origin. These monoclonal antibodies failed to react with strains which gave T1 maps unrelated to that of Sabin vaccine virus. However for type 1, five of the six antibodies examined in the study reacted only with strains with a T1 map indistinguishable from that of type 1 Sabin vaccine virus. In contrast, other monoclonal antibodies against poliovirus types 1, 2 and 3 reacted broadly within a serotype. PMID:3017595

Ferguson, M.; Magrath, D. I.; Minor, P. D.; Schild, G. C.



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

SciTech Connect

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.''

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



Six antigenic determinants in the surface layer of the archaebacterium Methanococcus vannielii revealed by monoclonal antibodies  

SciTech Connect

The immunogenicity and antigenic characteristics of the unique surface layer (S layer) of Methanococcus vannielii was studied with a panel of six monoclonal antibodies. Six surface determinants were identified for the first time, each recognized by one antibody exclusively. The determinants are proteins, located in the S layer, and accessible to antibody in whole, unfixed, as well as formalinized bacteria. Hence the six antigens and antibodies reported here should be useful for rapid identification of new isolates and for taxonomy of methanogens, notably Methanococcaceae. In this connection two novel applications of the slide immunoenzymatic assay were developed for analyses of monoclonal antibodies and their complementary sites in the bacterial envelope.

de Macario, E.C.; Koenig, H.; Macario, A.J.L.; Kandler, O.



Six antigenic determinants in the surface layer of the archaebacterium Methanococcus vannielii revealed by monoclonal antibodies.  


The immunogenicity and antigenic characteristics of the unique surface layer (S layer) of Methanococcus vanielii was studied with a panel of six monoclonal antibodies. Six surface determinants were identified for the first time, each recognized by one antibody exclusively. The determinants are proteins, located in the S layer, and accessible to antibody in whole, unfixed, as well as formalinized bacteria. Hence the six antigens and antibodies reported here should be useful for rapid identification of new isolates and for taxonomy of methanogens, notably Methanococcaceae. In this connection two novel applications of the slide immunoenzymatic assay were developed for analyses of monoclonal antibodies and their complementary sites in the bacterial envelope. PMID:6197477

Conway de Macario, E; König, H; Macario, A J; Kandler, O



Surface antigenic change during differentiation of a parasitic protozoan, Leishmania mexicana: Identification by monoclonal antibodies.  

PubMed Central

The fusion of SP2/0 myeloma cells with spleen cells from mice immunized with Leishmania mexicana amazonensis promastigotes produced hybridoma clones. Indirect immunofluorescent antibody assay with live leishmanias showed that the monoclonal antibody 6H12 recognized only the antigens bound to the surface of L. mexicana amazonensis promastigotes. It also showed that the antibody bound to neither amastigotes of this species nor to other Leishmania species--i.e., L. braziliensis braziliensis, L. tropica, and L. donovani. Monoclonal antibodies from three other clones (4D11, 4H9, and 6A11) were found to compete with 6H12 for binding to L. mexicana promastigotes. With lysates of [35S]methionine-labeled promastigotes, all four monoclonal antibodies precipitated the same triplet set of protein bands at the approximately equal to 68,000-dalton region, whereas another monoclonal antibody (6G5) precipitated a different band at approximately equal to 90,000 daltons. During differentiation of L. mexicana amazonensis from amastigotes to promastigotes, there was a 4- to 8-fold increase above the initial level in the binding of 6H12 monoclonal antibody to leishmanias, as detected by enzyme-linked immunosorbent assay and quantitative fluorometric assay, respectively. Thus, we have demonstrated the use of monoclonal antibodies as probes for antigens that change during leishmanial differentiation. Images PMID:6961414

Fong, D; Chang, K P



Surface antigenic change during differentiation of a parasitic protozoan, Leishmania mexicana: Identification by monoclonal antibodies.  


The fusion of SP2/0 myeloma cells with spleen cells from mice immunized with Leishmania mexicana amazonensis promastigotes produced hybridoma clones. Indirect immunofluorescent antibody assay with live leishmanias showed that the monoclonal antibody 6H12 recognized only the antigens bound to the surface of L. mexicana amazonensis promastigotes. It also showed that the antibody bound to neither amastigotes of this species nor to other Leishmania species--i.e., L. braziliensis braziliensis, L. tropica, and L. donovani. Monoclonal antibodies from three other clones (4D11, 4H9, and 6A11) were found to compete with 6H12 for binding to L. mexicana promastigotes. With lysates of [35S]methionine-labeled promastigotes, all four monoclonal antibodies precipitated the same triplet set of protein bands at the approximately equal to 68,000-dalton region, whereas another monoclonal antibody (6G5) precipitated a different band at approximately equal to 90,000 daltons. During differentiation of L. mexicana amazonensis from amastigotes to promastigotes, there was a 4- to 8-fold increase above the initial level in the binding of 6H12 monoclonal antibody to leishmanias, as detected by enzyme-linked immunosorbent assay and quantitative fluorometric assay, respectively. Thus, we have demonstrated the use of monoclonal antibodies as probes for antigens that change during leishmanial differentiation. PMID:6961414

Fong, D; Chang, K P



Binding of an Anti-Fullerene IgG Monoclonal Antibody to Single Wall  

E-print Network

Binding of an Anti-Fullerene IgG Monoclonal Antibody to Single Wall Carbon Nanotubes Bernard Fc shows antibodies on the nanotube and on mica. The mica antibody images are similar in height, New York 10027 Received June 19, 2001; Revised Manuscript Received July 1, 2001 Single wall carbon


Detection of Macrophage Migration Inhibitory Factor by Monoclonal Antibody in Sézary Syndrome  

Microsoft Academic Search

We have reported previously on the generation of a monoclonal antibody against human macrophage migration inhibitory factor (MIF), which is a mediator of cellular immunity. Macrophage migration inhibitory factor activity in the migration assay was closely correlated with antibody reactivity. Using this antibody called 1C5\\/B, we are now able to study the expression of MIF in situ. Here, we report

Christine Neumann; Renate Schlegel; Friedhelm Steckel; Clemens Sorg



Six antigenic determinants in the surface layer of the archaebacterium Methanococcus vannielii revealed by monoclonal antibodies  

Microsoft Academic Search

The immunogenicity and antigenic characteristics of the unique surface layer (S layer) of Methanococcus vannielii was studied with a panel of six monoclonal antibodies. Six surface determinants were identified for the first time, each recognized by one antibody exclusively. The determinants are proteins, located in the S layer, and accessible to antibody in whole, unfixed, as well as formalinized bacteria.

E. C. de Macario; H. Koenig; A. J. L. Macario; O. Kandler




EPA Science Inventory

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


Development of Oligodendrocytes and Schwann Cells Studied with a Monoclonal Antibody against Galactocerebroside  

Microsoft Academic Search

We have generated a hybridoma cell line secreting a monoclonal antibody that specifically binds to the surfaces of oligodendrocytes and Schwann cells, the cells involved in myelin formation in the central and peripheral nervous systems, respectively. Binding studies using purified sphingolipids showed that this antibody reacts strongly with galactocerebroside (GalC), the major galactosphingolipid of myelin. The antibody was used in

B. Ranscht; P. A. Clapshaw; J. Price; M. Noble; W. Seifert



Monoclonal Antibodies to Purified Muscarinic Receptor Display Agonist-Like Activity  

Microsoft Academic Search

Monoclonal antibody M-35, which immunoprecipitates native calf brain acetylcholine muscarinic receptor, mimics agonist stimulation of the intact guinea pig myometrium: the antibody, just like carbamoylcholine hydrochloride, causes a rise in intracellular cyclic GMP content, an inhibition of cyclic AMP accumulation due to prostacyclin, and induces uterine contractions. Another antibody, M-23, which reacts with the denatured muscarinic receptor, is devoid of

D. Leiber; S. Harbon; J.-G. Guillet; C. Andre; A. D. Strosberg



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

SciTech Connect

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

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



The application of monoclonal antibody panels to characterize pestivirus isolates from ruminants in Great Britain  

Microsoft Academic Search

Summary Monoclonal antibodies were prepared against bovine virus diarrhoea virus and hog cholera virus. They were used to test 101 field isolates of ruminant pestivirus in a simple binding assay using an indirect immunoperoxidase label on fixed cell cultures. The monoclonals were divided into three panels: (1) pestivirus group specific, (2) hog cholera specific, (3) selectively reactive with ruminant pestiviruses.

S. Edwards; J. J. Sands; J. W. Harkness



Crystal Structure of a Dimerized Cockroach Allergen Bla g 2 Complexed with a Monoclonal Antibody*  

E-print Network

Crystal Structure of a Dimerized Cockroach Allergen Bla g 2 Complexed with a Monoclonal Antibody allergen Bla g 2 and the Fab fragment of a monoclonal anti- body 7C11 was solved at 2.8-A° resolution. Bla of an -helix and a helical turn from each allergen monomer, exhibiting a novel dimerization


Phytochrome in Photosynthetically Competent Plants: Characterization by Monoclonal Antibodies. Progress Report.  

National Technical Information Service (NTIS)

New monoclonal antibodies have been prepared to 124-kdalton phytochrome from etiolated oats, to phytochrome from etiolated peas, and to alkaline phosphatase. Simultaneously, progress has been made in improving methodologies for purification of phytochrome...

L. H. Pratt




EPA Science Inventory

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


Tactical planning optimization for campaign scheduling of active pharmaceutical ingredient production based on monoclonal antibodies  

E-print Network

Monoclonal Antibodies (mAb's) are the fastest growing segment in the biopharmaceutical industry. They are used today as therapeutics and diagnostics for several medical applications, including various types of cancer, ...

Assia, Shai



Monoclonal Antibody 14F7, Which Recognizes a Stage-Specific Immature Oligodendrocyte  

E-print Network

Monoclonal Antibody 14F7, Which Recognizes a Stage-Specific Immature Oligodendrocyte Surface Molecule, Inhibits Oligodendrocyte Differentiation Mediated in Co-Culture With Astrocytes Kazunori Institute of Medical Science, Tokyo, Japan Cells at an intermediate stage of oligodendrocyte lineage

Kawato, Suguru


Human Monoclonal Antibodies Broadly Neutralizing against Influenza B Virus  

PubMed Central

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

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



Characterization of monoclonal antibodies against waterfowl parvoviruses VP3 protein  

PubMed Central

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. PMID:23176172



Assignment of a fibronectin gene to human chromosome 2 using monoclonal antibodies  

SciTech Connect

The locus coding for the presumed structural gene for fibronectin has been mapped to human chromosome 2 using human-mouse somatic cell hybrids. The assignment of fibronectin has been made by testing man-mouse somatic cell hybrids with two anti-human fibronectin monoclonal antibodies which recognize different antigenic determinants of human, but not mouse, fibronectin. Both monoclonal antibodies demonstrate a highly concordant association between the presence of two different human fibronectin antigens and human chromosome 2.

Koch, G.A. (Roswell Park Memorial Inst., Buffalo, NY); Schoen, R.C.; Klebe, R.J.; Shows, T.B.



Acetylcholinesterase Monoclonal Antibody-Induced Sympathectomy: Effects on Immune Status and Acute Morphine-Induced Immunomodulation  

Microsoft Academic Search

The present study examined the role of the sympathetic nervous system (SNS) in immunomodulation by using the acetylcholinesterase monoclonal antibody (AChE mAb)-induced sympathectomy model. As part of this investigation, the effects of AChE mAb treatment on the immune alterations produced by acute morphine treatment also were explored. Experimental rats received tail vein injections of murine monoclonal IgG2b antibodies against rat

Karamarie Fecho; Kimberly A. Maslonek; Linda A. Dykstra; Donald T. Lysle



Detection of the organophosphorus nerve agent soman by an ELISA using monoclonal antibodies  

Microsoft Academic Search

The development of a specific and sensitive immunologic ELISA detection system for methylphosphonoflouridic acid. 1,2,2-trimethylpropylester (soman) by the use of monoclonal antibodies (MAbs) is described. The monoclonal antibodies F71D7, F71H10, F71B12 and F71H9 originally produced against the soman derivative methyl phosphonic acid,p-aminophenyl 1,2,2-trimethylpropyldiester (MATP) also reacted with soman in a previously developed, direct competitive ELISA. After optimizing the ELISA system

Michael H. Erhard; Reiner Kiihlmann; Ladislaus Szinicz; Uli LiJsch



The Goodpasture antigen in Alport's syndrome: Studies with a monoclonal antibody  

Microsoft Academic Search

The Goodpasture antigen in Alport's syndrome: Studies with a monoclonal antibody. A mouse monoclonal antibody (MCA-P1), which recognizes an antigenic determinant in human glomerular basement membrane against which autoantibodies are directed in Goodpasture's syndrome, was used in indirect immunofluorescence studies to investigate glomerular basement membrane structure in Alport's syndrome. We found reduced or absent binding of MCA-P1 to glomerular and

Caroline O S Savage; Charles D Pusey; Michael J Kershaw; Steven J Cashman; Peter Harrison; Barry Hartley; David R Turner; J Stewart Cameron; David J Evans; C Martin Lockwood; C M Lockwood FRCP



[Targeted therapy and progressive multifocal leukoencephalopathy (PML): PML in the era of monoclonal antibody therapies].  


Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system and is associated with John Cunningham (JC) virus infection in the oligodendrocytes. The number of patients with PML increased after the pandemic of acquired immunodeficiency syndrome. Thereafter, an association between PML and monoclonal antibody therapy has come into light. Thus far, several monoclonal antibodies have been reported to cause PML. Currently, according to the Barts and the London School of Medicine and Dentistry, the number of PML cases due to natalizumab treatment for multiple sclerosis is 395 (incidence is 3.28/1,000). Moreover, the number of individuals with PML due to rituximab treatment is increasing (over 100 cases). Efalizumab, infliximab, adalimumab, etanercept, ibritumomab tiuxetan, bevacizumab, alemtuzumab, cetuximab, and brentuximab are also reported as risk factors of PML. The diagnosis of PML is based on clinical, neuroradiological, pathological, and molecular analyses. In clinical setting, magnetic resonance imaging provides the most important information in the diagnosis of PML. Patients with PML due to monoclonal antibody treatment may present clinical symptoms different from that of the classic PML, such as sensory disturbance and seizure. Once PML is identified in an individual receiving monoclonal antibody therapy, the monoclonal antibody must be immediately discontinued and removed from the body by plasmapheresis. Because most patients may present immune reconstitution inflammatory syndrome (IRIS), steroid therapy must be considered immediately. However, the prognosis of PML is still worse in patients receiving monoclonal antibody therapy. To prevent PML development, sophisticated and well-organized strategies must be established for monoclonal antibody treatment. Besides neurologists, physicians from other fields must be aware of PML associated with resulted from monoclonal antibody therapy. PMID:24200614

Takao, Masaki



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

NASA Astrophysics Data System (ADS)

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

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



Her2 Monoclonal Antibodies, Antibody Drug Conjugates, and Site Specific Antibody Conjugate Methods

Antibody drug conjugates (ADC) can demonstrate high efficacy as cancer therapeutics, however, much more can be done to improve their efficacy and safety profile. Site-specific antibody drug conjugation is a promising way to do this.


Antigenic heterogeneity in Mycoplasma iowae demonstrated with monoclonal antibodies.  


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

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



Characterization of a new monoclonal antibody against mercury (II)  

SciTech Connect

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

Marx, A.; Hock, B. [Technische Univ. Muenchen, Freising (Germany). Dept. of Botany



Generation of a Monoclonal Antibody Recognizing Mouse PD-1.  


Programmed death-1 (PD-1) is a transmembrane protein that shares homology with the B7/CD28 family of T cell signaling molecules. PD-1 interacts with its ligands PD-L1 and/or PD-L2 and provides a negative regulatory signal to CD4 and CD8 T cells that results ultimately in a phenotype termed T cell exhaustion. Here we expressed and purified mouse PD-1 protein and developed a monoclonal antibody (MAb) against mouse PD-1 by immunizing BALB/c mice with a specific region of the extracellular domains of PD-1 as antigen, which was expressed in Escherichia coli. A stable hybridoma cell line was established by animal immunization, cell fusion, and hybridoma screening. The MAb was then prepared from mouse ascites after inoculating the hybridoma cells. Different methods were used to analyze the characterization of the MAb, including ELISA, Western blotting, flow cytometry, and RT-PCR techniques. The results showed that the PD-1 MAb can bind to the PD-1 protein and promote lymphocyte proliferation. This PD-1 MAb will be a valuable tool for further investigation of programmed death-1 functions. PMID:25358006

Qin, Yu-E; Wang, Gongze; Yang, Jianlin; Liu, Chao-Qi



Adverse events to monoclonal antibodies used for cancer therapy  

PubMed Central

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

Baldo, Brian A



Haptens and monoclonal antibodies for immunoassay of imidazolinone herbicides.  


A set of haptens structurally resembling the herbicide imazethapyr (PURSUIT) was synthesized and used to derive monoclonal antibodies (MAbs) and direct and indirect competition enzyme immunoassays (EIAs) which could detect imazethapyr, imazaquin (SCEPTER), imazapic (CADRE), and imazamox (RAPTOR) in the 3-30 ng/mL (parts per billion) range, and imazapyr (ARSENAL) and imazamethabenz-methyl (ASSERT) in the 300-500 ppb range. Two MAbs, 3A2 and 3A5, had affinities of 10-75 nM for imazethapyr. MAbs 1A5, 1D2, and 3A5 were specific for the S isomers of the herbicides. Some MAbs were stable in solutions containing up to 15% methanol and 5% acetonitrile in indirect EIAs. Plates coated with hapten conjugates for indirect EIA could be stored frozen. Selectivity for the imidazolinones by some MAbs varied with different coating conjugates. These MAbs and haptens should prove useful in immunochemical analysis and residue recovery methods for imazethapyr and other imidazolinone herbicides. PMID:12033799

Chin, Tina E; Wong, Rosie B; Pont, Joseph L; Karu, Alexander E



Monoclonal antibodies for use in man: current regulatory situation in the Federal Republic of Germany.  


The article addresses the requirement to be met for approval of monoclonal antibodies with special emphasis on products coupled with radionuclides and on principles for the conduct of clinical trials. According to the German Drug Law monoclonal antibodies are considered as being sera. Therefore, the Paul-Ehrlich-Institut, Federal Office for Sera and Vaccines, is responsible for marketing authorizations. Sera and vaccines need a special manufacturing licence which is given by the competent authority of the Federal State. Batches of monoclonal antibodies can only be marketed if they have been released by the Paul-Ehrlich-Institut; in connexion with batch control the importance of reference preparations is stressed. The standard requirements for the data to be submitted with the applications for marketing authorizations are in accordance with the EEC Council Directives and Notes for Guidance. For the testing of radioactive monoclonal antibodies in clinical trials, compliance with both the Drug Law and The German Radiation Protection Ordinance must be ensured. In addition to the authorizations required for non-labelled monoclonal antibody products, the use of radioactive substances in diagnosis and therapy requires an authorization by the competent Federal State authority. The main purpose of the planning and performance of clinical trials with new monoclonal antibody in diagnosis and therapy must be the comparison with established diagnostic tools and/or established medicinal products of known effect. PMID:2205526

Haase, M



Cell lines of novel type derived from a diabetic secrete tissue-reactive human monoclonal antibodies.  


Human monoclonal antibodies have been produced from lymphocytes of an acute-onset insulin-dependent diabetic patient. Peripheral blood lymphocytes were hybridized with a fusion partner HMY-1320. Initial screening of human immunoglobulin secretion was made by a nitrocellulose dot blot assay. Ten stable cell lines of novel type, secreting human immunoglobulin, were obtained. These cell lines have been maintained in continuous culture over 6 months and cryopreserved in liquid nitrogen for 14 months. Human monoclonal antibodies of IgG and IgM class have been produced and are secreted at a rate of 150-650 ng/ml/10(6) cells/day. Monoclonal antibodies were tested for histological staining against a variety of endocrine and non-endocrine tissues. One monoclonal antibody, LT1E12, demonstrates a staining pattern in human, rat, and mouse tissues, similar to that of mitochondrial antibodies. Another antibody, LT3C4, demonstrates weak staining of smooth muscle in rat and mouse kidney sections. Neither specificities were detected in the diabetic patient's serum. The variety of immune tissue specificities obtained in this study demonstrates the potential value of human monoclonal antibodies as probes to analyze the complexity of autoimmunity in diabetes mellitus. PMID:1873494

De Silva, M G; Ebsworth, N M; Dodwell, L C; Moyle, S P; Swana, G T; Tan, K S



Development of an antigen microarray for high throughput monoclonal antibody selection.  


Monoclonal antibodies are valuable laboratory reagents and are increasingly being exploited as therapeutics to treat a range of diseases. Selecting new monoclonal antibodies that are validated to work in particular applications, despite the availability of several different techniques, can be resource intensive with uncertain outcomes. To address this, we have developed an approach that enables early screening of hybridoma supernatants generated from an animal immunised with up to five different antigens followed by cloning of the antibody into a single expression plasmid. While this approach relieved the cellular cloning bottleneck and had the desirable ability to screen antibody function prior to cloning, the small volume of hybridoma supernatant available for screening limited the number of antigens for pooled immunisation. Here, we report the development of an antigen microarray that significantly reduces the volume of supernatant required for functional screening. This approach permits a significant increase in the number of antigens for parallel monoclonal antibody selection from a single animal. Finally, we show the successful use of a convenient small-scale transfection method to rapidly identify plasmids that encode functional cloned antibodies, addressing another bottleneck in this approach. In summary, we show that a hybrid approach of combining established hybridoma antibody technology with refined screening and antibody cloning methods can be used to select monoclonal antibodies of desired functional properties against many different antigens from a single immunised host. PMID:24472540

Staudt, Nicole; Müller-Sienerth, Nicole; Wright, Gavin J



Development of an antigen microarray for high throughput monoclonal antibody selection  

PubMed Central

Monoclonal antibodies are valuable laboratory reagents and are increasingly being exploited as therapeutics to treat a range of diseases. Selecting new monoclonal antibodies that are validated to work in particular applications, despite the availability of several different techniques, can be resource intensive with uncertain outcomes. To address this, we have developed an approach that enables early screening of hybridoma supernatants generated from an animal immunised with up to five different antigens followed by cloning of the antibody into a single expression plasmid. While this approach relieved the cellular cloning bottleneck and had the desirable ability to screen antibody function prior to cloning, the small volume of hybridoma supernatant available for screening limited the number of antigens for pooled immunisation. Here, we report the development of an antigen microarray that significantly reduces the volume of supernatant required for functional screening. This approach permits a significant increase in the number of antigens for parallel monoclonal antibody selection from a single animal. Finally, we show the successful use of a convenient small-scale transfection method to rapidly identify plasmids that encode functional cloned antibodies, addressing another bottleneck in this approach. In summary, we show that a hybrid approach of combining established hybridoma antibody technology with refined screening and antibody cloning methods can be used to select monoclonal antibodies of desired functional properties against many different antigens from a single immunised host. PMID:24472540

Staudt, Nicole; Muller-Sienerth, Nicole; Wright, Gavin J.



8th Annual European Antibody Congress 2012  

PubMed Central

The 8th European Antibody Congress (EAC), organized by Terrapin Ltd., was again held in Geneva, Switzerland, following on the tradition established with the 4th EAC. The new agenda format for 2012 included three parallel tracks on: (1) naked antibodies; (2) antibody drug conjugates (ADCs); and (3) bispecific antibodies and alternative scaffolds. The meeting started and closed with three plenary lectures to give common background and to share the final panel discussion and conclusions. The two day event included case studies and networking for nearly 250 delegates who learned of the latest advances and trends in the global development of antibody-based therapeutics. The monoclonal antibody track was focused on understanding the structure-function relationships, optimization of antibody design and developability, and processes that allow better therapeutic candidates to move through the clinic. Discussions on novel target identification and validation were also included. The ADC track was dedicated to evaluation of the ongoing success of the established ADC formats alongside the rise of the next generation drug-conjugates. The bispecific and alternative scaffold track was focused on taking stock of the multitude of bispecific formats being investigated and gaining insight into recent innovations and advancements. Mechanistic understanding, progression into the clinic and the exploration of multispecifics, redirected T cell killing and alternative scaffolds were extensively discussed. In total, nearly 50 speakers provided updates of programs related to antibody research and development on-going in the academic, government and commercial sectors. PMID:23493119

Beck, Alain; Carter, Paul J.; Gerber, Hans-Peter; Lugovskoy, Alexey A.; Wurch, Thierry; Junutula, Jagath R.; Kontermann, Roland E; Mabry, Robert



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

SciTech Connect

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

Hermoso, R.; Fonolla, J.; Lopez-Gorge, J. (Estacion Experimental del Zaidin, Granada (Spain)); Ruiz-Cabello, F.; Garrido, F. (C.S. Virgen de las Nieves, Granada (Spain))



In vivo therapeutic synergism of anti-EGFR and anti-HER2 monoclonal antibodies against pancreatic carcinomas  

E-print Network

1 In vivo therapeutic synergism of anti-EGFR and anti-HER2 monoclonal antibodies against pancreatic contributed equally to this work. Running title: Anti-HER antibodies in pancreatic cancer Key words: EGFR, HER2, monoclonal antibodies, therapeutic synergism, pancreatic cancer Abbreviations: EGFR, EGF receptor

Boyer, Edmond


JC70: a new monoclonal antibody that detects vascular endothelium associated antigen on routinely processed tissue sections  

Microsoft Academic Search

A new monoclonal antibody, JC70, raised against a membrane preparation from a spleen affected by hairy cell leukaemia, recognises a membrane bound glycoprotein identical with that of the CD31 group of monoclonal antibodies. The antibody stains a fixation resistant epitope on endothelial cells in benign and malignant conditions in a wide variety of paraffin wax embedded tissue. JC70 stained malignant

D V Parums; J L Cordell; K Micklem; A R Heryet; K C Gatter; D Y Mason



Monoclonal antibodies distinguish phase variants of Coxiella burnetii.  

