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.