目的 以抗CD20全人单克隆抗体(ArzerraTM)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ArzerraTM的酶标板，先后加入生物素标记的ArzerraTM和酸解待测样品，包被药物(ArzerraTM)和生物素标记药物(Biotin-ArzerraTM)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(screening 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ArzerraTM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。
Abstract
OBJECTIVE To develop and validate an acid dissociated bridging ELISA assay for determination of antibodies (ADAs) against a human monoclonal antibody against CD20, ArzerraTM, in cynomulgus serum, which will be used to reflect the immunogenicity of ArzerraTM in cynomolgus serum. METHODS Biotin labeled ArzerraTM and acid treated unknown samples were added into the ArzerraTM pre-coated 96-well plates, sequentially. Positive controls (ADAs) in unknown samples bridged with coated ArzerraTM and biotin-labeled ArzerraTM (Biotin-Drug) to generate ADAs bridged compounds (Drug-ADAs-Biotin-Drug), and coloration was made with enzymatic reaction. RESULTS The sensitivity was 7.4 ng·mL-1, and the intra-assay and the inter-assay RSDs were less than 17.6% for positive controls. The screening cut point (SCP) for this assay was 1.23 and the confirmatory cut point (CCP) was 42.8%. The assay also showed high drug tolerance, which was 20 fold at 2.0 μg·mL-1 ADA level (Drug:ADA=40.0 μg·mL-1:2.0 μg·mL-1); and no “Hook effect” was detected within 3.2-100 000.0 ng·mL-1. Using this assay to determine ADAs against ArzerraTM in Cynomolgus serum, ADAs were detected at 21 d post administration. CONCLUSION The bridging ELISA is one of the best assays to determine ADAs against therapeutic monoclonal antibodies, because it shows high sensitivity and good drug tolerance.
关键词
酸解 /
桥式酶联免疫吸附分析法 /
抗药抗体 /
免疫原性
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Key words
acid dissociation /
bridging ELISA /
anti-drug antibody /
immunogenicity
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中图分类号：
R915
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参考文献
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脚注
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基金
“十三五”定向委托课题“生物类似药(Biosimilar)技术评价相关支撑体系的研究”资助项目(2015ZX09501008)
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