key: cord-1027700-x9x54fxw
authors: Flinck, Heini; Rauhio, Anne; Luukinen, Bruno; Lehtimäki, Terho; Haapala, Anna-Maija; Seiskari, Tapio; Aittoniemi, Janne
title: Comparison of two fully automated tests detecting antibodies against nucleocapsid N and spike S1/S2 proteins in COVID-19
date: 2020-08-29
journal: Diagn Microbiol Infect Dis
DOI: 10.1016/j.diagmicrobio.2020.115197
sha: fa18660c4966d42ed7b407124147aa6dec227c55
doc_id: 1027700
cord_uid: x9x54fxw

Automated assays for detecting SARS-CoV-2 antibodies in COVID-19 diagnostics have recently come available. We compared the performance of the Elecsys® Anti-SARS-CoV-2 and LIAISON® SARS-CoV-2 S1/S2 IgG tests. The seroconversion panel comprised of 120 samples from 13 hospitalized COVID-19 patients. For the sensitivity and specificity testing, samples from COVID-19 outpatients >15 days after positive NAAT result (n = 35), and serum control samples collected before the COVID-19 era (n = 161) were included in the material. Samples for the detection of possible cross-reactions were also tested. Based on our results, the SARS-CoV-2 antibodies can be quite reliable detected 2 weeks after NAAT positivity and 3 weeks after the symptom onset with both tests. However, since some COVID-19 patients were positive only with Elecsys®, the antibodies should be screened against N-antigen (Elecsys®), and reactive samples confirmed with S antigen (LIAISON®), but the both results should be reported. In some COVID-19 patients the serology can remain negative.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 , which was for the first time met in China in the end of the year 2019 (Zhu et al. 2020) . After that, the virus has caused a severe pandemic (Coronavirus COVID-19 Global Cases by the Center for Systems Science and Engineering (CSSE) at Johns Hopkins University, 2020). The acute COVID-19 is diagnosed by nucleic acid amplification test (NAAT). SARS-CoV-2 antibodies are formed in the blood usually within 2-3 weeks after infection (Okba et al. 2020) , and their determination can be used in epidemiological surveys and as a support in the diagnostics of prolonged and obscure cases. However, CE marked, in vitro diagnostics (IVD) suitable and US Food and Drug Administration (FDA) Emergency Use Authorized (EUA) SARS-CoV-2 antibody tests have not come on the market until recently, and there exists a few articles on the performance of fully automated test platforms (Egger et al. 2020; Kohmer et al. 2020; Merrill et al. 2020; Montesinos et al. 2020; Plebani et al. 2020a; Tang et al. 2020a; Tang et al. 2020b; Tré-Hardy et al. 2020) . In this paper, we compared the performance of the fully automated Elecsys ® Anti-SARS-CoV-2 test detecting antibodies against nucleocapsid N protein and LIAISON ® SARS-CoV-2 S1/S2 IgG test detecting antibodies against spike protein S1-and S2-antigens. 

SARS-CoV-2 antibodies were analysed using Elecsys ® Anti-SARS-CoV-2 test (Roche Diagnostics GmbH, Mannheim, Germany) detecting the antibodies against nucleocapsid N protein, and LIAISON ® SARS-CoV-2 S1/S2 IgG test (DiaSorin S.p.A., Saluggia, Italy) detecting the antibodies against spike (S) protein S1-and S2-antigens. Primary COVID-19 diagnosis had been based on in-house real time RT-PCR test detecting Egene target sequence according to Corman , Allplex TM 2019-nCoV Assay (Seegene

The antibody kinetics of the patients in the seroconversion panel is illustrated in Figure 1 . and the seroconversion times in Figure 2 . The time interval from the positive NAAT result to seroconversion was 5 days (median, range 0-11 days) with Elecsys ® and 7 days (median, range 2-13 days) with LIAISON ® . The seroconversion was first detected with Elecsys ® in ten and with LIAISON ® in two patients, and at the same time in one patient. In both methods, the seroconversion had happened in 85% (11/13) of the patients within 8 days and in all within 13 days after the positive NAAT result. The time interval from the onset of symptoms to seroconversion was 11 days with Elecsys ® (median, range 7-17 days) and 12 days (median, range 8-21)

with LIAISON ® , respectively. Although in some studies a decline in SARS-CoV-2 antibodies has been detected a few weeks after seroconversion (Favresse et al. 2020a : Seow et al. 2020 , in our seroconversion panel the antibody trend was rising in all patients with both methods. However, the follow-up time was quite short.

Elecsys ® detects total antibodies against N protein, while LIAISON ® detects only IgG antibodies against S1

and S2 antigens. The S protein is an immunogenic surface structure of the SARS-CoV-2 involved in the virus attachment to the host cells, and its functional subunits (S1 and S2) are used in the immunoassays. N protein is, in turn, the major structural protein of the SARS-CoV-2 involved in the replication processes of the virus (Infantino et al. 2020). The positive antibody test result refers to that the person has had COVID-19, but it does not definitely tell about protective immune response. N protein based tests may be more sensitive to detect past COVID-19, but S protein may be a possible target for neutralizing antibodies, and SARS-CoV-2 antibodies against that antigen may better predict the protective immunity (Walls et al. 2020 ). It has been suggested that the immune response against S antigens might become earlier than against N antigen ). However, in some clinical test comparisons the observed seroconversion time has been shorter with the tests detecting total antibodies to N antigen compared to those detecting IgG antibodies to S antigens, though the studies using systematic seroconversion panels with follow-up samples are sparse (Montesinos et al. 2020; Tang et al. 2020a ). In our seroconversion panel, the first positive result was detected in most cases earlier with Elecsys ® using N antigen than with LIAISON ® using S1/S2 antigens.