PubMed Central

Monoclonal antibodies (MAbs) directed against phase I and II variants of Coxiella burnetii were produced by fusing myeloma SP2/O-AG 14 cells with spleen cells from mice immunized with the chloroform-methanol extraction residue of phase I whole cells. Two hybridoma clones which distinguished the phase variants by microimmunofluorescence assay were isolated and characterized. The MAbs showing specificity for phase I cells (MAbI-1, immunoglobulin G, subclass 3 kappa) reacted with the hot phenol-water extract of phase I C. burnetii in immunodiffusion and enzyme-linked immunosorbent assays. MAbI-1 reacted with high-molecular-weight components from phase I phenol-water extract and whole cell in an immunoblot assay. Specificity of MAbI-1 for a carbohydrate epitope in the phenol-water extract was demonstrated by periodic acid inactivation of binding by a competitive enzyme-linked immunosorbent assay. Phase I antigenic sites were apparently well represented on the surface of cells as demonstrated by complete fluorescence and microagglutination. The MAb showing specificity for phase II cells (MAbII-1, immunoglobulin G, subclass 2b kappa) reacted with whole cells in the microimmunofluorescence assay, microagglutination test, complement fixation test, and the enzyme-linked immunosorbent assay. MAbII-1 reacted specifically with a 29,500-dalton surface protein as demonstrated by immunoprecipitation of 125I-surface-labeled cells. Although MAbII-1 reacted with detergent-solubilized protein, it did not react with sodium-dodecyl sulfate-denatured protein by immunoblot assay. This protein was not exposed on the surface of phase I cells, but chloroform-methanol extraction of phase I cells exposed the phase II epitope. Images PMID:6418662

Williams, J C; Johnston, M R; Peacock, M G; Thomas, L A; Stewart, S; Portis, J L



CCR5 Monoclonal Antibodies for HIV-1 Therapy  

PubMed Central

Purpose of review This report summarizes emerging clinical and preclinical data pertaining to the use of CCR5 monoclonal antibodies (mAbs) as therapies for HIV-1 infection. The epitope specificity of CCR5 mAbs is discussed in relation to its critical impact on antiviral activity and CCR5 antagonism. We compare and contrast mAbs and small-molecule CCR5 antagonists in terms of their binding and antiviral properties. Two CCR5 mAbs have entered clinical testing and have successfully completed proof-of-concept studies in HIV-infected individuals, providing initial information on the potential therapeutic utility of these agents. Recent findings New studies support the view that the most potently antiviral CCR5 mAbs recognize the second extracellular loop of CCR5 either exclusively or in combination with the amino terminus. Studies have revealed fundamental differences in how mAbs and small molecules bind CCR5 and inhibit HIV-1. CCR5 mAbs and small-molecule CCR5 antagonists have demonstrated consistent antiviral synergy and limited or no viral cross-resistance in independent studies. Single intravenous infusions of CCR5 mAbs significantly reduced HIV-1 RNA levels in infected individuals for 2–3 weeks without appreciable toxicity. Summary CCR5 mAbs have demonstrated broad and potent antiviral activity in vitro. Clinical studies have established CCR5 mAbs as potent antiretroviral agents with prolonged activity following a single dose. CCR5 mAbs represent both a distinct class of CCR5 inhibitor and a novel approach to HIV-1 therapy. PMID:19339948

Olson, William C.; Jacobson, Jeffrey M.



Molecular simulations of the pairwise interaction of monoclonal antibodies.  


Molecular simulations are employed to compute the free energy of pairwise monoclonal antibodies (mAbs) association using a conformational sampling algorithm with a scoring function. The work reported here is aimed at investigating the mAb-mAb association driven by weak interactions with a computational method capable of predicting experimental observations of low binding affinity. The simulations are able to explore the free energy landscape. A steric interaction component, electrostatic interactions, and a nonpolar component of the free energy form the energy scoring function. Electrostatic interactions are calculated by solving the Poisson-Boltzmann equation. The nonpolar component is derived from the van der Waals interactions upon close contact of the protein surfaces. Two mAbs with similar IgG1 framework but with small sequence differences, mAb1 and mAb2, are considered for their different viscosity and propensity to form a weak interacting dimer. mAb1 presents favorable free energy of association at pH 6 with 15 mM of ion concentration reproducing experimental trends of high viscosity and dimer formation at high concentration. Free energy landscape and minimum free energy configurations of the dimer, as well as the second virial coefficient (B22) values are calculated. The energy distributions for mAb1 are obtained, and the most probable configurations are seen to be consistent with experimental measurements. In contrast, mAb2 shows an unfavorable average free energy at the same buffer conditions due to poor electrostatic complementarity, and reversible dimer configurations with favorable free energy are found to be unlikely. Finally, the simulations of the mAb association dynamics provide insights on the self-association responsible for bulk solution behavior and aggregation, which are important to the processing and the quality of biopharmaceuticals. PMID:25350229

Lapelosa, Mauro; Patapoff, Thomas W; Zarraga, Isidro E



Safety and immunotoxicity assessment of immunomodulatory monoclonal antibodies  

PubMed Central

Most therapeutic monoclonal antibodies (mAbs) licensed for human use or in clinical development are indicated for treatment of patients with cancer and inflammatory/autoimmune disease and as such, are designed to directly interact with the immune system. A major hurdle for the development and early clinical investigation of many of these immunomodulatory mAbs is their inherent risk for adverse immune-mediated drug reactions in humans such as infusion reactions, cytokine storms, immunosuppression and autoimmunity. A thorough understanding of the immunopharmacology of a mAb in humans and animals is required to both anticipate the clinical risk of adverse immunotoxicological events and to select a safe starting dose for first-in-human (FIH) clinical studies. This review summarizes the most common adverse immunotoxicological events occurring in humans with immunomodulatory mAbs and outlines non-clinical strategies to define their immunopharmacology and assess their immunotoxic potential, as well as reduce the risk of immunotoxicity through rational mAb design. Tests to assess the relative risk of mAb candidates for cytokine release syndrome, innate immune system (dendritic cell) activation and immunogenicity in humans are also described. The importance of selecting a relevant and sensitive toxicity species for human safety assessment in which the immunopharmacology of the mAb is similar to that expected in humans is highlighted, as is the importance of understanding the limitations of the species selected for human safety assessment and supplementation of in vivo safety assessment with appropriate in vitro human assays. A tiered approach to assess effects on immune status, immune function and risk of infection and cancer, governed by the mechanism of action and structural features of the mAb, is described. Finally, the use of immunopharmacology and immunotoxicity data in determining a minimum anticipated biologic effect Level (MABEL) and in the selection of safe human starting dose is discussed. PMID:20421713

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



Response of a Concentrated Monoclonal Antibody Formulation to High Shear  

PubMed Central

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

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



Development and characterization of new rat monoclonal antibodies for procalcitonin.  


The development of selective and sensitive biological recognition elements, e.g., antibodies, for the detection of relevant blood markers is a great challenge in the field of biosensors. In this context, five new rat monoclonal antibodies (mAbs) for procalcitonin (PCT), a marker for bacterial infection and sepsis, were developed and characterized. One mAb, PROC1 3G3, was used as capture antibody. Four mAbs, PROC4 6C6, PROC4 6B2, PROC4 1G3, and PROC4 1D6, were used as detection mAbs, either as Protein G-purified or as biotinylated mAbs. A surface plasmon resonance (SPR) biosensor was used to characterize the antigen-antibody biomolecular interactions. The capture mAb (PROC1 3G3) has an equilibrium dissociation constant (K (D)) of 3.42 x 10(-8) M. All four detection mAbs (PROC4 6C6, PROC4 6B2, PROC4 1G3, and PROC4 1D6) are of high affinity (K (A) = 2.81-6.11 x 10(8) M(-1); K (D) = 1.64-3.56 x 10(-9) M) and have moderate dissociation rate constants (k (d) = 1.70-2.40 x 10(-3) s(-1)). Four different sandwich enzyme-linked immunosorbent assays (ELISAs) with standards of human recombinant (hr) PCT, using PROC1 3G3 as capture mAb and PROC4 mAbs as detection mAbs, respectively, led to highly specific determinations of PCT without cross-reactivities to calcitonin and katacalcin. The lower limits of quantification (LLOQ) for hrPCT (in 40 mM phosphate-buffered saline (PBS), pH 7.6) with these assays ranged from 2.3 to 12.8 microg L(-1). In addition, sandwich ELISAs were set up with biotinylated PROC4 mAbs, and with hrPCT in 4% human serum albumin (diluted 1:10 in 40 mM PBS, including 1:5 (v/v) LowCross Buffer(R)). The LLOQs of these sandwich assays ranged from 4.1 to 6.0 microg L(-1) and were thus much closer together for the different assays. With the latter assay setup (PROC1 3G3 as capture mAb, PROC4 6C6-biotin as detection mAb) a first collection of five serum samples was determined (healthy volunteers, unspiked, and spiked). Recovery rates for the spiked samples ranged from 98.3 to 115.7%. The newly developed anti-PCT mAbs should find broad applications in immunosensors for point-of-care diagnostics of sepsis and systemic inflammation processes. PMID:18712365

Krämer, Petra M; Gouzy, Marie-Françoise; Kess, Melanie; Kleinschmidt, Ulrike; Kremmer, Elisabeth



Monoclonal antibodies distinguish between storage and secreted forms of eosinophil cationic protein  

Microsoft Academic Search

The toxic effects of eosinophils on parasites1 and cells2 are due largely to the secretion of various granule proteins, following stimulation3. In order to study this secretory process (degranulation) further, we have raised mouse monoclonal antibodies against both human eosinophil granule extracts and secretion products. From immunocytochemical studies it appears that one antibody, EG1, recognized both the storage and secreted

Po-Chun Tai; Christopher J. F. Spry; Christer Peterson; Per Venge; Inge Olsson



Monoclonal antibodies to surface and cytoskeletal components of the spermatozoid of Pteridium aquilinum  

Microsoft Academic Search

Summary Detergent extracted spermatozoids of the fernPteridium aquilinum were used as mixed antigen preparations for raising monoclonal antibodies in order to obtain reagents for detecting as yet uncharacterized components of the plant cytoskeleton. Selected antibodies were studied by immunofluorescence microscopy of developing spermatids and mature spermatozoids. Some reacted directly with fixed cells, others required permeabilization treatments with cold methanol or

J. Marc; B. E. S. Gunning; A. R. Hardham; J. L. Perkin; S. M. Wick



Isolation and Characterization of Human Monoclonal Antibodies from Individuals Infected with West Nile Virus  

Microsoft Academic Search

Monoclonal antibodies (MAbs) neutralizing West Nile Virus (WNV) have been shown to protect against infection in animal models and have been identified as a correlate of protection in WNV vaccine studies. In the present study, antibody repertoires from three convalescent WNV-infected patients were cloned into an scFv phage library, and 138 human MAbs binding to WNV were identified. One hundred

Mark Throsby; Cecile Geuijen; Jaap Goudsmit; Arjen Q. Bakker; Jehanara Korimbocus; R. Arjen Kramer; Marieke Clijsters-van der Horst; Maureen de Jong; Mandy Jongeneelen; Sandra Thijsse; Renate Smit; Therese J. Visser; Nora Bijl; Wilfred E. Marissen; Mark Loeb; David J. Kelvin; Wolfgang Preiser; J. ter Meulen; J. de Kruif



The distribution of the therapeutic monoclonal antibodies cetuximab and trastuzumab within solid tumors  

Microsoft Academic Search

BACKGROUND: Poor distribution of some anticancer drugs in solid tumors may limit their anti-tumor activity. METHODS: Here we used immunohistochemistry to quantify the distribution of the therapeutic monoclonal antibodies cetuximab and trastuzumab in relation to blood vessels and to regions of hypoxia in human tumor xenografts. The antibodies were injected into mice implanted with human epidermoid carcinoma A431 or human

Carol M Lee; Ian F Tannock




EPA Science Inventory

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


A Monoclonal Antibody Selection for Immunohistochemical Examination of Lymphoid Tissues From Non-human Primates  

Microsoft Academic Search

Non-human primates (NHPs) offer valuable animal models for basic research into human diseases and for the preclinical validation of new therapeutics. Detailed in situ examination of the involved cell types using immunohistochemistry is often hampered by the lack of cross-reactive antibodies (Abs). In the current study, we have tested a large panel of monoclonal antibodies raised against human leukocyte differentiation

Yolanda S. Kap; Marjan van Meurs; Nikki van Driel; Gerrit Koopman; Marie-Jose Melief; Herbert P. M. Brok; Jon D. Laman; Bert A. t Hart



A Monoclonal Antibody Against Kinesin Inhibits Both Anterograde and Retrograde Fast Axonal Transport in Squid Axoplasm  

Microsoft Academic Search

One of our monoclonal antibodies against the heavy chain of bovine kinesin (H2) also recognized the heavy chain of squid kinesin. The immunofluorescence pattern of H2 in axoplasm was similar to that seen in mammalian cells with antibodies specific for kinesin light and heavy chains, indicating that squid kinesin is also concentrated on membrane-bounded organelles. Although kinesin is assumed to

Scott T. Brady; K. Kevin Pfister; George S. Bloom



Mathematical Analysis of the Pharmacokinetic-Pharmacodynamic (PKPD) Behaviour of Monoclonal Antibodies: Predicting in vivo  

E-print Network

Antibodies: Predicting in vivo Potency Philip J. Astona, , Gianne Derksa , Adewale Rajib,a,c , Balaji M the target affinity of a monoclonal antibody and its in vivo potency. The dynamics of the system is described mathematically by a target-mediated drug disposition model. As a measure of potency, we consider the minimum

Wirosoetisno, Djoko


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

SciTech Connect

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

Ratcliffe, D.R.



Epitope mapping of a monoclonal antibody against human thrombin by H/D-exchange mass spectrometry  

E-print Network

Epitope mapping of a monoclonal antibody against human thrombin by H/D-exchange mass spectrometry human thrombin has been determined by hydrogen/ deuterium exchange coupled to MALDI mass spectrometry. The antibody epitope was identified as the surface of thrombin that retained deuterium in the presence

Komives, Elizabeth A.


[Preparation and characterization of mouse monoclonal antibody against outer membrane protein P6 of Haemophilus influenzae].  


Objective To prepare and identify monoclonal antibody against Haemophilus influenzae(Hi) outer membrane protein P6. Methods Recombinant protein P6 as an immunogen was administered intraperitoneally to BALB/c mice. The splenocytes of the mouse were isolated from spleen and hybridized with Sp2/0 myeloma cells. Indirect ELISA was used for screening hybridoma and the number of chromosomes in hybridoma cells was determined by karyotype analysis. The titers and specificity of monoclonal antibodies in their culture supernatant were detected by indirect ELISA. The immunoglobulin class, subclasses and type of the monoclonal antibody were identified with colloidal gold labeled IsoQuick(TM) strips. Results Two hybridoma cell lines designated ?2G3 and ?2C4 were obtained. Karyotype analysis showed that the chromosome numbers of ?2G3 and ?2C4 were 103 and 95, respectively. The highest titers of antibodies in their culture supernatant were 1:256 and 1:512, respectively. Both monoclonal antibodies only reacted with standard or clinical isolated strains of Hi, and they both did not react with other bacteria. A2G3 was IgG2b, and ?2C4 was IgM, both of which were kappa light chains. They could recognize different antigen epitope of protein P6. Conclusion Two hybridoma cell lines producing the monoclonal antibodies against protein P6 of Hi outer membrane are obtained. PMID:25270206

Wen, Xiaoyan; Zhou, Fang; Zhang, Yanxia; Qiao, Haixia; Gao, Xue; Jia, Xiaohui; Zhang, Yutuo



A Spectrum of Monoclonal Antibodies Reactive with Human Mammary Tumor Cells  

NASA Astrophysics Data System (ADS)

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.

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



Simple diagnosis of Encephalitozoon sp. microsporidial infections by using a panspecific antiexospore monoclonal antibody.  

PubMed Central

Microsporidia (phylum Microsproa) have recently become recognized as common opportunistic protozoans in the United States and worldwide, particularly affecting immunodeficient patients. Microsporidian organisms within the genus Encephalitozoon are the cause of nephrologic, ophthalmic, pneumologic, gastroenteric, and systemic infections. However, diagnosis of the small spores by light microscopy is difficult, even with newly developed and improved staining techniques. We have developed an anti-Encephalitozoon species monoclonal antibody-based immunoassay for easy diagnosis. A hybridoma was produced and selected following one main criterion: recognition by immunofluorescence of all known Encephalitozoon spores affecting humans. The selected monoclonal antibody-secreting hybridomas were characterized by enzyme-linked immunosorbent assay, immunofluorescence, Western blot, and immunoelectron microscopy using Encephalitozoon species from fresh and fixed samples from patients and from in vitro cultures. In the immunofluorescence assay, one monoclonal antibody, termed 3B6, strongly recognized Encephalitozoon cuniculi, E. hellem, and E. intestinalis. Monoclonal antibody 3B6 bound to other microsporidia (Nosema and Vairimorpha spp.) without cross-reacting with any other parasite, including Enterocytozoon bieneusi, fungus, or bacterium tested. In immunoelectron microscopy assays, monoclonal antibody 3B6 bound to the exospore of Encephalitozoon species, while in Western blot assays, it recognized three to seven antigens with molecular masses ranging from 34 to 117 kDa. We have developed a sensitive and specific monoclonal antibody-based immunoassay to diagnose common microsporidian infections, particularly with Encephalitozoon species. This is a new tool for identifying spores in bodily fluids and biopsy samples and is an efficient diagnostic test. Additionally, monoclonal antibody 3B6 can serve to assess the prevalence of microsporidial infections in immunodeficient and immunocompetent patients. PMID:9041420

Enriquez, F J; Ditrich, O; Palting, J D; Smith, K



Ganglioside GD2 specificity of monoclonal antibodies to human neuroblastoma cell.  


Four monoclonal antibodies (3F8, 3A7, 3G6, and 2F7) against human neuroblastoma cells have been suggested to react with surface glycolipids of these cells. In this report these monoclonal antibodies were shown to be specific to the disialoganglioside GD2 using a thin-layer chromatography (TLC)-immunostaining method. When mixed human brain gangliosides were developed by TLC in two different solvent systems and incubated with each of the monoclonal antibodies, only GD2 was stained. These antibodies also reacted with highly purified GD2 on the plate. These findings suggest that GD2 provides an antigenic site on the surface of human neuroblastoma cells. PMID:2579648

Saito, M; Yu, R K; Cheung, N K



Radioimmunodiagnosis of ovarian cancer using /sup 123/I-labelled, tumor-associated monoclonal antibodies  

SciTech Connect

Monoclonal antibodies to epithelial cells, antigenic determinants labelled with /sup 123/I and /sup 125/I, were administered to 10 immunodeficient mice bearing subcutaneous xenografts of human ovarian cancer. Radio scans of the body taken with a gamma camera at various time intervals demonstrated the presence of the cancer in all the mice. The smallest detectable tumor was approximately 1 mm in diameter. In a subsequent clinical study using /sup 123/I-labelled monoclonal antibodies in 10 patients with ovarian cancer, tumor detection was achieved in 8 patients, with tumor uptake of labelled antibody ranging between 0.2-2.6%. As a complementary method to existing forms of diagnosis, the targeting of monoclonal antibodies to ovarian cancer cells in vivo raises the hope of achieving early diagnosis in otherwise undetectable ovarian cancer, and provides encouragement to the concept of selective therapy in oncology.

Epenetos, A.A.; Shepherd, J.; Britton, K.E.; Hawkins, L.; Nimmon, C.C.; Taylor-Papadimitriou, J.; Durbin, H.; Malpas, J.S.; Mather, S.; Granowska, M.



Redirected T-Cell Killing of Solid Cancers Targeted with an Anti-CD3/Trop-2-Bispecific Antibody Is Enhanced in Combination with Interferon-?.  


Trop-2 has limited presence on normal tissues but is highly expressed in diverse epithelial cancers. (E1)-3s is a T-cell-redirecting trivalent bispecific antibody (bsAb), comprising an anti-CD3 scFv covalently linked to a stabilized dimer of a Trop-2-targeting Fab using Dock-and-Lock. We show for the first time that bsAb-mediated bidirectional trogocytosis occurs between target and T cells and involves immunologic synapses. We studied the effects of interferon-? (INF?) on (E1)-3s-mediated T-cell killing of human gastric and pancreatic cancer cell lines. T-cell activation, cytokine induction, and cytotoxicity were evaluated ex vivo using peripheral blood mononuclear cells (PBMC) or T cells with NCI-N87 gastric cancer as target cells. In vivo activity was assayed with NCI-N87 and Capan-1 (pancreatic) xenografts. In the presence of target cells and PBMCs, (E1)-3s did not cause excess cytokine production. When combined with (E1)-3s, peginterferonalfa-2a-which alone did not increase T-cell activation or raise cytokine levels over baseline-increased CD69 expression but did not significantly increase cytokine induction. (E1) 3s mediated a highly potent T-cell lysis of NCI-N87 target cells in vitro. Inclusion of peginterferonalfa-2a or a more potent form of INF?, 20*-2b, significantly potentiated the activity of (E1)-3s by more than 2.5- or 7-fold, respectively. In vivo, combining peginterferonalfa-2a with (E1)-3s delayed Capan-1 growth longer than each single agent. Similarly, combination therapy delayed tumor proliferation of NCI-N87 compared with (E1)-3s or peginterferonalfa-2a single-treatment groups. (E1)-3s effectively induced T-cell-mediated killing of Trop-2-expressing pancreatic and gastric cancers, which was enhanced with INF?. Mol Cancer Ther; 13(10); 2341-51. ©2014 AACR. PMID:25053819

Rossi, Edmund A; Rossi, Diane L; Cardillo, Thomas M; Chang, Chien-Hsing; Goldenberg, David M



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

SciTech Connect

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

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



A Novel Proliferation-Associated Variant of CFR-1 Defined by a Human Monoclonal Antibody  

Microsoft Academic Search

The germline coded human monoclonal IgM antibody 103\\/51 was isolated from a gastric carcinoma patient. This antibody binds to a 130-kd membrane molecule and has a mitotic effect on tumor cells in vitro. To characterize the target, we sequenced the protein and showed that the antibody binds to the cysteine-rich fibroblast growth factor receptor (CFR)-1, which is highly homologous to

Frank Hensel; Stephanie Brändlein; Matthias Eck; Karsten Schmidt; Veit Krenn; Astrid Kloetzer; Angela Bachi; Matthias Mann; Hans Konrad Müller-Hermelink; H. Peter Vollmers



West Nile virus: characterization and diagnostic applications of monoclonal antibodies  

PubMed Central

Background Diagnosis of West Nile virus (WNV) infections is often difficult due to the extensive antigenic cross-reactivity among flaviviruses, especially in geographic regions where two or more of these viruses are present causing sequential infections. The purpose of this study was to characterize a panel of monoclonal antibodies (MAbs) produced against WNV to verify their applicability in WNV diagnosis and in mapping epitope targets of neutralizing MAbs. Methods Six MAbs were produced and characterized by isotyping, virus-neutralization, western blotting and MAb-epitope competition. The MAb reactivity against various WNVs belonging to lineage 1 and 2 and other related flaviviruses was also evaluated. The molecular basis of epitopes recognized by neutralizing MAbs was defined through the selection and sequencing of MAb escape mutants. Competitive binding assays between MAbs and experimental equine and chicken sera were designed to identify specific MAb reaction to epitopes with high immunogenicity. Results All MAbs showed stronger reactivity with all WNVs tested and good competition for antigen binding in ELISA tests with WNV-positive equine and chicken sera. Four MAbs (3B2, 3D6, 4D3, 1C3) resulted specific for WNV, while two MAbs (2A8, 4G9) showed cross-reaction with Usutu virus. Three MAbs (3B2, 3D6, 4D3) showed neutralizing activity. Sequence analysis of 3B2 and 3D6 escape mutants showed an amino acid change at E307 (Lys ? Glu) in the E protein gene, whereas 4D3 variants identified mutations encoding amino acid changed at E276 (Ser ? Ile) or E278 (Thr ? Ile). 3B2 and 3D6 mapped to a region on the lateral surface of domain III of E protein, which is known to be a specific and strong neutralizing epitope for WNV, while MAb 4D3 recognized a novel specific neutralizing epitope on domain II of E protein that has not previously been described with WNV MAbs. Conclusions MAbs generated in this study can be applied to various analytical methods for virological and serological WNV diagnosis. A novel WNV-specific and neutralizing MAb (4D3) directed against the unknown epitope on domain II of E protein can be useful to better understand the role of E protein epitopes involved in the mechanism of WNV neutralization. PMID:22500562



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

PubMed Central

We 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. None of the monoclonal autoantibodies appeared to bind to a significant percentage of cells of relatively small cell size, either before or after culture. 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. Further experiments, including those using aggregated Ig to block antibody binding, strongly indicated that anti-histone antibody binding was not Fc receptor mediated. 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 (0.25 micrograms/ml) 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. Images PMID:3876357

Holers, V M; Kotzin, B L



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


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

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




EPA Science Inventory

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


Preparation and characterization of a human aurora-A kinase monoclonal antibody  

Microsoft Academic Search

We have developed monoclonal antibodies against the human aurora-A serine\\/threonine kinase. After immunization of a mouse, a fusion was performed to obtain hybridomas that were selected because they produced immunoglobulin positively reacting against the protein used for immunization. We isolated one particular monoclonal that we named 35C1 using a series of selective assays. The first criteria of the screen for

Jean Yves Cremet; Simon Descamps; Frank Vérite; Anne Martin; Claude Prigent



The Role of B Cell-Mediated T Cell Costimulation in the Efficacy of the T Cell Retargeting Bispecific Antibody BIS20x31  

Microsoft Academic Search

In this study, we investigated the role of the naturally occurring B cell-mediated T cell costimulation in the antitumor efficacy of the bispecific Ab BIS20x3. BIS20x3 has a dual specificity for both CD20 and CD3 and has previously been shown to effectively direct the lytic potential of cytolytic T cells toward malignant, CD20 B cells. BIS20x3 instigated T cell-B cell

Alja J. Stel; Bart-Jan Kroesen; Susan Jacobs; Herman Groen; Hanneke C. Kluin-Nelemans; Sebo Withoff



The 39-kilodalton protein of Borrelia burgdorferi: a target for bactericidal human monoclonal antibodies.  

PubMed Central

Three human monoclonal immunoglobulin M antibodies against Borrelia burgdorferi, obtained from in vitro-stimulated peripheral blood lymphocytes, reacted in Western blots (immunoblots) with a prominent 39-kDa peptide and a faint band of approximately 66 kDa. Two of these antibodies showed bactericidal activity without addition of complement. All three antibodies were reactive in an enzyme immunoassay with cloned P39 (W.J. Simpson, M.E. Schrumpf, and T.G. Schwan, J. Clin. Microbiol. 28:1329-1337, 1990), suggesting that the target molecule of these antibodies is identical to the P39 protein. In addition, the majority of supernatants from human lymphocytes stimulated in vitro with crude B. burgdorferi antigen reacted in this assay, demonstrating that P39, although a minor component of B. burgdorferi, is an immunodominant antigen in these spirochetes. A fourth monoclonal antibody, reacting with OspA, also exhibited bactericidal activity. Images PMID:8406847

Scriba, M; Ebrahim, J S; Schlott, T; Eiffert, H



Prolongation of rat corneal graft survival by treatment with anti-CD4 monoclonal antibody.  

PubMed Central

A rat model of orthotopic corneal graft rejection was used to investigate the effect of depletion of subpopulations of immune cells by treatment with monoclonal antibodies. Though CD4+ cells were not eliminated completely by anti-CD4 monoclonal antibodies there was a profound delay in the rejection times of orthotopic corneal allografts. Furthermore a third of the CD4+ depleted animals failed to reject corneal allografts by 100 days post grafting. Despite an almost complete depletion of circulating CD8+ cells, the anti-CD8 antibody treated animals rejected corneal allografts in a similar time course to allografted controls treated with a non-reactive control antibody OX21. These results demonstrate that CD8+ T-cells are not required for rejection of corneal allografts whereas CD4+ T-cells play a critical role in the rejection response. Treatment with anti-CD4 antibodies may have a useful clinical application. Images PMID:1358194

Ayliffe, W; Alam, Y; Bell, E B; McLeod, D; Hutchinson, I V



Australian consensus guidelines for the safe handling of monoclonal antibodies for cancer treatment by healthcare personnel.  


