Electroblotting and immunohistochemical staining for identification of platelet antibodies

Abstract

Summary. The Western blot procedure has been adapted for use with a biotinylated antiglobulin reagent and a horseradish peroxidase complex (Vectastain ABC) to determine specific sites of antigen binding and permit discrimination between various types of platelet antibodies. Monospecific anti‐Pl^A1 antisera or sera containing mixtures of multispecific HLA and unidentified platelet specific antibodies were tested with Pl^A1 phenotyped platelets. Using monospecific anti‐Pl^A1, one intense band with relative molecular weight (M_r) of 88000 and corresponding to glycoprotein IIIa was seen with the Pl^A1+ platelets. With mixtures of antibodies, reactions were seen with platelet specific antibodies without interference from the HLA antibodies; with one serum a band of M_r approximately 135000 was identified with Bak^a+, but not Bak^a‐ platelets, indicating the presence of an anti‐Bak^a in the serum. The location of the Bak^a antigen corresponded to the area for GP IIb. With another serum, a band of M_r 88000 was revealed with Pl^A1‐and some Pl^A1+ platelets suggesting the presence of an anti‐Pl^A2. Two additional bands of M_r 160000 and 200000 were present on all preparations including autologous controls, probably due to the presence of non‐specific IgG. Thus, immunohistochemical staining is readily adaptable to the Western blot technique, and antibodies to platelet‐specific antigens can be easily differentiated from HLA antibodies.