An enzyme-linked immunosorbent assay (ELISA) for detergent solubilized Ia glycoproteins using nitrocellulose membrane discs.
An enzyme-linked immunosorbent assay has been developed for the quantitative detection of detergent solubilized murine Ia. Nitrocellulose membrane discs were used to bind membrane glycoproteins applied in solutions containing detergent. The bound antigen was detected by monoclonal antibodies and horseradish-peroxidase-coupled anti-IgG. The assay produced a linear response with respect to antigen concentration, and could readily detect partially purified Ia derived from 10(3) to 10(4) mitogen stimulated spleen cells. Nitrocellulose discs efficiently bound protein in the presence of deoxycholate, taurocholate, and octylglucoside. Less binding occurred in the presence of Triton X-100 or Tween 80, but 90% binding efficiency was obtained in 0.01% solutions of these detergents. The association of protein with the discs was stable under normal conditions for antigen detection, but could be further stabilized by briefly fixing with glutaraldehyde for more rigorous procedures. The ability of this method to detect antigen in detergent solutions makes it useful in monitoring fractions during the purification of cell membrane proteins.
D000818:Animals; D002417:Cattle; D002482:Cellulose / Q000494:pharmacology; D003601:Cytotoxicity Tests, Immunologic; D004797:Enzyme-Linked Immunosorbent Assay; D006023:Glycoproteins / Q000032:analysis* / Q000378:metabolism; D000949:Histocompatibility Antigens Class II / Q000032:analysis*; D007124:Immunoenzyme Techniques; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism; D051379:Mice; D011092:Polyethylene Glycols / Q000494:pharmacology; D011136:Polysorbates / Q000494:pharmacology; D012710:Serum Albumin, Bovine / Q000378:metabolism
D006023:Glycoproteins; D000949:Histocompatibility Antigens Class II; D008565:Membrane Proteins; D011136:Polysorbates; D012710:Serum Albumin, Bovine; D011092:Polyethylene Glycols; D002482:Cellulose