Use of protein A to remove immunoglobulins from serum in hybridoma culture media.
The levels of protein A-reactive immunoglobulin (PA-Ig) in foetal bovine serum were measured in commercial batches. For tissue culture media incorporating 10% foetal bovine serum, the levels of bovine PA-Ig were of a similar order to those of mouse monoclonal antibodies produced by hybridomas grown in such media. The equilibrium constants were calculated for the binding to protein A-Sepharose of a number of mouse monoclonal antibodies, and of PA-Ig in foetal bovine serum and normal mouse serum. The average affinity of the mouse PA-Ig was 10 times higher than that of the bovine PA-Ig, suggesting that the two could be separated by affinity chromatography on protein A-Sepharose. The mouse monoclonal antibodies, however, displayed a range of affinity 1.5-100 times that of the bovine PA-Ig, indicating that such separation could not be generally applied. The optimal technique involved removing PA-Ig from bovine serum before its inclusion in the culture medium and then purifying the monoclonal antibody on a second protein A-Sepharose column.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000302:isolation & purification*; D002417:Cattle; D002478:Cells, Cultured; D002846:Chromatography, Affinity / Q000379:methods; D003470:Culture Media; D005312:Fetal Blood / Q000276:immunology; D006825:Hybridomas / Q000276:immunology*; D007136:Immunoglobulins / Q000032:analysis* / Q000302:isolation & purification; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D013205:Staphylococcal Protein A / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; D003470:Culture Media; D007136:Immunoglobulins; D013205:Staphylococcal Protein A