A fluorescence-conjugated immunobinding assay for the detection of P-selectin on platelets.
P-selectin (GMP-140 or PADGEM) is translocated to the plasma membrane of platelets after platelet activation. P-selectin, therefore, may be a potential marker for evaluating platelet activation. A fluorescence-conjugated immunobinding assay (FCIBA) has been developed to detect specifically P-selectin on platelets. Platelets were isolated from fresh blood by centrifugation and stimulated with various doses of ADP before being fixed with 1% of paraformaldehyde. Fixed platelets were incubated with fluorescence-conjugated anti-P-selectin monoclonal antibody in the wells of fluoricon microtiter plates, and the fluorescence intensity was read on a fluorescence concentration analyzer. Once platelets were fixed, the procedures were completed in < 2 hours. The intra-assay coefficient of variation (CV) was 6.97% (n = 40), the time-based interassay CV was 8.11% (n = 16), and the sample-based inter-assay CV was 6.17% (n = 16). The FCIBA had an excellent correlation (r = 0.936, p < 0.001) with flow cytometry in the measurement of expressed P-selectin in platelets of 20 normal donors. Translocation of P-selectin in plasma-suspended platelets in response to increasing doses of adenosine diphosphate (ADP) occurred in a dose-dependent manner and correlated positively with ADP-induced platelet aggregation in terms of both stimulating doses of ADP (r = 0.99, p < 0.01) and time intervals (r = 0.92, p < 0.05). The findings show that FCIBA is a fast and convenient assay with good precision for the determination of P-selectin expression of human platelets.
Wen
D
D
Department of Endocrinology, Mayo Clinic, Rochester, Minnesota.
Nguyen
T T
TT
Plumhoff
E A
EA
Pineda
A A
AA
Bowie
E J
EJ
Kottke
B A
BA
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000328:Adult; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology; D001792:Blood Platelets / Q000737:chemistry* / Q000187:drug effects / Q000502:physiology; D015815:Cell Adhesion Molecules / Q000032:analysis; D004305:Dose-Response Relationship, Drug; D005260:Female; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D008297:Male; D019007:P-Selectin; D015539:Platelet Activation / Q000187:drug effects / Q000502:physiology; D010974:Platelet Aggregation / Q000187:drug effects / Q000502:physiology; D010980:Platelet Membrane Glycoproteins / Q000032:analysis* / Q000276:immunology; D013997:Time Factors
D000911:Antibodies, Monoclonal; D015815:Cell Adhesion Molecules; D019007:P-Selectin; D010980:Platelet Membrane Glycoproteins; D000244:Adenosine Diphosphate