A plug-and-play platform of ratiometric bioluminescent sensors for homogeneous immunoassays.

Heterogeneous immunoassays such as ELISA have become indispensable in modern bioanalysis, yet translation into point-of-care assays is hindered by their dependence on external calibration and multiple washing and incubation steps. Here, we introduce RAPPID (Ratiometric Plug-and-Play Immunodiagnostics), a mix-and-measure homogeneous immunoassay platform that combines highly specific antibody-based detection with a ratiometric bioluminescent readout. The concept entails analyte-induced complementation of split NanoLuc luciferase fragments, photoconjugated to an antibody sandwich pair via protein G adapters. Introduction of a calibrator luciferase provides a robust ratiometric signal that allows direct in-sample calibration and quantitative measurements in complex media such as blood plasma. We developed RAPPID sensors that allow low-picomolar detection of several protein biomarkers, anti-drug antibodies, therapeutic antibodies, and both SARS-CoV-2 spike protein and anti-SARS-CoV-2 antibodies. With its easy-to-implement standardized workflow, RAPPID provides an attractive, fast, and low-cost alternative to traditional immunoassays, in an academic setting, in clinical laboratories, and for point-of-care applications.

Diagnostic accuracy of serological tests and kinetics of severe acute respiratory syndrome coronavirus 2 antibody: A systematic review and meta-analysis.

This study aimed to assess the diagnostic test accuracy (DTA) of severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) serological test methods and the kinetics of antibody positivity. Systematic review and meta-analysis were conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guideline. We included articles evaluating the diagnostic accuracy of serological tests and the kinetics of antibody positivity. MEDLINE through PubMed, Scopus, medRxiv and bioRxiv were sources of articles. Methodological qualities of included articles were appraised using QUADAS-2 while Metandi performs bivariate meta-analysis of DTA using a generalized linear mixed-model approach. Stata 14 and Review Manager 5.3 were used for data analysis. The summary sensitivity/specificity of chemiluminescence immunoassay (CLIA), enzyme-linked immunosorbent assay (ELISA) and lateral flow immunoassay (LFIA) were 92% (95% CI: 86%-95%)/99% (CI: 97%-99%), 86% (CI: 82%-89%)/99% (CI: 98%-100%) and 78% (CI: 71%-83%)/98% (95% CI: 96%-99%), respectively. Moreover, CLIA-based assays produced nearly 100% sensitivity within 11-15 days post-symptom onset (DPSO). Based on antibody type, the sensitivity of ELISA-total antibody, CLIA-IgM/G and CLIA-IgG gauged at 94%, 92% and 92%, respectively. The sensitivity of CLIA-RBD assay reached 96%, while LFIA-S demonstrated the lowest sensitivity, 71% (95% CI: 58%-80%). CLIA assays targeting antibodies against RBD considered the best DTA. The antibody positivity rate increased corresponding with DPSO, but there was some decrement when moving from acute phase to convalescent phase of infection. As immunoglobulin isotope-related DTA was heterogeneous, our data have insufficient evidence to recommend CLIA/ELISA for clinical decision-making, but likely to have comparative advantage over RT-qPCR in certain circumstances and geographic regions.

Correlation of SARS-CoV-2 Neutralizing Antibodies to an Automated Chemiluminescent Serological Immunoassay.

INTRODUCTION: Neutralizing antibodies (NAbs) are capable of binding to a virus to render it incapable of infection. The ability of commercially available SARS-CoV-2 serological tests to detect NAbs has not been widely reported. We sought to correlate the antibodies detected by an automated chemiluminescent immunoassay with NAbs. METHODS: Residual serum samples from 35 patients that had a positive antibody test using the LIAISON® SARS-CoV-2 S1/S2 IgG chemiluminescent immunoassay and 2 antibody-negative control sera were tested for NAbs using a plaque reduction neutralization test (PRNT). RESULTS: NAbs were detected in 66% (23/35) of the antibody-positive samples. The immunoassay signal value ranged from 21.7 to 131.3 AU/mL (median, 90.5) with significant correlation between it and the PRNT (r = 0.61, P = 0.002). In the samples without NAbs, the immunoassay signal ranged from 16.3 to 66.2 AU/mL (median, 27.2). An immunoassay signal cutoff of >41 AU/mL was 91% sensitive and 92% specific for the detection of NAbs. DISCUSSION: It is important that correlates of immunity to SARS-CoV-2 be identified and NAbs are considered to be central indicators of such. PRNT is the gold-standard test for identifying NAbs but it cannot be used for large-scale testing of populations. It is necessary to establish relationships between it and widely used commercial serological assays for SARS-CoV-2.

