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目的以抗CD20全人单克隆抗体(Arzerra~(TM))为载体,建立并验证酸解桥式酶联免疫吸附分析法(Bridging ELISA),研究食蟹猴血清中该药物的抗药抗体(anti-drug antibodies,ADAs),了解该药物在猴体内的免疫原性。方法预先包被Arzerra~(TM)的酶标板,先后加入生物素标记的Arzerra~(TM)和酸解待测样品,包被药物(Arzerra~(TM))和生物素标记药物(Biotin-Arzerra~(TM))与样品中ADAs形成“药物-ADAs-生物素标记药物”桥接复合物并显色。结果验证方法灵敏度为7.4 ng·mL~(-1),阳性对照批内、批间精密度不大于17.6%,筛选阈值(sereening cut point,SCP)为1.23,确证实验阈值(confirmatory cut point,CCP)为42.8%;同时,方法具有20倍药物耐受性[Drug-ADAs(40.0μg·mL~(-1);2.0μg·mL~(-1))]及3.2~100 000.0 ng·mL~(-1)内无钩状效应等特性。利用该桥式酶联免疫吸附分析法测定食蟹猴血清中抗药抗体(抗Arzerra~(TM)抗体),于给药后21 d检测到ADAs的产生。结论针对治疗性单克隆抗体的ADAs检测,Bridging ELISA法具有较高的灵敏度及较好的耐药性等特征,故不失为该类药物ADA检测的理想方法之一。
OBJECTIVE To establish an anti-CD20 monoclonal antibody (Arzerra ~ (TM)) as a vector to establish and validate the Bridging ELISA for the detection of drug-resistant antibodies in cynomolgus monkey serum anti-drug antibodies, ADAs) to understand the immunogenicity of the drug in monkeys. Methods The plate of Arzerra ~ (TM) was pre-coated with biotin-labeled Arzerra ™ and acid-hydrolyzed test samples, then coated with Arzerra ™ and Biotin- Arzerra ™ forms a “drug-ADAs-biotin labeled drug” bridging complex with ADAs in the sample and develops color. The sensitivity of the method was 7.4 ng · mL -1. The intra-assay precision was no more than 17.6% in the positive control batch, and the cut-off point was 1.23. The confirmatory cut point (CCP ) Was 42.8%. In the meantime, the drug had 20 times drug resistance [Drug-ADAs (40.0μg · mL -1; 2.0μg · mL -1]] and 3.2 ~ 100 000.0 ng · mL -1 (-1) without hook-like effects and other characteristics. The anti-Arboreceptors (anti-ArzerraTM) antibodies in serum of cynomolgus monkeys were detected by this bridge enzyme-linked immunosorbent assay (ELISA), and the production of ADAs was detected 21 days after administration. Conclusion The ADA test of therapeutic monoclonal antibody, Bridging ELISA method has the characteristics of high sensitivity and good drug resistance, it is one of the ideal methods for the ADA detection of these drugs.