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目的建立WuTac血药浓度双抗体夹心定量ELISA检测方法,并进行初步临床应用。方法建立双抗体夹心ELISA法,定量检测血清中WuTac浓度,筛选最佳抗体包被浓度和酶标抗体稀释度,绘制标准曲线,并对该方法进行验证;应用该方法对36名健康志愿者单次注射不同剂量WuTac(0.05、0.1和0.2mg/kg)前后14个时间点的临床血清标本进行检测。结果最佳抗体包被浓度为0.2μg/ml,最佳酶标抗体稀释度为1∶15000,标准曲线的线性相关系数r≥0.99,线性范围为3.9～125ng/ml。高浓度WuTac标准品的变异系数小于15%;准确性为99.05%±5.00%(92.43%～110.02%);特异性良好。临床血清检测结果表明,WuTac在0.05～0.2mg/kg范围内呈线性药代动力学特征。结论已建立了WuTac血药浓度双抗体夹心定量ELISA检测方法,该方法的灵敏度、精密性和准确性均符合我国生物制品药代动力学研究的要求。
OBJECTIVE To establish a double antibody sandwich quantitative ELISA assay for detecting WuTac blood concentration and to conduct preliminary clinical application. Methods The double antibody sandwich ELISA method was established to detect the concentration of WuTac in serum, to screen the optimal antibody concentration and enzyme-labeled antibody dilution, to draw a standard curve and validate this method. By using this method, 36 healthy volunteers The clinical serum samples of 14 different time points before and after WuTac (0.05, 0.1 and 0.2 mg / kg) were injected. Results The optimal antibody concentration was 0.2μg / ml, the best dilution of ELISA was 1:15000, the linearity coefficient of the standard curve was more than or equal to 0.99 and the linear range was 3.9 ~ 125ng / ml. High concentration WuTac standard coefficient of variation of less than 15%; accuracy of 99.05% ± 5.00% (92.43% ~ 110.02%); good specificity. Clinical serum test results showed that WuTac linear pharmacokinetic characteristics in the range of 0.05 ~ 0.2mg / kg. Conclusion WuTac serum concentration double antibody sandwich quantitative ELISA assay has been established. The sensitivity, precision and accuracy of this method are in line with the requirements of the pharmacokinetics of biological products in China.