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Local antitumour treatment in carcinoma patients with bispecific-monoclonal-antibody-redirected T cells  

Microsoft Academic Search

In a pilot clinical study carcinoma patients with malignant ascites or pleural exudates have been treated locally with autologous lymphocytes activated ex vivo and redirected towards tumour cells with bispecific monoclonal antibodies. BIS-1, the bispecific monoclonal antibody used in this study, combines specificity against a tumour-associated antigen, AMOC-31, present on carcinomas, with a specificity against the CD3 complex on T

B. J. Kroesenl; A. ter Haar; H. Spakman; P. Willemse; D. Th. Sleijfer; E. G. E. de Vries; N. H. Mulder; H. H. Berendsen; P. C. Limburg; L. de Leij



Production of a bi-specific monoclonal antibody recognizing mouse kappa light chains and horseradish peroxidase  

Microsoft Academic Search

The production of a bi-specific monoclonal antibody that simultaneously recognizes mouse kappa light chains and horseradish peroxidase (HRP) for use as a general developing reagent in a wide variety of immunobased techniques is described. This antibody, named McC10, was produced by the fusion of an aminopterin-sensitive interspecies hybridoma which secretes rat monoclonal antibodies against HRP (RAP2·Ag) and splenocytes from a

R. L. Kenigsberg; A. C. Cuello



Antitumor immunity: easy as 1, 2, 3 with monoclonal bispecific trifunctional antibodies?  


Monoclonal antibodies occupy an increasing niche in the arsenal available to treat cancer. Several developments have rendered this the fastest growing sector in the pharmaceutical industry. Traditionally, antibodies were developed to block key signaling molecules implicated in tumor progression. However, antibodies also recruit additional immune effector mechanisms against tumors, a property that may be exploited for clinical benefit. Bispecific antibodies represent one such strategy in which elements derived from two monoclonal antibodies are incorporated into a single molecular species. Commonly, the bispecific approach is used to achieve simultaneous cross-linking of CD3 and a tumor antigen such as epithelial cell adhesion molecule (EpCAM), thereby recruiting T-cell activation to the tumor cell surface. A further sophistication involves the engineering of trifunctional derivatives such as the clinically approved agent, catumaxomab. Catumaxomab has antigen-binding arms that engage CD3 and EpCAM and a constant domain that recruits Fc receptor-bearing cells, notably monocytes, dendritic cells, and natural killer cells. Owing to this triangular binding capability, catumaxomab can activate both innate and adaptive immune effector mechanisms in addition to promoting immunologic memory. Recent data indicate that this agent can also promote immunogenic cell death, particularly when used in combination with selected chemotherapeutic agents such as oxaliplatin. Cancer Res; 73(18); 5613-7. ©2013 AACR. PMID:24014596

Maher, John; Adami, Antonella A



Generation of chimeric bispecific G250/anti-CD3 monoclonal antibody, a tool to combat renal cell carcinoma.  

PubMed Central

The monoclonal antibody (MAb) G250 binds to a tumour-associated antigen, expressed in renal cell carcinoma (RCC), which has been demonstrated to be a suitable target for antibody-mediated immunotherapy. A bispecific antibody having both G250 and anti-CD3 specificity can cross-link G250 antigen-expressing RCC target cells with T cells and can mediate lysis of such targets. Therapy studies with murine antibodies are limited by immune responses to the antibodies injected (HAMA response), which can be decreased by using chimeric antibodies. We generated a chimeric bispecific G250/anti CD3 MAb by transfecting chimeric genes of heavy and light chains for both the G250 MAb and the anti-CD3 MAb into a myeloma cell line. Cytotoxicity assays revealed that the chimeric bispecific MAb was capable of mediating lysis of RCC cell lines by cloned human CD8+T cells or by IL-2-stimulated peripheral blood lymphocytes (PBLs). Lysis mediated by the MAb was specific for target cells that expressed the G250 antigen and was effective at concentrations as low as 0.01 microgram ml-1. The chimeric bispecific G250/anti-CD3 MAb produced may be an effective adjuvant to the currently used IL-2-based therapy of advanced renal cell arcinoma. Images Figure 7

Luiten, R. M.; Coney, L. R.; Fleuren, G. J.; Warnaar, S. O.; Litvinov, S. V.



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



Quantitative and sensitive detection of the SARS-CoV spike protein using bispecific monoclonal antibody-based enzyme-linked immunoassay.  


The severe acute respiratory syndrome coronavirus (SARS-CoV) spike protein is known to mediate receptor interaction and immune recognition and thus it is considered as a major target for vaccine design. The spike protein plays an important role in virus entry, virus receptor interactions, and virus tropism. Sensitive diagnosis of SARS is essential for the control of the disease in humans. Recombinant SARS-CoV S1 antigen was produced and purified for the development of monoclonal and bi-specific monoclonal antibodies. The hybridomas secreting anti-S1 antibodies, F26G18 and P136.8D12, were fused respectively with the YP4 hybridoma to generate quadromas. The sandwich ELISA was formed by using F26G18 as a coating antibody and biotinylated F26G18 as a detection antibody with a detection limit of 0.037?g/ml (p<0.02). The same detection limit was found with P136.8D12 as a coating antibody and biotinylated F26G18 as a detection antibody. The sensitivity was improved (detection limit of 0.019?g/ml), however, when using bi-specific monoclonal antibody (F157) as the detection antibody. In conclusion, the method described in this study allows sensitive detection of a recombinant SARS spike protein by sandwich ELISA with bi-specific monoclonal antibody and could be used for the diagnosis of patients suspected with SARS. PMID:22995576

Sunwoo, Hoon H; Palaniyappan, Arivazhagan; Ganguly, Advaita; Bhatnagar, Pravin K; Das, Dipankar; El-Kadi, Ayman O S; Suresh, Mavanur R



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.



Chemical generation of bispecific antibodies  

PubMed Central

Bispecific antibodies (BsAbs) are regarded as promising therapeutic agents due to their ability to simultaneously bind two different antigens. Several bispecific modalities have been developed, but their utility is limited due to problems with stability and manufacturing complexity. Here we report a versatile technology, based on a scaffold antibody and pharmacophore peptide heterodimers, that enables rapid generation and chemical optimization of bispecific antibodies, which are termed bispecific CovX-Bodies. Two different peptides are joined together using a branched azetidinone linker and fused to the scaffold antibody under mild conditions in a site-specific manner. Whereas the pharmacophores are responsible for functional activities, the antibody scaffold imparts long half-life and Ig-like distribution. The pharmacophores can be chemically optimized or replaced with other pharmacophores to generate optimized or unique bispecific antibodies. As a prototype, we developed a bispecific antibody that binds both vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang2) simultaneously, inhibits their function, shows efficacy in tumor xenograft studies, and greatly augments the antitumor effects of standard chemotherapy. This unique antiangiogenic bispecific antibody is in phase-1 clinical trials.

Doppalapudi, Venkata R.; Huang, Jie; Liu, Dingguo; Jin, Ping; Liu, Bin; Li, Lingna; Desharnais, Joel; Hagen, Crystal; Levin, Nancy J.; Shields, Michael J.; Parish, Michelle; Murphy, Robert E.; Del Rosario, Joselyn; Oates, Bryan D.; Lai, Jing-Yu; Matin, Marla J.; Ainekulu, Zemeda; Bhat, Abhijit; Bradshaw, Curt W.; Woodnutt, Gary; Lerner, Richard A.; Lappe, Rodney W.



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


Catumaxomab, a rat/murine hybrid trifunctional bispecific monoclonal antibody for the treatment of cancer.  


Fresenius Biotech GmbH (licensed from TRION Pharma GmbH) is developing catumaxomab, a rat/murine hybrid, trifunctional, bispecific (anti-epithelial cell adhesion molecule and anti-CD3) mAb for the potential intravenous and intraperitoneal treatment of ovarian cancer, gastric cancer, malignant pleural effusion and other cancers, and also for malignant ascites associated with various cancers. In 2007, following the successful completion of a phase II/III clinical trial in patients with malignant ascites from various cancers, Fresenius filed for European approval. Catumaxomab is currently undergoing phase II clinical trials in patients with malignant ascites, ovarian and stomach cancer. PMID:18535935

Shen, Juqun; Zhu, Zhenping



Bispecific antibodies in cancer therapy  

Microsoft Academic Search

Based upon in vitro and animal studies, a number of Phase I and II clinical trials have been initiated to test whether bispecific antibodies could redirect immune effectors against tumor cells in cancer patients. Recently, results from those trials showed beneficial effects in some patients but it is clear many problems remain to be solved. In addition, molecular engineering approaches

David M Segal; George J Weiner; Louis M Weiner



Catumaxomab: a bispecific trifunctional antibody.  


The trifunctional bispecific monoclonal antibody catumaxomab has two binding specificities directed at epithelial cell adhesion molecule (EpCAM) and the T-cell antigen CD3. With its Fc-fragment, catumaxomab additionally binds accessory cells such as dendritic cells, macrophages and natural killer cells. The trifunctional approach thus leads to unrestricted but specific killing of epithelial tumor cells by major histocompatibility complex without the need for preactivation or external costimulation. The tumor-associated antigen EpCAM is strongly expressed in carcinomas of various origins including colon, rectum, ovarian, gastric, esophagus, lung, pancreas, breast and head and neck. Expression of EpCAM is often associated with an unfavorable prognosis in patients with breast cancer. Catumaxomab has been approved in Europe for the intraperitoneal treatment of malignant ascites in patients with EpCAM-positive epithelial tumors when standard therapy is not available or is no longer feasible. Basic preclinical and clinical findings with different routes of catumaxomab administration in various indications are summarized and discussed in this review. PMID:19927225

Sebastian, M; Kuemmel, A; Schmidt, M; Schmittel, A



Recombinant Bispecific Antibodies for Cancer Therapy  

Microsoft Academic Search

\\u000a Bispecific antibodies are molecules capable of simultaneously binding to two different antigens. While initially bispecific\\u000a antibodies have been developed mainly for cellular cancer immunotherapy through retargeting of effector cells to tumor cells,\\u000a recent developments include also dual targeting strategies and the retargeting of effector molecules, e.g. in radioimmunotherapy.\\u000a In addition to various applications, a plethora of bispecific antibody formats have

Dafne Müller; Roland E. Kontermann


Cancer therapy with bispecific antibodies: Clinical experience  

PubMed Central

The binding of at least two molecular targets simultaneously with a single bispecific antibody is an attractive concept. The use of bispecific antibodies as possible therapeutic agents for cancer treatment was proposed in the mid-1980s. The design and production of bispecific antibodies using antibody- and/or receptor-based platform technology has improved significantly with advances in the knowledge of molecular manipulations, protein engineering techniques, and the expression of antigens and receptors on healthy and malignant cells. The common strategy for making bispecific antibodies involves combining the variable domains of the desired mAbs into a single bispecific structure. Many different formats of bispecific antibodies have been generated within the research field of bispecific immunotherapeutics, including the chemical heteroconjugation of two complete molecules or fragments of mAbs, quadromas, F(ab’)2, diabodies, tandem diabodies and single-chain antibodies. This review describes key modifications in the development of bispecific antibodies that can improve their efficacy and stability, and provides a clinical perspective on the application of bispecific antibodies for the treatment of solid and liquid tumors, including the promises and research limitations of this approach.

Thakur, Archana; Lum, Lawrence G



Bispecific monoclonal antibody-mediated targeting of an indium-111-labeled DTPA dimer to primary colorectal tumors: pharmacokinetics, biodistribution, scintigraphy and immune response.  


Eleven patients with primary colorectal carcinoma tumors (4 +/- 2 cm) were given intravenous injections of 1-10 mg of an anti-CEA, anti-In-DTPA bispecific Fab'-Fab monoclonal antibody, and 2-8 days later, were injected with 1.2-4.2 nmol of an 111In-labeled DTPA dimer (6 mCi). The bispecific antibody exhibited good stability and F(ab)'2-like pharmacokinetics. After injection, the 111In-DTPA dimer distributed in a large volume (88 ml/kg-180 ml/kg) and cleared through the kidneys (mean residence time in the whole body: 9 hr-16 hr). Uptake of 111In by the tumor using this two-step technique (1.8%-17.5% injected dose ID/kg, measured from surgical samples 48 hr after hapten injection) was not found significantly lower than that achieved with our reference 111In-labeled anti-CEA F(ab)'2 1 to 4 days after injection in six patients with similar clinical status (5.5%-30.2% ID/kg). In addition, tumor-to-blood and tumor-to-liver uptake ratios were significantly improved (blood 7.8 versus 4.2, liver 2.8 versus 0.8). As a result, low background images allowed detection of 12 of 13 lesions, 4 hr and 24 hr after hapten injection. However, 7 of 11 patients developed HAMA. PMID:8410279

Le Doussal, J M; Chetanneau, A; Gruaz-Guyon, A; Martin, M; Gautherot, E; Lehur, P A; Chatal, J F; Delaage, M; Barbet, J



Time Resolved Native Ion-Mobility Mass Spectrometry to Monitor Dynamics of IgG4 Fab Arm Exchange and "Bispecific" Monoclonal Antibody Formation.  


Monoclonal antibodies (mAbs) and derivatives such as antibody-drug conjugates (ADC) and bispecific antibodies (bsAb), are the fastest growing class of human therapeutics. Most of the therapeutic antibodies currently on the market and in clinical trials are chimeric, humanized, and human immunoglobulin G1 (IgG1). An increasing number of IgG2s and IgG4s that have distinct structural and functional properties are also investigated to develop products that lack or have diminished antibody effector functions compared to IgG1. Importantly, wild type IgG4 has been shown to form half molecules (one heavy chain and one light chain) that lack interheavy chain disulfide bonds and form intrachain disulfide bonds. Moreover, IgG4 undergoes a process of Fab-arm exchange (FAE) in which the heavy chains of antibodies of different specificities can dissociate and recombine in bispecific antibodies both in vitro and in vivo. Here, native mass spectrometry (MS) and time-resolved traveling wave ion mobility MS (TWIM-MS) were used for the first time for online monitoring of FAE and bsAb formation using Hz6F4-2v3 and natalizumab, two humanized IgG4s which bind to human Junctional Adhesion Molecule-A (JAM-A) and alpha4 integrin, respectively. In addition, native MS analysis of bsAb/JAM-A immune complexes revealed that bsAb can bind up to two antigen molecules, confirming that the Hz6F4 family preferentially binds dimeric JAM-A. Our results illustrate how IM-MS can rapidly assess bsAb structural heterogeneity and be easily implemented into MS workflows for bsAb production follow up and bsAb/antigen complex characterization. Altogether, these results provide new MS-based methodologies for in-depth FAE and bsAb formation monitoring. Native MS and IM-MS will play an increasing role in next generation biopharmaceutical product characterization like bsAbs, antibody mixtures, and antibody-drug conjugates (ADC) as well as for biosimilar and biobetter antibodies. PMID:24007193

Debaene, François; Wagner-Rousset, Elsa; Colas, Olivier; Ayoub, Daniel; Corvaïa, Nathalie; Van Dorsselaer, Alain; Beck, Alain; Cianférani, Sarah



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.

Dhimolea, Eugen



Capture of endothelial progenitor cells by a bispecific protein\\/monoclonal antibody molecule induces reendothelialization of vascular lesions  

Microsoft Academic Search

Tissue injury is inevitably accompanied by disruption of the endothelium and exposure of the subendothelial matrix. To generate\\u000a a guidance molecule directing progenitor cells to sites of vascular lesions, we designed a bifunctional protein. The protein\\u000a consists of the soluble platelet collagen receptor glycoprotein VI and an antibody to CD133 (hereafter called GPVI-CD133).\\u000a In vitro and in vivo, this construct

Harald F. Langer; Jürgen W. von der Ruhr; Karin Daub; Tanja Schoenberger; Konstantinos Stellos; Andreas E. May; Hannah Schnell; Alexandra Gauß; Ramona Hafner; Peter Lang; Michael Schumm; Hans-Jörg Bühring; Karin Klingel; Sabine Conrad; Martin Schaller; Marc van Zandvoort; Gundram Jung; Stefanie Dimmeler; Thomas Skutella; Meinrad Gawaz



Monoclonal Antibodies  

Microsoft Academic Search

Over the last decades, progress has been made in diagnostic imaging, surgical techniques, radiotherapy, and chemotherapy for\\u000a the treatment of tumors of the central nervous system. However, the outcome for patients with high-grade gliomas (HGG) has\\u000a remained essentially unchanged. The use of monoclonal antibodies (MAbs), either unarmed or armed, to target and kill HGG cells\\u000a has appeared as a prospective

Abraham Boskovitz; David A. Reardon; Carol J. Wikstrand; Michael R. Zalutsky; Darell D. Bigner


Bispecific digoxigenin-binding antibodies for targeted payload delivery  

PubMed Central

Bispecific antibodies that bind cell-surface targets as well as digoxigenin (Dig) were generated for targeted payload delivery. Targeting moieties are IgGs that bind the tumor antigens Her2, IGF1R, CD22, or LeY. A Dig-binding single-chain Fv was attached in disulfide-stabilized form to C termini of CH3 domains of targeting antibodies. Bispecific molecules were expressed in mammalian cells and purified in the same manner as unmodified IgGs. They are stable without aggregation propensity and retain binding specificity/affinity to cell-surface antigens and Dig. Digoxigeninylated payloads were generated that retain full functionality and can be complexed to bispecific antibodies in a defined 2?1 ratio. Payloads include small compounds (Dig-Cy5, Dig-Doxorubicin) and proteins (Dig-GFP). Complexed payloads are targeted by the bispecifics to cancer cells and because these complexes are stable in serum, they can be applied for targeted delivery. Because Dig bispecifics also effectively capture digoxigeninylated compounds under physiological conditions, separate administration of uncharged Dig bispecifics followed by application of Dig payload is sufficient to achieve antibody-mediated targeting in vitro and in vivo.

Metz, Silke; Haas, Alexander K.; Daub, Karin; Croasdale, Rebecca; Stracke, Jan; Lau, Wilma; Georges, Guy; Josel, Hans-Peter; Dziadek, Sebastian; Hopfner, Karl-Peter; Lammens, Alfred; Scheuer, Werner; Hoffmann, Eike; Mundigl, Olaf; Brinkmann, Ulrich



Targeting T Cells with Bispecific Antibodies for Cancer Therapy  

PubMed Central

Bispecific antibodies (BiAbs) offer a unique opportunity to redirect immune effector cells to kill cancer cells. BiAbs combine the benefits of different binding specificities of two monoclonal antibodies (mAbs) into a single construct. This unique feature of BiAbs enables approaches that are not possible with single mAbs. Advances in antibody engineering and antigen profiling of malignant cells have led to the development of a number of BiAb formats and their combinations for redirecting effector cells to tumor targets. There have been significant advances in the design and application of BiAbs for intravenous and local injection. The initial barrier of cytokine storm has been partially overcome by more recent constructs that have improved clinical effectiveness without dose-limiting toxicities. Since the recent revival of BiAbs, there has been multiple, ongoing, phase I/II and III trials, and some promising clinical outcomes have been reported in completed clinical studies. This review focuses on arming T cells with BiAbs to create the ‘poor man's cytotoxic lymphocyte’.

Lum, Lawrence G.; Thakur, Archana



Bispecific antibodies with natural architecture produced by co-culture of bacteria expressing two distinct half-antibodies.  


By enabling the simultaneous engagement of two distinct targets, bispecific antibodies broaden the potential utility of antibody-based therapies. However, bispecific-antibody design and production remain challenging, owing to the need to incorporate two distinct heavy and light chain pairs while maintaining natural nonimmunogenic antibody architecture. Here we present a bispecific-antibody production strategy that relies on co-culture of two bacterial strains, each expressing a half-antibody. Using this approach, we produce 28 unique bispecific antibodies. A bispecific antibody against the receptor tyrosine kinases MET and EGFR binds both targets monovalently, inhibits their signaling, and suppresses MET and EGFR-driven cell and tumor growth. Our strategy allows rapid generation of bispecific antibodies from any two existing antibodies and yields milligram to gram quantities of bispecific antibodies sufficient for a wide range of discovery and preclinical applications. PMID:23831709

Spiess, Christoph; Merchant, Mark; Huang, Arthur; Zheng, Zhong; Yang, Nai-Ying; Peng, Jing; Ellerman, Diego; Shatz, Whitney; Reilly, Dorothea; Yansura, Daniel G; Scheer, Justin M



Quantification of cell surface proteins with bispecific antibodies  

PubMed Central

Flow cytometry is an established method for fast and accurate quantitation of cellular protein levels and requires fluorescently labeled antibodies as well as calibration standards. A critical step for quantitation remains the production of suitable detection antibodies with a precisely defined ratio of antigen-binding sites to fluorophores. Problems often arise as a consequence of inefficient and unspecific labeling which can influence antibody properties. In addition, the number of incorporated fluorophores necessitates a special normalization step for quantitation. To address these problems, we constructed different mono- and bivalent bispecific antibodies with binding site(s) for the cell surface antigens, cMET, EGFR1/HER1, ErbB2/HER2 or ErbB3/HER3 and with an additional digoxigenin-binding single-chain Fv fusion. The fluorophore Cy5 was covalently coupled to digoxigenin and quantitatively bound by the bispecific antibody. A panel of tumor cell lines was assessed under different culture conditions for absolute receptor expression levels of the indicated antigens and the data were set in relation to mRNA, gene count and immunoblot data. We could reproducibly quantify these receptors, omit the otherwise required normalization step and demonstrate the superiority of a 1 + 1 bispecific antibody. The same antibodies were also used to quantify the number of proteins in intracellular vesicles in confocal microscopy. The antibodies can be stored like regular antibodies and can be coupled with different digoxigenin-labeled fluorophores which makes them excellent tools for FACS and imaging-based experiments.

Panke, C.; Weininger, D.; Haas, A.; Schelter, F.; Schlothauer, T.; Bader, S.; Sircar, R.; Josel, H.P.; Baer, U.; Burtscher, H.; Mundigl, O.; Grote, M.; Brinkmann, U.; Sustmann, C.



Bispecific antibody-targeted phagocytosis of HER2\\/ neu expressing tumor cells by myeloid cells activated in vivo  

Microsoft Academic Search

Studies from our laboratory and others have established that both mononuclear phagocytes and neutrophils mediate very efficient cytotoxicity when targeted through Fc receptors using a suitable monoclonal or bispecific antibody (BsAb). Cross-linking an Fc receptor for IgG (Fc?R) triggers multiple anti-tumor activities including superoxide generation, cytokine and enzyme release, phagocytosis and antibody-dependent cellular cytotoxicity (ADCC). In this report, using unfractionated

Paul K. Wallace; Peter A. Kaufman; Lionel D. Lewis; Tibor Keler; Alice L. Givan; Jan L. Fisher; Mary G. Waugh; Andrea E. Wahner; Paul M. Guyre; Michael W. Fanger; Marc S. Ernstoff



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



Quantification of cell surface proteins with bispecific antibodies.  


Flow cytometry is an established method for fast and accurate quantitation of cellular protein levels and requires fluorescently labeled antibodies as well as calibration standards. A critical step for quantitation remains the production of suitable detection antibodies with a precisely defined ratio of antigen-binding sites to fluorophores. Problems often arise as a consequence of inefficient and unspecific labeling which can influence antibody properties. In addition, the number of incorporated fluorophores necessitates a special normalization step for quantitation. To address these problems, we constructed different mono- and bivalent bispecific antibodies with binding site(s) for the cell surface antigens, cMET, EGFR1/HER1, ErbB2/HER2 or ErbB3/HER3 and with an additional digoxigenin-binding single-chain Fv fusion. The fluorophore Cy5 was covalently coupled to digoxigenin and quantitatively bound by the bispecific antibody. A panel of tumor cell lines was assessed under different culture conditions for absolute receptor expression levels of the indicated antigens and the data were set in relation to mRNA, gene count and immunoblot data. We could reproducibly quantify these receptors, omit the otherwise required normalization step and demonstrate the superiority of a 1 + 1 bispecific antibody. The same antibodies were also used to quantify the number of proteins in intracellular vesicles in confocal microscopy. The antibodies can be stored like regular antibodies and can be coupled with different digoxigenin-labeled fluorophores which makes them excellent tools for FACS and imaging-based experiments. PMID:23960142

Panke, C; Weininger, D; Haas, A; Schelter, F; Schlothauer, T; Bader, S; Sircar, R; Josel, H P; Baer, U; Burtscher, H; Mundigl, O; Grote, M; Brinkmann, U; Sustmann, C



Cancer Imaging and Therapy with Bispecific Antibody Pretargeting  

PubMed Central

This article reviews recent preclinical and clinical advances in the use of pretargeting methods for the radioimmunodetection and radioimmunotherapy of cancer. Whereas directly-labeled antibodies, fragments, and subfragments (minibodies and other constructs) have shown promise in both imaging and therapy applications over the past 25 years, their clinical adoption has not fulfilled the original expectations due to either poor image resolution and contrast in scanning or insufficient radiation doses delivered selectively to tumors for therapy. Pretargeting involves the separation of the localization of tumor with an anticancer antibody from the subsequent delivery of the imaging or therapeutic radionuclide. This has shown improvements in both imaging and therapy by overcoming the limitations of conventional, or 1-step, radioimmunodetection or radioimmunotherapy. We focus herein on the use of bispecific antibodies followed by radiolabeled peptide haptens as a new modality of selective delivery of radionuclides for the imaging and therapy of cancer. Our particular emphasis in pretargeting is the use of bispecific trimeric (3 Fab’s) recombinant constructs made by a modular method of antibody and protein engineering of fusion molecules called Dock and Lock (DNL).

Goldenberg, David M.; Chatal, Jean-Francois; Barbet, Jacques; Boerman, Otto; Sharkey, Robert M.



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



Camel Single-domain Antibodies as Modular Building Units in Bispecific and Bivalent Antibody Constructs  

Microsoft Academic Search

Single-domain antibodies against various antigens are isolated from the unique heavy-chain antibodies of immunized camels and llamas. These minimal sized binders are very robust and bind the antigen with high affinity in a monomeric state. We evaluated the feasibil- ity to produce soluble, functional bispecific and bivalent antibodies in Escherichia coli with camel single-domain antibody fragments as building blocks. Two

Katja Els Conrath; Mark Lauwereys; Lode Wyns; Serge Muyldermans



Bispecific Antibodies that Mediate Killing of Cells Infected with Human Immunodeficiency Virus of Any Strain  

Microsoft Academic Search

Although AIDS patients lose human immunodeficiency virus (HIV)-specific cytotoxic T cells, their remaining CD8-positive T lymphocytes maintain cytotoxic function. To exploit this fact we have constructed bispecific antibodies that direct cytotoxic T lymphocytes of any specificity to cells that express gp120 of HIV. These bispecific antibodies comprise one heavy\\/light chain pair from an antibody to CD3, linked to a heavy

Jorg Berg; Erika Lotscher; Kathelyn S. Steimer; Daniel J. Capon; Jurg Baenziger; Hans-Martin Jack; Matthias Wabl



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



Targeting of anti-tumor responses with bispecific antibodies.  


T cells can be induced to specifically lyse tumor cells with bispecific antibodies containing anti-T cell receptor mAbs crosslinked to anti-tumor mAbs. Such "targeted cytolysis" requires that the target cell be bound directly to the cytotoxic cell. In addition, targeted T cells mediate a second activity, the secretion of factors that can block the growth of both tumor target cells and bystander tumor cells. When given to nude mice bearing intraperitoneal human ovarian carcinoma, targeted human T cells cause the rapid removal of most tumor cells from the peritoneum, and markedly prolong the times of survival of treated mice. The efficacy of targeted T cells for treating human cancer is currently being tested in clinical trials. PMID:1452212

Segal, D M; Qian, J H; Mezzanzanica, D; Garrido, M A; Titus, J A; Andrew, S M; George, A J; Jost, C R; Perez, P; Wunderlich, J R



Development of a Human IgG4 Bispecific Antibody for Dual Targeting of Interleukin-4 (IL-4) and Interleukin-13 (IL-13) Cytokines.  


Human bispecific antibodies have great potential for the treatment of human diseases. Although human IgG1 bispecific antibodies have been generated, few attempts have been reported in the scientific literature that extend bispecific antibodies to other human antibody isotypes. In this paper, we report our work expanding the knobs-into-holes bispecific antibody technology to the human IgG4 isotype. We apply this approach to generate a bispecific antibody that targets IL-4 and IL-13, two cytokines that play roles in type 2 inflammation. We show that IgG4 bispecific antibodies can be generated in large quantities with equivalent efficiency and quality and have comparable pharmacokinetic properties and lung partitioning, compared with the IgG1 isotype. This work broadens the range of published therapeutic bispecific antibodies with natural surface architecture and provides additional options for the generation of bispecific antibodies with differing effector functions through the use of different antibody isotypes. PMID:23880771

Spiess, Christoph; Bevers, Jack; Jackman, Janet; Chiang, Nancy; Nakamura, Gerald; Dillon, Michael; Liu, Hongbin; Molina, Patricia; Elliott, J Michael; Shatz, Whitney; Scheer, Justin M; Giese, Glen; Persson, Josefine; Zhang, Yin; Dennis, Mark S; Giulianotti, James; Gupta, Prateek; Reilly, Dorothea; Palma, Enzo; Wang, Jianyong; Stefanich, Eric; Scheerens, Heleen; Fuh, Germaine; Wu, Lawren C



Generating bispecific human IgG1 and IgG2 antibodies from any antibody pair.  


Bispecific antibodies and antibody fragments are a new class of therapeutics increasingly utilized in the clinic for T cell recruitment (catumaxomab anti-EpCAM/CD3 and blinatumomab anti-CD19/CD3), increase in the selectivity of targeting, or simultaneous modulation of multiple cellular pathways. While the clinical potential for certain bispecific antibody formats is clear, progress has been hindered because they are often difficult to manufacture, may suffer from suboptimal pharmacokinetic properties, and may be limited due to potential immunogenicity issues. Current state-of-the-art human IgG-like bispecific technologies require co-expression of two heavy chains with a single light chain, use crossover domains to segregate light chains, or utilize scFv (single-chain fragment variable)-Fc fusion. We have engineered both human IgG1 and IgG2 subtypes, with minimal point mutations, to form full-length bispecific human antibodies with high efficiency and in high purity. In our system, the two antibodies of interest can be expressed and purified separately, mixed together under appropriate redox conditions, resulting in a formation of a stable bispecific antibody with high yields. With this approach, it is not necessary to generate new antibodies that share a common light chain, therefore allowing the immediate use of an existing antibody regardless of whether it has been generated via standard hybridoma or display methods. We demonstrate the generality of the approach and show that these bispecific antibodies have properties similar to those of wild-type IgGs, and we further demonstrate the utility of the technology with an example of a CD3/CD20 bispecific antibody that effectively depletes B cells in vitro and in vivo. PMID:22543237

Strop, Pavel; Ho, Wei-Hsien; Boustany, Leila M; Abdiche, Yasmina N; Lindquist, Kevin C; Farias, Santiago E; Rickert, Mathias; Appah, Charles Takeshi; Pascua, Edward; Radcliffe, Teresa; Sutton, Janette; Chaparro-Riggers, Javier; Chen, Wei; Casas, Meritxell Galindo; Chin, Sherman Michael; Wong, Oi Kwan; Liu, Shu-Hui; Vergara, German; Shelton, Dave; Rajpal, Arvind; Pons, Jaume



Bispecific Antibodies that Mediate Killing of Cells Infected with Human Immunodeficiency Virus of Any Strain  

NASA Astrophysics Data System (ADS)

Although AIDS patients lose human immunodeficiency virus (HIV)-specific cytotoxic T cells, their remaining CD8-positive T lymphocytes maintain cytotoxic function. To exploit this fact we have constructed bispecific antibodies that direct cytotoxic T lymphocytes of any specificity to cells that express gp120 of HIV. These bispecific antibodies comprise one heavy/light chain pair from an antibody to CD3, linked to a heavy chain whose variable region has been replaced with sequences from CD4 plus a second light chain. CD3 is part of the antigen receptor on T cells and is responsible for signal transduction. In the presence of these bispecific antibodies, T cells of irrelevant specificity effectively lyse HIV-infected cells in vitro.

Berg, Jorg; Lotscher, Erika; Steimer, Kathelyn S.; Capon, Daniel J.; Baenziger, Jurg; Jack, Hans-Martin; Wabl, Matthias



Structural understanding of stabilization patterns in engineered bispecific Ig-like antibody molecules.  


Bispecific immunoglobulin-like antibodies capable of engaging multiple antigens represent a promising new class of therapeutic agents. Engineering of these molecules requires optimization of the molecular properties of one of the domain components. Here, we present a detailed crystallographic and computational characterization of the stabilization patterns in the lymphotoxin-beta receptor (LTbetaR) binding Fv domain of an anti-LTbetaR/anti-TNF-related apoptosis inducing ligand receptor-2 (TRAIL-R2) bispecific immunoglobulin-like antibody. We further describe a new hierarchical structure-guided approach toward engineering of antibody-like molecules to enhance their thermal and chemical stability. PMID:19626705

Jordan, Jacob L; Arndt, Joseph W; Hanf, Karl; Li, Guohui; Hall, Janine; Demarest, Stephen; Huang, Flora; Wu, Xiufeng; Miller, Brian; Glaser, Scott; Fernandez, Erik J; Wang, Deping; Lugovskoy, Alexey



Monoclonal Antibodies against Pectin  

PubMed Central

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

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



Cooperative mixtures of bispecific F(ab')2 antibodies for delivering saporin to lymphoma in vitro and in vivo  

SciTech Connect

We report that selected combinations of two or more monoclonal bispecific F(ab')2 antibodies (BsAbs) far outperform single derivatives in the delivery of the ribosome-inactivating protein, saporin, to guinea pig L2C leukemic cells. Throughout the work, BsAbs were constructed by thioether-linking the hinges of two Fab'gamma, one from monoclonal anti-L2C-idiotype antibody and the other from anti-saporin antibody. The latter was either affinity-purified rabbit polyclonal or one of a panel of five mouse monoclonal antibodies. In vitro cytotoxicity studies showed that, though all derivatives were effective, the BsAb made with the polyclonal antibody was always 10 to 20 times more potent than those made with a monoclonal antibody in yielding 50% inhibition of (3H)leucine uptake. This superior activity could be matched by selective mixtures of two or more of the monoclonal derivatives. Furthermore, in immunotherapeutic delivery of saporin to tumor, a pair of BsAbs performed significantly better than did either individually. Binding and uptake studies with radiolabeled saporin demonstrated a 20-fold increase in functional affinity when saporin was held at the cell surface by an appropriate BsAb mixture rather than by a single BsAb. In contrast, only small differences were recorded in the rate at which saporin was internalized as a result of the same maneuver. We conclude that the improved performance of combinations of BsAbs arises from their ability to provide multiple linkages between saporin molecules and cell surfaces, increasing the functional affinity with which saporin is tethered to the cell, but, in this system at least, having only a minor effect on the rate at which it is internalized. Cocktails of two or more BsAbs, selected to bind to multiple epitopes on ribosome-inactivating proteins could provide an important new strategy in immunotherapy.

French, R.R.; Courtenay, A.E.; Ingamells, S.; Stevenson, G.T.; Glennie, M.J. (General Hospital, Southampton (England))



Application of bispecific antibody against antigen and hapten for immunodetection and immunopurification  

PubMed Central

We present a bispecific antibody that recognizes an antigen and a hapten and can be applied to various biological assays, including immunoblotting and immunoprecipitation. In immunoblot analysis of serum, an anti-C5 × anti-cotinine bispecific tandem single-chain variable fragment (scFv)-Fc fusion protein and cotinine-conjugated horseradish peroxidase (HRP) generated a clean signal without the high background that was observed in a parallel experiment using HRP-conjugated goat anti-rabbit immunoglobulin G (Fc-specific) antibody. In immunoprecipitation analysis of serum, use of the bispecific tandem scFv-Fc fusion protein and cotinine-crosslinked magnetic beads significantly reduced the amount of protein contaminants compared with a parallel experiment done with protein A agarose beads. In subsequent immunoblot analysis, use of cotinine–HRP as the secondary probe instead of HRP-conjugated goat anti-rabbit IgG (Fc-specific) antibody successfully eliminated the band corresponding to the bispecific tandem scFv-Fc fusion protein.

Kim, Hyori; Park, Sunyoung; Lee, Hwa Kyoung; Chung, Junho



Application of bispecific antibody against antigen and hapten for immunodetection and immunopurification.  


We present a bispecific antibody that recognizes an antigen and a hapten and can be applied to various biological assays, including immunoblotting and immunoprecipitation. In immunoblot analysis of serum, an anti-C5 × anti-cotinine bispecific tandem single-chain variable fragment (scFv)-Fc fusion protein and cotinine-conjugated horseradish peroxidase (HRP) generated a clean signal without the high background that was observed in a parallel experiment using HRP-conjugated goat anti-rabbit immunoglobulin G (Fc-specific) antibody. In immunoprecipitation analysis of serum, use of the bispecific tandem scFv-Fc fusion protein and cotinine-crosslinked magnetic beads significantly reduced the amount of protein contaminants compared with a parallel experiment done with protein A agarose beads. In subsequent immunoblot analysis, use of cotinine-HRP as the secondary probe instead of HRP-conjugated goat anti-rabbit IgG (Fc-specific) antibody successfully eliminated the band corresponding to the bispecific tandem scFv-Fc fusion protein. PMID:24071736

Kim, Hyori; Park, Sunyoung; Lee, Hwa Kyoung; Chung, Junho



Bispecific antibodies for cancer therapy: the light at the end of the tunnel?  


With 23 approvals in the US and other countries and four approvals outside US, antibodies are now widely recognized as therapeutic molecules. The therapeutic and commercial successes met by rituximab, trastuzumab, cetuximab and other mAbs have inspired antibody engineers to improve the efficacy of these molecules. Consequently, a new wave of antibodies with engineered Fc leading to much higher effector functions such as antibody-dependent cell-mediated cytotoxicity or complement-dependent cytotoxicity is being evaluated in the clinic, and several approvals are expected soon. In addition, research on a different class of antibody therapeutics, bispecific antibodies, has recently led to outstanding clinical results, and the first approval of the bispecific antibody catumaxomab, a T cell retargeting agent that was approved in the European Union in April 2009. This review describes the most recent advances and clinical study results in the field of bispecific antibodies, a new class of molecules that might outshine conventional mAbs as cancer immunotherapeutics in a near future. PMID:20073127

Chames, Patrick; Baty, Daniel


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.

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.



Norovirus Monoclonal Antibodies and Peptides.  

National Technical Information Service (NTIS)

The present invention is drawn to monoclonal antibodies that bind to a Norovirus, peptides that inhibit monoclonal antibody binding to a Norovirus, and peptides that inhibit binding of a Norovirus to a cell. The compositions of the invention find use as N...

M. Hardy



Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies  

PubMed Central

We describe a generic approach to assemble correctly two heavy and two light chains, derived from two existing antibodies, to form human bivalent bispecific IgG antibodies without use of artificial linkers. Based on the knobs-into-holes technology that enables heterodimerization of the heavy chains, correct association of the light chains and their cognate heavy chains is achieved by exchange of heavy-chain and light-chain domains within the antigen binding fragment (Fab) of one half of the bispecific antibody. This “crossover” retains the antigen-binding affinity but makes the two arms so different that light-chain mispairing can no longer occur. Applying the three possible “CrossMab” formats, we generated bispecific antibodies against angiopoietin-2 (Ang-2) and vascular endothelial growth factor A (VEGF-A) and show that they can be produced by standard techniques, exhibit stabilities comparable to natural antibodies, and bind both targets simultaneously with unaltered affinity. Because of its superior side-product profile, the CrossMabCH1-CL was selected for in vivo profiling and showed potent antiangiogenic and antitumoral activity.

