Evaluation of nine commercial SARS-CoV-2 immunoassays

Due to urgency and demand, numerous severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoassays are rapidly being developed and placed on the market with limited validation on clinical samples. Thorough validation of serological tests are required to facilitate their use in the accurate diagnosis of SARS-CoV-2 infection, confirmation of molecular results, contact tracing, and epidemiological studies. This study evaluated the sensitivity and specificity of nine commercially available serological tests. These included three enzyme-linked immunosorbent assays (ELISAs) and six point-of-care (POC) lateral flow tests. The assays were validated using serum samples from: i) SARS-CoV-2 PCR-positive patients with a documented first day of disease; ii) archived sera obtained from healthy individuals before the emergence of SARS-CoV-2 in China; iii) sera from patients with acute viral respiratory tract infections caused by other coronaviruses or non-coronaviruses; and iv) sera from patients positive for dengue virus, cytomegalovirus and Epstein Barr virus. The results showed 100% specificity for the Wantai SARS-CoV-2 Total Antibody ELISA, 93% for the Euroimmun IgA ELISA, and 96% for the Euroimmun IgG ELISA with sensitivities of 90%, 90%, and 65%, respectively. The overall performance of the POC tests according to manufacturer were in the rank order of AutoBio Diagnostics > Dynamiker Biotechnology = CTK Biotech > Artron Laboratories > Acro Biotech ≥ Hangzhou Alltest Biotech. Overall, these findings will facilitate selection of serological assays for the detection SARS-CoV-2-specific antibodies towards diagnosis as well as sero-epidemiological and vaccine development studies.


Introduction
In December 2019, a novel coronavirus causing severe acute respiratory symptoms emerged in Wuhan, China [1]. The World Health Organization (WHO) termed the disease, coronavirus disease 2019 , and the causative virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As of 7 April, the virus has spread to 212 countries and territories with 1 279 722 confirmed cases and 72 614 deaths worldwide [2]. At present, the epidemic within the majority of countries have not yet reached its peak with the number of cases and deaths predicted to rise in the coming weeks and months.
Accurate diagnosis of COVID-19 is essential, not only to ensure appropriate patient care but also to facilitate identification of SARS-CoV-2 infected people, including asymptomatic carriers, who need to be isolated to limit virus spread. The WHO recommends nucleic acid detection of SARS-CoV-2 in respiratory samples for the diagnosis of COVID-19. Unfortunately, in the face of the rapidly growing epidemics worldwide, an increased demand for diagnostic tests has led to a critical shortage in operational material for respiratory sample collection and within the molecular diagnostic workflow [3,4]. This impedes rapid large scale testing, a necessity for controlling the epidemic. Moreover, the heterogeneity of respiratory sample material and anatomical location of sample collection, for example throat swab, saliva or endotracheal aspirate, affect the sensitivity of SARS-CoV-2 viral nucleic acid testing [5,6]. Overall, there is an urgent need to identify alternative diagnostic means.
Antibody testing, either using enzyme-linked immunosorbent assay (ELISA) or point-of-care (POC) lateral flow immunoassays, may overcome some of these challenges. SARS-CoV-2-specific antibodies can be detected in in serum of approximately 40% of COVID-19 patients as early as seven days after the onset of symptoms, with seroconversion rates rapidly increasing to >90% by day 14 [7]. In recent studies, antibody testing has been shown to be more sensitive than viral nucleic acid detection after approximately eight days of COVID-protein subunit 1 (S1) are detected in human serum or plasma. Briefly, 1:101 diluted serum samples were added to wells coated with recombinant SARS-CoV-2 antigen and incubated for 60 minutes at 37 °C. Wells were washed three times followed by the addition of HRP-conjugated anti-human IgA or IgG and subsequent incubation for 30 minutes at 37 °C. Wells were washed three times and a chromogen solution was added.
Following 30 minutes of incubation at room temperature, the reaction was stopped and the resultant absorbance was read on a microplate reader at 450 nm with reference at 620 nm. A ratio between the extinction of the sample and calibrator on each plate were calculated. According to the manufacturer's recommendations, a ratio <0.8 is considered negative, ≥0.8 and <1.1 borderline, and ≥1.1 positive. However, for sensitivity and specificity, 1.1 was used as a more stringent cut-off value for positive results and all values <1.1 were considered negative.

