An enzyme-linked immunosorbent assay for the detection of platelet antibodies using detergent-solubilized platelets...

A method is detailed for the solubilization of human platelets using a dialyzable detergent, decanoyl-N-methylglucamide (Mega-10). At a detergent/protein ratio of 1:12, the efficiency of solubilization was 27%. This platelet lysate (PLy) was then bound to nitrocellulose (NC) discs to assay retention of the native immunological functions of the platelet membrane antigens. Using alkaline phosphatase-coupled anti-IgG, the major platelet membrane glycoproteins GPIb, GPIIb, and GPIIb/IIIa were detectable with as little as 20 ng of monoclonal antibody. Antisera to the class I histocompatibility antigens HLA-A1, B7, B8, the PlA1 allodeterminant, and serum from multiply transfused, alloimmunized patients were reactive even after 100 days storage of the discs at 4 degrees C, and with as little as 1.0 micrograms of NC-bound PLy. The binding of the same antisera to intact, immobilized platelets as well as specific complement-mediated lymphocytotoxicity was also inhibited by PLy. PLy from HLA-A3- or B44-positive donors, however, did not inhibit cytotoxicity of lymphocytes expressing either antigen using several different antisera. Our results indicate that Mega-10 is an excellent solubilizing agent for the immunological study of platelet membranes. The fact that clinically relevant platelet membrane antigens are preserved, immunologically reactive, and stable over long periods of storage, makes this assay amenable to a routine crossmatching procedure for platelet transfusions.