An Improved Method for Isolation of H-2 and Ia Alloantigens with Immunoprecipitation Induced by Protein A-Bearing Staphylococci

Abstract

Mouse alloantigens, including the histocompatibility (H-2) and immune response linked (Ia) antigen molecules, can be readily isolated by a new precipitation technique utilizing protein A-bearing, fixed Staphylococci. The antigens are prepared by radiolabeling mouse spleen cells with 3H-leucine, solubilizing with the non-ionic detergent Non-Idet P-40, and purifying by affinity chromatography with lentil lectin coupled to Sapharose. The antigen preparations are mixed with appropriate alloantisera, and immune complexes formed in the mixture are then bound by the protein A sites on the Staphylococci. The organisms, Staphylococcus aureus, Cowan I strain (ATCC 12598), are heat inactivated and fixed, and are a stable, uniform IgG-binding reagent, especially when stored frozen. The antigen-antibody complexes are easily eluted from the organisms for electrophoretic analysis. The precipitation mediated by the organisms is more efficient, rapid, and artifact-free than the traditional “sandwich” precipitation technique involving anti-globulin reagents.