Immunoaffinity isolation of membrane antigens with biotinylated monoclonal antibodies and immobilized streptavidin matrices.
An efficient immunoaffinity method for isolating detergent-solubilized membrane antigens with murine and rat monoclonal antibodies is described that takes advantage of the high affinity (Ka approximately 10(14) M-1) of streptavidin for biotin. Streptavidin, a Mr approximately 60,000 biotin-binding protein secreted by Streptomyces avidinii, is covalently coupled to a high Mr exclusion agarose gel (streptavigel) and used to adsorb soluble immune complexes formed with affinity-purified, biotinylated monoclonal antibodies. The streptavigel matrix has the same high affinity and capacity for each unique biotinyl-monoclonal antibody. This results in maximal removal of antigen complexes with minimal nonspecific binding of cellular proteins, which is confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography of biosynthetically labeled murine spleen cell antigens (lymphocyte H-2 and Ia antigens). The utility of using biotinylated monoclonal antibodies and streptavigel is shown in experiments where direct comparisons are performed with Protein A-Sepharose. In addition, we have also compared streptavidin and avidin immobilized on a variety of other matrix materials for their physical and antigen-binding properties.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000954:Antigens, Surface / Q000302:isolation & purification*; D001426:Bacterial Proteins; D001710:Biotin; D002462:Cell Membrane / Q000276:immunology; D006183:H-2 Antigens / Q000276:immunology; D000949:Histocompatibility Antigens Class II / Q000276:immunology; D007163:Immunosorbent Techniques; D051379:Mice; D008810:Mice, Inbred C57BL; D012995:Solubility; D019809:Streptavidin
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D001426:Bacterial Proteins; D006183:H-2 Antigens; D000949:Histocompatibility Antigens Class II; D001710:Biotin; D019809:Streptavidin