WuTac血药浓度双抗体夹心定量ELISA检测方法的建立及其临床应用

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。

Development and Clinical Application of Double Antibody Sandwich ELISA for WuTac Concentration in Serum

Objective To develop a double antibody sandwich ELISA for WuTac concentration in serum and preliminarily apply in clinic. Methods The WuTac concentration in sera was determined quantitatively by double antibody sandwich ELISA, based on which the concentration of coating antibody and the dilution of enzyme-labeled antibody were optimized and a standard curve was plotted. The developed ELISA was verified and used for the determination of serum specimens from 36 healthy volunteers at 14 time points before and after injection with WuTac at various dosages(0. 05, 0. 1 and 0. 2 mg / kg). Results The optimal concentration of coating antibody was 0. 2 μg / ml, and the optimal dilution of enzyme-labeled antibody was 1 ∶ 15 000. The correlation coefficient (r value)of standard curve was not less than 0. 99. The linear determination range of developed double antibody sandwich ELISA was 3. 9 ～ 125 ng / ml. The variation coefficient(CV)of determination results of high concentration WuTac standard by the developed ELISA was less than 15%. The developed ELISA showed a accuracy of 99. 05% ± 5. 00%(92. 43% ～ 110. 02%)and good specificity. The determination results of clinical serum specimens proved that WuTac showed a characteristic of linear pharmacokinetics within a concentration range of 0. 05 ～ 0. 2 mg / kg. Conclusion A double antibody sandwich ELISA for WuTac concentration in serum was developed, with sensitivity, precision and accuracy meeting the requirements of pharmacokinetic study on biologics.