Simultaneous evaluation of antibodies that inhibit SARS-CoV-2 variants via multiplex assay.
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ABSTRACT:
SUBMITTER: Lopez E
PROVIDER: S-EPMC8409985 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Vaccines against SARS-CoV-2 have alleviated infection rates, hospitalization and deaths associated with COVID-19. In order to monitor humoral immunity, several serology tests have been developed, but the recent emergence of variants of concern has revealed the need for assays that predict the neutralizing capacity of antibodies in a fast and adaptable manner. Sensitive and fast neutralization assays would allow a timely evaluation of immunity against emerging variants and support drug and vaccine discovery efforts. Here we describe a simple, fast, and cell-free multiplexed flow cytometry assay to interrogate the ability of antibodies to prevent the interaction of Angiotensin-converting enzyme 2 (ACE2) and the receptor binding domain (RBD) of the original Wuhan-1 SARS-CoV-2 strain and emerging variants simultaneously, as a surrogate neutralization assay. Using this method, we demonstrate that serum antibodies collected from representative individuals at different time-points during the pandemic present variable neutralizing activity against emerging variants, such as Omicron BA.1 and South African B.1.351. Importantly, antibodies present in samples collected during 2021, before the third dose of the vaccine was administered, do not confer complete neutralization against Omicron BA.1, as opposed to samples collected in 2022 which show significant neutralizing activity. The proposed approach has a comparable performance to other established surrogate methods such as cell-based assays using pseudotyped lentiviral particles expressing the spike of SARS-CoV-2, as demonstrated by the assessment of the blocking activity of therapeutic antibodies (i.e. Imdevimab) and serum samples. This method offers a scalable, cost effective and adaptable platform for the dynamic evaluation of antibody protection in affected populations against variants of SARS-CoV-2.
| S-EPMC9730237 | biostudies-literature
Project description:ObjectivesDetection of antibodies to multiple SARS-CoV-2 antigens in a single assay could increase diagnostic accuracy, differentiate vaccination from natural disease, and aid in retrospective exposure determination. Correlation of binding antibody assessment in clinical assays with neutralizing antibodies is needed to better understand the humoral response to SARS-CoV-2 infection and establish of correlates of protection.MethodsA cohort of 752 samples was used to assess specificity, sensitivity, and comparison to 6 other Conformitè Europëenne serologic assays for the BioRad SARS-CoV-2 IgG multiplex assay which measures receptor binding domain IgG (RBD), spike-S1 IgG (S1), spike-S2 IgG (S2), and nucleocapsid IgG (N). A subset of serial specimens from 14 patients was also tested for neutralizing antibodies (n = 61).ResultsSpecificity for RBD and S1 IgG was 99.4% (n = 170) and 100% for S2 and N IgG (n = 170) in a cohort selected for probable interference. Overall assay concordance with other assays was >93% for IgG and total antibody assays and reached 100% sensitivity for clinical concordance at >14 days as a multiplex assay. RBD and S1 binding antibody positivity demonstrated 79-95% agreement with the presence of neutralizing antibodies.ConclusionsThe BioRad SARS-CoV-2 IgG assay is comparable to existing assays, and achieved 100% sensitivity when all markers were included. The ability to measure antibodies against spike and nucleocapsid proteins simultaneously may be advantageous for complex clinical presentations, epidemiologic research, and in decisions regarding infection prevention strategies. Additional independent validations are needed to further determine binding antibody and neutralizing antibody correlations.
| S-EPMC8387133 | biostudies-literature
Project description:BackgroundIn the context of the pandemic, the rapid emergency use authorisation of diagnostic assays for SARS-CoV-2 has meant there are few peer-reviewed published studies of clinical performance of commercial assays.AimsTo evaluate the clinical performance of AusDiagnostics respiratory multiplex tandem PCR assay including SARS-CoV-2.MethodsWe reviewed the results following implementation of AusDiagnostics respiratory multiplex tandem PCR assay including SARS-CoV-2, and compared with an in-house RT-PCR assay at our State Reference Laboratory.ResultsInitial validation using AusDiagnostics coronavirus multiplex tandem PCR assay including SARS-CoV-2 demonstrated good concordance with the State Reference Laboratory. After implementing the AusDiagnostics respiratory multiplex tandem PCR assay including SARS-CoV-2, we tested 7839 samples. 127 samples in which SARS-CoV-2 was detected using the AusDiagnostics assay were referred for testing at the State Reference Laboratory, with concordant results in 118/127 (92.9%) of samples. After resolution of discrepancies, 125/127 (98.4%) of AusDiagnostics results were determined to be true positive results. Out of 7839 samples tested for SARS-CoV-2 during this period, only 2 tests (0.02%) were indeterminate results.ConclusionThe AusDiagnostics respiratory MT-PCR assay is a reliable assay for detection of SARS-CoV-2.
