key: cord-340960-abanr641
authors: Brigger, D.; Horn, M.P.; Pennington, L.F.; Powell, A.E.; Siegrist, D.; Weber, B.; Engler, O.; Piezzi, V.; Damonti, L.; Iseli, P.; Hauser, C.; Froehlich, T.K.; Villiger, P.M.; Bachmann, M.F.; Leib, S.L.; Bittel, P.; Fiedler, M.; Largiadèr, C.; Marschall, J.; Stalder, H.; Kim, P.S.; Jardetzky, T.S.; Eggel, A.; Nagler, M.
title: Accuracy of serological testing for SARS‐CoV‐2 antibodies: first results of a large mixed‐method evaluation study
date: 2020-09-30
journal: Allergy
DOI: 10.1111/all.14608
sha: 
doc_id: 340960
cord_uid: abanr641

BACKGROUND: Serological immunoassays that can identify protective immunity against SARS‐CoV‐2 are needed to adapt quarantine measures, assess vaccination responses, and evaluate donor plasma. To date, however, the utility of such immunoassays remains unclear. In a mixed‐design evaluation study, we compared the diagnostic accuracy of serological immunoassays that are based on various SARS‐CoV‐2 proteins and assessed the neutralizing activity of antibodies in patient sera. METHODS: Consecutive patients admitted with confirmed SARS‐CoV‐2 infection were prospectively followed alongside medical staff and biobank samples from winter 2018/2019. An in‐house enzyme‐linked immunosorbent assay utilizing recombinant receptor‐binding domain (RBD) of the SARS‐CoV‐2 spike protein was developed and compared to three commercially available enzyme‐linked immunosorbent assays (ELISAs) targeting the nucleoprotein (N), the S1 domain of the spike protein (S1) and a lateral flow immunoassay (LFI) based on full‐length spike protein. Neutralization assays with live SARS‐CoV‐2 were performed. RESULTS: One‐thousand four‐hundred and seventy‐seven individuals were included comprising 112 SARS‐CoV‐2 positives (defined as a positive real‐time PCR result; prevalence 7.6%). IgG seroconversion occurred between day 0 and day 21. While the ELISAs showed sensitivities of 88.4% for RBD, 89.3% for S1, and 72.9% for N protein, the specificity was above 94% for all tests. Out of 54 SARS‐CoV‐2 positive individuals, 96.3% showed full neutralization of live SARS‐CoV‐2 at serum dilutions ≥1:16, while none of the 6 SARS‐CoV‐2 negative sera revealed neutralizing activity. CONCLUSIONS: ELISAs targeting RBD and S1 protein of SARS‐CoV‐2 are promising immunoassays which shall be further evaluated in studies verifying diagnostic accuracy and protective immunity against SARS‐CoV‐2.

Governments worldwide are facing a unique challenge: to save thousands of lives threatened by coronavirus disease 2019 (COVID- 19) , while minimising economic and social damage caused by lockdown and other strict measures. Serological immunoassays will play a central role in addressing these challenges for the following reasons 1 . First, serological tests might improve the rate of diagnosis as real-time RT-PCR is associated with a high number of false-negative results due to pre-analytical and other issues 2 . Second, antibody assays may support intensive surveillance measures such as universal testing, active case-finding, contact tracing, and linking clusters and thereby may facilitate an exit strategy from lockdown [3] [4] [5] [6] 9, 10 . In patients with severe disease extensive activation of cytokine-secreting cells from the innate and adaptive immune system has been reported to result in a cytokine storm contributing to acute respiratory distress syndrome and multiorgan failure [11] [12] [13] [14] [15] .

Antibody responses against different SARS-CoV-2 antigens have been described in serological samples of infected patients. Few patients with anti-viral antibodies have been identified in the first 5 days following symptom onset but the positive rate rapidly increases thereafter 16, 17 . To date, antibody testing has focused primarily on two highly abundant structural antigens of SARS-CoV-2, specifically the nucleoprotein (N) protein

This article is protected by copyright. All rights reserved and the spike (S) protein 18 . While the N phosphoprotein ensures the linkage of the viral RNA to the membrane 19 , the S glycoprotein binds to ACE2 and thereby initiates viral entry into the host cell 13, [20] [21] [22] . Neutralizing antibodies (NAb) are typically generated against the S protein and often target the receptor binding domain (RBD) 23, 24 . As demonstrated in a vaccination approach using inactivated virus, the RBD represents an immunodominant viral antigen since at least half of the detectable anti-S IgG antibodies were directed against the RBD 25 . In contrast, the amount of anti-N antibodies was 30-fold lower.

