基于开放式三明治酶联免疫分析的雌二醇检测方法的建立

Application of open sandwich ELISA on detection of estradiol

目的:通过建立开放式三明治酶联免疫分析方法(OE-ELISA),对雌二醇进行检测。方法:通过基因拼接构建质粒后在原核表达系统中制备小分子泛素相关修饰物蛋白-大肠杆菌麦芽糖结合蛋白-抗体轻链可变区(sumo-MBP-VL)和碱性磷酸酶-抗体重链可变区(ALP-VH)融合蛋白,并从产量和可溶性角度对两种蛋白的表达条件进行优化。将得到的两种蛋白纯化后分别作为包被抗体和酶标抗体,建立检测雌二醇的OE-ELISA方法。应用竞争ELISA和OE-ELISA测定不同浓度的雌二醇标准品,绘制标准曲线并计算两种方法的敏感性。结果:在质粒中加入sumo标签后,实现了sumo-MBP-VL融合蛋白的可溶性表达。在Rosetta宿主菌中37℃诱导后得到大量的ALP-VH和sumo-MBP-VL融合蛋白。OE-ELISA测定雌二醇标准品的标准曲线为y=0.0296x+1.3029,r^(2)=0.9961,敏感性为532 pg/mL,优于竞争ELISA 803 pg/mL的敏感性。结论:成功建立了检测雌二醇的OE-ELISA方法。

Objective:To detect estradiol by establishing open sandwich immunoassay(OE-ELISA).Methods:Alkaline phosphatase-heavy chain variable area(ALP-VH)and small ubiquitin-related modifier protein-maltose binding protein-light chain variable area(sumo-MBP-VL)fusion proteins were prepared in the prokaryotic expression system after gene splicing to construct the plasmid,and the expression conditions of the two proteins were optimized from the perspective of yield and solubility.The two proteins which were purified were used as coated antibodies and enzyme-conjugated antibodies to establish an OE-ELISA method for detection of estradiol.Competitive ELISA and OE-ELISA were used to determine different concentrations of estradiol standard products,and the sensitivity of the two methods was calculated by drawing standard curves.Results:The soluble expression of sumo-MBP-VL fusion protein was achieved by adding sumo tag to plasmid.A large number of ALP-VH and sumo-MBP-VL fusion proteins were induced at 37℃in Rosetta host bacteria.The standard curve of OE-ELISA for determination of estradiol standard products was y=0.0296x+1.3029,r^(2)=0.9961,and the sensitivity was 532 pg/mL,which was better than that of competitive ELISA(803 pg/mL).Conclusion:An OE-ELISA for estradiol detection was successfully established.