title: A high-throughput biotin-avidin-ELISA for studying expression of platelet membrane glycoproteins and its clinical application.
abstract: Platelet membrane glycoproteins (GPs) are critical for normal platelet adhesion, activation and aggregation. To define the abnormalities in surface GP expression on circulating platelets and provide a better biomarker of bleeding and thrombotic disorders, we have developed a accurate, time-saving and high-throughput biotin-avidin enzyme-linked immunosorbent assay (BA-ELISA) with the monoclonal antibodies (mAbs), 7E3 against the complex of GPIIb and GPIIIa (GPIIb/IIIa), SZ-51 against P-selectin, and SZ-2 against GPIb, respectively. The levels of P-selectin and GPIIb/IIIa were measured in patients with acute myocardial infarction (AMI), intracerebral hemorrhage (ICH), or diabetes mellitus (DM)) and healthy subjects. Inhibition of GP expression was evaluated with SZ-21, an inhibitory mAb against GPIIIa and aspirin, respectively. The sensitivity of BA-ELISA is high enough to detect platelet count as low as 3.13 x 10(9)/L in platelet-rich plasma (PRP). Both the inter-assay and intra-assay coefficient variation are less than 10%. Adenosine diphosphate (ADP)-induced or non-ADP-induced expression of P-selectin and GPIIb/IIIa was significantly higher in AMI, ICH or DM than that in controls (P < 0.01 for each). Either SZ-21 or aspirin can inhibit the ADP-induced expression of P-selectin and GPIIb/IIIa. Importantly, a high correlation was detected between BA-ELISA and flow cytometry methods. These observations indicate that BA-ELISA is a sensitive and high-throughput assay for evaluating platelet GP expression. The newly developed BA-ELISA can be popularized in community hospitals, because it does not require sophisticated equipments and reagents. This method is suitable for screening inhibitors of platelet activation and has a potential in use for diagnostic purposes.
authors.affiliation: Jiangsu Institute of Hematology, The First Affiliated Hospital of Soochow University, Key Laboratory of Thrombosis and Haemostasis, Ministry of Health, Suzhou, Jiangsu, China.
authors.forename: Youtao
authors.initials: Y
authors.lastname: Zhang
authors.forename: Yiming
authors.initials: Y
authors.lastname: Zhao
authors.forename: Shiqi
authors.initials: S
authors.lastname: Lu
authors.forename: Mingqing
authors.initials: M
authors.lastname: Zhu
authors.forename: Yang
authors.initials: Y
authors.lastname: He
authors.forename: Changgeng
authors.initials: C
authors.lastname: Ruan
mesh_terms: D000244:Adenosine Diphosphate; D000911:Antibodies, Monoclonal; D001360:Avidin; D015415:Biomarkers / Q000097:blood*; D001710:Biotin; D002543:Cerebral Hemorrhage / Q000175:diagnosis / Q000378:metabolism; D003920:Diabetes Mellitus / Q000175:diagnosis / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005434:Flow Cytometry; D006801:Humans; D009203:Myocardial Infarction / Q000175:diagnosis / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins / Q000032:analysis* / Q000378:metabolism*; D012680:Sensitivity and Specificity
chemical_list: D000911:Antibodies, Monoclonal; D015415:Biomarkers; D010980:Platelet Membrane Glycoproteins; D001360:Avidin; D000244:Adenosine Diphosphate; D001710:Biotin