Biotin-avidin immobilization of platelet glycoproteins (BAIPG): a new capture assay for the detection of anti-platelet antibodies.
Several 'capture' assays are currently employed to identify specific platelet antibodies, but all require the use of murine monoclonal antibodies (MoAbs) against the antigen of interest. We have developed a new antigen capture assay for the detection of platelet reactive antibodies, based on platelet surface sialoglycoprotein labelling with biotin hydrazide, and a following immobilization of the biotinylated platelet proteins to microtiter wells that had been coated with streptavidin. The resulting solid phase can then be used in a simple ELISA to detect serum and platelet associated antibodies. We describe here two versions of this biotin-avidin immobilization of platelet glycoproteins (BAIPG) assay. In BAIPG assay type I, the test sera are directly incubated in microtiter wells previously coated with streptavidin plus biotinylated platelet proteins. The BAIPG type II procedure involves the incubation of sera with biotinylated platelets before platelet solubilization, and, after platelet lysis, the immobilization of the immune complexes to streptavidin-coated wells. In both cases, the bound antibodies are determined by alkaline phosphatase conjugated anti-human IgG. Using BAIPG type I, positive results were obtained in 7/33 patients with idiopathic thrombocytopenic purpura (ITP), 1/10 patients with secondary immune thrombocytopenia (SIT) and 4/17 with non-immune thrombocytopenia (NIT). The BAIPG type II test was positive in 13 out of 33 patients with ITP, in six out of ten patients with SIT, and in three out of the 17 patients with NIT. A comparison between BAIPG and monoclonal antibody immobilization of platelet antigens (MAIPA) assays showed a high degree of correlation between the two methods. These results suggest that the BAIPG assay is a valuable new tool for the detection of anti-platelet antibodies.
Cordiano
I
I
Institute of Medical Semeiotic, University of Padua Medical School, Italy.
Steffan
A
A
Randi
M L
ML
Pradella
P
P
Girolami
A
A
Fabris
F
F
D001323:Autoantibodies / Q000097:blood*; D001360:Avidin; D001710:Biotin; D001792:Blood Platelets / Q000276:immunology*; D002462:Cell Membrane / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D008562:Membrane Glycoproteins / Q000276:immunology*; D013921:Thrombocytopenia / Q000276:immunology*
D001323:Autoantibodies; D008562:Membrane Glycoproteins; D001360:Avidin; D001710:Biotin