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目的建立纤维蛋白胶抗体间接ELISA检测方法,并进行初步应用。方法采用纤维蛋白胶作为包被抗原,建立检测纤维蛋白胶抗体的间接ELISA法,优化反应条件,并对方法的精密性及特异性进行验证。采用建立的间接ELISA法检测纤维蛋白胶免疫兔血清抗体。结果间接ELISA法的最佳反应条件为紫外灯管垂直照射酶标板20 min后,选择0.05 mol/L的碳酸氢钠溶液(pH 9.6)作为包被缓冲液,以10μg/ml的纤维蛋白胶4℃包被过夜,以含10%小牛血清的PBS封闭,0.1%PBST为抗体稀释液,纤维蛋白胶多克隆抗体和HRP标记的羊抗兔IgG的工作浓度分别为1∶2 500和1∶4 000,反应条件均为37℃孵育l h,底物显色条件为37℃孵育15 min,终止液为2 mol/L硫酸。该方法精密性良好,特异性较强。以建立的间接ELISA方法检测给药1、2、4、6周的兔血清抗体,发现给药2周后兔血清抗体水平明显升高,6周时开始下降。结论成功建立了纤维蛋白胶抗体间接ELISA检测方法,可用于兔免疫血清纤维蛋白胶抗体的检测。
Objective To establish an indirect ELISA method for detecting fibrin glue antibodies and to conduct preliminary application. Methods Fibrin glue was used as coating antigen, indirect ELISA method was established to detect fibrin glue antibodies, and the reaction conditions were optimized. The precision and specificity of the method were verified. Indirect ELISA was used to detect fibrin sera immune rabbit serum antibody. Results The optimal reaction conditions of indirect ELISA were as follows: 20 min after the UV lamp was irradiated vertically, 0.05 mol / L sodium bicarbonate solution (pH 9.6) was selected as coating buffer and 10 μg / ml fibrin glue Incubate overnight at 4 ° C, blocked with 10% fetal bovine serum in PBS, 0.1% PBST as antibody dilution, working concentrations of fibrin glue polyclonal antibody and HRP-labeled goat anti-rabbit IgG were 1: 250 and 1 : 4000, the reaction conditions were incubated at 37 ℃ for 1 h, the substrate was incubated at 37 ℃ for 15 min, and the final solution was 2 mol / L sulfuric acid. The method has good precision and strong specificity. Rabbit serum antibodies were detected by indirect ELISA at 1, 2, 4 and 6 weeks after administration. The serum antibody levels of rabbits increased significantly after 2 weeks of administration, and then decreased at 6 weeks. Conclusion The indirect ELISA method for detecting fibrin glue antibody was successfully established and could be used for the detection of rabbit serum immune fibrin glue antibodies.