Shalash Ahmed O, Azuar Armira, Madge Harrison Y R, Modhiran Naphak, Amarilla Alberto A, Liang Benjamin, Khromykh Alexander A, Watterson Daniel, Young Paul R, Toth Istvan, Skwarczynski Mariusz
School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, QLD 4072, Australia.
Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St. Lucia, QLD 4072, Australia.
Vaccines (Basel). 2021 Dec 16;9(12):1493. doi: 10.3390/vaccines9121493.
This protocol describes an ELISA-based procedure for accurate measurement of SARS-CoV-2 spike protein-receptor binding domain (RBD) neutralization efficacy by murine immune serum. The procedure requires a small amount of S-protein/RBD and angiotensin converting enzyme-2 (ACE2). A high-throughput, simple ELISA technique is employed. Plate-coated-RBDs are allowed to interact with the serum, then soluble ACE2 is added, followed by secondary antibodies and substrate. The key steps in this procedure include (1) serum heat treatment to prevent non-specific interactions, (2) proper use of blank controls to detect side reactions and eliminate secondary antibody cross-reactivity, (3) the addition of an optimal amount of saturating ACE2 to maximize sensitivity and prevent non-competitive co-occurrence of RBD-ACE2 binding and neutralization, and (4) mechanistically derived neutralization calculation using a calibration curve. Even manually, the protocol can be completed in 16 h for >30 serum samples; this includes the 7.5 h of incubation time. This automatable, high-throughput, competitive ELISA assay can screen a large number of sera, and does not require sterile conditions or special containment measures, as live viruses are not employed. In comparison to the 'gold standard' assays (virus neutralization titers (VNT) or plaque reduction neutralization titers (PRNT)), which are laborious and time consuming and require special containment measures due to their use of live viruses. This simple, alternative neutralization efficacy assay can be a great asset for initial vaccine development stages. The assay successfully passed conventional validation parameters (sensitivity, specificity, precision, and accuracy) and results with moderately neutralizing murine sera correlated with VNT assay results (R = 0.975, = 25), demonstrating high sensitivity.
本方案描述了一种基于酶联免疫吸附测定（ELISA）的程序，用于通过小鼠免疫血清准确测量严重急性呼吸综合征冠状病毒2（SARS-CoV-2）刺突蛋白受体结合域（RBD）的中和效力。该程序需要少量的S蛋白/RBD和血管紧张素转换酶2（ACE2）。采用了一种高通量、简单的ELISA技术。将包被在酶标板上的RBD与血清进行孵育，然后加入可溶性ACE2，接着加入二抗和底物。该程序的关键步骤包括：（1）血清热处理以防止非特异性相互作用；（2）合理使用空白对照以检测副反应并消除二抗交叉反应性；（3）加入适量的饱和ACE2以最大限度提高灵敏度，并防止RBD-ACE2结合与中和的非竞争性同时发生；（4）使用校准曲线进行基于机制的中和计算。即使手动操作，该方案也可在16小时内完成对30多个血清样本的检测；这包括7.5小时的孵育时间。这种可自动化的高通量竞争性ELISA检测方法可以筛选大量血清，并且由于不使用活病毒，因此不需要无菌条件或特殊的防护措施。与 “金标准” 检测方法（病毒中和滴度（VNT）或蚀斑减少中和滴度（PRNT））相比，后者既费力又耗时，且由于使用活病毒而需要特殊的防护措施。这种简单的替代性中和效力检测方法对于疫苗研发的初始阶段可能是一项非常有用的工具。该检测方法成功通过了常规验证参数（灵敏度、特异性、精密度和准确度），并且用具有中等中和能力的小鼠血清所得到的结果与VNT检测结果相关（R = 0.975，n = 25），显示出高灵敏度。