Point-of-care (POC) tests
Six POC tests for rapid detection of antibodies in blood, serum or plasma were evaluated: 2019-nCOV IgG/IgM For all tests, the recommended sample volume of 10 μl serum was added to the specimen well on the individual test cassettes followed by the addition of the supplied buffer. The buffer volume differed by manufacturer and was added accordingly (60 μl, three drops, two drops, two drops, two drops and 60 μl, respectively). The result was read visually after 10 minutes. Weak signals for IgM and IgG, together or separate, was considered positive.

Statistical analyses
Sensitivity was defined as the proportion of patients correctly identified as having SARS-CoV-2 infections, as initially diagnosed using nucleic acid detection of SARS-CoV-2 in respiratory samples. Specificity was defined as the proportion of SARS-CoV-2 immune naïve study participants accurately identified as negative for COVID-19. The clinical accuracies of the ELISA assays were examined by using Receiver Operator Characteristic (ROC) plots with GraphPad Prism version 8.0.2 (GraphPad Software, San Diego, CA, USA). ROC area under the curve (AUC) were calculated as the fraction "correctly identified to be positive" and the fraction "falsely identified to be positive" determined according to manufacturer cut-off values for positive results.
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Sensitivities and specificities of the ELISA assays
Three commercial CE-marked ELISA assays for detecting SARS-CoV-2 antibodies were evaluated using 30 serum samples from PCR-positive cases with SARS-CoV-2 and 82 control serum samples. Twenty nine of the 30 cases (97%) were positive for SARS-CoV-2-specific antibodies by at least one of the three ELISA assays. In one case, only a positive IgA result was detected, while in another case antibody responses were negative by all tests.
The distance of data points from the manufacturer recommended cut-off values and confidence in assigning a positive or negative status differed between the three assays ( Figure 1A). The distribution of positive and negative data points were distinct for the Wantai Total Ab assay, with a cut-off value above all the control sera samples, which allowed for unequivocal interpretation. Conversely, the Euroimmun IgA and IgG assays data had a less distinct separation, resulting in a 'grey zone' of borderline data points to which a positive or negative status could not be assigned. Both case and control sera had borderline or inconclusive data points.
The sensitivities and specificities are shown in table 1. The sensitivity of the Wantai Total Ab ELISA was equivalent to that of Euroimmun's IgA ELISA at 93% and was greater than that observed for Euroimmun's IgG ELISA at 67%. The specificity of the Wantai Total Ab ELISA was 100% compared to 93% and 96% for the Euroimmun IgA and IgG ELISAs, respectively. The positive predictive value and negative predictive value was 1). The positive predictive value of these tests were 100%, while the negative predictive values were 91%, 89%, 89%, and 74%, respectively. The Acro Biotech was evaluated on five case serum samples and had a specificity of 80%. One case serum sample was tested with the Hangzhou AllTest Biotech test and was positive for both IgM and IgG.
The specificity of the six POC tests were evaluated primarily on control samples that showed some crossreactivity in the SARS-CoV-2 ELISA assays; the number of control sera tested varied between the different POC tests ( Table 1). The POC tests manufactured by Dynamiker Biotechnology, CTK Biotech, AutoBio Diagnostics and Artron Laboratories had a 100% specificity, whereas the test from Acro Biotech and Hangzhou AllTest Biotech had a specificity of 80% and 87%, respectively. For the latter two tests, crossreactivity was only observed for IgM. The Acro Biotech test cross-reacted with a control serum sample from a human coronavirus HKU1 patient.

Antibody detection relative to duration of illness
To evaluate the sensitivities of the assays at different stages of COVID-19 disease, case sera were grouped according the duration of disease: early phase, 7 to 13 days after the onset of disease symptoms; middle phase, 14 to 20 days after the onset of disease symptoms; and late phase, ≥21 days after the onset of disease symptoms. The sensitivities of the assays ranged from 40 to 86% for the early phase samples, 67 to 100% for the middle phase samples, and 78 to 89% for the late phase ( Figure 2).
In the early phase, the Wantai Total Ab ELISA had a sensitivity of 71% that plateaued at 100% after 10 days of illness duration. The IgG ELISA had the lowest sensitivity at all three phases that showed a distinct increase with each consecutive phase i.e. 43% in the early phase, 67% in the middle phase, and 78% in the late phase.
While the four POC tests evaluated according to illness duration were often weakly positive or detected only IgG or IgM during the early phase (data not shown), their sensitivities were comparable to the Wantai Total Ab ELISA and Euroimmun IgA ELISA in all three phases. In the early phase, a case sample that was negative by both Total Ab and IgG was positive in the IgA ELISA.

