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目的建立牛血清Ig G(bovine immunoglobulin G,BIg G)抗原的双抗体夹心ELISA定量检测方法。方法用BIg G-Fc免疫BALB/c小鼠,制备抗BIg G单抗,确定包被抗体及酶标抗体后,建立定量检测BIg G抗原的双抗体夹心ELISA方法,并验证该方法的准确度、精密度、稳定性、特异性,确定该方法的线性范围和定量限度。应用建立的方法检测不同厂家、不同批次牛血清及纯化EV71样品中BIg G抗原残留量。结果建立了以3E12D2为包被单抗(8.0μg/ml,100μl/孔),4A2G1B1-HRP为酶标抗体(1∶1 000稀释,100μl/孔)定量检测BIg G的双抗体夹心ELISA方法,该方法的线性范围为0.3~9.6 ng/ml,R~2>0.99,定量限度为0.3 ng/ml;回收率在94.1%~108.5%之间,同一实验者重复检测和不同实验者检测的变异系数均<10%;37℃放置3和6 d的稳定性均>90%;该试剂样品与BIg G可发生特异性反应,与其他动物源血清无交叉反应。9批牛血清中BIg G含量均存在差异;精纯样品较粗纯样品BSA和BIg G均明显去除,但BIg G占牛血清残留量(BSA+BIg G)的比例却明显上升。结论建立的BIg G抗原的双抗体夹心ELISA方法符合定量检测需要,可用于牛血清的质量质控及残留量检测。
Objective To establish a double antibody sandwich ELISA for quantitative determination of bovine immunoglobulin G (BIgG) antigen. Methods BALB / c mice were immunized with BIg G-Fc to prepare anti-BIg G monoclonal antibody. After the coated antibody and enzyme-labeled antibody were determined, a double-antibody sandwich ELISA for quantitative determination of BIg G antigen was established and the accuracy of the method was verified , Precision, stability, specificity, to determine the method of linear range and quantitative limits. The established method was used to detect the residual amount of BIg G antigen in bovine serum and purified EV71 samples from different manufacturers and batches. Results A double-antibody sandwich ELISA was developed for the quantitative detection of BIg G using 3E12D2 as coating monoclonal antibody (8.0μg / ml, 100μl / well) and 4A2G1B1-HRP as enzyme-labeled antibody The linear range was 0.3 ~ 9.6 ng / ml, R ~ 2> 0.99, the limit of quantitation was 0.3 ng / ml. The recoveries ranged from 94.1% to 108.5%. The coefficient of variation All of which were less than 10%. The stability of the samples was> 90% at 37 ℃ for 3 and 6 d. The reagents reacted specifically with BIg G and did not cross-react with other animal serum. There were differences in the content of BIg G in nine batches of bovine serum. The crude samples of pure bovine serum samples were significantly removed, but the percentage of BSg to bovine serum albumin (BSA + BIg G) was significantly increased. Conclusion The established double antibody sandwich ELISA for BIg G antigen meets the requirement of quantitative detection and can be used for the quality control and residue detection of bovine serum.