Schaefer, Wolfgang; Regula, Jorg T.; Bahner, Monika; Schanzer, Jurgen; Croasdale, Rebecca; Durr, Harald; Gassner, Christian; Georges, Guy; Kettenberger, Hubert; Imhof-Jung, Sabine; Schwaiger, Manfred; Stubenrauch, Kay G.; Sustmann, Claudio; Thomas, Markus; Scheuer, Werner; Klein, Christian



A Bispecific Antibody Based Assay Shows Potential for Detecting Tuberculosis in Resource Constrained Laboratory Settings  

PubMed Central

The re-emergence of tuberculosis (TB) as a global public health threat highlights the necessity of rapid, simple and inexpensive point-of-care detection of the disease. Early diagnosis of TB is vital not only for preventing the spread of the disease but also for timely initiation of treatment. The later in turn will reduce the possible emergence of multi-drug resistant strains of Mycobacterium tuberculosis. Lipoarabinomannan (LAM) is an important non-protein antigen of the bacterial cell wall, which is found to be present in different body fluids of infected patients including blood, urine and sputum. We have developed a bispecific monoclonal antibody with predetermined specificities towards the LAM antigen and a reporter molecule horseradish peroxidase (HRPO). The developed antibody was subsequently used to design a simple low cost immunoswab based assay to detect LAM antigen. The limit of detection for spiked synthetic LAM was found to be 5.0 ng/ml (bovine urine), 0.5 ng/ml (rabbit serum) and 0.005 ng/ml (saline) and that for bacterial LAM from M. tuberculosis H37Rv was found to be 0.5 ng/ml (rabbit serum). The assay was evaluated with 21 stored clinical serum samples (14 were positive and 7 were negative in terms of anti-LAM titer). In addition, all 14 positive samples were culture positive. The assay showed 100% specificity and 64% sensitivity (95% confidence interval). In addition to good specificity, the end point could be read visually within two hours of sample collection. The reported assay might be used as a rapid tool for detecting TB in resource constrained laboratory settings.

Sarkar, Susmita; Tang, Xinli L.; Das, Dipankar; Spencer, John S.; Lowary, Todd L.; Suresh, Mavanur R.



Fragmentation of monoclonal antibodies  

PubMed Central

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

Vlasak, Josef



Construction and expression of D-dimer and GPIIb/IIIa single-chain bispecific antibody  

PubMed Central

The aim of this study was to construct a plasmid expressing glycoprotein IIb-IIIa (GPIIb/IIIa) and D-dimer single-chain bispecific antibody for the targeted therapy of thrombosis. The phosphorylated gene encoding the anti-GPIIb/IIIa single-chain variable fragment (scFv) and the gene encoding the anti-D-dimer scFv were amplified by PCR and linked in tandem by blunt-end ligation. The recombinant plasmid was transfected into the competent cell line HB2151 and identified by PCR and DNA sequencing. Then, the soluble recombinant antibody in bacterial lysates was purified by an NTA column and molecular sieve chromatography in turn. Finally, the binding specificity of the purified antibody was tested by enzyme-linked immunosorbent assay (ELISA). Results demonstrated that the construction of the expression plasmid was successful and the purified recombinant protein, which had a molecular weight of ?56 kDa, was specific to GPIIb/IIIa and D-dimer. In conclusion, a plasmid expressing a bispecific antibody was constructed by a new method of blunt-end ligation. The soluble recombinant protein is a promising platform for target-oriented thrombolytic therapy.




Optimization of multivalent bispecific antibodies and immunocytokines with improved in vivo properties.  


Multifunctional antibody-based biologics, such as bispecific antibodies and immunocytokines, can be difficult to produce with sufficient yield and stability, and often exhibit inferior pharmacokinetics. Dock-and-Lock (DNL) is a modular method that combines recombinant engineering with site-specific conjugation, allowing the construction of various complex, yet defined, biostructures with multivalency and multispecificity. The technology platform exploits the natural interaction between two interactive human protein binding domains that are modified to provide covalent fusion. We explored the potential application of a new class of IgG-based DNL modules with an anchor domain fused at the C-terminal end of the kappa light chain (C(k)), instead of the C-terminal end of the Fc. Two C(k)-derived prototypes, an anti-CD22/CD20 bispecific hexavalent antibody, comprising epratuzumab (anti-CD22) and four Fabs of veltuzumab (anti-CD20), and a CD20-targeting immunocytokine, comprising veltuzumab and four molecules of interferon-?2b, were compared to their Fc-derived counterparts. The Ck-based conjugates exhibited superior Fc-effector functions in vitro, as well as improved pharmacokinetics, stability, and anti-lymphoma activity in vivo. These results favor the selection of DNL conjugates with the C(k)-design for future clinical development. PMID:23116517

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



Monoclonal Antibodies Specific for Human Thymidylate Synthase.  

National Technical Information Service (NTIS)

The invention relates to monoclonal antibodies that are specific for the protein thymidylate synthase, and hybridomas producing these monoclonal antibodies. The invention further relates to methods of detection and diagnostic kits to test for the presence...

P. Johnston C. Allegra B. Chabner C. M. Liang



Uses of Monoclonal Antibody 8H9.  

National Technical Information Service (NTIS)

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 ...

N. K. Cheung



Synthesis and Evaluation of an Anti-MLC1 x Anti-CD90 Bispecific Antibody for Targeting and Retaining Bone-Marrow Derived Multipotent Stromal Cells in Infarcted Myocardium  

PubMed Central

A key issue regarding the use of stem cells in cardiovascular regenerative medicine is their retention in target tissues. Here, we have generated and assessed a bispecific antibody heterodimer designed to improve the retention of bone marrow–derived multipotent stromal cells (BMMSC) in cardiac tissue damaged by myocardial infarction. The heterodimer comprises an anti-human CD90 monoclonal antibody (mAb) (clone 5E10) and an anti-myosin light chain 1 (MLC1) mAb (clone MLM508) covalently cross-linked by a bis-aryl hydrazone. We modified the anti-CD90 antibody with a pegylated-4-formylbenzamide moiety to a molar substitution ratio (MSR) of 2.6 and the anti-MLC1 antibody with a 6-hydrazinonicotinamide moiety to a MSR of 0.9. The covalent modifications had no significant deleterious effect on mAb epitope binding. Furthermore, the binding of anti-CD90 antibody to BMMSCs did not prevent their differentiation into adipo-, chondro-, or osteogenic lineages. Modified antibodies were combined under mild conditions (RT, pH 6, 1 h) in the presence of a catalyst (aniline) to allow for rapid generation of the covalent bis-aryl hydrazone, which was monitored at A354. We evaluated epitope immunoreactivity for each mAb in the construct. Flow cytometry demonstrated binding of the bispecific construct to BMMSCs that was competed by free anti-CD90 mAb, verifying that modification and cross-linking were not detrimental to the anti-CD90 complementarity-determining region. Similarly, ELISA-based assays demonstrated bispecific antibody binding to plastic-immobilized recombinant MLC1. Excess anti-MLC1 mAb competed for bispecific antibody binding. Finally, the anti-CD90 × anti-MLC1 bispecific antibody construct induced BMMSC adhesion to plastic-immobilized MLC1 that was resistant to shear stress, as measured in parallel-plate flow chamber assays. We used mAbs that bind both human antigens and the respective pig homologues. Thus, the anti-CD90 × anti-MLC1 bispecific antibody may be used in large animal studies of acute myocardial infarction and may provide a starting point for clinical studies.

Gundlach, C. William; Caivano, Amy; Cabreira-Hansen, Maria da Graca; Gahremanpour, Amir; Brown, Wells S.; Zheng, Yi; McIntyre, Bradley W.; Willerson, James T.; Dixon, Richard A.F.; Perin, Emerson C.; Woodside, Darren G.



Monoclonal Antibodies in Diagnosis and Therapy  

NASA Astrophysics Data System (ADS)

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

Waldmann, Thomas A.



Development trends for monoclonal antibody cancer therapeutics  

Microsoft Academic Search

Monoclonal antibodies are now established as a key therapeutic modality for a range of diseases. Owing to the ability of these agents to selectively target tumour cells, cancer has been a major focus of development programmes for monoclonal antibodies so far. Here, we overview trends in the clinical development and regulatory approval of monoclonal antibodies for cancer since 1980, with

Viia E. Valge-Archer; Janice M. Reichert



Monoclonal antibodies to mouse butyrylcholinesterase.  


Our immunization strategy introduced recombinant mouse butyrylcholinesterase (BChE) to naïve BChE knockout mice. An extraordinarily strong immune reaction gave rise to a whole spectrum of antibodies with different properties. Two selective and highly efficient monoclonal anti-mouse BChE antibodies 4H1 (IgG1) and 4 C9 (IgG2a), with Kd values in the nanomolar range were generated. ELISA detected BChE in as little as 20-50 nl of mouse plasma using 2 ?g (4H1) or 4 ?g (4C9). Both antibodies cross-reacted with BChE in dog plasma but only 4 H1 reacted with rat BChE, suggesting that the antibodies are targeted towards different epitopes. Surprisingly, neither recognized human BChE. The anti-mouse BChE antibodies were used in immunohistochemistry analysis of mouse muscle where they specifically stained the neuromuscular junction. The antibodies enable visualization of the BChE protein in the mouse tissue, thus complementing activity assays. They can be used to study a long-lasting question about the existence of mixed acetylcholinesterase/BChE oligomers in mouse tissues. Moreover, monoclonal anti-mouse BChE antibodies can provide a simple, fast and efficient way to purify mouse BChE from small amounts of starting material by using a single-step immunomagnetic bead-based protocol. PMID:23099085

Mrvova, Katarina; Obzerova, Lucia; Girard, Emmanuelle; Krejci, Eric; Hrabovska, Anna



Radioimmunoguided surgery using monoclonal antibody  

SciTech Connect

The potential proficiency of radioimmunoguided surgery in the intraoperative detection of tumors was assessed using labeled monoclonal antibody B72.3 in 66 patients with tissue-proved tumor. Monoclonal antibody B72.3 was injected 5 to 42 days preoperatively, and the hand-held gamma-detecting probe was used intraoperatively to detect the presence of tumor. Intraoperative probe counts of less than 20 every 2 seconds, or tumor-to-adjacent normal tissue ratios less than 2:1 were considered negative (system failure). Positive probe counts were detected in 5 of 6 patients with primary colon cancer (83 percent), in 31 of 39 patients with recurrent colon cancer (79 percent), in 4 of 5 patients with gastric cancer (80 percent), in 3 of 8 patients with breast cancer (37.5 percent), and in 4 of 8 patients with ovarian cancer (50 percent) undergoing second-look procedures. Additional patients in each group were scored as borderline positive. Overall, radioimmunoguided surgery using B72.3 identified tumors in 47 patients (71.2 percent), bordered on positive in 6 patients (9.1 percent), and failed to identify tumor in 13 patients (19.7 percent). Improved selection of patients for antigen-positive tumors, the use of higher affinity second-generation antibodies, alternate routes of antibody administration, alternate radionuclides, and more sophisticatedly bioengineered antibodies and antibody combinations should all lead to improvements in radioimmunoguided surgery.

Martin, E.W. Jr.; Mojzisik, C.M.; Hinkle, G.H. Jr.; Sampsel, J.; Siddiqi, M.A.; Tuttle, S.E.; Sickle-Santanello, B.; Colcher, D.; Thurston, M.O.; Bell, J.G.



Phase I clinical trial of the bispecific antibody MDX-H210 (anti-Fc?RI × anti-HER-2\\/neu) in combination with Filgrastim (G-CSF) for treatment of advanced breast cancer  

Microsoft Academic Search

A phase I study of the bispecific antibody MDX-H210 in combination with granulocyte colony-stimulating factor (G-CSF) was performed in stage IV breast carcinoma patients, overexpressing HER-2\\/neu. MDX-H210, constructed by crosslinking antigen binding fragments (F(ab?) fragments) of monoclonal antibody (mAb) H22 to Fc gamma receptor I (Fc?RI), and mAb 520C9 to HER-2\\/neu, respectively, mediates the lysis of tumour cells in vitro,

R Repp; H H van Ojik; T Valerius; G Groenewegen; G Wieland; C Oetzel; B Stockmeyer; W Becker; M Eisenhut; H Steininger; Y M Deo; G H Blijham; J R Kalden; J G J van de Winkel; M Gramatzki



Monoclonal Antibodies to Cholesterol and Methods.  

National Technical Information Service (NTIS)

This patent application relates to monoclonal antibodies which demonstrate specific reactivity to cholesterol and methods for the detection of high levels of cholesterol by contacting biological specimens containing cholesterol with the monoclonal antibod...

C. R. Alving G. M. Swartz



Potent inhibition of local and disseminated tumor growth in immunocompetent mouse models by a bispecific antibody construct specific for Murine CD3  

Microsoft Academic Search

Bispecific single-chain antibody constructs specific for human CD3 have been extensively studied for antitumor activity in\\u000a human xenograft models using severe combined immunodeficient mice supplemented with human T cells. High efficacy at low effector-to-target\\u000a ratios, independence of T cell costimuli and a potent activation of previously unstimulated polyclonal T cells were identified\\u000a as hallmarks of this class of bispecific antibodies.

Bernd Schlereth; Petra Kleindienst; Iduna Fichtner; Grit Lorenczewski; Klaus Brischwein; Sandra Lippold; Antonio da Silva; Mathias Locher; Roman Kischel; Ralf Lutterbüse; Peter Kufer; Patrick A. Baeuerle



Radioimmunodetection of medullary thyroid cancer using a bispecific anti-CEA/anti-indium-DTPA antibody and an indium-111-labeled DTPA dimer.  


Two-step radioimmunotargeting using a bispecific anti-CEA/anti-in-DTPA monoclonal antibody and an 111In-labeled DTPA dimer (diDTPA-TL) was evaluated nine times in eight patients with medullary thyroid cancer (MTC). Immunoscintigraphy was performed 5 and 24 hr after injection of 111In-diDTPA-TL. For five patients, radioimmunoguided surgery (RIGS) was performed using a hand-held gamma probe (sodium iodine), and a biodistribution study was performed 48 hr (four times) and 24 hr (one time) after injection of 111In-diDTPA-TL. Mean tumor uptake (%ID/kg in tumor) was 39 (range 2.75-139). In these five patients, immunoscintigraphy visualized all known tumors and detected unknown foci (US and CT were negative) in the neck (once) and neck and liver (once). Immunoscintigraphy, performed four times in search of a recurrence, detected unknown localizations in the mediastinum and neck (twice) and was negative twice. There were no false-positives. In three of five patients who had surgery, RIGS localized tumor foci not detected by the surgeon. RIGS failed to detect two small lesions (10 x 10 mm) corresponding to sites of fibrosis and microscopic cancer infiltration. Bispecific anti-CEA/anti-In-DTPA mediated targeting of 111In-diDTPA-TL provided elevated tumor uptake and tumor-to-normal tissue ratios. Radioimmunodetection of small MTC lesions is thus possible even when morphological imaging techniques prove negative. PMID:8326383

Peltier, P; Curtet, C; Chatal, J F; Le Doussal, J M; Daniel, G; Aillet, G; Gruaz-Guyon, A; Barbet, J; Delaage, M



Systemic administration of a bispecific antibody targeting EGFRvIII successfully treats intracerebral glioma.  


Bispecific antibodies (bscAbs), particularly those of the bispecific T-cell engager (BiTE) subclass, have been shown to effectively redirect T cells against cancer. Previous efforts to target antigens expressed in both tumors and normal tissues have produced significant toxicity, however. Moreover, like other large molecules, bscAbs may be restricted from entry into the "immunologically privileged" CNS. A tumor-specific mutation of the epidermal growth factor receptor, EGFRvIII, is a constitutively activated tyrosine kinase not found in normal tissues but frequently expressed in glioblastomas and many other neoplasms. Because it is localized solely to tumor tissue, EGFRvIII presents an ideal target for immunotherapy. Here we report the preclinical evaluation of an EGFRvIII-targeted BiTE, bscEGFRvIIIxCD3. Our results show that bscEGFRvIIIxCD3 activates T cells to mediate potent and antigen-specific lysis of EGFRvIII-expressing gliomas in vitro (P < 0.001) at exceedingly low concentrations (10 ng/mL) and effector-to-target ratios (2.5:1). Treatment with i.v. bscEGFRvIIIxCD3 yielded extended survival in mice with well-established intracerebral tumors (P < 0.05) and achieved durable complete cure at rates up to 75%. Antitumor efficacy was significantly abrogated on blockade of EGFRvIII binding, demonstrating the need for target antigen specificity both in vitro and in vivo. These results demonstrate that BiTEs can be used to elicit functional antitumor immunity in the CNS, and that peptide blockade of BiTE-mediated activity may greatly enhance the safety profile for antibody-redirected T-cell therapies. Finally, bscEGFRvIIIxCD3 represents a unique advancement in BiTE technology given its exquisite tumor specificity, which enables precise elimination of cancer without the risk of autoimmune toxicity. PMID:23248284

Choi, Bryan D; Kuan, Chien-Tsun; Cai, Mingqing; Archer, Gary E; Mitchell, Duane A; Gedeon, Patrick C; Sanchez-Perez, Luis; Pastan, Ira; Bigner, Darell D; Sampson, John H



Development of humanized bispecific antibodies reactive with cytotoxic lymphocytes and tumor cells overexpressing the HER2 protooncogene  

Microsoft Academic Search

Summary The HER2 protooncogene encodes a 185-kD transmembrane phosphoglycoprotein, human epidermal growth factor receptor 2 (p185u~Rz), whose amplified expression on the cell surface can lead to malignant transformation. Overexpression of HER2\\/p185 urR2 is strongly correlated with progression of human ovarian and breast carcinomas. Recent studies have shown that human T cells can be targeted with bispecific antibody to react against

M. Refaat Shalaby; H. Michael Shepard; Len Presta; Maria L. Rodrigues; S Peter; C. L. Beverley; Marc Feldmann; Paul Carters



Production of Murine Monoclonal Antibodies using Traditional and Novel Technology.  

National Technical Information Service (NTIS)

Recent attempts to generate monoclonal antibodies against various biological immunogens have proven to be successful. This report discusses the traditional and novel technologies used to produce monoclonal antibodies. Hybridoma monoclonal antibodies are h...

M. M. Dixon



Monoclonal antibodies to Leptospira interrogans serovar pomona.  

PubMed Central

Three monoclonal antibodies produced against Leptospira interrogans serovar pomona have been studied for their diagnostic usefulness. All three monoclonals reacted strongly in the enzyme-linked immunosorbent assay and indirect fluorescent antibody test with serovar pomona and did not react with serovars grippotyphosa, canicola, icterohaemorrhagiae and hardjo.

Ainsworth, A J; Lester, T L; Capley, G



Monoclonal antibodies for bacterial identification and taxonomy  

Microsoft Academic Search

Some uses of monoclonal antibodies in bacteriology are discussed with emphasis on identification and classification of microorganisms. It is pointed out that monoclonal antibodies are useful in diagnostic purposes, i.e., identification of bacteria and bacterial antigens in unknowns and classification of new isolates. They are also important for studying relationships among bacteria with phylogenetic significance and for elucidating their ecological

E. Conway de Macario; A. J. L. Macario



Induction of antitumor immunity after cure of pulmonary metastases, using staphylococcal enterotoxin B and bispecific antibody.  


The combination of staphylococcal enterotoxin B (SEB) and anti-p97 x anti-CD3 bispecific antibody (bsAb) cures 60%-80% of mice with established pulmonary metastases of the syngeneic p97+ murine melanoma, CL62. We investigated the ability of cured mice to generate protective antitumor immunity. In tumor rechallenge experiments, CL62-cured mice developed protective immunity against rechallenge with CL62. The majority of mice also rejected the p97-negative parental cell line, K1735, indicating an immune response to tumor antigens common to both cell lines that were not bsAb-targeted. A significant humoral response developed against p97 antigen, but not against other antigens common to both CL62 and K1735. That the majority of cured mice nevertheless rejected K1735 suggests that tumor immunity is not antibody-dependent. Evidence of cellular immunity was obtained from the results of delayed-type hypersensitivity, proliferation and cytotoxicity assays, which revealed the presence of tumor-specific memory in bsAb-treated, CL62-cured mice. CD8+ T cells from cured, but not control mice were able to lyse tumor; however, memory CD4 cells had no cytolytic function. In vivo, however, both CD4 and CD8 T cells were required for effective protective immunity. These studies demonstrate that treatment with SEB and bsAb not only confers passive immune effects of tumor eradication, but also actively promotes the generation of a host antitumor immune response. PMID:10478639

Rice, D C; Chapoval, A I; Porter, L; Nelson, H



Monoclonal antibodies in solid tumours.  


Monoclonal antibodies (mAbs) have become an integral part of therapeutic strategies used to treat solid tumours. Directed against membrane-bound receptors or extracellular ligands with high specificity, they target elements upstream in the signal transduction pathways that contribute to malignant growth, proliferation, survival, angiogenesis and spread. Several mAbs have now received regulatory approval - trastuzumab, cetuximab, panitumumab, bevacizumab and catumaxomab-across multiple solid tumour types, including breast, colorectal, and non-small cell lung cancers, amongst others. Despite these successes there have been ample disappointments, which in turn is stimulating ongoing research and development efforts. Nevertheless, greater initiative and vision in this development process is needed, from intelligent compound design to enrichment of patient populations during clinical development, biomarker discovery and ultimately tailored, individualised treatment decisions. In this commentary we review those mAbs now in routine use for solid tumours, interesting aspects of their use and future directions. PMID:20406174

Markman, Ben; Tabernero, Josep



Preclinical evaluation of light-activatable, bispecific anti-human CD3 antibody conjugates as anti-ovarian cancer therapeutics  

PubMed Central

The administration of anti-CD3 antibodies, either unmodified or in bispecific formats, has been shown to kill tumors. However, their activity needs to be carefully controlled. We have approached this problem by inhibiting their anti-CD3 activity until it is required. Folated anti-human CD3 antibody bispecific conjugates were therefore synthesised in which the folate portion of the conjugates remained free to bind to folate receptor (FR) expressing cancer cells, whilst their anti-CD3 activity was reversibly inhibited. On irradiation with UV-A light, the T-cell binding activity of the anti-CD3 antibody can be restored only when and where it is required, i.e., adjacent to a tumor. Conjugate bound to FR expressed on normal tissues in other parts of the body remains inactive. This report describes the preclinical in vivo testing of these conjugates in transgenic mice whose T-cells express human CD3 molecules. When the ‘cloaked’ conjugates were reactivated in the region of the primary tumor, both primary tumor growth and liver metastasis were markedly reduced. That the deliberate targeting of T-cell activity locally to the primary tumor also resulted in reduced distant metastatic growth was a key finding. Light-activatable bispecific antibody conjugates similar to those described here offer a means to control T-cell targeting with a much higher degree of specificity to tumors because they minimize potentially dangerous and unwanted side effects in non-illuminated areas. The addition of light-specific targeting to the inherent tumor specific targeting of therapeutic antibody conjugates could result in the development of safer treatments for patients.

Dessi, John



[Antiviral humanized and human monoclonal antibodies].  


The paper considers the characteristics of monoclonal antibodies, methods for their experimental preparation, problems of their production, and possibilities of their use for the emergency prevention of viral infections and for the treatment of chronic diseases caused by human immunodeficiency virus, hepatitis B and C viruses, and herpes viruses. The future of experimentally produced or clinically trialed monoclonal antibodies is mainly determined by commercial considerations. It is possible that simplification of industrial production technologies and a reduction in the cost of evidence-based methods for evaluation of clinical effectiveness will allow monoclonal antibodies to be extensively used for the prevention and treatment of viral infections. PMID:22171470

Lashkevich, V A


Monoclonal antibodies in the treatment of cancer  

SciTech Connect

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

Dillman, R.O.



CD3 directed bispecific antibodies induce increased lymphocyte-endothelial cell interactions in vitro  

PubMed Central

Bispecific antibody (BsMAb) BIS-1 has been developed to redirect the cytolytic activity of cytotoxic T lymphocytes (CTL) to epithelial glycoprotein-2 (EGP-2) expressing tumour cells. Intravenous administration of BIS-1 F(ab?)2to carcinoma patients in a phase I/II clinical trial, caused immunomodulation as demonstrated by a rapid lymphopenia prior to a rise in plasma tumour necrosis factor-? and interferon-? levels. Yet, no lymphocyte accumulation in the tumour tissue and no anti-tumour effect could be observed. These data suggest a BsMAb-induced lymphocyte adhesion to blood vessel walls and/or generalized redistribution of the lymphocytes into tissues. In this study, we describe the effects of BIS-1 F(ab?)2binding to peripheral blood mononuclear cells (PBMC) on their capacity to interact with resting endothelial cells in vitro. Resting and pre-activated PBMC exhibited a significant increase in adhesive interaction with endothelial cells when preincubated with BIS-1 F(ab?)2, followed by an increase in transendothelial migration (tem). Binding of BIS-1 F(ab?)2to PBMC affected the expression of a number of adhesion molecules involved in lymphocyte adhesion/migration. Furthermore, PBMC preincubated with BIS-1 F(ab?)2induced the expression of endothelial cell adhesion molecules E-selectin, VCAM-1 and ICAM-1 during adhesion/tem. These phenomena were related to the CD3 recognizing antibody fragment of the BsMAb and dependent on lymphocyte–endothelial cell contact. Possibly, in patients, the BIS-1 F(ab?)2infusion induced lymphopenia is a result of generalized activation of endothelial cells, leading to the formation of a temporary sink for lymphocytes. This process may distract the lymphocytes from homing to the tumour cells, and hence prevent the occurrence of BIS-1 F(ab?)2– CTL-mediated tumour cell lysis. © 2000 Cancer Research Campaign

Molema, G; Tervaert, J W Cohen; Kroesen, B J; Helfrich, W; Meijer, D K F; Leij, L F M H de



Methods for Production of Monoclonal Antibodies.  

National Technical Information Service (NTIS)

The handbook includes detailed laboratory methods that will guide researchers, technicians, and students through their first experiences in producing monoclonal antibodies. It presents strategies and methods for immunizing mice, preparing lymphocytes from...

G. J. Letchworth J. A. Appleton



Preparation of astatine-labeled monoclonal antibodies  

SciTech Connect

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

Milesz, S.; Norseev, Yu.V.; Szucs, Z. [Joint Inst. for Nuclear Research, Dubna (Russian Federation)]|[Central Inst. of Physical Research, Budapest (Hungary)



Polyclonal and monoclonal antibodies in clinic.  


Immunoglobulins (Ig) or antibodies are heavy plasma proteins, with sugar chains added to amino-acid residues by N-linked glycosylation and occasionally by O-linked glycosylation. The versatility of antibodies is demonstrated by the various functions that they mediate such as neutralization, agglutination, fixation with activation of complement and activation of effector cells. Naturally occurring antibodies protect the organism against harmful pathogens, viruses and infections. In addition, almost any organic chemical induces antibody production of antibodies that would bind specifically to the chemical. These antibodies are often produced from multiple B cell clones and referred to as polyclonal antibodies. In recent years, scientists have exploited the highly evolved machinery of the immune system to produce structurally and functionally complex molecules such as antibodies from a single B clone, heralding the era of monoclonal antibodies. Most of the antibodies currently in the clinic, target components of the immune system, are not curative and seek to alleviate symptoms rather than cure disease. Our group used a novel strategy to identify reparative human monoclonal antibodies distinct from conventional antibodies. In this chapter, we discuss the therapeutic relevance of both polyclonal and monoclonal antibodies in clinic. PMID:24037837

Wootla, Bharath; Denic, Aleksandar; Rodriguez, Moses



Human monoclonal antibodies: the residual challenge of antibody immunogenicity.  


One of the major reasons for seeking human monoclonal antibodies has been to eliminate immunogenicity seen with rodent antibodies. Thus far, there has yet been no approach which absolutely abolishes that risk for cell-binding antibodies. In this short article, I draw attention to classical work which shows that monomeric immunoglobulins are intrinsically tolerogenic if they can be prevented from creating aggregates or immune complexes. Based on these classical studies two approaches for active tolerization to therapeutic antibodies are described. PMID:24037833

Waldmann, Herman



Immunocytochemistry with internally labeled monoclonal antibodies  

SciTech Connect

One of the advantages of the production of monoclonal antibodies by tissue culture methods is that they can be internally labeled by using appropriate radioactive amino acid in the culture fluid. Thus, radioactive immunological probes of high specific activity can be prepared. Here we report applications of these internally labeled monoclonal antibodies for the direct localization of immunoreactive sites in the central nervous system of the rat at both light and electron microscopic levels (radioimmunocytochemistry). We explored the combined use of radioimmunocytochemistry with immunoenzymatic methods for the simultaneous detection of two antigenic sites: substance P and serotonin or substance P and enkephalin. In neurobiology this procedure could help to clarify certain aspects of transmitter-specific synaptic interactions and the coexistence of neuroactive substances in single neuronal cell bodies or nerve terminals. We also describe the application of radioimmunocytochemistry with internally labeled monoclonal antibodies to quantify the immunoreactions in discrete microscopic areas.

Cuello, A.C. (Department of Pharmacology, Oxford, United Kingdom); Priestley, J.V.; Milstein, C.



Taking aim at cancer with monoclonal antibodies  

SciTech Connect

Conjugating radioisotopes to monoclonal antibodies could have certain advantages in cancer therapy. Radioactive compounds have the double-edged ability to kill cells that are up to centimeter or more away. This is a plausible way to overcome tumor heterogeneity, but it also means that normal cells near the tumor could be affected. Hybritech (San Diego, CA) has been supplying antibody linked to the radioisotope yttrium-90 for a number of clinical trials. Work at Johns Hopkins University (Baltimore, MD) has focused on polyclonal antibodies to hepatoma. Monoclonal antibodies will be used there soon, and trials could be expanded eventually to include breast, lung, and prostate cancer as well. Hybritech also expects that the yttrium-antibody conjugates developed with NCI will enter the clinic later this year for treating leukemia and lymphoma systems; treatments for melanomas should follow.

Klausner, A.



Rabbit monoclonal antibody: potential application in cancer therapy  

PubMed Central

By targeting antigens specifically, monoclonal antibodies represent a new class of therapeutic agents for the clinical management of various diseases including cancers. Monoclonal antibody technology has been greatly developed by reducing murine content in antibodies to minimize side effects in clinical applications. However, several intrinsic disadvantages of antibodies with murine origin limit the clinical efficacy of monoclonal antibodies based targeted therapy. The development of rabbit monoclonal antibody technology provides an alternative source of monoclonal antibodies with higher specificity and less cost for the development of routine targeted therapy against cancers.

Feng, Lifeng; Wang, Xian; Jin, Hongchuan



Methods for radiolabelling of monoclonal antibodies.  


The use of radionuclide labels allows to study the pharmacokinetics of monoclonal antibodies, to control the specificity of their targeting and to monitor the response to an antibody treatment with high accuracy. Selection of label depends on the processing of an antibody after binding to an antigen, and on the character of information to be derived from the study (distribution of antibody in the extracellular space, target occupancy or determination of sites of metabolism). This chapter provides protocols for labelling of antibodies with iodine-125 (suitable also for other radioisotopes of iodine) and with indium-111. Since radiolabelling might damage and reduce the immunoreactive fraction and/or affinity of an antibody, the methods for assessment of these characteristics of an antibody are provided for control. PMID:24037848

Tolmachev, Vladimir; Orlova, Anna; Andersson, Karl



A Monoclonal Antibody to Limbic System Neurons  

Microsoft Academic Search

A monoclonal antibody produced against hippocampal cell membranes labeled the surface of neurons in the rat limbic system. With a few exceptions, all nonlimbic components were unstained. This specific distribution of immunopositive neurons provides strong evidence of molecular specificity among functionally related neurons in the mammalian brain and supports the concept of a limbic system.

Pat Levitt



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)



Development trends for human monoclonal antibody therapeutics  

Microsoft Academic Search

Fully human monoclonal antibodies (mAbs) are a promising and rapidly growing category of targeted therapeutic agents. The first such agents were developed during the 1980s, but none achieved clinical or commercial success. Advances in technology to generate the molecules for study — in particular, transgenic mice and yeast or phage display — renewed interest in the development of human mAbs

Aaron L. Nelson; Eugen Dhimolea; Janice M. Reichert



Monoclonal Antibodies to Cytochrome B5.  

National Technical Information Service (NTIS)

Hybridomas obtained by the fusion of spleen cells from rat cytochrome b5-immunized mice with mouse myeloma cells produced five groups of monoclonal antibodies (MAbs) with three mouse immunoglobulin subtypes: IgGl, IgG2b and IgM. All of the MAbs bound stro...

S. S. Park H. V. Gelboin



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


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

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



Generation of a Monoclonal Antibody against Mycoplasma spp. following Accidental Contamination during Production of a Monoclonal Antibody against Lawsonia intracellularis  

PubMed Central

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

Hwang, Jeong-Min; Lee, Ji-Hye



Phase Separation in Solutions of Monoclonal Antibodies  

NASA Astrophysics Data System (ADS)

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

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



Stable IgG-like Bispecific Antibodies Directed toward the Type I Insulin-like Growth Factor Receptor Demonstrate Enhanced Ligand Blockade and Anti-tumor Activity  

PubMed Central

Bispecific antibodies (BsAbs) target multiple epitopes on the same molecular target or different targets. Although interest in BsAbs has persisted for decades, production of stable and active BsAbs has hindered their clinical evaluation. Here, we describe the production and characterization of tetravalent IgG-like BsAbs that combine the activities of allosteric and competitive inhibitors of the type-I insulin-like growth factor receptor (IGF-1R). The BsAbs, which were engineered for thermal stability, express well, demonstrate favorable biophysical properties, and recognize both epitopes on IGF-1R. Only one BsAb with a unique geometry, denoted BIIB4-5scFv, was capable of engaging all four of its binding arms simultaneously. All the BsAbs (especially BIIB4-5scFv) demonstrated enhanced ligand blocking over the single monoclonal antibodies (mAbs), particularly at high ligand concentrations. The pharmacokinetic profiles of two IgG-like BsAbs were tested in nude mice and shown to be comparable with that of the parental mAbs. The BsAbs, especially BIIB4-5scFv, demonstrated an improved ability to reduce the growth of multiple tumor cell lines and to inhibit ligand-induced IGF-1R signaling in tumor cells over the parental mAbs. BIIB4-5scFv also led to superior tumor growth inhibition over its parental mAbs in vivo. In summary, BsAbs that bridge multiple inhibitory mechanisms against a single target may generally represent a more effective strategy for intervention in oncology or other indications compared with traditional mAb therapy.

Dong, Jianying; Sereno, Arlene; Snyder, William B.; Miller, Brian R.; Tamraz, Susan; Doern, Adam; Favis, Michael; Wu, Xiufeng; Tran, Hon; Langley, Emma; Joseph, Ingrid; Boccia, Antonio; Kelly, Rebecca; Wortham, Kathleen; Wang, Qin; Berquist, Lisa; Huang, Flora; Gao, Sharon X.; Zhang, Ying; Lugovskoy, Alexey; Martin, Shelly; Gouvis, Heather; Berkowitz, Steven; Chiang, Gisela; Reff, Mitchell; Glaser, Scott M.; Hariharan, Kandasamy; Demarest, Stephen J.



SPECT assay of radiolabeled monoclonal antibodies  

SciTech Connect

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

Jaszczak, R.J.



Monoclonal antibody to chicken oviduct progesterone receptor.  

PubMed Central

Antibodies to molybdate-stabilized chicken oviduct progesterone receptor were raised in a Wistar rat and detected by interaction with homogeneous radioiodinated progesterone receptor. Spleen cells of this rat were then fused with mouse Sp2/0-Ag14 myeloma cells and the antibodies produced by the hybrid cells were detected by double immunoprecipitation using the 125I-labeled receptor. Cells of one of the positive cultures were then cloned by limiting dilution and one hybridoma cell line was studied. The monoclonal antibody produced was an IgG2b, and it reacted with the molybdate-stabilized "8-9S" form of the chicken oviduct progesterone receptor, labeled with either [3H]progesterone or [3H]ORG 2058 (a high-affinity synthetic progestin). Kd for the 8-9S progesterone receptor was approximately equal to 1 nM. Progesterone receptor-monoclonal antibody complexes were labeled with radioactive progesterone, suggesting that antibody does not prevent hormone binding. By using a [35S]methionine-labeled antibody, we were able to detect the progesterone receptor independently of its characteristic function of binding radioactive hormone. No crossreaction with human progesterone receptor was detected.

Radanyi, C; Joab, I; Renoir, J M; Richard-Foy, H; Baulieu, E E



Monoclonal Antibody Recognition of Abscisic Acid Analogs  

PubMed Central

Specificities of three monoclonal antibodies (15-I-C5, DBPA 1, and MAC 62) raised against the plant hormone (S)-(+)-abscisic acid (ABA) have been compared. Immunological cross-reactivities against fifteen biologically active analogs of ABA were measured. The ABA analogs were altered at one or more of four positions: the double bonds in the ring, at C-2 C-3 and at C-4 C-5, and in the oxidation level at C-1. Several analogs were optically active with chiral centers at C-1? and C-2?. For cross-reactivity, all three monoclonal antibodies required the carboxylic acid group, and the cis configuration of the double bond at C-2 C-3 of the ABA molecule. Monoclonals 15-I-C5 and DBPA 1 required the entire ABA sidechain from the C-1 to C-1?, but these monoclonals did cross-react with analogs with the ring double bond reduced and the C-2? methyl cis to the sidechain. Only MAC 62 recognized analogs containing an acetylene at C-4 C-5. MAC 62 had more strict requirements for the ring double bond, but gave some cross-reactivity with acetylenic analogs having a saturated ring. All three monoclonals had higher specificity for analogs having the same absolute configuration at C-1? as (S)-(+)-ABA. This work provides new information about the spatial regions of the ABA molecule that elicit immunological recognition, and serves as a basis for future investigations of the ABA receptor using ABA analogs and anti-idiotypic antibodies.

Walker-Simmons, Mary K.; Reaney, Martin J. T.; Quarrie, Stephen A.; Perata, Pierdomenico; Vernieri, Paolo; Abrams, Suzanne R.



Monoclonal antibody recognition of abscisic Acid analogs.  


Specificities of three monoclonal antibodies (15-I-C5, DBPA 1, and MAC 62) raised against the plant hormone (S)-(+)-abscisic acid (ABA) have been compared. Immunological cross-reactivities against fifteen biologically active analogs of ABA were measured. The ABA analogs were altered at one or more of four positions: the double bonds in the ring, at C-2 C-3 and at C-4 C-5, and in the oxidation level at C-1. Several analogs were optically active with chiral centers at C-1' and C-2'. For cross-reactivity, all three monoclonal antibodies required the carboxylic acid group, and the cis configuration of the double bond at C-2 C-3 of the ABA molecule. Monoclonals 15-I-C5 and DBPA 1 required the entire ABA sidechain from the C-1 to C-1', but these monoclonals did cross-react with analogs with the ring double bond reduced and the C-2' methyl cis to the sidechain. Only MAC 62 recognized analogs containing an acetylene at C-4 C-5. MAC 62 had more strict requirements for the ring double bond, but gave some cross-reactivity with acetylenic analogs having a saturated ring. All three monoclonals had higher specificity for analogs having the same absolute configuration at C-1' as (S)-(+)-ABA. This work provides new information about the spatial regions of the ABA molecule that elicit immunological recognition, and serves as a basis for future investigations of the ABA receptor using ABA analogs and anti-idiotypic antibodies. PMID:16667979

Walker-Simmons, M K; Reaney, M J; Quarrie, S A; Perata, P; Vernieri, P; Abrams, S R



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.



Recent developments in monoclonal antibody radiolabeling techniques  

SciTech Connect

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

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



Tracking fungi in soil with monoclonal antibodies  

Microsoft Academic Search

Species of the genus Trichoderma are ubiquitous soil-borne fungi that exhibit antagonism towards a number of economically important plant-pathogenic fungi\\u000a and oomycetes. This review discusses recent developments in the use of monoclonal antibodies to detect these fungi in their\\u000a natural soil environments and to quantify their population dynamics during antagonistic interactions with saprotrophic competitors\\u000a in soil-based systems. Immunological approaches to

Christopher R. Thornton


Tracking fungi in soil with monoclonal antibodies  

Microsoft Academic Search

Species of the genus Trichoderma are ubiquitous soil-borne fungi that exhibit antagonism towards a number of economically important plant-pathogenic fungi\\u000a and oomycetes. This review discusses recent developments in the use of monoclonal antibodies to detect these fungi in their\\u000a natural soil environments and to quantify their population dynamics during antagonistic interactions with saprotrophic competitors\\u000a in soil-based systems. Immunological approaches to

Christopher R. Thornton



Monoclonal antibodies to human glomerular antigens  

Microsoft Academic Search

Using cultured human fetal kidney cortical cells as antigen, two monoclonal antibodies (moAbs) against human glomeruli were produced. One of these moAbs, H-4, recognized the cell surface of glomerular epithelial cells, and the other, H-13, recognized the extracellular matrix present in the mesangial area. Both also reacted with liver, H-4 recognizing antigen present on the hepatocyte, and H-13 recognizing antigen

T. Nakamura; T. Oite; T. Kazama; S. Suzuki; M. Orikasa; M. Arakawa; F. Shimizu



Monoclonal antibodies against heparin and heparinoids.  