Agreement between serological assays
To determine the agreement between the different ELISAs and POC tests evaluate, the proportion of case sera that shared the same result between two assays were calculated. Despite comparable sensitivities of certain assays, the tests did not necessarily give the same result in all instances ( Figure 3A). The only tests that were 100% concordant were the Dynamiker Biotechnology and CTK Biotech POC tests ( Figure 3B) . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

Discussion
In the present study, three SARS-CoV-2-specific commercial ELISA assays and six POC rapid tests were evaluated using sera from hospitalized adult patients with PCR-confirmed diagnoses for SARS-CoV-2 and a collection of control serum samples taken before the emergence of the virus in China in December 2019.
Overall, the Wantai Total Ab ELISA had superior sensitivity and specificity compared to both Euroimmun IgA and IgG ELISAs. The POC tests varied notably, with the best performance observed for the test produced by AutoBio Diagnostics, followed by the tests produced by Dynamiker Biotechnology and CTK Biotech.
The differences observed for the sensitivity and specificity of the SARS-CoV-2-specific total antibody testing and antibody type ELISAs correspond to previous reports. . This notably lower sensitivity for SARS-CoV-2-specific IgG detection is in agreement to that observed here for the Euroimmun IgG ELISA (65%). In the present study the Wantai IgG and Euroimmun IgG ELISAs could not be compared due to unavailability of the former. The possibility that overall lower sensitivity of SARS-CoV-2 IgG ELISAs may be a more universal occurrence rather than manufacturer dependent warrants further investigation. In addition to lower sensitivities, the Euroimmun IgA and IgG ELISAs are also more prone to cross-react with negative sera as described in the present study and in a separate analysis of the beta-versions of these assays [10].
The differences between the assays may, in part, be explained by the SARS-CoV-2 antigen targeted and the ELISA format used. Both kits detect antibodies to the S1 subunit of the SARS-CoV-2 spike protein; however, the Wantai Total Ab ELISA only targets the RBD within the S1. The RBD represents approximately 33% of the S1 subunit, thus epitopes that may be recognized by cross-reacting epitopes outside of this domain are absent. Furthermore, the RBD is highly diverse between SARS-CoV-2 and other beta-coronaviruses (hCoV-OC43 and hCoV-HKU1), which may further reduce the likelihood of cross-reaction with these circulating coronaviruses. Moreover, the Wantai Total Ab ELISA uses an antigen-antibody-antigen(peroxidase) format whereas the Euroimmun ELISAs employ an antigen-antibody-antibody(peroxidase) format. The specificity of the former is determined by a single antibody, whereas the latter has a second antibody that may introduce additional specificities. The antigen-antibody-antibody format is required to distinguish between specific antibody types, but may not necessarily have lead to decreased specificity as shown for in-house ELISAs [10].
The clinical sensitivity of IgM for early diagnosis of COVID-19 is currently unclear. SARS-CoV-2-specific IgM does not consistently appear before its IgG counterpart, with some studies reporting detection of SARS-CoV-. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. In conclusion, our findings show that in an ELISA format the sensitivity of detecting total SARS-CoV-2 RBDspecific antibodies is higher than that of assays detecting spike-specific IgA or IgG only. It is important to note that the presence of SARS-CoV-2-specific antibodies does not necessarily correspond to protection against SARS-CoV-2 infection and disease. In order to define antibody-mediated protection, further investigation of virus-specific antibody functions that include neutralization and Fc-mediated effector functionality are needed. Sero-epidemiological investigations together with longitudinal studies on sequential samples taken from SARS-CoV-2 patients are necessary to characterize the spread of the virus and the long term protection of the antibodies measured.  Due to comparatively poorer assay performance in an initial round of testing, further testing were suspended.
. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.  . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
(which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.04.09.20056325 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
(which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.04.09.20056325 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.