间接酶联免疫吸附试验检测血清抗凝血酶原抗体

Detection of serum antiprothrombin antibody by indirect ELLSA

目的建立人抗凝血酶原抗体IgG、IgM的间接酶联免疫吸附试验(ELISA)检测方法。方法纯化人凝血酶原浓度10μg/ml包被于高吸附活性的聚氯乙烯微孔中,用山羊血清封闭,血清在Tirs盐酸缓冲生理盐水并含有1%牛血清清蛋白的溶液中按1:100稀释,第2抗体为辣根过氧化物酶标记的山羊抗人IgG或IgM,稀释度为1:10 000,以四甲基联苯胺为底物,1mmol/L HCl为终止液建立的ELISA反应体系检测血清中的抗凝血酶原抗体(aPT)-IgG与IgM。结果本实验建立检测血清中aPT-IgG、IgM的ELISA法,稳定性、特异性及重复性佳,与国外商品试剂盒有很好的相关性,有较好的准确度及精密度。结论本实验应用的间接ELISA半定量检测aPT,适合于实验研究及临床应用。

Objective To establish an indirect solid phase ELISA for the detection of IgG and IgM class autoantibodies directed against prothrombin. Methods High activated polyvinylchloride plates were coated with 100μl of 10μg/ml purified human prothrombin in Tris buffered saline（TBS） overnight at 4 ℃. To avoid non specific bind- ing, wells were blocked with 150μl goat serum. After washing with TBS containing 0.1% Tween 20, 100μl of serum samples diluted with TBS containing 1% bovine serum albumin（TBS 1% BSA） in 1 ： 100 were added in duplicate. Plates were incubated for lh at room temperature and after washing with TBS Tween, 100 μl of horse radish peroxidase （HRP）-conjugated goat antihuman IgG or IgM antibodies in TBS 1% BSA were added in the appropriate dilution. After half an hour incubation at room temperature followed by 3 washes, 100 μl tetramethylbenzidine （TMB）/H2O2 were added for 15 min, the reaction was terminated with 50μl 1 mmol/L HCI. Results were read at a wavelength of 450 nm against a background of 650 nm a plate reader. The sensitivity,specificity and reproducibility of ELISA, and its correlation to the imported kit from Germany were studied. Results The method for detecting IgG and IgM, aPT showed good sensitivity, specificity, reproducibility, and stabilization. Conclusion The ELISA kit is successfully developed, it is suitable to use in experimental and clinical application.