Background A critical step in immunoassay development is the selection of the appropriate capture and detection antibodies. The use of suboptimal antibodies can drastically affect the quality of the final assay. Traditional selection methods involve a direct testing of potential antibodies in the final assay setting; a time-consuming and material-intensive exercise that is often too complex to test all possibilities. Alternatives, such as abstract K-on/K-off determinations via SPR or BLI can be costly and often show limited translatability to the ultimate assay format. Here, we describe a novel systematic approach to rapidly screen antibody-antigen interactions for key assay specifications and test for translatability of the findings. Methods Nanoliter volumes of potential capture antibodies were spotted onto the bottom of ELISA wells, generating a microarray of antibodies in each well (>11,000 spots per plate). For the analysis of capture antibody-antigen interactions, a titration series of biotinylated analyte in negative sample matrix was applied. Binding was visualized using strep-polyHRP, precipitating TMB and high resolution scanning of each ELISA well. Antibody pair performance was investigated using unlabeled analyte and biotinylated detection antibody options. Sensitivity, specificity, dynamic range, binding speed, binding strength and matrix compatibility were investigated using time courses, concentration ranges, dilutions, interferent spikes and pH/salt/detergent challenges. Signals were quantified to calculate and statistically compare each possible interaction. Results Antibody-antigen interactions were investigated via parallel microarray ELISA for two example immunoassays: the HBsAg and cTnI test. For both analytes, a panel of antibodies was screened revealing antibodies with diverse characteristics. Striking differences were observed depending on the sample matrix composition. Without optimization, antibodies could be identified showing dynamic assay ranges of 3-4 log orders. Time course experiments revealed high-affinity antibodies suitable for rapid diagnostic tests. For HBsAg, levels down to 0.01 IU/mL (∼40 pg/mL) could be reproducibly detected, which is well below the minimal requirement of 0.13 IU/mL. Reactivity against multiple virus serotypes could be confirmed. Interestingly, for the cTnI test, a strong synergistic effect was observed when specific monoclonal antibodies were combined in the capture mix, resulting in a 6-fold sensitivity enhancement compared to their separate performances. Finally, we show that the results obtained with the parallel microarray ELISA approach are highly translatable to the final assay format, ranging from bead-based dry-chemistry assays to chemiluminescent immunoassays. Conclusions The parallel microarray ELISA evaluation presents a robust and reliable method for identifying the best antibodies for an immunoassay. It allows for a systematic and quantitative screen of numerous antibody options within 1-2 weeks under real immunoassay conditions with minimal antibody amounts. This method has shown to be highly translatable and is customizable to accommodate required assay specifications.

中文翻译：

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B-036 使用平行微阵列 ELISA 系统评估抗体-抗原相互作用在免疫测定中的适用性


背景 免疫测定开发的关键步骤是选择合适的捕获和检测抗体。使用次优抗体会极大地影响最终测定的质量。传统的选择方法涉及在最终测定环境中直接测试潜在的抗体；这是一项耗时且耗费材料的练习，通常过于复杂，无法测试所有可能性。替代方案（例如通过 SPR 或 BLI 进行抽象 K-on/K-off 测定）可能成本高昂，而且通常对最终检测格式的可转换性有限。在这里，我们描述了一种新颖的系统方法，可以快速筛选抗体-抗原相互作用的关键测定规范并测试结果的可翻译性。方法 将纳升体积的潜在捕获抗体点样到 ELISA 孔的底部，在每个孔中生成抗体微阵列（每板 >11,000 个点）。为了分析捕获抗体-抗原相互作用，应用了阴性样品基质中生物素化分析物的一系列滴定。使用 strep-polyHRP、沉淀 TMB 和每个 ELISA 孔的高分辨率扫描来可视化结合。使用未标记的分析物和生物素化的检测抗体选项来研究抗体对的性能。使用时间进程、浓度范围、稀释度、干扰物尖峰和 pH/盐/洗涤剂挑战来研究灵敏度、特异性、动态范围、结合速度、结合强度和基质相容性。对信号进行量化以计算和统计比较每种可能的相互作用。结果 通过平行微阵列 ELISA 研究了两种免疫测定实例的抗体-抗原相互作用：HBsAg 和 cTnI 测试。 对于这两种分析物，筛选了一组抗体，揭示了具有不同特征的抗体。根据样品基质组成观察到显着差异。如果不进行优化，可以识别出显示 3-4 个对数级动态测定范围的抗体。时程实验揭示了适合快速诊断测试的高亲和力抗体。对于 HBsAg，可以重复检测到低至 0.01 IU/mL (∼40 pg/mL) 的水平，这远低于 0.13 IU/mL 的最低要求。可以确认针对多种病毒血清型的反应性。有趣的是，对于 cTnI 测试，当特定单克隆抗体组合在捕获混合物中时，观察到强烈的协同效应，与单独的性能相比，灵敏度提高了 6 倍。最后，我们表明，通过平行微阵列 ELISA 方法获得的结果高度可转化为最终的测定形式，范围从基于微珠的干化学测定到化学发光免疫测定。结论 平行微阵列 ELISA 评估提供了一种稳健可靠的方法来识别免疫测定的最佳抗体。它允许在真实免疫测定条件下以最小的抗体量在 1-2 周内对众多抗体选项进行系统和定量筛选。该方法已被证明具有高度可转化性，并且可以定制以适应所需的测定规范。