A Biotin-Avidin-EL ISA for Studying on Platelet Membrane Surface P-Selectin and Its Clinical Application

To define the abnormalities in surface GPs expression on circulating platelets and provide better biomarker of bleeding and thrombotic disorders, we develop a high-throughput assay using biotin-avidin enzyme-linked immunosorbent assay (BA-ELISA) to test platelet responses to key platelet agonists and antagonists.

SZ51(anti-P-selectin mAb) coated-plate was added into 100μ l PRP with 10μ l ADP (27μ mol/L ADP)or 10μ l PBS without ADP and incubated for 1 h. Activated platelets captured were reacted with biotin-SZ-2 for 1 h. Then, the plate was re-incubated with avidin-HRP coiugated for 1h. Binding avidin was detected with OPD. The levels of P-selectin were detected in AMI group, ICH group, DM group or control group using the BA-ELISA method. This BA-ELISA was compared with flow cytometer (FCM). Inhibition of platelet GPs function was evaluated with inhibitory mAb, SZ21 and aspirin respectively.

The BA-ELISA can detect platelet count as low as 6.25 × 10⁹/L in PRP. Both of the inter-assay and intra-assay coefficient variation (C V) were less than 10%. The OD492nm values of the ADP-induced P-selectin in 30 AMI, 30 ICH and 30 DM platelets in PRP were 1.76± 0.56, 1.95±0.53 and 1.51±0.14 which was significantly higher than that in controls (0.68±0.21, P<0.01). The OD 492nm values of non ADP-induced P-selectin in 30 AMI, 30 ICH or 30 DM platelets in PRP were 0.72±0.25, 1.53±0.61 and 0.61±0.16 significantly higher than that in 30 controls (0.18±0.09, P<0.01). The correlation between the BA-ELISA and FCM was concordant (R=0.86). SZ21 or aspirin could inhibit the expression of P-selectin induced by ADP.

BA-ELISA is suitable for measuring platelet GP function and is also useful for diagnostic tests and for screening of inhibitors/activators of platelet activation.

No relevant conflicts of interest to declare.