The potential immunogenicity of therapeutic human and humanized monoclonal antibodies (mAb) is a significant concern, and so preclinical testing of therapeutic mAbs routinely includes assessment of anti-drug antibody (ADA) induction. Here, we report the development of automated screening and confirmatory bridging ELISAs for the detection of rat antibodies against DH1042, an engineered human mAb for the SARS-CoV-2 receptor-binding domain. The assays were evaluated for specificity, sensitivity, selectivity, absence of a prozone effect, linearity, intra- and inter- assay precision, and robustness, and found to be suitable for purpose. The assays were then used to evaluate anti-DH1042 antibodies in the sera of rats dosed with lipid-nanoparticle (LNP)-encapsulated mRNA encoding DH1042. Rats received two doses of 0.1, 0.4 or 0.6 mg/kg/dose LNP-mRNA 8 days apart. Twenty-one days after the second dose, 50-100% of rats had developed confirmed anti-DH1042 ADA depending on dose level. No animals in the control group developed anti-DH1042 ADA. These assays reflect new applications for a non-specialized laboratory automation platform, and the methodologies and approaches reported here provide a template that can be adapted for the automated detection and confirmation of ADA in preclinical testing of other biologics.

治疗性人类和人源化单克隆抗体 (mAb) 的潜在免疫原性是一个重要问题，因此治疗性 mAb 的临床前测试通常包括抗药物抗体 (ADA) 诱导的评估。在这里，我们报告了自动筛选和验证性桥接 ELISA 的开发，用于检测针对 DH1042 的大鼠抗体，DH1042 是一种针对 SARS-CoV-2 受体结合域的工程化人类单克隆抗体。对测定的特异性、灵敏度、选择性、无前带效应、线性、测定内和测定间精度以及鲁棒性进行了评估，并发现适合目的。然后使用该测定法评估服用脂质纳米颗粒（LNP）封装的编码 DH1042 mRNA 的大鼠血清中的抗 DH1042 抗体。大鼠接受两剂分别为 0.1、0.4 或 0.6 mg/kg/剂的 LNP-mRNA，间隔 8 天。第二次给药后 21 天，50-100% 的大鼠出现了确认的抗 DH1042 ADA，具体取决于剂量水平。对照组中没有动物产生抗DH1042 ADA。这些测定反映了非专业实验室自动化平台的新应用，这里报告的方法和方法提供了一个模板，可适用于其他生物制剂临床前测试中 ADA 的自动检测和确认。