Department of BioAnalytical Research & Development, Genentech, 1 DNA Way, South San Francisco, CA 94080, United States.
J Immunol Methods. 2010 Oct 31;362(1-2):101-11. doi: 10.1016/j.jim.2010.09.013. Epub 2010 Sep 22.
Electrochemiluminescence (ECL) assays have been widely used for the detection of anti-therapeutic antibodies (ATAs) against biotherapeutics. With the discontinuation of BioVeris (BV) ECL platform, an alternative technology was needed to replace BV assays to ensure continuous support of multi-year clinical studies. After evaluation of several immunoassay platforms, a novel homogeneous Biotin-digoxigenin (DIG) based bridging ELISA format was selected to develop an anti-rhuMAbX antibody screening assay to test serum samples from rheumatoid arthritis (RA) patients. With a homogeneous overnight sample incubation, the Biotin-DIG ELISA achieved comparable relative sensitivity and free drug tolerance to the previous BV ATA assay for rhuMAbX. To abrogate potential auto-antibody interference in RA sera, various assay conditions were thoroughly evaluated and a horseradish peroxidase (HRP)-conjugated chicken anti-DIG antibody was selected as the detection conjugate. Other potential interferences from serum Biotin, naturally occurring anti-avidin antibodies, and concomitant medications such as digoxin and hydrocortisone, which have similar structures to digoxigenin, were also investigated. Under optimized final assay conditions, the Biotin-DIG assay showed a relative sensitivity of approximately 11 ng/mL using a polyclonal anti-complementarity determining region (CDR) enriched positive control; the assay could detect 500 ng/mL of the positive control in the presence of approximately 27 μg/mL of rhuMAbX in RA serum. In addition, a confirmatory step was optimized for the assay based upon pre-incubating serum samples with an excess of free drug. Overall, the Biotin-DIG assay met the performance requirements for an ATA screening assay and had comparable sensitivity and drug tolerance to the BV assay; therefore this assay was a suitable replacement for the BV assay used for previous clinical studies of rhuMAbX. The Biotin-DIG based assay format can be broadly used as an effective screening platform for the detection of anti-therapeutic antibodies.
电化学发光（ECL）分析已广泛用于检测针对生物治疗药物的抗治疗性抗体（ATA）。随着 BioVeris（BV）ECL 平台的停用，需要一种替代技术来替代 BV 分析，以确保对多年临床研究的持续支持。在评估了几种免疫分析平台后，选择了一种新颖的均相生物素-地高辛（DIG）桥连 ELISA 格式，以开发一种抗 rhuMAbX 抗体筛选分析，以测试类风湿关节炎（RA）患者的血清样本。通过均相过夜样品孵育，Biotin-DIG ELISA 实现了与之前用于 rhuMAbX 的 BV ATA 分析相当的相对灵敏度和游离药物耐受性。为了消除 RA 血清中潜在的自身抗体干扰，彻底评估了各种分析条件，并选择辣根过氧化物酶（HRP）缀合的鸡抗 DIG 抗体作为检测缀合物。还研究了来自血清生物素、天然抗生物素蛋白抗体以及同时使用的药物（如地高辛和氢化可的松）的其他潜在干扰，这些药物与地高辛具有相似的结构。在优化的最终分析条件下，Biotin-DIG 分析使用多克隆抗互补决定区（CDR）富集的阳性对照物，相对灵敏度约为 11ng/mL；该分析可以在 RA 血清中存在约 27μg/mL 的 rhuMAbX 的情况下检测到 500ng/mL 的阳性对照物。此外，基于预孵育血清样品与过量游离药物的分析，优化了确认步骤。总体而言，Biotin-DIG 分析满足 ATA 筛选分析的性能要求，并且与 BV 分析具有相当的灵敏度和药物耐受性；因此，该分析是替代之前 rhuMAbX 临床研究中使用的 BV 分析的合适方法。Biotin-DIG 分析格式可广泛用作检测抗治疗性抗体的有效筛选平台。