Lateral flow immunoassays (LFI) 26, 27 as well as enzyme linked immunosorbent assays (ELISA) 28, 29 have been developed but not yet adequately evaluated. While LFIs are remarkably fast and only require minutes to perform, significant concern regarding their sensitivity and specificity has been raised 30 . ELISAs are considered more robust but require highly specialized laboratories with the capacity to run automated high-throughput measurements.

At the time of compiling this paper, the diagnostic performance of different immunoassays as well as their predictive value for protective immunity remains unclear.

Before a broad implementation of immunoassays can be justified, the following points need to be carefully assessed in adequately powered and designed diagnostic studies:

(1) diagnostic accuracy (or sensitivity/ specificity respectively) in the acute and subacute phase of the disease, (2) antibody kinetics over time in patients with confirmed COVID-19, (3) extent of cross-reactivity with other pathogens and patients with autoimmune disorders, (4) reliability between different assay settings and material characteristics, as well as (5) correlate of protective immunity 3 .

With the present study, we aimed to comprehensively establish the utility and diagnostic accuracy of serological immunoassays for SARS-CoV-2 infection and to explore protective immunity 12 as predicted by such immunoassays in a mixed-method observational study of hospital inpatients as well as medical personnel.

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International guidelines on study design were strictly followed 31 and cross-sectional, prospective observational, as well as case-control designs were used. Participants were recruited via three different routes: (i) inpatients with a SARS-CoV-2 test result (real-time PCR; RT-PCR), (ii) medical personnel of the Inselspital, and (iii) residual material from patients stored at the Liquid Biobank Bern (www.biobankbern.ch). Inclusion criteria of inpatients are (i) hospitalisation in Inselspital, (ii) tested positive for SARS-CoV-2 using RT-PCR (nasopharyngeal swab), (iii) aged 18 or older and (iv) signed general consent (exemption was granted for a few patients). For this manuscript, only inpatients who had tested positive for SARS-CoV-2 with more than 4 days of residual material available were considered. The temporal pattern of antibody response and seroconversion rate was assessed in a subgroup of inpatients; the first 25 consecutive patients were selected.

Inclusion criteria of medical personnel were (i) medical staff at Inselspital since February 2020, (ii) aged 18 or older, and (iii) signed informed consent. The personnel were recruited via mailing lists. A limited number of fully anonymized, residual biobank samples were also used for the purpose of this study with the inclusion criterion of having been collected from inpatients between December 2018 and February 2019. A total of 54 randomly selected sera from individuals who were tested positive in either of the three ELISA immunoassays as well as 6 negative controls were assessed in a live SARS-CoV-2 neutralization assay (all collected in April 2020).

The University Hospital Bern (Inselspital) is one of the largest tertiary hospitals in Switzerland covering a catchment area of more than 1 million inhabitants. With several associated smaller hospitals, it provides the full spectrum of general as well as highly specialised medical services. More than 10,000 employees work at the Insel Gruppe AG.

The study was supported by the local COVID-19 task force. The study protocol was approved by the appropriate ethics committee and the authorities of the University Hospital and conducted in accordance with the Declaration of Helsinki. The manuscript was prepared according to the Standards for Reporting Diagnostic accuracy studies (STARD) guideline 32 .

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Blood was taken following an established in-house protocol to ensure adequate preanalytical conditions and samples were collected using plastic syringes (serum or lithium heparin respectively, S-Monovette®, Sarstedt, Nümbrecht, Germany). Only residual material was used in the case of inpatients. Two tubes (serum and lithium heparin respectively) were drawn in the case of medical personnel. Samples were immediately transported to the central laboratory, processed using a GLP laboratory track and centrifuged within 30 minutes with an established protocol 33 .

With regard to inpatients, pseudonymized demographical, clinical as well as laboratory data were extracted and transferred by the Insel Data Science Center (IDSC) from electronic patient documentation. Limited data were collected for the purpose of this substudy: age, gender, time interval since RT-PCR (nasopharyngeal swab). A positive SARS-CoV-2 RT-PCR result was used as additional inclusion criterion. With regard to medical personnel, a RedCap database survey was constructed collecting demographical data, COVID-19 symptoms (presence, extent and date), comorbidities and risk factors, professional exposure, and date of RT-PCR.