The antigenicity of glycosaminoglycans and galactosaminoglycans is very weak. Accordingly, only a few reports on the successful production of monoclonal antibodies against glycosaminoglycans and galactosaminoglycans have been published. Antibodies have been raised against the heparinlike compounds heparan sulfate, chondroitin sulfate, and keratan sulfate. The production of heparin antibodies was reported. But further analysis showed that the antibodies were not directed against native heparin but against heparin conjugates or chemically modified heparins. After immunization with a heparin-bovine serum albumin conjugate, prepared by reductive amination, a monoclonal antibody against heparin and heparin fractions was obtained (Huhle et al: Semin Thromb Hemostas 20:193-204, 1995). For further analysis, tyramine, which was covalently bound to low-molecular-mass heparin (LMMH) by endpoint attachment (Malsch et al: Anal Biochem 217:255-264, 1994), was labeled with iodine-125 at the aryl residue. The tracer antibody complex was immunoprecipitated by goat anti-mouse immunoglobulin IgG. H1.18 recognized specifically intact heparin and heparin fractions. The lower detection limit for heparin preparations was 100 ng/mL. The H1.18 antibody was purified by ammonium sulfate precipitation. H1.18 remained biologically and functionally active after purification. The smallest disaccharide that was detected by H1.18 was found to be iduronic acid-anhydromannose, obtained by nitrous acid degradation of LMMH. Endpoint attachment of tyramine to anhydromannose did not modify the binding of the disaccharide to H1.18, indicating the importance of iduronic acid for binding of glycosaminoglycans to the antibody. PMID:9156406

Huhle, G; Harenberg, J; Malsch, R; Heene, D L



Monoclonal antibodies as catalysts for cyanide removal  

SciTech Connect

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

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



Targeting of Porphyromonas gingivaliswith a bispecific antibody directed to FcRI (CD89) improves in vitro clearance by gingival crevicular neutrophils  

Microsoft Academic Search

Phagocytosis and killing of pathogens by polymorphonuclear neutrophils (PMN) from gingival crevicular fluid (GCF) is diminished in chronic periodontitis patients. As an approach to improve targeting of PMN toward a periodontopathogen, Porphyromonas gingivalis , the efficacy of a bispecific antibody (BsAb) directed against both recombinant 130 kDa hemagglutinin domain (r130k-HMGD) of P. gingivalis, and PMN Fc receptor (FcR) was evaluated.

Tetsuo Kobayashia; Ayano Takauchia; Annemiek B. van Sprielc; Henriette A. Viled; Mitsuo Hayakawaf; Yasuko Shibataf; Yoshimitsu Abikof; Jan G. J. van de Winkeld; Hiromasa Yoshiea


The CEA\\/CD3-Bispecific Antibody MEDI565 (MT111) Binds a Nonlinear Epitope in the Full-Length but Not a Short Splice Variant of CEA  

Microsoft Academic Search

MEDI-565 (also known as MT111) is a bispecific T-cell engager (BiTE®) antibody in development for the treatment of patients with cancers expressing carcinoembryonic antigen (CEA). MEDI-565 binds CEA on cancer cells and CD3 on T cells to induce T-cell mediated killing of cancer cells. To understand the molecular basis of human CEA recognition by MEDI-565 and how polymorphisms and spliced

Li Peng; Michael D. Oberst; Jiaqi Huang; Philip Brohawn; Chris Morehouse; Kristen Lekstrom; Patrick A. Baeuerle; Herren Wu; Yihong Yao; Steven R. Coats; William Dall’Acqua; Melissa Damschroder; Scott A. Hammond



Therapeutic monoclonal antibody for sporotrichosis.  


Sporotrichosis is a chronic subcutaneous mycosis that affects both humans and animals worldwide. This subcutaneous mycosis had been attributed to a single etiological agent, Sporothrix schenckii. S. schenckii exhibits considerable genetic variability, and recently, it was suggested that this taxon consists of a complex of species. Sporotrichosis is caused by traumatic inoculation of the fungus, which is a ubiquitous environmental saprophyte that can be isolated from soil and plant debris. The infection is limited to cutaneous forms, but recently, more severe clinical forms of this mycosis have been described, especially among immunocompromised individuals. The immunological mechanisms involved in the prevention and control of sporotrichosis are not well understood. Some studies suggest that cell-mediated immunity plays an important role in protecting the host against S. schenckii. In contrast, the role of the humoral immune response in protection against this fungus has not been studied in detail. In a previous study, we showed that antigens secreted by S. schenckii induced a specific humoral response in infected animals, primarily against a 70-kDa molecule, indicating a possible role of specific antibodies against this molecule in infection control. In another study by our group, we produced a mAb against a 70-kDa glycoprotein of S. schenckii to better understand the effect of the passive immunization of mice infected with S. schenckii. The results showed a significant reduction in the number of CFUs in various mice organs when the mAb was injected before or during S. schenckii infection. Similar results were observed when T-cell-deficient mice were used. The drugs of choice in the treatment of sporotrichosis require long periods, and relapses are frequently observed, primarily in immunocompromised patients. The strong protection induced by the mAb against a 70-kDa glycoprotein makes it a strong candidate as a therapeutic vaccine against sporotrichosis. PMID:23226146

Almeida, Sandro R



Eradication of Tumors from a Human Colon Cancer Cell Line and from Ovarian Cancer Metastases in Immunodeficient Mice by a Single-Chain Ep-CAM-\\/CD3-Bispecific Antibody Construct  

Microsoft Academic Search

Bispecific T-cell engager (BiTE) are a class of bispecific single- chain antibodies that can very effectively redirect cytotoxic T cells for killing of tumor target cells. Here, we have assessed the in vivo efficacy of one representative, called bscEp- CAMxCD3, with specificity for tumors overexpressing epithe- lial cell adhesion molecule (Ep-CAM) in human xenograft models. Cells of the human colon

Bernd Schlereth; Iduna Fichtner; Grit Lorenczewski; Petra Kleindienst; Klaus Brischwein; Antonio da Silva; Peter Kufer; Ralf Lutterbuese; Ilse Junghahn; Sabine Kasimir-Bauer; Pauline Wimberger; Rainer Kimmig; Patrick A. Baeuerle


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


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



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.

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



Construction and Production of an IgG-Like Tetravalent Bispecific Antibody, IgG-Single-Chain Fv Fusion.  


In recent years, both laboratory and clinical studies have demonstrated that bispecific antibodies (BsAbs) may have significant potential application in cancer therapy either by targeting tumor cells with cytotoxic agents including effector cells, radionuclides, drugs, and toxins, or by simultaneously blocking two tumor-associated targets, e.g., tumor growth factors and/or their cell surface receptors. A major obstacle in the development of BsAb has been the difficulty of producing the materials in sufficient quality and quantity by traditional technologies such as the hybrid hybridoma and chemical conjugation methods. The development of recombinant BsAbs as therapeutic agents will depend heavily on the advances made in the design of the constructs (or formats) and production efficiency. Here we describe a recombinant method for the construction and production of a tetravalent IgG-like BsAb molecule, IgG-scFv fusion, in which, a single-chain Fv (scFv) antibody fragment of one antigen specificity is genetically fused to the c-terminal of a conventional IgG of a different antigen specificity. PMID:24037843

Lu, Dan; Zhu, Zhenping



Labeling of monoclonal antibodies with radionuclides  

SciTech Connect

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

Bhargava, K.K.; Acharya, S.A. (Albert Einstein College of Medicine-Montefiore Medical Center, Bronx, NY (USA))



Leukocyte analysis using monoclonal antibodies in human glomerulonephritis  

Microsoft Academic Search

Leukocyte analysis using monoclonal antibodies in human glomerulonephritis. The leukocyte subpopulations were analyzed within both the glomeruli and the interstitium in renal biopsies from 145 patients with various forms of glomerulonephritis. Cells were identified by monoclonal antibodies to leukocyte cell–surface antigens and immunoper-oxidase labelling. Leukocytes, as defined by a monoclonal antibody to the leukocyte common antigen (PHM1), were present in

David H Hooke; David C Gee; Robert C Atkins



A stable IgG-like bispecific antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor demonstrates superior anti-tumor activity  

PubMed Central

The epidermal growth factor receptor (EGFR) and the type I insulin-like growth factor receptor (IGF-1R) are two cell surface receptor tyrosine kinases known to cooperate to promote tumor progression and drug resistance. Combined blockade of EGFR and IGF-1R has shown improved anti-tumor activity in preclinical models. Here, we report the characterization of a stable IgG-like bispecific antibody (BsAb) dual-targeting EGFR and IGF-1R that was developed for cancer therapy. The BsAb molecule (EI-04), constructed with a stability-engineered single chain variable fragment (scFv) against IGF-1R attached to the carboxyl-terminus of an IgG against EGFR, displays favorable biophysical properties for biopharmaceutical development. Biochemically, EI-04 bound to human EGFR and IGF-1R with sub-nanomolar affinity, co-engaged the two receptors simultaneously, and blocked the binding of their respective ligands with similar potency compared to the parental monoclonal antibodies (mAbs). In tumor cells, EI-04 effectively inhibited EGFR and IGF-1R phosphorylation, and concurrently blocked downstream AKT and ERK activation, resulting in greater inhibition of tumor cell growth and cell cycle progression than the single mAbs. EI-04, likely due to its tetravalent bispecific format, exhibited high avidity binding to BxPC3 tumor cells co-expressing EGFR and IGF-1R, and consequently improved potency at inhibiting IGF-driven cell growth over the mAb combination. Importantly, EI-04 demonstrated enhanced in vivo anti-tumor efficacy over the parental mAbs in two xenograft models, and even over the mAb combination in the BxPC3 model. Our data support the clinical investigation of EI-04 as a superior cancer therapeutic in treating EGFR and IGF-1R pathway responsive tumors.

Sereno, Arlene; Aivazian, Dikran; Langley, Emma; Miller, Brian R; Snyder, William B; Chan, Eric; Cantele, Matt; Morena, Ronald; Joseph, Ingrid BJK; Boccia, Antonio; Virata, Cyrus; Gamez, James; Yco, Grace; Favis, Michael; Wu, Xiufeng; Graff, Christilyn P; Wang, Qin; Rohde, Ellen; Rennard, Rachel; Berquist, Lisa; Huang, Flora; Zhang, Ying; Gao, Sharon X; Ho, Steffan N; Demarest, Stephen J; Reff, Mitchell E; Hariharan, Kandasamy; Glaser, Scott M



Comparison of physical chemical properties of llama V HH antibody fragments and mouse monoclonal antibodies  

Microsoft Academic Search

Antigen specific llama VHH antibody fragments were compared to antigen specific mouse monoclonal antibodies with respect to specificity, affinity and stability. The llama VHH antibody fragments and the mouse monoclonal antibodies investigated were shown to be highly specific for the protein antigen hCG or the hapten antigen RR-6. The affinity of the interaction between monovalent llama VHH antibody fragments and

R. H. J. van der Linden; L. G. J. Frenken; B. de Geus; M. M. Harmsen; R. C. Ruuls; W. Stok; L. de Ron; S. Wilson; P. Davis; C. T. Verrips



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.

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



Effect of small-molecule-binding affinity on tumor uptake in vivo: a systematic study using a pretargeted bispecific antibody.  


Small-molecule ligands specific for tumor-associated surface receptors have wide applications in cancer diagnosis and therapy. Achieving high-affinity binding to the desired target is important for improving detection limits and for increasing therapeutic efficacy. However, the affinity required for maximal binding and retention remains unknown. Here, we present a systematic study of the effect of small-molecule affinity on tumor uptake in vivo with affinities spanning a range of three orders of magnitude. A pretargeted bispecific antibody with different binding affinities to different DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid)-based small molecules is used as a receptor proxy. In this particular system targeting carcinoembryonic antigen, a small-molecule-binding affinity of 400 pmol/L was sufficient to achieve maximal tumor targeting, and an improvement in affinity to 10 pmol/L showed no significant improvement in tumor uptake at 24 hours postinjection. We derive a simple mathematical model of tumor targeting using measurable parameters that correlates well with experimental observations. We use relations derived from the model to develop design criteria for the future development of small-molecule agents for targeted cancer therapeutics. PMID:22491799

Orcutt, Kelly Davis; Rhoden, John J; Ruiz-Yi, Benjamin; Frangioni, John V; Wittrup, K Dane



Monoclonal antibodies to Pseudomonas aeruginosa ferripyochelin-binding protein.  

PubMed Central

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

Sokol, P A; Woods, D E



Recombinant genetic libraries and human monoclonal antibodies.  


In order to comprehensively manipulate the human proteome we require a vast repertoire of pharmacological reagents. To address these needs we have developed repertoires of synthetic antibodies by phage display, where diversified oligonucleotides are used to modify the complementarity-determining regions (CDRs) of a human antigen-binding fragment (Fab) scaffold. As diversity is produced outside the confines of the mammalian immune system, synthetic antibody libraries allow us to bypass several limitations of hybridoma technology while improving the experimental parameters under which pharmacological reagents are produced. Here we describe the methodologies used to produce synthetic antibody libraries from a single human framework with diversity restricted to four CDRs. These synthetic repertoires can be extremely functional as they produce highly selective, high affinity Fabs to the majority of soluble human antigens. Finally we describe selection methodologies that allow us to overcome immuno-dominance in our selections to target a variety of epitopes per antigen. Together these methodologies allow us to produce human monoclonal antibodies to manipulate the human proteome. PMID:24037841

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



Peroxidase-Labelled Monoclonal Antibodies for use in Enzyme Immunoassay  

Microsoft Academic Search

Desirable characteristics of enzyme-antibody conjugates for use in enzyme immunoassay are labelling uniformity, permanent availability and stability. The use of monoclonal antibodies (MCABS) for preparation of enzyme conjugates, in place of poly-clonal antibodies, ensures labelling uniformity and permanent availability. The problem of stability still exists. Monoclonal antibody-horseradish peroxidase (McAB-HREQ) conjugates produced in our laboratory showed variable stability. After extensive testing

D. Henning; K. Nielsen



A Monoclonal Antibody Toolkit for C. elegans  

PubMed Central

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

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



Anti-infective monoclonal antibodies: perils and promise of development  

Microsoft Academic Search

So far, most monoclonal antibodies have been developed for treating cancer or immunological diseases. However, the global spread of infections such as West Nile and corona viruses, and the need to address the potential threat of bioterrorism, has boosted public interest in, and government support of, counter-measures for infectious diseases. The attractive features of monoclonal antibodies, such as high specificity

Matthew C. Dewitz; Janice M. Reichert



Detection of Rotavirus in Human Stools by Using Monoclonal Antibody.  

National Technical Information Service (NTIS)

A monoclonal antibody, 3F7, which reacts with the common rotavirus antigen on the sixth viral gene product was prepared. It was used in a direct monoclonal antibody radioimmunoassay (RIA) as a diagnostic reagent for detection in 3.5 hours of rotavirus in ...

G. Cukor D. M. Perron R. Hudson N. R. Blacklow



A Novel Protocol for Making of Monoclonal Antibody against AFP  

Microsoft Academic Search

Alpha-fetoprotein (AFP) is a major serum protein (Mr, =70,000) synthesized during fetal life. Reappearance of AFP in adult serum often signals pathological conditions, particularly hepatocarcinomas and teratocarcinomas. To obtain monoclonal antibodies against AFP for further study of the structure and biological function of AFP, a novel method for making hybridoma cell lines producing monoclonal antibodies for AFP by immunization of

Ren-Feng Li; Xiang-Qin TIAN; Xue-Bin Li; Zhi-Gang Guo; Zi-Liang Wang; Jin-Qing Jiang; Kun Zhao; Yu-Shu Zheng; San-Hu Wang



Human tumor-associated antigens identified by monoclonal antibodies  

Microsoft Academic Search

Monoclonal antibodies have been obtained that are specific for cell surface antigens of human tumors. Some of these antigens are tissue type specific tumor antigens, most strongly expressed on tumors of the same histological type. The antigens identified by the monoclonal antibodies are primarily normal differentiation antigens that are expressed more strongly on neoplastic cells than on most normal adult

Karl Erik Hellström; Ingegerd Hellström; Joseph P. Brown



Targeting of cancer stem cell marker EpCAM by bispecific antibody EpCAMxCD3 inhibits pancreatic carcinoma.  


Patients with pancreatic cancer have a poor survival rate, and new therapeutic strategies are needed. Epithelial cell adhesion molecule (EpCAM), suggested as a marker for cancer stem cells, is over-expressed on most pancreatic tumour cells but not on normal cells and may be an ideal therapeutic target. We evaluated the anti-tumour efficiency of bispecific EpCAMxCD3 antibody linking tumour cells and T lymphocytes. In NOD SCID mice, EpCAMxCD3 had a long serum half-life (t(1/2) approximately 7 days). EpCAMxCD3 significantly retarded growth of BxPC-3 pancreatic carcinoma xenografts. For mimicking a pancreatic cancer microenvironment in vitro, we used a three-dimensional tumour reconstruct system, in which lymphocytes were co-cultured with tumour cells and fibroblasts in a collagen matrix. In this in vivo-like system, EpCAMxCD3 potently stimulated production of the effector cytokines IFN-gamma and TNF-alpha by extracorporally pre-activated lymphocytes. Moreover, compared with a bivalent anti-CD3 antibody, EpCAMxCD3 more efficiently activated the production of TNF-alpha and IFN-gamma by non-stimulated peripheral blood mononuclear cells. Most excitingly, we demonstrate for the first time that EpCAMxCD3 induces prolonged contacts between lymphocytes and tumour cells, which may be the main reason for the observed anti-tumour effects. As an important prerequisite for future use in patients, EpCAMxCD3 did not alter lymphocyte migration as measured by time-lapse video microscopy. Our data may open a way to improve the immune response and treatment outcome in patients with pancreatic cancer. PMID:20196789

Salnikov, Alexei V; Groth, Ariane; Apel, Anja; Kallifatidis, Georgios; Beckermann, Benjamin M; Khamidjanov, Akmal; Ryschich, Eduard; Büchler, Markus W; Herr, Ingrid; Moldenhauer, Gerhard



Preparation and characterization of monoclonal antibodies to swine lymphocyte antigens  

SciTech Connect

A panel of hybridoma lines were produced by the fusion of Sp2/0 myeloma cells and spleen cells from mice immunized with pig peripheral blood lymphocytes. Thirty-three stable hybridomas were produced which secreted monoclonal antibodies that reacted with pig lymphocytes, as determined by an ELISA screening procedure. These monoclonal antibodies were characterized by a complement-mediated cytotoxicity test and by flow cytometric analysis. The molecular weights of the antigens recognized by the monoclonal antibodies were determined by immunoprecipitation of /sup 125/I surface labeled lymphocytes, followed by SDS-PAGE. One monoclonal antibody, 7-34-1 (IgG2a), which reacted to all peripheral blood lymphocytes, precipitated a MHC class I molecule composed of a 50 kd heavy chain and a 12 kd light chain (..beta../sub 2/ microglobulin). This monoclonal antibody may prove to be an important reagent for the detection of SLA antigens on pig cells.

Lie, W.R.; Rothschild, M.F.; Warner, C.M.



Monoclonal antibodies against human Tudor-SN.  


Tudor-SN is a multifunctional regulator of gene expression that has been shown to function as a transcriptional co-activator, regulator of miRNA processing, mRNA splicing, and stability. Tudor-SN has also been identified as a component in RNA-induced silencing complex. Here we have produced and characterized seven monoclonal antibody (MAb) clones against human Tudor-SN. Antibodies were generated against the fourth staphylococcal nuclease-like domain (SN4) and the Tudor domain of human Tudor-SN. The MAbs recognize the Tudor-SN protein in Western blot analysis and immunoprecipitation, and detect the specific antigen in immunohistochemistry assays. One of the antibody clones also recognizes the Drosophila melanogaster and Danio rerio Tudor-SN. Immunocytochemistry of HeLa cells revealed Tudor-SN localization in nucleolus, suggesting a possible new function for the protein in the compartment. An extensive expression analysis in human tissue arrays shows moderate to high expression of Tudor-SN in a wide range of organs and tissues, especially in epithelial cell types. PMID:20568998

Saarikettu, Juha; Ovod, Vladimir; Vuoksio, Milka; Grönholm, Juha; Yang, Jie; Silvennoinen, Olli



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.



Hexavalent bispecific antibodies represent a new class of anticancer therapeutics: 1. Properties of anti-CD20/CD22 antibodies in lymphoma  

PubMed Central

The dock and lock (DNL) method is a new technology for generating multivalent antibodies. Here, we report in vitro and in vivo characterizations of 20-22 and 22-20, a pair of humanized hexavalent anti-CD20/22 bispecific antibodies (bsAbs) derived from veltuzumab (v-mab) and epratuzumab (e-mab). The 22-20 was made by site-specific conjugation of e-mab to 4 Fabs of v-mab; 20-22 is of the opposite configuration, composing v-mab and 4 Fabs of e-mab. Each bsAb translocates both CD22 and CD20 into lipid rafts, induces apoptosis and growth inhibition without second-antibody crosslinking, and is significantly more potent in killing lymphoma cells in vitro than their parental antibodies. Although both bsAbs triggered antibody-dependent cellular toxicity, neither displayed complement-dependent cytotoxicity. Intriguingly, 22-20 and 20-22 killed human lymphoma cells in preference to normal B cells ex vivo, whereas the parental v-mab depleted malignant and normal B cells equally. In vivo studies in Daudi tumors revealed 20-22, despite having a shorter serum half-life, had antitumor efficacy comparable with equimolar v-mab; 22-20 was less potent than 20-22 but more effective than e-mab and control bsAbs. These results indicate multiple advantages of hexavalent anti-CD20/22 bsAbs over the individual parental antibodies and suggest that these may represent a new class of cancer therapeutics.

Cardillo, Thomas M.; Stein, Rhona; Chang, Chien-Hsing



Current status of cancer therapy with radiolabeled monoclonal antibody  

Microsoft Academic Search

Molecular targeting therapy has become a relevant therapeutic strategy for cancer. There are several monoclonal antibodies\\u000a used for the treatment of malignant tumors. Radioimmunoconjugate is composed of antibody and radionuclide showing a synergistic\\u000a effect of radiation and immunemediated cellular toxicity and thereby enabling increased efficacy and minimizing toxicity.\\u000a Radioimmunotherapy using131I- and90Y-labeled anti-CD20 monoclonal antibodies is now indicated for the treatment

Noboru Oriuchi; Tetsuya Higuchi; Hirofumi Hanaoka; Yasuhiko Iida; Keigo Endo



Cloned transgenic farm animals produce a bispecific antibody for T cell-mediated tumor cell killing  

Microsoft Academic Search

Complex recombinant antibody fragments for modulation of immune function such as tumor cell destruction have emerged at a rapid pace and diverse anticancer strategies are being developed to benefit patients. Despite improvements in molecule design and expression systems, the quantity and stability, e.g., of single-chain antibodies produced in cell culture, is often insufficient for treatment of human disease, and the

Ludger Grosse-Hovest; Sigrid Müller; Rosa Minoia; Eckhard Wolf; Valeri Zakhartchenko; Hendrik Wenigerkind; Caroline Lassnig; Urban Besenfelder; Mathias Müller; Simon D. Lytton; Gundram Jung; Gottfried Brem



Anti-CD3 x Anti-GD2 Bispecific Antibody Redirects T-Cell Cytolytic Activity to Neuroblastoma Targets  

PubMed Central

Background The ganglioside GD2 is an attractive target for immunotherapy of neuroectodermal tumors. We tested a unique bispecific antibody anti-CD3 × anti-GD2 (3F8BiAb) for its ability to redirect activated T cells (ATC) to target GD2-positive neuroblastomas. Procedure ATC were generated from normal human peripheral blood mononuclear cells (PBMC) by stimulating the PBMC with OKT3 and expanding the T cells in the presence of interleukin 2 (IL-2) for 14 days. ATC were armed with 3F8BiAb (100 ng/106 cells) or Her2BiAb (50 ng/106 cells) prior to use. 3F8 BiAb were tested for its dual-binding specificity to GD2 expressed on cancer cell lines and CD3 expressed on ATC. 3F8BiAb-armed ATC were further tested ex vivo for their cytotoxicity against GD2 positive tumor targets and its ability to induce cytokine response upon binding to targets. Results GD2 expression in neuroblastoma cells was confirmed by FACS analysis. Specific binding of 3F8BiAb to the tumor targets as well as to ATC was confirmed by FACS analysis. 3F8BiAb-armed ATC exhibited specific killing of GD2 positive neuroblastoma cell lines significantly above unarmed ATC (P < 0.001). GD2BiAb-armed ATC secreted significantly higher levels of Th1 cytokines and chemokines compared to unarmed ATC (P < 0.001). Conclusions These preclinical findings support the potential of a novel immunotherapeutic approach to target T cells to neuroblastoma.

Yankelevich, Maxim; Kondadasula, Sri Vidya; Thakur, Archana; Buck, Steven; Cheung, Nai-Kong V.; Lum, Lawrence G.



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.

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.

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



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



Clinical laboratory applications of monoclonal antibodies.  

PubMed Central

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

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



Rat monoclonal antibodies against Aspergillus galactomannan.  

PubMed Central

Monoclonal antibodies (MAbs) against Aspergillus fumigatus galactomannan were produced in rats. Seven of them, EB-A1 through EB-A7, were characterized in more detail. They were all immunoglobulin M antibodies, reacting in an indirect enzyme-linked immunosorbent assay with purified A. fumigatus galactomannan, with avidity constants of between 2 x 10(9) and 5 x 10(9)/M. Enzyme-linked immunosorbent assay inhibition experiments with modified galactomannan and synthetic oligomers of beta (1----5)galactofuranose demonstrated that the MAbs bound to an epitope located on the beta(1----5)galactofuranose-containing side chains of the galactomannan molecule. An identical or similar epitope also seemed to be present in other fungi. Immunofluorescence and immunoelectron microscopy experiments with EB-A2 revealed the presence of the antigen in the fungal wall and inside the cell. Immunoblotting experiments demonstrated that the epitope recognized by the MAbs was a common oligosaccharide moiety of a wide range of intracellular and extracellular glycoproteins in A. fumigatus. The characteristics of the MAbs justify their use in the diagnosis of invasive aspergillosis by antigen detection. Images

Stynen, D; Sarfati, J; Goris, A; Prevost, M C; Lesourd, M; Kamphuis, H; Darras, V; Latge, J P



Monoclonal antibodies in neuro-oncology  

PubMed Central

Monoclonal antibodies (mAbs) are used with increasing success against many tumors, but for brain tumors the blood-brain barrier (BBB) is a special concern. The BBB prevents antibody entry to the normal brain; however, its role in brain tumor therapy is more complex. The BBB is closest to normal at micro-tumor sites; its properties and importance change as the tumor grows. In this review, evolving insight into the role of the BBB is balanced against other factors that affect efficacy or interpretation when mAbs are used against brain tumor targets. As specific examples, glioblastoma multiforme (GBM), primary central nervous system lymphoma (PCNSL) and blood-borne metastases from breast cancer are discussed in the context of treatment, respectively, with the mAbs bevacizumab, rituximab and trastuzumab, each of which is already widely used against tumors outside the brain. It is suggested that success against brain tumors will require getting past the BBB in two senses: physically, to better attack brain tumor targets, and conceptually, to give equal attention to problems that are shared with other tumor sites.



Characterization of monoclonal antibodies against fowl poxvirus.  


Vaccines for the prevention of fowl pox in chickens and turkeys have been available for more than five decades. However, in recent years outbreaks have occurred in several previously vaccinated chicken flocks. Presumably, fowl poxviruses (FPVs) antigenically different from the attenuated vaccine strains are responsible for such occurrences. In support of this concept, we previously detected minor antigenic changes in field isolates based on comparative immunoblotting with polyclonal anti-FPV serum. Realizing the need for antibodies specific against the dominant antigens of FPV, monoclonal antibodies (MAbs) were produced by immunizing mice with either a field strain of FPV or a pigeon poxvirus, currently used for vaccination. Three hybridoma clones producing MAbs reacting with a specific FPV protein were selected from a total of 83 clones. In immunoblots, two of the MAbs, P1D9 and P2H10, recognized an antigen with an apparent molecular weight varying from 39 to 46 kD, depending on the FPV strain. The third MAb, P2D4, reacted with an approximately 80-kD protein, regardless of which FPV isolate was tested. Immunofluorescent staining with P1D9 and P2D4 revealed that these MAbs react with intracytoplasmic antigens in FPV-infected cells. PMID:10879917

Singh, P; Tripathy, D N


Therapeutic human monoclonal antibodies in inflammatory diseases.  


Monoclonal antibodies (mAbs) are antibodies of a single antigen specificity produced by identical immune cells, i.e., clones of a common germ cell. They offer unprecedented opportunities to drug development because of their ability to target almost any cell surface or secreted molecule with remarkable efficacy and safety. In this chapter, the application of human mAbs in the treatment of inflammatory diseases is reviewed. We discuss in detail several mAb-based drugs such as anti-tumor necrosis factor (anti-TNF), anti-interleukin-1 (anti-IL-1) receptor, anti-IL-6 receptor, anti-?4 integrin subunit, and anti-CD20 agents, all of which have been documented by clinical trials to be efficacious and have been approved for the therapy of several inflammatory and immune diseases, including rheumatoid arthritis, Crohn's disease, ulcerative colitis, spondyloarthropathies, juvenile arthritis, psoriasis, psoriatic arthritis, and others. These novel drugs can be used either as a monotherapy or in combination with other conventional therapeutic modalities, particularly if the disease under treatment is refractory to therapy using solely conventional techniques. As a large variety of mAb-based agents targeting a plethora of cytokines, chemokines, adhesion and co-stimulatory molecules, receptors, as well as diverse cell types, are presently under investigation, the therapeutic armamentarium of the clinician is expected to greatly broaden in the near future, providing improved patient care for a wide range of devastating diseases of our times. PMID:24037835

Kotsovilis, Sotirios; Andreakos, Evangelos



Isolation and characterization of an anti-CD16 single-chain Fv fragment and construction of an anti-HER2\\/ neu\\/anti-CD16 bispecific scFv that triggers CD16-dependent tumor cytolysis  

Microsoft Academic Search

Bispecific antibody (bsAb)-based clinical trials of cancer have been conducted primarily using intact murine monoclonal antibody (mAb)-derived molecules. In some of these trials, toxicity resulting from the interactions of antibody Fc domains with cellular Fc receptors has limited the doses of antibody (Ab) that can be employed. Furthermore, human anti-mouse Ab responses prohibit multiple therapy courses. These factors have decreased

A. M. McCall; G. P. Adams; A. R. Amoroso; U. B. Nielsen; L. Zhang; E. Horak; H. Simmons; R. Schier; J. D. Marks; L. M. Weiner



Engineering Fully Human Monoclonal Antibodies from Murine Variable Regions  

Microsoft Academic Search

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

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



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



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

NASA Astrophysics Data System (ADS)

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

Massey, Richard J.; Schochetman, Gerald



Allosteric control of acetylcholinesterase activity by monoclonal antibodies.  


Previous studies showed that monoclonal antibodies raised against phosphorylated fetal bovine serum acetylcholinesterase appeared to modulate the catalytic activity of the enzyme by binding to a conformational epitope located at or near the region of the peripheral anionic site. The mechanism of inhibition of acetylcholinesterase by these monoclonal antibodies was further investigated by determining their effect on (i) substrate inhibition due to the binding of excess substrate to the peripheral anionic site and (ii) binding of peripheral anionic site ligands, such as propidium and fasciculin. Results of these experiments demonstrate that the accessibility of substrate to the peripheral anionic site in these complexes was restricted but not completely blocked, as none of the monoclonal antibodies eliminated the phenomenon of excess substrate inhibition. The results also show that propidium clearly slowed the inhibition of fetal bovine serum acetylcholinesterase by all six inhibitory monoclonal antibodies but to different levels. Complexation of fetal bovine serum acetylcholinesterase with monoclonal antibodies 25B1, 4E5, 6H9, and 5E8 interfered with the binding of fasciculin to the complexed enzyme, suggesting that part of their epitope overlapped with the fasciculin binding site. These monoclonal antibodies bind, in part, at the peripheral anionic site, since polyclonal anti-idiotypic antibodies generated against two monoclonal antibodies, 25B1 and 6H9, bound stoichiometric amounts of propidium. Like fasciculin, binding of these monoclonal antibodies in the vicinity of the peripheral anionic site at the rim of the active site gorge allosterically affects the orientation of W86 located at the base of the gorge, resulting in inhibition of enzyme activity. PMID:9425034

Saxena, A; Hur, R; Doctor, B P



Intrahepatically transplanted human cord blood cells reduce SW480 tumor growth in the presence of bispecific EpCAM/CD3 antibody.  


Humanized mice were generated in order to investigate the anti-tumor efficacy of bispecific antibodies. The engraftment, distribution and differentiation of mononuclear cells (MNC) from cord blood transplanted into the liver of newborn non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice were measured. Using a human-specific polymerase chain reaction (PCR), human cells were found to be present in the liver for a time range from 5 min to 5 days. After long-term engraftment of 42 days, the highest level of human cells was measured in mouse thymus, with lower levels in spleen and bone marrow. Engrafted human cells in mouse organs showed T-cell differentiation only, as measured by CD3, CD4 and CD8 expression. The MNC transplanted intrahepatically into newborn mice were tested for T-cell mediated anti-tumor activity in vivo against subcutaneously transplanted human SW480 colon carcinoma in NOD/SCID mice. A delay of SW480 tumor growth in mice in the presence of a bispecific epithelial cell-adhesion molecule (EpCAM)/CD3 antibody was found to be associated with the presence of immunoreactive human CD3 cells within the SW480 tumor. Our data provide evidence that the intrahepatic transplantation of cord blood stem cells into newborn mice represents a valuable model for establishing functionally active human T cells with anti-tumor activity. PMID:20839999

Wulf-Goldenberg, Annika; Eckert, Klaus; Fichtner, Iduna



[Monoclonal antibodies from neurological and neuropsychological perspective].  


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

Piusi?ska-Macoch, Renata



Monoclonal antibodies against plant cell wall polysaccharides  

SciTech Connect

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

Hahn, M.G.; Bucheli, E.; Darvill, A.; Albersheim, P. (Univ. of Georgia, Athens (USA))



Technological progresses in monoclonal antibody production systems.  


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

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


Monoclonal Antibodies as Tools for Mapping Genetic Diversity in Plants.  

National Technical Information Service (NTIS)

The major objective of the project was to assess the production and use of monoclonal antibodies (MC Abs) as potential genetic chromosomal markers of soybean. The hypothesis that sufficient differences in antigens exist among genotypes of soybean to allow...

T. H. Ulrich




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....


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



Intraoperative probe-directed immunodetection using a monoclonal antibody  

Microsoft Academic Search

To assess monoclonal antibody (MAb) 17-1A and its F(ab')2 fragment in intraoperative radioimmunodetection and to evaluate further the clinical usefulness of a hand-held gamma-detecting probe (GDP), we injected radiolabeled monoclonal antibody 17-1A three to six days preoperatively or its F(ab')2 fragment two to three days preoperatively into 18 patients with colorectal cancer. Intraoperative GDP counts with tumor-tissue ratios of 1.5:1

P. J. ODwyer; C. M. Mojzisik; G. H. Hinkle; M. Rousseau; J. Olsen; S. E. Tuttle; R. F. Barth; M. O. Thurston; D. P. McCabe; W. B. Farrar



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

PubMed Central

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

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



Application of Quantitative Pharmacology in Development of Therapeutic Monoclonal Antibodies  

Microsoft Academic Search

The advancement of therapeutic monoclonal antibodies during various stages of the drug development process can be effectively\\u000a streamlined when appropriate translational strategies are applied. Design of successful translational strategies for development\\u000a of monoclonal antibodies should allow for understanding of the dose– and concentration–response relationships with respect\\u000a to both beneficial and toxic effects from early phases of drug development. Evaluation of

Mohammad Tabrizi; Cherryl Funelas; Hamza Suria



Plasmodium yoelii:Identification of Rhoptry Proteins Using Monoclonal Antibodies  

Microsoft Academic Search

Hienne, R., Ricard, G., Fusa??, T., Fujioka, H., Pradines, B., Aikawa, M., and Doury, J.-C. 1998.Plasmodium yoelii:Identification of rhoptry proteins using monoclonal antibodies.Experimental Parasitology90, 230–235. Thirteen monoclonal antibodies, obtained after immunization of mice withPlasmodium yoeliischizonts, were selected using immunofluorescence assay: they all presented typical fluorescence patterns of rhoptries. This antigen localization was confirmed by immunoelectron microscopy. The molecular weights of

Remi Hienne; Gerome Ricard; Hisashi Fujioka; Bruno Pradines; Masamichi Aikawa; Jean-Claude Doury




Microsoft Academic Search

Anti-solamargine and anti-ginsenoside Rb1 monoclonal antibodies were used for preparation of an immunoaffinity column. Total solasodine glycosides were separated directly from the crude extract of Solanum khasianum fruit, by the established immunoaffinity column. This method was specific for solasodine glycosides, which was detected by thin layer chromatography and eastern blotting. An immunoaffinity column, using anti-ginsenoside Rb1 monoclonal antibody, has made

Waraporn Putalun; Noriko Fukuda; Hiroyuki Tanaka; Yukihiro Shoyama



Development and regulation of monoclonal antibody products: Challenges and opportunities  

Microsoft Academic Search

Summary  An increasing number of monoclonal antibodies for cancer diagnosis and treatment are in clinical use and in the development\\u000a pipeline, with more expected as new molecular targets are identified. As with all drugs, product quality, an appropriate pre-clinical\\u000a pharmacology-toxicology testing program, and well-designed clinical trials are essential for a successful drug development\\u000a program. However, protein products such as monoclonal antibodies

Wendy C. Weinberg; Michelle R. Frazier-Jessen; Wen Jin Wu; Andrea Weir; Melanie Hartsough; Patricia Keegan; Chana Fuchs



Monoclonal Antibodies for Detection of Norwalk Virus Antigen in Stools  

Microsoft Academic Search

Monoclonal antibodies against the prototype 8FIIa strain of Norwalk virus were prepared and applied to an enzyme immunoassay (EIA) for detecting Norwalk virus in stool specimens. The monoclonal antibodies immunoprecipitated a 58-kDa protein which had been produced by in vitro transcription-translation of Norwalk virus cloned cDNA, and they reacted by EIA with recombinant Norwalk virus capsid protein at a sensitivity




Generation and characterization of monoclonal antibodies specific for vaccinia virus.  


Monoclonal antibodies to specific vaccinia virus (VACV) proteins are valuable reagents in studies of VACV. In this chapter, we describe methods of generating a panel of monoclonal antibodies that recognize a variety of VACV proteins in their native conformation in infected cells. The antibodies thus generated recognize mostly VACV proteins that are involved in virion assembly or/and are major antigens in smallpox vaccine. These antibodies are useful for tracking distinct steps in virion assembly and for studying the B cell epitopes in smallpox vaccine. PMID:22688770

Meng, Xiangzhi; Xiang, Yan



Use of monoclonal antibodies for the analysis of mycotoxins.  


Hybridomas producing monoclonal antibodies against aflatoxin M1, ochratoxin A, zearalenone, T-2 toxin, diacetoxyscirpenol, 3-acetyl-deoxynivalenol, fusarenon X, and roridin A were developed after immunization of BALB/c mice and fusion of the splenocytes with myeloma cells. The antibodies were characterized in terms of immunoglobulin subclass, sensitivity, and specificity. The use of these antibodies in competitive enzyme immunoassays, either as microtiter plate assays or membrane-based quick tests, as well as for the production of immunoaffinity columns is described. The advantages and disadvantages of monoclonal antibodies compared to polyclonal antisera for the improvement of mycotoxin analysis are discussed. PMID:7582631

Dietrich, R; Schneider, E; Usleber, E; Märtlbauer, E



Redirected Lysis of Human Melanoma Cells by a MCSP/CD3-bispecific BiTE Antibody that Engages Patient-derived T Cells  

PubMed Central

Summary Melanoma-associated chondroitin sulfate proteoglycan (MCSP; also called HMW-MAA, CSPG4, NG2, MSK16, MCSPG, MEL-CSPG, or gp240) is a well characterized melanoma cell-surface antigen. In this study, a new bispecific T-cell engaging (BiTE) antibody that binds to MCSP and human CD3 (MCSP-BiTE) was tested for its cytotoxic activity against human melanoma cell lines. When unstimulated peripheral mononuclear blood cells (PBMC) derived from healthy donors were co-cultured with melanoma cells at effector: target (E:T) ratios of 1:1, 1:5 or 1:10, and treated with MCSP-BiTE antibody at doses of 10, 100 or 1,000 ng/mL, all MCSP-expressing melanoma cell lines (n=23) were lysed in a dose- and E:T ratio-dependent fashion, whereas there was no cytotoxic activity against MCSP-negative melanoma cell lines (n=2). To investigate whether T cells from melanoma patients could act as effector cells, we co-cultured unstimulated PBMC with allogeneic melanoma cells from 13 patients (4 stage I/II, 3 stage III, and 6 stage IV) or with autologous melanoma cells from 2 patients (stage IV). Although cytotoxic activity varied, all 15 PBMC samples mediated significant redirected lysis by the BiTE antibody. When PBMC or CD8+ T cells were pre-stimulated by anti-CD3 antibody OKT3 and IL-2, the MCSP-BiTE concentrations needed for melanoma cell lysis decreased up to 1000-fold. Because MCSP is expressed on most human melanomas, immunotherapy with MCSP/CD3-bispecific antibodies merits clinical investigation.

