牛血清IgG双抗体夹心ELISA定量检测方法的建立及应用

Development and application of double antibody sandwich ELISA method for quantitative determination of bovine serum immunoglobulin G

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 develop a double antibody sandwich ELISA method for quantitative determination of bovine serum immunoglobulin G（BIg G）.Methods BALB / c mice were immunized BIg G-Fc to prepare monoclonal antibody（Mc Ab）,based on which the concentrations of coating antibody and enzyme-labeled antibody were optimized,and a double antibody sandwich ELISA method for quantitative determination of BIg G was developed,verified for accuracy,precision,stability and specificity,and determined for linear range and quantitative limit.The residual BIg G antigen contents in purified EV71 as well as bovine sera of various batches from various manufacturers were determined by the deve-loped method.Results The double antibody sandwich Q-ELISA for BIg G content was developed using 3E12D2 as coating antibody（8.0 μg / ml,100 μl / well）and 4A2G1B1-HRP as enzyme-labeled antibody（1 ∶ 1 000 diluted,100 μl / well）,of which the linear range was 0.3 ~ 9.6 ng / ml,the R2 value was more than 0.99,and quantitative detection limit was0.3 ng / ml.The recovery rate was 94.1% ~ 108.5%,and the CVs of determination results by one staff in repeat test and by various staff were less than 10%.The total recovery rate of samples after storage at 37 ℃ for 3 and 6 d were more than90%.The samples showed specific binding to BIg G while no cross reactions with other animal sera.The BIg G contents in nine batches of bovine sera were different.Both BSA and BIg G in purified samples were removed significantly as compared with that in crude samples,while the proportion of BIg G in residual bovine sera（BSA ＋ BIg G）increased significantly.Conclusion The developed double antibody sandwich Q-ELISA for BIg G met the requirements for quantitative determination,which might be used for the quality control and residual content determination of bovine serum.