key: cord-0729370-xtom8d3z authors: Schallier, Anneleen; De Baets, Sarah; De Bruyne, Dirk; Dauwe, Kenny; Herpol, Margaux; Couck, Pedro title: Assay dependence of long-term kinetics of SARS-CoV-2 antibodies date: 2021-04-22 journal: Diagn Microbiol Infect Dis DOI: 10.1016/j.diagmicrobio.2021.115403 sha: d4447981d9aeadad1ad3109fa804a7f0666b7c17 doc_id: 729370 cord_uid: xtom8d3z Since the worldwide outbreak of the novel coronavirus (SARS-CoV-2), the question raised whether infected patients would elicit long-lasting protective immunity. Several companies developed serological assays for the detection of SARS-CoV-2 antibodies. In this study, we compared four different serology assays in convalescents up to seven months post-infection. Both Abbott assays showed a significative decrease of IgG antibodies over time. Whereas the Elecsys Anti‑SARS‑CoV‑2 N assay (Roche) initially showed a significant increase, antibody titers significantly decreased at the latest timepoint. Although not significant, the Elecsys Anti‑SARS‑CoV‑2 S assay (Roche) showed tendency towards increasing titers overtime. Our data showed that results of SARS-CoV-2 serology should be interpreted with caution. Coronavirus disease 2019 , caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread rapidly around the world. First cases of COVID-19 appeared in Belgium in February 2020. As seen worldwide, testing capacity for the diagnosis of acute infection by molecular techniques was initially very low. Consequently, indications for testing was limited (e.g. severely ill patients, physicians) (Sciensano) . By the end of April 2020, several companies developed serological assays for the detection of SARS-CoV-2 antibodies. These assays are utile for epidemiological purposes and follow-up of the immune status of patients (post-infection and/or vaccination). Few publications were published on the longitudinal follow-up of these antibodies. Furthermore, these publications showed discordant results (Muecksch et al., 2020) . In this study, we compared four different serology assays in convalescents up to seven months post-infection. This study was approved by the standing Committee on Ethics of the University Hospital Gent. Experiments were performed in accordance with the guidelines and regulations. All participants signed an informed consent. Serum samples were collected from a cohort of 54 patients. Infection has been confirmed by polymerase chain reaction assay in 43 of the 54 participants. The other 11 patients had symptoms compatible with COVID-19 disease, lived in close contact with proven COVID-19 patients, but were not tested because of limited test availability. A questionnaire, mentioning the symptoms confer the National Guidelines, was completed. None of the patients was hospitalized. Serum samples were collected at 4 different time points: Timepoint 0 (median 34 days after positive PCR/symptoms; 95 CI 34.7 to 41.3; maximum 72 days; minimum 13 days; n=54) -Timepoint 1 (median 78 days; 95 CI 77.1 to 84.1; maximum 116 days; minimum 59 days; n=50) -Timepoint 2 (median 145 days; 95 CI 142 to 149; maximum 179 days; minimum 122 days; n=48) -Timepoint 3 (median 223 days; 95 CI 225 to 233; maximum 261 days; minimum 200 days; n=49). Patients were enrolled in the study when a sample was obtained for a minimum of 3 timepoints. Four different immunoassays were performed according to manufacturer's instructions. The first assay is the SARS-CoV-2 IgG assay (Abbott, 6R86-32, Sligo, Ireland). The second assay is the SARS-CoV-2 IgG II Quant assay (Abbott, 6S60-22, Sligo, Ireland). Both assays are chemiluminescent microparticle immunoassays (CMIA). The third and fourth assay are respectively the Elecsys Anti-SARS-CoV-2 (Roche, 09203079190, Mannheim, Germany) and Elecsys Anti-SARS-CoV-2 S (Roche, 09289275190, Mannheim, Germany) assay. Both assays are based on electrochemiluminescence immunoassay "ECLIA" technique. Wilks' lambda MANOVA test was performed to test whether there are differences between the means of the antibody response in function of time. P-values <0,05 were considered statistically significant. As shown in figure 1 and 2, antibody responses for both Abbott assays showed a significant decrease in function of time. For assay 1, P-values were <0,001 between all timepoints. Furthermore, 30/49 patients showed false negative results on timepoint 3. Indeed, antibody titers of these patients dropped below the cut-off. For assay 2, P-values were <0,001, except between timepoints 1 and 2 and between timepoints 2 and 3. P-values were <0,01 for the latter timepoints. At the latest timepoint measured, 4/49 patients showed false negative results. Significant difference (P<0,001) was observed for different timepoints for the Elecsys Anti-SARS-CoV-2 assay (Roche). As shown in figure 1 and 2, an initial significant increase of antibody titers could be detected for this third assay. This increase is followed by a significant decrease towards timepoint 3. Only 1/49 patients showed false negative result at the latest timepoint. Although not significant, a tendence towards increasing titers was observed overtime with the Elecsys Anti-SARS-CoV-2 S assay (Roche). Since the start of the worldwide spread of COVID-19 virus, serology assays were developed to measure circulating antibody levels. These assays are useful for epidemiological surveys, vaccination strategy and prediction of immunity (Muecksch et al., 2020) . Although several studies showed initially promising sensitivity and specificity