The S1 protein and RBD are regarded as ideal candidates for the development of diagnostic tests and vaccines targeting SARS-CoV-2 34 . The pCAGGS plasmid containing the human codon-optimized sequence of the SARS CoV-2 S protein receptor binding domain (RBD, amino acids R319-F541) with native S signal sequence (amino acids M1-S14) and a C-terminal hexahistidine tag was kindly provided by Prof. Florian Krammer.

Plasmid DNA was prepared using the Gene Elute HP Plasmid Maxiprep Kit (Sigma-Aldrich). Prior to transfection Expi293F cells (Thermo-Fisher) were grown to a density of 3.0 x 10 6 cells/ml in culture medium (a mixture of 33% Expi293 and 66% FreeStyle-293 media from Thermo-Fisher). For each liter of transfection, 0.5mg of plasmid DNA was diluted in 100ml of culture medium, mixed with 1.3ml FectoPro transfection reagent (Polyplus), and incubated for 10 minutes at room temperature prior to addition to cells.

Immediately following transfection cells were supplemented with 100x D-glucose (400g/l)

This article is protected by copyright. All rights reserved and 100 x Valproic acid (300mM) boost solutions. Three days post transfection the cell culture supernatants were harvested by centrifugation at 7,000 x g for 15min.

Supernatants were passed through a 0.22µm filter and 1:1 diluted with PBS containing 10 mM imidazole. For purification of his-tagged RBD protein 5 ml Ni-NTA resin (HisPur NiNTA ThermoFisher) was washed three times with washing buffer (PBS with 10mM imidazole) and incubated on a stir plate at 4°C for 1 hour. Subsequently, the mixture was poured into a glass column with a frit and washed 3 times with 5 column volumes of washing buffer. The protein was then eluted three times with 15 ml PBS containing 250mM imidazole. Elutions were pooled and dialyzed overnight against PBS using 3.5 kDa cutoff SnakeSkin dialysis tubing. The final protein concentration was determined by NanoDrop measurement at A 280. The quality of recombinant RBD protein was analyzed by SDS-PAGE and analytical size-exclusion chromatography.

All ELISA assays were performed on a DSX automated ELISA system device (DYNEX Technologies). The in-house assay was prepared as follows: 96-well plates were coated overnight at 4°C with 100µl of 1µg/ml RBD protein in PBS. The following day, each well was blocked with 300µl of PBS/0.15% casein at 4°C until use and at least overnight.

Subsequently plates were washed twice with PBS and 100µl sera were added at a 1:100 dilution in PBS/0.15% casein for 1 hour at RT. After five washes with 300µl PBS/0.1% Tween 100µl of HRP-labeled secondary polyclonal anti-human IgM (Sigma, A0420) and anti-human IgG (Sigma, A0170) antibodies were added in a 1:10'000 dilution for 30 minutes at RT. Again, the plates were washed 5 times with PBS/0.1% Tween and 100µl of TMB substrate solution (Sigma, T4444) was added for 15 minutes at RT. The development was stopped by adding 100µl of 0.5M H 2 SO 4 and results were measured at OD450-620nm. All samples with an OD > 0.5 were assigned as positive.

Several commercial tests were conducted according to the manufacturers' instructions.

An ELISA produced by Euroimmun AG, Lübeck, Germany targeting the S1 protein as the

This article is protected by copyright. All rights reserved immobilized antigen for the detection of IgG antibodies was employed. Briefly, samples were diluted 1:100 in sample buffer and 100l of diluted samples, pre-diluted positive and negative controls, as well as pre-diluted calibrator were added for 1 hour at 37°C. After three wash steps with 300µl wash buffer, 100µl of HRP-labeled secondary anti-human IgG antibodies were added for 30 minutes at 37°C. The plates were washed again three times with wash buffer and 100µl of TMB solution was added for 20 minutes at RT. The development was stopped by adding 100µl of 0.5M H 2 SO 4 and results were measured at OD450-620nm. Antibody values were expressed as a ratio (OD sample /OD calibrator ). All samples with a ratio >1.1 were assigned as positive. Comorbidities and risk factors, which will be used as covariables in subsequent phases of this study, will be extracted from electronic patient records and asked in the RedCap

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At University Hospital Bern we first established a carefully designed mixed-method diagnostic accuracy study (Fig. 1) . 