Multicenter evaluation of two chemiluminescence and three lateral flow immunoassays for the diagnosis of COVID-19 and assessment of antibody dynamic responses to SARS-CoV-2 in Taiwan.

This multicenter, retrospective study included 346 serum samples from 74 patients with coronavirus disease 2019 (COVID-19) and 194 serum samples from non-COVID-19 patients to evaluate the performance of five anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody tests, i.e. two chemiluminescence immunoassays (CLIAs): Roche Elecsys® Anti-SARS-CoV-2 Test (Roche Test) and Abbott SARS-CoV-2 IgG (Abbott Test), and three lateral flow immunoassays (LFIAs): Wondfo SARS-CoV-2 Antibody Test (Wondfo Test), ASK COVID-19 IgG/IgM Rapid Test (ASK Test), and Dynamiker 2019-nCoV IgG/IgM Rapid Test (Dynamiker Test). We found high diagnostic sensitivities (%, 95% confidence interval [CI]) for the Roche Test (97.4%, 93.4-99.0%), Abbott Test (94.0%, 89.1-96.8%), Wondfo Test (91.4%, 85.8-94.9%), ASK Test (97.4%, 93.4-99.0%), and Dynamiker Test (90.1%, 84.3-94.0%) after >21 days of symptom onset. Meanwhile, the diagnostic specificity was 99.0% (95% CI, 96.3-99.7%) for the Roche Test, 97.9% (95% CI, 94.8-99.2%) for the Abbott Test, and 100.0% (95% CI, 98.1-100.0%) for the three LFIAs. Cross-reactivity was observed in sera containing anti-cytomegalovirus (CMV) IgG/IgM antibodies and autoantibodies. No difference was observed in the time to seroconversion detection of the five serological tests. Specimens from patients with COVID-19 pneumonia demonstrated a shorter seroconversion time and higher chemiluminescent signal than those without pneumonia. Our data suggested that understanding the dynamic antibody response after COVID-19 infection and performance characteristics of different serological test are crucial for the appropriate interpretation of serological test result for the diagnosis and risk assessment of patient with COVID-19 infection.

Diagnostic accuracy of an automated chemiluminescent immunoassay for anti-SARS-CoV-2 IgM and IgG antibodies: an Italian experience.

A pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been spreading throughout the world. Though molecular diagnostic tests are the gold standard for COVID-19, serological testing is emerging as a potential surveillance tool, in addition to its complementary role in COVID-19 diagnostics. Indubitably quantitative serological testing provides greater advantages than qualitative tests but today there is still little known about serological diagnostics and what the most appropriate role quantitative tests might play. Sixty-one COVID-19 patients and 64 patients from a control group were tested by iFlash1800 CLIA analyzer for anti-SARS CoV-2 antibodies IgM and IgG. All COVID-19 patients were hospitalized in San Giovanni di Dio Hospital (Florence, Italy) and had a positive oro/nasopharyngeal swab reverse-transcription polymerase chain reaction result. The highest sensitivity with a very good specificity performance was reached at a cutoff value of 10.0 AU/mL for IgM and of 7.1 for IgG antibodies, hence near to the manufacturer's cutoff values of 10 AU/mL for both isotypes. The receiver operating characteristic curves showed area under the curve values of 0.918 and 0.980 for anti-SARS CoV-2 antibodies IgM and IgG, respectively. iFlash1800 CLIA analyzer has shown highly accurate results for the anti-SARS-CoV-2 antibodies profile and can be considered an excellent tool for COVID-19 diagnostics.