Kolesov Denis E, Sinegubova Maria V, Dayanova Lutsia K, Dolzhikova Inna V, Vorobiev Ivan I, Orlova Nadezhda A
Laboratory of Mammalian Cell Bioengineering, Skryabin Institute of Bioengineering, Research Center of Biotechnology of the Russian Academy of Sciences, 117312 Moscow, Russia.
Laboratory of Glycoproteins Biotechnology, Institute of Bioorganic Chemistry of the Russian Academy of Sciences, 117997 Moscow, Russia.
Diagnostics (Basel). 2022 Feb 3;12(2):393. doi: 10.3390/diagnostics12020393.
The humoral response to the SARS-CoV-2 S protein determines the development of protective immunity against this infection. The standard neutralizing antibodies detection method is a live virus neutralization test. It can be replaced with an ELISA-based surrogate virus neutralization test (sVNT), measuring the ability of serum antibodies to inhibit complex formation between the receptor-binding domain (RBD) of the S protein and the cellular ACE2 receptor. There are conflicting research data on the sVNT methodology and the reliability of its results. We show that the performance of sVNT dramatically improves when the intact RBD from the Wuhan-Hu-1 virus variant is used as the plate coating reagent, and the HRP-conjugated soluble ACE2 is used as the detection reagent. This design omits the pre-incubation step in separate tubes or separate microplate and allows the simple quantification of the results using the linear regression, utilizing only 3-4 test sample dilutions. When this sVNT was performed for 73 convalescent plasma samples, its results showed a very strong correlation with VNT (Spearman's Rho 0.83). For the RBD, bearing three amino acid substitutions and corresponding to the SARS-CoV-2 beta variant, the inhibitory strength was diminished for 18 out of 20 randomly chosen serum samples, and the magnitude of this decrease was not similar to the change in overall anti-RBD IgG level. The sVNT assay design with the ACE2-HRP is preferable over the assay with the RBD-HRP reagent and is suitable for mass screening of neutralizing antibodies titers.
对严重急性呼吸综合征冠状病毒2（SARS-CoV-2）刺突蛋白（S蛋白）的体液反应决定了针对这种感染的保护性免疫的发展。标准的中和抗体检测方法是活病毒中和试验。它可以被基于酶联免疫吸附测定（ELISA）的替代病毒中和试验（sVNT）所取代，后者用于测量血清抗体抑制S蛋白的受体结合域（RBD）与细胞血管紧张素转换酶2（ACE2）受体之间形成复合物的能力。关于sVNT方法及其结果的可靠性存在相互矛盾的研究数据。我们发现，当使用来自武汉-1病毒株的完整RBD作为酶标板包被试剂，并使用辣根过氧化物酶（HRP）偶联的可溶性ACE2作为检测试剂时，sVNT的性能会显著提高。这种设计省略了在单独试管或单独酶标板中的预孵育步骤，并且仅使用3-4个测试样品稀释度，就能通过线性回归对结果进行简单定量。当对73份康复期血浆样本进行这种sVNT检测时，其结果与病毒中和试验（VNT）显示出非常强的相关性（斯皮尔曼等级相关系数为0.83）。对于携带三个氨基酸替换且对应于SARS-CoV-2β变异株的RBD，在随机选择的20份血清样本中，有18份的抑制强度降低，且这种降低的幅度与总体抗RBD IgG水平的变化不同。与使用RBD-HRP试剂的检测方法相比，采用ACE2-HRP的sVNT检测设计更优，适用于中和抗体滴度的大规模筛查。