These consensus guidelines provide recommendations for the safe handling of monoclonal antibodies. Definitive recommendations are given for the minimum safe handling requirements to protect healthcare personnel. The seven recommendations cover: (i) appropriate determinants for evaluating occupational exposure risk; (ii) occupational risk level compared with other hazardous and non-hazardous drugs; (iii) stratification of risk based on healthcare personnel factors; (iv) waste products; (v) interventions and safeguards; (vi) operational and clinical factors and (vii) handling recommendations. The seventh recommendation includes a risk assessment model and flow chart for institutions to consider and evaluate clinical and operational factors unique to individual healthcare services. These guidelines specifically evaluated monoclonal antibodies used in the Australian cancer clinical practice setting; however, the principles may be applicable to monoclonal antibodies used in non-cancer settings. The guidelines are only applicable to parenterally administered agents. PMID:25302720

Alexander, M; King, J; Bajel, A; Doecke, C; Fox, P; Lingaratnam, S; Mellor, J D; Nicholson, L; Roos, I; Saunders, T; Wilkes, J; Zielinski, R; Byrne, J; MacMillan, K; Mollo, A; Kirsa, S; Green, M



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

NASA Technical Reports Server (NTRS)

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

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



A direct competitive enzyme linked immunosorbent assay for progesterone using monoclonal antibody.  


A direct competitive enzyme-linked immunosorbent assay (ELISA) with monoclonal antibody (MAb) was diagnosed with progesterone (P) level of human serum. In high concentrations and large amounts of displacer effect, this monoclonal antibody (MAb) can retain biological activity so that it can be specially combined with progesterone. Under conditions of the existing displaced agent, monoclonal antibody 11F8(3)H5 can maintain high specificity and affinity and can specifically bind progesterone in serum. Progesterone ELISA standard curve was calculated according to the following formula: Logit(y) = -1.358Log(x) + 0.4961, r = 0.9944. The serum progesterone values obtained by this method correlated well with those obtained by chemiluminescent immunoassay (CLIA): the correlative equation was y = 0.7804x + 0.7600, r = 0.9126. PMID:24555930

Wang, Bin; Liu, Yi-bing; Zhang, Xue-feng; Feng, Ting-ting; Xu, Wen-ge; Han, Shi-quan



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

PubMed Central

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

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



Epitope and functional specificity of monoclonal antibodies to mouse gamma interferon: the synthetic peptide approach  

SciTech Connect

Four anti-recombinant mouse gamma interferon (..cap alpha..-IFN..gamma..) monoclonal antibodies were generated using hamster spleen cells. Binding of /sup 125/I-IFN..gamma.. by these protein A-bound antibodies was specifically blocked by cold IFN..gamma... Binding by three of these antibodies was also blocked by a synthetic peptide corresponding to the N-terminal 1-39 amino acids of IFN..gamma.., while a corresponding C-terminal (95-133) peptide had no effect on binding. One of the N-terminal specific monoclonal antibodies inhibited both the antiviral and macrophage priming (for tumor cell killing) activities of IFN..gamma.., while the other two had no effect on either biological function. Blocking experiments with cold IFN..gamma.. and N-terminal peptide suggest that the epitope specificities of the monoclonal antibodies could be determined by the conformational or topographic structure of IFN..gamma... Polyclonal antibodies to either the N-terminal or C-terminal peptides also inhibited both the antiviral and macrophage priming activities of IFN..gamma... All of the antibodies that inhibited IFN..gamma.. function also blocked binding of IFN..gamma.. to membrane receptor on cells, while antibodies that did not inhibit function also did not block binding. The data suggest that both the N-terminal and C-terminal domains of IFN..gamma.. play an important role in its antiviral and macrophage priming functions, possibly in a cooperative manner.

Russell, J.K.; Hayes, M.P.; Carter, J.M.; Torres, B.A.; Dunn, B.M.; Johnson, H.M.



Additive cytotoxicity of different monoclonal antibody-cobra venom factor conjugates for human neuroblastoma cells.  


Insufficient numbers of antigen molecules and heterogeneity of antigen expression on tumor cells are major factors limiting the immunotherapeutic potential of the few clinically useful monoclonal antibodies capable of mediating complement cytotoxicity and antibody-dependent cellular cytotoxicity. To overcome this limitation, we converted two non-cytotoxic monoclonal anti-neuroblastoma antibodies, designated 3E7 (IgG2b) and 8H9 (IgG1), and the non-cytotoxic F(ab')2 fragment of the cytotoxic monoclonal anti-GD2 antibody 3F8 (IgG3) into cytotoxic antibody conjugates by covalent attachment of cobra venom factor (CVF), a structural and functional homologue of the activated third component of complement. Competitive binding experiments confirmed the different specificities of the three antibodies. In the presence of human complement, all three antibody-CVF conjugates mediated selective complement-dependent lysis of human neuroblastoma cells. Consistent with the kinetics of the alternative pathway of complement, approximately seven hours incubation were required to reach maximum cytotoxicity of up to 25% for the 3E7-CVF conjugate, up to 60% for the 8H9-CVF conjugate, and up to 95% for the 3F8 F(ab')2-CVF conjugate. The different extent of maximal cytotoxic activity of the three conjugates was reflected by corresponding differences in the extent of binding of both unconjugated antibodies and the respective conjugates. Any combination of the three antibody-CVF conjugates caused an additive effect in complement-mediated lysis. Using a cocktail of all three conjugates, the extent of complement-mediated killing could be increased up to 100%. These data demonstrate that by coupling of CVF the relative large number of non-cytotoxic monoclonal anti-tumor antibodies of interesting specificity can be used to design cocktails of cytotoxic conjugates and, thereby, to overcome the problem of insufficient and heterogeneous antigen expression on tumor cells for immunotherapy. PMID:9413745

Juhl, H; Petrella, E C; Cheung, N K; Bredehorst, R; Vogel, C W



Comparison of In Vitro Monoclonal Antibody Production Methods with an In Vivo Ascites Production Technique.  


Economic, technical, legislative, and ethical issues influence decisions concerning alternatives to the use of animals in biomedical research. Since the development of cell hybridoma technology, production of ascites in mice has been a popular technique for generating high concentrations of monoclonal antibodies. However, the availability of several in vitro methods that can be performed in most laboratories make these alternative methods of monoclonal antibody production an attractive option. To evaluate the practicality of the use of in vitro techniques, three tissue culture methods and a technique for production of ascites were compared on the basis of yield, material costs, and time requirements. Analysis of results revealed that the time and material costs to produce 100 mg of monoclonal antibody 7.16.4 by inducing ascites in irradiated mice was similar or slightly more than that for tissue culture in standard plastic flasks and hollow fiber cartridge bioreactors; however, tissue culture in gas permeable bags was slightly less expensive than these methods in terms of cost and time. When ascites production in irradiated mice was compared with tissue culture in plastic flasks or gas permeable bags for monoclonal antibody 225, the in vitro methods were approximately five times more expensive than the ascites production technique. Information reported here outlines factors that should be considered and evaluated when choosing a monoclonal antibody production method. Furthermore, these results documented that in vitro technology for the production of monoclonal antibodies can be adapted by most conventional laboratories to provide sufficient resources for the most commonly performed experimental protocols. PMID:12456135

Peterson, Norman C.; Peavey, Jennifer E.



Novel monoclonal antibody inhibits tumor growth in breast cancer and angiosarcoma

A monoclonal antibody targeting a protein known as SFPR2 has been shown by researchers at the University of North Carolina and its Lineberger Comprehensive Cancer Center to inhibit tumor growth in pre-clinical models of breast cancer and angiosarcoma. In a paper published in the April 19 issue of Molecular Cancer Therapeutics, a team used a monoclonal antibody to target SFRP2 expressed in cells from triple-negative breast cancer and the aggressive blood-vessel malignancy angiosarcoma, reducing the rate of tumor growth.


Monoclonal antibodies to type X collagen. Biosynthetic studies using an antibody to the amino-terminal domain.  


Monoclonal antibodies to chick type X collagen have been used to study the structure, biosynthesis, and location of type X in cartilage. The antibodies were produced by injecting purified type X collagen into female SJL/J mice and then fusing their spleen cells with Sp2/0 myeloma cells. Hybridoma culture supernatants were screened for antibodies to type X collagen by enzyme-linked immunosorbent assay and Western blots. Positive supernatants did not cross-react with other collagen types (I, II, IX, XI) or with fibronectin. Three monoclonal antibodies were chosen for further characterization. Two of them (1A6 and 6F6) recognize a pepsin-sensitive domain of type X collagen. Rotary shadowing showed that 1A6 and 6F6 both recognize the same end of type X, probably the aminoterminal non-triple helical domain. Amino acid sequencing of the intact protein and of the epitope-containing peptide confirmed that the antibody recognition sites for 1A6 and 6F6 are within the amino-terminal domain. Monoclonal antibody 2B3 reacts with the pepsinized (45 kDa) and weakly with the nonpepsinized (59 kDa) forms of type X collagen. The monoclonal antibodies were used for immunolocalization of type X in hypertrophic chondrocytes and reacted only with tissue samples from areas undergoing endochondral ossification, e.g. growth plate and fracture callus. Antibody 6F6, when coupled to Sepharose, selectively binds to type X collagen from cell and organ cultures. In a pulse-chase experiment, no processing of the 59-kDa form of type X could be detected. Two components with molecular masses of approximately 70 and 85 kDa, arising from a disulfide-bonded aggregate, were synthesized by both the permanent and calcifying cartilage organ cultures but did not react with the antibody, suggesting that these proteins are not related to type X. In summary, the pulse-chase results and the immune precipitation with monoclonal antibody 6F6 did not detect biosynthetic precursors larger than 59 kDa or proteolytically processed forms of type X. PMID:2826450

Summers, T A; Irwin, M H; Mayne, R; Balian, G



Development of monoclonal antibodies to human microsomal epoxide hydrolase and analysis of "preneoplastic antigen"-like molecules  

E-print Network

Development of monoclonal antibodies to human microsomal epoxide hydrolase and analysis), 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

Hammock, Bruce D.


Human anti-anthrax protective antigen neutralizing monoclonal antibodies derived from donors vaccinated with anthrax vaccine adsorbed  

Microsoft Academic Search

BACKGROUND: Potent anthrax toxin neutralizing human monoclonal antibodies were generated from peripheral blood lymphocytes obtained from Anthrax Vaccine Adsorbed (AVA) immune donors. The anti-anthrax toxin human monoclonal antibodies were evaluated for neutralization of anthrax lethal toxin in vivo in the Fisher 344 rat bolus toxin challenge model. METHODS: Human peripheral blood lymphocytes from AVA immunized donors were engrafted into severe

Ritsuko Sawada-Hirai; Ivy Jiang; Fei Wang; Shu Man Sun; Rebecca Nedellec; Paul Ruther; Alejandro Alvarez; Diane Millis; Phillip R Morrow; Angray S Kang



Monoclonal antibodies to adenosine receptor by an auto-anti-idiotypic approach  

SciTech Connect

BALB/c mice were immunized with adenosine 6-aminocaproyl-BSA. Hybridoma cell lines that secreted anti-idiotypic antibodies were identified by their binding to rabbit anti-adenosine antibodies, but not to normal rabbit immunoglobulins. Two such monoclonal antibodies, AA18 and AA21, also inhibited the binding of ({sup 3}H)adenosine to the rabbit anti-adenosine antibodies. Therefore, both appeared to recognize idiotypic determinants on the rabbit anti-adenosine antibodies. The monoclonal antibodies AA18 and AA21 were established as being directed at adenosine receptors by the following criteria: (1) they bound to both rat and bovine brain membranes, and binding could be inhibited by CHA, an adenosine receptor agonist, (2) they inhibited the binding of ({sup 3}H)R-PIA, an adenosine receptor agonist, to rat brain membranes; and (3) they inhibited the adenylate cyclase of rat brain membranes. The monoclonal antibodies were used to screen cDNA libraries in lambda gt11.

Ku, Hsing-Hsu.



Hybridoma-derived monoclonal immunoglobulin M antibodies to Legionella pneumophila serogroup 1 with diagnostic potential.  

PubMed Central

Mouse hybridomas were isolated by fusing P3-X63-Ag 8.653 myeloma cells with spleen cells from mice that had been repeatedly immunized with Legionella pneumophila serogroup 1 organisms. In one fusion, three independent hybridoma cultures which secreted antibodies that reacted with the immunizing strain in the indirect immunofluorescent-antibody test were selected for cloning. Representative continuously growing clones, one of each hybridoma, which remained stable in producing high-titer antibodies were examined in detail. Extensive specificity tests revealed that these hybridoma-derived monoclonal antibodies were specifically directed against L. pneumophila serogroup 1 organisms and showed no cross-reactions in the indirect immunofluorescent-antibody test either with the other known serogroups of L. pneumophila or with other unrelated bacterial species. The three monoclonal antibodies F4/CB5/K18, F/4CB5/K104, and F4/JD3.8/K101 belonged to the immunoglobulin M class and were capable of agglutinating serogroup 1 organisms of L. pneumophila exquisitely. These monoclonal antibodies against L. pneumophila with defined fine specificity should enable purification and subsequent analysis of the corresponding antigenic determinant(s) and can also be used for the preparation of unlimited supplies of standard diagnostic reagents for the identification of L. pneumophila in the tissues and body fluids. PMID:6874913

Sethi, K K; Drueke, V; Brandis, H



Production and characterization of monoclonal antibodies to budgerigar fledgling disease virus major capsid protein VP  

NASA Technical Reports Server (NTRS)

Eleven hybridoma cell lines producing monoclonal antibodies (MAbs) against intact budgerigar fledgling disease (BFD) virions were produced and characterized. These antibodies were selected for their ability to react with BFD virions in an enzyme-linked immunosorbent assay. Each of these antibodies was reactive in the immunofluorescent detection of BFD virus-infected cells. These antibodies immunoprecipitated intact virions and specifically recognized the major capsid protein, VP1, of the dissociated virion. The MAbs were found to preferentially recognize native BFD virus capsid protein when compared with denatured virus protein. These MAbs were capable of detecting BFD virus protein in chicken embryonated cell-culture lysates by dot-blot analysis.

Fattaey, A.; Lenz, L.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)



Human Monoclonal Antibodies Against a Plethora of Viral Pathogens From Single Combinatorial Libraries  

NASA Astrophysics Data System (ADS)

Conventional antibody generation usually requires active immunization with antigen immediately prior to the preparation procedure. Combinatorial antibody library technology offers the possibility of cloning a range of antibody specificities at a single point in time and then accessing these specificities at will. Here we show that human monoclonal antibody Fab fragments against a plethora of infectious agents can be readily derived from a single library. Further examination of a number of libraries shows that whenever antibody against a pathogen can be detected in the serum of the donor, then specific antibodies can be derived from the corresponding library. We describe the generation of human Fab fragments against herpes simplex virus types 1 and 2, human cytomegalovirus, varicella zoster virus, rubella, human immunodeficiency virus type 1, and respiratory syncytial virus. The antibodies are shown to be highly specific and a number are effective in neutralizing virus in vitro.

Williamson, R. Anthony; Burioni, Roberto; Sanna, Pietro P.; Partridge, Lynda J.; Barbas, Carlos F., III; Burton, Dennis R.



Monoclonal antibodies to human hemoglobin S and cell lines for the production thereof  


The present invention provides monoclonal antibodies specific to and distinguishing between hemoglobin S and hemoglobin A and methods for their production and use. These antibodies are capable of distinguishing between two hemoglobin types which differ from each other by only a single amino acid residue. The antibodies produced according to the present method are useful as immunofluorescent markers to enumerate circulating red blood cells which have the property of altered expression of the hemoglobin gene due to somatic mutation in stem cells. Such a measurement is contemplated as an assay for in vivo cellular somatic mutations in humans. Since the monoclonal antibodies produced in accordance with the instant invention exhibit a high degree of specificity to and greater affinity for hemoglobin S, they are suitable for labeling human red blood cells for flow cytometric detection of hemoglobin genotype. 4 figs.

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



Monoclonal antibodies to human hemoglobin S and cell lines for the production thereof  


The present invention provides monoclonal antibodies specific to and distinguish between hemoglobin S and hemoglobin A and methods for their production and use. These antibodies are capable of distinguishing between two hemoglobin types which differ from each other by only a single amino acid residue. The antibodies produced according to the present method are useful as immunofluorescent markers to enumerate circulating red blood cells which have the property of altered expression of the hemoglobin gene due to somatic mutation in stem cells. Such a measurement is contemplated as an assay for in vivo cellular somatic mutations in humans. Since the monoclonal antibodies produced in accordance with the instant invention exhibit a high degree of specificity to and greater affinity for hemoglobin S, they are suitable for labeling human red blood cells for flow cytometric detection of hemoglobin genotype.

Jensen, Ronald H. (Livermore, CA); Vanderlaan, Martin (San Ramon, CA); Bigbee, William L. (Livermore, CA); Stanker, Larry H. (Livermore, CA); Branscomb, Elbert W. (Walnut Creek, CA); Grabske, Robert J. (Berkeley, CA)



Monoclonal antibodies against hepatitis B e antigen: production, characterization, and use for diagnosis.  


Five different hybridoma clones secreting anti-HBeAg antibody were constructed by fusing cells of mouse myeloma line SP2/0 with splenocytes from BALB/c mice immunized with recombinant HBeAg. The monoclonal antibodies obtained were characterized immunologically and one was used to develop ELISA for detection of HBeAg and anti-HBeAg antibody. These monoclonal assays enabled the detection of 3 U HBeAg/ml and 1 U anti-HBeAg/ml with reference to standards of the Paul Ehrlich Institute, Frankfurt, F.R.G. Both assays compared well with a commercially available kit (Abbott Laboratory) and were used for detection of HBeAg and anti-HBeAg antibody in clinical serum samples. PMID:2370287

Korec, E; Dostálová, V; Korcová, J; Mancal, P; König, J; Borisova, G; Cibinogen, V; Pumpen, P; Gren, E; Hlozánek, I



Combined active and passive immunization against nicotine: Minimizing monoclonal antibody requirements using a target antibody concentration strategy  

PubMed Central

Nicotine vaccines have shown preliminary evidence of efficacy for enhancing smoking cessation rates, but the serum nicotine-specific antibody (NicAb) concentrations produced are highly variable and many subjects do not develop effective levels. As an alternative to vaccination, passive immunization with nicotine-specific monoclonal antibodies could produce more uniform serum NicAb concentrations, but its use is limited by their high cost and shorter elimination half-life. This study investigated supplementing vaccination with monoclonal antibodies in a targeted fashion to increase vaccine efficacy while minimizing the required monoclonal antibody dose. Rats were vaccinated and then given individualized supplemental doses of the nicotine-specific monoclonal antibody Nic311 to achieve a target total serum NicAb concentration known to be effective for blocking locomotor sensitization (LMS) to nicotine. Rats received vaccine, Nic311, both, or neither, followed by 0.3 mg/kg nicotine s.c. for 10 days to produce LMS. Combination immunotherapy completely blocked the development of LMS, while monotherapy with vaccine or Nic311 alone were only minimally effective. Lower brain nicotine levels were associated with reduced locomotor activity averaged over days 7-10. Despite its greater efficacy, combination immunotherapy did not reduce the variability in the resulting total serum NicAb concentrations. Variability in total serum NicAb concentrations was contributed to by both vaccine-generated antibody and by Nic311. These data show that combination immunotherapy, using a Nic311 dose that is by itself only minimally effective, can substantially enhance nicotine vaccine efficacy. However, variability in serum NicAb levels with combination immunotherapy may make translation of this approach challenging. PMID:21802529

Cornish, Katherine E.; Harris, Andrew C.; LeSage, Mark G.; Keyler, Dan E.; Burroughs, Danielle; Earley, Cathy; Pentel, Paul R.



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

Microsoft Academic Search

BACKGROUND: Antibodies produced in response to infection with any of the four serotypes of dengue virus generally provide homotypic immunity. However, prior infection or circulating maternal antibodies can also mediate a non-protective antibody response that can enhance the course of disease in a subsequent heterotypic infection. Naturally occurring human monoclonal antibodies can help us understand the protective and pathogenic roles

John S Schieffelin; Joshua M Costin; Cindo O Nicholson; Nicole M Orgeron; Krystal A Fontaine; Sharon Isern; Scott F Michael; James E Robinson



Inhibition of kinesin-driven microtubule motility by monoclonal antibodies to kinesin heavy chains  

PubMed Central

We have prepared and characterized seven mouse monoclonal antibodies (SUK 1-7) to the 130-kD heavy chain of sea urchin egg kinesin. On immunoblots, SUK 3 and SUK 4 cross-reacted with Drosophila embryo 116- kD heavy chains, and SUK 4, SUK 5, SUK 6, and SUK 7 bound to the 120-kD heavy chains of bovine brain kinesin. Three out of seven monoclonal antikinesins (SUK 4, SUK 6, and SUK 7) caused a dose-dependent inhibition of sea urchin egg kinesin-induced microtubule translocation, whereas the other four monoclonal antibodies had no detectable effect on this motility. The inhibitory monoclonal antibodies (SUK 4, SUK 6, and SUK 7) appear to bind to spatially related sites on an ATP- sensitive microtubule binding 45-kD chymotryptic fragment of the 130-kD heavy chain, whereas SUK 2 binds to a spatially distinct site. None of the monoclonal antikinesins inhibited the microtubule activated MgATPase activity of kinesin, suggesting that SUK 4, SUK 6, and SUK 7 uncouple this MgATPase activity from motility. PMID:2974459



Crystal Structure of a Dimerized Cockroach Allergen Bla g 2 Complexed with a Monoclonal Antibody  

Microsoft Academic Search

The crystal structure of a 1:1 complex between the German cockroach allergen Bla g 2 and the Fab' fragment of a monoclonal antibody 7C11 was solved at 2.8-â« resolution. Bla g 2 binds to the antibody through four loops that include residues 60-70, 83-86, 98-100, and 129-132. Cation-Ï interactions exist between Lys-65, Arg-83, and Lys-132 in Bla g 2 and

Mi Li; Alla Gustchina; Jerry Alexandratos; Alexander Wlodawer; S. Wunschmann; Christopher L. Kepley; Martin D. Chapman; Anna Pomes



Synthesis of potent taxoids for tumor-specific delivery using monoclonal antibodies.  


The targeted delivery of taxoids, in the form of taxane-antibody immunoconjugates, requires the preparation of taxoids containing moieties suitable for their conjugation to monoclonal antibodies. A series of taxoids incorporating a disulfide-containing linker at various positions of the taxoid framework have been prepared to investigate the most suitable position for conjugation. A second series of taxoids modified at the C-2 position aimed at increasing the potency of these taxanes has also been prepared. PMID:15225730

Miller, Michael L; Roller, Elizabeth E; Wu, Xinyaun; Leece, Barbara A; Goldmacher, Victor S; Chari, Ravi V J; Ojima, Iwao



Immunocytochemical staining of cells in pleural and peritoneal effusions with a panel of monoclonal antibodies  

Microsoft Academic Search

A panel of seven monoclonal antibodies was applied to smears of cell deposit from 70 pleural and peritoneal fluids, using an immunoalkaline phosphatase (IAP) procedure. The cases were chosen to show typical cytological patterns, both benign and malignant, and in this way the diagnostic value of the method could be assessed. The antibodies used were 2D1 (anti-leucocyte), Ca 1, HMFG-2

A K Ghosh; A I Spriggs; J Taylor-Papadimitriou; D Y Mason



Removal of digoxin from plasma using monoclonal anti-digoxin antibodies immobilized on agarose  

Microsoft Academic Search

Monoclonal anti-digoxin antibodies (dig-Ab) have been covalently coupled to agarose supports to evaluate them as part of an extracorporeal device for removal of digoxin from the circulation. The agarose supports studied were Sepharose CL-6B, agarose-polyacrolein microsphere (APAM) beads, Bio Gel A-5m and Affi-gel 15 (Bio-Rad). Antibody concentrations between 2 and 4 mg\\/g gel were coupled to the agarose beads which

M. Brizgys; S. Pincus; D. E. Rollins



In vivo Therapy with Monoclonal Anti-I-A Antibody Suppresses Immune Responses to Acetylcholine Receptor  

NASA Astrophysics Data System (ADS)

A monoclonal antibody to I-A gene products of the immune response gene complex attenuates both humoral and cellular responses to acetylcholine receptor and appears to suppress clinical manifestations of experimental autoimmune myasthenia gravis. This demonstrates that use of antibodies against immune response gene products that are associated with susceptibility to disease may be feasible for therapy in autoimmune conditions such as myasthenia gravis.

Waldor, Matthew K.; Sriram, Subramaniam; McDevitt, Hugh O.; Steinman, Lawrence



Monoclonal Antibody Against Human Tyrosine and Reactive With Melanotic and Amelanotic Melanoma Cells  

Microsoft Academic Search

Human tyrosinase was partially purified from the lysate of a melanoma cell line and used to immunize BALB\\/c mice. Spleen cells from the immunized mice were fused with a murine myeloma cell line (NS-1), yielding a hybridoma (5C12) that produced monoclonal antibody directed against tyrosinase. 5C12 antibody reacted with normal human melanocytes, neval cells, primary cultures of melanoma biopsies, and

Max McEwan; Peter G. Parsons; Denis J. Moss



Separation of functional subsets of human t cells by a monoclonal antibody  

Microsoft Academic Search

A monoclonal antibody was produced to human peripheral blood T cells. This hybridoma antibody, termed OKT4, was reactive by indirect immunofluorescence with only 55 to 60% of the peripheral blood T cell population (OKT4\\/sup +\\/) and unreactive with normal B cells, null cells, and macrophages. With cell-sorter separation of OKT4\\/sup +\\/ and OKT4⁻ cells, it was shown that these T

E. L. Reinherz; P. C. Kung; G. Goldstein; S. F. Schlossman



Monoclonal Antibodies against Der s 1, a Major Allergen of Dermatophagoides siboney  

Microsoft Academic Search

Six stable clones secreting murine monoclonal antibodies (Mabs) against Der s 1 were obtained. The binding of Mabs showed cross-reactivity with Dermatophagoides farinae, as determined by enzyme-linked immunosorbent assay (ELISA). In a Western blot assay, antibodies reacted with a 24-kD protein considered to represent the major allergen Der s 1. The repertoire of antigenic sites on Der s 1 was

Minerva Sewer; Keiko Uyema; Mayrel Labrada; Maritza González; Marcos Coca



Tissue-Specific and Species-Specific Monoclonal Antibodies to Avian Red Cell Nuclear Proteins  

Microsoft Academic Search

In order to identify potential red cell-specific regulatory proteins and to define additional red cell-specific markers, we have isolated a series of hybridomas that produce monoclonal antibodies that react with nuclear preparations from avian red blood cells. Several antibodies have been well characterized for their tissue- and species-specific reactions by using solid-phase and protein-transfer radioimmunoassays as well as immunofluorescence. These

Caroline M. Kane; Pei Feng Cheng; John B. E. Burch; Harold Weintraub



Monoclonal antibody analysis of neutralization and antibody-dependent enhancement of feline infectious peritonitis virus.  

