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Establishment Of Biotin-avidin-ELISA To Study Platelet Membrane Glycoprotein Function And Its Clinical Applications

Posted on:2011-09-29Degree:MasterType:Thesis
Country:ChinaCandidate:Y T ZhangFull Text:PDF
GTID:2154360305476497Subject:Clinical Laboratory Science
Abstract/Summary:PDF Full Text Request
Background:Platelet membrane surface glycoproteins (GP) are critical for normal platelet adhesion to the vessel wall and subsequent platelet aggregation. Changes of platelet surface GPs may occur in circulating platelets that could increase the risk for bleeding or thrombosis. The accurate definition of surface GP abnormalities in circulating platelets may provide better understanding of bleeding and thrombotic disorders. The primary GPs studied were GP Ib and GPIIb/IIIa, two of the major intrinsic plasma membrane GPs; CD62p (P-selectin), aα-granule membrane glycoprotein that becomes exposed on the platelet surface following secretion. Thrombin or ADP-induced secretion in normal platelets caused the appearance of CD62p on the platelet surface, increased exposure of GPIIb/IIIa. Patients with acute myocardial infarction (AMI), acute cerebral infarction(ACI) and diabetes mellitus (DM) had an increased expression of CD62p and GP IIb/IIIa on the surface of their platelets. Platelet surface GPs were measured on intact platelets in whole blood and platelet membrane microparticles were assayed in cell-free plasma using gel electrophoresis, immunoradiometric assay and flow cytometry. These classical detection methods will help to define acquired abnormalities of platelet surface glycoproteins. However, These classical detection methods also have many disadvantages. In this study, we establish here a noncompetitive, novel biotin-avidin enzyme-linked immunosorbent assay (BA-ELISA) for quantifying human platelet membrane surface glycoproteins by two different monoclonal antibodies and the avidin-biotin technique.Objective:(1) Using monoclonal antibodies and biotin-avidin system to establish a BA-ELISA method and the sensitivity and repeatability of BA-ELISA for platelet function were detected.(2) By using the BA-ELISA method, the level of platelet membrane CD62p and GPIIb/IIIa were measured in patients (AMI, ACI, DM), and its clinical using value was evaluated.Methods:A monoclonal antibody (mAb) to GPIIb/IIIa, 7E3 and a mAb to CD62p, SZ51 were immobilized onto microtiter plate wells, respectively, and then platelet-rich plasma (PRP) with or without ADP-induced was added to the wells, respectively. After incubated with biotin-labeled anti-GPIb mAb, SZ2 (biotin-SZ2), the conjugated biotin-SZ2 was checked by avdin-labeled horseradish peroxidase (Avdin-HRP). The sensitivity and repeatability of BA-ELISA for platelet function were detected. Inhibitions of platelet membrane glycoprotein function were evaluated with inhibitory mAb, SZ21 and aspirin respectively. By using the BA-ELISA method, the level of platelet membrane CD62p and GPIIb/IIIa were measured in patients (acute myocardial infarction(AMI), acute cerebral infarction(ACI), diabetes mellitus(DM)) and controls (healthy people).This method was compared with flow cytometric immunoflurescence (FCM)method.Results:(1) level of sensitivityThe BA-ELISA can detect platelet count as low as 6.25×109 / L (SZ 51 coated plates) and 3.13×109 / L (7E3 coated plates) in PRP of the specimens.(2) RepeatabilityBoth of the inter-assay and intra-assay coefficient variation(C V) were less than 10?.(3) Comparison of MethodsThe result between the BA-ELISA and FCM was concordant(R=0.86).(4) Inhibition of platelet function was tested by BA-ELISASZ21 or aspirin can inhibit the expression of CD62p and GPIIb/IIIa which ADP-induced. (5) Clinical applications of the BA-ELISA The ADP-induced (non-induced) CD62p and GPIIb/IIIa of 30AMI, 30 ACI, 30 DM and 50 healthy platelets in PRP were detected by BA-ELISA respectively. The absorbance (A492/620nm) value were 1.76?0.56(0.72?0.25, 1.95?0.53(1.53?0.61);1.51?0.14(0.61?0.16), 1.88?0.42(1.46?0.57)and 1.38?0.26(0.43?0.21), 1.61?0.42(1.23?0.32), significantly higher than that in controls (0.68?0.21(0.18?0.09)and 0.87?0.29(0.44?0.14), P<0.01).Conclusions:(1) The BA-ELISA method for human platelet membrane glycoprotein function assay was first established in our country.(2) This assay is suitable for measuring platelet surface glycoproteins and is also more sensitive and precise than the previously published immunoassays based on competitive binding assay.(3) This method can be used to evaluate the influence of platelet activators/inhibitors on platelet function and detect platelet activation level caused by thrombus or its associated disease.(4) The CD62p and GPIIb/IIIa levels on platele in the AMI, ACI and DM patients are markedly higher than those in the congtrols. The detection of platelet CD62p and GPIIb/IIIa can estimate accurately the function of platelet. They provided a evidence for clinic prevention, diagnosis, therapy and prognosis estimation in thrombotic disorders disease...
Keywords/Search Tags:platelet, membrane glycoprotein, activation, enzyme-linked immunosorbent assay, mononclonal antibody
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