Lagousi Theano, Routsias John, Spoulou Vana
First Department of Paediatrics, "Aghia Sophia" Children's Hospital, Immunology and Vaccinology Research Laboratory and Infectious Diseases Department "MAKKA", Athens Medical School, 11527 Athens, Greece.
University Research Institute for the Study of Genetic & Malignant Disorders in Childhood, First Department of Paediatrics, Aghia Sofia Children's Hospital, Athens Medical School, 11527 Athens, Greece.
Diagnostics (Basel). 2021 Oct 23;11(11):1970. doi: 10.3390/diagnostics11111970.
Prompt COVID-19 diagnosis is urgently required to support infection control measures. Currently available serological tests for measuring SARS-CoV-2 antibodies use different target antigens, although their sensitivity and specificity presents a challenge. We aimed to develop an "in-house" serological ELISA to measure antibodies against SARS-CoV-2 by combining different protein antigens. Sera ( = 44) from COVID-19-confirmed patients were evaluated against different SARS-CoV-2 protein antigens and all potential combinations using ELISA. Patients' sera were also evaluated against commercially available ELISA diagnostic kits. The mixture containing RBD 2.5 μg/mL, S2 1 μg/mL and N 1.5 μg/mL was found to be the most potent. Plates were incubated with patients' sera (1:100), and goat anti-human alkaline phosphatase-conjugated IgG, ΙgM and IgA antibody was added. The cut-off value for each assay was determined using the mean optical density plus two standard deviations of pre-pandemic controls. The "in-house" ELISA displayed 91% sensitivity and 97% specificity for IgG antibodies, whereas its sensitivity and specificity for IgM and IgA were 75% and 95% and 73% and 91%, respectively. The "in-house" ELISA developed here combined three SARS-CoV-2 antigens (RBD, S2 and N) as capture antigens and displayed comparable and even higher sensitivity and specificity than otherwise quite reliable commercially available ELISA diagnostic kits.
快速进行新冠病毒病（COVID-19）诊断对于支持感染控制措施至关重要。目前用于检测严重急性呼吸综合征冠状病毒2（SARS-CoV-2）抗体的血清学检测使用不同的靶抗原，尽管其敏感性和特异性存在挑战。我们旨在开发一种“内部”血清学酶联免疫吸附测定（ELISA），通过组合不同的蛋白质抗原来检测抗SARS-CoV-2抗体。使用ELISA对44份来自COVID-19确诊患者的血清针对不同的SARS-CoV-2蛋白质抗原以及所有可能的组合进行评估。患者血清也使用市售ELISA诊断试剂盒进行评估。发现含有2.5μg/mL受体结合域（RBD）、1μg/mL S2和1.5μg/mL核衣壳蛋白（N）的混合物效果最佳。将酶标板与患者血清（1:100）孵育，然后加入山羊抗人碱性磷酸酶标记的IgG、IgM和IgA抗体。每个检测的临界值使用大流行前对照的平均光密度加上两个标准差来确定。“内部”ELISA对IgG抗体的敏感性为91%，特异性为97%，而其对IgM和IgA的敏感性和特异性分别为75%和95%以及73%和91%。这里开发的“内部”ELISA将三种SARS-CoV-2抗原（RBD、S2和N）作为捕获抗原，与其他相当可靠的市售ELISA诊断试剂盒相比，显示出相当甚至更高的敏感性和特异性。