PubMed Central

Fifty-four monoclonal antibodies (MAbs) to feline infectious peritonitis virus (FIPV) were characterized according to protein specificity, immunoglobulin subclass, virus neutralization, reactivity with different coronaviruses, and ability to induce antibody-dependent enhancement (ADE) of FIPV infection in vitro. The MAbs were found to be specific for one of three structural proteins of FIPV. A total of 47 MAbs were specific for the 205-kDa spike protein (S), 3 MAbs were specific for the 45-kDa nucleocapsid protein (N), and 4 MAbs were specific for the 26- to 28-kDa membrane protein (M). The S-specific MAbs showed various degrees of cross-reactivity with strains of FIPV, feline enteric coronavirus, canine coronavirus, and porcine transmissible gastroenteritis virus. Nineteen S-specific MAbs neutralized FIPV. A total of 15 of the neutralizing MAbs induced ADE, and all but 1 were of the immunoglobulin G2a subclass. The remaining four neutralizing MAbs that did not induce ADE were of the immunoglobulin G1 subclass. Two S-specific MAbs induced ADE but were nonneutralizing. None of the N- or M-specific MAbs was neutralizing or induced ADE. On the basis of the reactivity patterns of the MAbs with FIPV and related coronaviruses, it was concluded that there is a minimum of five neutralizing sites on S. In most instances, neutralizing MAbs were able to induce ADE, demonstrating a direct relationship between neutralization and enhancement. The difference in immunoglobulin subclass between neutralizing MAbs that induced ADE and those that did not induce ADE suggests that there may be a restriction in the immunoglobulin subclasses capable of mediating ADE. Images PMID:1383568

Corapi, W V; Olsen, C W; Scott, F W



Cross-reactive and specific monoclonal antibodies against cellobiohydrolases I and II and endoglucanases I and II of trichoderma reesei  

SciTech Connect

Splenocytes derived from mice inoculated with a commercial cellulase preparation or purified cellulases were fused with a stable myeloma cell line (SP2/0). Specific monoclonal antibodies to cellobiohydrolases I and II and endoglucanases I and II were established. In addition to specific monoclonal antibodies, we were also able to establish stable hybridoma cell lines which produced monoclonal antibodies that recognized similar epitopes possessed by two or more of the above cellulases. By obtaining monospecific antibodies for all four individual cellulases, the role and function of the individual cellulases can thus be studied in greater detail.

Nieves, R.A.; Todd, R.J.; Ellis, R.P. (Colorado State Univ., Fort Collins (USA)); Himmel, M.E. (Solar Energy Research Institute, Golden, CO (USA))



Rapid production of antigen-specific monoclonal antibodies from a variety of animals  

PubMed Central

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. PMID:23017270



A syngeneic monoclonal anti-idiotype antibody against HNK-1: characterization and cytotoxic activity in vitro.  

PubMed Central

A syngeneic, monoclonal, anti-idiotype antibody, G6D9, was raised against the mouse monoclonal IgM HNK-1. G6D9, characterized as an IgG1-kappa, inhibits the binding of HNK-1 to the myelin-associated glycoprotein (MAG). G6D9 does not interfere with the binding of various human monoclonal anti-MAG IgM to their specific antigen. G6D9 binds HNK-1 hybridoma cells on their surface and within their cytoplasm, as demonstrated using indirect immunofluorescence. In the presence of complement, G6D9 is cytotoxic for HNK-1-secreting cells. A cell lysis of 32% was observed and compared to lysis obtained with other antibodies directed against mouse lymphocyte antigens. The use of G6D9 as a tool to study specific cytotoxicity and immunotherapy in vivo is discussed. PMID:2649290

Schluep, M; Page, N; Burger, D; Steck, A J



Monoclonal antibodies with neutralizing activity segregate isolates of bovine viral diarrhea virus into groups  

Microsoft Academic Search

Summary Isolates of bovine viral diarrhea (BVD) virus were differentiated by monoclonal antibodies (MoAbs) reactive with the 56kD viral polypeptide. Patterns of neutralizing activity of the MoAbs indicate that multiple epitopes are involved in virus neutralization.

S. Bolin; V. Moennig; N. E. Kelso Gourley; J. Ridpath



Targeted Monoclonal Antibody Therapy for a Rare T-Cell Leukemia

This study will assess the safety of MiK-beta-1, a monoclonal antibody, in patients with T-cell large granular lymphocyte leukemia (T-LGLL). If the treatment is tolerated, it may eventually be used to treat not only T-LGLL, but also certain autoimmune disorders such as rheumatoid arthritis and multiple sclerosis.


A new xenogenic monoclonal antibody detecting class I H-2 molecules  

Microsoft Academic Search

In an attempt to produce monoclonal antibodies which recognize surface antigens of splenic lymphocytes of NZB mice, female Lewis rats were injected intraperitoneally with 10 v NZB spleen cells three times at weekly intervals and boosted 1-2 months later with the same dose. Three days after the last injection, the recipient spleen cells were hybridized in the presence of polyethylene

Vijaya Manohar; Elinor M. Brown; William M. Leiserson; Thomas M. Chused



A monoclonal antibody against tetrodotoxin that reacts to the active group for the toxicity  

Microsoft Academic Search

A monoclonal antibody against tetrodotoxin was produced. Tetrodotoxin coupled with keyhole limpet hemocyanin was used as an immunogen to BALB\\/c mice. These mice had no clinical signs for the toxicity of tetrodotoxin during the immunization. The reason may be that the guanidyl group of tetrodotoxin which is an important group for the toxicity was hidden by coupling with keyhole limpet

Kendo Matsumura



Treatment of neoplastic meningitis by targeted radiation using 131 I-radiolabelled monoclonal antibodies  

Microsoft Academic Search

Between 1984 and 1993, monoclonal antibodies (MAbs) radiolabelled with 131I were administered into the CSF of 52 patients with neoplastic meningitis (meningosis) with progressive disease despite active conventional therapy. Selection of MAbs was based on immunoreactivity with patients' tumour and lack of binding to normal central nervous system (CNS) tissue.

Hugh B. Coakham; John T. Kemshead



Application of monoclonal antibodies to investigate plant cell wall deconstruction for biofuels production  

E-print Network

Application of monoclonal antibodies to investigate plant cell wall deconstruction for biofuels, or microbes. Lignocellulosic biomass is typically pretreated prior to enzymatic hydrolysis to disrupt cell.1039/c1ee02112e To better understand how hydrothermal pretreatment reduces plant cell wall recalcitrance

California at Riverside, University of


Human lipoprotein lipase: relationship of activity, heparin affinity, and conformation as studied with monoclonal antibodies  

Microsoft Academic Search

The objective of this study was to investigate how a conformational change in lipoprotein lipase (LPL) affects its molecular functions. Monoclonal antibodies (MAbs) were raised against purified bovine milk lipoprotein lipase. MAb 5D2 bound to human and bovine LPL both before and after denaturation of LPL. MAb 5F9 also recognized LPL from both species, but only after denaturation of the

Jonas Peterson; Wilfred Y. Fujimoto; John D. Brunzelll



EPA Science Inventory

Stachybotrys chartarum is known to produce the hemolysin stachylysin and its detection in human serum has been proposed as a biomarker for exposure to the fungus. In this study we report the initial characterization of monoclonal antibodies (mAbs) against stachylysin and the dev...


Radiolabelled monoclonal antibodies in tumour imaging and therapy: out of fashion?  

Microsoft Academic Search

The initial enthusiasm for the development of diagnostic and therapeutic studies involving the use of monoclonal antibodies was replaced by scepticism as hopes remained unfulfilled. Against this background one needs to ask whether immunoscintigraphy (IS) serves clinical needs effectively and whether radioimmunotherapy (RIT) has a future. The current review considers these questions by reference to relevant studies. Taking colorectal cancer

Angelika Bischof Delaloye; Bernard Delaloye



A monoclonal antibody against meningococcus group B polysaccharides distinguishes embryonic from adult N-CAM  

Microsoft Academic Search

The neural cell adhesion molecules (N- CAM) occur chiefly in two molecular forms that are selectively expressed at various stages of development. Highly sialylated forms prevalent in embryonic and neonatal brain are gradually replaced by less sialylated forms as development proceeds. Here we describe a monoclonal antibody raised against the capsular poly- saccharides of meningococcus group B (Men B) which

Genevieve Rougon; Catherine Dubois; Noel Buckley; John L. Magnani; Wendell Zollinger



Detection of Substance P in the Central Nervous System by a Monoclonal Antibody  

Microsoft Academic Search

Peptides with transmitter-like characteristics are being found in many brain areas. The application of immunocytochemical and radioimmunoassay methods has contributed much to the clarification of these neuronal systems. Here we report the development of a rat monoclonal antibody produced by a hybrid myeloma and its application to the study of one of these peptides, substance P. The hybrid clone, isolated

A. C. Cuello; G. Galfre; C. Milstein



Monoclonal Antibodies Against Herpes Simplex Virus Types 1 and 2 Nucleocapsids and Kit.  

National Technical Information Service (NTIS)

The method of producing clinical assays for use of monoclonal antibodies in the diagnosis of herpes simplex virus (HSV) infections and the differentiation of herpes simplex virus types 1 and 2 as a diagnostic kit for differentiating HSV-1 and HSV-2 utiliz...

B. Hampar, M. Zweig, H. Rabin



Monoclonal antibodies reveal multiple forms of expression of human microsomal epoxide hydrolase  

E-print Network

January 2012 Keywords: Microsomal epoxide hydrolase Drug metabolism Monoclonal antibodies Flow cytometry.3.2.9) is a drug-metabolizing enzyme that catalyzes the conversion of epoxides formed during phase I metabolism acid transporter (von Dippe et al., 1993). It is speculated that efficient execution of such multiple

Hammock, Bruce D.


Topographical analysis of the G virion of Aleutian mink disease parvovirus with monoclonal antibodies  

Microsoft Academic Search

Summary The topography of the Aleutian mink disease parvovirus (ADV) G virion was analyzed with monoclonal antibodies and polyclonal antiserum. There was homology between the two major structural proteins as others have previously reported. Trypsin treatment of the virion with subsequent immunoblotting revealed that VP2 represents the main peptide on the exterior of virion and that VP1 is probably embedded

D. L. Barnard; F. B. Johnson



Proliferative activity of cells in the synovium as demonstrated by a monoclonal antibody, Ki67  

Microsoft Academic Search

The presence of proliferating cells has been sought in the synovium of rheumatoid arthritic (RA) and osteoarthritic (OA) joints using the monoclonal antibody Ki67, which marks a nuclear antigen present in all stages of the cell cycle apart from Go. Synovia were studied from 21 RA and 14 OA cases using the indirect immunoperoxidase technique. Double-staining was performed on 18

Peggy A. Lalor; P. I. Mapp; P. A. Hall; P. A. Revell



Production and characterization of monoclonal antibodies against different epitopes of Ebola virus antigens  

Microsoft Academic Search

Ebola virus (EBOV) causes hemorrhagic fever in humans and nonhuman primates with up to 90% mortality rate. In this study, Ebola virus like particles (EVLPs) and the aglycosyl subfragment of glycoprotein (GP(1) subfragment D) were used to generate monoclonal antibodies (MAbs) against different epitopes of the viral antigens. Such MAbs could be useful in diagnostics and potential therapeutics of viral

Soraya Shahhosseini; Dipankar Das; Xiangguo Qiu; Heinz Feldmann; Steven M. Jones; Mavanur R Suresh



Ebola GP-Specific Monoclonal Antibodies Protect Mice and Guinea Pigs from Lethal Ebola Virus Infection  

Microsoft Academic Search

Ebola virus (EBOV) causes acute hemorrhagic fever in humans and non-human primates with mortality rates up to 90%. So far there are no effective treatments available. This study evaluates the protective efficacy of 8 monoclonal antibodies (MAbs) against Ebola glycoprotein in mice and guinea pigs. Immunocompetent mice or guinea pigs were given MAbs i.p. in various doses individually or as

Xiangguo Qiu; Lisa Fernando; P. Leno Melito; Jonathan Audet; Heinz Feldmann; Gary Kobinger; Judie B. Alimonti; Steven M. Jones



Protective Efficacy of Neutralizing Monoclonal Antibodies in a Nonhuman Primate Model of Ebola Hemorrhagic Fever  

Microsoft Academic Search

Ebola virus (EBOV) is the causative agent of severe hemorrhagic fever in primates, with human case fatality rates up to 90%. Today, there is neither a licensed vaccine nor a treatment available for Ebola hemorrhagic fever (EHF). Single monoclonal antibodies (MAbs) specific for Zaire ebolavirus (ZEBOV) have been successfully used in passive immunization experiments in rodent models, but have failed

Andrea Marzi; Reiko Yoshida; Hiroko Miyamoto; Mari Ishijima; Yasuhiko Suzuki; Megumi Higuchi; Yukie Matsuyama; Manabu Igarashi; Eri Nakayama; Makoto Kuroda; Masayuki Saijo; Friederike Feldmann; Douglas Brining; Heinz Feldmann; Ayato Takada



Specificity of a Monoclonal Antibody for Alkaline Phosphatase in Escherichia coli and Shigella Species  

Microsoft Academic Search

The specificity of monoclonal antibody 2E5 for the alkaline phosphatase of Escherichia coli was studied against the alkaline phosphatases of 251 other bacterial strains. The organisms used included members of the six species of the genus Escherichia (E. coli, E. fergusonii, E. hermannii, E. blattae, E. vulneris, E. adecarboxylata), 41 species representing the family Enterobacteriaceae, and, in addition, Pseudomonas aeruginosa,



Preclinical safety testing of monoclonal antibodies: the significance of species relevance  

Microsoft Academic Search

Selecting a pharmacologically relevant animal species for testing the safety and toxicity of novel monoclonal antibody (mAb) therapies to support clinical testing can be challenging. Frequently, the species of choice is the primate. With the increased number of mAbs in the pharmaceutical pipeline, this has significant implications for primate use, and so raises several important scientific, ethical and economic issues.

Nick Pullen; Mark Graham; Ian Ragan; Kathryn Chapman



Two subpopulations of differentiated chondrocytes identified with a monoclonal antibody to keratan sulfate  

Microsoft Academic Search

We have prepared a monoclonal antibody, named MZ15, that specifically binds keratan sulfate. Immunofluorescence studies showed that the distribution of keratan sulfate in articular cartilage was not uniform: the amount of keratan sulfate increased with distance from the articular surface. Two subpopulations of chondrocytes could be distinguished after isolation from cartilage by the presence or absence of cell surface keratan




Bacterial Expression of a Human Monoclonal Antibody-Alkaline Phosphatase Conjugate Specific for Entamoeba histolytica  

Microsoft Academic Search

We previously produced human monoclonal antibody Fab fragments specific to Entamoeba histolytica in Escherichia coli. In order to use these Fab fragments for diagnostic purposes, an expression vector to produce a fusion protein of Fab and alkaline phosphatase (PhoA) in E. coli was designed and constructed. The E. coli PhoA gene was fused to the 3? terminus of the gene

Hiroshi Tachibana; Masataka Takekoshi; Xun-Jia Cheng; Yuta Nakata; Tsutomu Takeuchi; Seiji Ihara



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

Microsoft Academic Search

Chikungunya fever is an arbovirosis of major impact in public health in Asia and Africa. Chikungunya (CHIK) virus is member of the genus Alphavirus and belongs to the Semliki Forest (SF) antigenic complex. We describe for the first time a panel of monoclonal antibodies (MAbs) reactive to CHIK envelope E2 glycoprotein. For the screening of E2-specific MAbs, we expressed a

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



Immunological relationships between phocid and canine distemper virus studied with monoclonal antibodies  

Microsoft Academic Search

The immunological relationships between distemper viruses, isolated from a seal and mink in Denmark and from a dog in Greenland, were investigated with 39 previously developed monoclonal antibodies (MAbs) directed against four major structural proteins of canine distemper virus (CDV). They were also investi- gated with 16 newly developed MAbs directed against the fusion (F) and large glycoprotein (named H

C. Orvell; M. Blixenkrone-Moller; Vilhjalmur Svansson; P. Have



Phytochrome in photosynthetically competent plants characterization by monoclonal antibodies: Progress report  

SciTech Connect

Monoclonal antibodies to oat and to pea phytochrome have been characterized and immunopurified. In addition a fully automated, microcomputer-based, dual wavelength spectrophotometer was designed and constructed. An ELISA that is sensitive to subfemtomol levels of phytochrome has been developed. 1 fig.

Pratt, L.H.



Prevalence, specificity and functionality of anti-ganglioside antibodies in neuropathy associated with IgM monoclonal gammopathy.  


IgM antibodies against gangliosides and their complexes were studied in sera from 54 patients with polyneuropathy and IgM monoclonal gammopathy (IgM-PNP) without anti-MAG antibodies. Anti-ganglioside antibodies were found in 19 (35%) patients. Five (9%) patients had antibodies against ganglioside complexes. IgM antibodies against gangliosides activated complement in vitro. Light chain usage was restricted to kappa or lambda in most, but not all patients. In conclusion, anti-ganglioside antibodies in IgM-PNP are common, display pathogenic properties and do not always arise from a monoclonal B cell proliferation. PMID:24529728

Stork, Abraham C J; Jacobs, Bart C; Tio-Gillen, Anne P; Eurelings, Marijke; Jansen, Marc D; van den Berg, Leonard H; Notermans, Nicolette C; van der Pol, W-Ludo



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

PubMed Central

Background 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 envelope and nonstructural 1 proteins. Phylogenetic distances between the four serotypes of DENV were as different as those of other flaviviruses, such as Japanese encephalitis virus and West Nile virus. Large variations in the DENV serotypes were comparable with the differences between species of flavivirus. Furthermore, the diversity of flavivirus capsid protein was much greater than that of envelope and nonstructural 1 proteins. Conclusion In this study, we produced specific monoclonal antibodies that can be used to detect DENV-2 capsid protein, but not a cross-reactive one with all serotypes of DENV capsid protein. The high diversity of the DENV capsid protein sequence by phylogenetic analysis supported the low cross-reactivity of monoclonal antibodies against DENV capsid protein. PMID:23209363

Noda, Megumi; Masrinoul, Promsin; Punkum, Chaweewan; Pipattanaboon, Chonlatip; Ramasoota, Pongrama; Setthapramote, Chayanee; Sasaki, Tadahiro; Sasayama, Mikiko; Yamashita, Akifumi; Kurosu, Takeshi; Ikuta, Kazuyoshi; Okabayashi, Tamaki



Development of VHH Antibodies against Dengue Virus Type 2 NS1 and Comparison with Monoclonal Antibodies for Use in Immunological Diagnosis  

PubMed Central

The possibility of using variable domain heavy-chain antibodies (VHH antibodies) as diagnostic tools for dengue virus (DENV) type 2 NS1 protein was investigated and compared with the use of conventional monoclonal antibodies. After successful expression of DENV type 2 NS1 protein, the genes of VHH antibodies against NS1 protein were biopanned from a non-immune llama library by phage display. VHH antibodies were then expressed and purified from Escherichia coli. Simultaneously, monoclonal antibodies were obtained by the conventional route. Sequence analysis of the VHH antibodies revealed novel and long complementarity determining regions 3 (CDR3). Epitope mapping was performed via a phage display peptide library using purified VHH and monoclonal antibodies as targets. Interestingly, the same region of NS1, which comprises amino acids 224HWPKPHTLW232, was conserved for both kinds of antibodies displaying the consensus motif histidine-tryptophan-tryptophan or tryptophan-proline-tryptophan. The two types of antibodies were used to prepare rapid diagnostic kits based on immunochromatographic assay. The VHH antibody immobilized rapid diagnostic kit showed better sensitivity and specificity than the monoclonal antibody immobilized rapid diagnostic kit, which might be due to the long CDR3 regions of the VHH antibodies and their ability to bind to the pocket and cleft of the targeted antigen. This demonstrates that VHH antibodies are likely to be an option for developing point-of-care tests against DENV infection. PMID:24751715

Fatima, Aneela; Wang, Haiying; Kang, Keren; Xia, Liliang; Wang, Ying; Ye, Wei; Wang, Jufang; Wang, Xiaoning



A human recombinant monoclonal antibody to cocaine: Preparation, characterization and behavioral studies.  


Cocaine abuse remains prevalent worldwide and continues to be a major health concern; nonetheless, there is no effective therapy. Immunopharmacotherapy has emerged as a promising treatment strategy by which anti-cocaine antibodies bind to the drug blunting its effects. Previous passive immunization studies using our human monoclonal antibody, GNCgzk, resulted in protection against cocaine overdose and acute toxicity. To further realize the clinical potential of this antibody, a recombinant IgG form of the antibody has been produced in mammalian cells. This antibody displayed a high binding affinity for cocaine (low nanomolar) in line with the superior attributes of the GNCgzk antibody and reduced cocaine-induced ataxia in a cocaine overdose model. PMID:25205191

Eubanks, Lisa M; Ellis, Beverly A; Cai, Xiaoqing; Schlosburg, Joel E; Janda, Kim D



Production and evaluation of monoclonal antibodies for the detection of beet mild yellowing luteovirus and related strains  

Microsoft Academic Search

A sugar-beet-infecting isolate of beet mild yellowing luteovirus (BMYV), and aBrassica-infecting isolate of beet western yellows luteovirus (BWYV) were used to produce monoclonal antibodies for epidemiological studies with BMYV and related field strains. Thirty-four monoclonal antibodies were tested for their reaction with 9 luteoviruses in triple-antibody-sandwich enzyme-linked immunosorbent assay. One (MAFF 24) is now routinely used in the UK for

H. G. Smith; I. Barker; G. Brewer; M. Stevens; P. B. Hallsworth



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


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 ?1,4-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 ?1,4-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 possible. Moreover, real-time, or near real-time control can be enabled by facile, rapid measurement of cell surface biomarkers of cellular biosynthetic capability. PMID:23737295

Grainger, Rhian K; James, David C



Current and emerging monoclonal antibody treatments for chronic lymphocytic leukemia: state of the art.  


Anti-CD20 monoclonal antibodies (mAbs), rituximab, ofatumumab and obinutuzumab, have a significant impact in the treatment of chronic lymphocytic leukemia (CLL), particularly in combination with chemotherapy. Over the last few years, several new mAbs have been developed and investigated in CLL. The most promising newer mAbs are directed against CD20, CD19, CD37 and CD40. Combinations of antibodies with targeted drugs like ibrutinib, idelalisib or lenalidomide will probably replace chemotherapy-based combinations in the near future. This review gives a critical overview of established mAbs as well as new antibodies potentially useful in CLL. PMID:25249370

Robak, Tadeusz



Monoclonal antibodies for the separate detection of halodeoxyuridines and method for their use  


Monoclonal antibodies are described which have specific affinities for halogenated nucleoside analogs and are preferentially selective for one particular halogen. Such antibodies, when incorporated into immunochemical reagents, may be used to identify and independently quantify the cell division character of more than one population or subpopulation in flow cytometric measurements. Independent assessment of division activity in cell sub-populations facilitates selection of appropriate time and dose for administration of anti-proliferative agents. The hybridomas which secrete halogen selective antibodies and the method of making them are described.

Vanderlaan, Martin (Danville, CA); Watkins, Bruce E. (Livermore, CA); Stanker, Larry H. (Livermore, CA)



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

SciTech Connect

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

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



Development and characterisation of monoclonal antibodies reactive with porcine CSF1R (CD115).  


Macrophage colony-stimulating factor (CSF1) controls the proliferation and differentiation of cells of the mononuclear phagocyte system. CSF1, alongside a second ligand, interleukin-34 (IL-34), acts by binding to a cell surface receptor (CSF1R). We previously cloned and expressed pig CSF1 and IL-34. Here we produced a pig CSF1R-Ig+pFUSE Fc fusion protein and used it as an immunogen to produce three monoclonal antibodies (ROS8G11, ROS3A5 and ROS3B10) targeted against porcine CSF1R. Specific binding of each monoclonal antibody was confirmed by ELISA, Western blot, flow cytometry and immunocytochemistry. The antibodies did not block CSF1 signalling. The surface expression of CSF1R in pig peripheral blood was restricted to CD14-positive monocytes and was also detected on lung macrophages. These antibodies provided an opportunity to investigate the increase of available CSF1R during pig BMDM differentiation. The new monoclonal antibodies provide useful reagents to support the study of monocyte and macrophage biology in the pig. PMID:25020194

Moffat, L; Rothwell, L; Garcia-Morales, C; Sauter, K A; Kapetanovic, R; Gow, D J; Hume, D A



Development of Rabbit Monoclonal Antibodies for Detection of Alpha-Dystroglycan in Normal and Dystrophic Tissue  

PubMed Central

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. PMID:24824861

Fortunato, Marisa J.; Ball, Charlotte E.; Hollinger, Katrin; Patel, Niraj B.; Modi, Jill N.; Rajasekaran, Vedika; Nonneman, Dan J.; Ross, Jason W.; Kennedy, Eileen J.; Selsby, Joshua T.; Beedle, Aaron M.



EP1: a novel rabbit monoclonal antibody for detection of oestrogen receptor ?  

PubMed Central

Aims Assessment of hormone receptor expression is part of routine examination of every breast cancer. In this study, we report the characterisation of a novel rabbit monoclonal antibody, clone EP1, directed against oestrogen receptor (ER) ?. Additionally, its immunohistochemical performance characteristics in archival tissues are evaluated in normal tissues and two distinct cohorts of breast cancer patients. Methods Comparative analyses between EP1 and the anti-ER? component of the ER/PR pharmDx kit (cocktail of mouse monoclonal antibody clones 1D5 and ER-2-123) and between EP1 and another commercially available rabbit monoclonal antibody, clone SP1, are described. Results Clone EP1 specifically detects nuclear ER in all tissues examined; cytoplasmic staining was not observed. The analysis shows a high degree of concordance (?95%) between EP1 and both the ER? component of the Dako ER/PR pharmDx kit and Ventana clone SP1. However, the use of EP1 antibody together with Dako EnVision FLEX detection system resulted in a stronger staining intensity as compared with SP1 antibody using the Ventana ultraView DAB detection system resulting in better ‘ease of use.’ Conclusions The use of EPI can result in better interpretation of the results of the ER analysis. PMID:23894168

Badve, Sunil; Vladislav, I Tudor; Spaulding, Betsy; Strickland, Anna; Hernandez, Sylvia; Bird-Turner, Lisa; Dodson, Cecelia; Elleby, Bjorn; Phillips, Therese



New monoclonal-antibody two-site solid-phase immunoradiometric assay for human thyrotropin evaluated  

SciTech Connect

The authors compared results with a commercial solid-phase two-site immunoradiometric assay kit for human thyrotropin in which monoclonal antibodies are used with those by our radioimmunoassay, which is optimized for measurement of low concentrations of thyrotropin. In the immunoradiometric assay a specific antibody to the beta subunit of human thyrotropin is immobilized on a polystyrene bead, and a radiolabeled monoclonal antibody directed against the alpha subunit provides a measure of bead-immobilized hormone. The mean thyrotropin concentrations in 70 euthyroid serum samples were similar in the two assays. Values for hypothyroid patients were clearly higher in both assays than values for euthyroid individuals. In commercial assays the major source of error in measurement of thyrotropin response to thyroliberin in terms of the increment over the basal concentration of thyrotropin has been systematic errors in the measurement of those basal concentrations. With the present assay, however, basal values are obtained with good precision and accuracy.

