Hybridoma最新文献

Since the hybridoma technique to produce monoclonal antibodies (MAbs) was discovered, thousands of MAbs with predefined protein specificity have been produced, and a natural or recombinant protein as antigen is necessary for inducing MAbs in the conventional hybridoma technique. To induce epitope-specific MAbs, we suggest an epitope vaccine as a new technique to induce MAbs with predefined epitope specificity. ELDKWA was identified as an important neutralizing epitope on HIV-1 gp41. The MAb 2F5, recognizing ELDKWA epitope, has shown broad neutralizing activity to many HIV strains, including primary isolates, but the mutant in ELNKWA epitope results in escape 2F5-based neutralization. To produce MAbs recognizing this mutated epitope for consideration of passive immunotherapy against the mutant bearing the ELNKWA epitope, MAbs with predefined ELNKWA epitope specificity were induced by synthetic epitope-peptide instead of a natural or recombinant gp41 bearing this epitope. Three MAbs were identified to recognize ELNKWA epitope on the synthetic epitope-peptide, and interestingly could bind the recombinant gp41 with ELDKWA epitope in an ELISA assay and immunoblotting analysis.

Human Herpesvirus 8 (HHV-8) is clearly associated with Kaposi's sarcoma (KS), body cavity-based lymphomas (BCBL), and certain forms of multifocal Castleman's disease (MCD). It appears to be the sexually transmissible agent involved in the development of AIDS-associated KS. HHV-8 genomes are invariably present in BCBL-derived cell lines where lytic replication of the virus can be induced by phorbol esters (PE). First-generation HHV-8 serological assays were based on these cell lines. More recently, several genes encoding HHV-8 antigens have been identified. One of the most reactive antigens is encoded by HHV-8 open reading frame K8.1. Although K8.1 does not exhibit overt sequence homology to any other known gene, it is likely to be analogous to gp220/350 of Epstein-Barr or gp150 of murine herpesvirus-68, virion-envelope glycoproteins involved in target cell recognition. Mice were immunized with purified GST-K8.1 fusion protein expressed in E. coli. After fusion of murine plasma cells with the myeloma cell line P3-X63-Ag8. monoclonal antibodies (MAbs) were generated, which are specifically directed against K8.1 protein. The binding site for each MAb was identified by deletion mutant analysis using recombinant GST-K8.1 mutants and K8.1-specific peptides. Without exception, the epitopes recognized by these MAbs were located within the N-terminal part of the protein [amino acids (aa) 29 to 80], thus identifying a highly immunogenic region. These antibodies will not only be useful tools for HHV-8 diagnostics, but will also facilitate the analysis of K8.1 function.

Spleen cells from BALB/c mice immunized with human thyroid stimulating hormone (beta-subunit) were fused with mouse myeloma cells (P3/X63-Ag8) and five hybridomas secreting monoclonal antibodies (MAbs) were obtained. These hybridomas specifically recognize (hTSH) and do not cross-react with the other human glycoprotein hormones such as: luteinizing hormone (LH), follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hcG). The MAbs were of the IgG1 subclass and ascitic fluid from these hybridomas was purified by affinity chromatography on Protein A-sepharose CL-4B column to isolate the IgG1 active fraction. The affinity constant of these MAbs ranged from 3.2 x 10(10) to 1.5 x 10(11) M(-1).

We report on the rapid generation of two monoclonal antibodies, ATM A16.35 and ATM D16.11, that bind to the kinase domain of mutated ataxia telangiectasia (ATM). These antibodies were generated against E. coli-expressed recombinant protein using the RIMMS strategy. We show that ATM A16.35 binds ATM by Western blot analysis, and ATM D16.11 forms immune complexes with native ATM in immunoprecipitations without neutralizing kinase activity.

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

Prostate-specific membrane antigen (PSMA) is a 750-amino acid glycoprotein highly expressed in malignant prostate tissues. PSMA reacts with the murine monoclonal antibody 7E11.C5, whose binding epitope has been mapped to the N-terminal of the protein distributed on the cytoplasmic side of the plasma membrane. We have developed murine monoclonal antibodies specific for extracellular epitopes of PSMA. Three of these antibodies--1G9, 3C6, and 4D4--display distinct binding properties consistent with their recognition of conformational epitopes within native PSMA. Results indicate this panel of antibodies binds to native full-length PSMA, but not to fusion proteins containing portions of the linear sequence of the protein. Antibody binding is greatly reduced upon heat denaturation of native PSMA, and these antibodies do not detect PSMA by Western blot. Immunoprecipitation experiments demonstrate the ability of each to bind to full-length PSMA as well as PSM', a form of the protein missing the first 57 amino acids. These results indicate each antibody is specific for an epitope within the extracellular domain, a region spanning residues 44-750. Flow cytometric experiments indicate strong specific binding to live LNCaP cells. Antibody inhibition studies demonstrate that these antibodies recognize at least two distinct epitopes. Taken together, the results demonstrate that these antibodies are specific for native protein conformational epitopes within the extracellular domain. Their properties, in particular strong binding to live cancer cells, make them ideal candidates that are clearly superior to linear sequence epitope specific antibodies for in vivo applications.

The binding specificities of a panel of mouse monoclonal antibodies (MAbs) to human nerve growth factor (hNGF) were determined by epitope mapping using chimeric and point mutants of NGF. Subsequently, the MAbs were used to probe NGF structure-function relationships. Six MAbs, which recognize distinct or partially overlapping regions of hNGF, were evaluated for their ability to block the binding of hNGF to the TrkA and p75 NGF receptors in various in vitro assays, which included blocking of TrkA autophosphorylation and blocking of NGF-dependent survival of dorsal root ganglion sensory neurons. Three MAbs (911,912,938) were potent blockers of all activities. Potent blocking of p75 binding occurs only with MAb 909, which recognizes an NGF region identified by mutagenesis as important for NGF-p75 binding. These results are consistent with recently proposed models of binding regions involved in NGF-TrkA and NGF-p75 interactions generated through mutagenic analysis and structure determination of the NGF-TrkA complex. These studies provide insight to the epitope specificities and potency of MAbs that would be useful for physiological NGF blocking studies.