Torisu-Itakura, Hitoe; Schoellhammer, Hans F.; Sim, Myung-Shin; Irie, Reiko F.; Hausmann, Susanne; Raum, Tobias; Baeuerle, Patrick A.; Morton, Donald L.



Monoclonal antibodies: new agents for cancer detection and targeted therapy  

SciTech Connect

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

Baldwin, R.W.; Byers, V.S. (Univ. of Nottingham (United Kingdom))



Monoclonal antibodies distinguish identifiable neurones in the leech  

NASA Astrophysics Data System (ADS)

Monoclonal antibodies were isolated by screening 475 hybridomas obtained from mice immunized with whole leech nerve cords. The majority (about 300) reacted with leech nervous tissue, but only about 40 made antibodies that identified single kinds or small sets of cells. Twenty of the antibodies which react with specific neurones were studied in greater detail and are described here. They include antibodies against identified sensory neurones and motor neurones as well as against numerous unidentified cells.

Zipser, Birgit; McKay, Ronald



Boronated monoclonal antibody conjugates for neutron capture therapy  

SciTech Connect

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

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



Radiocurability by Targeting Tumor Necrosis Factor-{alpha} Using a Bispecific Antibody in Carcinoembryonic Antigen Transgenic Mice  

SciTech Connect

Purpose: Tumor necrosis factor-{alpha} (TNF-{alpha}) enhances radiotherapy (RT) killing of tumor cells in vitro and in vivo. To overcome systemic side effects, we used a bispecific antibody (BsAb) directed against carcinoembryonic antigen (CEA) and TNF-{alpha} to target this cytokine in a CEA-expressing colon carcinoma. We report the evaluation of this strategy in immunocompetent CEA-transgenic mice. Methods and Materials: The murine CEA-transfected colon carcinoma MC-38 was used for all experiments. In vitro, clonogenic assays were performed after RT alone, TNF-{alpha} alone, and RT plus TNF-{alpha}. In vivo, the mice were randomly assigned to treatment groups: control, TNF-{alpha}, BsAb, BsAb plus TNF-{alpha}, RT, RT plus TNF-{alpha}, and RT plus BsAb plus TNF-{alpha}. Measurements of endogenous TNF-{alpha} mRNA levels and evaluation of necrosis (histologic evaluation) were assessed per treatment group. Results: In vitro, combined RT plus TNF-{alpha} resulted in a significant decrease in the survival fraction at 2 Gy compared with RT alone (p < 0.00001). In vivo, we observed a complete response in 5 (50%) of 10, 2 (20%) of 10, 2 (18.2%) of 11, and 0 (0%) of 12 treated mice in the RT plus BsAb plus TNF-{alpha}, RT plus TNF-{alpha}, RT alone, and control groups, respectively. This difference was statistically significant when TNF-{alpha} was targeted with the BsAb (p = 0.03). The addition of exogenous TNF-{alpha} to RT significantly increased the endogenous TNF-{alpha} mRNA level, particularly when TNF-{alpha} was targeted with BsAb (p < 0.01). The percentages of necrotic area were significantly augmented in the RT plus BsAb plus TNF-{alpha} group. Conclusion: These results suggest that targeting TNF-{alpha} with the BsAb provokes RT curability in a CEA-expressing digestive tumor syngenic model and could be considered as a solid rationale for clinical trials.

Larbouret, Christel; Robert, Bruno [INSERM, Centre de Recherche en Cancerologie de Montpellier, Universite de Montpellier, and CRLC Val d'Aurelle-Paul Lamarque, Montpellier (France); Linard, Christine [IRSN, Fontenay-aux-Roses (France); Teulon, Isabelle [INSERM, Centre de Recherche en Cancerologie de Montpellier, Universite de Montpellier, and CRLC Val d'Aurelle-Paul Lamarque, Montpellier (France); Gourgou, Sophie M.Sc. [Unite de Biostatistiques en Oncologie, CRLC Val d'Aurelle-Paul Lamarque, Montpellier (France); Bibeau, Frederic [Departement d'Anatomie Pathologique, CRLC Val d'Aurelle-Paul Lamarque, Montpellier (France); Martineau, Pierre [Institut de Biotechnologie et Pharmacologie, CRLC Val d'Aurelle-Paul Lamarque, Montpellier (France); Santoro, Lore [INSERM, Centre de Recherche en Cancerologie de Montpellier, Universite de Montpellier, and CRLC Val d'Aurelle-Paul Lamarque, Montpellier (France); Departement d'Oncologie Radiotherapie, CRLC Val d'Aurelle-Paul Lamarque, Montpellier (France); Pouget, Jean-Pierre; Pelegrin, Andre [INSERM, Centre de Recherche en Cancerologie de Montpellier, Universite de Montpellier, and CRLC Val d'Aurelle-Paul Lamarque, Montpellier (France); Azria, David [INSERM, Centre de Recherche en Cancerologie de Montpellier, Universite de Montpellier, and CRLC Val d'Aurelle-Paul Lamarque, Montpellier (France); Departement d'Oncologie Radiotherapie, CRLC Val d'Aurelle-Paul Lamarque, Montpellier (France)], E-mail:



Monoclonal Antibodies and Cancer. Studies on the Use of Monoclonal Antibodies for Imaging and Therapy of Cancer, with Special Emphasis on Ovarian Carcinoma.  

National Technical Information Service (NTIS)

The usefulness of radiolabeled monoclonal antibodies for imaging and treatment of human (ovarian) cancer was investigated. A review of tumor imaging with monoclonal antibodies is presented. Special attention is given to factors that influence the localiza...

H. J. Haisma



Function-blocking antithrombospondin-1 monoclonal antibodies  

PubMed Central

Summary Background Thrombospondin-1 (TSP-1) has been implicated in many different processes based in part on inhibitory activities of anti-TSP-1 monoclonal antibodies (mAbs). Objective To map epitopes of 13 anti-TSP-1 mAbs to individual modules or groups of modules spanning TSP-1 and the closely related TSP-2 homolog. Results The mapping has led to assignment or reassignment of the epitopes of four mAbs, refinement of the epitopes of six mAbs, and confirmation of the epitopes of the remaining three mAbs. ESTs10, P12, and MA-II map to the N-terminal domain; 5G11, TSP127.6, and ESTs12 to the third properdin module; C6.7, HB8432, and P10 to epidermal growth factor (EGF)-like modules 1 and/or 2; and A6.1, mAb133, MA-I, and D4.6 to the calcium-binding wire module. A6.1, which recognizes a region of the wire that is identical in mouse and human TSP-1, reacts with TSP-1 from both species, and also reacts weakly with human TSP-2. Two other mouse antihuman TSP-1 mAbs, A4.1 and D4.6, also react with mouse TSP-1. Conclusions Consideration of previous literature and mapping of epitopes of inhibitory mAbs suggest that biological activities are present throughout TSP-1, including the EGF-like modules that have not been implicated in the past. Because the epitopes for 10 of the antibodies likely are within 18 nm of one another in calcium-replete TSP-1, some of the inhibitory effects may result from steric hindrance. Such seems to be the case for mAb133, which binds the calcium-binding wire but is still able to interfere with the activation of latent TGF-? by the properdin modules.




Monoclonal antibodies: versatile platforms for cancer immunotherapy  

Microsoft Academic Search

Antibodies are important therapeutic agents for cancer. Recently, it has become clear that antibodies possess several clinically relevant mechanisms of action. Many clinically useful antibodies can manipulate tumour-related signalling. In addition, antibodies exhibit various immunomodulatory properties and, by directly activating or inhibiting molecules of the immune system, antibodies can promote the induction of antitumour immune responses. These immunomodulatory properties can

Rishi Surana; Shangzi Wang; Louis M. Weiner



A monoclonal antibody for the specific diagnosis of plague*  

PubMed Central

A stable mouse-cell hybridoma was obtained that secretes an IgA monoclonal antibody reactive with the fraction 1 (F1) envelope antigen of Yersinia pestis. Titres of the antibody typically ranged from 1:32 768 to 1:65 536 in mouse ascitic fluids. The monoclonal antibody formed a line of precipitation when run against F1 antigen in Ouchterlony gel diffusion tests. In tests of 235 strains of Y. pestis, lines of identity occurred between the precipitates formed with a solution of purified F1 antigen and the F1 antigen produced by the plague strains. No precipitates formed for 65 strains that were incapable of elaborating F1 antigen. Specificity of the monoclonal antibody for strains of Y. pestis producing F1 was also indicated by negative results for 50 yersinia strains other than Y. pestis tested by an ELISA that used the antibody to capture antigen. Experiments to determine the shelf-life of the antibody were conducted over 3-4 years. When the monoclonal antibody was freeze-dried in vials, titre was retained for three years when the vials were stored at -70 °C but only for two months when they were stored at ambient temperatures. When the antibody was freeze-dried in wells of ELISA plates, sensitivity of the plates for capture of F1 antigen was preserved for four years when the plates were stored at -70 °C compared with two weeks for plates stored at room temperature. When a solution of the antibody was sealed in wells of ELISA plates and refrigerated at 4 °C, reactivity of the antibody and sensitivity of the plates were retained for a year. Alternatives for the application of this monoclonal antibody in ELISA and other plague diagnostic procedures are discussed.

Williams, J. E.; Gentry, M. K.; Braden, C. A.; Tyndal, G. L.; Altieri, P. L.; Berman, S.; Robinson, D. M.



Monoclonal antibodies to drosophila cytochrome P-450's  

SciTech Connect

Hybridomas producing monoclonal antibodies were prepared by the fusion of SP2/0 myeloma cells and spleen cells from a female BALB/c mouse immunized by cytochrome P-450-A and P-450-B purified from Drosophila Hikone-R (BG) microsomes. P-450-A and P-450-B are electrophoretically distinct subsets of Drosophila P-450. P-450-A is ubiquitous among strains tested, while P-450-B is present in only a few strains displaying unique enzyme activities and increased insecticide resistance. The Oregon-R strain contains only cytochromes P-450-A and is susceptible to insecticides. The authors Hikone-R (BG) strain expresses both cytochromes P-450-A and P-450-B and is insecticide resistant. Antibody producing hybridomas were detected in a solid-phase radioimmunoassay (RIA) by binding to Hikone-R (BG) or Oregon-R microsomes. Four independent hybridomas were identified as producing monoclonal antibodies that recognized proteins in the P-450 complex by immunoblot experiments. Three monoclonal antibodies recognized P-450-A proteins, while one monoclonal antibody bound predominantly P-450-B. This monoclonal antibody also recognized southern armyworm (Spodoptera eridania, Cramer) microsomal proteins.

Sundseth, S.S.; Kennel, S.J.; Waters, L.C.



Research and Diagnostic Applications of Monoclonal Antibodies to 'Coccidioides immitis'.  

National Technical Information Service (NTIS)

The authors have been assembling a panel of mouse monoclonal antibodies to antigens on the surface of endospores and spherules, and in culture filtrates and soluble extracts of spherule-phase C. immitis. These antibodies are being used to develop antigen-...

A. E. Karu D. J. Gennevois J. W. Hoffman S. J. Kraeger H. B. Levine



Monoclonal antibody to cytokeratin for use in routine histopathology  

Microsoft Academic Search

CAM 5.2 is a murine monoclonal antibody, raised against the colon carcinoma cell line HT29, which recognises lower molecular weight intracellular cytokeratin proteins within secretory epithelia. Extensive indirect immunohistochemical studies have confirmed that this antibody stains formalin fixed (and freshly frozen) normal and malignant human tissue in a consistent manner. Reliable staining of conventionally processed pathological tissues provides more accurate

C A Makin; L G Bobrow; W F Bodmer



Human Monoclonal Antibodies for Neutralization of Botulinum Neurotoxin.  

National Technical Information Service (NTIS)

The purpose of this work is to generate neutralizing human monoclonal antibodies to Botulinum neurotoxins (BoNT) A, B, and E. To generate a large panel of antibodies, mice transgenic for the human immunoglobulin were immunized with BoNT/A, B, and E bindin...

J. D. Marks



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



Specific Endocrine Tissue Marker Defined by a Monoclonal Antibody  

Microsoft Academic Search

One of two mouse monoclonal antibodies (LK2H10) produced by hybridoma technology against a human endocrine tumor (pheochromocytoma) demonstrated specific immunoreactivity for 69 normal and neoplastic endocrine cells and tissues known to contain secretory granules. This immunoreactivity was specific, since other normal tissues, tumors from endocrine cells without granules, and tumors from other nonendocrine tissues were negative when tested with antibody

Ricardo V. Lloyd; Barry S. Wilson



Identification and serotyping of Microsporum canis isolates by monoclonal antibodies.  

PubMed Central

Hybridoma cells were produced by fusing mouse myeloma cells with spleen cells from mice primed with an exoantigen of Microsporum canis. Three clones produced antibodies which were examined by the Western blot technique for their potential usefulness in the identification of M. canis isolates and differentiation of strains within the species. Based on reactions with immunological determinants, all of the M. canis isolates tested presented either species- or strain-specific domains. Monoclonal antibodies proved to be useful reagents for the identification of M. canis isolates and for the differentiation of strains within the species. A purified antigen depleted of common antigenic determinants was obtained in affinity chromatography by using monoclonal antibody. Images

Polonelli, L; Castagnola, M; Morace, G



Monoclonal antibodies to damaged and regenerating vascular endothelium.  


Monoclonal antibodies were raised by injecting mice with cultured bovine, porcine or human vascular endothelial cells, and tested in an in vitro model of endothelial damage and regrowth. Antibodies were identified which produced four distinct and reproducible immunoperoxidase staining patterns, and these were further characterized by Western blotting and I125 labelling. Antibodies which recognize damaged endothelial cells probably do so by reacting with intracellular antigens made accessible by increased membrane permeability. Other antibodies give a diffuse staining of intact cells, by reacting with a membrane component, or a "negative stain" pattern by reacting with extracellular matrix material. A small number of antibodies recognize new or spreading endothelium perhaps by recognizing selectively expressed antigens on these cells. Appropriate monoclonal antibodies may be useful in studying endothelial behaviour in vivo. PMID:2462052

Pringle, S; de Bono, D P



Monoclonal Antibodies in Gynecological Cancer: A Critical Point of View  

PubMed Central

During the last decades, several improvements in treating gynecological malignancies have been achieved. In particular, target therapies, mostly monoclonal antibodies, have emerged as an attractive option for the treatment of these malignancies. In fact, various molecular-targeted agents have been developed for a variety of malignancies with the objective to interfere with a precise tumor associated receptor, essential for cancer cell survival or proliferation, blocking its function, of the cancer cells. Alternatively, monoclonal antibodies have been developed to block immune suppression or enhance functions of immune effector cells. So far, several monoclonal antibodies have been tested for clinical efficacy for the treatment of gynecological cancers. Antibodies against Vascular Endothelial Growth Factor (VEGF) and Epidermal Growth Factor Receptor (EGFR) have been used in different neoplasms such as ovarian and cervical cancer. Catumazumab, a bivalent antibody against CD3 and EpCAM, is effective in the treatment of neoplastic ascites. Other antibodies are peculiar for specific cancer-associated antigen such as Oregovomab against CA125 or Farletuzumab against the folate receptor. Here we describe the preclinical and clinical experience gained up to now with monoclonal antibodies in tumors of the female genital tract and trace future therapeutic and research venues.

Bellati, Filippo; Napoletano, Chiara; Gasparri, Maria Luisa; Visconti, Valeria; Zizzari, Ilaria Grazia; Ruscito, Ilary; Caccetta, Jlenia; Rughetti, Aurelia; Benedetti-Panici, Pierluigi; Nuti, Marianna



Serological classification of Neisseria gonorrhoeae with monoclonal antibodies.  

PubMed Central

Hybrid cells producing monoclonal antibodies against antigens of Neisseria gonorrhoeae were obtained by the polyethylene glycol-mediated fusion of mouse myeloma cells and lymphocytes from mice immunized with gonococcal protein I or outer membrane proteins. From four fusions, 16 phenotypically stable, independently cloned hybrid cell lines were selected for continued study. Each of the cell lines produced a characteristically different monoclonal antibody which reacted in immunoprecipitation assays with a unique antigenic determinant on protein I of the outer membrane complex of the bacteria. In antibody binding, immunofluorescence, and coagglutination assays these antibodies each reacted with a restricted group of N. gonorrhoeae strains. None of the monoclonal antibodies reacted with 17 other different species of Neisseria or with Branhamella catarrhalis. When tested on 34 N. gonorrhoeae reference serotyping strains, the monoclonal antibodies demonstrated serological relationships between the strains which paralleled those observed with conventional polyvalent antisera. These antibodies now provide standardized reagents for the rapid and precise serological characterization of many strains of N. gonorrhoeae. Images FIG. 1 FIG. 2 FIG. 3 FIG. 4 FIG. 5

Tam, M R; Buchanan, T M; Sandstrom, E G; Holmes, K K; Knapp, J S; Siadak, A W; Nowinski, R C



Immunolocalization of Keratin Polypeptides Epidermis Using Monoclonal Antibodies in Human  

Microsoft Academic Search

Three monoclonal antibodies (AE1, AE2, and AE3) were prepared against human epidermal keratins and used to study keratin expression during normal epidermal differentia- tion. Immunofluorescence staining data suggested that the antibodies were specific for keratin- type intermediate filaments. The reactivity of these antibodies to individual human epidermal keratin polypeptides (65-67, 58, 56, and 50 kdaltons) was determined by the immunoblot




Characterization of Monoclonal Antibodies Against Human Low Density Lipoprotein  

Microsoft Academic Search

Seven monoclonal antibodies against human low density lipoprotein (LDL) have been characterized as to their specificity and ability to interfere with the LDL pathway in cultured human flbroblasts. The Immunoreactivity with LDL of two of the antibodies (2D8 and 4G3) was particularly sensitive to modification of lysine and arginlne resi- dues In LDL. Cotitration experiments indicated that the antibodies 3A8

Ross W. Milne; Richard Theolis; Roy B. Verdery; Yves L Marcel



Immunocytochemistry of brain-reactive monoclonal antibodies in peripheral tissues  

Microsoft Academic Search

Among a total of 135 tissue-reactive monoclonal antibodies previously prepared, 81 were brain-selective and were classified into neuronal and non-neuronal categories. The neuronal antibodies were again subdivided into antineurofibrillar, antiperikaryonal-neurofibrillar, and antisynapse-associated groups. On the basis of morphologic, developmental, biochemical, and pathologic criteria, the antibodies in at least two of these groups were found to detect heterogeneous antigens (called “neurotypes”)

Elisabeth Östermann; Nancy H. Sternberger; Ludwig A. Sternberger



Development and characterization of new rat monoclonal antibodies for procalcitonin  

Microsoft Academic Search

The development of selective and sensitive biological recognition elements, e.g., antibodies, for the detection of relevant\\u000a blood markers is a great challenge in the field of biosensors. In this context, five new rat monoclonal antibodies (mAbs)\\u000a for procalcitonin (PCT), a marker for bacterial infection and sepsis, were developed and characterized. One mAb, PROC1 3G3,\\u000a was used as capture antibody. Four

Petra M. Krämer; Marie-Françoise Gouzy; Melanie Keß; Ulrike Kleinschmidt; Elisabeth Kremmer



A monoclonal antibody-linked immunoassay for hemoglobin H disease  

Microsoft Academic Search

Summary A murine monoclonal antibody (mAb) was generated that recognizes hemoglobin (Hb) H, the tetrameric form (ß4) of human ß-globin chains. The antibody ß4-1 (?1, ?) does not react with Hbs A, F, Bart's, or isolated ß chains, indicating that the antibody recognizes an epitope comprised of multiple ß chains. A simple, rapid, and sensitive enzyme immunoassay was established to

M. Shyamala; C. R. Kiefer; H. Moscoso; F. A. Garver



Mechanism of action of therapeutic monoclonal antibodies: promises and pitfalls of in vitro and in vivo assays.  


Therapeutic monoclonal antibodies (mAbs) are mostly used in cancer, as anti-infectious agents and as immunomodulatory drugs, and are amongst the most active area of research and development in the pharmaceutical industry. This class of drugs comprises unconjugated antibodies or antibody fragments, antibody-drug conjugates, radio-immunoconjugates and bispecific/trispecific molecules. A better understanding of the mechanism of action of successful mAbs is fundamental for the selection of more active and less toxic mAbs of new generation. Furthermore reliable screening of new compounds at an early stage of preclinical development, for both efficacy and toxicity, should allow the selection of the best molecules at an early stage, and improve the rate of success of this class of drugs. Here we review the major methods that are employed for testing the activity of therapeutic mAbs in vitro and in vivo in small animal models and point out to some of the pitfalls in these assays. PMID:22387378

Golay, Josée; Introna, Martino



Monoclonal antibodies as disease modifying therapy in multiple sclerosis.  


Multiple sclerosis (MS), a demyelinating disease of the central nervous system, was untreatable until the mid-1990s when beta-interferons and glatiramer acetate were introduced. These agents, while effective, were relatively nonspecific in action. Over the last 10 years, research has focused toward developing more targeted therapies for the disease. Monoclonal antibodies (mAbs) have been central to these efforts and many of the mAbs studied in MS have been singularly effective. We review here the 6 monoclonal antibodies that have been approved for MS or are in late-stage clinical trials, focusing on the drugs' efficacy and safety. Additionally, we review several monoclonal antibodies that were studied in MS but were found to be ineffective or even deleterious in this patient population. PMID:24027005

Longbrake, Erin E; Parks, Becky J; Cross, Anne H



Passive Intranasal Monoclonal Antibody Prophylaxis against Murine Pneumocystis carinii Pneumonia  

PubMed Central

Passive antibody immunoprophylaxis is one method used to protect patients against infection if they are unable to mount an adequate active immune response. Topical application of antibody may be effective against infections at mucosal sites. Using a SCID mouse model of Pneumocystis carinii pneumonia, we were able to demonstrate protection against an airborne challenge with P. carinii by intranasal administration of antibody. Immunoglobulin M (IgM) monoclonal antibodies to an epitope shared by mouse and human P. carinii organisms reduced organism numbers by more than 99% under the conditions described. An IgG1 switch variant of one of the IgM monoclonal antibodies was also protective. These experiments provide a model for exploring the utility of this approach in protecting at-risk patients from infection with P. carinii.

Gigliotti, Francis; Haidaris, Constantine G.; Wright, Terry W.; Harmsen, Allen G.



Properties and characterization of monoclonal antibodies to Tacaribe virus.  


Monoclonal antibodies prepared against Tacaribe and Junin viruses have been used to define further the serological relationships between arenaviruses of the Tacaribe complex. A close relationship was found between these two viruses and the heterologous Amapari and Machupo viruses, with Pichinde virus and Parana virus being more distantly related. Among the antibodies specific for Tacaribe virus, five were found to react with viral antigens at the surface of infected cells and to neutralize virus infectivity in vitro. These five antibodies could be differentiated by competitive immunoassay as recognizing at least two antigenically distinct epitopes. The kinetics of reaction between antibody and virus were examined for all five neutralizing antibodies. One antibody (2.25.4) effectively neutralized all infectious virus. The remaining four directed against a second epitope gave significant persistent fractions which could be reduced by addition of complement, anti-mouse immunoglobulin, or antibody 2.25.4. Variants of Tacaribe virus resistant to neutralization by antibody 2.25.4 were obtained by growth in the presence of this antibody and neutralization kinetics were reexamined using the heterologous monoclonal neutralizing antibodies. Several different neutralization profiles were obtained, suggesting that point mutations resulted in conformational changes at topographically selected distinct epitopes recognized by the remaining antibodies. PMID:2410550

Howard, C R; Lewicki, H; Allison, L; Salter, M; Buchmeier, M J



Purification of human monoclonal antibodies and their fragments.  


This chapter summarizes the most common chromatographic mAb and mAb fragment purification methods, starting by elucidating the relevant properties of the compounds and introducing the various chromatography modes that are available and useful for this application. A focus is put on the capture step affinity and ion exchange chromatography. Aspects of scalability play an important role in judging the suitability of the methods. The chapter introduces also analytical chromatographic methods that can be utilized for quantification and purity control of the product. In the case of mAbs, for most purposes the purity obtained using an affinity capture step is sufficient. Polishing steps are required if material of particularly high purity needs to be generated. For mAb fragments, affinity chromatography is not yet fully established, and the capture step potentially may not provide material of high purity. Therefore, the available polishing techniques are touched upon briefly. In the case of mAb isoform and bispecific antibody purification, countercurrent chromatography techniques have been proven to be very useful and a part of this chapter has been dedicated to them, paying tribute to the rising interest in these antibody formats in research and industry. PMID:24037849

Müller-Späth, Thomas; Morbidelli, Massimo



The clinical application of monoclonal antibody therapies in renal transplantation.  


Monoclonal antibodies have become valuable tools for the precise clinical manipulation of the immune system. These highly specific proteins have proven their usefulness in both the treatment and prevention of organ transplant rejection. Indeed, they are the centrepieces of many evolving regimens designed to reduce or eliminate the need for chronic immunosuppression. This manuscript will review the monoclonal antibodies that have made their way into the clinic either as experimental therapies or approved drugs. It will provide a general overview of this class of agents and their mechanisms of action. Standard therapies and potential new applications will be described. PMID:15155134

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



[Monoclonal antibody therapy for non-Hodgkin's lymphoma].  


Anti-CD20 monoclonal antibodies are currently used for non-Hodgkin's lymphoma treatment and Rituximab (MabThera) and ibritumomab-tiuxetan (Zevalin) currently approved for clinical use. Rituximab has improved survival of patients with follicular and diffuse large B cells lymphoma. Rituximab is safe with uncommon side effects mainly observed during the first infusion. It is basically used associated with chemotherapy every three weeks and also as maintenance therapy for relapsed follicular lymphoma. A better understand of its mechanisms of action has allowed to develop new generation of monoclonal antibody currently tested in clinical trials. PMID:20222313

Paul, Franciane; Rossi, Jean-François; Cartron, Guillaume



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

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



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


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



Purification of monoclonal antibodies by simulated moving-bed chromatography.  


A simulated moving bed (SMB) system has been developed for the biospecific purification of monoclonal antibodies. Adsorption and desorption of the desired product is performed under different conditions. To increase the purity and yield of the antibodies, two purge steps have to be introduced. The steady-state performance of the SMB system was modelled by solving the governing differential equations using a linear driving force approximation. The model parameters were determined independently in batch experiments. They were used to determine the operating conditions of the SMB system for the purification of monoclonal antibodies from cell culture supernatant. The antibodies could be isolated with a yield of > or = 90. SDS gel electrophoresis of the feed and product stream showed that more than 99% of the contaminating proteins were removed in a single step by SMB chromatography. PMID:9129308

Gottschlich, N; Kasche, V



Phage display technology for human monoclonal antibodies.  


During the last 15 years in vitro technologies opened powerful routes to combine the generation of large libraries together with fast selection procedures to identify lead candidates. One of the commonest methods is based on the use filamentous phages. Antibodies (Abs) can be displayed successfully on the surface of phage by fusing the coding sequence of the antibody variable (V) regions to the phage minor coat protein pIII. By creating large libraries, antibodies with affinities comparable to those obtained using traditional hybridomas technology can be selected by a series of cycles of selection on antigen. As in this system antibody genes are cloned simultaneously with selection they can be easily further engineered for example by increasing their affinity (to levels unobtainable in the immune system), modulating their specificity or their effector function (by recloning into a full-length immunoglobulin scaffold). This chapter describes the basic protocols for antibody library construction, handling, and selection. PMID:24037846

Deantonio, Cecilia; Cotella, Diego; Macor, Paolo; Santoro, Claudio; Sblattero, Daniele



Monoclonal Antibodies as Probes of the Organization of the Eukaryotic Cell Nucleus.  

National Technical Information Service (NTIS)

As an approach to exploring nuclear organization and function, we have isolated monoclonal antibodies directed against determinants present in a subfraction of rat liver nuclei. Certain of these monoclonal antibodies recognize proteins of the residual nuc...

D. D. Newmeyer



Isolation of Monoclonal Antibodies with Predetermined Conformational Epitope Specificity  

Microsoft Academic Search

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

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



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



An immunocytochemical investigation with monoclonal antibodies to somatostatin  

Microsoft Academic Search

Four monoclonal antibodies specific for somatostatin have been produced and characterized. These antibodies were used to assess the anatomical relationship of somatostatin-containing cells in the pancreas and gastrointestinal tract of man, baboon and rat with ten other peptide-containing endocrine cells. The peptides investigated were gastrin, cholecystokinin, motilin, secretin, neurotensin, gastric inhibitory polypeptide, gut-glucagon, pancreatic glucagon, pancreatic polypeptide and insulin.

A. M. J. Buchan; L. K. J. Sikora; J. G. Levy; C. H. S. McIntosh; I. Dyck; J. C. Brown



Monoclonal Antibody Specific to Urochordate Botryllus schlosseri Pyloric Gland  

Microsoft Academic Search

We describe here the development of a new hybridoma cell line, CF12F6, that produces a specific antibody to Botryllus schlosseri (a colonial tunicate). The monoclonal antibody was isotyped as IgG1 (by enzyme-linked immunosorbent assay), and the cellular localization of the antigenic epitope that reacts specifically with CF12F6 was confined to the cells of the pyloric gland of the zooid (by

Ziva Lapidot; Guy Paz; Baruch Rinkevich



Identification of two antigenic determinants in pseudomurein by monoclonal antibodies  

SciTech Connect

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

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



Fully human antibodies specific to CADM1  

US Patent & Trademark Office Database

The present disclosure provides isolated monoclonal antibodies, particularly human monoclonal antibodies, more particularly engineered antibodies resulting in increased binding to Fc receptors and/or increased potency for ADCC or immunoconjugates, which specifically bind to CADM1 with high affinity. Nucleic acid molecules encoding CADM1 antibodies, expression vectors, host cells and methods for expressing the CADM1 antibodies are also provided. Bispecific molecules and pharmaceutical compositions comprising the CADM1 antibodies are also provided. Methods for detecting CADM1, as well as methods for treating various cancers, including lung cancer and pancreatic cancer, are disclosed.



Biological characterization of human monoclonal antibodies to rabies virus.  

PubMed Central

Rabies virus antigen-specific human monoclonal antibodies (MAbs) that recognized either viral glycoprotein, ribonucleoprotein, or matrix proteins were generated. Only glycoprotein-specific MAb neutralized a variety of rabies viruses and protected laboratory rodents against lethal rabies virus infection. The determinant recognized by this MAb does not appear to reside in previously defined antigenic sites of the viral glycoprotein. Images

Dietzschold, B; Gore, M; Casali, P; Ueki, Y; Rupprecht, C E; Notkins, A L; Koprowski, H



Characterization of Cryptococcus neoformans capsular glucuronoxylomannan polysaccharide with monoclonal antibodies.  

PubMed Central

Mice were immunized with Cryptococcus neoformans serotype A capsular glucuronoxylomannan (GXM) conjugated to bovine serum albumin-adipic dihydrazide. Two splenocyte fusions yielded two monoclonal antibodies (MAbs) that were highly reactive in dot enzyme immunoassay, immunofluorescence, and sandwich enzyme immunoassay. The first MAb, BD-1 [immunoglobulin G1 (kappa) [IgG1(kappa)

Todaro-Luck, F; Reiss, E; Cherniak, R; Kaufman, L



Solidphase enzyme immunoassay of urokinase using monoclonal antibodies  

Microsoft Academic Search

Using two monoclonal antibodies directed against urokinase, we have developed a micro enzyme-linked immunosorbant assay (ELISA) to detect and measure urokinase in biological fluids. The system presents the following characteristics: simple and rapid procedure, reproducibility, sensitivity (urokinase levels down to 1 ng\\/ml) and evaluation of the enzyme in biological fluids such as urine, pleural elfusions, and ascitic fluids without preliminary

P. Hérion; D. Portetelle; J.-D. Franssen; J. Urbain; A. Bollen



Monoclonal antibodies to the alternative oxidase of higher plant mitochondria  

Microsoft Academic Search

The higher plant mitochondrial electron transport chain contains, in addition to the cytochrome chain which terminates with cytochrome oxidase, an alternative pathway that terminates with an alternative oxidase. The alternative oxidase of Sauromatum guttatum Schott has recently been identified as a cluster of proteins with apparent M{sub r} of 37, 36, and 35 kilodaltons (kD). Monoclonal antibodies have now been

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



[Immunoenzyme analysis of the Willebrand factor using monoclonal antibodies].  


A method for measuring Willebrand's factor protein has been developed, based on indirect solid-phase enzyme immunoassay with Soviet monoclonal antibodies to this factor. Normal Willebrand's factor level in the plasma has been found 55-161 percent. The method has been tried in patients with Willebrand's disease and with oncologic diseases. PMID:1710696

Toropova, B G; Gornostaev, V S; Danilov, A O; Niagulov, D F; Okulov, V B; Olimpieva, A B; Papaian, L P; Serebrianaia, N B; Chistiakova, Z M; Golovina, O G



Monoclonal antibodies in allergy; current applications and promising trials.  


Allergic disorders, including asthma, allergic rhinoconjunctivitis, atopic dermatitis, food allergies, urticaria and anaphylaxis have significant impacts on our daily lives. Innovation of novel treatment modalities targeting effector cells, cytokines, receptors or signaling pathways may bring new alternatives in the management of diseases, including allergic disorders. Understanding the mechanisms of allergic immune response is a requisite to plan or switch to a new or an adjunctive treatment course instead of or in addition to current conventional therapies, which are almost effective and safe in most of the cases. Monoclonal antibodies are major candidates that may act on the specific targets of allergic immune response in the treatment of these disorders. As monoclonal antibodies may contribute to our designing of new treatment options within the next years, specific concerns have to be considered such as efficacy, safety, long-term tolerability of these molecules as well as identifying associations with the mechanisms of disease pathogenesis. Hereby, it is aimed to review most of the currently published clinical studies in which some monoclonal antibodies were trialed for the management of allergic disorders. This review also addressed some recently patented monoclonal antibodies and their uses. PMID:19925446

Ozdemir, Cevdet



Internal radiation dosimetry for clinical testing of radiolabeled monoclonal antibodies  

Microsoft Academic Search

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

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



Monoclonal antibodies raised against post-translational domains of the electroplax sodium channel  

Microsoft Academic Search

Summary Eleven monoclonal antibodies were identified that recognized eel electroplax sodium channels. All the monoclonal antibodies specifically immunostained the mature TTX-sensitive sodium channel (Mr 265,000) on immunoblots. None of the monoclonal antibodies would precipitate the in vitro translated channel core polypeptide in solution. One monoclonal antibody, 3G4, was found to bind to an epitope involving terminal polysialic acids. Extensive digestion

Stan Ivey; William B. Thornhill; Simon R. Levinson



Targeting of Porphyromonas gingivalis with a bispecific antibody directed to FcalphaRI (CD89) improves in vitro clearance by gingival crevicular neutrophils.  


Phagocytosis and killing of pathogens by polymorphonuclear neutrophils (PMN) from gingival crevicular fluid (GCF) is diminished in chronic periodontitis patients. As an approach to improve targeting of PMN toward a periodontopathogen, Porphyromonas gingivalis, the efficacy of a bispecific antibody (BsAb) directed against both recombinant 130 kDa hemagglutinin domain (r130k-HMGD) of P. gingivalis, and PMN Fc receptor (FcR) was evaluated. GCF PMN exhibited higher IgA FcR (FcalphaRI) levels, and lower IgG FcR (FcgammaRIIa and FcgammaRIIIb) levels than PB PMN. Functional studies revealed that GCF PMN exhibited a higher capacity to phagocytose and kill P. gingivalis opsonized with a BsAb targeting P. gingivalis r130k-HMGD to FcalphaRI as compared to an anti-r130k-HMGD antibody. However, phagocytosis and killing activity of PB PMN that were incubated with the two antibodies proved comparable. These data support targeting of pathogens toward FcalphaRI as an option to improve antibacterial immunity in chronic periodontitis patients. PMID:15542178

Kobayashi, Tetsuo; Takauchi, Ayano; van Spriel, Annemiek B; Vilé, Henriette A; Hayakawa, Mitsuo; Shibata, Yasuko; Abiko, Yoshimitsu; van de Winkel, Jan G J; Yoshie, Hiromasa



Monoclonal antibodies to outer membrane antigens of Vibrio cholerae.  

PubMed Central

Hybridoma-derived monoclonal antibodies were prepared against outer membrane antigens of four strains of Vibrio cholerae that were cultivated under iron-limited conditions, and these antibodies were partially characterized. We established a library of 66 hybridomas which produced monoclonal antibodies defining 16 different V. cholerae antigens. Two antigens (molecular weights, 18,000 and 112,000) were heat modifiable, whereas the reacting epitope of a third antigen (40,000-dalton-18,000-dalton doublet) was completely destroyed when it was heated at 100 degrees C. The 112,000-dalton heat-modifiable protein was an iron-regulated outer membrane protein. This protein bound 59Fe in vitro when it was combined with the V. cholerae siderophore-iron complex 59Fe-vibriobactin; it was also found in in vivo grown V. cholerae, as were three other antigens. A total of 26 hybridomas produced antibody to V. cholerae lipopolysaccharide. Of these, 12 were cross-reactive with lipopolysaccharides of other gram-negative bacteria, including 2 which recognized lipid A. Several of these anti-lipopolysaccharide monoclonal antibodies appeared to be lipopolysaccharide region specific. Some membrane antigens were strain specific, whereas others were common to both O group 1 and non-O group 1 vibrios. Images

Sciortino, C V; Yang, Z S; Finkelstein, R A



Monoclonal antibodies as probes of epithelial membrane polarization  

PubMed Central

Monoclonal antibodies directed against antigens in the apical plasma membrane of the toad kidney epithelial cell line A6 were produced to probe the phenomena that underlie the genesis and maintenance of epithelial polarity. Two of these antibodies, 17D7 and 18C3, were selected for detailed study here. 17D7 is directed against a 23-kD peptide found on both the apical and basolateral surfaces of the A6 epithelium whereas 18C3 recognizes a lipid localized to the apical membrane only. This novel observation of an apically localized epithelial lipid species indicates the existence of a specific sorting and insertion process for this, and perhaps other, epithelial plasma membrane lipids. The antibody-antigen complexes formed by both these monoclonal antibodies are rapidly internalized by the A6 cells, but only the 18C3-antigen complex is recycled to the plasma membrane. In contrast to the apical localization of the free antigen, however, the 18C3-antigen complex is recycled to both the apical and basolateral surface of the epithelium, which indicates that monoclonal antibody binding interferes in some way with the normal sorting process for this apical lipid antigen.



Library of monoclonal antibodies against brush border membrane epithelial antigens  

SciTech Connect

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

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



Monoclonal antibody cross-reactions between Drosophila and human brain.  

PubMed Central

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

Miller, C A; Benzer, S



A Versatile Monoclonal Antibody Specific to Human SERPINB5  

PubMed Central

Maspin (SERPINB5) is a member of the Clade B subgroup of the large superfamily of serine protease inhibitors. It is proposed that maspin is a tumor suppressor; however, its molecular role remains to be elucidated. Here we report the characterization of a mouse monoclonal antibody directed against human maspin. This antibody, 16F7, recognizes maspin in both its native and denatured form, unlike several other commercial antibodies tested in this study. It will be a useful and versatile tool for future analyses of the biological function of maspin.

Teoh, Sonia S.Y.; Wang, Hong; Risbridger, Gail P.; Bird, Phillip I.