Pekary, A.E.; Hershman, J.M.



Purification of bovine thyroid-stimulating hormone by a monoclonal antibody  

SciTech Connect

A monoclonal antibody directed against bovine TSH was obtained by hybridoma technology. This antibody was specific for TSH and did not react with bovine LH and FSH. Affinity chromatography of crude TSH was performed on anti-TSH Sepharose. Bovine TSH was purified in a single step to near homogeneity by this technique, as shown by cation exchange chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified TSH. The biological activity of the hormone was not affected during the purification, as determined by (/sup 3/H)thymidine incorporation of the TSH-dependent FRTL5 cell line. The results indicate that affinity purification of TSH by means of a monoclonal antibody is a simple one-step procedure for the production of biologically active, highly purified TSH.

Lock, A.J.; van Denderen, J.; Aarden, L.A.



Monoclonal Antibodies to the Apical Chloride Channel in Necturus Gallbladder Inhibit the Chloride Conductance  

NASA Astrophysics Data System (ADS)

Monoclonal antibodies raised by injecting Necturus gallbladder cells into mice were tested for their ability to inhibit the apical chloride conductance induced by elevation of cellular cAMP. Five of these monoclonal antibodies bound to the apical cells, as shown by indirect immunofluorescence microscopy, and inhibited the chloride conductance; one antibody that bound only to subepithelial smooth muscle, by indirect immunofluorescence microscopy, showed no inhibition of chloride transport. The channel or a closely related molecule is present in the membrane whether or not the pathway is open, since, in addition to inhibiting the conductance of the open channel, the antibody also bound to the membrane in the resting state and prevented subsequent opening of the channel. The antibody was shown to recognize, by ELISA, epitopes from the Necturus gallbladder and small intestine. Finally, by Western blot analysis of Necturus gallbladder homogenates, the antibody was shown to recognize two protein bands of Mr 219,000 and Mr 69,000. This antibody should permit isolation and characterization of this important ion channel.

Finn, Arthur L.; Tsai, Lih-Min; Falk, Ronald J.



Immuno-histological diagnosis of lymphoproliferative diseases by selected combinations of antisera and monoclonal antibodies.  


Tissue sections of frozen biopsy specimens obtained from normal and hyperplastic human lymphoid tissues, 33 cases of non-Hodgkin lymphomas as well as various forms of immunoregulatory disorders (angioimmunoblastic and dermatopathic lymphadenopathy) were analysed in immunofluorescence tests (using red TRITC and green FITC double-labelling). A panel of antisera including well-characterized conventional reagents to immunoglobulin classes, T lymphoid and Ia-like antigens, and monoclonal antibodies was used. In selected cases the results were compared with the observations of membrane-marker staining on viable cells in suspension. the findings show that the immunological methods can give a very accurate analysis of the normal and malignant lymphoid cells, and can provide complementary information to conventional histology. The investigator can choose the reagent combinations which give answers to various specific questions: e.g. antisera to light chains establish the monoclonality of lymphomas, whilst staining combinations for human T and Ia-like antigens are particularly useful in various immunoregulatory disorders. Monoclonal antibodies will be particularly useful in various immunoregulatory disorders. Monoclonal antibodies will be particularly useful reagents for analysing the tissue distribution of lymphoid subpopulations and ancillary cells in tissue biopsy specimens. PMID:6775656

Janossy, G; Thomas, J A; Pizzolo, G; Granger, S M; McLaughlin, J; Habeshaw, J A; Stansfeld, A G; Sloane, J



Monoclonal antibodies specific for the carboxy terminus of simian virus 40 large T antigen.  


Three mouse hybridomas secreting antibodies against the undecapeptide Lys-Pro-Pro-Thr-Pro-Pro-Pro-Glu-Pro-Glu-Thr, corresponding to the carboxy terminus of simian virus 40 large T antigen, were isolated and cloned. A sensitive enzyme-linked immunosorbent assay was used to characterize the properties of the monoclonal antibodies. All three hybridomas, designated KT1, KT3, and KT4, produced antibodies that immunoprecipitated large T. The antibodies differed in their affinities for the peptide and for the native protein. Antibodies from KT3 precipitated large T better than those from KT1 or KT4. KT3 antibodies also had the highest affinity for the free peptide (5.2 X 10(6) M-1) as determined by radioimmunoassay; KT1 and KT4 antibodies had ca. 5- and 1,000-fold lower affinities, respectively. Inhibition studies with shorter peptides, overlapping the undecapeptide, revealed the approximate regions recognized by the different monoclonal antibodies. KT3 antibodies bound to a region within the carboxy-terminal six amino acids of large T. Antibodies from KT1 and KT4 reacted with sequences located further towards the amino terminus of the undecapeptide. Surprising results were obtained with KT4 antibodies. Their binding to the undecapeptide was completely inhibited by the undecapeptide itself or the carboxy-terminal hexapeptide. The carboxy-terminal pentamer, on the other hand, slightly enhanced binding, and the carboxy-terminal tetramer, Glu-Pro-Glu-Thr, was strongly stimulatory. A model for this effect is proposed. Using the enzyme-linked immunosorbent assay, we confirmed previous studies (W. Deppert and G. Walter, Virology 122:56-70, 1982) which found that antiserum against sodium dodecyl sulfate-denatured large T reacts strongly with the carboxy terminus of large T. By inhibition studies, we identified the approximate region within the undecapeptide recognized by anti-sodium dodecyl sulfate-denatured large T and compared this region with the region identified by antipeptide serum. PMID:6208378

MacArthur, H; Walter, G



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

SciTech Connect

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

Eisenberg, R.J. (Univ. of Pennsylvania, Philadelphia, PA); Long, D.; Pereira, L.; Hampar, B.; Zweig, M.; Cohen, G.H.



Heterobifunctional reagents: A new approach to radiolabeling of monoclonal antibodies  

Microsoft Academic Search

The use of bifunctional chelate such as the cyclic anhydride of DTPA for radiolabeling antibodies (Abs) may lead to homopolymerization, and intra- or intermolecular cross-linking, with resulting denaturation and decrease immunoreactivity of Abs. The authors, therefore, investigated the use of heterobifunctional reagents, whereby one group selectively couples to the amino group of the Ab and the other group to the

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



Reagent Target Request for Monoclonal Antibody Production and Characterization

Requests will be reviewed and considered for merit based on their justification and contribution to existing NCI-funded projects. Priority will be given to projects applying the antibodies to proteomic research. Requests should be submitted electronically by completing the accompanying form.


Radioimmunodetection of prostate cancer by 111In-labeled monoclonal antibody against prostatic acid phosphatase.  


Purified human prostate acid phosphatase (PAP) was used to generate a specific monoclonal antibody (FC 3001) for detection of PAP expressed by some prostatic carcinomas. DTPA derivatives of MoAb-F(ab')2-fragments were labeled with indium-111 chloride. This labeled antibody was tested in 15 prostate cancer patients who underwent staging pelvic lymphadenectomy; 9 of them received labeled antibody alone whereas 6 received simultaneous injections of labeled and unlabeled antibody with two dose levels (40 or 80 mg). Biodistribution data obtained by direct blood measurements and imaging procedures indicated that simultaneous injection of unlabeled antibody reduced both the blood elimination rate and the accumulation in the liver. Accumulation of the radionuclide in pelvic lymph node metastases was observed in some patients but in a couple of patients accumulation was noted also in normal lymph nodes. The method cannot in its present design replace staging pelvic lymphadenectomy and further studies are needed for elaboration of clinically useful radioimmunodetection methods. PMID:8305218

Ahonen, A; Kairemo, K; Karnani, P; Heikkilä, J; Nurmi, M; Teräs, M; Lukkarinen, O



Specificity mapping of human anti-T cell receptor monoclonal natural antibodies: defining the properties of epitope recognition promiscuity  

Microsoft Academic Search

The classical concept of antibody bind- ing is defined as an exclusive and high-affinity interac- tion with one epitope. The emerging reality about antibody combing sites, however, is that some can bind unrelated determinants. The studies presented here define this quality as epitope recognition promiscuity by analyzing the capacity of monoclonal human auto- antibodies to bind sets of overlapping peptides




Radioimmunoassay of circulating schistosome antigen with a radiation-immobilized monoclonal antibody : Preliminary results  

NASA Astrophysics Data System (ADS)

A two-site immunoradiometric assay with a mouse monoclonal antibody to a circulating schistosome antigen was comparatively investigated using the monoclonal antibody either absorbed to microtiter plates (reference IRMA) or immobilized by several techniques. Radiation polymerization methods were carried out at Takasaki Radiation Chemistry Research Establishment, Takasaki, Gunma (I. Kaetsu, M. Kumakura), using 2-hydroxyethyl methacrylate monomers and 1 Mrad irradiation. A significant correlation was obtained with the reference IRMA and the assay using radiation polymerization-immobilized antibody ( r = 0.94), although non-specific binding to the polymer discs was higher (x 10) than with microtiter plates. Immobilization of the monoclonal antibody onto polypropylene/polyethylene copolymer films grafted with methacrylic acid irradiated at 0.68 Mrads and treated with carbodiimide/N-hydroxysuccinimide, was carried out at the Dept of Bioengineering, University of Washington, Seattle, Washington (A.S. Hoffman, W.R. Gombotz, S. Uenoyama). A significant correlation ( r = 0.90) was obtained with the reference IRMA. Non-specific binding was also higher than with microtiter plates (x 6). An important result was the increased shelf life of the immobilized reagent.

Dessaint, J. P.; Nogueira-Queiroz, J. A.; Capron, A.


Discovery of a chemical modification by citric acid in a recombinant monoclonal antibody.  


Recombinant therapeutic monoclonal antibodies exhibit a high degree of heterogeneity that can arise from various post-translational modifications. The formulation for a protein product is to maintain a specific pH and to minimize further modifications. Generally Recognized as Safe (GRAS), citric acid is commonly used for formulation to maintain a pH at a range between 3 and 6 and is generally considered chemically inert. However, as we reported herein, citric acid covalently modified a recombinant monoclonal antibody (IgG1) in a phosphate/citrate-buffered formulation at pH 5.2 and led to the formation of so-called "acidic species" that showed mass increases of 174 and 156 Da, respectively. Peptide mapping revealed that the modification occurred at the N-terminus of the light chain. Three additional antibodies also showed the same modification but displayed different susceptibilities of the N-termini of the light chain, heavy chain, or both. Thus, ostensibly unreactive excipients under certain conditions may increase heterogeneity and acidic species in formulated recombinant monoclonal antibodies. By analogy, other molecules (e.g., succinic acid) with two or more carboxylic acid groups and capable of forming an anhydride may exhibit similar reactivities. Altogether, our findings again reminded us that it is prudent to consider formulations as a potential source for chemical modifications and product heterogeneity. PMID:25136741

Chumsae, Chris; Zhou, Liqiang Lisa; Shen, Yang; Wohlgemuth, Jessica; Fung, Emma; Burton, Randall; Radziejewski, Czeslaw; Zhou, Zhaohui Sunny



Murine monoclonal antibodies to type Ib polysaccharide of group B streptococci bind to human milk oligosaccharides.  

PubMed Central

The chemical structures of the repeating units of the type Ib polysaccharide of group B streptococci and of the desialylated form of this antigen are almost identical to those of some oligosaccharides in human milk and certain fetal antigens. The structural similarities suggested that the molecules may be immunologically cross-reactive. Mouse monoclonal antibodies to the sialylated and nonsialylated forms of the type Ib polysaccharide were produced and tested for their ability to bind to immobilized human milk oligosaccharides. One antibody, SMB19, reacted specifically with the sialylated form of the type Ib polysaccharide and was also bound by an affinity column containing immobilized sialyllacto-N-tetraose a. The antibody was eluted from the affinity column with EDTA, since its binding to the antigen was calcium dependent. A second monoclonal antibody. SIbD2, bound specifically to the nonsialylated form of the type Ib polysaccharide and also to immobilized lacto-N-tetraose. The antibody was eluted from the affinity column at an acidic pH and retained immunologic activity. These results further extend our previous observations that certain antibodies raised against group B streptococci can also react with normal human glycoconjugates. Images PMID:1548081

Pritchard, D G; Gray, B M; Egan, M L



Immunochemical identification of three proteinic determinants on the archaebacterium Methanococcus vannielii by monoclonal antibodies  

SciTech Connect

Archaebacteria evolved independently from Eubacteria and Eukaryotes and present unique biochemical features. One group, the methanogens, show a variety of cell-walls paralleling their own evolutionary diversity. Chemical analyses revealed proteins in the cell-wall of Methanococcacea but no murein or pseudomurein. Immunochemical studies were begun using a panel of monoclonal antibodies against M. vannielii SB and quantitative micro-immunoenzymatic methods developed for this purpose. Direct binding and inhibition-blocking assays using sugars and aminosugars also indicated absence of these residues in the SB's cell-wall. Three antibodies, however, recognized three different antigenic determinants involving amino acids: Lys, Phe and Thr (antibody 5A); Ser, Try and Tyr (antibody 5B); and Arg, Lys and Phe (antibody 5F). Each site was recognized by one antibody only, and vice versa. These results agree with those provided by well established chemical methods and support the notion that SB possesses a peculiar cell-wall with proteins only. The data also show the resolution power and reliability of monoclonal probes for structural analysis of the bacterial envelope.

de Macario, E.C.; Macario, A.J.L.; Magarinos, M.C.



Rat monoclonal antitubulin antibodies derived by using a new nonsecreting rat cell line  

PubMed Central

Hybrid myeloma cell lines secreting monoclonal antibodies to tubulin have been prepared using rat myelomas and spleen cells from rats immunized with yeast tubulin. A comparison between the results obtained with the rat myeloma Y3-Ag 1.2.3., which secretes a light chain, and a new line, YB2/O, which does not, shows that they are both excellent parental lines and that the second produces hybrids with no myeloma chain components. The antitubulin antibodies in the serum of rats bearing two of the hybrid myeloma tumors gave titers of up to 1:10(6) from which large amounts of monoclonal antibodies could be easily purified. They recognized tubulin from yeast as well as from birds and mammals. The two antibodies gave clear immunofluorescent staining of yeast mitotic spindles as well as the interphase microtubule network of tissue culture cells. Some difference in the pattern of immunofluorescence staining of yeast cells and nuclei was observed between the two antibodies. The purified antibodies could be conjugated to colloidal gold particles and used for direct labeling of yeast microtubules for electron microscopy. PMID:6811596



Monoclonal antibodies to cytomegalovirus: rapid identification of clinical isolates and preliminary use in diagnosis of cytomegalovirus pneumonia.  

PubMed Central

Two monoclonal antibodies which react specifically with cells infected by cytomegalovirus (CMV) are described. One antibody, 6-E3, reacts with a 72,000-dalton protein that appears early in infection and remains localized in the cell nucleus. The other antibody, 6-C5, reacts with an 80,000-dalton protein that appears late in infection and remains localized in cytoplasmic inclusion bodies. Both monoclonal antibodies react with conventional laboratory strains of CMV and can be used in immunofluorescence assays to identify clinical isolates of CMV in culture. Preliminary tests on lung tissues from patients with CMV pneumonia show that only antibody 6-C5 detects CMV infection in primary clinical specimens. A comparison of culture, histological, and immunological methods demonstrates that the monoclonal antibodies possess sufficient specificity and sensitivity to warrant their continued development as immunodiagnostic tools for the detection of CMV infection in both tissue culture and tissues obtained directly from patients. Images PMID:6292094

Goldstein, L C; McDougall, J; Hackman, R; Meyers, J D; Thomas, E D; Nowinski, R C



Isolation and characterization of monoclonal antibodies to cytoskeletal and membrane proteins of the Paramecium cortex.  


Monoclonal antibodies have been generated against whole cortical fragments of Paramecium to provide tools for the analysis of cortex morphogenesis as well as for the biochemical dissection of this complex membrane-cytoskeleton structure. Of 80 positive hybridomas in ELISA tests, 17 monoclonal were characterized by immunoblotting, immunofluorescence tests on sectioned as well as on Triton-permeabilized cells (including confocal microscopy), and by EM immunocytochemistry on permeabilized cells. Five classes of monoclonals were obtained directed, respectively, against the epiplasm (or elements tightly associated with it), the trichocysts, the microtubules, the surface membranes, and poorly defined intracellular antigens. Of these, the newest and most promising appear to be a set of monoclonals decorating both intensely and sharply some specific parts of the epiplasm (outer periphery, outer central part or core). These antibodies therefore provide the first demonstration of a molecular heterogeneity of composition at the level of individual epiplasmic scales in Paramecium. In addition, they offer powerful tools to follow the biogenesis of these structures during cell division. Finally, they have allowed the identification of a number of previously uncharacterized protein components of the cortex. PMID:23195645

Jeanmaire-Wolf, R; Clérot, J C; Nahon, P; Iftode, F; Fleury, A; Adoutte, A



Fine Specificity, Idiotypy, and Nature of Cloned Heavy-Chain Variable Region Genes of Murine Monoclonal Rheumatoid Factor Antibodies  

Microsoft Academic Search

We investigated the immunochemical and molecular characteristics of murine monoclonal rheumatoid factors. Study of the fine specificity of 20 monoclonal rheumatoid factor antibodies shows a wide degree of heterogeneity. However, many express an interstrain cross-reactive idiotype. We show that our rheumatoid factors utilize a restricted set of the heavy-chain variable region (VH) repertoire representing the more 3' VH families. Preferential

Audrey J. Manheimer-Lory; Marc Monestier; Blanche Bellon; Frederick W. Alt; Constantin A. Bona



Production and characterization of monoclonal antibodies specific for Shewanella colwelliana exopolysaccharide.  

PubMed Central

Six monoclonal antibodies were produced to whole cells of Shewanella colwelliana (Aco1 to Aco6) and two (Aco22 to Aco23) to purified exopolysaccharide (EPS). Aco1, -4 to -6, -22, and -23 bound to both the cell surface and the purified EPS, while Aco2 and -3 bound to cells only. The EPS of S. colwelliana was antigenically unique from those of nine other species of marine bacteria that were tested. Mapping studies revealed that all of the EPS-specific monoclonal antibodies bound to the same epitope. This EPS epitope was sensitive to cleavage of ester bonds, but neither pyruvate, acetate, nor terminal nonreducing sugars were required for antigenicity. When S. colwelliana was grown on rich media, most of its EPS was loosely associated with the cell surface. Images PMID:7686001

Sledjeski, D D; Weiner, R M



Demonstration of antigenic variation among rabies virus isolates by using monoclonal antibodies to nucleocapsid proteins.  

PubMed Central

Rabies virus isolates from terrestrial animals in six areas of the United States were examined with a panel of monoclonal antibodies to nucleocapsid proteins. Characteristic differences in immunofluorescence reactions permitted the formation of four antigenically distinct reaction groups from the 231 isolates tested. The geographic distribution of these groups corresponded well with separate rabies enzootic areas recognized by surveillance of sylvatic rabies in the United States. Distinctive reaction patterns were also identified for viral proteins from four infected bat species, and identical patterns were found in eight isolated cases of rabies in terrestrial animals. These findings suggest that monoclonal antibodies can be used to study the prevalence, distribution, and transmission of rabies among wildlife species. PMID:2429983

Smith, J S; Reid-Sanden, F L; Roumillat, L F; Trimarchi, C; Clark, K; Baer, G M; Winkler, W G



Monocyte/macrophage-reactive monoclonal antibody Ki-M6 recognizes an intracytoplasmic antigen.  

PubMed Central

A monoclonal antibody, termed Ki-M6, is described, which shows a restricted reactivity to cells of the monocyte/macrophage system. On light- and electron-microscopic immunoperoxidase staining Ki-M6 recognizes monocytes and the phagocytosing compartment of macrophages residing in different tissue sites; granulocytes and the so-called immune accessories of B- and T-cell immune response as closely monocyte/macrophage related cell populations do not reveal any reactivity. This is shown by comparison with the monoclonal antibodies Ki-M4 and Ki-M1 or OKT6 recognizing immune accessory cells by immunohistochemical methods. Ki-M6 binds to a lysosomal membrane-restricted antigen of 60,000 daltons without influencing significantly lysosome-related functions as far as the chemiluminescence response is concerned. Images Figure 2 Figure 4 Figure 1 Figure 3 Figure 6 PMID:3777131

Parwaresch, M. R.; Radzun, H. J.; Kreipe, H.; Hansmann, M. L.; Barth, J.



Synergistic neutralization of human immunodeficiency virus type 1 by combinations of human monoclonal antibodies.  

PubMed Central

The ability of antibodies to the V3 region and the CD4-binding domain (CD4bd) of human immunodeficiency virus type 1 (HIV-1) to act in synergy to neutralize HIV has been demonstrated previously. However, synergy between antibodies to other HIV-1 epitopes has not been studied. We have used 21 combinations of human monoclonal antibodies (MAbs) directed against different epitopes of the gp120 and gp41 proteins of HIV-1 to evaluate their ability to act in synergy to neutralize HIV-1. Combinations of anti-V3 and anti-CD4bd antibodies, anti-V3 and anti-gp120 C-terminus antibodies, anti-CD4bd and anti-C-terminus antibodies, anti-V3 and anti-gp41 antibodies, and anti-CD4bd and anti-gp41 antibodies were tested. Our results show that some, but not all anti-V3 antibodies can act in synergy with anti-CD4bd antibodies. In addition, for the first time, antibodies to the C-terminus region have been found to act in synergy with the anti-CD4bd antibodies. Various anti-CD4bd MAbs also act in synergy when used together. The use of such cocktails of human MAbs for passive immunization against HIV-1 may prove to be important for therapy in postexposure settings and for prevention of maternal-fetal transmission of the virus. The results also provide information on the types of antibodies that should be elicited by an effective vaccine. PMID:7514683

Laal, S; Burda, S; Gorny, M K; Karwowska, S; Buchbinder, A; Zolla-Pazner, S



Evaluating delivery of a monoclonal antibody using a linear Lorentz-force actuated needle-free injector  

E-print Network

The medical application of injection of monoclonal antibodies using a controllable auto-loading needle-free jet injector has been evaluated for two potentially limiting factors: viscosity of the formulation and shearing ...

Jin, Tiffany



Application of phase partitioning and thiophilic adsorption chromatography to the purification of monoclonal antibodies from cell culture fluid.  


A two-step method for the isolation of an IgG1 monoclonal antibody against horseradish peroxidase from hybridoma cell culture supernatant is described. Purification was achieved using an aqueous two-phase extraction procedure in conjunction with thiophilic adsorption chromatography. In an aqueous two-phase system composed of 5% PEG 1540 and 22% phosphate the monoclonal antibody preferentially associates with the PEG-rich top phase whereas proteins such as albumin and transferrin partition into the salt-rich bottom phase. Final purification of the monoclonal antibody was achieved by subjecting the PEG-rich top phase to thiophilic adsorption chromatography. The monoclonal antibody purified to homogeneity retained its specificity for horseradish peroxidase as revealed by polyacrylamide gel electrophoresis and an enzyme-linked immunosorbent assay. The potential of this purification protocol for large scale applications is discussed. PMID:1593132

Sulk, B; Birkenmeier, G; Kopperschläger, G



Murine Monoclonal Anti-Idiotope Antibody Breaks Unresponsiveness and Induces a Specific Antibody Response to Human Melanoma-Associated Proteoglycan Antigen in Cynomolgus Monkeys  

Microsoft Academic Search

The mouse monoclonal antibody MEM136 (mAb1) is directed against an epitope on human melanoma-associated proteoglycan antigen (MPG). This epitope is also present on various normal human and subhuman tissues. A monoclonal murine anti-idiotope (anti-Id) antibody (mAb2), designated I-Mel-2, was generated against MEM136 and used as a surrogate antigen for the MPG molecule. I-Mel-2 was tested in cynomolgus monkeys (Macaca fascicularis)

Panchanon Chattopadhyay; Jean Starkey; W. John W. Morrow; Syamal Raychaudhuri



In vitro photothermal destruction of neuroblastoma cells using carbon nanotubes conjugated with GD2 monoclonal antibody  

Microsoft Academic Search

Despite aggressive multimodality therapy, most neuroblastoma-bearing patients relapse and survival rate remains poor. Exploration of alternative therapeutic modalities is needed. Carbon nanotubes (CNTs), revealing optical absorbance in the near-infrared region, warrant their merits in photothermal therapy. In order to specifically target disialoganglioside (GD2) overexpressed on the surface of neuroblastoma stNB-V1 cells, GD2 monoclonal antibody (anti-GD2) was conjugated to acidified CNTs.

Chung-Hao Wang; Yao-Jhang Huang; Chia-Wei Chang; Wen-Ming Hsu; Ching-An Peng



The Airways, a Novel Route for Delivering Monoclonal Antibodies to Treat Lung Tumors  

Microsoft Academic Search

Purpose  Lung cancer is the leading cause of cancer-related death worldwide. The efficacy of current systemic treatments is limited,\\u000a with major side effects and only modest survival improvements. Aerosols routinely used to deliver drugs into the lung for\\u000a treating infectious and inflammatory lung diseases have never been used to deliver monoclonal antibodies to treat lung cancer.\\u000a We have shown that cetuximab,

Agnès Maillet; Laurent Guilleminault; Etienne Lemarié; Stéphanie Lerondel; Nicolas Azzopardi; Jérôme Montharu; Nicolas Congy-Jolivet; Pascale Reverdiau; Brigitte Legrain; Christelle Parent; Dominique-Henri Douvin; José Hureaux; Yves Courty; Michèle De Monte; Patrice Diot; Gilles Paintaud; Alain Le Pape; Hervé Watier; Nathalie Heuzé-Vourc’h


Further characterization of the binding properties of two monoclonal antibodies recognizing human Tn red blood cells  

Microsoft Academic Search

Summary The terminal a anomeric GalNAc residue is an essential sugar for the Tn glycotope, human blood group A determinant, and Forssman antigen. In a previous study [King M.J., Parson S.F., Wu A.M., Jones N., Transfusion 31: 142–149, 1991], we defined two monoclonal antibodies (MoAbs, BRIC66 and BRIC111) reacting with human Tn red blood cells. However, more advanced studies of

Albert M. Wua; June H. Wub; Hsiang-Wei Kuoa; Anthony Herpa



Binding and signalling properties of a growth hormone enhancing monoclonal antibody  

Microsoft Academic Search

We have used a sequential, qualitative biosensor based assay to demonstrate that OA15, a monoclonal antibody which enhances in vivo the activity of bovine growth hormone (bGH) does not disrupt the interaction between bGH and its cognate receptor (as represented by recombinant bovine GH binding protein -rbGHBP). We have confirmed this using a classical cell-based radio-receptor assay with the GH-responsive

James Beattie; Silvia Bramani; Camillo Secchi; James Mockridge



S-Antigen immunoreactivity in human pineal glands and pineal parenchymal tumors. A monoclonal antibody study  

Microsoft Academic Search

Using a four-step immunoperoxidase (PAP) method and the monoclonal antibody MAbA9-C6 (MAbA9-C6), which defines an epitope of the retinal S-antigen (S-Ag), we investigated the S-Ag immunoreactivity in human fetal, newborn, infantile and adult pineal glands and in 13 human pineal parenchymal tumors. S-Ag immunoreactivity was demonstrated in a few cells in one of the four fetal and in both infantile

E. Perentes; L. J. Rubinstein; M. M. Herman; L. A. Donoso



Specificity of polycllonal and monoclonal antibodies for the identification of Xanthomonas campestris pv. campestris  

Microsoft Academic Search

Polyclonal and monoclonal antibodies (PCAs and MCAs), produced to whole cells and flagellar extracts ofXanthomonas campestris pv.campestris (Xcc), respectively, were tested for specificity. In immunofluorescence microscopy (IF) the three PCAs tested, reacted at low dilutions with all Xcc strains, some other xanthomonads and non-xanthomonads. At higher dilutions most cross-reactivity with non-xanthomonad strains disappeared. However, the cross-reactivity with strains ofX. c.

