流式免疫微球芯片技术分析特异性血小板自身抗体

目的建立流式免疫微球芯片技术（FCIBA）分析特异性血小板自身抗体的新方法。方法使用不同红色荧光强度的微球，包被不同血小板膜蛋白单克隆抗体（抗GPⅡb／Ⅲa、抗GPⅠa／Ⅱa、抗GPⅣ、抗GPⅠb／Ⅸ和抗HLA-ABC单克隆抗体），制备特异性血小板自身抗体检测微球，用流式细胞仪检测微球捕获的血小板抗原-自身抗体复合物，检测结果与微孔板改进抗原捕获ELISA（MACE）进行比较，并检测了部分免疫性以及非免疫性血小板减少性紫癜患者血清中的5种特异性血小板自身抗体。结果FCIBA分析抗GPⅡb／Ⅲa、抗GPⅠa／Ⅱa、抗GPⅣ、抗GPⅠb／Ⅸ和抗HLA-ABC5种特异性血小板自身抗体，批内重复性变异系数（CV）分别为4．82％、6．09％、5．04％、5．73％、5．30％；稀释试验呈对数线性相关，相关系数（r）分别为0．9972．0．9966．0．9988．0．9965、0．9982；新方法对5种特异性血小板自身抗体的分析结果均与MACE试验结果高度相关，r值分别为0．9289、0．9224、0．8894、0．9100、0．9134（P均〈0．01）。新方法分析98例免疫性血小板减少性紫癜患者的血清样本，血小板特异性自身抗体检出的阳性率为51．69％；其中，抗GPⅡb／Ⅲa为40．82％，抗GPⅠa／，Ⅱa为24．45％，抗GPⅣ为19．39％，抗GPⅠb／Ⅸ为32．65％，抗HLA-ABC为17．35％。新方法分析40例非免疫性血小板减少性紫癜患者的血清样本，阳性率为0。结论本研究建立了FCIBA分析特异性血小板自身抗体的新方法，可同时分析抗血小板GPⅡb／Ⅲa、GPⅠa／，Ⅱa、GPⅣ、GPⅠb／Ⅸ和HLA-ABC5种特异性血小板自身抗体。

Flow cytometric immnno-bead array assay for detection of platelet-specific autoantibodies

Objective To establish a new flow cytometric immuno-bead array assay (FCIBA)to detect several platelet-specific autoantibodies simultaneously in a single serum sample.Methods A series of beads of different red fluorescent intensity were used for coating with different anti-platelet membrane protein monoclonal antibodies(anti-GP Ⅱb/a,anti-GP Ⅰ a/Ⅱ a,anti-GP Ⅳ,anti-GP Ⅰ b/Ⅸ and anti-HLAABC)to detect five platelet-specific autoantibodies in serum. The beads captured platelet antigenautoantiboay complex.Subsequently,different platelet-specific autoantibodies in a patient serum can be detected simultaneously by the flow cytometry.In addition,we evaluated the new FCIBA,and compared its results with modified antigen capture ELISA method(MACE).The new FCIBA was used to detect five platelet-specific autoantibodies in serums of autoimmune thrombocytopenic purpura(AITP)and nonautoimmune thromboeytopenic purpura patients.Results The new FCIBA can be used to detect five plateletspecific autoantibodies simultaneously(Anti-GP Ⅱ b/Ⅲ a,Anti-GP Ⅰ a/Ⅱ a,Anti-GP Ⅳ,Anti-GP Ⅰ b/Ⅸand Anti-HLA-ABC).The coefficient of variation(CV)of intra-repetition are 4.82%,6.09%,5.04%,5.73%and 5.30%,respectively.The dilution test results are in good logarithm linearity which are 0.997 2,0.996 6,0.998 8,0.996 5 and 0.998 2,respectively. The resuhs of the new FCIBA are highly correlated with those with MACE method,and the coefficient correlation were 0.928 9,0.922 4,0.889 4,0.910 0 and 0.913 4,respectively(P＜0.01).51.69%samples of AITP patients show positive for platelet-specific autoantibodies as detected by the new FCIBA.Among the AITP patients,the positivity of specific autoantibodies for anti-GPⅡb/ Ⅲ a,anti-GP Ⅰ a/Ⅱ a,anti-GP Ⅳ,anti-GP Ⅰb/Ⅸ and anti-HLA-ABC were 40.82%,24.45%,19.39%,32.65%and 17.35%.Among the 40 non-autoimmune thrombocytopenic purpura patients,none of platelet-specific autoantibodies in serum samples can be detected.Conclusion A new FCIBA is established successfully to detect five platelet-specific autoantibodies coefficient correlation.