Protocol for head-to-head evaluation of SARS-CoV-2 immunoassays (June 5th 2020) (original) (raw)
Validation of a commercially available SARS-CoV-2 serological Immunoassay
Aims: To validate the diagnostic accuracy of a Euroimmun SARS-CoV-2 IgG and IgA immunoassay for COVID-19 disease. Methods: In this unmatched (1:1) case-control validation study, we used sera of 181 laboratory-confirmed SARS-CoV-2 cases and 176 negative controls collected before the emergence of SARS-CoV-2. Diagnostic accuracy of the immunoassay was assessed against a whole spike protein-based recombinant immunofluorescence assay (rIFA) by receiver operating characteristic (ROC) analyses. Discrepant cases between ELISA and rIFA were further tested by pseudo-neutralization assay. Results: COVID-19 patients were more likely to be male and older than controls, and 50.3% of them were hospitalized. ROC curve analyses indicated that IgG and IgA had a high diagnostic accuracy with AUCs of 0.992 (95% Confidence Interval [95%CI]: 0.986-0.996) and 0.977 (95%CI: 0.963-0.990), respectively. IgG assays outperformed IgA assays (p=0.008). Considering optimized cut-offs taking the 15% inter-assay im...
2020
ObjectivesSerological tests detect antibodies against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in the ongoing coronavirus disease-19 (COVID-19) pandemic. Independent external clinical validation of performance characteristics is of paramount importance.MethodsFour fully automated assays, Roche Elecsys Anti-SARS-CoV-2, Abbott SARS-CoV-2 IgG, Siemens SARS-CoV-2 total (COV2T) and SARS-CoV-2 IgG (COV2G) were evaluated using 350 pre-pandemic samples and 700 samples from 245 COVID-19 patients (158 hospitalized, 87 outpatients).ResultsAll tests showed very high diagnostic specificity. Sensitivities in samples collected at least 14 days after disease onset were slightly lower than manufacturers’ claims for Roche (93.04%), Abbott (90.83%), and Siemens COV2T (90.26%), and distinctly lower for Siemens COV2G (78.76%). Concordantly negative results were enriched for immunocompromised patients. ROC curve analyses suggest a lowering of the cut-off index for the Siemens COV2G as...
Evaluation of antibody testing for SARS-Cov-2 using ELISA and lateral flow immunoassays
2020
Background: The SARS-CoV-2 pandemic caused >1 million infections during January-March 2020. There is an urgent need for robust antibody detection approaches to support diagnostics, vaccine development, safe individual release from quarantine and population lock-down exit strategies. The early promise of lateral flow immunoassay (LFIA) devices has been questioned following concerns about sensitivity and specificity. Methods: We used a panel of plasma samples designated SARS-CoV-2 positive (from SARS-CoV-2 RT-PCR-positive individuals; n=40) and negative (samples banked in the UK prior to December-2019 (n=142)). We tested plasma for SARS-Cov-2 IgM and IgG antibodies by ELISA and using nine different commercially available LFIA devices. Results: ELISA detected SARS-CoV-2 IgM or IgG in 34/40 individuals with an RT-PCR-confirmed diagnosis of SARS-CoV-2 infection (sensitivity 85%, 95%CI 70-94%), vs 0/50 pre-pandemic controls (specificity 100% [95%CI 93-100%]). IgG levels were detected in 31/31 RT-PCRpositive individuals tested ≥10 days after symptom onset (sensitivity 100%, 95%CI 89-100%). IgG titres rose during the 3 weeks post symptom onset and began to fall by 8 weeks, but remained above the detection threshold. Point estimates for the sensitivity of LFIA devices ranged from 55-70% versus RT-PCR and 65-85% versus ELISA, with specificity 95-100% and 93-100% respectively. Within the limits of the study size, the performance of most LFIA devices was similar. Conclusions: The performance of current LFIA devices is inadequate for most individual patient applications. ELISA can be calibrated to be specific for detecting and quantifying SARS-CoV-2 IgM and IgG and is highly sensitive for IgG from 10 days following symptoms onset. .