A. A. J. M. Franken; J. F. Zilverentant; P. M. Boonekamp; A. Schots



A human monoclonal antibody against varicella-zoster virus glycoprotein III  

Microsoft Academic Search

Hybridomas producing human monoclonal antibodies (HMAbs) against varicella-zoster virus (VZV) were generated by fusing murine myeloma cells with human lymphocytes immunized in vitro. An assay system was developed to select anti-glycoprotein (gp)III HMAbs from the pool of anti-VZV HMAbs. A murine anti- gplII MAb, 4B7, did not react with a VZV-infected cell homogenate, but did react with a VZV-infected cell

Toru Sugano; Takami Tomiyama; Yoh-ichi Matsumoto; Satoshi Sasaki; Tsuyoshi Kimura; Bagher Forghani; Yasuhiko Masuho



CD34 monoclonal antibody-immobilized electrospun polyurethane for the endothelialization of vascular grafts  

Microsoft Academic Search

Targeting endothelial progenitor cells (EPC) for in vivo endothelialization is an emerging and promising approach for the development of cardiovascular medical devices. This study\\u000a examined the efficacy of capturing CD34 positive EPC onto polyurethane (PU) immobilized with CD34 monoclonal antibodies (mAbs)\\u000a (a biomecial polymer for cardiovascular devices). Electrospun PU matrices were fabricated and heparin was immobilized along\\u000a with CD34 mAb.

Yoon Ki Joung; In Kyu Hwang; Ki Dong Park; Chan Woo Lee



Monoclonal antibodies recognizing larval and pupal-specific cuticular proteins of Tenebrio molitor (Insecta, Coleoptera)  

Microsoft Academic Search

To study the sequential expression of insect epidermal cells during metamorphosis, a library of monoclonal antibodies (MABs) was prepared against the water-soluble proteins from preecdysial pupal cuticle of Tenebrio molitor. Six selected MABs recognizing only larval and pupal cuticular proteins (CPs) in immunoblot analysis were classified into three types. Type 1 recognized a 21.5 and a 22 kDa polypeptide, type

Aleth Lemoine; Claire Millot; Geneviève Curie; Valérie Massonneau; Jean Delachambre



Regulatable systemic production of monoclonal antibodies by in vivo muscle electroporation  

Microsoft Academic Search

The clinical application of monoclonal antibodies (mAbs) potentially concerns a wide range of diseases including, among others, viral infections, cancer and autoimmune diseases. Although intravenous infusion appears to be the simplest and most obvious mode of administration, it is very often not applicable to long-term treatments because of the restrictive cost of mAbs certified for human use and the side

Norma Perez; Pascal Bigey; Daniel Scherman; Olivier Danos; Marc Piechaczyk; Mireia Pelegrin



Mapping of antigenic sites on the bovine ephemeral fever virus glycoprotein using monoclonal antibodies  

Microsoft Academic Search

Monoclonal antibodies (MAbs) were produced against the G, M2 and N proteins of bovine ephemeral fever virus (BEFV) and 29 were selected for further study. Thirteen neutralizing MAbs were assigned to one conformation-independent and at least two conforma- tion-dependent antigenic sites on the G protein by a competitive binding ELISA. The panel of MAbs were tested by neutralization and immunofluorescence

D. H. Cybinski; P. J. Walker; K. A. Byrne; H. Zakrzewski



A Monoclonal Antibody against a-Smooth Muscle Actin: A New Probe for Smooth Muscle Differentiation  

Microsoft Academic Search

A monoclonal antibody (anti-ctsm-1) recog- nizing exclusively a-smooth muscle actin was selected and characterized after immunization of BALB\\/c mice with the NH2-terminal synthetic decapeptide of a-smooth muscle actin coupled to keyhole limpet hemocyanin. Anti-ctsm-1 helped in distinguishing smooth muscle cells from fibroblasts in mixed cultures such as rat dermal fibroblasts and chicken embryo fibroblasts. In the aortic media, it recognized

Omar Skalli; Patricia Ropraz; Arnold Trzeciak; Gilbert Benzonana; Dieter Gillessen; Giulio Gabbiani



The use of monoclonal antibodies for the treatment of epithelial ovarian cancer (review)  

Microsoft Academic Search

The prognosis for patients with ovarian cancer is still poor and more effective therapeutic modalities are needed. (Radio)immunotherapy using monoclonal antibodies (Mabs) could be one of these approaches. Here, we review the status of (radio)immunotherapy using Mabs for the treatment of ovarian cancer. The Pubmed database was searched for clinical trials investigating the effect of (radio)immunotherapy in ovarian cancer published

A. L. M. Oei; F. C. Sweep; C. M. G. Thomas; O. C. Boerman; L. F. A. G. Massuger



Glycan optimization of a human monoclonal antibody in the aquatic plant Lemna minor  

Microsoft Academic Search

N-glycosylation is critical to the function of monoclonal antibodies (mAbs) and distinguishes various systems used for their production. We expressed human mAbs in the small aquatic plant Lemna minor, which offers several advantages for manufacturing therapeutic proteins free of zoonotic pathogens. Glycosylation of a mAb against human CD30 was optimized by co-expressing the heavy and light chains of the mAb

Jason D Sterling; Jeffrey T Regan; John R Gasdaska; Karen K Frantz; Charles G Peele; Amelia Black; David Passmore; Cristina Moldovan-Loomis; Mohan Srinivasan; Severino Cuison; Pina M Cardarelli; Lynn F Dickey; Kevin M Cox



Immunochemical identification of three proteinic determinants on the archaebacterium Methanococcus vannielii by monoclonal antibodies  

Microsoft Academic Search

Archaebacteria evolved independently from Eubacteria and Eukaryotes and present unique biochemical features. One group, the methanogens, show a variety of cell-walls paralleling their own evolutionary diversity. Chemical analyses revealed proteins in the cell-wall of Methanococcacea but no murein or pseudomurein. Immunochemical studies were begun using a panel of monoclonal antibodies against M. vannielii SB and quantitative micro-immunoenzymatic methods developed for

E. C. de Macario; A. J. L. Macario; M. C. Magarinos



New Hematopoietic Differentiation Antigens Detected by Anti-K562 Monoclonal Antibodies1  

Microsoft Academic Search

Following immunizations of BALB\\/c mice with K562 cells, we have obtained seven original monoclonal antibodies (MoAbs): (a) One MoAb, GA3, defines an antigen essentially restricted to the red cell series. This antigen is expressed on immature erythroblasts but is not detectable on the surface of early and late erythroid progenitors. GA3 MoAb immu- noprecipitates a M, 105,000 glycoprotein on K562

Marie Christine Dokhelar; Yvonne Finale; Nelly Kieffer; Jeanine Breton-Gorius; William Vainchenker; Thomas Tursz



Radioimmunoscintigraphy of ovarian tumors using a new monoclonal antibody, SM3  

Microsoft Academic Search

Radioimmunoscintigraphy with SM-3 monoclonal antibody produces results similar to those obtained with ¹²³I-labeled HMFG-2. The tumor specificity of SM-3 in vitro is not as marked as that in vivo. SM-3 is, however, much easier to produce in tissue culture and, with the availability of {sup 99m}Tc labelling, has allowed radioimmunoscintigraphy to be done as an outpatient procedure. This technique has

T. W. Jobling; M. Granowska; K. E. Britton; D. G. Lowe; S. J. Mather; J. Burchell; M. Naeem; J. H. Shepherd



lmmunolocalization of a Unique Form of Maize Kernel Glutamine Synthetase Using a Monoclonal Antibody  

Microsoft Academic Search

lhe pedicel (basal maternal tissue) of maize (Zea mays 1.) kernels contains a physically and kinetically unique form of glutamine synthetase (GS,,) that is involved in the conversion of transport forms of nitrogen into glutamine for uptake by the developing endosperm (M.J. Muhitch (1989) Plant Physiol 91: 868-875). A monoclonal antibody has been raised against this kernel-specific CS that does

Michael J. Muhitch; Frederick C. Felker; Earl W. Taliercio; Prem S. Chourey


Vasculogenesis in the early quail blastodisc as studied with monoclonal-antibody recognizing endothelial-cells  

E-print Network

Development 100, 339-349 (1987) Printed in Great Britain ? The Company of Biologists Limited 1987 339 Vasculogenesis in the early quail blastodisc as studied with a monoclonal antibody recognizing endothelial cells LUC PARDANAUD 1 , CURTIS ALTMANN 2... , PAUL KITOS 3 , FRANCOISE DIETERLEN-LIEVRE 1 and CLAYTON A. BUCK 2 'Insritut d'Embryologie du College de France, el du CNRS, 94736 Nogent-sur-Mame, Cedex, France 2 Wisiar Institute, 36th Street at Spruce, Philadelphia, PA 19104, USA ^Department...

Pardanaud, L.; Altmann, C.; Kitos, Paul A.; Dieterlenlievre, F.; Buck, C. A.



[Canakinumab, a monoclonal antibody against IL-1?, with potential utility in different inflammatory processes].  


Canakinumab is a fully human monoclonal antibody targeted at IL-1? which has shown to be effective in the control the symptoms of patients affected by CAPS and other autoinflammatory diseases. Its effect is rapid and sustained. In clinical trials conducted up until now, the most common adverse effects reported with the use of this drug have been different types of infections, migraines and vertigo. PMID:21596185

Carné, Xavier



Studies on the regulation of 1-aminocyclopropane-1-carboxylate synthase in tomato using monoclonal antibodies  

Microsoft Academic Search

A partially purified preparation of 1-aminocyclopropane-1-carboxylate (ACC) synthase (EC from tomato (Lycopersicon esculentum (Mill.) fruit tissue was used to generate monoclonal antibodies (MAb) specific for the two different MAbs yielded a 50-kDa polypeptide as shown by sodium dodecylsulfate-polyacrylamide gel electrophoresis. An enzyme-linked immunosorbent assay (ELISA) capable of detecting 35S]methionine followed by extraction and immunopurification in the presence of various

Anthony B. Bleecker; Gail Robinson; Hans Kende



Detection and identification of a serine to arginine sequence variant in a therapeutic monoclonal antibody  

Microsoft Academic Search

Sequence variants, also known as unintended amino acid substitutions in the protein primary structure, are one of the critical quality attributes needed to be monitored during process development of monoclonal antibodies (mAbs). Here we report on analytical methods for detection and identification of a sequence variant in an IgG1 mAb expressed in Chinese hamster ovary (CHO) cells. The presence of

Diya Ren; Jian Zhang; Ross Pritchett; Hongbin Liu; Jennifer Kyauk; Jun Luo; Ashraf Amanullah



Monoclonal Antibodies Reveal Receptor Specificity among G-Protein-Coupled Receptor Kinases  

Microsoft Academic Search

Guanine nucleotide-binding regulatory protein (G protein)-coupled receptor kinases (GRKs) constitute a family of serine\\/threonine kinases that play a major role in the agonist-induced phosphorylation and desensitization of G-protein-coupled receptors. Herein we describe the generation of monoclonal antibodies (mAbs) that specifically react with GRK2 and GRK3 or with GRK4, GRK5, and GRK6. They are used in several different receptor systems to

Martin Oppermann; Maria Diverse-Pierluissi; Mark H. Drazner; Sara L. Dyer; Neil J. Freedman; Karsten C. Peppel; Robert J. Lefkowitz



Factors Affecting Passive Monoclonal Antibody Therapy of Moloney Sarcoma in BALB\\/c Mice1  

Microsoft Academic Search

We have developed a syngeneic monoclonal antibody (MoAb) (244-19A) which retards growth and contributes to cures of BALB\\/c mice bearing Moloney sarcoma cell (MSC) tumors (S. J. Kennel, T. Lankford, and K. M. Flynn, Cancer Res., 43: 2843- 2847, 1983). The 244-19A epitope has not been detected in normal tissue or in any cultured cell (other than MSC) tested, including

Stephen J. Kennel; P. K. Lankford; K. M. Flynn; R. Winegar


Epitope-defined monoclonal antibodies against type-IV collagen for diagnosis of Alport's syndrome  

Microsoft Academic Search

chains of type-IV collagen (1-3 ). The genes of the Background. Alport's syndrome can be diagnosed by a3( IV ) and a4 (IV ) chains are on chromosome 2 in a staining the a5 chain of type IV collagen in kidney head-to-head fashion (4 ), whereas the gene for the biopsy specimens with a monoclonal antibody. Because a5( IV )

M. Kagawa; Y. Kishiro; I. Naito; T. Nemoto; H. Nakanishi; Y. Ninomiya; Y. Sado



Potency test of inactivated Newcastle disease vaccines by monoclonal antibody blocking ELISA  

Microsoft Academic Search

The potency of 27 different inactivated Newcastle disease (ND) vaccines was determined by immunization and challenge tests. Three- and four-week-old SPF chickens were immunized with 1\\/50 dose of vaccines. The chickens were bled and challenged three weeks later. The protected\\/challenged ratio was determined. Serum samples were tested by the haemagglutination inhibition (HI) test and by a monoclonal antibody (MAB) blocking

E. Horváth; G. Czifra; E. Nagy; B. Engström; M. Mérza



Inhibition of kinesin-driven microtubule motility by monoclonal antibodies to kinesin heavy chains  

Microsoft Academic Search

Abstract, We have prepared and characterized seven mouse,monoclonal,antibodies (SUK 1-7) to the 130-kD heavy chain of sea urchin egg kinesin. On immuno- blots, SUK 3 and SUK 4 cross-reacted with Drosoph- ila embryo ll6-kD heavy chains, and SUK 4, SUK 5, SUK 6, and SUK 7 bound to the 120-kD heavy chains of bovine brain kinesin. Three out of seven

Amie L. Ingold; Stanley A. Cohn; Jonathan M. Scholey



An overview of the current clinical use of the anti-CD20 monoclonal antibody rituximab  

Microsoft Academic Search

The chimeric anti-CD20 monoclonal antibody rituximab has become part of the standard therapy for patients with non-Hodgkin's lymphoma (NHL). To date, more than 300 000 patients have been treated with rituximab worldwide, including patients with indolent and aggressive NHL, Hodgkin's disease and other B-cell malignancies. Combination of rituximab with cytotoxic agents or cytokines has been explored in a number of

J. Boye; T. Elter; A. Engert



Targeting cancer cells using PLGA nanoparticles surface modified with monoclonal antibody  

Microsoft Academic Search

Targeting drugs to their sites of action is still a major challenge in pharmaceutical research. In this study, polylactic-co-glycolic acid (PLGA) immuno-nanoparticles were prepared for targeting invasive epithelial breast tumour cells. Monoclonal antibody (mAb) was used as a homing ligand and was attached to the nanoparticle surface either covalently or non-covalently. The presence of mAb on the nanoparticle surface, its

Petra Kocbek; Nataša Obermajer; Mateja Cegnar; Janko Kos; Julijana Kristl



Phytochrome in photosynthetically competent plants characterization by monoclonal antibodies. Progress report  

SciTech Connect

Detailed information concerning the physicochemical properties of phytochrome is sought, but since only trace quantities are present in plant tissues, it is extremely labile to modification in crude plant extracts, efficient and sensitive methods for its purification and characterization will be required. Towards this end immune serums directed towards oat phytochrome have been prepared. Unfortunately the phytochrome in green oats is immunochemically distinct from phytochrome in etiolated oats. Consequently, effort has been directed at preparation of monoclonal antibodies for green-oat phytochrome.

Pratt, L.H.



Radiometric assay for direct quantitation of rat liver cytochrome P-450b using monoclonal antibodies.  


A simple and sensitive assay has been developed that is capable of detecting as little as 0.2 ng of the major isozyme of cytochrome P-450 (P-450b) isolated from the livers of phenobarbital-induced rats. This assay employs monoclonal antibodies generated against cytochrome P-450b to directly quantify the levels of this enzyme in various tissues. Separation of bound from free labeled antibody is achieved by using 6,9-diaminoacridine lactate (Rivanol). The useful range of the assay is between 1 and 100 ng of P-450b. PMID:3935002

Rothwell, C E; Khazaeli, M B; Bernstein, I A



Reactivity of monoclonal antibodies to Rickettsia rickettsii with spotted fever and typhus group rickettsiae.  

PubMed Central

Analysis of 15 spotted fever group (SFG) and 2 typhus group strains of rickettsiae with a panel of monoclonal antibodies revealed a number of shared and unique epitopes of the 120- and 155-kilodalton surface proteins. All of the SFG strains but neither of the typhus group strains reacted with antibody to the lipopolysaccharidelike antigen of Rickettsia rickettsii; possibly the lipopolysaccharidelike antigen is the common antigen which defines the SFG. North Carolina and Montana strains of R. rickettsii known to differ slightly in virulence for guinea pigs differed in at least one epitope of the 120-kilodalton protein. Images PMID:2432081

Anacker, R L; Mann, R E; Gonzales, C



The DA6-147 monoclonal antibody raised against the HLA-DR? chain identifies a cryptic epitope  

E-print Network

of BoLA molecules with monoclonal antibodies raised against 2 different major histocompatibility class characterized an epitope con- served on BoLA- and HLA-DRa chains. This epitope, not accessible on intact cellsLA-DQ monoclonal antibody. BoLA / MHC / class II product / bovine Résumé ― Mise en évidence d'un épitope

Paris-Sud XI, Université de


A human monoclonal antibody specific to placental alkaline phosphatase, a marker of ovarian cancer.  


Placental alkaline phosphatase (PLAP) is a promising ovarian cancer biomarker. Here, we describe the isolation, affinity-maturation and characterization of two fully human monoclonal antibodies (termed B10 and D9) able to bind to human PLAP with a dissociation constant (Kd) of 10 and 30 nM, respectively. The ability of B10 and D9 antibodies to recognize the native antigen was confirmed by Biacore analysis, FACS and immunofluorescence studies using ovarian cancer cell lines and freshly-frozen human tissues. A quantitative biodistribution study in nude mice revealed that the B10 antibody preferentially localizes to A431 tumors, following intravenous administration. Anti-PLAP antibodies may serve as a modular building blocks for the development of targeted therapeutic products, armed with cytotoxic drugs, radionuclides or cytokines as payloads. PMID:24247025

Ravenni, Niccolò; Weber, Marcel; Neri, Dario



Monoclonal antibodies to three separate domains on 124 kilodalton phytochrome from Avena  

SciTech Connect

Forty-six monoclonal antibodies have been prepared against 124 kilodalton phytochrome from Avena sativa cv Garry. Clones grown in mice have yielded ascites fluids wtih antibodies which bind to three distinct regions of the molecule, as visualized by immunoblot analysis of proteolytically produced peptides of the protein. One antibody group (type 1) recognizes an antigenic domain(s) that lies within 6 kilodaltons of the amino terminus of the molecule, a region critical to correct protein-chromophore interaction. The second group (type 2) binds to an antigenic site(s) present within the chromophore-containing half of the molecule that is adjacent to the domain recognized by the type 1 antibodies. The third group (type 3) recognizes an antigenic site(s) that resides in the nonchromophoric, carboxy terminal end.

Daniels, S.M.; Quail, P.H.



Characterization of monoclonal antibodies against human prorenin profragment and identification of active prorenins in plasma.  


Monoclonal antibodies were raised against a synthetic peptide (43 amino acid residues) that corresponds to the complete profragment of human prorenin. Seven monoclonal antibodies were chosen for further characterization. Two antibodies, 2-X-C1 and 4-X-E1, reacted with the middle region and C-terminus of the profragment and were isotyped IgG1. The affinity constants of these antibodies against the human profragment were 7.6 x 10(8) and 3.0 x 10(7) M-1, respectively. Immunoaffinity columns containing the antibodies 2-X-C1 and 4-X-E1, respectively, were used for the characterization of active prorenin in human plasma. This active prorenin strongly bound to the 4-X-E1 column and eluted as two separate peaks which corresponded to fully and partially active prorenin, respectively. The partially active prorenin had higher activity with a small substrate, tridecapeptide, than with a large one, angiotensinogen, although the fully active prorenin had the same renin activity irrespective of the size of the substrate. These data suggest that new forms of prorenin, active prorenin, exist in human plasma and that their active sites are completely or partially exposed to the substrates. Moreover, the active prorenin in plasma was found not only in human but also in all tested mammalians. Cross-reactivity among the profragments of mammalian plasma prorenins can be explained by conservation of the amino acid sequence (epitope) of the combining site. PMID:2691500

Ishizuka, Y; Iizumi, S; Akiyama, F; Hori, H; Poorman, R A; Lobl, T J; Murakami, K



Diagnosis of myelomonocytic and macrophage neoplasms in routinely processed tissue biopsies with monoclonal antibody KP1.  

PubMed Central

A new monoclonal antibody, KP1, against the CD68 antigen, which labels macrophages and other members of the mononuclear phagocyte lineage in routinely processed tissue sections, has been used to stain a range of lymphoid, histiocytic, and myelomonocytic proliferations. All 20 neoplasms of myeloid, myelomonocytic, and presumed macrophage derivation reacted with antibody KP1. None of the 22 cases of T cell neoplasia had positive reactions. Although 14 of 41 B lineage lymphomas and leukaemias were stained by antibody KP1, staining was usually confined to small dots of reactivity, in contrast to the strong and extensive cytoplasmic staining seen in the neoplasms of myeloid and macrophage/monocyte origin. Furthermore, positive B cell neoplasms were almost all small cell proliferations, which are unlikely to be confused with myelomonocytic malignancies. It was concluded that antibody KP1 is a valuable addition to a panel of monoclonal antibodies for phenotyping lymphomas, particularly in routinely fixed tissues. It should assist the pathologist in the recognition of extramedullary presentation of leukaemia, aid in the diagnosis of suspected cases of true histiocytic neoplasia, and allow for quantitation of macrophages infiltrating lymphomas and other solid tumors. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:2688430

Warnke, R. A.; Pulford, K. A.; Pallesen, G.; Ralfkiaer, E.; Brown, D. C.; Gatter, K. C.; Mason, D. Y.



Human monoclonal antibodies produced by primary in vitro immunization of peripheral blood lymphocytes.  

PubMed Central

A general procedure is described for the production of human monoclonal antibodies from peripheral blood lymphocytes immunized in vitro against T-cell-dependent antigens. These lymphocytes immunized in culture were used to produce human-human or human-mouse hybridomas secreting monoclonal antibodies specific for digoxin, hemocyanin, a recombinant fragment of the gp120 envelope glycoprotein of human immunodeficiency virus (PB1), or a melanoma-associated antigen (p97). Depletion of a lysosome-rich cell population, containing large granular lymphocytes, monocytes, cytotoxic T cells, and a subset of CD8-positive T cells, was shown to be crucial before the cells could be immunized in vitro. This depletion was accomplished by treating the peripheral blood lymphocytes with the lysosomotropic agent L-leucine methyl ester. In addition, the in vitro immunization had to be supported by interleukin 2, gamma-interferon, and B-cell growth and differentiation factors, derived from irradiated, pokeweed-mitogen-stimulated human T cells. The production of human monoclonal antibodies from primary, antigen-specifically activated peripheral lymphocytes might obviate the need to immunize volunteers or patients. PMID:3131770

Borrebaeck, C A; Danielsson, L; Moller, S A



Immune reactivity of four monoclonal and two polyclonal antibodies raised against recombinant pneumococcal surface adhesin A.  


In this study four monoclonal antibodies (MAbs) and two polyclonal antisera were raised against pneumococcal surface adhesion A (psaA), a 37?kDa cell wall protein, and were characterized. The MAbs were purified, isotyped, and epitope mapped with peptide microarrays. All monoclonals and polyclonals underwent testing in immunodot blot, Western blot, and ELISA assays to assess their specificity. All monoclonal antibodies belonged to isotype IgG1 ?. Peptide microarray mapping identified two likely epitopes, which could not be confirmed using synthetic peptides of the identified amino acid sequence. All four MAbs detected the 53 pneumococcal serotypes tested in the immunodot blot and only reacted with Streptococcus pseudopneumoniae in cross-reactivity studies by Western blot. The polyclonal antisera also recognized all tested pneumococcal serotypes and cross-reacted with S. pseudopneumoniae and Streptococcus oralis by Western blot. The MAbs cross-reacted with S. pseudopneumoniae in the ELISA. Both polyclonal antisera cross-reacted with all isolates tested in the ELISA. These antisera should be suitable to establish a diagnostic ELISA platform for pneumococcal antigen detection with polyclonal antisera as the capture antibody. The specificity of the four MAbs appears to be high as they only recognized S. pneumoniae and S. pseudopneumoniae. However, further testing of closely related streptococcal species is necessary to confirm this finding. PMID:22741580

Werno, Anja M; Lewis, John G; George, Peter M; Murdoch, David R



Immunosuppression with monoclonal antibodies and CTLA4-Ig after myoblast transplantation in mice.  


Various combinations of monoclonal antibodies specific for lymphocyte cell surface antigens and a recombinant molecule (CTLA4-Ig) were used to immunosuppress mice after transplantation of MHC-incompatible myoblasts. To assess the effectiveness of the immunosuppression, the donor myoblasts were obtained from a transgenic mouse (TnI LacZ1/29) containing a beta-galactosidase (beta-gal) reporter gene under the control of a muscle-specific promoter. No muscle fibers expressing beta-gal were observed 1 month after the myoblast transplantation, when the animals were not immunosuppressed or were treated with CTLA4-Ig alone. Approximately 50% of the muscle fibers expressed the beta-gal reporter gene 1 month after transplantation in mice treated with CTLA4-Ig combined with an anti-CD4 monoclonal antibody and in mice treated with a combination of anti-CD4, anti-CD8, and anti-lymphocyte function-associated antigen-1. The percentage of beta-gal-labeled muscle fibers increased to 94% when this combination of the three monoclonal antibodies was administrated weekly for 3 weeks. Although excellent graft survival rates were obtained 1 month after transplantation, reflecting an effective immunosuppression by these three treatments, no beta-gal-positive fibers were found 2 months after the transplantation, indicating the inability of these immunosuppressive agents to maintain long-term graft survival and induce tolerance to the myoblasts and muscle fibers of donor origin. PMID:8878391

Guerette, B; Gingras, M; Wood, K; Roy, R; Tremblay, J P



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


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

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



Production and characterization of high-affinity monoclonal antibodies against morphine.  


