抗重组高效抗肿瘤抗病毒蛋白抗药抗体ELISA检测方法的建立及应用

Validation and its application of an ELISA method for the detection of anti-drug antibody against recombinant potent anti-tumor and anti-virus protein in patient sera

目的:建立受试者血清中重组高效抗肿瘤抗病毒蛋白抗药抗体的检测方法,考察用药后不同组别受试者的抗药抗体产生情况。方法:首先建立了检测血清中抗药抗体ELISA方法并进行了方法学的验证;然后筛选了3个给药组血清样本中的抗药抗体,用结果为阳性的受试者血清最大稀释倍数表示抗药抗体的滴度。结果:该ELISA方法检测乐复能血清抗药抗体的特异性好,检测限为204 ng·mL-1,高、中、低及阴性样本批内、批间重复性CV值均小于6%,达到常规ELISA的标准。10μg连续给药组7例受试者首次用药3周后有3例产生抗体,用药5周后均产生抗体;10μg隔日给药组6例受试者首次用药4周后均产生抗体;20μg隔日连续给药组10例受试者首次用药2周后有1例产生抗体,用药4周后8例产生抗体,用药6周后9例产生抗体。结论:建立了重组高效抗肿瘤抗病毒蛋白免疫原性的ELISA检测方法。首次用药4周后3个组的抗药抗体阳性率达到高值,组别之间无明显差别。比较而言,第3组(20μg隔日给药)的抗药抗体滴度较低。

Objective： To establish a method to detect anti-drug antibody in the sera of HBeAg positive patients injected with a recombinant potent anti-tumor and anti-virus protein. Methods： The established ELISA method was validated,and used to screen the anti-drug antibody in the serum samples from the three groups. The largest dilution was determined as the titer of anti-drug antibody in each positive sample. Results： The established ELISA method was specific to detect the anti-drug antibody in the serum with the detection limit of 204 ng·mL^-1. All the intra-and inter-batch variation coefficients of all detected concentrations were below 6%,fitting the standard of conventional ELISA. In the 10 μg qd group,3 of 7 subjects emerged anti-drug antibodies at 3 weeks,while all subjects emerged anti-drug antibodies at 5 weeks after the first injection. In the 10 μg qod group,all the 6 subjects emerged anti-drug antibodies at 4 weeks. In the 20 μg qod group,1 of 10 subjects produced antibodies at 2 weeks,and 8 subjects produced antibodies at 4 weeks,while 9 subjects produced antibodies at 6 weeks after the first injection. Conclusion： We have established an ELISA method to detect the antibody against a recombinant potent antitumor and anti-virus protein in the patients injected with this protein. The three groups reached high value of antidrug antibodies at 4 weeks after injection without significant difference between groups. In comparison,the third group（ 20 μg qod） has a lower anti-drug antibody activity.