Radioimmunodetection of human melanoma with indium-111-labeled monoclonal antibody  

SciTech Connect

The purpose of the study was threefold: (1) to evaluate the efficacy of an /sup 111/In-labeled murine monoclonal antibody (ZME-018) directed against a heavy molecular weight melanoma associated glycoprotein in localizing metastatic disease; (2) to determine the effect of unlabeled antibody mass (2.5, 5, 10, 20, and 40 mg) on labeled antibody blood clearance, biodistribution and lesion detection; (3) to estimate radiation dosimetry. Twenty-five patients with previously documented disease received an intravenous infusion of 2.5 to 40 mg of monoclonal antibody with 1 mg of the antibody labeled with 5 mCi of /sup 111/In. There were no acute reactions. Patients were scanned without computer enhancement or background subtraction techniques at 24 and 72 hr after injection. Imaging detected tumor in 14/18 (78%) patients with active disease, identified 24/44 (77%) of lesions greater than 1 cm and changed or specifically directed patient management in 22% (4/18) patients with tumor. There was a prolongation in blood clearance associated with decreased liver and spleen activity following administration of 20 and 40 mg of antibody compared to the three lower antibody dose levels. Assuming a biodistribution similar to (/sup 111/In)ZME-018, the radiation dose delivered to normal tissues by (90Y)ZME-018 would restrict its use as a routine vehicle for radioimmunotherapy; however, it may be possible to deliver substantial tumor doses in selected patients.

Taylor, A. Jr.; Milton, W.; Eyre, H.P.; Christian, P.; Wu, F.; Hagan, P.; Alazraki, N.; Datz, F.L.; Unger, M.



Use of Heteropolymeric Monoclonal Antibodies to Attach Antigens to the C3b Receptor of Human Erythrocytes: A Potential Therapeutic Treatment  

NASA Astrophysics Data System (ADS)

We have prepared bispecific, cross-linked monoclonal antibodies (heteropolymers) with specificity for both targeted antigens and the human erythrocyte (RBC) complement receptor. These heteropolymers facilitate binding of target antigens (human IgG and dinitrophenylated bovine ? globulin) to human RBCs under conditions that either allow or preclude complement activation. Quantitative analyses of this binding agree well with the number of complement receptors per RBC. In vitro "whole-blood" model experiments indicate heteropolymer-facilitated binding of antigens to RBCs is rapid and stable at 37^circC. It may be possible to extend these prototype experiments to the in vivo situation and use heteropolymer-attached RBCs for the safe and rapid binding, neutralization, and removal from the circulation of pathogenic antigens associated with infectious disease.

Taylor, Ronald P.; Sutherland, William M.; Reist, Craig J.; Webb, Donna J.; Wright, Eleanor L.; Labuguen, Ronald H.



Use of heteropolymeric monoclonal antibodies to attach antigens to the C3b receptor of human erythrocytes: A potential therapeutic treatment  

SciTech Connect

The authors prepared bispecific, cross-linked monoclonal antibodies (heteropolymers) with specificity for both targeted antigens and the human erythrocyte (RBC) complement receptor. These heteropolymers facilitate binding of target antigens (human IgG and dinitrophenylated bovine {gamma} globulin) to human RBCs under conditions that either allow or preclude complement activation. Radioimmuno-assay analyses of this binding agree well with the number of complement receptors per RBC. In vitro whole-blood model experiments indicate heteropolymer-facilitated binding of antigens to RBCs is rapid and stable at 37C. It may be possible to extend these prototype experiments to the in vivo situation and use heteropolymer-attached RBCs for the safe and rapid binding, neutralization, and removal from the circulation of pathogenic antigens associated with infectious disease.

Taylor, R.P.; Sutherland, W.M.; Reist, C.J.; Webb, D.J.; Wright, E.L.; Labuguen, R.H. (Univ. of Virginia, Charlottesville (United States))



Production and preliminary characterization of monoclonal antibodies directed at two surface proteins of rhesus rotavirus.  

PubMed Central

A series of monoclonal antibodies was isolated which reacted with one of two major surface proteins of rhesus rotavirus. Thirty-six monoclonal antibodies immunoprecipitated the 82-kilodalton outer capsid protein, the product of the fourth gene, the viral hemagglutinin. These monoclonal antibodies exhibited hemagglutination inhibition activity and neutralized rhesus rotavirus to moderate or high titer. Three monoclonal antibodies immunoprecipitated the 38-kilodalton outer capsid glycoprotein, the eighth or ninth gene product. These three monoclonal antibodies neutralized rhesus rotavirus to high titer and also inhibited viral hemagglutination. Images

Greenberg, H B; Valdesuso, J; van Wyke, K; Midthun, K; Walsh, M; McAuliffe, V; Wyatt, R G; Kalica, A R; Flores, J; Hoshino, Y



Targeting of murine radiolabeled monoclonal antibodies in the lymphatics  

SciTech Connect

The tissue localization of a radiolabeled monoclonal antibody directed against a mouse Class I major histocompatibility antigen has been determined in mice following i.v. and s.c. administration. When labeled antibody was given s.c., radioactivity rapidly accumulated in regional lymph nodes draining the injection site, allowing visualization of the nodes by gamma camera imaging within minutes of injection. At 2 h after s.c. injection, radioactivity in regional nodes was present largely as intact antibody, but considerable degradation of antibody present in nodes was noted by 12 h after injection. Since little of the radioactivity reached the blood stream, visualization of regional nodes was possible for long periods after dosing. In contrast, antibody given i.v. showed no significant accumulation in lymph nodes at any time after dosing.

Parker, R.J.; Keenan, A.M.; Dower, S.K.; Steller, M.A.; Holton, O.D.; Sieber, S.M.; Weinstein, J.N.



Monoclonal antibodies to rabbit liver cathepsin B  

Microsoft Academic Search

Three stable hybridoma cell lines (AF8, BC11, CE2) have been produced that secrete antibodies specific for cathepsin B. These have been characterized by ELISA, SDS-PAGE immunostaining, immunoprecipitation and immunofluorescent staining. CE2 immunoprecipitated native cathepsin B with retention of enzymic activity, but failed to cross-react with the alkali-denatured enzyme. BC11 bound only to the denatured form of cathepsin B and AF8

R. John Wardale; Rose A. Maciewicz; David J. Etherington



Stimuli-responsive magnetic nanoparticles for monoclonal antibody purification.  


Monoclonal antibodies (mAbs) are important therapeutic proteins. One of the challenges facing large-scale production of monoclonal antibodies is the capacity bottleneck in downstream processing, which can be circumvented by using magnetic stimuli-responsive polymer nanoparticles. In this work, stimuli-responsive magnetic particles composed of a magnetic poly(methyl methacrylate) core with a poly(N-isopropylacrylamide-co-acrylic acid) (P(NIPAM-co-AA)) shell cross-linked with N, N'-methylenebisacrylamide were prepared by miniemulsion polymerization. The particles were shown to have an average hydrodynamic diameter of 317 nm at 18°C, which decreased to 277 nm at 41°C due to the collapse of the thermo-responsive shell. The particles were superparamagnetic in behavior and exhibited a saturation magnetization of 12.6 emu/g. Subsequently, we evaluated the potential of these negatively charged stimuli-responsive magnetic particles in the purification of a monoclonal antibody from a diafiltered CHO cell culture supernatant by cation exchange. The adsorption of antibodies onto P(NIPAM-co-AA)-coated nanoparticles was highly selective and allowed for the recovery of approximately 94% of the mAb. Different elution strategies were employed providing highly pure mAb fractions with host cell protein (HCP) removal greater than 98%. By exploring the stimuli-responsive properties of the particles, shorter magnetic separation times were possible without significant differences in product yield and purity. PMID:23420794

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



Acrosomal status evaluation in human ejaculated sperm with monoclonal antibodies.  


An important question in mammalian gamete physiology concerns how capacitation and the occurrence of acrosome reactions in motile sperm relate to fertility. Evaluation of these relationships has been restricted by practical limitations because rapid, quantitative assays are unavailable. We have developed a rapid, reproducible assay for the evaluation of acrosomal status utilizing monoclonal antibodies specific to antigens localized in the acrosomal cap region of the sperm head. Mice were immunized with human ejaculated sperm preparations and the resultant hybridomas producing antisperm antibody were selected by solid-phase radioimmunoassay and indirect immunofluorescence (IIF). Two monoclonal antibodies (HS-19, HS-21) recognized target antigens restricted to the acrosomal cap by IIF, and 87 +/- 8.5% of the sperm in fresh ejaculates from 10 different sperm donors showed positive cap fluorescence with these reagents. Loss of HS-21 binding as measured by IIF was correlated with disappearance of the acrosomal cap as observed directly by transmission electron microscopy. Acrosomal disappearance, artificially induced in vitro using the calcium ionophore A23187, also resulted in a loss of HS-21 binding. The induction of acrosomal loss by ionophore was dependent upon extracellular calcium. The data presented suggest that specific monoclonal antibodies can be used for the rapid evaluation of acrosomal status in mammalian sperm. PMID:3926014

Wolf, D P; Boldt, J; Byrd, W; Bechtol, K B



Antibody-dependent cell cytotoxicity in monoclonal antibody-mediated tumor immunotherapy  

PubMed Central

Antibody-dependent cell cytotoxicity (ADCC) is critical in monoclonal antibody (mAb)-mediated cancer therapy. We recently showed that a tumor-specific mAb in combination with cyclophosphamide inhibited tumor cell growth and induced ADCC-synapses between tumor and effector cells in vivo, opening perspectives to enhance anti-tumor responses by manipulating the immune system.

Hubert, Pascale; Amigorena, Sebastian



Transfer of monoclonal antibodies into mammalian cells by electroporation.  


A simple rapid and reproducible procedure for transferring monoclonal antibodies into mammalian cells by electroporation is described. Two functionally different monoclonal antibodies (Mab 3F3 and Mab 2B4) specific for asparagine synthetase (EC were used for electroporation into HeLa, HT-5, and L5178Y D10/R (L-asparaginase-resistant) cells. The conditions were optimized so that the viability of the electroporated cells was very high (80-90%), and 90% of the viable cells had antibody incorporated. Electropermeabilized cells were structurally intact, and the high voltage electric pulse had no inhibitory effect on overall cellular DNA and protein synthesis. Incorporated immunoglobulins showed unaltered structural integrity and were functionally active. L5178Y D10/R cells incorporated with an antibody (Mab 3F3) known to be a potent inhibitor of tumor asparagine synthetase showed increased dependence on an exogenous source of asparagine in the culture medium, while the growth of cells incorporated with a control (noninhibitory) antibody (Mab 2B4) remained unaffected. These studies demonstrate that electroporation can be employed successfully for large scale transfer of antibodies into cultured mammalian cells for the study of cellular metabolism. PMID:2768274

Chakrabarti, R; Wylie, D E; Schuster, S M



Toxicities associated with monoclonal antibody infusions in cancer patients.  


The toxicity during and following 291 infusions of 19 murine and three human monoclonal antibodies (MoAB) in 177 cancer patients with 10 different malignancies was assessed. Doses ranged from 0.5 to 500 mg administered over 0.25 to 24 hours. Various reactions in varying degrees were observed in 45 (28%) patients during their first MoAb infusion. Nine additional patients experienced toxicity following a subsequent antibody infusion. Antibodies that reacted with circulating cells were associated with toxicity in 20 of 28 (71%) of the first infusions, compared to 24 of 127 (19%) for patients receiving antibodies that did not react with circulating cells. Fevers, rigors, chills, and diaphoresis were observed in 10% to 12% of the patients and were associated with binding to circulating cells. Presumed hypersensitivity reactions, including urticaria, pruritus, bronchospasm, and anaphylaxis occurred in 20 patients (11%). There were five episodes of bronchospasm and a single episode of anaphylaxis. Liver transaminases were elevated in 14%. There was no correlation between dose or infusion rate and toxicity. Murine monoclonal antibodies that are not conjugated to cytotoxic agents can be given with an acceptable frequency of side effects and serious allergic reactions. There is a small risk of anaphylaxis, and one should avoid rapid infusion of high antibody doses in the presence of circulating target cells and/or circulating free antigen. PMID:3269251

Dillman, R O; Beauregard, J C; Jamieson, M; Amox, D; Halpern, S E



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



Monoclonal antibodies to the thyrotropin receptor: the identification of blocking and stimulating antibodies.  


Monoclonal antibodies to the thyrotropin (TSH) receptor have been obtained from fusions of mouse myeloma cells with spleen cells immunized with solubilized thyroid membrane preparations. Two monoclonal antibodies which inhibit 125I-TSH binding and are reactive with the glycoprotein component of the bovine TSH receptor (11E8 and 13D11), are shown to inhibit basal and TSH stimulated adenylate cyclase activity in bovine thyroid membranes and human thyroid cells. Both antibodies also inhibit 125I-TSH binding in vitro, whether binding is measured at pH 6.0 in low salts and at 0-4 C or at pH 7.4 in 50 mM NaCl and at 37 C. The glycoprotein component is thus a portion of the physiologic TSH receptor in vivo and 125I-TSH binding studies apparently measure the high affinity glycoprotein component under nonphysiologic conditions and conditions more representative of the physiologic milieu. A third monoclonal antibody whose interaction with thyroid membranes is prevented by TSH is shown to stimulate adenylate cyclase activity in bovine thyroid membranes and human thyroid cells. This stimulating antibody only weakly inhibits 125I-TSH binding to thyroid membranes or to the glycoprotein component of the TSH receptor. The 22A6 antibody does, however, immunoprecipitate mixed brain gangliosides, in distinct contrast to the monoclonal antibodies to the glycoprotein receptor component, i.e., 11E8 and 13D11. The results support the speculation that autoimmune antibodies which inhibit TSH binding to thyroid membranes are not necessarily identical to antibodies which stimulate function; that antibodies directed at the high affinity initial site of TSH interaction with a cell can behave as blocking rather than stimulating antibodies and that a possible relationship exists between stimulating antibodies and the low affinity TSH binding sites (gangliosides) on thyroid membranes. PMID:6296219

Valente, W A; Yavin, Z; Yavin, E; Grollman, E F; Schneider, M; Rotella, C M; Zonefrati, R; Toccafondi, R S; Kohn, L D


Differentiating between isolates of Vibrio vulnificus with monoclonal antibodies  

Microsoft Academic Search

Monoclonal antibodies (MAbs) against Vibrio vulnificus (isolate I, VVC and isolate II, VVB) were raised using heat-killed and heat-killed plus SDS–mercaptoethanol treated forms of VVC and VVB for immunizing Swiss mice. Twenty three hybridomas producing MAbs against V. vulnificus were selected and divided into five groups according to their specificities to different V. vulnificus isolates and apparent protein antigens which

Sirirat Rengpipat; Suttinee Pusiririt; Sombat Rukpratanporn



Modulation of tumor immunity by therapeutic monoclonal antibodies  

Microsoft Academic Search

The surveillance of tumors by the immune system of cancer patients and its impact on disease progression and patient survival\\u000a have been largely documented over the last years. In parallel, the use of therapeutic monoclonal antibodies (mAbs) in oncology\\u000a has gained a widespread recognition as it has made it possible to increase patient survival and to ameliorate the quality\\u000a of

Riad Abès; Jean-Luc Teillaud



Monoclonal antibodies to human brain acetylcholinesterase: properties and applications  

Microsoft Academic Search

1.Acetylcholinesterase (AChE) was purified 20,000-fold in a 43% yield from 90 g of human cerebellum by combined immunoaffinity and ligand affinity chromatography. The purified enzyme migrated as a 68,000-dalton band during polyacrylamide gel electrophoresis under denaturing and reducing conditions.2.Balb\\/c mice were immunized with multiple 10-µg injections of this material in order to raise monoclonal antibodies to human brain AChE. Three

Zoltan Rakonczay; Stephen Brimijoin



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



Monoclonal antibody refolding and assembly: Protein disulfide isomerase reaction kinetics  

Microsoft Academic Search

The protein disulfide isomerase (PDI) reaction kinetics has been studied to evaluate its effect on the monoclonal antibody\\u000a (MAb) refolding and assembly which accompanies disulfide bond formation. The MAbin vitro assembly experiments showed that the assembly rate of heavy and light chains can be greatly enhanced in the presence of PDI\\u000a as compared to the rate of assembly obtained by

Dewey D. Y. Ryu



Antigenic diversity of Akabane virus detected by monoclonal antibodies  

Microsoft Academic Search

The antigenic properties of 21 Japanese field isolates and two Australian strains of Akabane (AKA) virus (Simbu serogroup, bunyavirus) isolated from 1959 to 1990 were compared by enzyme-linked immunosorbent assay (ELISA), plaque-reduction neutralization (PRNT) and hemagglutination inhibition (HI) tests using monoclonal antibodies (Mabs) to the OBE-1 strain of AKA virus. Sixteen Mabs were established by fusing P3X63Ag8U1 mouse myeloma cells

H. Akashi; Y. Inaba



Biosimilar monoclonal antibodies: a science-based regulatory challenge.  


Monoclonal antibodies (MAs) are complex biotherapeutics as their molecular mechanism of action depends on multiple domains. Consequently regulatory approval of biosimilars of MAs is subjected to specific, science-based guidelines. An extensive comparative in vitro characterization to evaluate the biosimilarity of the various functional domains is required. The exquisite species specificity of MAs precludes reliable in vivo non-clinical evaluations and means that adequately designed clinical studies are extremely critical to confirm the biosimilarity. To date no biosimilar MAs have been approved. Taking into account the expected high development costs for biosimilar MAs, their use may well be superseded by alternative antibody formats and next-generation MAs. PMID:23286777

Declerck, Paul J



Recovery and purification process development for monoclonal antibody production  

PubMed Central

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

Ma, Junfen; Winter, Charles; Bayer, Robert



Application of monoclonal antibodies as cancer therapy in solid tumors.  


Monoclonal antibodies have become an important new class of therapeutic agents approved for use in solid tumors. They function through several different mechanisms including inhibition of tumor-related signal transduction, induction of apoptosis, inhibition of angiogenesis, enhancing host immune response against cancer and targeted delivery of cytotoxic agents to the tumor site. Several monoclonal antibodies have now received regulatory approval--trastuzumab, cetuximab, panitumumab, bevacizumab, catumaxomab, ipilimumab and denosumab--across multiple solid tumor types, including breast, colorectal, head and neck, non-small cell lung cancers and melanomas, amongst others. These agents are employed clinically in some neoadjuvant/adjuvant and radical treatment settings, as well as more extensively in the metastatic and palliative settings. Current research is focused on innovative compound design, novel targets, predictive biomarker discovery, enriched patient populations, and combination strategies in order to overcome resistance and prolong disease control. Here we provide an overview of monoclonal antibodies approved for use in clinical oncology and those currently in clinical development. PMID:22432839

Dienstmann, Rodrigo; Markman, Ben; Tabernero, Josep



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.

Huang, Zhi-Qiang; Buchsbaum, Donald J



Monoclonal antibodies to three strains of hantaviruses: Hantaan, R22, and Puumala  

Microsoft Academic Search

Summary Thirty hybrid cell lines that produce monoclonal antibodies to three strains of hantaviruses have been generated and characterized. One clone specific to Hantaan 76–118 strain, four clones specific to Rattus strains and one clone specific to Puumala virus have been identified. Most of the monoclones produced antibodies specific to nucleoproteins. Only two monoclones were found to produce glycoprotein specific,

S. L. Ruo; A. Sanchez; L. H. Elliott; L. S. Brammer; J. B. McCormick; S. P. Fisher-Hoch



Role of Immunogen Design in Induction of Soman-Specific Monoclonal Antibodies.  

National Technical Information Service (NTIS)

The study of monoclonal antibodies raised against defined hapten epitopes has been a useful approach to understanding antibody repertoire. The situation in which antibodies are raised against different epitopes of the same hapten but have some common reco...

J. K. Johnson D. M. Cerasoli D. E. Lenz



A monoclonal antibody detaches embryonic skeletal muscle from extracellular matrices  

PubMed Central

We have described a monoclonal antibody that rounds and detaches chick skeletal myoblasts and myotubes from extracellular substrata. The antibody also inhibits the attachment of myogenic cells to a gelatin- coated substratum but has no detectable effect on myoblast fusion. The cellular response to antibody treatment varies with differentiation and cell type. Young myoblasts and myotubes are rapidly rounded and detached by the antibody. Older myotubes require longer incubation times or higher antibody titers for rounding and detachment. Chick embryo fibroblasts, cardiac cells, and neurons are not similarly rounded and remain attached. Since the antibody also detaches cells from embryonic muscle tissue explants, the cell-substratum interaction perturbed by the antibody appears relevant to the in vivo interaction of myogenic cells with their extracellular matrices. Binding studies using iodinated antibody revealed 2-4 x 10(5) sites per myoblast with an apparent Kd in the range of 2-5 x 10(-9) molar. Embryo fibroblasts bind antibody as well and display approximately twice the number of binding sites per cell. The fluorescence distribution of antigen on myoblasts and myotubes is somewhat punctate and particularly bright along the edge of the myotube. The distribution on fibroblasts was also punctate and was particularly bright along the cell periphery and portions of stress fibers. For both cell types the binding was distinctly different than that reported for collagen, fibronectin, and other extracellular molecules. The antigen, as isolated by antibody affinity chromatography, inhibits antibody-induced rounding. SDS PAGE reveals two unique polypeptides migrating in the region of approximately 120 and 160 kilodaltons (kd). The most straightforward mechanism for the antibody-induced rounding and detachment is the perturbation of a membrane molecule involved in adhesion. The hypothesized transmembrane link between extracellular macromolecules and the cytoskeleton provides an obvious candidate.



Isolation of human monoclonal antibodies from peripheral blood B cells.  


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

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



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.



Transformation-Related Antigens Identified by Monoclonal Antibodies  

NASA Astrophysics Data System (ADS)

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

Strand, Mette



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

Montano, X; Lane, D P



Efficient elimination of chronic lymphocytic leukaemia B cells by autologous T cells with a bispecific anti-CD19/anti-CD3 single-chain antibody construct.  


Recently, we have shown that a novel recombinant bispecific single-chain antibody construct (bscCD19 x CD3), induces highly efficacious lymphoma-directed cytotoxicity mediated by unstimulated peripheral T lymphocytes. Functional analysis of bscCD19 x CD3 has so far been exclusively performed with human B lymphoma cell lines and T cells from healthy donors. Here we analysed the properties of bscCD19 x CD3 using primary B cells and autologous T cells from healthy volunteers or patients with B-cell chronic lymphocytic leukaemia (B-CLL). We show that bscCD19 x CD3 induces T-cell-mediated depletion of nonmalignant B cells in all four cases and depletion of primary lymphoma cells in 22 out of 25 cases. This effect could be observed at low effector-to-target (E:T) ratios and in the majority of cases without additional activation of autologous T cells by IL-2. Even in samples derived from patients heavily pretreated with different chemotherapy regimens, strong cytotoxic effects of bscCD19 x CD3 could be observed. The addition of bscCD19 x CD3 to patients' cells resulted in an upregulation of activation-specific cell surface antigens on autologous T cells and elevated levels of CD95 on lymphoma B cells. Although anti-CD95 antibody CH-11 failed to induce apoptosis in lymphoma cells, we provide evidence that B-CLL cell depletion by bscCD3 x CD3 is mediated at least in part by apoptosis via the caspase pathway. PMID:12750704

Löffler, A; Gruen, M; Wuchter, C; Schriever, F; Kufer, P; Dreier, T; Hanakam, F; Baeuerle, P A; Bommert, K; Karawajew, L; Dörken, B; Bargou, R C



Differential induction of lupus associated antinuclear antibodies in MRL mice by monoclonal anti-Sm antibodies.  


The effect of monoclonal autoantibodies on immunoregulation was investigated in MRL/MpJ-lpr/lpr mice. Passive transfer of KSm2 (a monoclonal IgG2a antibody directed against the 16 kD polypeptide of Sm) induced IgG antibodies to the other major immunoreactive polypeptides of Sm (28 and 29 kD) in all mice studied, and to polypeptides of the closely related antigen nRNP/Sm in 63% of the mice. In addition an increment in IgG anti-dsDNA antibodies, and in IgA and IgM anti-Sm antibodies, over control levels was observed. These effects were not due to polyclonal activation since anti-histone antibody levels were unaffected. Two other IgG2a monoclonal antibodies: KSm5 (directed against the 28 and 29 kD Sm polypeptides) and OX 12 (directed against an irrelevant antigen) failed to modulate the autoimmune responses of the mice in any way. These results demonstrate specific antibody-mediated connectivity between B cell clones producing autoantibodies against three distinct antigens. PMID:3496996

Stocks, M R; Williams, D G; Maini, R N



Production and characterisation of monoclonal antibodies to cell wall components of the flax rust fungus  

Microsoft Academic Search

Monoclonal antibodies have been raised against an haustorium-enriched sample prepared from flax leaves infected with the biotrophic flax rust pathogen Melampsora lini. The monoclonal antibodies were produced following conventional and co-immunisation procedures and the range of antibody specificities was compared. The preparation used as immunogen for the conventional protocol was a crude isolate of haustoria consisting of approx. 65% fungal

Leanne J. Murdoch; Issei Kobayashi; Adrienne R. Hardham



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

PubMed Central

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

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



Monoclonal antibodies to antigens associated with transitional cell carcinoma of the human urinary bladder  

Microsoft Academic Search

The cellular target structures for six monoclonal antibodies raised against cultured human bladder carcinoma cells (TCC) were investigated. The specificities of these antibodies when tested against a large panel of cells have been described in the companion paper. Radiolabeled cell lysates were precipitated with the different monoclonal antibodies bound to protein A (Staphylococcus aureus) on a matrix of Sepharose beads.

Staffan Paulie; Hannu Koho; Hedi Ben-Aissa; Yngve Hansson; Marie-Louise Lundblad; Peter Perlmann



Development of a humanized monoclonal antibody with therapeutic potential against West Nile virus  

Microsoft Academic Search

Neutralization of West Nile virus (WNV) in vivo correlates with the development of an antibody response against the viral envelope (E) protein. Using random mutagenesis and yeast surface display, we defined individual contact residues of 14 newly generated monoclonal antibodies against domain III of the WNV E protein. Monoclonal antibodies that strongly neutralized WNV localized to a surface patch on

Theodore Oliphant; Michael Engle; Grant E Nybakken; Chris Doane; Syd Johnson; Ling Huang; Sergey Gorlatov; Erin Mehlhop; Anantha Marri; Kyung Min Chung; Gregory D Ebel; Laura D Kramer; Daved H Fremont; Michael S Diamond



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

Microsoft Academic Search

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

S. C. Srivastava; G. L. Buraggi



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

Sternberger, N H; Sternberger, L A



Precipitation of a Monoclonal Antibody by Soluble Tungsten  

PubMed Central

Tungsten microparticles may be introduced into some pre-filled syringes during the creation of the needle hole. In turn, these microcontaminants may interact with protein therapeutics to produce visible particles. We found that soluble tungsten polyanions formed in acidic buffer below pH 6.0 can precipitate a monoclonal antibody within seconds. Soluble tungsten in pH 5.0 buffer at about 3 ppm was enough to cause precipitation of a mAb formulated at 0.02 mg/mL. The secondary structure of the protein was near-native in the collected precipitate. Our observations are consistent with the coagulation of a monoclonal antibody by tungsten polyanions. Tungsten-induced precipitation should only be a concern for proteins formulated below about pH 6.0 since tungsten polyanions are not formed at higher pHs. We speculate that the heterogenous nature of particle contamination within the poorly mixed syringe tip volume could mean that a specification for tungsten contamination based on the entire syringe volume is not appropriate. The potential potency of tungsten metal contamination is highlighted by the small number of particles that would be required to generate soluble tungsten levels needed to coagulate this antibody at pH 5.0.

Bee, Jared S.; Nelson, Stephanie A.; Freund, Erwin; Carpenter, John F.; Randolph, Theodore W.



Intraoperative probe-directed immunodetection using a monoclonal antibody  

SciTech Connect

To assess monoclonal antibody (MAb) 17-1A and its F(ab')2 fragment in intraoperative radioimmunodetection and to evaluate further the clinical usefulness of a hand-held gamma-detecting probe (GDP), we injected radiolabeled monoclonal antibody 17-1A three to six days preoperatively or its F(ab')2 fragment two to three days preoperatively into 18 patients with colorectal cancer. Intraoperative GDP counts with tumor-tissue ratios of 1.5:1 or greater were obtained from 15 (75%) of 20 tumor sites, with ratios averaging 2.3:1 for fragments and 3.4:1 for whole antibody. The GDP counts contributed to intraoperative decision making in three patients, either by localization of tumor not identified by inspection or palpation or by mapping margins of resection with histologic confirmation of a local/regional recurrence. These preliminary data demonstrate that probe-directed, intraoperative radioimmunodetection can assist the surgeon in detecting subclinical tumor deposits and thus better evaluate the extent of primary or recurrent colorectal cancers intraoperatively.

O'Dwyer, P.J.; Mojzisik, C.M.; Hinkle, G.H.; Rousseau, M.; Olsen, J.; Tuttle, S.E.; Barth, R.F.; Thurston, M.O.; McCabe, D.P.; Farrar, W.B.



Identification of Vibrio vulnificus O serovars with antilipopolysaccharide monoclonal antibody.  

PubMed Central

A serotyping scheme for Vibrio vulnificus predicated on the detection of lipopolysaccharide (LPS) antigens is proposed. The serovar O typing scheme used to type V. vulnificus employs polyclonal antisera raised in rabbits immunized with heat-killed whole-cell vaccines. Polyclonal typing sera produced in this manner cross-react with heterologous strains. Affinity purification of polyclonal antisera with LPS affinity columns resolved some of these cross-reactions; however, affinity-purified polyclonal antisera still showed cross-reactions that were nonreciprocal. On the basis of the serological patterns that were obtained with affinity-purified polyclonal antisera, V. vulnificus strains were selected as vaccine strains for production of monoclonal antibody. Spleen cells harvested from BALB/c mice immunized with formalin-killed V. vulnificus cells were fused with SP2/O-Ag 14 myeloma cells. Hybridomas were screened by using LPS and whole-cell enzyme-linked immunosorbent assay to identify clones secreting LPS-specific antibodies. Monoclonal antibodies identified five LPS serological varieties of V. vulnificus and a single serovar each for Vibrio damsela and Vibrio hollisae. No cross-reactions between V. vulnificus and V. hollisae or V. damsela were observed.

Martin, S J; Siebeling, R J



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

Microsoft Academic Search

Our studies examined pharmacokinetic mechanisms involved in high-affinity (Kd?11 nM) monoclonal antibody-based antagonism of (+)-methamphetamine-induced locomotor effects. Male rats received (+)-methamphetamine (0.3, 1, or 3 mg\\/kg i.v.) followed 30 min later by saline or anti-(+)-methamphetamine monoclonal antibody. All groups received a constant dose of monoclonal antibody that was equimolar in binding sites to the body burden of a 1 mg\\/kg

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



Monoclonal and specific polyclonal antibodies for immunoassay of Clostridium difficile toxin A.  

PubMed Central

Monoclonal antibody, affinity-purified antibody, and monospecific antiserum against toxin A were produced. The monoclonal antibody was an immunoglobulin G2a kappa chain isotype that immunoprecipitated toxin A, as shown by crossed immunoelectrophoresis. These antibodies were compared by counterimmunoelectrophoresis, latex agglutination, and indirect enzyme-linked immunosorbent assay for their sensitivity in detecting toxin A. Our findings indicate that these antibodies may be useful as immunodiagnostic reagents for Clostridium difficile disease. Images

Lyerly, D M; Phelps, C J; Wilkins, T D



Monoclonal antibody based immunoassay for human aminoacylase-1.  


Aminoacylase-1 is expressed in a wide variety of cell types. Although the exact role of this enzyme in cellular physiology remains unclear, it has been postulated to function in the salvage of acylated aminoacids. The enzyme is also of interest due to its gene map location on chromosome 3p21, a region deleted in several neoplasms. We have produced mouse monoclonal antibodies and rabbit antisera which recognize human aminoacylase-1. A sandwich ELISA has been developed which allows the measurement of human aminoacylase-1. PMID:2745715

Miller, Y E; Kao, B



Monoclonal antibody detection of proteins on sperm related to fertility  

US Patent & Trademark Office Database

Heparin binding proteins (HBP) are produced by male accessory glands and bind to sperm at ejaculation. The ability of accessory glands to produce HBP and sperm to bind HBP differs among males. Identifying the presence of a 21.5, 24 and 30 kDa heparin binding proteins in sperm membranes with a monoclonal antibody resulted in separating groups of bulls by 40% difference in fertility. Thus, fertility potential of a bull can be predicted by heparin binding protein content of sperm membranes.



Monoclonal antibody based immunoassays for cooking-induced meat mutagens  

SciTech Connect

We report here new monoclonal antibodies (Mabs) numbered AIA-8 through AIA-12 produced using the same methods used to produce AIA-1, and a new Mab, IQ-7, produced with the same methods used for IQ-1. Our motivation in seeking these new clones was to increase the repertoire of available Mabs to insure adequate coverage of all known AIAs. Also, the mice used to produce these new hybridoma clones had been immunized about six months longer than those used to generate the first clones. Longer immunization is often associated with higher affinity Mabs and therefore more sensitive competition immunoassays. 15 refs., 2 figs., 2 tabs.

Vanderlaan, M.; Hwang, M.; Knize, M.G.; Watkins, E.; Felton, J.S.



Characterization of Monoclonal Antibodies that Inhibit the Catalytic Activity of Acetylcholinesterases.  

National Technical Information Service (NTIS)

Monoclonal antibodies were generated against fetal bovine serum acetylcholinesterase and fetal bovine serum acetylcholinesterase inhibited by diisopropyl fluorophosphate or 7-(methylethoxyphosphinyloxy) -1-methylquinolinium iodide. Six monoclonalantibodie...

M. K. Gentry D. R. Moorad R. S. Hur A. Saxena Y. Ashani



Tumour targeting of the anti-ovarian carcinoma x anti-CD3/TCR bispesific monoclonal antibody OC/TR and its parental MOv18 antibody in experimental ovarian cancer.  


The anti-tumour x anti-T-cell bispecific monoclonal antibody (biMAb) OC/TR is a biologically produced biMAb combining the anti-ovarian carcinoma activity of the MOv18 MAb with anti-CD3/T-cell receptor (TCR) complex activity. In this study, the in vitro binding characteristics of the OC/TR biMAb and its tumour targeting potential in nude mice with Hela tumours was studied. Scatchard analysis revealed that the affinity constant of the biMAb was 7 times lower than the affinity of the parental MOv18 antibody. Uptake of the OC/TR antibody in the Hela xenografts in nude mice was significantly higher than the tumour uptake of an irrelevant control antibody, indicating that the radioiodinated OC/TR biMAb specifically localized in the tumour xenografts. However, tumour uptake was significantly lower than the tumour uptake of the parental MOv18 antibody. This reduced tumour uptake most likely is a result of its reduced affinity. We conclude that, despite the loss of bivalent tumour cell binding, the biMAb OC/TR can still specifically localize in tumours. This indicates that the first prerequisite of an effective therapeutic approach using systemically applied biMAb can be met. Whether the interaction with human T-cells will affect the tumour targeting potential of the biMAb in patients remains to be investigated. PMID:8572619

Boerman, O C; Tibben, J G; Massuger, L F; Claessens, R A; Corstens, F H


Monoclonal antibody-based candidate therapeutics against HIV type 1.  


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

Chen, Weizao; Dimitrov, Dimiter S



Monoclonal Antibody-Based Candidate Therapeutics Against HIV Type 1  

PubMed Central

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

Dimitrov, Dimiter S.



Glycoengineered Pichia-based expression of monoclonal antibodies.  


Currently, mammalian cells are the most commonly used hosts for the production of therapeutic monoclonal antibodies (mAbs). These hosts not only secrete mAbs with properly assembled two heavy and two light chains but also deliver mAbs with a glycosylation profile that is compatible with administration into humans. GlycoFi, a wholly owned subsidiary of Merck & Co., Inc., humanized the Pichia glycosylation pathway which allows it to express glycoproteins with a human-like glycan profile. This offers an alternative mAb production platform similar to mammalian hosts and in some cases it even provides more homogenous product and better efficacy, such as enhanced effector function. This chapter describes a protocol for using glycoengineered Pichia to produce full-length mAbs. It covers a broad spectrum of mAb expression technologies in yeast including expression vector construction, yeast transformation, high-throughput strain selection to fermentation, and antibody purification. PMID:23475712

Zha, Dongxing



Monoclonal antibody to native P39 protein from Borrelia burgdorferi.  

PubMed Central

We have produced, by using a sonicate of Borrelia burgdorferi, a monoclonal antibody (MAb), NYSP39H, that is specific for the P39 protein band. This MAb reacted with 13 isolates of B. burgdorferi but not with eight different spirochetes (four borrelias, two leptospiras, and two treponemas). Surface labeling of B. burgdorferi with biotin and subsequent treatment with Nonidet P-40 showed that P39 was not biotinylated but was extracted with Nonidet P-40, indicating that it is present within the outer membrane, but not on the surface of the spirochete. Immunoelectron microscopy revealed the immunogold probe primarily at the cytoplasmic membrane region of the spirochete. The MAb detected B. burgdorferi in the indirect fluorescent-antibody test only when the spirochetes from a culture or in a tick homogenate were fixed with polylysine and not with acetone. NYSP39H appears to be an appropriate probe for use in the specific detection of B. burgdorferi. Images

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



Generation and characterization of anti-chitinase monoclonal antibodies.  


Class IV chitinase, an allergenic protein of Vitis vinifera (grape), was purified by anion exchange chromatography and used for immunization of Balb/c mice. Monoclonal antibodies (MAbs) were raised by hybridoma technology using Sp2/0 myeloma cells. Finally after three limiting dilutions, six stable clones were generated. Antibody isotyping showed that IgG(2a), IgG(2b), and IgM were produced by one, two, and three of the clones, respectively. All of the MAbs had kappa light chain. The affinities were in the range of 3?×?10(8) to 1.2?×?10(9) M(-1). The MAbs were specific for grape chitinase as confirmed by Western blotting. In conclusion, we successfully produced several MAbs against grape class IV chitinase, which could be used for assessment of this allergen in different grape cultivars. PMID:21529287

Soukhtanloo, Mohammad; Falak, Reza; Sankian, Mojtaba; Varasteh, A-Reza



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.



Characterization of monoclonal antibodies against a human chondrocyte surface antigen.  


Chondrocytes express a number of cell-surface molecules that mediate cell-cell or cell-matrix interactions. Identification and full characterization of new chondrocyte surface molecules will lead to a better understanding of the function of the chondrocyte. Researchers used primary human chondrocytes as an immunogen, and various monoclonal antibodies (MAbs) were generated using standard hybridoma technology. A monoclonal antibody named 5D2 was selected for further characterization. The antigen recognized by 5D2 MAb is expressed by primary human chondrocytes, primary synovial fibroblasts, synovial fibroblast cell lines (SW982), primary skin fibroblasts, and osteoblasts, but not expressed in blood cells. Biochemical analysis revealed that the 5D2 antigen is a protein with a molecular weight of approximately 25-35?kDa. Protein identification by mass spectrometry and molecular cloning revealed that 5D2 antigen is identical to the Thy-1 molecule. Furthermore we confirmed this specificity of the antibody by the isolated and cloned Thy-1 gene to the COS-7 and probed it with the 5D2 antibody using Western blot analysis. We examined the role of the Thy-1 molecule in arthritis models and tissue; one was papain-induced rat arthritis, the other was immunohistological staining of osteoarthritic (OA) human articular cartilage. OA cartilage showed a higher expression of Thy-1 as compared with normal tissue in all experimental approaches. The in vitro studies showed that the inflammatory cytokine interleukin-1? up-regulated Thy-1 molecule expression in the cartilage tissue. It can be concluded that the Thy-1 might be a potential biomarker for cartilage pathogenesis, degradation, and metabolic turnover. PMID:23750475

Chanmee, Theerawut; Phothacharoen, Peraphan; Thongboonkerd, Visith; Kasinrerk, Watchara; Kongtawelert, Prachya



Eradication of tumors from a human colon cancer cell line and from ovarian cancer metastases in immunodeficient mice by a single-chain Ep-CAM-/CD3-bispecific antibody construct.  