Journal of Clinical Microbiology, 2021
Commercially available SARS-CoV-2-directed antibody assays may assist in diagnosing past exposure to SARS-CoV-2 antigens. We cross-compared the following eight immunoassays detecting antibodies against SARS-CoV-2 nucleocapsid (N) or spike (S) antigens in three cohorts consisting of 859 samples from 622 patients: (i) EDI novel coronavirus COVID-19 (Epitope), (ii) RecomWell SARS-CoV-2 (Mikrogen), (iii) COVID-19 ELISA (VirCell), (iv) Elecsys anti-SARS-CoV-2 N (Roche), (v) Liaison SARS-CoV-2 S1/S2 (DiaSorin), (vi) anti-SARS-CoV-2 ELISA (EuroImmun), (vii) Elecsys anti-SARS-CoV-2 S (Roche), and (viii) Liaison SARS-CoV-2 TrimericS (DiaSorin).
Evaluation of nine commercial SARS-CoV-2 immunoassays
medRxiv (Cold Spring Harbor Laboratory), 2020
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 noncoronaviruses; 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.
Improvements and limits of anti SARS-CoV-2 antibodies assays by WHO (NIBSC 20/136) standardization
Diagnosis
Objectives A few CLIA automated immunoassays for the recognition of anti S1-RBD SARS-CoV-2 antibodies have recently been placed on the market. Preliminary data demonstrate a high correlation between methods but wide differences in absolute concentrations. A new WHO international standard for anti-SARS-CoV-2 immunoglobulin, NIBSC code 20/136, has been recently introduced to reduce the differences. The aim of this study is thus to verify the harmonization made by NIBSC 20/136 on Ab anti S1-RBD measurement on real samples. Methods The following assays were studied: LIAISON® SARS-CoV-2 TrimericS IgG (DiaSorin); Elecsys® anti-SARS-CoV-2 S (ROCHE); Atellica IM SARS-CoV-2 IgG (sCOVG) (Siemens); MAGLUMI® SARS-CoV-2 S-RBD IgG (Snibe), measuring 210 samples from 70 health workers with no previous SARS-CoV2 infection, during their Pfizer-BioNTech’s BNT162b2 vaccination period. Results The recalculation of concentrations based on the NIBSC 20/136 standardization improve the analytical and diagn...
Comparative evaluation of six immunoassays for the detection of antibodies against SARS-CoV-2
medRxiv, 2020
Objectives: Serologic techniques can serve as a complement to diagnose SARS-CoV-2 infection. The objective of our study was to compare the diagnostic performance of six immunoassays to detect antibodies against SARS-CoV-2: three lateral flow immunoassays (LFAs), one ELISA and two chemiluminescence assays (CLIAs). Methods: We evaluated three LFAs (Alltest, One Step and SeroFlash), one ELISA (Dia.Pro) and two CLIAs (Elecsys and COV2T). To assess the specificity, 60 pre-pandemic sera were used. To evaluate the sensitivity, we used 80 serum samples from patients with positive PCR for SARS-CoV-2. Agreement between techniques was evaluated using the kappa score (k). Results: All immunoassays showed a specificity of 100% except for SeroFlash (96.7%). Overall sensitivity was 61.3%, 73.8%, 67.5%, 85.9%, 88.0% and 92.0% for Alltest, One Step, SeroFlash, Dia.Pro, Elecsys and COV2T, respectively. Sensitivity increased throughout the first two weeks from the onset of symptoms, reaching sensitivi...
Performance Assessment of First-Generation Anti-SARS-CoV-2 Serological Assays
ABSTRACTThe clinical and epidemiological use of SARS-CoV-2 antibody assays is under debate with urgent need to validate and verify the performance of SARS-CoV-2 serologic assays. We aim to assess the clinical and analytical performance of three commercial serological assays of SARS-CoV-2, comparing three anti-SARS-CoV-2-IgG ELISA and identifying the seroconversion and seroprevalence in our population.A cross sectional study conducted from April 2020 to July 2020 at National Institute of Blood disease and Bone Marrow Transplantation Karachi, Pakistan with sample size of 404, enrolled consecutively. Participants were categorized into four groups’ namely convalescent plasmadonors (CPDs n=239), health care professionals (HCPs n=44), healthy blood donors (HBDs n=70) and from community (n=51).We evaluated the performance of Elecsys anti-SARS-CoV-2 electrochemiluminescence (ECLIA) assay on Cobas-e411 by Roche, three qualitative anti-SARS-CoV-2-IgG enzyme linked imunosorbant assay (ELISA) b...
