key: cord-0833927-ndr4lpme
authors: Reza, Soleimani; Mehdi, Khourssaji; Antoine, Aupaix; Hector, Rodriguez-Villalobos; Anaïs, Scohy; Benoît, Kabamba-Mukadi
title: Usefulness of a multiplex immunodot in case of discordant results between automated COVID-19 serological assays
date: 2021-03-18
journal: J Virol Methods
DOI: 10.1016/j.jviromet.2021.114129
sha: 7580999911c8bbfa85a5f423e3d662aebea9f66e
doc_id: 833927
cord_uid: ndr4lpme

BACKGROUND: At present, the only reliable test for COVID-19 diagnosis is RT-qPCR. Serological assays have been widely used to increase the detection sensitivity of infected population. Hereby, we report the performance of a new pan-IgG multiplex Enzyme Immunoassay (immunodot) method for exploration of discrepant SARS-COV-2 serological results. METHODS: A retrospective study on 38 residual serum samples from recovered COVID-19 subjects with discordant serological results on Roche and Snibe platforms, were reanalyzed on a new semi-automated pan-IgG immunodot Enzyme Immunoassay, namely COVIDOT-TEST, in order to find the source of discrepancies and to evaluate the latter method. All samples were analyzed on the BlueDiver® Instrument and all strips were read by the BlueScan® Scanner using Dr DOT® Software. Results: Based on our data, subject samples showed specific IgG reactions on [Formula: see text] 2 different antigens on immunodot strips. Of these 38 samples, 97.4% of samples showed specific IgG reaction against S1 + S2 antigens, 89.5% showed against RBD antigen, 86.8% against S2 antigen reaction on the COVIDOT-TEST kit. Specific IgG-S1 antigen and IgG-N antigen reactions were detected in 73.7% and 65.8% of the samples, respectively. CONCLUSION: The new semi-automated pan-IgG immunodot Enzyme Immunoassay method appeared to be a reliable assay to confirm suspicious COVID-19 serological screening results.

preliminary data show that the semi-automated immunodot kit is a reliable assay to confirm suspicious COVID-19 serological screening results and may be of interest in the vaccine response evaluation. 

Background: At present, the only reliable test for COVID-19 diagnosis is RT-qPCR.

Serological assays have been widely used to increase the detection sensitivity of infected population. Hereby, we report the performance of a new pan-IgG multiplex Enzyme Immunoassay (immunodot) method for exploration of discrepant SARS-COV-2 serological results.

Methods: A retrospective study on 38 residual serum samples from recovered COVID-19 subjects with discordant serological results on Roche and Snibe platforms, were reanalyzed on a new semi-automated pan-IgG immunodot Enzyme Immunoassay, namely COVIDOT-TEST, in order to find the source of discrepancies and to evaluate the latter method. All samples were analyzed on the BlueDiver ® Instrument and all strips were read by the BlueScan ® Scanner using Dr DOT ® Software.

Results: Based on our data, subject samples showed specific IgG reactions on ≥ 2 different antigens on immunodot strips. Of these 38 samples, 97.4% of samples showed specific IgG reaction against S1+S2 antigens, 89.5% showed against RBD antigen, 86.8% against S2 antigen reaction on the COVIDOT-TEST kit. Specific IgG-S1 antigen and IgG-N antigen reactions were detected in 73.7% and 65.8% of the samples, respectively.

J o u r n a l P r e -p r o o f

In the last days of 2019, a cluster of pneumonia cases of unknown origin was observed in several local hospitals of the city of Wuhan (Hubei, China). Soon after, the Chinese center for disease control and prevention (CDC) informed the World Health Organization (WHO) of a new threat to the humanity due to severe acute respiratory infection (SARI) [1] [2] [3] [4] with human-to-human transmission and in some cases with an acute respiratory distress syndrome (ARDS), or organ failures, and eventually deaths [4] [5] [6] . As of 7 January, a new virus was isolated and the genome sequence was identified by Lu, et al. showing that the virus belongs to the family of coronaviruses [7] . The WHO named the new virus as the '2019 novel coronavirus' (2019-nCoV) [8] and studies showed animal origin with more than 90% genome sequence similarity to MERS-CoV, SARS-CoV and Bat coronaviruses [7, 9] The COVID-19 diagnosis has been challenging as the RT-qPCR is the only reliable test [11] to diagnose an ongoing infection. One other way is investigating the humoral immune response against SARS-CoV-2 which could increase the detection of infected population, particularly in J o u r n a l P r e -p r o o f asymptomatic, pauci-symptomatic or recovered/immunized subjects [12] . For the latter purpose, in our laboratory we are using 2 fully automated tests, namely Roche Elecsys ® Anti SARS-CoV-2 (ani-N antibodies) as the screening test and Snibe MAGLUMI TM 2019-nCoV (separate IgG and IgM detection) to identify a specific immune reactivity. Discordant results between these two methods, approximately 10% of the analyzed samples, caused interpretation difficulties for patients and physicians.