of the available serology assays, other publications announced rapid decay of anti-SARS-CoV-2 antibodies (Tanis et al., 2021 , Ibarrondo et al., 2020 . Studies investigating the long-term kinetics of antibody titers are crucial for the COVID-19 strategy. Serology assays for SARS-CoV-2 employ viral nucleoside (N) or spike surface protein (S) antigen. The viral spike protein is considered to be the preferred antigen, because it shows high specificity and shows to be the main antigen provoking neutralizing antibodies (Petherick, 2020). Therefore, S-based may be preferred to N-based assays (Muecksch et al., 2020) . In this study, we compared 4 different serology assays from 2 different companies. Both Abbott and Roche launched their first serology assay in April/May 2020. Both kits, the SARS-CoV-2 IgG assay (assay 1, Abbott) and Elecsys Anti-SARS-CoV-2 assay (assay 3, Roche) utilize N antigen. With the start of vaccination, both companies developed assays directed against the S protein: SARS-CoV-2 IgG II Quant assay (assay 2, Abbott) and SARS-CoV-2 S assay (assay 4, Roche). As demonstrated by Muecksch et al., the Abbott SARS-CoV-2 assay shows decreasing antibody levels in function of time, making this test not useful for epidemiological purposes. In concordance with Muecksch et al. and Gudbjartsson et al., we describe an initial increase in antibodies until 145 days (min 122 -max 179 days) for Elecsys Anti-SARS-CoV-2 assay (assay 3). But, at the latest timepoint in this study (about 7 months after the initial infection), the antibody titers of the latter assay decrease. This observation is in discordance with Gaebler et al. and Favresse et al., who showed increased levels up until respectively 6.2 months and 32 weeks. Overall, our results indicate that both N-protein based assays show decreasing antibody levels. Although not significant, a tendency towards increasing antibody titers was observed for the S-based Roche assay (assay 4). These data are in concordance with reports that describe increasing total antibodies, using pan-immunoglobulin assays (Schaffner et al., 2020; Gaebler et al., 2021) . To our knowledge, no other publication evaluated the S-based Abbott assay (assay 2). Although the decline was slower in time, compared to the N-based assay, we also observed the risk of false negative results at the latest timepoint by using this test. It has been described that most antibody responses in COVID-19 patients target in particular the S1 subunit and RBD region of the S viral protein. These regions are thought to elicit the most potent neutralizing effect (Kim et al., 2020) . Furthermore pan-immunoglobulin assays show better performance than isotype-specific assays (Schaffner et al., 2020) . In combination with the longlasting detection of antibody titers observed, one could state that the SARS-CoV-2 S assay (Roche) is the superior assay for epidemiologic purposes in this study. Unfortunately, up until now, there is no proof that these persistent levels of antibody titers will induce protection against a second COVID-19 infection. Promising data were published by Deng et al. Re-exposure to SARS-CoV-2 virus in convalescent monkeys showed no recurrence of COVID-19 disease. Further studies are needed to investigate which serology assays show the best prediction towards protective antibodies against subsequent exposures to SARS-CoV-2. Several companies urgently marketed COVID-19 serology assays. In this longitudinal study, we explored the kinetics of SARS-CoV-2 antibodies until 223 days post positive PCR or presence of symptoms by means of 4 different assays. We showed that results of these assays should be interpreted with caution. Although this study lacks information on correlation of antibodies with neutralizing activity, we could show that the pan-immunoglobulin SARS-CoV-2 S assay of Roche showed the best performance for epidemiological purposes. Primary exposure to SARS-CoV-2 protects against reinfection in rhesus macaques Long-term kinetics of anti-SARS-CoV-2 antibodies in a cohort of 197 hospitalized and non-hospitalized COVID-19 patients Evolution of antibody immunity to SARS-CoV-2. Nature Humoral Immune Response to SARS-CoV-2 in Iceland Rapid Decay of Anti-SARS-CoV-2 Antibodies in Persons with Mild Covid-19 Will SARS-CoV-2 Infection Elicit Long-Lasting Protective or Sterilising Immunity? Implications for Vaccine Strategies (2020). Front Immunol Longitudinal analysis of clinical serology assay performance and neutralising antibody levels in COVID19 convalescents Petherick A Developing antibody tests for SARS-CoV-2. Lancet Characterization of a Pan-Immunoglobulin Assay Quantifying Antibodies Directed against the Receptor Binding Domain of the SARS-CoV-2 S1-Subunit of the Spike Protein: A Population-Based Study Evaluation of four laboratory-based SARS-CoV-2 IgG antibody immunoassays Anneleen Schallier: conceptualization, supervision, writing-original draft Sarah De Baets: formal analysis, investigation, writing -review and editing Dirk De Bruyne: formal analysis, investigation formal analysis, writing -review and editing; Margaux Herpol: conceptualization, writing -review and editing; Pedro Couck: writing -review and editing Anneleen Schallier: conceptualization, supervision, writing-original draft Sarah De Baets: formal analysis, investigation, writing -review and editing Dirk De Bruyne: formal analysis, investigation formal analysis, writing -review and editing; Margaux Herpol: conceptualization, writing -review and editing