Since it has been shown that SARS-CoV-2 IgG and IgM antibodies can occur simultaneously or sequentially either IgG or IgM first (Long et al. 2020) , it can be speculated whether the IgM in the Elecsys ® total antibody composition causes the earlier positive reaction in some cases compared to LIAISON ® . However, it is also possible that it is a result of differences in the test chemistry or the antibody response to different antigens J o u r n a l P r e -p r o o f Journal Pre-proof (Long et al. 2020; Tang et al. 2020b) . The seroconversion times with Elecsys ® and LIAISON ® in our study were well in line with the other studies (Okba et al. 2020; Egger et al. 2020 . We applied also a receiver operating characteristic (ROC) curve performance analysis, and determined the optimal cutoff for the tests in our material using Youden index. The optimal cut-off for Elecsys ® was 0.137 COI with the sensitivity and specificity of 97.5% and 96.9%, and for LIAISON ® 11.9 AU/mL with the sensitivity and specificity of 90.0% and 96.9%, respectively. According to our results the optimized cuf-off for Elecsys ® was well in line with the other studies, but for LIAISON ® it was somewhat higher. These optimized cut-offs needs Cross-reactivities were tested for several conditions as shown in Table 2 . Two human coronavirus (HCoV) -OC43 positive patients had also a positive LIAISON ® test result 60 and 70 days after HCoV-OC43 diagnosis (levels 19 and 21 AU/mL, respectively). However, antibodies against SARS-CoV-2 N protein by Elecsys ® were totally negative (0.076 and 0.084 COI, respectively). Since the samples had been collected in April and May 2020, it is possible that these patients may also have had a non-diagnosed COVID-19 and thus specific SARS-CoV-2 antibodies, but it is unlikely, since the COVID-19 morbidity rates in Finland have so far remained considerably low (134 diagnosed cases/100 000 persons until July 31, 2020), and the seroprevalence has been below 0.5% in general population determined by a golden standard method i.e. Otherwise all the cross-reactivity results were negative. SARS-CoV-2 belongs to Betacoronaviruses like HCoV-OC43 and HCoV-HKU1. HCoV-229E and HCoV-NL63 belong to Alphacoronaviruses. The crossreactions are usually seen within Alpha-and Betacoronavirus genera but not between them (Huang et al. 2020) .

In this study, we compared the performance of the fully automated Elecsys ® Anti-SARS-CoV-2 test detecting antibodies against nucleocapsid N protein and LIAISON ® SARS-CoV-2 S1/S2 IgG test detecting antibodies against spike protein S1-and S2-antigens. The seroconversion was detected in most cases earlier with Elecsys ® than with LIAISON ® , but the antibodies could be quite reliable detected 2 weeks after NAAT positivity and 3 weeks after the symptom onset with both methods. Elecsys ® was somewhat more sensitive and specific than LIAISON ® , but the differences were minor. However, since some patients were seropositive only with Elecsys ® , we conclude that the SARS-CoV-2 antibodies should be screened against N antigen (Elecsys ® ), and reactive samples confirmed with S antigen test (LIAISON ® ), but the both test results should be reported parallel for clinical evaluation and related to the patient's clinical picture. Furthermore, in some patients the COVID-19 serology may remain completely negative. Because clear guidelines for the use of SARS-CoV-2 serology are lacking at the moment, these aspects should be taken into consideration when these processes are assessed. 

Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR

Coronavirus COVID-19 Global Cases by the Center for Systems Science and Engineering

Comparison of the Elecsys® Anti-SARS-CoV-2 immunoassay with the EDI™ enzyme linked immunosorbent assays for the detection of SARS-CoV-2 antibodies in human plasma

Unexpected kinetics of anti-SARS

CoV-2 total antibodies in two patients with chronic lymphocytic leukemia

Clinical performance of the Elecsys electrochemiluminescent immunoassay for the detection of SARS-CoV-2 total antibodies

Response of anti-SARS-CoV-2 total antibodies to nucleocapsid antigen in COVID-19 patients: a longitudinal study

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Microcrystalline chitosan is ineffective to decrease plasma lipids in both apolipoprotein E epsilon 4 carriers and non-carriers: a long-term placebocontrolled trial in hypercholesterolaemic volunteers

Evaluation of Nucleocapsid and Spike Protein-Based Enzyme-Linked Immunosorbent Assays for Detecting Antibodies against SARS-CoV-2

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Towards the rational utilization of SARS-CoV-2 serological tests in clinical practice

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Serological population study of the coronavirus epidemic by Finnish Institute for Health

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We thank Nina Peltonen for delivering the chitosan study control samples, Auni Collings for checking the written language, and all others who have enabled this study and taken part in developing COVID-19 diagnostics in Fimlab Laboratories operation region.