Roy Daisy R, Kemp Troy J, Haynesworth Katarzyna, Loftus Sarah A, Pinto Ligia A
Vaccine, Immunity, and Cancer Directorate, Frederick National Laboratory for Cancer Research, Frederick, Maryland, USA.
Microbiol Spectr. 2023 Mar 16;11(2):e0389822. doi: 10.1128/spectrum.03898-22.
SARS-CoV-2 antibody testing is important for seroprevalence studies and for evaluating vaccine immune responses. We developed and validated a Luminex bead-based multiplex serology assay for measuring IgG levels of anti-SARS-CoV-2 antibodies against full-length spike (S), nucleocapsid (N), and receptor-binding domains (RBDs) of wild-type, RBD N501Y mutant, RBD E484K mutant, RBD triple mutant SARS-CoV-2 proteins, Sars-CoV-1, MERS-CoV, and common human coronaviruses, including SARS-CoV-2, OC43, 229E, HKU1, and NL63. Assay cutoff values, sensitivity, and specificity were determined using samples from 160 negative controls and 60 PCR-confirmed, SARS-CoV-2-infected individuals. The assay demonstrated sensitivities of 98.3%, 95%, and 100% and specificities of 100%, 99.4%, and 98.8% for anti-(S), -N, and -RBD, respectively. Results are expressed as IgG antibody concentrations in BAU/mL, using the WHO international standard (NIBSC code 20/136) for anti-SARS-CoV-2 IgG antibodies. When the multiplex assay was performed and compared with singleplex assays, the IgG antibody measurement geometric mean ratios were between 0.895 and 1.122, and no evidence of interference was observed between antigens. Lower and upper IgG concentration limits, based on accuracy (between 80% and 120%), precision (percent relative standard deviation, ≤25%), and sample dilutional linearity (between 75% and 125%), were used to establish the assay range. Precision was established by evaluating 24 individual human serum samples obtained from vaccinated and SARS-CoV-2-infected individuals. The assay provided reproducible, consistent results with typical coefficients of variation of ≤20% for all assays, irrespective of the run, day, or analyst. Results indicate the assay has high sensitivity and specificity and thus is appropriate for use in measuring SARS-CoV-2 IgG antibodies in infected and vaccinated individuals. The SARS-CoV-2 pandemic resulted in the development and validation of multiple serology tests with variable performance. While there are multiple SARS-CoV-2 serology tests to detect SARS-CoV-2 antibodies, the focus is usually either on only one antigen at a time or multiple proteins from only one SARS-CoV-2 variant. These tests usually do not evaluate antibodies against viral proteins from different SARS-CoV-2 variants or from other coronaviruses. Here, we evaluated a multiplex serology test based on Luminex technology, where antibodies against multiple domains of SARS-CoV-2 wild type, SARS-CoV-2 mutants, and common coronavirus antibodies are detected simultaneously in a single assay. This Luminex-based multiplex serology assay can enhance our understanding of the immune response to SARS-CoV-2 infection and vaccination.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）抗体检测对于血清流行率研究和评估疫苗免疫反应至关重要。我们开发并验证了一种基于Luminex微珠的多重血清学检测方法，用于测量针对野生型、受体结合域（RBD）N501Y突变体、RBD E484K突变体、RBD三重突变体SARS-CoV-2蛋白、严重急性呼吸综合征冠状病毒1（Sars-CoV-1）、中东呼吸综合征冠状病毒（MERS-CoV）以及包括SARS-CoV-2、OC43、229E、HKU1和NL63在内的常见人类冠状病毒的全长刺突（S）、核衣壳（N）的抗SARS-CoV-2抗体的IgG水平。使用来自160名阴性对照和60名经PCR确认的SARS-CoV-2感染个体的样本确定检测临界值、敏感性和特异性。该检测方法针对抗S、抗N和抗RBD的敏感性分别为98.3%、95%和100%，特异性分别为100%、99.4%和98.8%。结果以抗SARS-CoV-2 IgG抗体的BAU/mL表示，采用世界卫生组织国际标准（NIBSC编号20/136）。当进行多重检测并与单重检测进行比较时，IgG抗体测量几何平均比值在0.895至1.122之间，未观察到抗原之间的干扰迹象。基于准确性（80%至120%之间）、精密度（相对标准偏差百分比，≤25%）和样本稀释线性（75%至125%之间）确定IgG浓度下限和上限，以建立检测范围。通过评估从接种疫苗和感染SARS-CoV-2的个体获得的24份个体人血清样本确定精密度。无论运行、日期或分析人员如何，该检测方法对于所有检测均提供可重复、一致的结果，典型变异系数≤20%。结果表明该检测方法具有高敏感性和特异性，因此适用于测量感染和接种疫苗个体中的SARS-CoV-2 IgG抗体。SARS-CoV-2大流行导致开发并验证了多种性能各异的血清学检测方法。虽然有多种SARS-CoV-2血清学检测方法来检测SARS-CoV-2抗体，但重点通常要么一次仅针对一种抗原，要么仅针对一种SARS-CoV-2变体的多种蛋白。这些检测方法通常不评估针对不同SARS-CoV-2变体或其他冠状病毒的病毒蛋白的抗体。在此，我们评估了一种基于Luminex技术的多重血清学检测方法，该方法可在一次检测中同时检测针对SARS-CoV-2野生型、SARS-CoV-2突变体的多个结构域以及常见冠状病毒抗体的抗体。这种基于Luminex的多重血清学检测方法可以增强我们对SARS-CoV-2感染和疫苗接种免疫反应的理解。
