Highly sensitive immunoadsorption procedure for detection of low-abundance proteins.
A procedure that virtually eliminates nonspecific adsorption of radiolabeled proteins during immunoprecipitation was devised utilizing staphylococcal cells containing protein A (Staph A). Immunoprecipitates (antigen-antibody complexes) were solubilized from Staph A pellets into detergent micelles by incubation in a small volume of 1% sodium dodecyl sulfate (SDS) at 23 degrees C for 10 min. To allow re-formation of immunocomplexes and rebinding to new Staph A, the SDS-solubilized material was diluted 20-fold in buffer containing 1% Triton X-100 and 0.5% sodium deoxycholate. Specific conductance measurements revealed that this solubilization and subsequent reimmunoadsorption of antibody-antigen complexes occur at SDS concentrations that are first above and then below its critical micelle concentration. This procedure lowered the nonspecific background from approximately 2250 parts per million (ppm) to less than 25 ppm with a final recovery of 30-50% depending on the antigen and antibody. Chaotropic agents such as 2 M urea, 0.2 M KOH, and 3.5 M MgCl2 (as well as combinations of urea and SDS) can substitute for 1% SDS, although the final recovery is somewhat lower. Fluorography of radiolabeled proteins obtained in this manner displays virtually undetectable background even for exposures as long as 2 months. These methods allowed the unambiguous detection of low-abundance antigens at a high level of sensitivity, for example, mouse mammary tumor virus protein products and epidermal growth factor receptor.
D011232:Chemical Precipitation; D007163:Immunosorbent Techniques; D008324:Mammary Tumor Virus, Mouse; D008823:Micelles; D008832:Microchemistry; D011506:Proteins / Q000032:analysis*; D012967:Sodium Dodecyl Sulfate; D012995:Solubility; D013205:Staphylococcal Protein A; D013696:Temperature; D014764:Viral Proteins / Q000032:analysis
D008823:Micelles; D011506:Proteins; D013205:Staphylococcal Protein A; D014764:Viral Proteins; D012967:Sodium Dodecyl Sulfate