Performance evaluation of a Sars-CoV-2 rapid test and two automated immunoassays
Jornal Brasileiro de Patologia e Medicina Laboratorial, 2021
Introduction: Due to urgency and demand of a response to the Covid-19 pandemic, numerous Sars-CoV-2 immunoassays have been rapidly developed. Objective: This study aimed at assessing the performance of rapid Sars-CoV-2 antibody test in comparison to high-throughput serological assays. Methods: A total of 86 serum samples were evaluated in the three assays: a lateral flow immunoassay-Wondfo Sars-CoV-2 Antibody Test (WRT)-and two chemiluminescence immunoassays: Elecsys Anti-Sars-CoV-2 (ECLIA), and Sars-CoV-2 IgG (CMIA-IgG). Results: The estimated diagnostic sensitivities of serological tests in the evaluation of serum samples from the epidemiological survey were: WRT 59% [95% confidence interval (CI) 43.4%-72.9%], ECLIA 66.7% (51%-79.4%), and CMIA-IgG 61.5% (47.1%-73%). Meanwhile, the estimated diagnostic specificity was for WRT 78.7% (95% CI 65.1%-88%), ECLIA 72.3% (58.2%-83.1%), and CMIA-IgG 76.6% (74%-95.5%). The sensitivity and specificity values were lower than manufacturers' claimed. Although 16.2% (14/86) of serological results were discordant among the three Sars-CoV-2 serological assays, the degree of agreement by the kappa index was adequate: WRT/CMIA-IgG [0.757 (95% CI 0.615-0.899)], WRT/ECLIA [0.715 (0.565-0.864)], and ECLIA/CMIA-IgG [0.858 (0.748-0.968)]. Conclusion: The serological testing may be a useful diagnostic tool, which reinforces its careful evaluation, and, as well as the correct time to use it.
Japanese Journal of Infectious Diseases, 2022
Comparative validation data and clinical performance data are essential for the reliable interpretation of SARS-CoV-2 antibody test results. This study aimed to assess the performance of six SARS-CoV-2 IgG immunoassays in different disease severity settings. Four automated chemiluminescence immunoassays Access (Beckman Coulter), Architect (Abbott), Atellica-IM (Siemens) and Elecsys (Roche) and two ELISA assays (SARS-CoV-2 IgG-S1-based and NCP IgG, Euroimmun) were evaluated in 143 patients and 50 pre-pandemic control sera. Accuracy and precision tests were performed for validation. Overall sensitivity differed between 73.38-88.65%, being higher in spike protein-based assays. Specificity was > 98% in all immunoassays. IgG response was lower for the samples taken <20 days post-symptom onset (87.30%) than for the samples taken >20 days postsymptom onset (94.80%). Higher rate of antibody was detected in the clinically moderate disease group. In the asymptomatic and mild group more antibody positivity was detected with spike protein-based assays. Clinical performance of the immunoassays differs according to disease severity and antigen targeted; moderate disease leading to highest rate of IgG response. All the assays tested were eligible for the detection of SARS-CoV-2 IgG however, spike-based assays revealed relatively higher sensitivity than the nucleoprotein-based assays particularly in the asymptomatic and mild disease severity.
2022
Severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) is a novel human coronavirus that was identified in 2019. SARS-CoV-2 infection results in an acute, severe respiratory disease called coronavirus disease 2019 (COVID-19). The emergence and rapid spread of SARS-CoV-2 has led to a global public health crisis, which continues to affect populations across the globe. Real time reverse transcription polymerase chain reaction (rRT-PCR) is the reference standard test for COVID-19 diagnosis. Serological tests are valuable tools for serosurveillance programs and establishing correlates of protection from disease. This study evaluated the performance of one in-house enzyme linked immunosorbent assay (ELISA) utilizing the pre-fusion stabilized ectodomain of SARS-CoV-2 spike (S), two commercially available chemiluminescence assays Ortho VITROS Immunodiagnostic Products Anti-SARS-CoV-2 Total Reagent Pack and Abbott SARS-CoV-2 IgG assay and one commercially available Surrogate Virus Neutralization Test (sVNT), GenScript USA Inc., cPass SARS-CoV-2 Neutralization Antibody Detection Kit for the detection of SARS-CoV-2 specific antibodies. Using a panel of rRT-PCR confirmed COVID-19 patients' sera and a negative control group as a reference standard, all three immunoassays demonstrated high comparable positivity rates and low discordant rates. All three immunoassays were highly sensitive with estimated sensitivities ranging from 95.4%-96.6%. ROC curve analysis indicated that all three immunoassays had high diagnostic accuracies with area under the curve (AUC) values ranging from 0.9698-0.9807. High positive correlation was demonstrated among the conventional microneutralization test (MNT) titers and the sVNT inhibition percent values. Our study indicates that independent evaluations are necessary to optimize the overall utility and the interpretation of the results of serological tests. Overall, we demonstrate that all serological tests evaluated in this study are suitable for the detection of SARS-CoV-2 antibodies.