To explore these discrepancies, we re-analyzed these samples with ALPHADIA S.A./N.E.

COVIDOT-TEST, a semi-quantitative multiplex immunodot kit intended for the in-vitro panimmunoglobin G (IgG) detection against different SARS-CoV-2 antigens.

A retrospective study was conducted on sera from 38 recovered COVID-19 subjects (17 male, All subjects were immunocompetent, without any comorbidity, between 18 to 60 years old, with normal renal function (≥ 90 mL/min). Out of 52 candidates, 14 were excluded because of low residual sample volume (n= 6), advanced age (n= 4), chronic renal failure (n= 2) and hemorrhagic or lipemic samples (n= 2). Samples were stored at +4° C until analysis. Testing J o u r n a l P r e -p r o o f was performed with the laboratory technologist being blinded to the prior results. Our study fulfilled the ethical principles provided by the Declaration of Helsinki. manufacturer's data, the COVIDOT-TEST reaches 100% sensitivity by the fourth week after the onset of symptoms. Anti-N, anti-S1+S2 and anti-S2 appeared to be the earliest antibodies and specificities were estimated more than 99% for S1+S2, S2 and RBD antigens, at 99% for S1 and at 96% for N antigens. 

Means and 95% confidence intervals were determined in COVID-19 recovered subjects. The agreement between the methods was determined by the kappa index. A P < .05 was considered as statically significant. Data analysis was performed using GraphPad Prism software (Version 8.0; California, CA).

All tested samples (selected for discrepancy between screening methods) had positive anti-N antibodies on the Elecsys ® Anti-SARS-CoV-2 platform while they were negative with separate %) is also shown in Table 1 .

The results of 4 EQC (2 positive and 2 negative controls) are shown in Table 2 . One positive EQC sample showed IgG reactivity with all 5 SARS-CoV-2 specific antigens, while the other positive EQC showed IgG reactivity with RBD, S1+S2, S2 and N antigens but not with S1

antigen on the COVIDOT TEST kit. The two negative EQC were correctly classified. All 4

EQC showed an IgG-N-OC43 antigen reaction, but only the 2 positive EQC showed antibody-J o u r n a l P r e -p r o o f antigen reaction with N-SARS (CoV-1). Overall, a complete agreement between Roche and ALPHADIA platforms was achieved (Cohen's Kappa = 1.00).

Here, we showed that a reactive humoral immune response against different SARS-CoV-2 specific antigens could be determined in COVID-19 recovered subject using a new semiquantitative pan-IgG multiplex immunodot kit. In our study, RBD and S (S1+S2) proteins appeared to be the most often recognized antibody targets, while anti-S1 IgG showed the lowest sensitivity. Other human coronaviruses proteins, particularly N proteins from SARS, MERS, HKU1, OC43, 229E, NL63 are known to have amino acid sequence similarities with SARS-CoV-2 N protein [15] , which is likely to be subject of cross-reactivity [15] . Based on our data, it appears that an IgG reactivity against N-SARS-CoV-2 antigen might be the cause of N-SARS cross-reactivity, as we observed a high correlation in positive results for these 2 antigens (N-SARS vs N-SARS-CoV-2). This is not surprising since these 2 nucleocapsid proteins share the highest amino acid sequence homology among human coronaviruses. Interestingly, even though there is a significant N proteins similarity between MERS and SARS-CoV-2, all samples were negative for N-MERS antigen. N-OC43 dots were positive in all samples and N-HKU1 in 24 samples. These immune reactivities could be explained, on the one side, by the high prevalence (more than 50% [16, 17] ) of antibodies against common coronaviruses in the general population, and on the other side by the probable cross-reactivity of SARS-CoV-2 antibodies with antigens of coronaviruses from the same genus (beta-coronaviruses). The other two N protein antigens, N-229E and N-NL63, showed less than 10% positive reactions, which is in line with the fact that these coronaviruses have a lower prevalence in the general population. Moreover, they belong to a different virus genus (alpha-coronaviruses) and thus are less likely to share cross-reactive epitopes. All results were in agreement with the data provided J o u r n a l P r e -p r o o f by the manufacturer. The Elecsys ® Anti-SARS-CoV-2 assay appeared to be more sensitive than

