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 sp ecifically P-selectin on platelets. Platelets were isolated from fresh blood by centrifugation and stimulated with various doses of ADP befo re being fixed with 1% of paraformaldehyde. Fixed platelets were incub ated with fluorescence-conjugated anti-P-selectin monoclonal antibody in the wells of fluoricon microtiter plates, and the fluorescence inte nsity was read on a fluorescence concentration analyzer. Once platelet s were fixed, the procedures were completed in <2 hours. The intra-ass ay coefficient of variation (CV) was 6.97% (n = 40), the time-based in terassay CV was 8.11% (n = 16), and the sample-based inter-assay CV wa s 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-select in in platelets of 20 normal donors. Translocation of P-selectin in pl asma-suspended platelets in response to increasing doses of adenosine diphosphate (ADP) occurred in a dose-dependent manner and correlated p ositively with ADP-induced platelet aggregation in terms of both stimu lating 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 assa y with good precision for the determination of P-selectin expression o f human platelets.