Preferred use of primary radiolabeled anti-platelet monoclonal antibodies. Comparison of immunoblotting methods for the analysis of functional domains on human platelets.
Several methodologies used for the identification and characterization of platelet receptors for antiplatelet monoclonal antibodies are compared. Two antiplatelet monoclonal antibodies, are investigated due to their potent effects on human platelet function. A platelet-activating monoclonal antibody, called M.Ab.F11, is able to induce platelet aggregation and granular secretion. A platelet-inhibitory monoclonal antibody, named G10, strongly blocks the platelet aggregation and granular secretion induced by M.Ab.F11, as well as by physiological agonists. In order to identify the specific antigens recognized by these monoclonal antibodies, we tested a number of immunostaining methods. Comparison of various procedures revealed that a high degree of nonspecific interactions with platelet proteins occurred when the commonly used secondary reagents, protein A and radiolabeled or enzyme-conjugated secondary antibodies interacted with the platelet proteins either in the presence or absence of primary monoclonal antibodies. On the other hand, we observed a high degree of specificity and selectivity when only radiolabeled anti-platelet monoclonal antibodies were used as single reagents. It is established that M.Ab.F11 interacts with the platelet membrane proteins of 32 and 35 Kd, and M.Ab.G10 recognizes 100 Kd protein, which corresponds to GPIIIa molecule.
Walkowiak
B
B
Department of Anatomy and Cell Biology, State University of New York, Brooklyn 11203.
Naik
U P
UP
Lange
M
M
Kornecki
E
E
D000911:Antibodies, Monoclonal; D001345:Autoradiography; D001792:Blood Platelets / Q000737:chemistry*; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D007457:Iodine Radioisotopes; D008722:Methods; D010980:Platelet Membrane Glycoproteins / Q000032:analysis*; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D007457:Iodine Radioisotopes; D010980:Platelet Membrane Glycoproteins