Chemiluminescent enhanced CD47 detection on Western blotting

CD47 is a leucocyte antigen of wide tissue distribution including platelets and erythrocytes. It has been found that anti‐CD47 antibodies may cause platelet activation. Sensitive techniques are therefore needed for biochemical investigation of platelet glycoproteins including CD47. Conventional Western blotting techniques using horseradish peroxidase‐conjugated antibody and 3,3′‐diaminobenzidine (DAB) substrate resulted in unsatisfactory results with CD47 due to low sensitivity. When chemiluminescent detection with horseradish peroxidase‐conjugated antibody was used in conjunction with Western blotting, detection of the CD47 molecule was greatly enhanced. A protein of 47–55 kDa was immunoblotted from platelets by anti‐CD47 while the mAb immunoblotted a broader band (45–60 kDa) from erythrocyte membranes. Thus, there may be different forms of CD47 present on platelets and erythrocytes. The results indicate that Western blotting with the chemiluminescent detection technique is a useful and sensitive alternative for the study of human platelet antigens.