This article is protected by copyright. All rights reserved breathlessness, coughing, or loss of smell 

Recombinantly expressed RBD has been used to establish an in-house ELISA for the detection of IgM and IgG anti-SARS-CoV-2 antibodies in human serum samples (supplementary Fig. 1a,b) . Optimal serum dilutions were determined by titration of sera derived from six SARS-CoV-2+ and six SARS-CoV-2-individuals. The serum dilution of 1:100 allowed efficient discrimination between positive and negative outcome (supplementary Fig. 2) . After automatization on a DYNEX DSX device, the intra-assay (within-run) and inter-assay (day-to-day) precisions of the in-house RBD ELISA was assessed (supplementary Fig. 3a- 3e,f) . Overall, the in-house RBD ELISA assay showed high intra-and inter-assay reproducibility and demonstrated a high degree of agreement between plasma and serum samples.

Among a subgroup of 25 SARS-CoV-2+ inpatients, seroconversion for IgM and IgG antibodies was observed between day 0 and day 21 after the RT-PCR result and between day 2 and day 21 after the start of symptoms (Fig. 2) . Interestingly, IgM and IgG antibody responses against RBD and S1 were substantially more pronounced as compared to N. Assessment of the longitudinal dynamics of patient sera revealed a marked and consistent increase of IgG antibodies for RBD and S1 (Fig. 3a) . IgM antibodies were measured in the RBD and N ELISA and detectable at least for two weeks after seroconversion (Fig. 3b) . Interestingly, the individual temporal IgG and IgM patterns showed a high degree of inter-individual variability with one group of patients

This article is protected by copyright. All rights reserved Of these samples, all were negative for anti-RBD IgM and IgG, as well as anti-S1 IgG.

However, two biobank samples tested positive for anti-N IgG (ELISA; 6.2%), and one tested positive for anti-N IgM (ELISA; 3%). All samples were negative for anti-S IgG and

IgM (100%) as tested by LFI.

The pooled study population consisted of 1477 individuals, 112 of whom tested as RT-PCR positive (prevalence 7.6%). Sera from all individuals were tested in the three different ELISA setups for IgG and IgM anti-SARS-CoV-2 antibodies (Fig. 4a) . A subgroup of samples (n=159) was additionally assessed on LFI (Fig. 4b) .

Both assay formats showed high specificity above 94% for IgG and IgM measurements (supplementary Table 4 ). However, the sensitivity between assays and formats varied considerably. The highest sensitivities were reached for IgG measurements with the S1 (89.3%) and RBD (88.4%) ELISA, followed by IgG measurements on N (72.9%) ELISA.

Sensitivities for IgM measurements were all considerably lower for both ELISA and LFI formats, which could be due to the more transient detectability of IgM upon infection.

To detect potential sources of variability, we additionally studied the antibody response in salient subgroups of RT-PCR positive individuals (Fig. 4c) 

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In the tested inpatient population, we observed three "false-negative" (negative in S1

ELISA despite positive RT-PCR) outcomes. Among three false-negative inpatients (P07, P041, and P042), two were measured at an early time-point (Patient 7 and 41), and one patient (P042) might have experienced seroconversion at a very late time-point because of a significant increase of antibody titers at day 24 (supplementary Fig. 3 and   supplementary Fig. 4) . In the assessed hospital staff, seven were classified as "falsenegative". All of these reported mild diseases and had symptoms clearly associated with COVID-19 (fever, breathlessness, cough, and loss of taste or smell). Twenty-two individuals in the hospital staff group tested "false-positive" (positive S1 ELISA results despite negative RT-PCR). Fourteen of them experienced one or more symptoms clearly associated with COVID-19. The remaining eight individuals were clearly positive in at least three assays. All other individuals were either classified as "true-positive" (positive in S1 ELISA, and positive in RT-PCR), or as "true-negative" (negative in S1 ELISA, and negative in RT-PCR).

In terms of performance, the calculated area under the receiver operating characteristic (Fig. 5b) .