Bispecific T-cell engager (BiTE) are a class of bispecific single-chain antibodies that can very effectively redirect cytotoxic T cells for killing of tumor target cells. Here, we have assessed the in vivo efficacy of one representative, called bscEp-CAMxCD3, with specificity for tumors overexpressing epithelial cell adhesion molecule (Ep-CAM) in human xenograft models. Cells of the human colon carcinoma line SW480 were mixed at a 1:1 ratio with unstimulated human peripheral mononuclear cells, s.c. injected in nonobese diabetes/severe combined immunodeficiency (NOD/SCID) mice, and animals were treated with bscEp-CAMxCD3. Five daily i.v. injections of as little as 100 ng per mouse of bscEp-CAMxCD3 completely prevented tumor outgrowth when treatment was started at the day of tumor cell inoculation. BscEp-CAMxCD3 was also efficacious when administered up to 8 days after xenograft injection. Established tumors could be eradicated in all animals by five 10 microg doses given between days 8 and 12 after tumor cell inoculation. To test the efficacy of bscEp-CAMxCD3 in a more physiologic model, pieces of primary metastatic tumor tissue from ovarian cancer patients were implanted in NOD/SCID mice. Partial tumor engraftment and growth was observed with four of six patient samples. Treatment of established tumors with daily 5 microg doses led to a significant reduction and, in some cases, eradication of human tumor tissue. These effects obviously relied on the tumor-resident T cells reactivated by bscEp-CAMxCD3. Our data show that the class of single-chain bispecific antibodies has very high antitumor efficacy in vivo and can use previously unstimulated T cells at low effector-to-target ratios. PMID:15805290

Schlereth, Bernd; Fichtner, Iduna; Lorenczewski, Grit; Kleindienst, Petra; Brischwein, Klaus; da Silva, Antonio; Kufer, Peter; Lutterbuese, Ralf; Junghahn, Ilse; Kasimir-Bauer, Sabine; Wimberger, Pauline; Kimmig, Rainer; Baeuerle, Patrick A



Creatine kinase-inhibiting monoclonal antibodies: preparation and characterization of porcine MM isoenzyme-specific antibodies.  


Five monoclonal antibodies (CKM-B07, F12, D08, H09 and G01) against porcine creatine kinase (CK; EC MM isoenzyme, which inhibit the enzymatic activity, were prepared. The hybridomas which produced monoclonal antibodies were screened by direct measurement of the inhibitory activity of their culture supernatant. Only two of them, however, were found to be measurable by an enzyme-linked immunosorbent assay with porcine CK-MM as an antigen. CKM-G01 inhibited 100% porcine CK-MM activity, while the others, 73-87%. On the other hand, only CKM-H09 inhibited porcine CK-BB activity (15%). CKM-F12 and D08 inhibited more than 50% CK-MB activity, whereas they did not inhibit CK-BB activity. The monoclonal antibodies were also tested for bovine, rabbit and human CK-MM. All the antibodies inhibited bovine and human CK-MM activity as well. In particular, CKM-G01 was found to exhibit more than 98% inhibition of all CK-MM activity tested, indicating that a common or very similar epitope which affects the activity is present on these enzymes. Admixing of CKM-B07 with other antibodies effected synergisms in inhibition, not only to porcine CK-MM activity but also to human CK-MM activity. A mixture of CK-B07 and G01 inhibited 100% human CK-MM activity, suggesting applicability of these monoclonal antibodies to clinical laboratory diagnosis. PMID:3173359

Suzuki, T; Tomita, K; Murachi, T



[Monoclonal antibody mimicking the structure of an immunoprotective epitope against Fasciola hepatica].  


The ES-78 monoclonal antibodiy recognizes excretory-secretory antigens of Fasciola hepatica. This monoclonal antibody is used for diagnosis of humans and cattle fasciolosis. The production of 4 anti-idiotype monoclonal antibodies (3G5, 5G1, 3H4 and 1H6) inhibiting the antigen-monoclonal antibody ES 78 reaction was reported. This antibody recognizes excretion-secretion antigens of Fasciola hepatica and it is used for the diagnosis in faeces of fascioliasis in the bovine cattle and in humans. When the 3G5 was used in rabbits as an immunogen there was a response of antibodies against the excretion-secretion antigen of Fasciola hepatica without a previous contact wih it. The utilization of this anti-idiotype monoclonal antibody as an immunogen mimmicking a protective epitope against fascioliasis together with appropriate adjuvants may be a new way of reducing the parasitic burden in experimental models of fascioliasis. PMID:23427435

Marcet Sánchez, Ricardo; Figueredo Pino, Mabel; Sarracent Pérez, Jorge


SPECT assay of radiolabeled monoclonal antibodies. Third yearly progress report, September 1991--February 1992.  

National Technical Information Service (NTIS)

The accurate determination of the biodistribution of radiolabeled monoclonal antibodies (MoAbs) is important for calculation of dosimetry and evaluation of pharmacokinetic variables such as antibody dose and route of administration. The hypothesis of this...

R. J. Jaszczak



Preparation and Characterization of Monoclonal Antibodies to Enteric Adenovirus Types 40 and 41.  

National Technical Information Service (NTIS)

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

J. E. Herrmann D. M. Perron-Henry D. Stobbs-Walro N. R. Blacklow



High Affinity Monoclonal Antibodies to Bowman-Birk Inhibitor and Immunoassay Methods.  

National Technical Information Service (NTIS)

Hybrid cell lines (hybridomas) which produce and secrete high affinity monoclonal antibodies specific for Bowman-Birk inhibitor (BBI) are described. High affinity antibodies to BBI are described that have one or more of the following additional characteri...

D. L. Brandon A. H. Bates M. Friedman



Antigen-specific in vitro immunization: a source for human monoclonal antibodies.  


Human monoclonal antibody has great potential for treatment of various diseases utilizing their specificity against antigens. We have shown an in vitro immunization (IVI) protocol inducing antigen-specific immune responses in human peripheral blood mononuclear cells (PBMCs) for efficient production of human monoclonal antibodies. By using IVI method antigen specific antibody genes can be efficiently obtained because of increasing production of antigen-specific antibodies from in vitro immunized PBMCs. This IVI protocol will be widely applied for combination with several display methods and enhance the production of human monoclonal antibodies. PMID:24037847

Tomimatsu, Kosuke; Shirahata, Sanetaka



Intraperitoneal yttrium-90-labeled monoclonal antibody in ovarian cancer  

SciTech Connect

From March 1987 to March 1988, a phase I to II study was carried out in 25 patients with ovarian cancer. They received escalating doses of intraperitoneally (IP) administered yttrium-90 (Y-90)-labeled monoclonal antibody, HMFG1, against a tumor cell-surface antigen. Myelosuppression prevented an escalation of the administered Y-90 activity above 25 mCi. Y-90-labeled antibody was absorbed from the peritoneal cavity into the circulation. Maximum blood Y-90 activity was observed 40 hours after the IP injection with a mean of 21% of the injected activity (range, 14.2% to 26.4%) in the circulation. The radiation dose the bone marrow received from circulating Y-90-labeled antibody (the blood radiation dose) was calculated by applying the Medical Internal Radiation Dose (MIRD) formulation to the measured Y-90 activity in patients blood. Myelosuppression occurred following calculated blood radiation doses to bone marrow of only 10 to 30 cGy. The excessive myelosuppression following such modest radiation doses from circulating Y-90-labeled antibody could be explained by the uptake of Y-90 by bone. In an attempt to reduce bone absorption of Y-90, seven patients received an intravenous (IV) infusion of EDTA. This increased the urinary excretion of Y-90 from a mean of 11.1% to 32.3% of the injected activity (P = .0001). Fourteen patients had assessable tumor at laparoscopy. Tumor regression was observed in one patient, and palliation of ascites in a further patient.

Stewart, J.S.; Hird, V.; Snook, D.; Dhokia, B.; Sivolapenko, G.; Hooker, G.; Papadimitriou, J.T.; Rowlinson, G.; Sullivan, M.; Lambert, H.E. (Royal Postgraduate Medical School, London (England))



Mouse Leukemia: Therapy with Monoclonal Antibodies against a Thymus Differentiation Antigen  

NASA Astrophysics Data System (ADS)

Monoclonal antibodies against a thymus cell differentiation antigen (Thy-1.1) were effective in the therapy of a transplanted mouse leukemia. Passive immunization resulted in high titers of cytotoxic antibody in the serum of treated mice and the suppression of metastatic tumor cells. The tumor-suppressive effects of the monoclonal antibodies were amplified by the administration of exogenous complement. This combined antibody and complement therapy resulted in the cure of leukemia in a significant proportion of the treated animals.

Bernstein, Irwin D.; Tam, Milton R.; Nowinski, Robert C.



Mouse Leukemia: Therapy with Monoclonal Antibodies against a Thymus Differentiation Antigen  

Microsoft Academic Search

Monoclonal antibodies against a thymus cell differentiation antigen (Thy-1.1) were effective in the therapy of a transplanted mouse leukemia. Passive immunization resulted in high titers of cytotoxic antibody in the serum of treated mice and the suppression of metastatic tumor cells. The tumor-suppressive effects of the monoclonal antibodies were amplified by the administration of exogenous complement. This combined antibody and

Irwin D. Bernstein; Milton R. Tam; Robert C. Nowinski



Production and Characterization of Monoclonal Antibodies against the Hemolysin BL Enterotoxin Complex Produced by Bacillus cereus  

Microsoft Academic Search

A total of five hybridoma cell lines that produced monoclonal antibodies against the components of the hemolysin BL (HBL) enterotoxin complex and sphingomyelinase produced by Bacillus cereus were established and characterized. Monoclonal antibody 2A3 was specific for the B component, antibodies 1A12 and 8B12 were specific for the L2 component, and antibody 1C2 was specific for the L1 protein of




Production and characteristics of monoclonal antibodies against staphylococcal enterotoxin A  

SciTech Connect

Four hybridomas (designated B/sub 2/I, B/sub 2/II, CV/sub 3/ and C/sub 5/) secreting monoclonal antibodies (McAb) to staphylococcal enterotoxin A (SEA) were produced by fusion of NS-1 myeloma cells with spleen cells of BALB/c mice hyperimmunized against SEA. Immunization consisted of a intraperitoneal injection of 50 SEA in phosphate-buffered saline (PBS, pH 7.2) and complete Freund adjuvant, followed four weeks later with 150 SEA in PBS and incomplete Freund adjuvant. Three days prior to fusion an intravenous injection of 150 SEA was given per day. Monoclonal antibodies from all clones (tissue culture supernatant) showed high end-point titers (>10/sup -7/) in an indirect enzyme-linked immunosorbent assay (ELISA) with 1 SEA as the coating antigen in microtiter plate wells. Even nanogram levels of purified SEA yielded high absorbance with purified IgG/sub 1/ from clone B/sub 2/I. Saturation analysis with competitive ELISA indicated the McAb of all clones to be of similar and high affinity to SEA. Cross-reactivity with SEB, SEC/sub 1/, SED, and SEE was observed with McAb from all the clones. Specific reactivity of all McAb with each enterotoxin was noted in autoradiographs of electroimmunoblots (of SDS-PAG) of crude SEA, SEB, SEC/sub 1/, SED and SEE from growth of enterotoxigenic Staphylococcus aureus strains (265, 243, 293, 494 and ET-10 respectively) even when present in nanogram levels. Autoradiographs of crude SEB showed a 30,000 dalton SE band and an additional larger molecular weight band. Both polyclonal and monocloal antibodies demonstrated this second band with radiolabelled (/sup 125/I) Protein A and sheep anti-mouse F(ab/sup 1/)/sub 2/ probes.

Edwin, C.



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

Technology Transfer Automated Retrieval System (TEKTRAN)

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


Monoclonal antibodies to cyclodiene insecticides and method for detecting the same  


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

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




Microsoft Academic Search

Monoclonal antibodies (mAbs) recognizing different regions of PrP are potential tools in the study of prion diseases and immunotherapy. We used shuffled recombinant prion protein containing elk and mouse PrP as antigen to produce monoclonal antibodies in mice. We found that mAb 5C6 mapped to a discontinuous epitope comprised of amino acid 132 and 158 (mouse numbering). Monoclonal anibody 9E9

Chu-Chun Weng



Immunenzymometric assay for the heart specific glycogen phosphorylase BB in human serum using monoclonal antibodies.  


An enzyme-linked immunosorbent assay for the determination of the human glycogen phosphorylase isoenzyme BB (GP BB) using two murine monoclonal antibodies was developed. A series of hybridoma clones producing monoclonal antibodies to GP BB were obtained by the standard lymphocyte hybridoma technique. Two of the selected clones synthesizing monoclonal antibodies, which recognize different epitopes, were employed for the immunenzymometric assay. The first monoclonal antibody was immobilized on microtiter plates and the bound glycogen phosphorylase BB was detected with the second monoclonal antibody conjugated with horse radish peroxidase. This assay enables a specific and sensitive measurement of GP BB in the range of 0.5-150 ng/ml phosphate-buffered saline containing 0.5% bovine serum albumin in less than 3 hours. The lower limit in human serum amounts about 3 ng/ml. Preliminary data obtained with human sera from patients after aorto-coronary artery bypass surgery are demonstrated. PMID:2658982

Hofmann, U; Rabitzsch, G; Löster, K; Handschack, W; Noll, F; Krause, E G



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.

Brand, Toni M; Iida, Mari



Microchip assays for screening monoclonal antibody product quality.  


Microchip CE-SDS was evaluated as a high-throughput alternative to conventional CE-SDS for monitoring monoclonal antibody protein quality. A commercial instrument (LabChip) 90) was used to separate dodecyl sulfate coated proteins through a sieving polymer based on the proteins' sizes. Under reducing conditions, the microchip CE-SDS separation was similar to that of conventional CE-SDS, providing reasonable resolution of the non-glycosylated and the glycosylated heavy chains. The fluorescence detection on LabChip 90 using non-covalent fluorescent labeling method was about as sensitive as the 220 nm UV detection used in a conventional CE instrument. A simple glycan typing assay was developed for the reducing microchip CE-SDS format. Antibodies, either pure or in crude cell culture media are treated with Endoglycosidase H, which specifically cleaves the hybrid and high mannose type glycans. A heavy chain migration shift on reducing CE-SDS resulting from the loss of glycan is used to measure the level of high mannose/hybrid type glycans as a percentage of the total glycans. Microchip CE-SDS, under both non-reducing and reducing conditions, can be used in a variety of antibody product screening assays. The microchip analyses provide sufficient resolution and sensitivity for this purpose but on a time scale approximately 70 times faster (41 s versus 50 min per sample) than conventional CE separation under typical operational conditions. PMID:19130579

Chen, Xiaoyu; Tang, Kaiyan; Lee, Maximilian; Flynn, Gregory C



Generation and characterization of new HER2 monoclonal antibodies.  


Antibodies are among the most commonly used research and diagnostic tools. Antibody type and clonality are important in any assay as they can influence epitope detection. HER2 oncoprotein is overexpressed or undergoes gene amplification in approximately 30% of invasive breast carcinomas and 20% of gastric adenocarcinomas. Overexpression of HER2 is primarily detected by immunohistochemistry (IHC) on neoplastic tissue sections. We produced five murine hybridoma clones secreting monoclonal antibodies (MAbs) against HER2 protein. For hybridoma production, spleen cells from BALB/c mice immunized with a recombinant fragment of the extracellular portion of HER2 (rHER2) were fused to SP2/O-Ag14 cells, selected in HAT medium and screened by indirect ELISA. MAbs secreted were characterized according to isotypes, functional affinity constants, reaction with the native protein in MCF-7 cells by indirect immunofluorescence and in tissue sections from HER2 positive breast cancer specimens by IHC. Two MAbs were IgG2b and three were IgG1, and their affinity constants ranged from 6×10(7) to 1×10(9)M(-1). All MAbs reacted with the native protein and two stained strongly the membrane of neoplastic cells overexpressing HER2. These two MAbs could be useful in assaying HER2 overexpression in human tissues for research and possibly diagnostic purposes after a proper large-scale validation study. PMID:22901624

Vasconcellos, Flávia Aleixo; Aleixo, Pedro Bandeira; Stone, Simone Cardozo; Conceição, Fabricio Rochedo; Dellagostin, Odir Antônio; Aleixo, José Antonio Guimarães



Novel Designs of Multivalent Anti-CD20 Humanized Antibodies as Improved Lymphoma Therapeutics  

Microsoft Academic Search

Multivalent antibodies, either monospecific or bispecific, may improve the efficacy of current therapeutic interventions involving a single monoclonal antibody (mAb). We have applied the Dock-and-Lock (DNL) method, a new platform technology for the site-specific and covalent assembly of modular components into stably tethered complexes of defined composition, to prepare a hexavalent, anti-CD20 antibody, designated Hex-hA20, which comprises six Fabs with

Edmund A. Rossi; David M. Goldenberg; Thomas M. Cardillo; Rhona Stein; Yang Wang; Chien-Hsing Chang


T lymphocytes can be effectively recruited for ex vivo and in vivo lysis of AML blasts by a novel CD33/CD3-bispecific BiTE antibody construct.  


Patients with acute myelogenous leukemia (AML) are in high need of novel targeted therapies. Here we explored the ex vivo activity of AMG330, a novel T-cell-engaging BiTE (bi-specific T-cell engagers) antibody (Ab) construct, that is bispecific for the myeloid differentiation antigen, CD33 and CD3, in primary samples from AML patients (N=23) and AML cell lines. KG-1 and U937 cells were lysed in co-culture with healthy donor T-cells at AMG330 concentrations as low as 0.1?ng/ml (1.8?pM). T-cells derived from AML patient samples were found to be as active in redirected lysis by AMG330 as T-cells from healthy donors. In an autologous setting, AMG330 could activate and expand T-cells in primary AML patient samples, and effectively mediated the redirected lysis of AML blasts and normal myeloid cells. A deficiency in target-cell lysis was only observed in samples with very low initial effector-to-target (E:T) ratio. However, this could be overcome if previously stimulated autologous T-cells were tested in patient samples at a higher E:T ratio. In vivo experiments in immunodeficient mice demonstrated significant inhibition of tumor growth by AMG330 and an inducible infiltration of human T-cells into subcutaneous HL60 tumors. The activities of the CD33/CD3-bispecific BiTE Ab construct AMG330 warrant further development for the treatment of AML. PMID:23178753

Aigner, M; Feulner, J; Schaffer, S; Kischel, R; Kufer, P; Schneider, K; Henn, A; Rattel, B; Friedrich, M; Baeuerle, P A; Mackensen, A; Krause, S W



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.

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



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


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

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



Purification and identification of cell surface antigens using lamprey monoclonal antibodies  

PubMed Central

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

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



An Antibody Delivery System for Regulated Expression of Therapeutic Levels of Monoclonal Antibodies In Vivo  

Microsoft Academic Search

Monoclonal antibody (mAb) delivery by gene transfer in vivo may be an attractive alternative to current mAb therapies for applications that require long-term therapy. This article describes a transfer system that allows inducible high-level expression of unmodified mAbs in vivo. A recombinant adeno-associated viral (rAAV) vector is used that comprises an expression cassette consisting of a dimerizer-regulated promoter that drives

Jianmin Fang; Saili Yi; Andrew Simmons; Guang Huan Tu; Minh Nguyen; Thomas C Harding; Melinda VanRoey; Karin Jooss



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.



Development of monoclonal antibodies specific to urochordate intracellular epitopes.  


Sixteen monoclonal antibodies (MAbs) specific to 2 urochordate genera (Botryllus schlosseri and Botrylloides) intracellular epitopes were generated in mice immunized with a mixture of fresh and paraformaldehyde-fixed cells obtained from animal's blood and cells from dissociated organs. Hybridoma clones were selected by ELISA tests and immunohistochemistry assays on paraffin-embedded animal tissues. Five MAbs were tested for reactions with different zooidal organs and cell compartments; 7 MAbs were tested, separately, on 5 different botryllid colonies (3 Botryllus and 2 Botrylloides). The results revealed high polymorphism. Whereas some of the MAbs recognized, specifically, only part of the botryllid genotypes tested, others recognized only part of the cellular compartments. These MAbs will be used as an important tool in the study of botryllid ascidian immunology and developmental biology, revealing the first wide panel of MAbs specific to urochordate intracellular antigens. PMID:16271304

Lapidot, Z; Rinkevich, B



Hierarchical clustering of monoclonal antibody reactivity patterns in nonhuman species.  


Monoclonal antibodies (Mab) are an important resource for defining molecular expression and probing molecular function. The characterization of Mab reactivity patterns, however, can be costly and inefficient in nonhuman experimental systems. To develop a computational approach to the pattern analysis of Mab reactivity, we analyzed a panel of 128 Mab recognizing sheep antigens. Quantitative single parameter flow cytometry histograms were obtained from five cell types isolated from normal animals. The resulting 640 histograms were smoothed using a Gaussian kernel over a range of bandwidths. Histogram features were selected by SiZer--an analytic tool that identifies statistically significant features. The extracted histogram features were compared and grouped using hierarchical clustering. The validity of the clustering was indicated by the accurate pairing of externally verified molecular reactivity. We conclude that our computational algorithm is a potentially useful tool for both Mab classification and molecular taxonomy in nonhuman experimental systems. PMID:19639632

Pratt, Juan Pablo; Zeng, Qing; Ravnic, Dino; Huss, Harold; Rawn, James; Mentzer, Steven J



Monoclonal antibody to proteoglycan derived from Grifola frondosa (Maitake).  


A murine monoclonal antibody (MAb) was prepared by immunizing BALB/c mice with a proteoglycan fraction derived from Grifola frondosa (Maitake mushroom), followed by the hybridization of spleen cells with mouse myeloma cells. The MAb (subclass; Ig G2b), designated MPG2, reacted with schizophyllan (SPG), curdlan, scleroglucan, laminarin and lentinan, but not with dextran, pullulan, mannan and xylan. Immunohistochemistry (ABC-GO method) showed that MAb MPG2 reacted with lysosomal proteoglycan and (1-->6)-beta-branched laminaritriose taken up by rabbit peritoneal macrophages. These results suggest that this MAb may recognize mainly (1-->3)-beta-D-glucan, and may be useful for determining the immunological properties of Grifola frondosa-derived proteoglycan. PMID:8069265

Hirata, A; Adachi, Y; Itoh, W; Komoda, M; Tabata, K; Sugawara, I



New Monoclonal Antibody Specific for Candida albicans Germ Tube  

PubMed Central

Hydrophobic components of the germ tube of the dimorphic pathogenic fungus Candida albicans were used as immunogens to prepare monoclonal antibodies (MAbs). Among the resulting MAbs, one (MAb 16B1-F10) was shown by indirect immunofluorescence to be specific to the surface of the mycelium phase of the C. albicans and C. stellatoidea species. No labeling of any other genera and Candida species tested was observed, including C. dubliniensis, a newly described species which has many phenotypic similarities to C. albicans. This phase-specific epitope resides on a protein moiety. The molecular mass of the antigen released by Zymolyase digestion was determined by gel filtration and ranges from 25 to 166 kDa. The antigen was also shown to be highly hydrophobic. This anti-C. albicans cell wall surface-specific MAb may be a good candidate for use in tests for the rapid differentiation of the two closely related species C. albicans and C. dubliniensis.

Marot-Leblond, Agnes; Grimaud, Linda; Nail, Sandrine; Bouterige, Sandrine; Apaire-Marchais, Veronique; Sullivan, Derek J.; Robert, Raymond



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.

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



Monoclonal antibodies specific to thermostable proteins in animal blood.  


Bovine plasma proteins are used as high-quality protein supplements in animal feed and as binders or colorants in food for human consumption. Religious observance, as well as recent fears of epidemic bovine spongiform encephalopathy, highlights the need for methods to detect bovine blood in processed food and animal feed for regulatory purposes, as the currently available methods are neither species-specific, blood-specific, nor valid for excessively heat-processed samples. This paper reports the development of monoclonal antibodies (MAbs) raised against bovine thermostable plasma proteins that display a unique species specificity pattern for plasma proteins. Immunoblotting revealed several thermostable antigenic proteins (10, 25, 40, and 60 kDa) in bovine plasma sterilized at 121 degrees C for 15 min. These MAbs can be employed individually or combined in immunoassays for analytical purposes and investigations of the chemical and biological properties of the thermostable plasma proteins identified here. PMID:17622154

Hsieh, Yun-Hwa P; Ofori, Jack A; Rao, Qinchun; Bridgeman, C Roger



A novel monoclonal antibody specific to peptide pdnaelvlltlgqawqg.  


Cis-aconitic acid decarboxylase (CAD) has been assumed to be a key enzyme in the production of itaconic acid. Here we aimed to efficiently generate the monoclonal antibody against the CAD protein. We synthesized the peptide "pdnaelvlltlgqawqg" based on the published CAD cDNA sequences. The peptide was chemically linked with the carrier protein keyhole limpet hemocyanin and then injected into Balb/c mice. Hybridomas were screened by indirect enzyme-linked immunosorbent assay (ELISA) using either purified 6 x His-CAD fusion protein or the peptide. One MAb named K2 (IgG1), effective in detecting the native CAD protein, was characterized by ELISA and Western immunoblotting. By using the MAb, we found that the CAD protein was more highly expressed in poorly differentiated gastric cancer tissues than in well-differentiated and moderately differentiated tissues. Taken together, the MAb K2 would be helpful for understanding the functions of CAD in gastric carcinogenesis. PMID:19857120

Ji, Genlin



Concentrations of EpCAM ectodomain as found in sera of cancer patients do not significantly impact redirected lysis and T-cell activation by EpCAM/CD3-bispecific BiTE antibody MT110  

PubMed Central

Ectodomains of target antigens for antibody-based therapies can be shed from the target cell surface and found in sera of patients. Shed ectodomains of therapeutic targets not only pose the risk of sequestering therapeutic antibodies but, in a multimeric form, of triggering T cell activation by bispecific antibodies binding to CD3 on T cells. Recently, epithelial cell adhesion molecule (EpCAM) has been shown to be activated by release of its ectodomain, called EpEX. Here, we show that only very low amounts of EpEX are detectable in sera of cancer patients. Among 100 cancer patient samples tested, only 17 (17%) showed serum levels of EpEX in excess of 0.05 ng/ml with highest EpEX concentrations of 5.29, 1.37 and 0.52 ng/ml. A recombinant form of human EpEX (recEpEX) was produced to assess its possible effect on redirected lysis and T cell activation by EpCAM/CD3-bispecific BiTE antibody MT110, currently being tested in patients with solid tumor malignancies. RecEpEX had a very minor effect on redirected lysis by MT110 with an approximate IC50 value of 3,000 ng/ml, which is a concentration close to three orders of magnitude higher than the highest EpEX concentration found in cancer patients. Concentrations of 30 ng/ml EpEX in combination with 250 ng/ml MT110 were minimally required to induce a detectable activation of CD4+ and CD8+ T cells. We conclude that soluble EpEX in sera of cancer patients is unlikely to pose an issue for the efficacy or safety of MT110, and perhaps other antibodies binding to N-terminal epitopes of EpCAM.

Petsch, Silke; Gires, Olivier; Ruttinger, Dominik; Denzel, Sabine; Lippold, Sandra; Wolf, Andreas



Anti-idiotype monoclonal antibody elicits broadly neutralizing anti-gp120 antibodies in monkeys.  

PubMed Central

Murine monoclonal antibodies (mAbs) were raised against human, polyclonal, anti-gp120 antibodies (Ab1) and were selected for binding to broadly neutralizing anti-gp120 antibodies in sera positive for human immunodeficiency virus (HIV). One anti-idiotype mAb (Ab2), 3C9, was found to be specific for human anti-gp120 antibodies directed against an epitope around the conserved CD4 attachment site of gp120. The 3C9 reactive human anti-gp120 antibodies (3C9+ Ab) neutralized MN, IIIB, RF, and four primary isolates of HIV type 1 (HIV-1). Cynomolgus monkeys were immunized with 3C9 in adjuvant to test whether this anti-idiotype mAb could induce neutralizing anti-gp120 antibodies. The results show that purified anti-anti-idiotype antibodies (Ab3) from 3C9 immune sera bind to an epitope around the CD4 attachment site of gp120SF and gp120IIIB. Furthermore, purified gp120-specific Ab3 neutralize MN, IIIB, and RF isolates. These results demonstrate that primates immunized with an anti-idiotype mAb produce broadly neutralizing anti-HIV-1 antibodies. Since this anti-idiotype mAb was selected by identifying a clonotypic marker, its biological activity can be explained as the results of clonotypic B-cell stimulation.

Kang, C Y; Nara, P; Chamat, S; Caralli, V; Chen, A; Nguyen, M L; Yoshiyama, H; Morrow, W J; Ho, D D; Kohler, H



Specificities of antibodies to acetylcholine receptors in sera from myasthenia gravis patients measured by monoclonal antibodies.  

PubMed Central

The pattern of antibody specificities in sera from patients with myasthenia gravis (MG) was determined by the ability of monoclonal antibodies against defined determinants on the acetylcholine receptor molecule to inhibit binding of the serum antibodies to receptor from human muscle. We found that MG patients produce fundamentally the same pattern of specificities as that produced by animals immunized with receptor purified from fish electric organs or mammalian muscle. Most of the antibodies are directed at the "main immunogenic region' which is located on the extracellular surface of the alpha subunit and is distinct from the acetylcholine binding site. Regions on the beta and gamma subunits near the main immunogenic region are also significantly immunogenic. In one patient the proportions of antibodies to various regions are constant over time despite changes in total antibody amount and clinical state. Between patients there is no obvious correlation between antibody specificities and clinical state. These data suggest that the autoimmune response in MG is stimulated by human receptor rather than a crossreacting (e.g., viral) antigen and that in both MG and experimental autoimmune MG the pattern of specificities produced is determined by the inherently immunogenic structural features of the receptor molecule. They also suggest that the wide differences in clinical state sometimes observed between patients with the same total concentration of antireceptor antibody are due primarily to differences in endogenous factors which affect the safety factor for neuromuscular transmission rather than to the presence of especially pathogenic antireceptor specificities.

Tzartos, S J; Seybold, M E; Lindstrom, J M



The CEA/CD3-Bispecific Antibody MEDI-565 (MT111) Binds a Nonlinear Epitope in the Full-Length but Not a Short Splice Variant of CEA  

PubMed Central

MEDI-565 (also known as MT111) is a bispecific T-cell engager (BiTE®) antibody in development for the treatment of patients with cancers expressing carcinoembryonic antigen (CEA). MEDI-565 binds CEA on cancer cells and CD3 on T cells to induce T-cell mediated killing of cancer cells. To understand the molecular basis of human CEA recognition by MEDI-565 and how polymorphisms and spliced forms of CEA may affect MEDI-565 activity, we mapped the epitope of MEDI-565 on CEA using mutagenesis and homology modeling approaches. We found that MEDI-565 recognized a conformational epitope in the A2 domain comprised of amino acids 326–349 and 388–410, with critical residues F326, T328, N333, V388, G389, P390, E392, I408, and N410. Two non-synonymous single-nucleotide polymorphisms (SNPs) (rs10407503, rs7249230) were identified in the epitope region, but they are found at low homozygosity rates. Searching the National Center for Biotechnology Information GenBank® database, we further identified a single, previously uncharacterized mRNA splice variant of CEA that lacks a portion of the N-terminal domain, the A1 and B1 domains, and a large portion of the A2 domain. Real-time quantitative polymerase chain reaction analysis of multiple cancers showed widespread expression of full-length CEA in these tumors, with less frequent but concordant expression of the CEA splice variant. Because the epitope was largely absent from the CEA splice variant, MEDI-565 did not bind or mediate T-cell killing of cells solely expressing this form of CEA. In addition, the splice variant did not interfere with MEDI-565 binding or activity when co-expressed with full-length CEA. Thus MEDI-565 may broadly target CEA-positive tumors without regard for expression of the short splice variant of CEA. Together our data suggest that MEDI-565 activity will neither be impacted by SNPs nor by a splice variant of CEA.

Huang, Jiaqi; Brohawn, Philip; Morehouse, Chris; Lekstrom, Kristen; Baeuerle, Patrick A.; Wu, Herren; Yao, Yihong; Coats, Steven R.; Dall'Acqua, William; Damschroder, Melissa; Hammond, Scott 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



Monoclonal Antibody to Thy-1 Enhances Regeneration of Processes by Rat Retinal Ganglion Cells in Culture  

NASA Astrophysics Data System (ADS)

Ganglion cells were dissociated from postnatal rat retinas, identified by specific fluorescent labels, and maintained in culture on a variety of substrates. Regeneration of processes by retinal ganglion cells was enhanced when the cells were plated on glass coated with a monoclonal antibody against the Thy-1 determinant. Plain glass and glass coated with polylysine, collagen, fibronectin, or other monoclonal antibodies supported the growth of neural processes, but were less effective than antibody to Thy-1.

Leifer, Dana; Lipton, Stuart A.; Barnstable, Colin J.; Masland, Richard H.



Autoradiographic analysis of monoclonal antibody distribution in human colon and breast tumor xenografts  

Microsoft Academic Search

The targeting of monoclonal antibodies to human tumor xenografts in nude mice was investigated by analysis of the cellular distribution of two radioiodinated monoclonal antibodies, B6.2 and B72.3, which recognize different tumor-associated antigens. The time course of distribution of each antibody within Clouser human mammary carcinoma (B6.2 positive, B72.3 negative) and LS174T human colorectal carcinoma (B6.2 positive, B72.3 positive) following

Peter L. Jones; Brian M. Gallagher; Howard Sands



Monoclonal antibody production using a new supermacroporous cryogel bioreactor.  


A supermacroporous cryogel bioreactor has been developed to culture hybridoma cells for long-term continuous production of monoclonal antibodies (mAb). Hybridoma clone M2139, secreting antibodies against J1 epitope (GERGAAGIAGPK; amino acids, 551-564) of collagen type II, are immobilized in the porous bed matrix of a cryogel column (10 mL bed volume). The cells got attached to the matrix within 48 h after inoculation and grew as a confluent sheet inside the cryogel matrix. Cells were in the lag phase for 15 days and secreted mAb into the circulation medium. Glucose consumption and lactic acid production were also monitored, and during the exponential phase (approximately 20 days), the hybridoma cell line consumed 0.75 mM day-1 glucose, produced 2.48 mM day-1 lactic acid, and produced 6.5 microg mL-1 day-1 mAb during the exponential phase. The mAb concentration reached 130 microg mL-1 after continuous run of the cryogel column for 36 days. The yield of the mAb after purification was 67.5 mg L-1, which was three times greater than the mAb yield obtained from T-flask batch cultivation. Even after the exchange of medium reservoir, cells in the cryogel column were still active and had relatively stable mAb production for an extended period of time. The bioreactor was operated continuously for 55 days without any contamination. The results from ELISA as well as arthritis experiments demonstrate that the antibodies secreted by cells grown on the cryogel column did not differ from antibodies purified from the cells grown in commercial CL-1000 culture flasks. Thus, supermacroporous cryogels can be useful as a supporting material for productive hybridoma cell culture. Cells were found to be viable inside the porous matrix of the cryogel during the study period and secreted antibodies continuously. The antibodies thus obtained from the cryogel reactor were found to be functionally active in vivo, as demonstrated by their capacity to induce arthritis in mice. PMID:17590012

Nilsang, Suthasinee; Nandakumar, Kutty Selva; Galaev, Igor Yu; Rakshit, Sudip Kumar; Holmdahl, Rikard; Mattiasson, Bo; Kumar, Ashok



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

SciTech Connect

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

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

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.

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.



A monoclonal antibody specific for cells of the melanocyte lineage.  

PubMed Central

A monoclonal antibody, NKI/beteb, was prepared against membranes from a human melanoma metastasis, and in immunoprecipitates of melanoma cell lysates specific 100- and 7-kd glycoproteins were found. The large glycoproteins were also present in conditioned medium of melanoma cell lines. The antigen is located on the inner side of membranes of (pre)melanosomes and premelanosomelike vesicles. The antibody reacted in the immunoperoxidase test on frozen tissue sections with 27 of 28 nevocellular nevi (15/16 common, 12/12 dysplastic), 39/39 primary melanomas (3 intraepidermal, 24 cutaneous, 12 choroidal), 56/63 melanoma metastases, and 4/4 clear-cell sarcomas (melanoma of soft tissue). With sections of formalin-fixed paraffin-embedded tissues, the reaction was less sensitive. No reactivity was detected with frozen sections of 185 other tumors, except for 1 case of non-Hodgkin's lymphoma in which macrophages were positive. With the exception of melanocytes, all frozen sections of adult tissues that were tested were negative with NKI/beteb. On the basis of its tissue distribution so far, the antigen recognized by NKI/beteb seems to be a specific and sensitive diagnostic marker for cells of the melanocyte lineage. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7

Vennegoor, C.; Hageman, P.; Van Nouhuijs, H.; Ruiter, D. J.; Calafat, J.; Ringens, P. J.; Rumke, P.



Tumor size: effect on monoclonal antibody uptake in tumor models  

SciTech Connect

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

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



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.



An antifibrin monoclonal antibody useful in immunoscintigraphic detection of thrombi  

SciTech Connect

Balb/c mice were immunized with human plasmin-generated fibrinogen degradation product Y. Spleen cells were fused with P3X63-Ag8.653 myeloma cells. A clone (Y22) was found that produces monoclonal antibodies (MoAbs) with a strong reactivity with human fibrin and only a weak reactivity with fibrinogen in an enzyme immunoassay (EIA). Y22 also reacts with fibrin of rabbits, rats, sheep, and dogs. The antibodies are of the IgG1 kappa-type and appear to be directed against a conformation-dependent epitope in the D-domain of fibrin. Experiments with {sup 99m}Tc-labeled Y22 in vitro show that Y22 binds rapidly to forming clots. {sup 99}mTc-Y22 also binds to preformed plasma clots in a plasma milieu, even in the presence of high concentrations of heparin. Clot localization experiments in rabbits and rats confirm the high fibrin specificity and the potential of {sup 99m}Tc-Y22 for thrombus imaging in vivo.

Wasser, M.N.; Koppert, P.W.; Arndt, J.W.; Emeis, J.J.; Feitsma, R.I.; Pauwels, E.K.; Nieuwenhuizen, W. (Gaubius Institute TNO, Leiden (Netherlands))



Monoclonal antibody to cardiac myosin: imaging of experimental myocardial infarction  

SciTech Connect

Monoclonal antibody R11D10 to human cardiac myosin, which also cross-reacted with canine cardiac myosin, was used to demonstrate in vivo localization and visualization by gamma scintigraphy of experimental myocardial infarction. R11D10 Fab with a Ka of 5 X 10(8) M-1 was labeled with technetium-99m (99mTc) by the dithionite reduction method of technetium pertechnetate, via a bifunctional chelating agent, diethylene triamine pentaacetic acid (DTPA). Uptake of 99mTc R11D10 Fab in the infarct can be visualized as early as 2 h after intravenous administration. Comparison of R11D10 uptake to thallium-201, an analogue of potassium which is sequestered by normal myocardium, showed an inverse relation (r . -0.75, -0.87, -0.89), similar to that obtained with 125I labeled polyclonal antimyosin Fab. Ratios of R11D10 Fab in the infarct to normal myocardium were as high as 30:1 where access of antibody to antigen was not blood flow limited. However, with severe blood-flow restriction, the ratios were lower at about 10:1. Despite the theoretical limitation of a single epitope per myosin molecule available for binding by R11D10 Fab, the immense excess of myosin in the infarcted myocardium allowed adequate concentration of radiolabeled R11D10 for visualization of the infarct by external gamma scintiscanning.

Khaw, B.A.; Mattis, J.A.; Melincoff, G.; Strauss, H.W.; Gold, H.K.; Haber, E.



Profiling formulated monoclonal antibodies by (1)h NMR spectroscopy.  


Nuclear magnetic resonance (NMR) is arguably the most direct methodology for characterizing the higher-order structure of proteins in solution. Structural characterization of proteins by NMR typically utilizes heteronuclear experiments. However, for formulated monoclonal antibody (mAb) therapeutics, the use of these approaches is not currently tenable due to the requirements of isotope labeling, the large size of the proteins, and the restraints imposed by various formulations. Here, we present a new strategy to characterize formulated mAbs using (1)H NMR. This method, based on the pulsed field gradient stimulated echo (PGSTE) experiment, facilitates the use of (1)H NMR to generate highly resolved spectra of intact mAbs in their formulation buffers. This method of data acquisition, along with postacquisition signal processing, allows the generation of structural and hydrodynamic profiles of antibodies. We demonstrate how variation of the PGSTE pulse sequence parameters allows proton relaxation rates and relative diffusion coefficients to be obtained in a simple fashion. This new methodology can be used as a robust way to compare and characterize mAb therapeutics. PMID:24006877

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



Serotyping of Chlamydia psittaci isolates using serovar-specific monoclonal antibodies with the microimmunofluorescence test.  

PubMed Central

A panel of 10 serovar-specific monoclonal antibodies that could distinguish 10 distinct serovars of Chlamydia psittaci was prepared. The panel included one monoclonal antibody to each of the 10 serovars. Monoclonal antibodies were selected for their specificity in the indirect microimmunofluorescence test. Each of the monoclonal antibodies had a titer of 1:1,280 or higher to the homologous strain, with only two showing any cross-reactivity at a dilution of 1:10. Chlamydial antigen derived from organisms growing in tissue culture of one well of a 96-well multiwell dish was usually sufficient for the serotyping of an isolate. Infected yolk sac preparations were also suitable for serotyping. The panel of monoclonal antibodies was used to serotype 55 mammalian and avian strains. All except five of the strains were successfully serotyped; these five strains are presumed to represent at least two additional serovars. The use of a panel of monoclonal antibodies in the indirect microimmunofluorescence test provides a rapid and reliable method for serotyping new isolates. Monoclonal antibodies to new serovars can easily be added to the panel.