medRxiv, 2021
Background: Serological testing for SARS-CoV-2 complements nucleic acid tests for patient diagnosis and enables monitoring of population susceptibility to inform the COVID-19 pandemic response. As we move into the era of vaccines, the detection of neutralising antibody will become increasingly important. Many serological tests have been developed under emergency use authorization, but their reliability remains unclear. Methods: We evaluated the performance of six commercially-available Enzyme-linked Immunosorbent Assays (ELISAs), including a surrogate virus neutralization test, for detection of SARS-CoV-2 immunoglobulins (IgA, IgM, IgG), total or neutralising antibodies and a subset of results were compared to microneutralisation. Results: For sera collected > 14 days post-symptom onset the Wantai total Ab performed best with highest sensitivity 100% (95% confidence interval: 94.6-100) followed by 93.1% for Euroimmun NCP-IgG, 93.1% for GenScript Surrogate Virus Neutralization Tes...
Comparison of sixteen serological SARS-CoV-2 immunoassays in sixteen clinical laboratories
2020
Serological SARS-CoV-2 assays are needed to support clinical diagnosis and epidemiological investigations. Recently, assays for the large-volume detection of total antibodies (Ab) and immunoglobulin (Ig) G and M against SARS-CoV-2 antigens have been developed, but there are limited data on the diagnostic accuracy of these assays. This study was organized as a Danish national collaboration and included fifteencommercial and one in-house anti-SARS-CoV-2 assays in sixteen laboratories. Sensitivity was evaluated using 150 serum samples from individuals diagnosed with asymptomatic,mild or moderate nonhospitalized (n=129) or hospitalized (n=31) COVID-19, confirmed bynucleic acid amplification tests, collected 13-73 days from symptom onset. Specificity and cross-reactivity were evaluated in samples collected prior to the SARS-CoV-2 epidemic from > 586 blood donors and patients with autoimmune diseases or CMV or EBV infections. Predefined specificity criteria of ≥ 99% were met by all tot...
Comparison of 16 Serological SARS-CoV-2 Immunoassays in 16 Clinical Laboratories
Journal of Clinical Microbiology
Serological assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are needed to support clinical diagnosis and epidemiological investigations. Recently, assays for large-scale detection of total antibodies (Ab), immunoglobulin G (IgG), and IgM against SARS-CoV-2 antigens have been developed, but there are limited data on the diagnostic accuracy of these assays.
PLOS ONE, 2021
Background In the ongoing pandemic situation of COVID-19, serological tests can complement the molecular diagnostic methods, and can be one of the important tools of sero-surveillance and vaccine evaluation. Aim To develop and evaluate a rapid SARS-CoV-2 specific ELISA for detection of anti-SARS-CoV2 IgG from patients’ biological samples. Methods In order to develop this ELISA, three panels of samples (n = 184) have been used: panel 1 (n = 19) and panel 2 (n = 60) were collected from RT-PCR positive patients within 14 and after 14 days of onset of clinical symptoms, respectively; whereas panel 3 consisted of negative samples (n = 105) collected either from healthy donors or pre-pandemic dengue patients. As a capturing agent full-length SARS-CoV2 specific recombinant nucleocapsid was immobilized. Commercial SARS-CoV2 IgG kit based on chemiluminescent assay was used for the selection of samples and optimization of the assay. The threshold cut-off point, inter-assay and intra-assay var...