Twelve hybridoma cell lines producing MAbs against morphine were established by using morphine hemisuccinate-conjugated bovine serum albumin as an immunogen. The MAbs belonged to the IgG1 subclass with kappa- or lambda-chains. The association constants of the antibodies ranged from 4.6 x 10(8) to 4.7 x 10(10) (M-1). These antibodies revealed slightly different cross-reactivities with various agonistic opiates and antagonists. In general, the antibodies were strongly cross-reactive with the opiate agonists, codeine, ethylmorphine, dihydromorphine and dihydrocodeine, while their cross-reactivities with norcodeine and the opiate antagonists, naloxone and naltrexone, were weak. The cross-reactivities with dihydromorphinone, dihydrocodeinone, naloxone, naltrexone, dextromethorphan and homatropine varied from clone to clone. Interestingly, certain MAbs displayed weak but significant cross-reactivities with the synthetic opiate, meperidine. However, none of the antibodies was cross-reactive with the opioid peptides, beta-endorphin, Met-enkephalin, and D-Ala2-D-Leu5-enkephalinamide. Radioimmunoassay for morphine using one of the antibodies (MOR 131.5.13) was shown to be sufficiently sensitive (IC50 = 0.1 nM) for the purposes of forensic analysis of morphine. This set of monoclonal anti-opiate antibodies is assumed to be suitable for analyzing the structure-function relationship in the hapten-antibody interaction, since the antibodies revealed similar but not identical cross-reactivities with various morphine related compounds. PMID:3211162

Sawada, J; Janejai, N; Nagamatsu, K; Terao, T



The use of a neutralizing monoclonal antibody to detect infections of equine herpesvirus type 2 (EHV-2).  


A blocking enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies to equine herpesvirus 2 in serum samples of horses. By measuring the binding to a single epitope, this blocking ELISA gives a good picture of the antibody status in the animal. The test is based on a monoclonal antibody with neutralizing activity and had a sensitivity of 94% and a specificity of 100%. Antibodies due to newly acquired infection in foals were successfully detected with this blocking ELISA. PMID:11580059

Nordengrahn, A; Klingeborn, B; Lindholm, A; Merza, M



Eradication of Established Tumors by a Fully Human Monoclonal Antibody to the Epidermal Growth Factor Receptor without Concomitant Chemotherapy  

Microsoft Academic Search

A fully human IgG2k monoclonal antibody (MAb), E7.6.3, specific to the human epidermal growth factor (EGF) receptor (EGFr) was gener- ated from human antibody-producing XenoMouse strains engineered to be deficient in mouse antibody production and to contain the majority of the human antibody gene repertoire on megabase-sized fragments from the human heavy and k light chain loci. The E7.6.3 MAb

Xiao-Dong Yang; Xiao-Chi Jia; Jose R. F. Corvalan; Ping Wang; C. Geoffrey Davis; Aya Jakobovits


Monoclonal antibody blocking the recognition of an insulin peptide-MHC complex modulates type 1 diabetes  

PubMed Central

The primary autoantigen triggering spontaneous type 1 diabetes mellitus in nonobese diabetic (NOD) mice is insulin. The major T-cell insulin epitope lies within the amino acid 9–23 peptide of the ?-chain (B:9–23). This peptide can bind within the peptide binding groove of the NOD MHC class II molecule (MHCII), IAg7, in multiple positions or “registers.” However, the majority of pathogenic CD4 T cells recognize this complex only when the insulin peptide is bound in register 3 (R3). We hypothesized that antibodies reacting specifically with R3 insulin–IAg7 complexes would inhibit autoimmune diabetes specifically without interfering with recognition of other IAg7-presented antigens. To test this hypothesis, we generated a monoclonal antibody (mAb287), which selectively binds to B:9–23 and related variants when presented by IAg7 in R3, but not other registers. The monoclonal antibody blocks binding of IAg7-B:10–23 R3 tetramers to cognate T cells and inhibits T-cell responses to soluble B:9–23 peptides and NOD islets. However, mAb287 has no effect on recognition of other peptides bound to IAg7 or other MHCII molecules. Intervention with mAb287, but not irrelevant isotype matched antibody, at either early or late stages of disease development, significantly delayed diabetes onset by inhibiting infiltration by not only insulin-specific CD4 T cells, but also by CD4 and CD8 T cells of other specificities. We propose that peptide–MHC-specific monoclonal antibodies can modulate autoimmune disease without the pleiotropic effects of nonselective reagents and, thus, could be applicable to the treatment of multiple T-cell mediated autoimmune disorders. PMID:24550292

Zhang, Li; Crawford, Frances; Yu, Liping; Michels, Aaron; Nakayama, Maki; Davidson, Howard W.; Kappler, John W.; Eisenbarth, George S.



Study of rat kidney transamidinase structure and regulation with monoclonal antibodies and the purification and characterization of human kidney transamidinase  

SciTech Connect

The isolation of monoclonal antibodies to transamidinase made possible the development of an immunosorbent inhibition assay for transamidinase protein using a /sup 125/I-labeled monoclonal antibody. This assay is a more direct measurement of transamidinase protein than the determination of the amount of polyclonal antibody required to precipitate the transamidinase activities. Rats were fed diets supplemented with creatine and/or glycine, and the amounts of transamidinase protein were determined with the assay using the monoclonal antibody. The transamidinase activities of kidneys from the rats fed the various supplemented diets ranged from 10 to 40% of the control values, whereas, the amounts of transamidinase protein were, in all instances no lower than 66% of the control values. Purified homogeneous rat kidney transamidinase and rat kidney supernatants were subjected to isoelectric focussing and four to five fractions of the enzyme were obtained. Polyclonal antibodies, but not the monoclonal antibodies were found by Western blotting experiments to recognize all the forms of the enzyme obtained by the isoelectric focussing. The author concluded that the monoclonal antibodies recognized forms of the enzyme that changed very little in amount, relative to the alterations in enzyme activities, when rats were fed a diet containing creatine.

Gross, M.D.



Evaluation of the use of monoclonal antibodies to hemagglutinin and fusion glycoproteins of Newcastle disease virus for virus identification and strain differentiation purposes  

Microsoft Academic Search

Summary Monoclonal antibodies detect evident antigenic variations in NDV HN and F protein. However, the A\\/PMV-1 viruses can be identified by HI test using a preparation made of the combination of two different monoclonals.

G. Meulemans; M. Gonze; M. C. Carlier; P. Petit; A. Burny; Le Long



Production and characterization of monoclonal antibodies against rat brush border antigens of the proximal convoluted tubule.  

PubMed Central

In order to understand further the processes involved in immunological injury of the kidney, we have prepared monoclonal antibodies against brush border (BB) antigens of rat proximal tubule. The 27 antibodies which constitute the basis of this report have been cloned, characterized immunochemically, and classified in three specificity groups on the basis of tissue reactivity. The first group is made up of six antibodies reacting with antigens simultaneously present on BB and glomerulus: three are directed against a high molecular weight (MW) protein which migrates with an apparent MW of 330,000; two react with a 90,000 MW protein that is present diffusely on renal and intestinal BB as well as on endothelial cells; one recognizes an antigen exclusively present on superficial tubules and glomerular epithelial cells, which could not be chemically characterized. The second group is made up of eight antibodies present on renal and intestinal BB: five react with a 120,000 MW antigen, one with a 300,000 MW antigen. The third group comprises 13 antibodies. Two are directed against antigens present within the cytoplasm or the basolateral membranes of renal tubules. Eleven react with intracellular antigens probably related to the cytoskeleton. Since they have been identified through several fusions, some of the monoclonal antibodies described are probably directed against immunodominant proteins of the BB. They open new possibilities for purifying the corresponding antigens by affinity chromatography as well as for obtaining BB preparations selectively depleted of the strongest immunogens thus favouring antibody production to previously unrecognized antigens. Images Figure 1 Figure 2 Figure 3 PMID:6381294

Ronco, P; Melcion, C; Geniteau, M; Ronco, E; Reininger, L; Galceran, M; Verroust, P



Human monoclonal antibodies specific to hepatitis B virus generated in a human/mouse radiation chimera: the Trimera system.  

PubMed Central

An approach to develop fully human monoclonal antibodies in a human/mouse radiation chimera, the Trimera system, is described. In this system, functional human lymphocytes are engrafted in normal strains of mice which are rendered immuno-incompetent by lethal total body irradiation followed by radioprotection with severe combined immunodeficient (SCID) mouse bone marrow. Following transplantation, human lymphocytes colonize murine lymphatic organs and secrete human immunoglobulins. We have established this system as a tool to develop fully human monoclonal antibodies, and applied it for the generation of monoclonal antibodies specific for hepatitis B virus surface antigen. A strong memory response to hepatitis B surface antigen was elicited in Trimera engrafted with lymphocytes from human donors positive for antibodies to hepatitis B surface antigen. The human specific antibody fraction in the Trimera was 10(2)-10(3)-fold higher as compared with that found in the donors. Spleens were harvested from Trimera mice showing high specific-antibody titres and cells were fused to a human-mouse heteromyeloma fusion partner. Several stable hybridoma clones were isolated and characterized. These hybridomas produce high-affinity, IgG, anti-hepatitis B surface antigen antibodies demonstrating the potential of the Trimera system for generating fully human monoclonal antibodies. The biological function and the neutralizing activity of these antibodies are currently being tested. Images Figure 1 Figure 2 Figure 4 PMID:9616363

Eren, R; Lubin, I; Terkieltaub, D; Ben-Moshe, O; Zauberman, A; Uhlmann, R; Tzahor, T; Moss, S; Ilan, E; Shouval, D; Galun, E; Daudi, N; Marcus, H; Reisner, Y; Dagan, S



Rapid selection of cell subpopulation-specific human monoclonal antibodies from a synthetic phage antibody library.  

PubMed Central

Peripheral blood leukocytes incubated with a semisynthetic phage antibody library and fluorochrome-labeled CD3 and CD20 antibodies were used to isolate human single-chain Fv antibodies specific for subsets of blood leukocytes by flow cytometry. Isolated phage antibodies showed exclusive binding to the subpopulation used for selection or displayed additional binding to a restricted population of other cells in the mixture. At least two phage antibodies appeared to display hitherto-unknown staining patterns of B-lineage cells. This approach provides a subtractive procedure to rapidly obtain human antibodies against known and novel surface antigens in their native configuration, expressed on phenotypically defined subpopulations of cells. This approach does not depend on immunization procedures or the necessity to repeatedly construct phage antibody libraries. Images Fig. 1 PMID:7537380

de Kruif, J; Terstappen, L; Boel, E; Logtenberg, T



Mechanisms of Allergen-Antibody Interaction of Cockroach Allergen Bla g 2 with Monoclonal Antibodies That Inhibit IgE Antibody Binding  

PubMed Central

Background Cockroach allergy is strongly associated with asthma, and involves the production of IgE antibodies against inhaled allergens. Reports of conformational epitopes on inhaled allergens are limited. The conformational epitopes for two specific monoclonal antibodies (mAb) that interfere with IgE antibody binding were identified by X-ray crystallography on opposite sites of the quasi-symmetrical cockroach allergen Bla g 2. Methodology/Principal Findings Mutational analysis of selected residues in both epitopes was performed based on the X-ray crystal structures of the allergen with mAb Fab/Fab? fragments, to investigate the structural basis of allergen-antibody interactions. The epitopes of Bla g 2 for the mAb 7C11 or 4C3 were mutated, and the mutants were analyzed by SDS-PAGE, circular dichroism, and/or mass spectrometry. Mutants were tested for mAb and IgE antibody binding by ELISA and fluorescent multiplex array. Single or multiple mutations of five residues from both epitopes resulted in almost complete loss of mAb binding, without affecting the overall folding of the allergen. Preventing glycosylation by mutation N268Q reduced IgE binding, indicating a role of carbohydrates in the interaction. Cation-? interactions, as well as electrostatic and hydrophobic interactions, were important for mAb and IgE antibody binding. Quantitative differences in the effects of mutations on IgE antibody binding were observed, suggesting heterogeneity in epitope recognition among cockroach allergic patients. Conclusions/Significance Analysis by site-directed mutagenesis of epitopes identified by X-ray crystallography revealed an overlap between monoclonal and IgE antibody binding sites and provided insight into the B cell repertoire to Bla g 2 and the mechanisms of allergen-antibody recognition, including involvement of carbohydrates. PMID:21789239

Glesner, Jill; Wünschmann, Sabina; Li, Mi; Gustchina, Alla; Wlodawer, Alexander; Himly, Martin; Chapman, Martin D.; Pomés, Anna



A monoclonal antibody based capture ELISA for botulinum neurotoxin serotype B: toxin detection in food.  


Botulism is a serious foodborne neuroparalytic disease, caused by botulinum neurotoxin (BoNT), produced by the anaerobic bacterium Clostridium botulinum. Seven toxin serotypes (A-H) have been described. The majority of human cases of botulism are caused by serotypes A and B followed by E and F. We report here a group of serotype B specific monoclonal antibodies (mAbs) capable of binding toxin under physiological conditions. Thus, they serve as capture antibodies for a sandwich (capture) ELISA. The antibodies were generated using recombinant peptide fragments corresponding to the receptor-binding domain of the toxin heavy chain as immunogen. Their binding properties suggest that they bind a complex epitope with dissociation constants (KD's) for individual antibodies ranging from 10 to 48 × 10-11 M. Assay performance for all possible combinations of capture-detector antibody pairs was evaluated and the antibody pair resulting in the lowest level of detection (L.O.D.), ~20 pg/mL was determined. Toxin was detected in spiked dairy samples with good recoveries at concentrations as low as 0.5 pg/mL and in ground beef samples at levels as low as 2 ng/g. Thus, the sandwich ELISA described here uses mAb for both the capture and detector antibodies (binding different epitopes on the toxin molecule) and readily detects toxin in those food samples tested. PMID:24253240

Stanker, Larry H; Scotcher, Miles C; Cheng, Luisa; Ching, Kathryn; McGarvey, Jeffery; Hodge, David; Hnasko, Robert



A Monoclonal Antibody Based Capture ELISA for Botulinum Neurotoxin Serotype B: Toxin Detection in Food  

PubMed Central

Botulism is a serious foodborne neuroparalytic disease, caused by botulinum neurotoxin (BoNT), produced by the anaerobic bacterium Clostridium botulinum. Seven toxin serotypes (A – H) have been described. The majority of human cases of botulism are caused by serotypes A and B followed by E and F. We report here a group of serotype B specific monoclonal antibodies (mAbs) capable of binding toxin under physiological conditions. Thus, they serve as capture antibodies for a sandwich (capture) ELISA. The antibodies were generated using recombinant peptide fragments corresponding to the receptor-binding domain of the toxin heavy chain as immunogen. Their binding properties suggest that they bind a complex epitope with dissociation constants (KD’s) for individual antibodies ranging from 10 to 48 × 10?11 M. Assay performance for all possible combinations of capture-detector antibody pairs was evaluated and the antibody pair resulting in the lowest level of detection (L.O.D.), ~20 pg/mL was determined. Toxin was detected in spiked dairy samples with good recoveries at concentrations as low as 0.5 pg/mL and in ground beef samples at levels as low as 2 ng/g. Thus, the sandwich ELISA described here uses mAb for both the capture and detector antibodies (binding different epitopes on the toxin molecule) and readily detects toxin in those food samples tested. PMID:24253240

Stanker, Larry H.; Scotcher, Miles C.; Cheng, Luisa; Ching, Kathryn; McGarvey, Jeffery; Hodge, David; Hnasko, Robert



Generation and characterization of monoclonal antibodies to mTOR kinase.  


Abstract Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that plays a critical role in the regulation of basic cellular functions, including cellular growth and proliferation. In this study we describe the generation and characterization of novel monoclonal antibodies directed against mTOR protein kinase. A GST-tagged fragment of mTOR expressed in bacteria was used as an antigen. Antibody-producing hybridoma cells were obtained by fusing SP2/0 myeloma cells with splenocytes from immunized mice. Anti-mTOR antibody-producing hybridoma cell lines were first identified by enzyme-linked immunosorbent assay and then subcloned by limiting dilution. Antibodies produced by selected clones were further tested for their reactivity towards the GST/mTOR 1334-1504 recombinant protein. Furthermore, antibody produced by F11 clone was shown to recognize specifically mTOR in different tissues and cell lines in Western blotting, immunoprecipitation, and immunohistochemistry. In addition, mTOR F11 antibody was suitable for immunoprecipitating and testing mTOR activity in in vitro kinase assay. In summary, generated antibodies will be useful for investigating mTOR signaling complexes in normal and pathological states. PMID:18803507

Nemazanyy, Ivan; Breus, Oksana; Gout, Ivan; Filonenko, Valeriy; Panasyuk, Ganna



Production and specificity of monoclonal antibodies against nodularin conjugated through N-methyldehydrobutyrine.  


Nodularin (Nod) is a cyclic pentapeptide hepatotoxin produced by the cyanobacterial genus Nodularia living in brackish waters and coastal lagoons. The toxicity of Nod is due to specific inhibition of the type-1 and type-2A intracellular protein phosphatases (PP1 and PP2A, respectively). We have developed a monoclonal antibody against Nod using chemical modification (aminoethylation) of one of its core amino acids, N-methyldehydrobutyrine. The developed antibody is highly specific for Nod, with negligible reactivity to the closely related cyanobacterial toxin microcystin (MC). The monoclonal antibody was employed for quantitative competitive ELISA assay. The analytical sensitivity of the assay was up to 0.2 ng/ml. Comparison of the developed ELISA test with HPLC-based measurements of Nod, with both laboratory and field samples, showed a good correspondence between the results yielded by these two methods. The antibodies developed by this technique provide means for developing extremely sensitive and specific analytical assays for direct measurement of nodularin and related toxins in cyanobacterial or water samples. PMID:11478952

Mikhailov, A; Härmälä-Braskén, A S; Polosukhina, E; Hanski, A; Wahlsten, M; Sivonen, K; Eriksson, J E



Monoclonal antibodies specific to Streptococcus mutans GS-5 glucosyltransferase-C inhibit bacterial glucosyltransferase.  


Glucosyltransferase-C (GTFC) is a virulence factor of Streptococcus mutans. Additionally, GTFC represents an essential element required for bacterial cell coherence, allowing for the formation of dental plaque, which leads to dental caries. As such, monoclonal antibodies (MAbs) against S. mutans are believed to offer some protection against dental caries. In the current study, we amplified an approximately 1.5 kb fragment of the N-terminal half of the S. mutans gtfC gene by PCR, then induced expression of this gene. This protein was designated GTFCN. After the expressed protein was purified, it was used as an immunogen and injected into BALB/c mice. We selected and established two MAbs by producing hybridomas (HCN17 and HCN37). The anti-GTFCN antibody isotype was confirmed as IgG2a for HCN17 and IgG2b for HCN37. The anti-GTFCN antibody was found to specifically react with the GTFCN protein. The enzymatic activity of the crude glucosyltransferase of S. mutans GS-5 was significantly inhibited at a concentration of 350 ng of MAb/mL. These results suggest that the monoclonal anti-GTFCN antibodies could represent an alternative modality for passive immunization to prevent S. mutans aggregation and dental plaque. PMID:24111864

Kim, Mi-Ah; Lee, Kyung-Yeol; Kim, Jae-Gon



Rapid detection of members of the family Enterobacteriaceae by a monoclonal antibody.  

PubMed Central

Six monoclonal antibodies directed against enterobacteria were produced and characterized. The specificity of one of these antibodies (CX9/15; immunoglobulin G2a) was studied by indirect immunofluorescence against 259 enterobacterial strains and 125 other gram-negative bacteria. All of the enterobacteria were specifically recognized, the only exception being Erwinia chrysanthemi (one strain tested). Bacteria not belonging to members of the family Enterobacteriaceae were not detected, except for Plesiomonas shigelloides (two strains tested), Aeromonas hydrophila (five strains tested), and Aeromonas sobria (one strain tested). This recognition spectrum strongly suggested that CX9/15 recognized the enterobacterial common antigen. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) experiments, the six antienterobacteria antibodies presented similar specificities; they all revealed only one band with an apparent molecular weight of about 20,000 from the crude extract of an enterobacterium. The six monoclonal antibodies, and especially CX9/15, can be used to develop new tests for rapid and specific detection of enterobacteria. Images PMID:1622220

Levasseur, S; Husson, M O; Leitz, R; Merlin, F; Laurent, F; Peladan, F; Drocourt, J L; Leclerc, H; Van Hoegaerden, M



Production of monoclonal antibody against doxycycline for immunoassay of seven tetracyclines in bovine muscle and milk.  


The objective of this study was to produce a generic monoclonal antibody for multi-determination of the residues of tetracycline drugs in bovine muscle and milk. Two new immunogens of doxycycline were prepared that were used to produce the monoclonal antibodies. Results showed the obtained antibodies simultaneously recognized seven tetracycline drugs (doxycycline, tetracycline, chlortetracycline, oxytetracycline, minocycline, methacycline, demeclocycline). The obtained antibodies and three coating antigens were arranged into six combinations to optimize the reagents combination. After comparison of the performances of these combinations, a heterologous indirect competitive ELISA was then used to determine the seven tetracyclines in bovine muscle and milk. The crossreactivities to the seven analytes were in the range of 47%-102% and the limits of detection were in the range of 1.5-6.9 ng/mL depending on the compound. The recoveries of the seven drugs from fortified blank samples were in the range of 75.3%-106.8% with coefficients of variation lower than 10.9%. Therefore, this method could be used as a multi-analytes screen tool for routine monitoring of the residues of these tetracycline drugs in bovine muscle and milk. PMID:23305276

Gao, Feng; Zhao, Guo X; Zhang, Hui C; Wang, Ping; Wang, Jian P



Antimyosin monoclonal antibodies for early detection of cardiac allograft rejection  

SciTech Connect

Sixty-eight indium 111-labeled antimyosin Fab-DTPA imaging studies (0.5 mg intravenously with a radioactivity of 65 to 75 MBq) were executed on 37 of 116 patients undergoing heart transplantation to assess diagnostic accuracy and clinical utility. As controls, 21 patients with cardiomyopathy (n = 8), unstable angina (n = 9), and myocardial infarction (n = 4) were selected. After 48 hours, single photon emission computed tomographic images were evaluated visually, and heart/lung ratios were measured, using the region of interest technique. They were compared with echocardiographic and endomyocardial biopsy results. In 40 studies a heart/lung ratio less than or equal to 1.6 corresponded to a negative biopsy result in 98% (40/41). Echocardiography enabled correct identification of 95% of the patients with normal biopsy findings. In 91% (22/24) a positive biopsy finding correlated with a heart/lung ratio greater than 1.6 including 20 mild rejections, but in only 64%, with an increase in wall thickness and/or decrease of fractional diameter shortening seen on echocardiogram. In addition, the various stages of rejection episodes determined the amount of the heart-lung ratio. There was a significant relationship between the histologic findings and the antimyosin uptake. In 13 patients a second investigation was performed after rejection therapy. All patients had a negative biopsy result, and the heart/lung ratio decreased to normal ranges (less than or equal to 1.6). Five antimyosin antibody studies were excluded, as in these cases, negative uptake results were found during rejection therapy with high-dose steroids. The overall sensitivity was calculated at 93% and the specificity at 98%.

Schuetz, A.; Fritsch, S.; Kemkes, B.M.; Kugler, C.; Angermann, C.; Spes, C.; Anthuber, M.; Weiler, A.; Wenke, K.; Gokel, J.M. (Univ. of Munich-Grosshadern, Munich (Germany, F.R.))



A Novel Rabbit Monoclonal Antibody Platform To Dissect the Diverse Repertoire of Antibody Epitopes for HIV-1 Env Immunogen Design  

PubMed Central

The majority of available monoclonal antibodies (MAbs) in the current HIV vaccine field are generated from HIV-1-infected people. In contrast, preclinical immunogenicity studies have mainly focused on polyclonal antibody responses in experimental animals. Although rabbits have been widely used for antibody studies, there has been no report of using rabbit MAbs to dissect the specificity of antibody responses for AIDS vaccine development. Here we report on the production of a panel of 12 MAbs from a New Zealand White (NZW) rabbit that was immunized with an HIV-1 JR-FL gp120 DNA prime and protein boost vaccination regimen. These rabbit MAbs recognized a diverse repertoire of envelope (Env) epitopes ranging from the highly immunogenic V3 region to several previously underappreciated epitopes in the C1, C4, and C5 regions. Nine MAbs showed cross-reactivity to gp120s of clades other than clade B. Increased somatic mutation and extended CDR3 were observed with Ig genes of several molecularly cloned rabbit MAbs. Phylogenic tree analysis showed that the heavy chains of MAbs recognizing the same region on gp120 tend to segregate into an independent subtree. At least three rabbit MAbs showed neutralizing activities with various degrees of breadth and potency. The establishment of this rabbit MAb platform will significantly enhance our ability to test optimal designs of Env immunogens to gain a better understanding of the structural specificity and evolution process of Env-specific antibody responses elicited by candidate AIDS vaccines. PMID:23864612

Chen, Yuxin; Vaine, Michael; Wallace, Aaron; Han, Dong; Wan, Shengqin; Seaman, Michael S.; Montefiori, David; Wang, Shixia



A novel bispecific antibody targeting tumor necrosis factor ? and ED-B fibronectin effectively inhibits the progression of established collagen-induce arthritis.  


Specific delivery of TNF-? antagonist to the inflamed site can increase its efficacy and reduce the side effects. In this study, we constructed a bispecific diabody (BsDb) that targets TNF-? and ED-B-containing fibronectin (B-FN), a fibronectin isoform specifically expressed in the pannus of the inflamed joint in rheumatoid arthritis. BsDb was produced in Escherichia coli as inclusion bodies, purified to homogeneity, and refolded to the functional form. Our data demonstrate that BsDb could simultaneously bind to human TNF-? and B-FN and neutralize TNF-? action. In the collagen-induced arthritis mouse model, we compared the biodistrubtion and therapeutic efficacy of BsDb with those of the anti-TNF-? scFv (TNF-scFv). Similar to TNF-scFv, BsDb penetrated into the synovial tissue quickly, showing a rapid clearance from blood and normal organs. In contrast, BsDb could selectively accumulate and retain in arthritic joints of mice for a long period time, resulting in a much stronger inhibition of arthritis progression in mice than TNF-scFv. The findings described herein indicate that BsDb has a good specificity to the inflamed joint, with low toxicity to normal organs and seems to be an ideal biological agent for the treatment of RA and other chronic inflammatory disease. PMID:24992210

Liu, Mengyuan; Xie, Mian; Jiang, Sijing; Liu, Guoping; Li, Lujun; Liu, Dongxu; Yang, Xiaosong



Mechanistic insights into the neutralization of cytotoxic abrin by the monoclonal antibody D6F10.  


