Selective production of hybridoma cells: antigenic-based pre-selection of B lymphocytes for electrofusion with myeloma cells.
Methods for pre-selecting B lymphocytes were studied and investigated. First, biotinylated antigen was used for selecting B lymphocytes. These pre-selected B lymphocytes were then combined with biotinylated myeloma cells by adding streptavidin. The final formula of the selected B cell-myeloma cell was as follows: B cell-(antigen-biotin-strept-avidin-biotin)-myeloma cell. Then, this B cell-myeloma cell conjugate was fused by the pulsed electric field (PEF) method, which fused only those conjugated cells. The fusion efficiency obtained by this method was 3-15-times higher than that obtained by the non-specific poly(ethylene glycol) (PEG) fusion method. Second, avidin-antigen conjugate was used to select B lymphocytes. For this purpose, bifunctional cross-linkers such as N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) and m-maleimidobenzoyl N-hydroxysuccinimide (MBS) were chosen. Each reagent contains two heterofunctional groups which can make covalent bond with both Lys and Cys residues. Typical avidin-antigen conjugate is expressed as avidin-SPDP (or MBS)-antigen. Thus, final B cell-myeloma cell conjugate was B cell-antigen-SPDP (or MBS)-avidin-biotin-myeloma cell. The yield of this procedure was of the order of 10(-2). Here, we suggest that the pre-selection of B lymphocytes by biotinylated antigen or avidin-antigen conjugate is a new method of obtaining selected hybridoma cells which produce specific monoclonal antibodies against the antigen used for selecting B lymphocytes.
Tomita
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Faculty of Pharmaceutical Sciences, Nagoya City University, Japan.
Tsong
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D000818:Animals; D000941:Antigens / Q000378:metabolism; D001360:Avidin; D001402:B-Lymphocytes / Q000276:immunology*; D001710:Biotin; D002459:Cell Fusion; D002469:Cell Separation / Q000379:methods*; D003432:Cross-Linking Reagents; D004560:Electricity; D006825:Hybridomas / Q000276:immunology*; D051379:Mice; D009101:Multiple Myeloma; D013388:Succinimides; D014407:Tumor Cells, Cultured
D000941:Antigens; D003432:Cross-Linking Reagents; D013388:Succinimides; D001360:Avidin; C018151:N-succinimidyl 3-(2-pyridyldithio)propionate; C013097:3-maleimidobenzoyl N-hydroxysuccinimide; D001710:Biotin