Department of Biological and Biomedical Sciences, Aga Khan University, Karachi, Pakistan.
Department of Pathology and Laboratory Medicine, Aga Khan University, Karachi, Pakistan.
PLoS One. 2021 Dec 10;16(12):e0259551. doi: 10.1371/journal.pone.0259551. eCollection 2021.
Individuals recovering from COVID-19 are known to have antibodies against the Spike and other structural proteins. Antibodies against Spike have been shown to display viral neutralization. However, not all antibodies against Spike have neutralizing ability although they may be cross-reactive. There is a need for easy-to-use SARS-CoV-2 neutralizing assays for the determination of virus-neutralizing activity in sera of individuals. Here we describe a PCR-based micro-neutralization assay that can be used to evaluate the viral neutralization titers of serum from SARS-CoV-2 infected individuals.
The SARS-CoV-2 strain used was isolated from a nasopharyngeal specimen of a COVID-19 case. The limiting dilution method was used to obtain a 50% tissue culture infective dose (TCID50) of Vero cells. For the micro-neutralization assay, 19 serum samples, with positive IgG titers against Spike Receptor-Binding Domain (RBD) were tested. After 24 hours, infected cells were inspected for the presence of a cytopathic effect, lysed and RNA RT-PCR conducted for SARS-CoV-2. PCR target Ct values were used to calculate percent neutralization/inhibition of SARS-CoV-2.
Out of 19 samples, 13 samples gave 100% neutralization at all dilutions, 1 sample showed neutralization at the first dilution, 4 samples showed neutralization at lower dilutions, while one sample did not demonstrate any neutralization. The RBD ODs and neutralization potential percentages were found to be positively correlated.
We describe a rapid RT-PCR-based SARS-CoV-2 microneutralization assay for the detection of neutralizing antibodies. This can effectively be used to test the antiviral activity of serum antibodies for the investigation of both disease-driven and vaccine-induced responses.
已知从 COVID-19 中康复的个体具有针对 Spike 和其他结构蛋白的抗体。已证明针对 Spike 的抗体具有病毒中和作用。但是，并非所有针对 Spike 的抗体都具有中和能力，尽管它们可能具有交叉反应性。需要易于使用的 SARS-CoV-2 中和测定法来确定个体血清中的病毒中和活性。在这里，我们描述了一种基于 PCR 的微中和测定法，可用于评估 SARS-CoV-2 感染个体血清中的病毒中和滴度。
使用从 COVID-19 病例的鼻咽标本中分离的 SARS-CoV-2 株。使用有限稀释法获得 Vero 细胞的 50％组织培养感染剂量（TCID50）。对于微中和测定，测试了 19 份针对 Spike 受体结合域（RBD）具有阳性 IgG 滴度的血清样品。24 小时后，检查感染细胞是否存在细胞病变效应，裂解并进行 SARS-CoV-2 的 RNA RT-PCR。PCR 目标 Ct 值用于计算 SARS-CoV-2 的中和/抑制百分比。
在 19 个样本中，有 13 个样本在所有稀释度下均达到 100％的中和作用，1 个样本在第一稀释度下显示中和作用，4 个样本在较低稀释度下显示中和作用，而一个样本则没有显示出任何中和作用。RBD OD 和中和潜力百分比呈正相关。
我们描述了一种快速基于 RT-PCR 的 SARS-CoV-2 微中和测定法，用于检测中和抗体。这可以有效地用于测试血清抗体的抗病毒活性，以研究疾病驱动和疫苗诱导的反应。
