Development And Performance Evaluation of A Rapid In-House ELISA for Retrospective Serosurveillance of SARS-CoV-2

Abstract

BackgroundIn 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.AimTo develop and evaluate a rapid SARS-CoV-2 specific ELISA for detection of anti-SARS-CoV2 IgG from patient’s biological samples.MethodsIn order to develop the 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 variations were determined. The total assay time for this in-house ELISA was set for 30 minutes.ResultsThe assay time was set at a total of 30 minutes with the sensitivity of 84% (95% confidence interval, CI, 60.4%, 96.6%) and 98% (95% CI, 91.1%, 100.0%), for panel 1 and 2 respectively, with over all 94.9% sensitivity (95% CI 87.5%, 98.6%). Moreover, the clinical specificity is 97.1% (95% CI, 91.9%, 99.4%) with no cross reaction with dengue sample. The overall positive and negative predictive values are 96.2% (95% CI 89.2%, 99.2%) and 96.2% (95% CI, 90.6% 99.0%) respectively. In-house ELISA demonstrated 100% positive and negative percent agreement with ROCHE (Elecsys; Anti-SARS-CoV-2), with a Cohen’s kappa value of 1.00 (very strong agreement), while comparing 13 positive and 17 negative confirmed cases.ConclusionThe assay is rapid and can be applied as one of the early and retrospective sero-monitoring tools in all over the affected areas.

Detection of SARS-CoV-2 by antigen ELISA test is highly swayed by viral load and sample storage condition

“…Immunoplate 96-well flat-bottom plate (Extra Gene, USA) was coated with different dilution (1:50, 1:100, and 1:200) 100 µL/ well of mouse SARS-CoV-2 NCP specific monoclonal antibody using coating buffer (sodium bicarbonate, pH >9) and incubated either overnight at 4°C or 37°C for an hour following our previously established ELISA methods [22,23]. The unbound…”

Current diagnostic approaches to detect two important betacoronaviruses: Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

“…For SARS-CoV-2, several studies had reported the use of ELISA to detect antibodies against N protein of SARS-CoV and the sensitivity and specificity of such test ranged from 90 to 94%, and, above 97%, respectively ( Table 11 ) [202] , [203] . Compared to ELISA assay that was aimed to detect antibodies like IgG against the N protein of SARS-CoV-2 [202] , [203] , the use of ELISA test that was focused to detect antibodies against S protein of SARS-CoV-2 showed wide range values of sensitivity and specificity, according to the findings reported from various studies [199] , [204] , [205] , [206] , [207] .…”

“…For SARS-CoV-2, several studies had reported the use of ELISA to detect antibodies against N protein of SARS-CoV and the sensitivity and specificity of such test ranged from 90 to 94%, and, above 97%, respectively ( Table 11 ) [202] , [203] . Compared to ELISA assay that was aimed to detect antibodies like IgG against the N protein of SARS-CoV-2 [202] , [203] , the use of ELISA test that was focused to detect antibodies against S protein of SARS-CoV-2 showed wide range values of sensitivity and specificity, according to the findings reported from various studies [199] , [204] , [205] , [206] , [207] . Some studies showed that S protein-based ELISA test was less sensitive to detect IgG and IgA against SARS-CoV-2 (<70%) [206] , [207] while few other studies reported that such test was highly sensitive (at least 90%) to detect IgG, IgA, or IgM against the virus S antigen [199] , [204] , [205] , [208] .…”

“…Some studies showed that S protein-based ELISA test was less sensitive to detect IgG and IgA against SARS-CoV-2 (<70%) [206] , [207] while few other studies reported that such test was highly sensitive (at least 90%) to detect IgG, IgA, or IgM against the virus S antigen [199] , [204] , [205] , [208] . Therefore, N protein-based ELISA assay could be a better choice of ELISA test to predict serological response in the SARS-CoV-2 patients as the test results of this assay seem to be more consistent and vary less in different reported studies [202] , [203] as compared to the S protein-based ELISA test [199] , [204] , [206] , [207] , [208] , [209] . On the other hand, some studies had reported the use of ELISA test that was aimed to detect different antibodies against different proteins of SARS-CoV-2, and examples of these tests were tests that were focusing to detect antibodies against N and S protein of SARS-CoV-2 [196] , [204] , [209] , [210] , [211] , [212] , [213] , [214] , [215] .…”

Assessment of a rapid pan-antibody dot test for detection of antibodies against SARS-CoV-2

“…Though nucleic acid detection has the advantages of earlystage sensitivity, high specificity, and easy operation, the accuracy of nucleic acid detection results needs to be comprehensively analyzed from influencing factors such as sample type, quality, experimental factors, kit performance, patient infection cycle, especially for difficult-to-diagnose cases, the accuracy of detection should be more strictly controlled 8 . Therefore, complementary serological immunoassays such as enzyme-linked immunosorbent assay (ELISA), lateral flow immunoassay (LFIA), and loop-mediated isothermal amplification (LAMP), rapid highthroughput flow-through membrane immunoassay (FMIA) are utilized nowadays for the pathogen detection, infection progression evaluation, and transmission dynamics analysis [9][10][11] .…”