纤维蛋白胶抗体间接ELISA检测方法的建立及初步应用  

Development and preliminary application of indirect ELISA method for fibrin glue antibody

作　　者：蔡阳[1] 任真[1] 何学军[1] 吴华[1] 乔红群[1] 

机构地区：[1]南京工业大学药学院,南京210009

出　　处：《中国生物制品学杂志》2012年第2期237-240,共4页Chinese Journal of Biologicals

摘　　要：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 develop and preliminarily apply an indirect ELISA method for fibrin glue antibody. Methods An indirect ELISA method for fibrin glue antibody was developed using fibrin glue as coating antigen, of which the reaction condition was optimized, and precision and specificity were verfied. The serum antibody of rabbits immunized with fibrin glue was determined by the developed method. Results The reaction condition for the developed indirect ELISA method was optimized as follows： microtiter plate was irradiated vertically with UV light for 20 min, then coated with fibrin glue at a concentration of 10 I-Lg/ml, diluted with 0. 05 tool/L bicarbonate solution （pH 9. 6） as coating buffer solution, at 4℃ overnight; the PBS containing 10% fetal bovine serum was served as blocking solution, while 0. 1% PBST as antibody diluent; the optimal dilutions of polyelonal antibody against fibrin glue and HRP-labeled goat anti-rabbit IgG were 1 ： 2 500 and 1 ： 4 000 respectively. The optimal temperature and time for reaction were 37℃and 1 h, while those for substrate coloration were 37℃ and 15 min, respectively. The sulfuric acid at a concentration of 2 mol/L was served as stop solution. The method showed high precision and specificity. The antibodies in sera of rabbits 1, 2, 4 and 6 weeks after immunization were determined, and the result showed that the serum antibody level increased significantly 2 weeks while started to decrease 6 weeks after immunization. Conclusion An indirect ELISA method for fibrin glue antibody was success- fully developed, which might be used for the determination of fibrin glue antidoy in immune sera of rabbits.

关 键 词：纤维蛋白组织黏着剂 抗体 酶联免疫吸附测定 

分 类 号：R392.33[医药卫生—免疫学]