2021
ObjectiveTo investigate the performance of a rapid point-of-care antibody test, the BioMedomics COVID-19 IgM/IgG Rapid Test, in comparison with a high-quality, validated, laboratory-based platform, the Roche Elecsys Anti-SARS-CoV-2 assay.MethodsSerological testing was conducted on 708 individuals. Concordance metrics were estimated. Logistic regression was used to assess associations with seropositivity.ResultsSARS-CoV-2 seroprevalence was 63.4% (449/708; 95% CI 59.8%-66.9%) using the BioMedomics assay and 71.9% (509/708; 95% CI 68.5%-75.1%) using the Elecsys assay. There were 62 discordant results between the two assays. One specimen was seropositive in the BioMedomics assay, but seronegative in the Elecsys assay, while 61 specimens were seropositive in the Elecsys assay, but seronegative in the BioMedomics assay. Positive, negative, and overall percent agreements between the two assays were 88.0% (95% CI 84.9%-90.6%), 99.5% (95% CI 97.2%-99.9%), and 91.2% (95% CI 88.9%-93.1%), res...
The Journal of Applied Laboratory Medicine, 2021
Background Serological testing provides a record of prior infection with SARS-CoV-2, but assay performance requires independent assessment. Methods We evaluated 3 commercial (Roche Diagnostics pan-IG, and Epitope Diagnostics IgM and IgG) and 2 non-commercial (Simoa and Ragon/MGH IgG) immunoassays against 1083 unique samples that included 251 PCR-positive and 832 prepandemic samples. Results The Roche assay registered the highest specificity 99.6% (3/832 false positives), the Ragon/MGH assay 99.5% (4/832), the primary Simoa assay model 99.0% (8/832), and the Epitope IgG and IgM 99.0% (8/830) and 99.5% (4/830), respectively. Overall sensitivities for the Simoa, Roche pan-IG, Epitope IgG, Ragon/MGH IgG, and Epitope IgM were 92.0%, 82.9%, 82.5%, 64.5% and 47.0%, respectively. The Simoa immunoassay demonstrated the highest sensitivity among samples stratified by days postsymptom onset (PSO), <8 days PSO (57.69%) 8–14 days PSO (93.51%), 15–21 days PSO (100%), and > 21 days PSO (95.1...
Comparison of Serological Assays for the Detection of SARS-CoV-2 Antibodies
Viruses
SARS-CoV-2 virus was first detected in late 2019 and circulated globally, causing COVID-19, which is characterised by sub-clinical to severe disease in humans. Here, we investigate the serological antibody responses to SARS-CoV-2 infection during acute and convalescent infection using a cohort of (i) COVID-19 patients admitted to hospital, (ii) healthy individuals who had experienced ‘COVID-19 like-illness’, and (iii) a cohort of healthy individuals prior to the emergence of SARS-CoV-2. We compare SARS-CoV-2 specific antibody detection rates from four different serological methods, virus neutralisation test (VNT), ID Screen® SARS-CoV-2-N IgG ELISA, Whole Antigen ELISA, and lentivirus-based SARS-CoV-2 pseudotype virus neutralisation tests (pVNT). All methods were able to detect prior infection with COVID-19, albeit with different relative sensitivities. The VNT and SARS-CoV-2-N ELISA methods showed a strong correlation yet provided increased detection rates when used in combination. ...
IFCC Interim Guidelines on Serological Testing of Antibodies against SARS-CoV-2
Clinical Chemistry and Laboratory Medicine (CCLM)
Serological testing for the detection of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is emerging as an important component of the clinical management of patients with coronavirus disease 2019 (COVID-19) as well as the epidemiological assessment of SARS-CoV-2 exposure worldwide. In addition to molecular testing for the detection of SARS-CoV-2 infection, clinical laboratories have also needed to increase testing capacity to include serological evaluation of patients with suspected or known COVID-19. While regulatory approved serological immunoassays are now widely available from diagnostic manufacturers globally, there is significant debate regarding the clinical utility of these tests, as well as their clinical and analytical performance requirements prior to application. This document by the International Federation for Clinical Chemistry and Laboratory Medicine (IFCC) Taskforce on COVID-19 provides interim guidance on: (A) clinical indications an...