抗康柏西普抗体筛选用ELISA检测方法的建立

Development of an ELISA for screening anti-conbercept antibody

目的:为评价康柏西普免疫原性,建立一种恒河猴血清中的抗康柏西普抗体检测方法。方法:建立一种以康柏西普为包被蛋白、羊抗人Ig G Kappa light chain抗体为检测抗体的ELISA检测方法,并验证其精密度和专属性。同时运用细胞增殖实验确定阳性样品抗康柏西普抗体的活性。结果:所建立的检测方法最佳包被浓度为5μg·m L-1,检测抗体的最佳稀释倍数为2 500倍。阳性判断阈(cut point)为0.162,批内和批间变异系数均未超过15%。康柏西普浓度在小于10μg·m L-1时对检测无干扰。24个血清样品中,抗康柏西普抗体阳性率为62.5%,HUVEC细胞增殖实验显示无阳性血清具有中和康柏西普生物学活性的能力。结论:经验证,该方法可应用于抗康柏西普抗体的检测,为临床前及临床研究提供支持。

Objective： To develop an ELISA method for assessing immunogenicity of conbercept in Rhesus monkey. Methods： An ELISA method was developed using conbercept and goat-anti-human HRP-Ig G Kappa light chain as capture and detector protein,respectively. Validation of precision and specialization of the method were conducted then. To further characterize anti-conbercept antibodies,positive serum were analyzed by cell proliferation assay. Results： The best coating concentration was 5 μg·mL^- 1,and the optimal dilution ratio of the detected antibody was 1∶ 2 500. The cut point of screening was 0. 162,and inter- and intra-batch coefficient variations were less than 15%. Conbercept concentration of 10 μg·mL^- 1or less had no effect on detection. Anti-conbercept antibody was positively detected in 15 out of the 24 serum samples（ 62. 5%）. HUVEC cell proliferation assay indicated that all the positive serum can not neutralize conbercept. Conclusion： The method for analyzing anti-conbercept antibody is validated; it is valuable for pre-clinical and clinical applications.