Evaluation of platelet surface antigens: localization of the PlA1 alloantigen.
An approach is described for localizing antigenic determinants to specific platelet proteins. Platelets are solubilized, electrophoresed and transferred to nitrocellulose paper where they are immobilized. After incubation with radiolabelled specific antibody, the antigenic determinants are localized by radioautography. Purified proteins ranging in size from 50000 to 540000 could be studied in this manner and still retain their antigenicity. Solubilized immobilized platelet proteins were reacted with serum IgG from two patients with post-transfusion purpura known to have anti-PlA1 antibodies. The radioactivity was associated with a major protein band with an apparent molecular weight of 100000 daltons. However, when purified anti-PlA1 antibody eluted from PlA1 (+) platelets was used, at least six additional minor bands were noted with molecular weights ranging from about 86000 to 175000. No radioactive bands were noted using PlA1 negative platelets or reduced PlA1 (+) platelet proteins. Reaction of the antibody from one patient wih proteins from thrombasthenic platelets resulted in a marked reduction in the intensity of all radioactive bands. In addition, a new high molecular weight band was noted which was not present in reactions containing PlA1 (+) platelets. These data suggest that the PlA1 antigen is primarily but not entirely localized to glycoprotein IIIa. This approach is relatively simple, requires only minimal amounts of antibody and should be generally useful in the study of cellular antigens and other complex mixtures of proteins.
D000954:Antigens, Surface / Q000276:immunology*; D001792:Blood Platelets / Q000276:immunology*; D001798:Blood Proteins / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000276:immunology; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D007519:Isoantigens / Q000276:immunology*; D008565:Membrane Proteins / Q000276:immunology; D008970:Molecular Weight
D000954:Antigens, Surface; D001798:Blood Proteins; D000939:Epitopes; D007074:Immunoglobulin G; D007519:Isoantigens; D008565:Membrane Proteins