Pharma Research Penzberg, Roche Diagnostics GmbH, Nonnenwald 2, 82377 Penzberg, Germany.
J Pharm Biomed Anal. 2010 Jun 5;52(2):249-54. doi: 10.1016/j.jpba.2009.12.029. Epub 2010 Jan 4.
Current state of the art bridging ELISA technologies for detection of anti-drug antibodies (ADAs) against therapeutic antibodies bear the risk of false-negative results due to interference by circulating drug. Methods to remove the drug in the sample or sample pre-treatment techniques such as acid dissociation of the immune complexes are limited, laborious and may destroy ADAs resulting again in false-negative results. The immune complex ELISA described in this publication provides a simple solution. It is designed to analyze samples from cynomolgus monkeys dosed with human antibodies; it can be used for all human antibodies since it is independent of the specific antibody and its target. The generic applicability of the ADA assay is enabled by the use of (1) a murine anti-human Fc monoclonal antibody (MAb) as capture reagent; (2) a murine anti-cynomolgus monkey IgG MAb as detection reagent; and (3) an ADA positive control conjugate consisting of cynomolgus IgG complexed with human IgG. In its basic version, the generic ADA ELISA specifically detects only immune complexes formed in vivo. Validation of the ADA assay revealed a lower limit of quantitation of 15.6 ng/mL in serum samples. Intra-assay and inter-assay precision was characterized by a coefficient of variation of less than 10% and accuracy was within 8%. Matrix effects were low as evidenced by a mean recovery of 95%. In vitro pre-incubation of the serum samples with drug makes also the free ADA in the sample amenable to measurement by the immune complex ELISA as demonstrated by analysis of ADAs from two cynomolgus monkey studies with two different antibodies. The generic and versatile nature of this ADA assay favors its use in pilot pharmacokinetic and safety studies in cynomolgus monkeys during candidate selection of antibodies. The assay can help to explain unexpected drug clearance profiles, loss of efficacy or safety events caused by immune complexes and guide further development.
当前用于检测治疗性抗体的抗药物抗体（ADA）的桥接 ELISA 技术现状存在因循环药物干扰而导致假阴性结果的风险。去除样品中药物的方法或样品预处理技术（例如免疫复合物的酸解离）受到限制，既费力又可能破坏 ADA，从而再次导致假阴性结果。本文所述的免疫复合物 ELISA 提供了一种简单的解决方案。它旨在分析用人类抗体给药的食蟹猴的样品；由于它独立于特定的抗体及其靶标，因此可用于所有人类抗体。ADA 测定的通用适用性是通过使用 (1) 鼠抗人 Fc 单克隆抗体（MAb）作为捕获试剂；(2) 鼠抗食蟹猴 IgG MAb 作为检测试剂；和 (3) 由食蟹猴 IgG 与人 IgG 复合而成的 ADA 阳性对照缀合物来实现的。在其基本版本中，通用 ADA ELISA 特异性仅检测体内形成的免疫复合物。ADA 测定的验证表明，血清样品的定量下限为 15.6ng/mL。日内和日间精密度的特征是变异系数小于 10%，准确性在 8%以内。如通过对两种不同抗体的两项食蟹猴研究的 ADA 分析所示，基质效应较低，平均回收率为 95%。如通过对两种不同抗体的两项食蟹猴研究的 ADA 分析所示，如通过对两种不同抗体的两项食蟹猴研究的 ADA 分析所示，在药物与血清样品体外孵育的情况下，该免疫复合物 ELISA 也可用于测量游离 ADA。该 ADA 测定法的通用性和多功能性有利于在候选抗体的选择过程中在食蟹猴中进行初步药代动力学和安全性研究。该测定法有助于解释由免疫复合物引起的意外药物清除曲线、疗效丧失或安全性事件，并指导进一步的开发。