Abrin, an A/B toxin obtained from the Abrus precatorius plant is extremely toxic and a potential bio-warfare agent. Till date there is no antidote or vaccine available against this toxin. The only known neutralizing monoclonal antibody against abrin, namely D6F10, has been shown to rescue the toxicity of abrin in cells as well as in mice. The present study focuses on mapping the epitopic region to understand the mechanism of neutralization of abrin by the antibody D6F10. Truncation and mutational analysis of abrin A chain revealed that the amino acids 74-123 of abrin A chain contain the core epitope and the residues Thr112, Gly114 and Arg118 are crucial for binding of the antibody. In silico analysis of the position of the mapped epitope indicated that it is present close to the active site cleft of abrin A chain. Thus, binding of the antibody near the active site blocks the enzymatic activity of abrin A chain, thereby rescuing inhibition of protein synthesis by the toxin in vitro. At 1?10 molar concentration of abrin:antibody, the antibody D6F10 rescued cells from abrin-mediated inhibition of protein synthesis but did not prevent cell attachment of abrin. Further, internalization of the antibody bound to abrin was observed in cells by confocal microscopy. This is a novel finding which suggests that the antibody might function intracellularly and possibly explains the rescue of abrin's toxicity by the antibody in whole cells and animals. To our knowledge, this study is the first report on a neutralizing epitope for abrin and provides mechanistic insights into the poorly understood mode of action of anti-A chain antibodies against several toxins including ricin. PMID:23922965

Bagaria, Shradha; Ponnalagu, Devasena; Bisht, Shveta; Karande, Anjali A



Generation of a monoclonal antibody for INI1/hSNF5/BAF47.  


INI1/hSNF5/BAF47, which has an SNF5 domain, belongs to the SWI/SNF family. This family is known as ATP-dependent regulators of gene expression by remodeling chromatin structure during cell differentiation. However, the detailed function of INI1/hSNF5/BAF47 is unclear. Here we report the generation of a specific monoclonal antibody for INI1/hSNF5/BAF47 by the mouse iliac lymph node method. The obtained antibody recognized two isoforms of INI1/hSNF5/BAF47 in immunoblotting and precisely recognized the nuclear localization of INI1/hSNF5/BAF47 in immunostaining. This antibody can contribute to further elucidation of the mechanisms of gene expression regulation by INI1/hSNF5/BAF47 during cell differentiation. PMID:24555937

Harada, Akihito; Hayashi, Masayasu; Kuniyoshi, Yuuki; Semba, Yuichiro; Sugahara, Satoko; Tachibana, Taro; Ohkawa, Yasuyuki; Fujita, Masatoshi



Monoclonal antibody against the N-terminal end of human plasma fibronectin.  

PubMed Central

Purified human plasma fibronectin was digested with cathepsin G and the degradation products were tested for reactivity towards a monoclonal antibody. In an immunoblotting assay, after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the digestion products, the 85 000-Mr and 72 000-Mr gelatin- and heparin-binding fragments as well as the N-terminal 30 000-Mr heparin-binding fragment reacted with the antibody, whereas the 64 000-Mr gelatin- and heparin-binding fragment did not. In enzyme immunoassay the antibody reacted with intact fibronectin and the 30 000-Mr fragment but not with a 40 000-Mr gelatin-binding fragment. The alignment of the binding domains in these fragments and in the intact molecule [Vartio (1982) Eur. J. Biochem. 123, 223-233] localizes the antigenic determinant to the 21 000 Da N-terminal Staphylococcus aureus-binding region of fibronectin. Images Fig. 1. Fig. 2. PMID:6194791

Vartio, T; Salonen, E M; De Petro, G; Barlati, S; Miggiano, V; Stahli, C; Virgallita, G; Takacs, B; Vaheri, A



Production and characterization of monoclonal antibodies to wall-localized peroxidases from corn seedlings  

NASA Technical Reports Server (NTRS)

A library of 22 hybridomas, which make antibodies to soluble wall antigens from the coleoptiles and primary leaves of etiolated corn (Zea mays L.) seedlings, was raised and cloned three times by limit dilution to assure monoclonal growth and stability. Two of these hybridomas made immunoglobulin G antibodies, designated mWP3 and mWP19, which both effectively immunoprecipitated peroxidase activity from crude and partially purified preparations of wall peroxidases. Direct peroxidase-binding assays revealed that both antibodies bound enzymes with peroxidase activity. As judged by immunoblot analyses, mWP3 recognized a Mr 98,000 wall peroxidase with an isoelectric point near 4.2, and mWP19 recognized a Mr 58,000 wall peroxidase. Immunogold localization studies showed both peroxidases are predominately in cell walls.

Kim, S. H.; Terry, M. E.; Hoops, P.; Dauwalder, M.; Roux, S. J.



Human Monoclonal Antibodies to Pf 155, a Major Antigen of Malaria Parasite Plasmodium falciparum  

NASA Astrophysics Data System (ADS)

Pf 155, a protein of the human malaria parasite Plasmodium falciparum, is strongly immunogenic in humans and is believed to be a prime candidate for the preparation of a vaccine. Human monoclonal antibodies to Pf 155 were obtained by cloning B cells that had been prepared from an immune donor and transformed with Epstein-Barr virus. When examined by indirect immunofluorescence, these antibodies stained the surface of infected erythrocytes, free merozoites, segmented schizonts, and gametocytes. They bound to a major polypeptide with a relative molecular weight of 155K and to two minor ones (135K and 120K), all having high affinity for human glycophorin. The antibodies strongly inhibited merozoite reinvasion in vitro, suggesting that they might be appropriate reagents for therapeutic administration in vivo.

Udomsangpetch, Rachanee; Lundgren, Katarina; Berzins, Klavs; Wahlin, Birgitta; Perlmann, Hedvig; Troye-Blomberg, Marita; Carlsson, Jan; Wahlgren, Mats; Perlmann, Peter; Bjorkman, Anders



Monoclonal antibodies specific for Clostridium difficile toxin B and their use in immunoassays.  

PubMed Central

Five mouse monoclonal antibodies (MAbs) against Clostridium difficile toxin B have been raised and characterized. Three of them were immunoglobulin M (IgM) antibodies (6B10, 6G3, and 10B9), and the other two were of the IgG1 isotype (9E5 and 17G2), recognizing specifically two distinct epitopes on the toxin B molecule. No MAb was able to neutralize cytotoxic activity significantly. The two IgG1 MAbs were purified and applied to various immunodiagnostic assays. MAbs coupled to latex beads were used for specific removal of toxin B from cytotoxic samples and for agglutination assay. An indirect sandwich enzyme-linked immunosorbent assay with MAb 9E5 or 17G2 as the capture antibody was established for identification of toxin B with a lower detection limit of 5 ng/ml. Images PMID:1378062

Muller, F; Stiegler, C; Hadding, U



E4, a new monoclonal antibody identifying a human prostatic cell surface antigen  


The monoclonal antibody E4 (IgG2a, kappa) was raised by immunizing mice with dispersed cells obtained from human benign prostatic hyperplasia (BPH). The antibody identifies an antigen abundantly expressed in normal prostate epithelial cells, in benign epithelial prostatic cells and in well- and moderately well differentiated adenocarcinomas of the prostate, whereas poorly differentiated prostatic adenocarcinomas display somewhat less expression. Investigation of the human prostatic adenocarcinoma cell line DU 145 revealed E4 immunoreactivity localized to the cell surface. SDS-PAGE analysis under reducing conditions demonstrated an approximate molecular weight of 70,000 for the antigen. The highly specific reactivity with prostate tissue, as well as intense surface staining, especially in well- and moderately well differentiated prostatic adenocarcinomas, makes the E4 antibody a useful immunohistochemical marker and a possible candidate for future immunoscintigraphy and/or targeted radiotherapy. PMID:10851493

Nilsson; Essand; Logdahl; Larson; Nordgren; Juhlin



Explanation of the Reaction of Monoclonal Antibodies with Candida Albicans Cell Surface in Terms of Compound Poisson Process  

Microsoft Academic Search

Surprisingly, still very little is known about the mathematical modeling of peaks in the binding affinities distribution function. In general, it is believed that the peaks represent antibodies directed towards single epitopes. In this paper, we refer to fluorescence flow cytometry experiments and show that even monoclonal antibodies can display multi-modal histograms of affinity distribution. This result take place when

Miroslaw R. Dudek; Józef Mleczko



Production of monoclonal antibodies and development of an antigen capture ELISA directed against the envelope glycoprotein GP of Ebola virus  

Microsoft Academic Search

Ebola virus (EBOV) causes severe outbreaks of Ebola hemorrhagic fever in endemic regions of Africa and is considered to be of impact for other parts of the world as an imported viral disease. To develop a new diagnostic test, monoclonal antibodies to EBOV were produced from mice immunized with inactivated EBOV species Zaire. Antibodies directed against the viral glycoprotein GP

Andreas Lucht; Roland Grunow; Christian Otterbein; Peggy Möller; Heinz Feldmann; Stephan Becker



Characterization of Monoclonal Antibodies to Acanthamoeba Myosin-I that Cross-react with Both Myosin-II and Low  

E-print Network

Characterization of Monoclonal Antibodies to Acanthamoeba Myosin-I that Cross-react with Both Myosin-II and Low Molecular Mass Nuclear Proteins Susan J. Hagen, Daniel P. Kiehart, Donald A. Kaiser antibod- ies that bind to the heavy chain of Acanthamoeba myosin-IA. Eight of these antibodies bind


Identification of phage-displayed peptides which bind to the human HnRNPA1 protein-specific monoclonal antibody.  


We have screened a peptide phage display library to examine if monoclonal antibody-binding phages could be isolated from the library and thereby predict the antigenic epitopes of the antibodies from the isolated phages. The library was screened for high-avidity binding to monoclonal antibodies by an affinity purification technique called biopanning. Among the monoclonal antibodies examined, the human hnRNPA1 protein-specific monoclonal antibody 9H10 showed selective binding of phages. After two rounds of the biopanning, twelve clones of high-avidity-binding phages were chosen and their inserts were sequenced. Nucleotide sequence comparison of the 12 clones showed that there were 5 different species, with two species containing four members, implying that they were predominantly selected by the biopanning. The amino acid sequences of the inserts of the 12 clones were compared with that of the human hnRNPA1 protein in order to find the putative epitope of the human hnRNPA1 protein for 9H10. The C-terminal region of the human hnRNPA1 protein shows significant homology with the peptide sequences of the selected phage clones. These results show that this peptide phage display library can be useful in defining the epitope of some monoclonal antibodies. PMID:10515612

Kim, J H; Lee, J H; Kim, J; Kim, J K



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

SciTech Connect

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

Muto, M.G.; Finkler, N.J.; Kassis, A.I.; Lepisto, E.M.; Knapp, R.C. (Brigham and Women's Hospital, Boston, MA (USA))



Detection of hepatitis A virus and antibody by solid-phase radioimmunoassay and enzyme-linked immunosorbent assay with monoclonal antibodies.  

PubMed Central

Monoclonal antibodies (K3-2F2 and K3-4C8) raised against hepatitis A virus were used to develop a solid-phase radioimmunoassay and enzyme-linked immunosorbent assay for the detection of hepatitis A virus and antibody. Assays with this pair of monoclonal antibodies were compared in parallel with similarly constructed solid-phase radioimmunoassays and enzyme-linked immunosorbent assays in which human polyclonal serum was used. The monoclonal antibody assay proved to be more sensitive for the detection of hepatitis A virus from fecal specimens as well as for anti-hepatitis A virus immunoglobulin G (IgG) and IgM in sera. PMID:2991329

Coulepis, A G; Veale, M F; MacGregor, A; Kornitschuk, M; Gust, I D



Monoclonal antibody purification using cationic polyelectrolytes: an alternative to column chromatography.  


The potential of cationic polyelectrolytes to precipitate host cell and process related impurities was investigated, to replace one or more chromatography steps in monoclonal antibody purification. The impact of antibody isoelectric point, solution properties (pH and ionic strength), and polyelectrolyte properties (structure, molecular weight and pK(a)) on the degree of precipitation was studied. At neutral pH, increasing solution ionic strength impeded the ionic interaction between the polyelectrolyte and impurities, reducing impurity precipitation. Increasing polyelectrolyte molecular weight and pK(a) enabled precipitation of impurities at higher ionic strength. PoIy(arginine) was selected as the preferred polyelectrolyte in unconditioned cell culture fluid. PoIy(arginine) precipitation achieved consistent host cell protein clearance and antibody recovery for multiple antibodies across a wider range of polyelectrolyte concentrations. Poly(arginine) precipitation was evaluated as a flocculant and as a functional replacement for anion exchange chromatography in an antibody purification process. Upstream treatment of cell culture fluid with poly(arginine) resulted in flocculation of solids (cells and cell debris), and antibody recovery and impurity clearance (host cell proteins, DNA and insulin) comparable to the downstream anion exchange chromatography step. PMID:20945489

Peram, Thanmaya; McDonald, Paul; Carter-Franklin, Jayme; Fahrner, Robert



Characterization by enzyme-linked immunosorbent assay of monoclonal antibodies to Pisum and Avena phytochrome  

SciTech Connect

Nine monoclonal antibodies to pea (Pisum sativum L.) and 16 to oat (Avena sativa L.) phytochrome are characterized by enzyme-linked immunosorbent assay against phytochrome from six different sources: pea, zucchini (Cucurbita pepo L.), lettuce (Lactuca sativa L.), oat, rye (Secale cereale L.), and barley (Hordeum vulgare L.). All antibodies were raised against phytochrome with a monomer size near 120,000 daltons. Nevertheless, none of them discriminated qualitatively between 118/114-kilodalton oat phytochrome and a photoreversible, 60-kilodalton proteolytic degradation product derived from it. In addition, none of the 23 antibodies tested discriminated substantially between phytochrome - red-absorbing form and phytochrome - far red-absorbing form. Two antibodies to pea and six to oat phytochrome also bound strongly to phytochrome from the other species, even though these two plants are evolutionarily widely divergent. Of these eight antibodies, two bound significantly to all of the six phytochrome preparations tested, indicating that these two may recognize highly conserved regions of the chromoprotein. Since the molecular function of phytochrome is unknown, these two antibodies may serve as unique probes for regions of this pigment that are important to its mode of action. 27 references, 3 figures, 1 table.

Cordonnier, M.M.; Greppin, H.; Pratt, L.H.



Defining Influenza A Virus Hemagglutinin Antigenic Drift by Sequential Monoclonal Antibody Selection  

PubMed Central

SUMMARY Human influenza A virus (IAV) vaccination is limited by “antigenic drift,” rapid antibody-driven escape reflecting amino acid substitutions in the globular domain of hemagglutinin (HA), the viral attachment protein. To better understand drift, we used anti-hemagglutinin monoclonal Abs (mAbs) to sequentially select IAV escape mutants. Twelve selection steps, each resulting in a single amino acid substitution in the hemagglutinin globular domain, were required to eliminate antigenicity defined by monoclonal or polyclonal Abs. Sequential mutants grow robustly, showing the structural plasticity of HA, although several hemagglutinin substitutions required an epistatic substitution in the neuraminidase glycoprotein to maximize growth. Selecting escape mutants from parental versus sequential variants with the same mAb revealed distinct escape repertoires, attributed to contextual changes in antigenicity and the mutation landscape. Since each hemagglutinin mutation potentially sculpts future mutation space, drift can follow many stochastic paths, undermining its unpredictability and underscoring the need for drift-insensitive vaccines. PMID:23498956

Das, Suman R.; Hensley, Scott E.; Ince, William L.; Brooke, Christopher B.; Subba, Anju; Delboy, Mark G.; Russ, Gustav; Gibbs, James S.; Bennink, Jack R.; Yewdell, Jonathan W.



Controlling trisulfide modification in recombinant monoclonal antibody produced in fed-batch cell culture.  


Molecular heterogeneity was detected in a recombinant monoclonal antibody (IgG1 mAb) due to the presence of a trisulfide linkage generated by the post-translational insertion of a sulfur atom into disulfide bonds at the heavy-heavy and heavy-light junctions. This molecular heterogeneity had no observable effect on antibody function. Nevertheless, to minimize the heterogeneity of the IgG1 mAb from run-to-run, an understanding of the impact of cell culture process conditions on trisulfide versus disulfide linkage formation was desirable. To investigate variables that might impact trisulfide formation, cell culture parameters were varied in bench-scale bioreactor studies. Trisulfide analysis of the samples from these runs revealed that the trisulfide content in the bond between heavy and light chains varied considerably from <1% to 39%. Optimizing the culture duration and feeding strategy resulted in more consistent trisulfide levels. Cysteine concentration in the feed medium had a direct correlation with the trisulfide level in the product. Systematic studies revealed that cysteine in the feed and the bioreactor media was contributing hydrogen sulfide which reacted with the IgG1 mAb in the supernatant leading to the insertion of sulfur atom and formation of a trisulfide bond. Cysteine feed strategies were developed to control the trisulfide modification in the recombinant monoclonal antibody. PMID:22473825

Kshirsagar, Rashmi; McElearney, Kyle; Gilbert, Alan; Sinacore, Marty; Ryll, Thomas



Increasing parvovirus filter throughput of monoclonal antibodies using ion exchange membrane adsorptive pre-filtration.  


Pre-filtration using ion exchange membrane adsorbers can improve parvovirus filter throughput of monoclonal antibodies (mAbs). The membranes work by binding trace foulants, and although some antibody product also binds, yields > or =99% are easily achieved by overloading. Results show that foulant adsorption is dependent on pH and conductivity, but independent of scale and adsorber brand. The ability to use ion exchange membranes as pre-filters is significant because it provides a clean, well defined, chemically stable option for enhancing throughput. Additionally, ion exchange membranes facilitate characterization of parvovirus filter foulants. Examination of adsorber elution samples using sedimentation velocity analysis and SEC-MALS/QELS revealed the presence of high molecular weight species ranging from 8 to 13 nm in hydrodynamic radius, which are similar in size to parvoviruses and thus would be expected to plug the pores of a parvovirus filter. A study of two identical membranes in-series supports the hypothesis that the foulants are soluble, trace level aggregates in the feed. This study's significance lies in a previously undiscovered application of membrane chromatography, leading to a more cost effective and robust approach to parvovirus filtration for the production of monoclonal antibodies. PMID:20229510

Brown, Arick; Bechtel, Charity; Bill, Jerome; Liu, Hui; Liu, Jun; McDonald, Dan; Pai, Satyan; Radhamohan, Asha; Renslow, Ryan; Thayer, Brooke; Yohe, Stefan; Dowd, Chris



Evaluation of a monoclonal antibody-based immunoradiometric assay for prostatic acid phosphatase  

SciTech Connect

This report evaluates a new immunoradiometric assay for prostatic acid phosphatase in serum, based on a dual monoclonal antibody reaction system (Hybritech-TANDEM). A solidphase antibody binds the acid phosphatase molecule and a second monoclonal antibody to a different antigenic site serves as the /sup 125/I-radiolabel. The method was tested on 67 patients with various stages of prostatic carcinoma and 134 patients without the disease. It also was compared with a conventional polyclonal radioimmunoassay (NEN) and an enzymatic activity method (duPont aca). The upper limit for the TANDEM assay on nondiseased male patients was found to be 2.0 microgram/L. Based on this upper limit of normal, the diagnostic sensitivity of the method for all cases of prostatic carcinoma was 60%. Researchs could not distinguish the enzyme released in abnormal amounts due to benign prostatic hypertrophy and certain nonprostatic malignant diseases from that of prostatic carcinoma. The diagnostic specificity was calculated at 95%. For the clinically undetectable Stage 1 disease, sensitivity was 44% (four abnormal values out of nine cases). The TANDEM procedure is simple to use and reproducible.

Davies, S.N.; Gochman, N.



Isolation of Trichinella-specific antigens for diagnosis by gradient monoclonal antibody affinity chromatography.  


Monoclonal antibodies were generated for the isolation of specific antigens from Trichinella spiralis. Two monoclonal antibodies (7G6-2 and 10B6-1) of class IgG2b and IgG1 were selected according to their reactivities in an enzyme-linked immunosorbent assay and western blot. Clone 7G6-2 reacted with an antigen with molecular mass of approximately 60 kDa, and clone 10B6-1 bound to multiple antigens ranging from 49 to 62 kDa on western blot. Antibodies of each clone were purified partially from mouse ascites fluid by ammonium sulfate precipitation and were coupled to CNBr-activated Sepharose 4B. Antigens with molecular masses of 49 kDa and 57 kDa (P49/57), 52-62 kDa (P52/62), and 60 kDa (P60) were isolated from larval excretory-secretory products and crude worm extract with column 10B6-1 and column 7G6-2, respectively, in part by changing the pH of elution buffers. These antigens were mostly glycoproteins, strongly immunogenic, and specific to the parasite. PMID:2254818

Su, X Z; Prestwood, A K



Monoclonal antibodies as probes for plasma membrane domains in the exocrine pancreas.  


Monoclonal antibodies (mAb) were generated as probes for the plasma membrane domains of pancreatic acinar cells. Primary monolayer cultures of mouse pancreatic acinar cells, which have an expanded apical surface relative to normal pancreas, were used to immunize rats. With conventional immunization and fusion protocols, 3% of the hybridomas were positive against the acinar lumen by indirect immunofluorescence of mouse pancreas cryosections. Culturing of spleen cells from an immunized rat on the apical surface of acinar cell monolayer cultures before fusion with the myeloma (an in vitro boost) doubled the percentage of hybridomas producing apical membrane-specific mAb. Monoclonal antibodies were characterized by immunofluorescence, ultrastructural immunoperoxidase cytochemistry, immunoprecipitation, and immunoblotting. One antibody, acinar-1 (IgG2a), labeled the apical membranes of pancreatic acinar cells, hepatocytes, salivary and lacrimal gland acinar cells, and the brush border of small intestine enterocytes. This mAb precipitated and blotted a protein of 94 KD. Acinar-2 (IgM) also labeled pancreatic acinar cell apical membranes but did not label other tissues and did not precipitate or blot. Acinar-3 labeled pancreatic acinar cell lateral membranes. Duct-1 (IgM) labeled pancreatic duct apical membrane and ducts in liver and salivary glands but did not precipitate or blot. These domain-specific mAb demonstrate that common antigenic determinants occur in the apical surfaces of several exocrine epithelia and may be important in secretion. PMID:3292643

De Lisle, R C; Logsdon, C D; Hootman, S R; Williams, J A



Purification of monoclonal antibodies from whole hybridoma fermentation broth by fluidized bed adsorption.  


To achive the coarse purification of a monoclonal antibody from whole hybridoma fermentation broth a fluidized bed cation exchange process was used. The procedure consisted of application of the crude sample and washing of the bed in a fluidized mode and elution in a fixed bed mode. A completely clarified eluate was obtained with purification factors between 4 and 8 and a concentration of the desired product (monoclonal antibody) by a factor of more than 3 was achived. Thus, a combination of the three early steps of the downstream process clarification, concentration and coarse purification was possible. Two different materials were tested: a commercially available agarose-based matrix (Stream-line-SP), and a self-derivatized material based on controlled-pore glass (Bioran). Initial experiments were performed to describe the fluidization of the glass material. Comparison with the agarose material showed several differences, the agarose matrix allowing liquid flow closer to plug flow than the glass material. Increased backmixing in the liquid phase was detected when fluidizing the glass adsorbent compared with the agarose-based matrix. Despite this fact, comparison of the two materials with respect to antibody binding and elution demonstrated a similar performance. (c) 1995 John Wiley & Sons, Inc. PMID:18623139

Thömmes, J; Halfar, M; Lenz, S; Kula, M R



HLA-DR monoclonal antibodies inhibit the proliferation of normal and chronic granulocytic leukaemia myeloid progenitor cells.  


We studied the capacity of murine monoclonal antibodies with HLA-DR specificity to inhibit the proliferation in vitro of erythroid (BFU-E and CFU-E) and granulocyte-macrophage (CFU-GM) progenitor cells in normal bone marrow and the blood of patients with chronic granulocytic leukaemia (CGL). Two IgG2 antibodies (CA 2.06 and L243) inhibited the proliferation of normal BFU-E and CFU-GM at relatively high dilution; a third antibody, DA2, had no effect on either progenitor cell. A complement-fixing monoclonal antibody with T-cell activity (OKT3) produced only minor reduction in progenitor cel proliferation. Further studies with L243 showed that BFU-E and CFU-GM from the blood of patients with CGL were inhibited to the same degree as normal marrow progenitor cells. The inhibition of progenitor cell proliferation by a given antibody was always complement dependent and was therefore presumed to be due to a direct cytotoxic effect. The inhibitory effect of monoclonal antibodies is a valuable approach to the characterization of antigenic determinants on myeloid progenitor cells and the differential cytotoxicity of selected monoclonal antibodies might be exploitable for therapy. PMID:6957244

Goldman, J M; Hibbin, J; Kearney, L; Orchard, K; Th'ng, K H



Detection of hypoxic cells by monoclonal antibody recognizing 2-nitroimidazole adducts.  


Hypoxic cells in tissue pose many medical problems, and there is a need for more accurate measurements of tissue hypoxia. However, measurement of the pO2 and the extent of hypoxia within normal and tumor tissue have proven difficult. One of the most sensitive of the currently available methodologies involves the oxygen-dependent metabolic activation of nitroheterocyclic drugs, leading to adducts between the drugs and cellular macromolecules. Limitations of the present drugs and adduct-detection methods prompted the present studies. A pentafluorinated derivative [EF5; 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetam ide] of etanidazole was synthesized with the expectation of lessening some of the non-oxygen-dependent variability in adduct formation observed previously with other nitroaromatic compounds. EF5-protein conjugates, prepared by radiochemical reduction, were found to be immunogenic and allowed the development of monoclonal antibodies. One of these antibodies, ELK2-4, has been characterized and found to be highly specific for the EF5 adducts whether produced radiochemically or by cellular bioreductive metabolism. 9L rat glioma cells pretreated with EF5 under hypoxic, compared with aerobic, conditions were readily discriminated immunochemically using fluorochrome-conjugated secondary antibodies which recognize the ELK2-4 antibody subtype (IgG1). Similarly, the central region of multicenter spheroids, composed of EMT6 mouse mammary sarcoma cells, was selectively visualized by immunohistochemistry after the spheroids were incubated for 4 h in 0.5 mM EF5. Tumor biopsy, preparation, and immunohistochemical staining 24 h after treatment of tumor-bearing animals with drug also demonstrated high contrast regions within EMT6 mouse or Morris 7777 hepatoma rat tumors. The use of this new compound and its highly specific monoclonal antibody may allow elucidation of bioreductive metabolism of the nitroheterocyclics and significantly improve technologies for the quantitation of tissue pO2. PMID:8242628

Lord, E M; Harwell, L; Koch, C J



Analysis of Memory B Cell Responses and Isolation of Novel Monoclonal Antibodies with Neutralizing Breadth from HIV1Infected Individuals  

Microsoft Academic Search

BackgroundThe isolation of human monoclonal antibodies (mAbs) that neutralize a broad spectrum of primary HIV-1 isolates and the characterization of the human neutralizing antibody B cell response to HIV-1 infection are important goals that are central to the design of an effective antibody-based vaccine.Methods and FindingsWe immortalized IgG+ memory B cells from individuals infected with diverse clades of HIV-1 and

Davide Corti; Johannes P. M. Langedijk; Andreas Hinz; Michael S. Seaman; Fabrizia Vanzetta; Blanca M. Fernandez-Rodriguez; Chiara Silacci; Debora Pinna; David Jarrossay; Sunita Balla-Jhagjhoorsingh; Betty Willems; Maria J. Zekveld; Hanna Dreja; Eithne O'Sullivan; Corinna Pade; Chloe Orkin; Simon A. Jeffs; David C. Montefiori; David Davis; Winfried Weissenhorn; Áine McKnight; Jonathan L. Heeney; Federica Sallusto; Quentin J. Sattentau; Robin A. Weiss; Antonio Lanzavecchia; Derya Unutmaz