IgG/IgM MAGLUMI 2019-nCoV method. Such discrepancies between methods have been previously reported [18, 19] . The later variation in the test performance could partially be explained by assay design and targeted N protein epitopes by each manufacturer [20] . As the patients were not immunocompromised and the period between RT-qPCR and serology was about 6 moths, we assumed all patients had enough time to develop antibodies against SARS-CoV-2. Therefore, negative results by MAGLUMI, are probably false negatives.

Even though we could not analytically verify the COVIDOT-TEST, the SARS-CoV-2 serological results for the EQC provided by the national Belgian institute for health, Sciensano, were in agreement with the true results. A complete agreement was also observed between results of the COVIDOT-TEST and the Roche platform, suggesting the new pan-IgG immunodot kit is at least as sensitive as Elecsys ® anti-N antibodies kit. The new semi-automated COVIDOT-TEST appeared to be a reliable assay to confirm suspicious COVID-19 serological screening results and may be of interest in the vaccine response evaluation.

Our study was preliminary conducted on a limited number of samples and has limitations. First, we could not determine the exact date of symptom onset because of the heterogenicity in clinical data. Hence, the delay between symptom onset and serology sampling could not be exactly determined but was at least 6 months before sample collection for serology. Secondly, in our cohort we did not observe any inverse discrepancies as all selected samples appeared to be positive on the Elecsys ® Anti-SARS-CoV-2 platform while they were negative with MAGLUMI 2019-nCoV method. At last, we did not verify analytical performance of the new Pan-IgG COVIDOT-TEST assay. Further studies are needed to confirm such discrepancies and our observations. 

The authors have nothing to disclose and there was no conflict of interest. 

A Novel Coronavirus from Patients with Pneumonia in China

Recommendation for the review of biological reference intervals in medical laboratories

Characteristics of and Important Lessons From the Coronavirus Disease 2019 (COVID-19) Outbreak in China: Summary of a Report of 72314 Cases From the Chinese Center for Disease Control and Prevention

Early Transmission Dynamics in Wuhan, China, of Novel Coronavirus-Infected Pneumonia

Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study

Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus-Infected Pneumonia in

Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding

Coronavirus disease (COVID-19) Pandemic

A pneumonia outbreak associated with a new coronavirus of probable bat origin

The origin, transmission and clinical therapies on coronavirus disease 2019 (COVID-19) outbreak -an update on the status

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

The Promise and Peril of Antibody Testing for COVID-19

Clinical usefulness of fully automated chemiluminescent immunoassay for quantitative antibody measurements in COVID-19 patients

Development and Validation of the Elecsys Anti-SARS-CoV-2 Immunoassay as a Highly Specific Tool for Determining

Serological differentiation between COVID-19 and SARS infections

Epidemiology characteristics of human coronaviruses in patients with respiratory infection symptoms and phylogenetic analysis of HCoV-OC43 during 2010-2015 in Guangzhou

Epidemiology of seasonal coronaviruses: Establishing the context for COVID-19 emergence

Evaluation of SARS-CoV-2 IgG antibody response in PCR positive patients: Comparison of nine tests in relation to clinical data

Comparison of sixteen serological SARS-CoV-2 immunoassays in sixteen clinical laboratories

Clinical Performance of the Roche SARS-CoV-2 Serologic Assay

The authors acknowledge ALPHADIA S.A./N.V product's managers, M. Bonnet & B.Noirhomme for providing and performing all tests. The authors would like to thank Alain Vigneron for proofreading.

The data that supports the findings of this study are available in the main manuscript of this article. The detailed clinical/biological data of COVID-19 recovered patients are not publicly available due to privacy or ethical restrictions.