Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A.
The Cowan I strain of the bacterium Staphylococcus aureus has been used as an adsorbent for antibodies complexed with radiolabeled antigens from cell lysates. This application is advanced as a superior alternative to other methods of immune precipitation for the isolation of antigens. It exploits the high adsorption capacity for IgG molecules by protein A molecules on the cell walls of certain strains of staphylococci, along with the advantageous sedimentation properties of the bacteria. The interaction of immune complexes with the adsorbent was defined initially using a model system of bovine serum albumin with a high excess of rabbit anti-bovine serum albumin antibodies (IgG). The uptake of immune complexes under these conditions was extremely rapid, occurring within seconds, whereas maximum binding of free IgG was much slower. In addition, once bound the complexed antigen could not be displaced from the adsorbent either by large amounts of normal IgG or by extra free antibody. Antigen could be eluted almost completely from the inert adsorbent for analytic or preparative purposes with a variety of solvent systems, such as the detergent SDS in combination with urea and high temperature, and neutral salts with strong lyotropic salting in properties. The efficacy of the protein A-antibody adsorption technique was tested in direct comparisons with a conventional double antibody precipitation method for the isolation of mouse lymphocyte IgM. The bacterial adsorbent not only had a distinct advantage in speed of antigen isolation, but analyses by polyacrylamide gel electrophoresis in SDS also revealed consistently higher antigen recoveries, lower levels of background radioactivity, and an absence of other cell components which may nonspecifically bind to and complicate analyses using conventional immune precipitates.
D000327:Adsorption; D000818:Animals; D000907:Antibodies, Bacterial; D000936:Antigen-Antibody Complex; D000941:Antigens / Q000302:isolation & purification*; D001666:Binding Sites, Antibody; D007074:Immunoglobulin G / Q000032:analysis; D007158:Immunologic Techniques; D008214:Lymphocytes / Q000276:immunology; D051379:Mice; D008808:Mice, Inbred CBA; D012710:Serum Albumin, Bovine / Q000276:immunology; D013211:Staphylococcus aureus / Q000276:immunology*
D000907:Antibodies, Bacterial; D000936:Antigen-Antibody Complex; D000941:Antigens; D007074:Immunoglobulin G; D012710:Serum Albumin, Bovine