A total of 54 randomly selected sera from individuals who were tested positive in either of the three ELISA immunoassays as well as 6 negative controls were assessed in a live SARS-CoV-2 neutralization assay using ACE2-expressing Vero-E6 cells (34 inpatient samples, and 26 samples of medical personnel). Full neutralization of viral infection has been determined based on 100% inhibition of the cytopathic effect in a serial dilution of the sera (supplementary Fig. 6) . The means of highest serum dilutions at which full neutralization was observed correlated remarkably well with the measured antibody responses in the ELISA immunoassays (Fig. 6a-c) . Importantly, 96.3% of the sera from ELISA positive individuals showed full inhibition at serum dilutions ≥1:16. The two sera that did not show neutralization (P037 and P042) were drawn at an early time point

This article is protected by copyright. All rights reserved where the patients did not yet show antiviral antibodies. Both patients, however, fully neutralized the virus after seroconversion at a later time point (Fig. 6d) . Further, all 6 sera from ELISA negative individuals showed no neutralizing activity. Of note, one or two ELISA assays were negative in 17 samples with full neutralization.

We report first results of a large, mixed-design evaluation study which was implemented to compare the diagnostic accuracy of serological immunoassays for SARS-CoV-2

antibodies. While the time to seroconversion varied substantially between infected individuals, the mounted IgG responses were robust and stable over time in all assays relying on RBD, S1 as well as N. With regards to the ELISA assays, the overall diagnostic accuracy was adequate with a high specificity. Some "false-positive" results are likely due to a rather narrow diagnostic window and limited sensitivity of the RT-PCR as well as asymptomatic disease course 36 . "False-negative" results may be caused by a long seroconversion period observed in some patients and mild disease course in other individuals. The accuracy measures of LFI and N were inferior compared to ELISA targeting S1 and RBD. Strikingly, there is a high degree of correlation between antibody responses to these viral surface proteins and the neutralizing activity against live SARS-

A few other studies have previously assessed the diagnostic accuracy of serological immunoassays. Recently, Long and colleagues studied the antibody response in 285 patients with COVID-19 using a magnetic chemiluminescent immunoassay. 37 In accordance with their results, we observed high inter-individual variation in the time to seroconversion. In contrast to their study, we confirmed these findings with an appropriate diagnostic accuracy protocol using different serological immunoassays. In another case-control study, Infantino et al. analyzed 61 COVID-19 inpatient samples and 64 selected patients collected before 2020 using a magnetic chemiluminescent immunoassay 38 . In agreement with their results, we found limited sensitivity but high specificity of the serological SARS-CoV-2 immunoassays. In further study conducted at the Geneva University Hospital, 181 samples of COVID-19 patients were included as well as 176 controls collected before 2020, and analyzed with the same S1 ELISA that we

This article is protected by copyright. All rights reserved used in our study. Similar to our results they report a high specificity for IgG, particularly with an adjusted cut-off value 39 . In line with other studies the accuracy and performance

LFIs was rather weak 40,41 .

The study presented here adds important value to previous reports as it (i) was designed as a comprehensive diagnostic accuracy study combining different research methods, (ii) directly compares major assay approaches, (iii) was fully approved by all appropriate authorities, (iv) was independently conducted at a University Hospital, (v and indicate that such serological tests might even be used to predict protective immunity in near future 45, 46 . To draw further conclusions, however, SARS-CoV-2 positive patients have to be followed over an extended time period in future studies.

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In line with previous studies 47 , we observed that the antibody response is more pronounced in patients with severe disease than patients without (Figure 4 , panel C;

inpatients, hospitalized patients, older patients). However, the response was similar in patients with mechanical ventilation and hospitalized patients. This is most likely due to limitations in sensitivity, which does not contradict our general observations.

In summary, we report the first results of a large, mixed-design evaluation study that has been conducted in an independent academic setting at the University Hospital Bern to assess the diagnostic accuracy of various immunoassays to determine antibody responses against SARS-CoV-2. While antibody responses of individual COVID-19

patients against RBD and S1 protein were similar, a weaker reactivity against N protein became apparent. The time to seroconversion varied substantially between COVID-19 patients but the IgG response was robust and stable in all three ELISA setups. Their overall diagnostic accuracy was adequate with a high specificity but limited sensitivity.

The antibody responses measured in these ELISAs correlated remarkably well with SARS-CoV-2 neutralizing activity of the sera. On the other hand, accuracy measures of S protein based LFIs were poor. Together, our results emphasize that appropriate serological immunoassays represent a valuable tool to identify a good portion of patients with previous SARS-CoV-2 infection, will help to facilitate exit strategies from lockdown and might even be used to predict immunity to SARS-CoV-2 in near future.

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This article is protected by copyright. All rights reserved This article is protected by copyright. All rights reserved Accepted Article

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All authors declare that there is no conflict of interests.

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