目的:建立一种快速、灵敏、高效和易操作的方法,用于检测重组人源化抗TNF-α单抗WLB303猴重复给药毒性试验血清中产生的中和抗体.方法:对食蟹猴静脉输注WLB303,连续给药4周.采取给药期和恢复期不同时间的血清,随后采用桥式酶联免疫吸附法对血清中的抗药抗体进行定性筛选,对筛选为阳性的血清样本进行预处理,然后以WLB303生物学活性测定法为基础的中和反应法对中和抗体进行确证.结果:建立了WLB303中和抗体的确证方法,确定了检测的临界点.确证方法的检出率为100％,在血清中检测到具有中和活性的抗体,中和抗体的中和作用强度总体与给药时间、给药剂量呈正相关.结论:建立的检测方法能够直接体现出剂量差异性和个体差异性,也能反映中和抗体的滴度差别.该方法的特异性、灵敏度和有效性能够满足试验要求,可以用于单抗药物重复给药动物毒性试验血清中和抗体的检测.%Objective:To establish a rapid,sensitive,highly-efficient and easy-to-use method for detection of neutralizing antibodies against WLB303 in serum of animals undergoing repeated dose toxicity study.Methods:The cynomolgus monkeys were continuously infused with recombinant humanized anti-TNF-α monoclonal antibodies intravenously for 4 weeks.Serum samples were taken at different time points before and after administration and at different stages of recovery period.Then a bridging-ELISA method was used to qualitatively screen anti-drug antibody (ADA).A sample pre-treatment procedure was conducted to eliminate the non-specific interference in serum samples prior to verification of neutralizing activity.Neutralization activity was verified in a cell-based neutralization antibody assay.Results:A cell-based neutralizing antibody assay was established and the cut-off point was confirmed.The detection rate of the confirmed neutralizing antibodies was 100％.Specific neutralizing antibodies were detected in serum samples,and the neutralization activity of neutralizing antibodies had positive correlation with the time and dosage of administration.Conclusion:The established assay has proven to be capable of demonstrating dosedependent effect difference and inter-individual difference.It also reflects difference in titers of neutralizing antibodies.The cell-based neutralizing antibody assay was found to have desired properties of specificity,sensitivity and validity,and can be used as a feasible method for detection of neutralizing antibodies in repeated dose toxicity study.
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