Andersen, A A



Rapid immunotyping of Chlamydia trachomatis with monoclonal antibodies in a solid-phase enzyme immunoassay.  

PubMed Central

The technical complexity of determining the serovar of Chlamydia trachomatis strains has limited the use of serotyping in clinical and epidemiologic studies. We developed a simple method for rapidly serotyping isolates of C. trachomatis by using monoclonal antibodies in a dot-enzyme-linked immunosorbent assay (ELISA) system. Isolates were passaged three to six times in shell vial cultures to greater than 50% monolayer infection, and chlamydial elementary bodies were isolated by sonication and microcentrifugation. Chlamydial antigen was spotted onto a series of replicate nitrocellulose membrane patches and reacted with C. trachomatis-specific monoclonal antibodies. Bound antibody was detected visually by a color reaction by using peroxidase-conjugated anti-mouse immunoglobulins. This method can be routinely applied to 60 or more specimens concurrently. We compared dot-ELISA serotyping with monoclonal antibody microimmunofluorescence serotyping of 124 clinical C. trachomatis isolates and found that dot-ELISA has sensitivity and serotyping accuracy comparable to that of monoclonal antibody microimmunofluorescence. Images

Barnes, R C; Wang, S P; Kuo, C C; Stamm, W E



Generation and characterization of monospecific and bispecific hexavalent trimerbodies.  


Here, we describe a new class of multivalent and multispecific antibody-based reagents for therapy. The molecules, termed "trimerbodies," use a modified version of the N-terminal trimerization region of human collagen XVIII noncollagenous 1 domain flanked by two flexible linkers as trimerizing scaffold. By fusing single-chain variable fragments (scFv) with the same or different specificity to both N- and C-terminus of the trimerizing scaffold domain, we produced monospecific or bispecific hexavalent molecules that were efficiently secreted as soluble proteins by transfected mammalian cells. A bispecific anti-laminin x anti-CD3 N-/C-trimerbody was found to be trimeric in solution, very efficient at recognizing purified plastic-immobilized laminin and CD3 expressed at the surface of T cells, and remarkably stable in human serum. The bispecificity was further demonstrated in T cell activation studies. In the presence of laminin-rich substrate, the bispecific anti-laminin x anti-CD3 N-/C-trimerbody stimulated a high percentage of human T cells to express surface activation markers. These results suggest that the trimerbody platform offers promising opportunities for the development of the next-generation therapeutic antibodies, i.e., multivalent and bispecific molecules with a format optimized for the desired pharmacokinetics and adapted to the pathological context. PMID:23221741

Blanco-Toribio, Ana; Sainz-Pastor, Noelia; Álvarez-Cienfuegos, Ana; Merino, Nekane; Cuesta, Ángel M; Sánchez-Martín, David; Bonet, Jaume; Santos-Valle, Patricia; Sanz, Laura; Oliva, Baldo; Blanco, Francisco J; Álvarez-Vallina, Luis



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

PubMed Central

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

Arcuri, Santo; Galletti, Silvia; Faldella, Giacomo



New and old monoclonal antibodies for the treatment of chronic lymphocytic leukemia.  


Over the last few years, several new agents have been under evaluation in preclinical studies and clinical trials, showing promise in treating chronic lymphocytic leukemia (CLL). Among these agents, monoclonal antibodies (mAbs) such as rituximab and alemtuzumab have changed the natural course of the disease. Nowadays there are several new promising monoclonal antibodies under investigation against the CD20, CD23, CD37 and CD40 molecules. Application of newer monoclonal antibodies represents an area of ongoing clinical research in CLL. PMID:21561405

Laurenti, L; De Padua, L; D'Arena, G; Vannata, B; Innocenti, I; Tarnani, M; Deaglio, S; Sica, S; Efremov, D G; Leone, G



Targeting rat anti-mouse transferrin receptor monoclonal antibodies through blood-brain barrier in mouse.  


Drug targeting through the brain capillary endothelium, which forms the blood-brain barrier (BBB) in vivo, may be achieved with peptidomimetic monoclonal antibodies that target peptide transcytosis systems on the BBB in vivo. Murine monoclonal antibodies to the rat transferrin receptor, such as the OX26 monoclonal antibody, are targeted through the BBB on the transferrin receptor in the rat. However, the present studies show the OX26 monoclonal antibody is not an effective brain delivery vector in mice. The emergence of transgenic mouse models creates a need for brain drug-targeting vectors for this species. Two rat monoclonal antibodies, 8D3 and RI7-217, to the mouse transferrin receptor were evaluated in the present studies. Both the RI7-217 and the 8D3 antibody had comparable permeability-surface area products at the mouse BBB in vivo. However, owing to a higher plasma area under the concentration curve, the mouse brain uptake of the 8D3 antibody was higher, 3.1 +/- 0.4% of injected dose [(ID)/g] compared with the brain uptake of the RI7 antibody, 1.6 +/- 0.2% ID/g, at 60 min after i.v. injection. Conversely, the mouse brain uptake of the OX26 antibody, which does not recognize the mouse transferrin receptor, was negligible, 0.06 +/- 0.01% ID/g. The RI7-127 antibody was more selective for brain because this antibody was not measureably taken up by liver. The capillary depletion technique demonstrated transcytosis of the RI7-217 antibody through the mouse BBB in vivo. The brain uptake of the 8D3 antibody was saturable, consistent with a receptor-mediated transport process. In conclusion, these studies indicate rat monoclonal antibodies to the mouse transferrin receptor may be used for brain drug-targeting studies in mice such as transgenic mouse models. PMID:10688622

Lee, H J; Engelhardt, B; Lesley, J; Bickel, U; Pardridge, W M



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

NASA Astrophysics Data System (ADS)

A monoclonal antibody to an eosinophil specific determinant is very important in assessing eosinophil function during helminthic infection. Eosinophils induced by Schistosoma mansoni infection in BALB/c mice were used to induce C57B1/6 immunocytes for production of hybridomas secreting eosinophil monoclonal antibodies. These antibodies were shown to react with an eosinophil surface epitope but not with neutrophils or macrophages as determined by ELISA, immunodiffusion, immunofluorescence, and immunoblot assay. Affinity chromatography with eosinophil chemotactic factor-sepharose consistently selected out a { rm M_ R} 67,000 protein from solubilized eosinophil membrane antigens but not from neutrophil and macrophage antigens. In vitro studies showed that the eosinophil-specific monoclonal antibodies abrogated antibody-dependent eosinophil -mediated killing of S. mansoni schistosomula using mouse, rat or human eosinophils. Neutrophil and macrophage killing activities were unaffected. The monoclonal antibodies effected complement-dependent lysis of mouse and rat eosinophils but not of human eosinophils. ECF-treated eosinophils showed enhanced killing of schistosomula which was blocked by the monoclonal antibody. Murine and human eosinophils preincubated with monoclonal antibody exhibited decreased chemotaxis to ECF at optimal chemotactic concentrations. The monoclonal antibody also blocked eosinophil binding to ECF- sepharose beads. In vivo induction of peripheral blood eosinophilia by injection of S. mansoni eggs was suppressed by injections of monoclonal antibodies 2CD13 and 2QD45 in mouse and rat experimental models. Eosinophilia induced by keyhole limpet hemocyanin- cyclophosphamide treatment was also suppressed by monoclonal antibody in both murine and rat systems. Pulmonary granulomas in mice given egg injection and monoclonal antibody were smaller and contained fewer eosinophils than those granulomas from mice given eggs only. In immuno-biochemical studies, the monoclonal antibody 2QD45 specifically immunoprecipitated the {rm M_ R} 67,000 ECF-binding protein from ^{125}{rm I}-labeled mouse, rat, and human eosinophils as assessed by SDS-PAGE and autoradiography. Two-dimensional gel electrophoresis showed that this ECF-binding protein has a lower PI point than either mouse or bovine albumin.

Minkoff, Marjorie Sue


A Human Thymus-Leukemia Antigen Defined by Hybridoma Monoclonal Antibodies  

Microsoft Academic Search

A series of mouse hybridomas producing monoclonal antibodies against human acute lymphocytic leukemia (ALL) cells was generated and screened for tumor specificity. Among 1200 primary cultures, 60 produced an antibody that could distinguish between the immunizing leukemia cells and an isologous B lymphoblastoid cell line. Of these, two produced an antibody that detects an antigen expressed preferentially on ALL cells

Ronald Levy; Jeanette Dilley; Robert I. Fox; Roger Warnke



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 three separate domains on 124 kilodalton phytochrome from Avena  

Microsoft Academic Search

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

S. M. Daniels; P. H. Quail




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...


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

PubMed Central

Monoclonal antibodies directed against human renin were obtained by the fusing of myeloma cells with spleen cells from Balb/c or high-responder Biozzi mice injected with pure tumoral or highly purified renal renin. These procedures resulted in the production of seven stable monoclonal antibodies to human renin. Antibodies in the hybridoma culture medium were screened by binding to pure iodinated renin or insolubilized renin in a solid phase assay. The concentration of purified antibodies that provided a 50% binding to iodinated renin varied from 1 X 10(-10) to 1 X 10(-7) M. Two monoclonal antibodies were found to be potent inhibitors of renin enzymatic activity in vitro, behaving as noncompetitive inhibitors (Ki, 1 to 4 X 10(-10) M). They were specific for primate renin. Three monoclonal antibodies provided suitable immunoadsorbants for renin purification. One of these immunoadsorbants was used for large-scale purification of the renal enzyme, resulting in an 825-fold renin enrichment in a single step. Two antibodies were able to distinguish between active and inactive renin and enabled concomitant separation and purification of the two enzyme forms in various biological fluids. Monoclonal antibodies also stained human and monkey renal renin when indirect immunofluorescence and peroxidase-antiperoxidase techniques were used. A highly sensitive radioimmunometric assay of renin was constructed with two monoclonal antibodies. The sensitivity of this improved assay should permit the detection of renin in normal human plasma. Monoclonal antibodies have been shown to be superior to polyclonal antibodies in the following areas: the separation of active from inactive renin, the purification of renin from biological fluids, and the setting up of a direct assay of plasma renin. Images

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



Serum antibodies and monoclonal antibodies secreted by thymic B-cell clones from patients with myasthenia gravis define striational antigens.  


The biochemical identities of several antigens to which striational antibodies bind were determined by using serum antibodies and monoclonal antibodies from two patients with myasthenia gravis. The monoclonal antibodies were secreted by EBV-transformed B-lymphocyte clones obtained from thymus and thymoma. Serum and monoclonal antibodies reacted with discrete components of the skeletal muscle sarcomere, giving rise to several different patterns of immunofluorescence staining. Immunoblot analyses and enzyme-linked immunosorbent assays revealed three different antibody specificities: myosin, alpha-actinin, and/or actin. Individual monoclonal StrAb reacted with both muscle and nonmuscle isotypes of actin or myosin. It is noteworthy that contractile proteins (1) are associated with acetylcholine receptors (AChR) in plasma membranes, and (2) are biochemically altered in transformed cells. It is therefore conceivable that the release of neoantigenic AChR-associated contractile proteins from thymic epithelial cells undergoing neoplastic transformation may provide the immunogenic stimulus for production of StrAb. More precise definition of StrAb specificities in individual patients with MG and/or thymoma might provide a basis for diagnostic and/or prognostic classification of these diseases. Furthermore, the monoclonal antibodies will be useful in experimentally testing the potential pathogenicity of StrAb. PMID:3500666

Williams, C L; Lennon, V A; Momoi, M Y; Howard, F M



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.



Development of panel of monoclonal antibodies specific to urochordate cell surface antigens.  


Monoclonal antibodies are an important tool in the study of botryllid ascidians' immunology and developmental biology. Here we describe the development of a panel of 38 monoclonal antibodies that are specific to Botryllus schlosseri (Ascidiacea; subfamily Botryllinae) cell surface antigens. Many of these hybridomas recognize (by enzyme-linked immunosorbent assay and immunohistochemistry) epitopes of Botrylloides subpopulations (SP) II and III from the Mediterranean coast of Israel and show, on blood cell smear assays, reactions with subsets of Botryllus circulating blood cells. Fluorescence-activated cell sorting analyses using antibodies positive for botryllid tissues revealed up to 3.6% positive cells. ELISA screenings were performed with 64 new monoclonal antibodies on 5 different individual botryllid ascidian colonies (B. schlosseri, Botrylloides). The positive antibodies in this panel identified a large number of different antigenic determinants, some of which distinguish Botryllus versus Botrylloides colonies, and other, different colonies within these two species, or different cell types within tissues, embryos, and buds of individual colonies. Only 21 monoclonal antibodies tested positive with all colonies. Cross-reactivity with at least one Botrylloides colony was recorded in 49 hybridomas that identified Botryllus cells. This wide panel of monoclonal antibodies is the first such detailed set of monoclonals available for studies on botryllid ascidians. PMID:15988630

Lapidot, Ziva; Rinkevich, Baruch



Differentiating between isolates of Vibrio vulnificus with monoclonal antibodies.  


Monoclonal antibodies (MAbs) against Vibrio vulnificus (isolate I, VVC and isolate II, VVB) were raised using heat-killed and heat-killed plus SDS-mercaptoethanol treated forms of VVC and VVB for immunizing Swiss mice. Twenty three hybridomas producing MAbs against V. vulnificus were selected and divided into five groups according to their specificities to different V. vulnificus isolates and apparent protein antigens which ranged from approximately 3-50 kDa. Four groups were specific to V. vulnificus without cross reactivity to either other Vibrio spp. or other bacterial species. In dot blot based assays, one group of MAbs were specific to VVC, with a sensitivity of approximately 1.6 x 10(7) CFU ml(-1) (approximately 1.6 x 10(4) cells spot(-1)), and bound to proteins of approximately 50 and approximately 39 kDa. Other MAbs, binding to proteins ranging from approximately 3-14 and approximately 40 kDa, detected VVB (but not VVC) with high sensitivity at approximately 1.6 x 10(5) and 4 x 10(6) CFU ml(-1) (approximately 1.6 x 10(2) and 4 x 10(3) cells spot(-1)), respectively. In addition, certain MAbs were able to recognize V. vulnificus in tissues by means of immunohistochemistry. The remaining groups demonstrated cross reactivity to Vibrio fluvialis. MAbs from this study can, therefore, detect the difference between some isolates of V. vulnificus and in addition to pathogen detection may, with further antibodies, form the basis of serovar typing isolates in the future. PMID:18706941

Rengpipat, Sirirat; Pusiririt, Suttinee; Rukpratanporn, Sombat



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.

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



Monoclonal antibody R5 for detection of putatively coeliac-toxic gliadin peptides  

Microsoft Academic Search

Monoclonal antibody R5 against rye secalin was recently suggested to be useful in analysis of gluten in food. The epitope specificity of R5 was characterized and compared with those of eight other monoclonal antibod- ies (mabs) against gliadins (gli) and secalins. Mabs were tested for binding to synthetic peptides spanning in over- lapping manner sequences of gli. In a luminescence

Franka Kahlenberg; Daniel Sanchez; Ingolf Lachmann; Ludmila Tuckova; Helena Tlaskalova; Enrique Mendez; Thomas Mothes



Competitive enzyme immunoassay with monoclonal antibody for homovanillic acid measurement in human urine samples  

Microsoft Academic Search

A fast competitive enzyme immunoassay (EIA) for mea- suring homovanillic acid in human urine samples was developed with a monoclonal antibody and acetylcho- linesterase as enzyme label. Enzyme detection was per- formed by an easy colorimetric assay. Monoclonal anti- bodies were screened on the basis of sensitivity, specificity, and correlation studies. EIA has a detection limit of 0.5 mmol\\/L, a

Frederic Taran; Yveline Frobert; Christophe Creminon; Jacques Grassi; Didier Olichon; Charles Mioskowski; Philippe Pradelles


Antigenicity of the carboxyl terminus of insulin: isolation of human insulin-specific monoclonal antibodies.  

PubMed Central

Monoclonal technology was used to isolate antibodies binding the B30 (carboxy) terminal residue in the polyclonal-provoked immune response to human insulin. Although both spleen and lymph node cell fusions were carried out, only the latter were successful in isolating monoclonal antibodies that bound the carboxy terminal of human insulin. The binding of such antibodies was abolished or diminished by substitutions of the B30 residue. Studies with insulin species variants showed that the molecular binding between antibody and insulin may be critically determined by a subresidue feature, e.g. presence or absence of a single methyl group, as shown by the binding of the monoclonal antibody D10 to human insulin (threonine at B30) but not to rabbit insulin (serine at B30). Such studies are of interest in the study of the molecular basis of antibody-antigen interaction.

Mirza, I H; Wilkin, T J



Will immunogenicity limit the use, efficacy, and future development of therapeutic monoclonal antibodies?  

PubMed Central

While monoclonal antibodies show promise for use in the treatment of a variety of disease states, including cancer, autoimmune disease, and allograft rejection, generation of anti-antibody responses still remains a problem. For example, 50% of the patients who receive OKT3 produce blocking antibodies that interfere with its binding to T cells, thus decreasing the therapeutic effect (51). HAMA responses have also interfered with tumor imaging (39,40) and radioimmunotherapy (56). The generation of an anti-antibody response is dependent on many factors. These include the dose of antibody, the number of injections of antibody, the immunogenicity of the antibody, the form of the antibody, and the immunocompetence of the recipient. Predictably, both the number of injections of antibody and the dosage are influential in the generation of an anti-antibody response. It is apparent that human antibodies, chimeric antibodies, and mouse Fab fragments are much less likely to induce anti-antibody responses than intact mouse monoclonal antibodies or mouse F(ab')2 fragments when one injection is administered. Injections of human or chimeric antibodies appears to reduce immunogenicity, but the probability that anti-antibody responses can still be induced on multiple injections must be considered and appropriately evaluated. Several areas demand extensive investigation to enhance the clinical utility of monoclonal antibodies. First, results of thorough clinical trials with human or chimeric antibodies need to be evaluated for the induction of anti-antibodies after multiple injections of antibodies. Second, less immunogenic forms of antibodies (Fab, Fv) need to be studied for their clinical efficacies and for their abilities to induce anti-antibody responses.

Kuus-Reichel, K; Grauer, L S; Karavodin, L M; Knott, C; Krusemeier, M; Kay, N E



Therapeutic monoclonal antibodies: strategies and challenges for biosimilars development.  


Biosimilar medicines already on the market may have a primary structure identical to their reference products (e.g., amino acid sequences should be identical). In the case of monoclonal antibodies (mAbs), and due to their more complex structure, a greater level of demand would be in order and identity at other levels (e.g., post-translational modifications within the Fc region of the molecule) should be proved to establish "similarity". These requirements would lead to a greater development in the process and tighter quality controls during the production of biosimilar mAbs. The following issues should be taken into account in the comparability exercise: - The designs of the studies carried out to obtain approval of the reference product are not always adequate to show that safety and efficacy of the biosimilar mAbs are comparable. A similar efficacy does not necessarily imply a similar safety profile between the innovator and biosimilar products. - The design of clinical tests to demonstrate comparability must be flexible and adaptable throughout the development of the product. - The European Medicines Agency (EMA) will consider suitable goals in the evaluation of biosimilar mAbs for their approval (e.g., to specify whether their goal is to check similarity with the reference product or to show that the treatment is effective at a clinical level). PMID:22978327

Calvo, Begoña; Zuñiga, Leyre



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



Monoclonal antibodies that react with live Listeria spp.  

PubMed Central

Seven monoclonal antibodies (MAbs) against Listeria spp. that were reactive with live Listeria spp. were developed. Two of these MAbs (55-8 and 55-37) were members of the immunoglobulin M class, and all other MAbs were members of the immunoglobulin G class. MAb 55-23 reacted with 148 of 157 strains tested. MAb 34-51 reacted with serotype 1/2a, 1/2b, and 1/2c strains and exhibited a scattered reaction pattern with strains belonging to other serotypes. MAb 55-44 reacted with all of the strains belonging to serotype 4b tested. MAb 55-4 reacted with all of the serotype 1/2a isolates tested, although reactivity with other isolates also was observed. The other MAbs exhibited scattered reaction patterns. No correlation of reactivity pattern with serotype was found. Marked differences were observed between the reactivities of MAbs as determined by a magnetic immunoluminescence assay and a whole-cell enzyme-linked immunosorbent assay. Only MAb 55-23 exhibited minor reactivity with three Streptococcus spp. isolates, while no reactivity was observed with six Bacillus spp. strains, one Escherichia coli strain, and one Citrobacter sp. strain. In Western blots (immunoblots) MAbs 55-23, 55-44, and 34-9 exhibited reactivity; all other MAbs were negative in this assay.

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



Production of monoclonal antibody to herbicide fenoxaprop-ethyl.  


Fenoxaprop-ethyl is a selective aryloxyphenoxypropionate herbicide used widely to control annual and perennial grasses. A monoclonal antibody (MAb) against fenoxaprop-ethyl (FE), designated as 3E6B9C, was produced and had very low cross-reactivity with some of its structural analogs, such as clodinafop-propargyl, diclofop-methyl, lactofen, and quizalofop-p-ethyl. An indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed. The concentration of R-(+)-fenoxaprop-ethyl (R-FE) producing 50% of inhibition (IC(50)) and the working range of icELISA were 3.1?ng/mL and 0.6-29?ng/mL, respectively. This assay is also sensitive to R-fenoxaprop, S-(-)-fenoxaprop-ethyl, and metamifop with IC(50) of 3.4, 2.7, and 3.5?ng/mL, respectively. The recoveries of R-FE in soil samples with the icELISA were 86-102%. PMID:22008074

Cui, Yongliang; Nan, Tiegui; Tan, Guiyu; Li, Qing X; Wang, Baomin; Liu, Shangzhong



Monoclonal antibodies in the treatment of neuroimmunological diseases.  


Over the past 25 years, monoclonal antibodies (mAb) have become important elements in the therapeutic concepts for numerous clinical specialities, including oncology, gastroenterology, hemostaseology and endocrinology. One of the most dynamic fields of their use is the treatment of autoimmune diseases. Although the number of existing mAb interfering with the immune system has increased remarkably and many studies have yielded encouraging results in the treatment of neuroimmunological diseases, their clinical use is still limited compared with standard treatments. The only mAb which has been approved for a neuroimmunological disease by now is natalizumab for the treatment of relapsing-remitting multiple sclerosis (RRMS). This article gives an overview on mAb that are currently in use or under investigation for treating neuroimmunological diseases like multiple sclerosis (MS), neuromyelitis optica (NMO), chronic inflammatory demyelinating polyneuropathy (CIDP), inclusion body myositis (IBM), dermatomyositis, polymyositis, opsoclonusmyoclonus syndrome (OMS), multifocal motor neuropathy (MMN), anti-myelin-glycoprotein neuropathy (Anti-MAG), stiff person syndrome and myasthenia gravis (MG). PMID:22612751

Rommer, Paulus S; Patejdl, Robert; Zettl, Uwe K



Antigen-specific human monoclonal antibodies from transgenic mice.  


Due to the difficulties found when generating fully human monoclonal antibodies (mAbs) by the traditional method, several efforts have attempted to overcome these problems, with varying levels of success. One approach has been the development of transgenic mice carrying immunoglobulin (Ig) genes in germ line configuration. The engineered mouse genome can undergo productive rearrangement in the B cell population, with the generation of mouse B lymphocytes expressing human Ig (hIg) chains. To avoid the expression of mouse heavy or light chains, the endogenous mouse Ig (mIg) loci must be silenced by gene-targeting techniques. Subsequently, to obtain antigen-specific mAbs, conventional immunization protocols can be followed and the mAb technique used (fusion of activated B cells with mouse myeloma cells, screening, cloning, freezing, and testing) with these animals expressing human Ig genes. This chapter describes the type of transgenic knockout mice generated for various research groups, provides examples of human mAbs developed by research groups and companies, and includes protocols of immunization, generation, production, and purification of human mAbs from such mice. In addition, it also addresses the problems detected, and includes some of the methods that can be used to analyze functional activities with human mAbs. PMID:24037845

Mompó, Susana Magadán; González-Fernández, Africa



Molecular structure of eight human autoreactive monoclonal antibodies  

PubMed Central

The heavy (H) and light (L) chain V-region sequences of eight human autoreactive immunoglobulin M (IgM) monoclonal antibodies (mAbs: BY-4, BY-7, BY-12, IRM-3, IRM-7, IRM-8, IRM-10 and CDC-1) were determined at the cDNA level. All VH and VL families were identified. Four different VH families were represented, VH3 being the most common as five of the mAbs (BY-7, BY-12, IRM-3, IRM-8 and CDC-1) used genetic elements of this family, whereas VH1, VH2 and VH4 were only present in IRM-7, BY-4 and IRM-10, respectively. BY-4, BY-7, BY-12, IRM-7 and IRM-10 reacted with a variety of self as well as non-self antigens, thus exhibiting polyreactive behaviour. Comparison of the gene segments utilized by these mAbs with their germline counterparts revealed that the gene segments were close to germline configuration. The length of H-CDR3 was found to be relatively long (27–60 nucleotides) among the polyreactive mAbs and the presence of Tyr and Trp residues in this region seems to be of vital importance for polyreactivity. We have analysed the utilization of gene elements and the presence of amino acid residues in regions particularly important for antigen binding, such as CDR. Common molecular features relating to the function of the mAbs are discussed.

Aguilera, I; Melero, J; Nunez-Roldan, A; Sanchez, B



Polyelectrolyte Nanogels Decorated with Monoclonal Antibody for Targeted Drug Delivery  

PubMed Central

Novel surface-functionalized cross-linked nanogels were developed as a platform to allow conjugation of monoclonal antibodies (mAb) for targeted drug delivery. Well-defined diblock copolymers of poly(ethylene glycol)-b-poly(methacrylic acid) (PEG-b-PMA) with PEG terminal aldehyde functionality were synthesized by atom transfer radical polymerization (ATRP) and characterized by GPC and 1H NMR. These copolymers were used to prepare nanogels via condensation of PEG-b-PMA with Ca2+ ions into micelle-like aggregates, cross-linking of the PMA/Ca2+ cores and removal of Ca2+ ions. The resulting nanogels represent highly swollen spherical polyelectrolyte particles with free terminal aldehyde functionalities at the nonionic PEG chains. A reductive amination reaction between aldehyde groups and amino groups of mAb resulted in effective conjugation to the nanogels of mAb CC49 against tumor-associated glycoprotein 72 (TAG-72). The mAb retained the binding affinity to bovine submaxillary mucin after conjugation as shown by surface plasmon resonance (SPR). Therefore, aldehyde functionalized nanogels can be linked to mAb using a simple, one-step approach. They may have potential for targeted delivery of diagnostic and therapeutic agents to tumors.

Nukolova, Nataliya V.; Yang, Zigang; Kim, Jong Oh; Kabanov, Alexander V.; Bronich, Tatiana K.



Monoclonal antibodies: potential new therapeutic treatment against multiple myeloma.  


Despite recent treatments, such as bortezomib, thalidomide, and lenalidomide, therapy of multiple myeloma (MM) is limited, and MM remains an incurable disease associated with high mortality. The outcome of patients treated with cytotoxic therapy has not been satisfactory. Therefore, new therapies are needed for relapsed MM. A new anticancer strategy is the use of monoclonal antibodies (MoAbs) that represent the best available combination of tumor cytotoxicity, environmental signal privation, and immune system redirection. Clinical results in patients with relapsed/refractory MM suggest that MoAbs are likely to operate synergistically with traditional therapies (dexamethasone), immune modulators (thalidomide, lenalidomide), and other novel therapies (bortezomib); in addition, MoAbs have shown the ability to overcome resistance to these therapies. It remains to be defined how MoAb therapy can most fruitfully be incorporated into the current therapeutic paradigms that have achieved significant survival earnings in patients with MM. This will require careful consideration of the optimal sequence of treatments and their clinical position as either short-term induction therapy, frontline therapy in patients ineligible for ASCT, or long-term maintenance treatment. PMID:23506222

Allegra, Alessandro; Penna, Giuseppa; Alonci, Andrea; Russo, Sabina; Greve, Bruna; Innao, Vanessa; Minardi, Viviana; Musolino, Caterina



Synthesis of Phosphorus Based Haptens for Monoclonal Catalytic Antibody Production. Phase 2.  

National Technical Information Service (NTIS)

Our objective in this Phase II project is to synthesize haptens for production of monoclonal antibodies which will catalyse the hydrolysis of phosphorus-based nerve agents. Our syntheses followed three strategies. The first is synthesis of stable pentacoo...

K. Liu S. T. Tuladhar



Production and characterization of monoclonal antibodies specific to sweet clover necrotic mosaic virus.  


Monoclonal antibodies were produced in vitro by fusing mouse myeloma cells P3-X63-AgU1 with spleen cells derived from BALB/c mice immunized with purified sweet clover necrotic mosaic virus (SCNMV). Twenty-one out of 47 clones which secreted monoclonal antibodies of high titres against SCNMV were injected intraperitoneally into mice previously primed with Pristane. The ascites fluid harvested 10 to 14 days later showed a strong and specific anti-SCNMV activity in reverse passive haemagglutination inhibition, passive haemagglutination and indirect enzyme linked immunosorbent assay. The monoclonal antibodies of the 21 clones did not react with red clover necrotic mosaic virus (Swedish isolate). The class and subclass of immunoglobulins of the monoclonal antibodies secreted by established cultures were determined to be IgG2a for 15 clones and IgG3 for 6 clones. PMID:6470091

Hiruki, C; Figueiredo, G; Inoue, M; Furuya, Y



Monoclonal Antibodies Passively Protect BALB/c Mice Against Burkholderia mallei Aerosol Challenge.  

National Technical Information Service (NTIS)

Glanders is a debilitating disease with no vaccine available. Murine monoclonal antibodies were produced against Burkholderia mallei, the etiologic agent of glanders, and were shown to be effective in passively protecting mice against a lethal aerosol cha...

S. R. Trevino A. R. Permenter M. J. England N. Parthasarathy P. H. Gibbs



SPECT assay of radiolabeled monoclonal antibodies. Comprehensive progress report, September 1989--February 1992.  

National Technical Information Service (NTIS)

The long-term goal of this research project is to develop methods to improve the utility of single photon emission computed tomography (SPECT) to quantify the biodistribution of monoclonal antibodies (MoAbs) labeled with clinically relevant radionuclides ...

R. J. Jaszczak



Murine Monoclonal Antibody Defines a Unique Epitope Shared by Klebsiella Lipopolysaccharides. (Reannouncement with New Availability Information).  

National Technical Information Service (NTIS)

A hybridoma secreting a monoclonal antibody (MAb) directed against Klebsiella lipopolysaccharide (LPS) was derived from spleen cells of mice immunized with a smooth, nonencapsulated Klebsiella strain (Friedlaender 201; serogroup O1). The MAb, called V/9-5...

M. Trautmann K. Vogt C. Hammack A. S. Cross



Novel Neutrophil Chemotactic Factor, Cloned cDNA and Monoclonal Antibodies Thereto.  

National Technical Information Service (NTIS)

The invention relates to an isolated, synthetic preparation of a novel neutrophil-specific chemotactic factor (NCF), monoclonal antibodies having specific binding affinity for NCF and a clone containing the complete coding sequence for NCF.

K. Matsushima T. Yoshimur E. J. Leonard J. Oppenheimer E. Appella



[Recombinant proteins or monoclonal antibodies: comparative properties and interest in systemic lupus erythematosus].  


The emergence of biologic therapies, such as monoclonal antibodies or recombinant fusion proteins, have revolutionized the management of autoimmune disorders, in particular rheumatoid arthritis. These biologic agents have been engineered to deplete key cellular populations or to block cytokines or molecules involved in the activation and/or the differentiation of immune cells, such as T cells or B cells. In systemic lupus erythematosus (SLE), a monoclonal antibody directed against the B-cell activating factor of the TNF family (BAFF or BLyS), belimumab, has demonstrated its efficacy in large, randomized and placebo-controlled studies, whereas rituximab, a monoclonal antibody directed against the CD20 expressed by B cells, failed to achieve his primary endpoint in renal and non-renal SLE. Studies on the safety and the efficacy of monoclonal antibodies or recombinant fusion proteins directed against other key molecules involved in the pathogenesis of SLE are ongoing. PMID:23351696

Terrier, Benjamin; Mouthon, Luc



Radioimmundarstellung experimenteller Gliome mittels radioaktiv markierter monoklonaler Antikoerper. (Radioimmunoimaging of experimental gliomas using radiolabelled monoclonal antibodies).  

National Technical Information Service (NTIS)

The biodistribution and tumour uptake of radiolabelled (131 I) glioma-seeking monoclonal antibodies (14 AC1) and their F(ab')(sub 2) fragments were investigated in nude mice having received glioma transplants. Radioimmunoimaging by external scintigraphy a...

H. Glaessner



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


Alveolar echinococcosis is a rare but potentially fatal disease. Immunodiagnosis based on antibodies or antigens plays an important role in its diagnosis. In this study, metacestode somatic antigens of Echinococcus multilocularis were used to immunize BALB/c mice, and hybridomas were formed by cell fusion. Making use of the inherent effect of monoclonal antibody techniques to isolate different epitopes, we obtained a repertoire of 32 monoclonal antibodies against the metacestode somatic antigens. These monoclonal antibodies were used to investigate the specificity and localization of the metacestode antigens by enzyme-linked immunosorbent assay and immunohistochemistry, respectively. Nine antibodies specifically reacted with E. multilocularis, while 14 and ten cross-reacted with Echinococcus granulosus and Taenia saginata, respectively. Twenty-five antibodies stained the laminated layer. Eight reacted with the tegument of the protoscolex. Fourteen antibodies recognized the germinal layer. Most of the monoclonal antibodies can react with the antigen Em2. One antibody can react with antigen Em2 and Em10. One antibody that cross-reacted with T. saginata stained the germinal layer and protoscolex, especially its hooklets and suckers, but could not react with Em2 and Em10 antigens. It detected protein bands at 26 and 52 kDa. Two E. multilocularis-specific monoclonal antibodies stained both the germinal and laminated layers and could be used not only to purify specific antigens but also for immunohistochemical studies of E. multilocularis. In summary, these 32 monoclonal antibodies could have potential applications as useful tools in further studies of E. multilocularis antigen profiles. PMID:20397027

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



Monoclonal Antibodies in Allogeneic Hematopoietic Stem Cell Transplantation for Hematologic Malignancies  

Microsoft Academic Search

\\u000a Monoclonal antibodies have revolutionized the treatment of hematologic malignancies. The monoclonal antibody rituximab plays\\u000a an integral part in the upfront treatment of both aggressive and indolent CD20-expressing non-Hodgkin’s lymphoma (NHL). Although\\u000a allogeneic hematopoietic stem cell transplantation (HSCT) can be a curative therapy for many hematologic malignancies, much\\u000a improvement remains to be made to reduce rates of regimen related-transplant toxicity, disease

Maria Corinna Palanca-Wessels


A monoclonal antibody detecting an Ia specificity mapping in the IA or IE subregion  

Microsoft Academic Search

An H-2s anti-H-2d monoclonal la antibody (CE197) is described which appears to detect a new Ia specificity that maps either in the I-A or the I-E subregion, depending on the haplotype examined. Binding inhibition studies with radiolabeled, monoclonal I-A-and I-E-specific antibodies imply that CE197 reacts with a public determinant shared by a variety of Aa-Aß and Ea-Eß dimers.

Frank W. Symington; Jonathan Sprent



A monoclonal antibody against GBM heparan sulfate induces an acute selective proteinuria in rats  

Microsoft Academic Search

A monoclonal antibody against GBM heparan sulfate induces an acute selective proteinuria in rats. After immunization of mice with partially-purified heparan sulfate proteoglycan (HSPG) isolated from rat glomeruli, a monoclonal antibody (mAb JM-403) was obtained, which was directed against heparan sulfate (HS), the glycosaminoglycan side chain of HSPG. In ELISA it reacted with isolated human glomerular basement membrane (GBM) HSPG,

Jacob van den Born; Lambert P W J van den Heuvel; Marinka A H Bakker; Jacques H Veerkamp; Karel J M Assmann; Jo H M Berden



Production and characterization of monoclonal antibodies to Actinobacillus pleuropneumoniae serotype 5b.  

PubMed Central

Monoclonal antibodies specific for capsular polysaccharide (CPS) and lipopolysaccharide (LPS) of A. pleuropneumoniae serotype 5b were generated by hybridoma cells and selected by indirect ELISA of culture supernatants with purified and structurally defined LPS and CPS preparations and their synthetic conjugates. It was shown in this study that at least one monoclonal antibody, 3B4, presented 100% specificity and recognized all A. pleuropneumoniae serotype 5 field strains tested in a dot-ELISA assay. Images Fig. 1. Fig. 2.

Altman, E; Gidney, M A; Harrison, B A; Gottschalk, M



Determination of Ochratoxin a in coffee beans and coffee products by monoclonal antibody affinity chromatography  

Microsoft Academic Search

A clean?up method using a monoclonal antibody affinity column was developed for the analysis of ochratoxin A (OTA) in coffee products. Monoclonal antibody specific for OTA was covalently bound to Sepharose?4B and the resultant affinity column was used for the clean?up of sample extracts. OTA was quantitatively recovered from the affinity column. The subsequent high performance liquid chromatography (HPLC) of

M. Nakajima; H. Terada; K. Hisada; H. Tsubouchi; K. Yamamoto; T. Uda; Y. Itoh; O. Kawamura; Y. Ueno



Monoclonal antibodies to a higher-plant nitrate reductase: Differential inhibition of enzyme activities  

Microsoft Academic Search

A set of monoclonal antibodies has been raised against NADH-nitrate reductase (NR; EC from spinach (Spinacea oleracea L.) leaves. Antibodies were screened by enzyme-linked immunosorbent assay and by their ability to inhibit various activities of the enzyme. The six monoclonals selected (AFRC MAC 74 to 79) are all gamma globulins; four (MAC 74 to 77) inhibit all terminal donating

B. A. Notton; R. J. Fido; G. Galfre



Monoclonal antibodies which identify carbohydrate-defined MHC Class I epitopes  

Microsoft Academic Search

Eleven different monoclonal antibodies specific for H-2K- and H-2D-encoded Class I molecules have been screened to determine Class I epitopes dependent on both carbohydrate and protein structures. Monoclonal antibodies have been identified which bind to carbohydrate-defined antigens encoded by both the H-2K and H-2D gene regions. Sensitivity to glycosidases versus pronase has been used to classify antigens both expressed as

Helen C O'Neill



Monoclonal antibody R5 for detection of putatively coeliac-toxic gliadin peptides  

Microsoft Academic Search

Monoclonal antibody R5 against rye secalin was recently suggested to be useful in analysis of gluten in food. The epitope\\u000a specificity of R5 was characterized and compared with those of eight other monoclonal antibodies (mabs) against gliadins (gli)\\u000a and secalins. Mabs were tested for binding to synthetic peptides spanning in overlapping manner sequences of gli. In a luminescence\\u000a assay R5

Franka Kahlenberg; Daniel Sanchez; Ingolf Lachmann; Ludmila Tuckova; Helena Tlaskalova; Enrique Méndez; Thomas Mothes



Intraglomerular T cells and monocytes in nephritis: Study with monoclonal antibodies  

Microsoft Academic Search

Glomerular T cells and monocytes in nephritis: study with monoclonal antibodies. Intraglomerular T cells, monocytes, total leucocytes and other mononuclear subsets were sought in renal biopsies from patients with glomerulonephritis, using monoclonal antibodies and immunoperoxidase techniques. Twenty–four biopsies with no significant glomerular proliferation on optical microscopy, thirty–two with only endocapillary hypercellularity, and twenty–one with extra capillary crescentic glomerular disease were

Fernando E B Nolasco; John Stewart Cameron; Barrie Hartley; Adolfo Coelho; Gillian Hildreth; Rowena Reuben



A human myeloma cell line suitable for the generation of human monoclonal antibodies  

Microsoft Academic Search

Ever since monoclonal antibodies were produced in 1975 with mouse myeloma cells there has been interest in developing human myeloma cultures for the production of monoclonal antibodies. However, despite multiple attempts, no human myeloma line suitable for hybridoma production has been described. Here we report the derivation of a hypoxanthine-aminopterin-thymidine-sensitive and ouabain-resistant human myeloma cell line (Karpas 707H) that contains

Abraham Karpas; Alla Dremucheva; Barbara H. Czepulkowski



Cell, tissue-, and position-specific monoclonal antibodies against the planarian Dugesia (Girardia) tigrina  

Microsoft Academic Search

To obtain specific immunological probes for studying molecular mechanisms involved in cell renewal, cell differentiation,\\u000a and pattern formation in intact and regenerating planarians, we have produced a hybridoma library specific for the asexual\\u000a race of the freshwater planarian Dugesia (Girardia) tigrina. Among the 276 monoclonal antibodies showing tissue-, cell-, cell subtype-, subcellular- and position-specific staining,\\u000a we have found monoclonal antibodies

D. Bueno; Jaume Baguñà; Rafael Romero



RadioimmunoPET: Detection of colorectal carcinoma with positron-emitting copper-64-labeled monoclonal antibody  

Microsoft Academic Search

Detection of tumor foci may be improved by combining the selective tumor-targeting properties of a monoclonal antibody with the superior sensitivity and contrast resolution of PET. An anti-colorectal carcinoma monoclonal antibody (MAb 1A3) was labeled with ⁶⁴Cu, a positron-emitting radionuclide, by use of a bifunctional chelate (bromoacetamidobenzyl-TETA) and evaluated in 36 patients with suspected advanced primary or metastatic colorectal cancer.

