key: cord-0878956-7raieam7 authors: Weidner, Lisa; Gänsdorfer, Simon; Unterweger, Stephan; Weseslindtner, Lukas; Drexler, Camilla; Farcet, Maria; Witt, Volker; Schistal, Elisabeth; Schlenke, Peter; Kreil, Thomas R.; Jungbauer, Christof title: Quantification of SARS-CoV-2 antibodies with eight commercially available immunoassays date: 2020-07-06 journal: J Clin Virol DOI: 10.1016/j.jcv.2020.104540 sha: c373e7bffb9d14be5a4119cbd60d2496200e1984 doc_id: 878956 cord_uid: 7raieam7 Since the emergence of SARS-CoV-2 numerous antibody assays have become available, demonstrating different performance characteristics. This study focused on a quantitative correlation between different commercial assays and a neutralization test (NT). Comparative data is needed as a basis for the production of convalescent plasma and potential interpretations COVID-19 immunity. Sera of 100 SARS-CoV-2 convalescent plasma donors were collected and SARS-CoV-2 antibodies were characterized using three different IgG-ELISAs EUROIMMUN IgG and NCP-IgG ELISA, Wantai ELISA), two CLIA (Elecsys, LIAISON) and two lateral flow tests (MEDsan IgM/IgG-Rapid-Test, Wantai Rapid Test) and subsequently correlated to neutralization titers. The Wantai ELISA and the Elecsys provide the highest sensitivities in this sample (98 and 95 percent respectively). Titers with the best overall quantitative correlation to the NT titer were obtained with the Euroimmun IgG ELISA assay (Rho=0.759) and the Wantai ELISA assay (Rho=0.729). An infection without fever and negative or weakly positive reactions in the Wantai Rapid test were negative predictive factors for NT titers >1:200 (negative predictive value of 92% and 92% respectively, combination of both 100%). The Wantai ELISA titer could be a suitable substitute for NT. An adequate pooling strategy of plasma units additionally could compensate deviations of individual antibody titers. 2 patients during seroconversion. 3, 4, 5, 6, 7, 8, 9 However, there are still numerous unknown characteristics, correlations and performance of the available tests. 10 In recent months convalescent plasma has gained attention as a treatment option for COVID-19. 11, 12 Currently it is being used in countries around the world with 102 live studies registered. 13 Hence there is a growing demand for high-titer plasma donations as an neutralization test (NT) titer of at least 1:320 for therapeutic plasma is suggested. 14 NT, seen as gold standard for assessing specific immunity and a benchmark for other antibody assays, is a biological assay requiring individual tests in several laboratories with incubation times of 5 -7 days. This complexity and the need for increased biosafety level 3 precautions makes it difficult for routine testing on a large scale. 15, 16 However, it remains the only test which demonstrates the neutralization performance of antibodies instead of just indicating their presence. Until now, no readily available alternative has been identified as a substitute for the virus neutralization test titer. The approach of this study is to find an alternative assay that is simple and fast to perform, delivers acceptable correlation to the NT and is commercially available. Moreover, this paper examines factors that predict high or low titers in individuals which can be used as selection criteria for SARS-CoV 2 convalescent plasma donors, and to increase the collection of units with sufficient NT titer without the need for advanced screening using NT. Therefore, a new approach was established in which immunoassays were performed using serial dilutions of the samples. Additionally the rapid tests (lateral flow) were rated optically by the strength of their reaction. J o u r n a l P r e -p r o o f METHODS Sera of 100 convalescent plasma donors collected between 26 and 61 days (median 47 days, standard deviation 6.6 days) after onset of COVID-19 symptoms were tested using NT, 3 ELISA assays, 2 CLIA and 2 lateral flow tests. All donors have tested NAT (Nucleic Acid Test) positive for SARS-CoV-2 from nasopharyngeal or pharyngeal swab during initial diagnostics. The WHO progression scale 17 for COVID-19 was used and common symptoms 18 were noted. The donors were asked retrospectively about fever, cough, loss of taste and smell, headache, fatigue, gastrointestinal symptoms, body aches and sore throat during the period of their infection. The Euroimmun SARS-CoV-2 IgG ELISA (Euroimmun, Lübeck, Germany) (EI IgG ELISA) which uses a recombinant protein of the S1 domain (spike protein) as a target was performed on an Results with a ratio greater than 1 are considered positive and there are no borderline or equivocal results for this test. Titers of 3 samples had to be extrapolated due to a lack of material for further dilution above 1:320. 21 All samples were tested in serial dilutions beginning at 1:2.5 for the Elecsys and the Wantai ELISA and 1:5 for the EI IgG ELISA. For the EI NCP ELISA, samples, as recommended in the package insert, should be diluted 1:101 for testing, however a dilution of 1:20 was chosen. The diluent used was in accordance with assay instructions (dilution buffer (EI ELISAs), PBS (Wantai ELISA) or Multi Assay Diluent (Elecsys)). The titer was designated as the last dilution that yielding a positive or borderline result. For both Euroimmun SARS-CoV-2 IgG ELISA and Euroimmun SARS-CoV-2 NCP IgG ELISA (Euroimmun, Lübeck, Germany) a dilution of 1:101 is recommended. The ratios for this dilution were linearly interpolated based on the results obtained for 1:80 and 1:160. Roche Elecsys Anti-SARS-CoV-2 (Roche Diagnostics, Rotkreuz, Switzerland) (Elecsys) was performed on the Cobas e801. SARS-CoV-2 nucleocapsid (N) antigen is used as a target and detected with an electrochemilumescence sandwich assay (ECLIA). Results are semi quantitative and are expressed as qualitative statements (reactive/non-reactive). A Cut-off-Index (COI) ≥ 1 is considered reactive. 22 The LIAISON® SARS-CoV-2 S1/S2 IgG (DiaSorin S.p.A., Saluggia, Italy) (LIAISON) is a CLIA which detects antibodies reactive with the spike protein (S1/S2 domain). The results of neutralization test and Euroimmun IgG ELISA 1:100 Ratios of these samples were described earlier as Dispatch. 26 Statistical analysis was performed using SPSS Version 23. All metric data were tested for normal distribution using a Kolmogorov-Smirnov-Test at a level of significance of 0.05. All rations except those derived from Euroimmun SARS-CoV-2 NCP IgG ELISA lacked normal distribution. Therefore all bivariate correlations were calculated using Spearman analysis and were assumed significant when p<0.05. For determining negative (NPV) and positive predictive values (PPV), Crosstabs and a Chi 2 test were used. In total 93 individuals of this group experienced no or mild symptoms classified as 1 or 2 according to WHO progression scale. Only 6 donors had symptoms that classified them as WHO 3 -6. Included donors stated that they experienced following symptoms in in descending order: 63% fever, 48% headache, 44% body aches, 43% loss of taste and smell, 40% cough, 31% J o u r n a l P r e -p r o o f fatigue, 23% gastrointestinal symptoms, 29% sore throat. Fever was more common amongst men with 73.8% in comparison to women with 47.4% (T-Test p<0.05). A symptom that positively correlated with the titer of the neutralization test was fever (p<0.01). The mean NT50 titer was 1:305 in the group of 63 donors who experienced fever during COVID-19 and therefore significantly higher (p=0.001) than in the group lacking fever with a mean NT titer of 1: 107. The negative predictive value was 91.7% for donors who did not experience a fever to also have an NT titer below 1:200. The WHO progression scale correlated with the NT titer as well, but slightly weaker than the symptom fever  = 0.304). The neutralization test showed a sensitivity of 99.00%. 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