On the caveats of a multiplex test for SARS-CoV-2 to detect seroconversion after infection or vaccination

The Covid-19 pandemic, caused by SARS-CoV-2, resulted in more than 5 million deaths being one of the biggest challenges the world faces today. Here we present optimizations to all steps of an enzyme-linked immunosorbent assay (ELISA)-based test to detect IgG, IgA and IgM against the trimeric spike (S) protein, receptor binding domain (RBD), and N terminal domain of the nucleocapsid (N-NTD) protein of SARS-CoV-2. We discuss how to determine the specific thresholds for antibody positivity and its limitations according to the antigen used. We applied the assay in a cohort of 126 individuals from Rio de Janeiro, Brazil, consisting of 23 PCR-positive individuals; and 103 individuals without confirmed diagnosis for SARS-CoV-2 infection. To illustrate the differences in serological responses to vaccinal immunization, we applied the test in 18 individuals of our cohort before and after receive ChAdOx-1 nCoV-19 or CoronaVac vaccines. Taken together, our results show that the test can be customized at different stages depending on its application, enabling the user to analyze different cohorts, saving time, reagents, or samples. It also represents a valuable tool for elucidating the immunological consequences of new viral strains and for monitoring vaccination coverage and the duration of response to different immunization regimens.

由严重急性呼吸综合征冠状病毒2型（SARS-CoV-2）引发的新型冠状病毒肺炎（COVID-19）疫情已造成超500万人死亡，是当今全球面临的最重大公共卫生挑战之一。本研究针对基于酶联免疫吸附试验（ELISA）的检测方法全流程进行优化，该检测可靶向检测针对SARS-CoV-2三聚体刺突（S）蛋白、受体结合域（RBD）以及核衣壳蛋白N端结构域（N-NTD）的IgG、IgA及IgM抗体。本研究探讨了如何依据所用抗原确定抗体阳性检测的特异性阈值，以及该检测方法的局限性。我们将该检测方法应用于巴西里约热内卢的126名个体组成的研究队列，其中包括23名聚合酶链式反应（PCR）阳性个体，以及103名未确诊SARS-CoV-2感染的个体。为阐明疫苗免疫后的血清学反应差异，我们对队列中的18名个体在接种ChAdOx-1 nCoV-19或CoronaVac疫苗前后分别开展了检测。综合来看，本研究结果表明，该检测方法可根据应用场景在不同阶段进行定制，能够支持研究者分析不同队列，节省时间、试剂与样本。同时，该方法也可作为一项有价值的研究工具，用于阐明新型病毒株的免疫学影响，以及监测疫苗接种覆盖率和不同免疫方案下的抗体应答持续时长。