G. W. Philpott; S. W. Schwarz; C. J. Anderson



Monoclonal antibodies to human germ cell tumors from “routine” paraffin-embedded pathological specimens  

Microsoft Academic Search

We have established a method for monoclonal antibody (MoAb) preparation from routine paraffin-embedded tissue of human seminoma as an immunogen. Three 40-µm thich sections were deparaffinized and rehydrated. An eight-week-old BALB\\/c mouse was immunized intraperitoneally with this extract, which showed no detectable protein bands on sodium laurylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Five monoclonal antibodies (MoAbs) with different characteristics were obtained; one

A. Takenaka; S. Kitazawa; S. Maeda; S. Kamidono



Application of monoclonal anti-HY antibody for human H-Y typing  

Microsoft Academic Search

We have successfully produced monoclonal anti-H-Y antibody by fusing NS-1 myeloma cells with splenocytes from C57BL\\/6 females immunized with syngeneic male splenocytes. We have proved that the antibody is male specific and that it cross reacts with human H-Y. We have further tested 80 normal individuals for H-Y antigen and obtained significant differences between males and females. Therefore, the monoclonal

Gloria C. Koo; Nobuhiko Tada; Raju Chaganti; Ulrich Hammerling



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.



Thrombus radioimmunoscintigraphy: an approach using monoclonal antiplatelet antibody.  

PubMed Central

Thrombus detection and localization is of cardinal importance in clinical medicine. The currently available method using autologous 111In-labeled platelets is too lengthy and complex for everyday use. It requires careful separation of the platelets prior to labeling and visualization of the thrombus becomes possible only 24 hr after injection. An approach to thrombus imaging using a monoclonal antiplatelet antibody labeled with 111In or 123I is described. The antibody (7E3) prepared against human platelets inhibits the interaction between fibrinogen-coated beads and both human and dog platelets. 7E3 is an IgG1 that binds to the complexed glycoprotein IIb/IIIa. Ninety percent of a tracer dose of radiolabeled 7E3 binds to human platelets and 50% binds to dog platelets. In vitro studies showed that virtually all of the platelet-bound radioactivity becomes incorporated into clots formed by adding thrombin to whole blood. 7E3 was labeled with 111In by the cyclic anhydride diethylenetriaminepentaacetic acid method or by radioiodination with 123I. At a ratio of 1:50 (anhydride:7E3) the specific activity ranged between 10 and 40 muCi/micrograms (1 Ci = 37 GBq) without change in the antibody characteristics. In vivo studies in dogs were performed by preincubating for 1 hr the radiolabeled 7E3 with citrated blood or by directly injecting the radiolabeled 7E3 intravenously. Experimental thrombi were induced by transcatheter placement of copper coils into peripheral arteries and veins as well as in the superior vena cava and pulmonary artery. With gamma camera, visualization of venous and arterial thrombi as well as sites of intimal injury without visible thrombi, could be observed 1-1.5 hr after injection. There was no need for delayed imaging because of the fast clearance of radioactivity from the circulation nor was there need for blood pool subtraction. Two to 10-hr thrombi could be imaged but 48-hr thrombi were not detectable with this method. No change in platelet counts before and after the injection of labeled 7E3 nor increased bleeding tendency occurred. The advantages of this method are a shorter preimaging preparation time, faster visualization after injection, and no need for delayed imaging or subtraction techniques. For these reasons human investigations seem to be warranted. Images

Oster, Z H; Srivastava, S C; Som, P; Meinken, G E; Scudder, L E; Yamamoto, K; Atkins, H L; Brill, A B; Coller, B S



Monoclonal antibodies to TEM-1 plasmid-mediated beta-lactamase.  

PubMed Central

At least 28 plasmid-mediated beta-lactamases have been described in gram-negative bacteria. To assess the relationship among these enzymes, we produced and characterized 28 murine monoclonal antibodies to the TEM-1 plasmid-mediated beta-lactamase. Radial immunodiffusion identified 3 monoclonal antibodies as immunoglobulin M (IgM), 18 as subclass IgG1, 2 as IgG2a, and 5 as IgG2b. Using a newly described enzyme immunoassay, cross-reactivity of 16 of these monoclonal antibodies was tested against 24 plasmid-determined beta-lactamases. The 16 monoclonal antibodies cross-reacted with TEM-2 and TLE-1 and, to a certain extent, SHV-1. Different levels of cross-reactivity were also observed with OXA-3 (11 of 16), OXA-7 (8 of 16), OXA-1 (2 of 16), OXA-6 (2 of 16), and AER-1 (2 of 16). Six monoclonal antibodies demonstrated partial neutralization of beta-lactamase activity. This study suggests that common epitopes are shared by nine biochemically distinct plasmid-mediated beta-lactamases. On the basis of cross-reactivities with these monoclonal antibodies, we identified four epitopes on TEM-1, TEM-2, TLE-1, and SHV-1 beta-lactamases. Images

Morin, C J; Patel, P C; Levesque, R C; Letarte, R



Monoclonal antibody to transferrin receptor blocks transferrin binding and inhibits human tumor cell growth in vitro.  

PubMed Central

A murine hybridoma has been obtained that produces a monoclonal antibody against the human transferrin receptor. In contrast to previously characterized monoclonal antibodies that recognize the transferrin receptor, this antibody, designated 42/6, blocks the binding of transferrin to its receptor and inhibits the growth of the human T leukemic cell line, CCRF-CEM, in vitro. Inhibition of cell growth was dose dependent, and as little as 2.5 micrograms of purified antibody per ml had a detectable effect, even though transferrin was present in the tissue culture medium in large molar excess. Cells grown in the presence of antibody for 7 days accumulated in S phase of the cell cycle. The addition of iron to antibody-treated cultures in the form of ferric complexes or ferrous sulfate did not overcome the growth inhibitory effects of the anti-transferrin-receptor antibodies. This result suggests that either transferrin is the only means by which CCRF-CEM leukemic cells can be provided with sufficient iron in vitro or that other factors in addition to iron starvation are involved in the antibody-mediated growth inhibition. The inhibition of cell growth by 42/6 monoclonal antibody suggests that monoclonal antibodies against proliferation-associated cell surface antigens, such as the transferrin receptor, may be useful pharmacological reagents to modify cell growth in vitro. Images

Trowbridge, I S; Lopez, F



Human monoclonal antibody with dual GM2/GD2 specificity derived from an immunized melanoma patient.  

PubMed Central

GM2 ganglioside is a common cell surface constituent of human melanoma and other tumors of neuroectodermal origin, and vaccination with GM2 ganglioside results in high levels of anti-GM2 antibodies in patients with melanoma. Lymphocytes from a GM2-vaccinated patient (VS) were transformed by Epstein-Barr virus and tested for production of antibodies with reactivity for GM2-positive tumor cells. A high percentage of antibody-producing B cells was detected, but antibody reactivity was generally lost during culture expansion. Two cultures, however, remained stable for antibody productivity and one was used to develop a stable hybrid line with mouse myeloma. The monoclonal antibody (designated 3-207) derived from patient VS has dual specificity for GM2 and GD2, despite the fact that only GM2 antibody could be detected in the patient's serum. Monoclonal antibody 3-207 shows high-titered reactivity with a range of melanoma, astrocytoma, neuroblastoma, and leukemia cell lines, cells with prominent cell surface expression of GM2 and GD2. The cell surface reactivity of monoclonal antibody 3-207 was not abolished by treatment of target cells with neuraminidase, as the enzyme converted GD2 to GM2, which was still detected by monoclonal antibody 3-207. Images

Yamaguchi, H; Furukawa, K; Fortunato, S R; Livingston, P O; Lloyd, K O; Oettgen, H F; Old, L J



A Monosialoganglioside is a Monoclonal Antibody-Defined Antigen of Colon Carcinoma  

Microsoft Academic Search

The antigen of a monoclonal antibody that is specific for cells of human carcinoma of the colon is a monosialoganglioside as determined by the direct binding of antibody to thin-layer chromatograms of total lipid extracts of tissues. Binding of antibody to chromatograms is detected by autoradiography after the application of iodine-125-labeled F(ab')2 of rabbit immunoglobulin G antibodies to mouse immunoglobulins.

John L. Magnani; Manfred Brockhaus; David F. Smith; Victor Ginsburg; Magdalena Blaszczyk; Kenneth F. Mitchell; Zenon Steplewski; Hilary Koprowski



Rapid Identification of Monospecific Monoclonal Antibodies Using a Human Proteome Microarray*  

PubMed Central

To broaden the range of tools available for proteomic research, we generated a library of 16,368 unique full-length human ORFs that are expressible as N-terminal GST-His6 fusion proteins. Following expression in yeast, these proteins were then individually purified and used to construct a human proteome microarray. To demonstrate the usefulness of this reagent, we developed a streamlined strategy for the production of monospecific monoclonal antibodies that used immunization with live human cells and microarray-based analysis of antibody specificity as its central components. We showed that microarray-based analysis of antibody specificity can be performed efficiently using a two-dimensional pooling strategy. We also demonstrated that our immunization and selection strategies result in a large fraction of monospecific monoclonal antibodies that are both immunoblot and immunoprecipitation grade. Our data indicate that the pipeline provides a robust platform for the generation of monoclonal antibodies of exceptional specificity.

Jeong, Jun Seop; Jiang, Lizhi; Albino, Edisa; Marrero, Josean; Rho, Hee Sool; Hu, Jianfei; Hu, Shaohui; Vera, Carlos; Bayron-Poueymiroy, Diane; Rivera-Pacheco, Zully Ann; Ramos, Leonardo; Torres-Castro, Cecil; Qian, Jiang; Bonaventura, Joseph; Boeke, Jef D.; Yap, Wendy Y.; Pino, Ignacio; Eichinger, Daniel J.; Zhu, Heng; Blackshaw, Seth



The future role of monoclonal antibody therapy in childhood acute leukaemias.  


Leukaemia is the single most common childhood malignancy. With modern treatment regimens, survival in acute lymphoblastic leukaemia (ALL) approaches 90%. Only about 70% of children with acute myeloid leukaemia (AML) achieve long term survival. Patients who relapse have a dismal prognosis. Novel therapeutic approaches are needed to improve treatment outcomes in newly-diagnosed patients with a poor prognosis and for patients with relapsed/refractory disease that have limited treatment options. One promising approach in treating haematological malignancies has been the use of monoclonal antibodies to target cell surface antigens expressed on malignant cells. Most success with monoclonal antibody therapy in the treatment of haematological malignancies has come in the setting of adult B-cell non-Hodgkin lymphoma with the addition of the anti-CD20 monoclonal antibody rituximab to standard treatment regimens. In order to further advance treatment of haematological malignancies, novel monoclonal antibodies continue to be developed that target a variety of cell surface antigens. Several antibodies continue to be investigated in childhood leukaemias. This review will discuss the development of monoclonal antibodies that target a variety of cell surface antigens for the treatment of childhood ALL and the use of the anti-CD33 antibody gemtuzumab ozogamicin in the treatment of childhood AML. PMID:22881237

Barth, Matthew; Raetz, Elizabeth; Cairo, Mitchell S



Demonstration of two distinct antigenic determinants on hepatitis B e antigen by monoclonal antibodies  

SciTech Connect

Mice were immunized against hepatitis B e antigen (HBeAg) isolated from sera of asymptomatic carriers of hepatitis B virus. Their spleen cells were fused with mouse myeloma (NS-1) cells, and 5 clones of hybridoma cells secreting antibody against HBeAg (anti-HBe) were isolated. For the production of anti-HBe in large scale, cells were cultivated both in vitro and in the peritoneal cavity of ascitic mice. Although monoclonal antibodies produced by these clones showed a strong reactivity of anti-HBe in hemagglutination tests, individual monoclonal anti-HBe did not reveal any precipitin line in immunodiffusion. When 2 of the 5 monoclonal antibodies were mixed together, however, some combinations showed a precipitin line against HBeAg, whereas others did not. Utilizing solid-phase radioimmunoassay involving a number of combinations of monoclonal antibodies used for solid-phase and radiolabeling, the 5 antibodies were classified into 2 groups. Three of the anti-HBe antibodies were found to be directed to 1 determinant of HBeAg (determinant a); the remaining 2 to the other determinant (determinant b). Determinants a and b were detected on HBeAg in the serum, as well as on the polypeptide of 19,000 daltons (P19) derived from the nucleocapsid of hepatitis B virus. Monoclonal anti-HBe antibodies with different specificities may provide useful tools in delineating the antigenic structure of HBeAg and also in evaluating immune responses of the host directed to its subdeterminants.

Imai, M.; Nomura, M.; Gotanda, T.; Sano, T.; Tachibana, K.; Miyamoto, H.; Takahashi, K.; Toyama, S.; Miyakawa, Y.; Mayumi, M.



Accessing of recombinant human monoclonal antibodies from patient libraries by eukaryotic ribosome display  

PubMed Central

What are effective antibodies and when do they arise to prevent or delay disease onset during a natural infection or in the course of vaccination? To address these questions at a molecular level requires longitudinal studies, capturing and analyzing the antibody repertoire at regular intervals following exposure or sero-conversion. Such studies require a method that allows the rapid generation and evaluation of monoclonal antibodies from relatively small volumes of blood. Here we describe an approach for rapidly generating human monoclonal antibodies in vitro by directly screening single-chain antibody repertories derived from donor peripheral blood mononuclear cells using ribosome display. Two single-chain antibody libraries were constructed using RNA extracted from peripheral blood mononuclear cells of two HIV-1 long-term non-progressor donors (K530 and M325). Both libraries were subjected to a single round of in vitro ribosome display for enrichment of human monoclonal antibodies against recombinant gp120K530, derived from virus isolated from donor K530. This study has validated a novel, in vitro method for the rapid generation of human monoclonal antibodies. An antibody library could be constructed from as little as 3 ?g of total RNA, the equivalent of 3–5 mL of human blood.

Tang, Jie; Wang, Lin; Markiv, Anatoliy; Jeffs, Simon A.; Dreja, Hanna; McKnight, Aine; He, Mingyue; Kang, Angray S.



Enzyme-Linked Immunosorbent Assay Using Glycoprotein and Monoclonal Antibody for Detecting Antibodies to Vesicular Stomatitis Virus Serotype New Jersey  

Microsoft Academic Search

In this study, an enzyme-linked immunosorbent assay (ELISA) using glycoprotein and a monoclonal antibody (MAb) was developed for the detection of antibodies to vesicular stomatitis virus (VSV) serotype New Jersey (NJ). The glycoprotein to be used as a diagnostic antigen was extracted from partially purified VSV-NJ, and a neutralizing MAb specific to VSV-NJ was incorporated to compete with antibodies in

Hyang-Sim Lee; Eun-Jeong Heo; Hye-Young Jeoung; Hyo-Rim Ko; Chang-Hee Kweon; Hee-Jeong Youn; Young-Joon Ko



Antibody-dependent cell-mediated cytotoxicity using a murine monoclonal antibody against human colorectal cancer in cancer patients  

Microsoft Academic Search

Antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by a murine monoclonal antibody against human colerectal carcinoma, antibody 19–9, with human effector cells was tested in 33 patients with various carcinomas, 16 patients with benign lesions, and 13 normal controls, using a 12-h 51Cr release assay using human colorectal cancer cells as targets. Peripheral blood mononuclear cells (PBM) from these groups of patients

Kiyoshi Takamuku; Tsuyoshi Akiyoshi; Hideo Tsuji



A rat model for imaging the effect of anti mouse antibody responses on the biodistribution of radiolabelled mouse monoclonal antibodies  

Microsoft Academic Search

Rats were injected both intradermally and intravenously with an IgG2b mouse monoclonal antibody (791T\\/36) and subsequently the biodistribution of intravenously injected 111In labelled antibody was examined by gamma camera imaging in these and control rats. The majority of pretreated rats showed a marked perturbation of the biodistribution of the radiolabelled antibody with a marked increase of tracer in the liver.

M. V. Pimm; A. C. Perkins; L. G. Durrant; R. W. Baldwin



Production and characterization of monoclonal antibodies against the lethal factor component of Bacillus anthracis lethal toxin.  

PubMed Central

The lethal toxin of Bacillus anthracis consists of two components, protective antigen and lethal factor. Protective antigen is cleaved after binding to cell receptors, yielding a receptor-bound fragment that binds lethal factor. Sixty-one monoclonal antibodies to the lethal factor protein have been characterized for specificity, antibody subtype, and ability to neutralize lethal toxin. Three monoclonal antibodies (10G3, 2E7, and 3F6) neutralized lethal toxin in Fisher 344 rats. However, in a macrophage cytolysis assay, monoclonal antibodies 10G3, 2E7, 10G4, 10D4, 13D10, and 1D8, but not 3F6, were found to neutralize lethal toxin. Binding studies showed that five of the monoclonal antibodies that neutralized lethal toxin in the macrophage assay (10G3, 2E7, 10G4, 10D4, and 13D10) did so by inhibiting the binding of lethal factor to the protective antigen fragment bound to cells. Monoclonal antibody 1D8, which was also able to neutralize lethal toxin activity after lethal factor was prebound to cell-bound protective antigen, only partially inhibited binding of lethal factor to protective antigen. Monoclonal antibody 3F6 did not inhibit the binding of lethal factor to protective antigen. A competitive-binding enzyme-linked immunosorbent assay showed that at least four different antigenic regions on lethal factor were recognized by these seven neutralizing hybridomas. The anomalous behavior of 3F6 suggests that it may induce a conformational change in lethal factor. Differences in neutralizing activity of monoclonal antibodies were related to their relative affinity and epitope specificity and the type of assay.

Little, S F; Leppla, S H; Friedlander, A M



Monoclonal antibodies: longitudinal prescribing information analysis of hypersensitivity reactions.  


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

Kleyman, Konstantin; Weintraub, Debra S



Biotherapies in inflammatory ocular disorders: Interferons, immunoglobulins, monoclonal antibodies.  


Biotherapies used in clinical practice for the treatment of ophthalmologic manifestations of systemic diseases include interferons (IFN), intravenous immunoglobulins (IVIG) and monoclonal antibodies (anti-TNF, anakinra, tocilizumab and rituximab). Several open prospective studies have shown the effectiveness of IFN-? (78 to 98% complete remission) for the treatment of severe uveitis in Behcet's disease. IFN is capable of inducing prolonged remission and continued after his arrest, in 20-40% of patients. Side effects (flu-like, psychological effects) limit its use in practice. Anti-TNF? (infliximab and adalimumab) represents an attractive alternative therapeutic in severe uveitis refractory to immunosuppressants, especially in Behcet's disease. They are almost always (>90% of cases) and rapidly effective but their action is often suspensive. Anti-TNF? requires an extended prescription or takes over from another immunosuppressant once ocular inflammation has been controlled. IVIG are used for the treatment of Kawasaki disease and Birdshot disease. Several open or retrospective studies showed their effectiveness for the treatment of severe and refractory cicatricial pemphigoid. Tolerance of IVIG is good but their efficacy is transient. Rituximab showed an efficacy in few observations of various inflammatory eye diseases (uveitis, scleritis and idiopathic inflammatory pseudo-tumors or associated with granulomatosis with polyangiitis) and cicatricial pemphigoid. The risk of infection associated with this biotherapy limits its use in refractory diseases to conventional therapy. Anakinra (a soluble antagonist of IL-1R) showed interesting results in terms of efficiency in one small open study in Behcet's disease. Its safety profile is good and with a quick action that could be interesting for the treatment of severe uveitis. PMID:23470459

Saadoun, D; Bodaghi, B; Bienvenu, B; Wechsler, B; Sene, D; Trad, S; Abad, S; Cacoub, P; Kodjikian, L; Sève, P



Murine anti-cytomegalovirus monoclonal antibodies with autoreactivity.  

PubMed Central

Certain murine monoclonal antibodies (mAb) raised against structural proteins of mouse cytomegalovirus (MCMV) display distinct patterns of multiple organ-autoreactivity in addition to their viral specificities. We analysed the autoreactivity of five such mAb by immunoperoxidase histochemistry, western immunoblot and enzyme-linked immunosorbent assay (ELISA). Four mAb recognized cellular autoantigens in the salivary gland, lung, heart, liver, kidney, ileum, striated muscle and brain, as detected by immunoperoxidase histochemistry. However, the mAb showed different specificities for nuclear, cytoplasmic and surface membrane antigens on various cell types in addition to common autoreactivities. Immunoblot analyses showed that some of the mAb recognized polypeptides of various molecular weights obtained from 100,000 g supernatants of normal BALB/c liver, brain, striated and cardiac muscle homogenates. Reactivity of the mAb with a 200,000 molecular weight (MW) polypeptide was similar to our previous finding of the reaction of late immune polyclonal sera with a 200,000 MW polypeptide, the heavy chain of myosin. The mAb reacted to the cardiac isoform of myosin as determined by ELISA and immunoblot. Reactivity of mAb with cardiac myosin, as detected by immunoblot, was removed by absorption with cardiac myosin and recovered in the eluate. However, cardiac myosin used in a competitive inhibition ELISA did not abrogate the reactivity of the mAb with MCMV antigens. These anti-MCMV mAb appear to be multispecific for both virus and self-antigens, including cardiac myosin, and possibly recognize these different antigens through partly similar or distinct antigen-binding sites. Images Figure 1 Figure 2 Figure 3 Figure 4

Lawson, C M; O'Donoghue, H L; Farrell, H E; Shellam, G R; Reed, W D



Monoclonal antibodies: Longitudinal prescribing information analysis of hypersensitivity reactions  

PubMed Central

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

Kleyman, Konstantin; Weintraub, Debra S.



[Monoclonal antibodies in diabetes: almost approaching the dream?].  


Type 1 diabetes is characterized by the autoimmune-mediated destruction of the insulin-producing beta cells of the pancreatic islets of Langerhans. Several cells are potentially implicated in the selective destruction of beta cells, including the beta cells themselves, and T-lymphocytes and B-lymphocytes that are working as antigen-presenting cells. Both types of lymphocytes play also a role in the progressive loss of graft function after islet transplantation. Therefore, immunotherapy may represent a great opportunity to prevent, treat or even cure type 1 diabetes, and the input of monoclonal antibodies (mAb) appears crucial in such a strategy. The concept has first been validated in various animal models, especially the classical one of the NOD mouse. During recent years, promising results of a few clinical trials have been published with the administration of anti-CD3 mAbs targeting T lymphocytes at the time of diagnosis of type 1 diabetes. Results showed a more sustained residual insulin secretion during the following months associated with a reduction in insulin needs. Interesting results may also be expected from the use of anti-CD20 mAbs targeting B lymphocytes. Finally, when considering immunosuppressive therapies after beta-cell transplantation, mAbs, especially those blocking interleukin-2, are already used in clinical practice, but new trials are expected with mAbs targeting T or B lymphocytes. Thus, mAbs might be efficacious in a near future in the prevention (when administered early in the natural course of the disease, in high risk patients) and the treatment of type 1 diabetes, and therefore could avoid, or at least minimize, the constraints of intensive subcutaneous insulin therapy. PMID:19642469

Philips, J C; Keymeulen, B; Mathieu, C; Scheen, A J


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))



Production and characterization of an Mls-1-specific monoclonal antibody  

PubMed Central

Superantigens (SAGs) represent a new class of antigens, characterized as T cell receptor (TCR) V beta-reactive elements. Bacterial toxins constitute the major group of exogenous SAGs, while the mouse mammary tumor virus (MMTV)-encoded Mls molecules represent the endogenous SAGs. Mls-1 is the prototype of the latter SAGs, because it elicits a very potent T cell stimulatory response in vitro in unprimed T cells expressing the TCR V beta 6 or 8.1 chains. In vivo, Mls-1 causes deletion of immature T cells bearing the V beta 6, 7, 8.1, or 9 chains. Although Mls-1 was functionally discovered > 20 yr ago, it has not been possible to raise antibodies against this molecule. We have previously cloned and sequenced the Mtv-7 sag gene, which encodes Mls-1. Sequence comparisons with other MMTV sag genes suggested that the polymorphic 3' end encodes the TCR V beta specificity of these SAGs. We have, therefore, immunized hamsters with a 14-amino acid peptide from the deduced COOH-terminal sequence of the Mtv-7 sag gene. We describe here the production of a monoclonal antibody (mAb), 3B12, which is peptide specific and reacts with a recombinant baculovirus product of Mtv-7 sag. This mAb blocks Mls-1-specific T cell recognition and detects the Mls-1 protein on the surface of the B cell hybridoma LBB.A, but not on LBB.11, which is an Mtv-7 loss variant of LBB.A. Transfection of the Mtv-7 sag gene into LBB.11 renders this cell functionally Mls-1+ as well as positive for 3B12 binding, confirming the specificity of this mAb. It is well documented that B cells and CD8+ T cells express T cell stimulatory Mls-1 determinants, and we show here that this functional profile correlates with the expression of MMTV-specific mRNA. However, primary lymphocytes derived from Mls-1+ mice do not stain with 3B12, even after in vitro activation with mitogens or phorbol ester.



Production and characterization of an Mls-1-specific monoclonal antibody.  


Superantigens (SAGs) represent a new class of antigens, characterized as T cell receptor (TCR) V beta-reactive elements. Bacterial toxins constitute the major group of exogenous SAGs, while the mouse mammary tumor virus (MMTV)-encoded Mls molecules represent the endogenous SAGs. Mls-1 is the prototype of the latter SAGs, because it elicits a very potent T cell stimulatory response in vitro in unprimed T cells expressing the TCR V beta 6 or 8.1 chains. In vivo, Mls-1 causes deletion of immature T cells bearing the V beta 6, 7, 8.1, or 9 chains. Although Mls-1 was functionally discovered > 20 yr ago, it has not been possible to raise antibodies against this molecule. We have previously cloned and sequenced the Mtv-7 sag gene, which encodes Mls-1. Sequence comparisons with other MMTV sag genes suggested that the polymorphic 3' end encodes the TCR V beta specificity of these SAGs. We have, therefore, immunized hamsters with a 14-amino acid peptide from the deduced COOH-terminal sequence of the Mtv-7 sag gene. We describe here the production of a monoclonal antibody (mAb), 3B12, which is peptide specific and reacts with a recombinant baculovirus product of Mtv-7 sag. This mAb blocks Mls-1-specific T cell recognition and detects the Mls-1 protein on the surface of the B cell hybridoma LBB.A, but not on LBB.11, which is an Mtv-7 loss variant of LBB.A. Transfection of the Mtv-7 sag gene into LBB.11 renders this cell functionally Mls-1+ as well as positive for 3B12 binding, confirming the specificity of this mAb. It is well documented that B cells and CD8+ T cells express T cell stimulatory Mls-1 determinants, and we show here that this functional profile correlates with the expression of MMTV-specific mRNA. However, primary lymphocytes derived from Mls-1+ mice do not stain with 3B12, even after in vitro activation with mitogens or phorbol ester. PMID:8381154

Mohan, N; Mottershead, D; Subramanyam, M; Beutner, U; Huber, B T



Monoclonal antibodies against avian paramyxovirus-3: antigenic mapping and functional analysis of the haemagglutinin-neuraminidase protein and the characterization of nonspecific monoclonal antibodies  

Microsoft Academic Search

Summary Sixteen monoclonal antibodies (Mabs) which immunoprecipitated the haemagglutinin neuraminidase (HN) of chorio-allantoic membrane-grown avian paramyxovirus-3 (PMV-3) of British turkeys were produced. Thirteen were PMV-3 specific. Three were nonspecific because they also bound to other viral proteins and to bovine kidney cells treated with neuraminidase enzyme or infected with influenza virus.

C. L. Anderson; P. H. Russell



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



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.



Monoclonal antibody 2G13, a new axonal growth cone marker  

Microsoft Academic Search

Growth cones are specialized sensorimotor structures at the tips of neurites implicated in pathfinding decisions and axonal outgrowth during neuronal development. We generated a mouse monoclonal antibody (mAb 2G13) against chick tectum and found that the antibody exclusively labelled axonal growth cones, particularly their filopodia and lamellipodia, in developing rat CNS and in embryonic neurons in culture. The high fidelity

Olivier Stettler; Maxwell S. Bush; Melissa Kasper; Burkhard Schlosshauer



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



Production and purification of monoclonal antibodies to Pisum and Avena phytochrome  

Microsoft Academic Search

At least nine monoclonal antibodies against phytochrome from Pisum sativum L. and 20 against phytochrome from Avena sativa L. have been obtained from mouse hybridomas that were produced by fusion of spleen cells with SP 2\\/O-Ag14 myeloma cells. Hybridomas were selected and cloned in a single step by plating on a semisolid methylcellulose medium. Eight antibodies to Pisum and one

Marie-Michèle Cordonnier; Cecile Smith; Hubert Greppin; Lee H. Pratt



Use of a Resin Bound Monoclonal Antibody in the Purification of Various Components in Anthrax Vaccines.  

National Technical Information Service (NTIS)

Lethal factor from B. anthracis (Vollum 1B strain) has been purified 1130 fold by immunosorbent chromatography using a mouse anti-lethal factor monoclonal antibody Sepharose-4B column. The antibody and was eluted with buffer containing 4 M NaSCN with 77% ...

J. W. Burnett G. J. Calton



Use of monoclonal antibody against human neutrophil elastase in normal and leukaemic myeloid cells  

Microsoft Academic Search

A monoclonal antibody, NP57, was produced and used against the neutrophil granule protein elastase, which selectively stain neutrophils in cryostat and paraffin wax sections. The antibody stains neutrophils and a subpopulation of monocytes in blood smears and neutrophil precursors in bone marrow smears, and gives positive reactions with the cell lines HL60 and U-937. It labelled the blast cells in

K A Pulford; W N Erber; J A Crick; I Olsson; K J Micklem; K C Gatter; D Y Mason



Monoclonal Antibody ERY-1 Identifies an Antigen in Erythroid Cells, Hepatocellular and Renal Cell Carcinomas  

Microsoft Academic Search

We have identified a monoclonal antibody (ERY-1), which reacts with erythrocytes, erythroid precursor cells, and with embryonal yolk sac, and normal liver and kidney. The antibody also decorates the neoplastic cells of hepatocellular, renal, and yolk sac carcinomas. No reactivity was seen in a variety of other epithelial or mesenchymal neoplasms. It is possible that ERY-1 recognizes an erythropoiesis-associated antigen

Mehrdad Nadji; Shinobu Matsuo; Parvin Ganjei; Jocelyn Ziegels-Weissman; Neal S. Penneys



Colloidal gold conjugated monoclonal antibodies, studied in the BIAcore biosensor and in the Nycocard immunoassay format.  


Interactions between immobilized capture monoclonal antibodies (mAbs), analyte molecules and colloidal gold conjugated second monoclonal antibodies have been investigated in the BIAcore biosensor and in the Nycocard immunoassay format. This report focuses on six monoclonal antibodies against human heart myoglobin, although, results with other antigens are also discussed. The BIAcore was used to screen monoclonal antibodies as antigen capture reagents, and for their function as colloidal gold conjugated second antibodies in the Nycocard. Some antibodies with low affinity caused by a rapid antigen dissociation rate, showed high affinity kinetics when used unlabelled or as gold conjugated detector reagents. One gold conjugated mAb with excellent properties in the Nycocard, showed double binding to one epitope, when tested in the BIAcore. The real time visualization of association and dissociation rates was a unique tool in the elucidation of antigen-antibody interactions. Our study confirmed that good antibody candidates selected with the BIAcore must always be tested in their actual conjugation situation before final optimization. PMID:7602134

Johne, B; Hansen, K; Mørk, E; Holtlund, J



Monoclonal antibodies as molecular probes for cell wall antigens of the brown alga, Fucus  

Microsoft Academic Search

Monoclonal antibodies to cell wall carbohydrates were produced against carbohydrates extracted from the brown alga, Fucus distichus ssp. edentatus (de la Pyl.) Powell. Mouse spleen cells were immunized in vitro with alginate and fucans, and hybridoma cultures were screened by enzyme immunoassay. Most antibodies were immunoglobulin (Ig)M and one was IgA. Antigens were localized on methacrylate sections of Fucus tissues

V. Vreeland; M. Slomich; W. M. Laetsch



Human specific anti-type IV collagen monoclonal antibodies, characterization and immunohistochemical application  

Microsoft Academic Search

This paper describes two new monoclonal antibodies reactive with human specific type IV collagen epitopes in frozen as well as routinely fixed and processed tissue sections. The antibodies (1042 and 1043) were raised against human placental type IV collagen and were shown by immunoblotting and ELISA tests to react exclusively with type IV collagen determinants. Extensive immunohistochemical survey studies on

M. G. Havenith; J. P. M. Cleutjens; C. Beek; E. v. d. Linden; A. F. P. M. Goeij; F. T. Bosman



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.



Relationships between Monoclonal Antibody-binding Sites on the Measles Virus Haemagglutinin  

Microsoft Academic Search

SUMMARY Twenty-one monoclonal antibodies directed against the measles virus haemagglu- tinin have recently been obtained. These were known to fall into five groups, each defined by its effects on the biological functions of the H protein. A representative of each group was selected and examined by competitive radioimmunoassay in an at- tempt to deduce the relationships between antibody-binding sites on




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



A monoclonal antibody with binding and inhibiting activity towards human pancreatic carcinoma cells  

Microsoft Academic Search

The murine monoclonal antibody (MAb) BW 494 was characterized in relation to its tissue specificity, the epitope recognized, in vitro and in vivo radiolocalization and its potential to mediate antibody dependent cellular cytotoxicity (ADCC) and complement mediated cytolysis (CMC). The MAb defined carbohydrate epitope located on a >200 k daltons glycoprotein was mainly expressed on the majority of well differentiated

K. Bosslet; H. F. Kern; E. J. Kanzy; A. Steinstraesser; A. Schwarz; G. Liiben; H. U. Schorlemmer; H. H. Sedlacek



Monoclonal Antibody Mapping of the Envelope Glycoprotein of the Dengue 2 Virus, Jamaica  

Microsoft Academic Search

Although dengue (DEN) virus is the etiologic agent of dengue fever, the most prevalent vector-borne viral disease in the world, precise information on the antigenic structure of the dengue virion is limited. We have prepared a set of murine monoclonal antibodies (MAbs) specific for the envelope (E) glycoprotein of DEN 2 virus and used these antibodies in a comprehensive biological

John T. Roehrig; Richard A. Bolin; Robert G. Kelly



A sensitive monoclonal antibody-based enzyme-linked immunosorbent assay for chlorpyrifos residue determination  

Technology Transfer Automated Retrieval System (TEKTRAN)

A monoclonal antibody-based competitive antibody-coated enzyme-linked immunosorbent assay (ELISA) was developed and optimized for determining chlorpyrifos residue in agricultural products. The IC50 and IC10 of this ELISA were 3.3 ng/mL and 0.1 ng/mL respectively. The average recoveries recovery rate...


Selective macrophage activation by muramyldipeptide bound to monoclonal antibodies specific for mouse tumor cells  

Microsoft Academic Search

IgM monoclonal antibodies directed against tumor cells which do not mediate antibody-dependent macrophage cytotoxicity (ADMC) even when they are cytotoxic in the presence of complement, have been shown to render macrophages tumoricidal when they carry an immunomodulating agent, i.e., muramyldipeptide (MDP).

Annie-Claude Roche; Pascal Bailly; Patrick Midoux; Michel Monsigny



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.

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



Secretory leukocyte protease inhibitor (SLPI) might contaminate murine monoclonal antibodies after purification on protein G.  


The large scale production of a monoclonal anti-progesterone antibody in serum free medium followed by affinity chromatography on protein G lead to a contamination of the antibody sample with a protein of about 14 kDa. This protein was identified by mass spectrometry as secretory leukocyte protease inhibitor (SLPI). This SLPI contamination lead to a failure of the fiber-optic based competitive fluorescence assay to detect progesterone in milk. Purification of the monoclonal antibody using protein A columns circumvented this problem. PMID:22285640

Schenk, Jörg A; Fettke, Joerg; Lenz, Christine; Albers, Katharina; Mallwitz, Frank; Gajovic-Eichelmann, Nenad; Ehrentreich-Förster, Eva; Kusch, Emely; Sellrie, Frank



Preparation and experimentation of an antidigitalin monoclonal antibody: interest in human treatment.  


A monoclonal antidigitalin antibody was obtained following the immunization of BALB/mice with digitalin coupled to BSA, and the fusion of lymphocytes from these mice with murin myeloma 8653. The supernatants were screened by a radioimmunoassay in which the supernatant was incubated with digitalin coupled to I125. This monoclonal antibody "Dig 278" has an antigen binding ratio of 95%; is an IgGl; has a titre of 1/1000 and its affinity constant, as determined by a Scatchard plot, was 1,20(9) L/M. By experimenting with the monoclonal antibodies on 15 rabbits, we have obtained the reversal of heart rhythm disorders and the survival of animals injected with a lethal dose of digitalin. All the controls died. This antibody could prove useful in treating advanced cases of digitalin intoxication and for which no satisfactory treatment is available at present. PMID:6526144

Edelman, L; Colignon, A; Shermann, J M; Girre, C; Dally, S; Reviron, J



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


Tumor spheroids of HT-29 human colon adenocarcinoma and A375 melanoma were established to investigate the uptake and clearance kinetics of TNT-1, a monoclonal antibody that targets necrotic cells of tumors. Our data reveal that there was rapid uptake of TNT-1 and its F(ab')2 fragment in both spheroid models, whereas an antibody of irrelevant specificity, Lym-1, and its F(ab')2 fragment bound poorly to the spheroids. Unlike previously reported monoclonal antibodies to tumor cell-surface antigens, TNT-1 showed 1) a linear uptake that increased over time without saturation in tumor spheroids and 2) an unexpected uptake by a subpopulation of cells in the viable outer rim of the spheroids. These preclinical studies provide important information concerning the therapeutic potential of TNT monoclonal antibodies for the treatment of cancer and micrometastases. PMID:1988705

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



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.



An ELISA-inhibition test using monoclonal antibody for the serology of leprosy.  

PubMed Central

In this study a mouse monoclonal antibody (47-9) is described, which recognized an epitope on the 36 kD protein antigen of M. leprae. The monoclonal antibody showed specificity for M. leprae. An ELISA-inhibition test based on the competitive inhibition by antibodies from human test sera of the binding of the enzyme labelled monoclonal antibody to M. leprae was developed. Seropositivity was found in 100% of the multibacillary leprosy patients group and in 91% of the paucibacillary patients. Only 5% of the 223 control sera were positive. Because of the high seropositivity found in both multi- and paucibacillary patients, it is suggested that the epitope on the 36 kD antigen is immuno-dominant. Therefore the ELISA-inhibition test described herein might well be a suitable tool for diagnosis of leprosy. Images Fig. 1

Klatser, P R; De Wit, M Y; Kolk, A H



Monoclonal antibodies against LDL progressively oxidized by myeloperoxidase react with ApoB-100 protein moiety and human atherosclerotic lesions  

Microsoft Academic Search

Oxidized-LDL are involved in atherosclerosis pathogenesis, while the production of anti-ox-LDL monoclonal antibodies is critical for the development of diagnostic tools. This work reports the production of four monoclonal antibodies raised against human LDL, oxidized at different levels by the myeloperoxidase system. Characterization of these monoclonal antibodies showed that they do not cross-react with neither native LDL, VLDL nor hydrogen

N. Moguilevsky; K. Zouaoui Boudjeltia; S. Babar; P. Delrée; I. Legssyer; Y. Carpentier; M. Vanhaeverbeek; J. Ducobu