| S-EPMC7236671 | biostudies-literature
Project description:Serological assays for SARS-CoV-2 antibodies must be validated for performance with a large panel of clinical specimens. Most existing assays utilize a single antigen target and may be subject to reduced diagnostic specificity. This study evaluated a multiplex assay that detects antibodies to three SARS-CoV-2 targets. Human serum specimens (n = 323) with known previous SARS-CoV-2 exposure status were tested on a commercially available multiplex bead assay (MBA) measuring IgG to SARS-CoV-2 spike protein receptor-binding domain (RBD), nucleocapsid protein (NP), and RBD/NP fusion antigens. Assay performance was evaluated against reverse transcriptase PCR (RT-PCR) results and also compared with test results for two single-target commercial assays. The MBA had a diagnostic sensitivity of 89.8% and a specificity of 100%, with serum collection at >28 days following COVID-19 symptom onset showing the highest seropositivity rates (sensitivity: 94.7%). The MBA performed comparably to single-target assays with the ability to detect IgG against specific antigen targets, with 19 (5.9%) discrepant specimens compared to the NP IgG assay and 12 (3.7%) compared to the S1 RBD IgG assay (kappa coefficients 0.92 and 0.88 compared to NP IgG and S1 RBD IgG assays, respectively. These findings highlight inherent advantages of using a SARS-CoV-2 serological test with multiple antigen targets; specifically, the ability to detect IgG against RBD and NP antigens simultaneously. In particular, the 100.0% diagnostic specificity exhibited by the MBA in this study is important for its implementation in populations with low SARS-CoV-2 seroprevalence or where background antibody reactivity to SARS-CoV-2 antigens has been detected. IMPORTANCE Reporting of SARS-CoV-2 infections through nucleic acid or antigen based diagnostic tests severely underestimates the true burden of exposure in a population. Serological data assaying for antibodies against SARS-CoV-2 antigens offers an alternative source of data to estimate population exposure, but most current immunoassays only include a single target for antibody detection. This report outlines a direct comparison of a multiplex bead assay to two other commercial single-target assays in their ability to detect IgG against SARS-CoV-2 antigens. Against a well-defined panel of 323 serum specimens, diagnostic sensitivity and specificity were very high for the multiplex assay, with strong agreement in IgG detection for single targets compared to the single-target assays. Collection of more data for individual- and population-level seroprofiles allows further investigation into more accurate exposure estimates and research into the determinants of infection and convalescent responses.
| S-EPMC9241621 | biostudies-literature
Project description:BackgroundNumerous serology assays are available for detection of SARS-CoV-2 antibodies but are limited in that only 1 or 2 target antigen(s) can be tested at a time. Here, we describe a novel multiplex assay that simultaneously detects and quantifies IgG antibodies to SARS-CoV-2 antigens, spike (S), nucleocapsid (N), receptor-binding domain (RBD), and N-terminal domain (NTD) in a single well.MethodsSensitivity was determined using samples (n = 124) from confirmed SARS-CoV-2 RT-PCR positive individuals. Prepandemic (n = 100) and non-COVID respiratory infection positive samples (n = 100) were used to evaluate specificity. Samples were analyzed using COVID-19 IgG multiplex serology assay from Meso Scale Discovery (MSD) and using commercial platforms from Abbott, EUROIMMUN, and Siemens.ResultsAt >14 days post-PCR, MSD assay displayed >98.0% sensitivity [S 100% (95% CI 98.0%-100.0%); N 98.0% (95% CI 97.2%-98.9%); RBD 94.1% (95% CI 92.6%-95.6%); NTD 98.0% (95% CI, 97.2%-98.9%)] and 99% specificity (95% CI 99.3%-99.7%) for antibodies to all 4 antigens. Parallel assessment of antibodies to more than 1 antigen improved the sensitivity to 100% (95% CI 98.0%-100.0%) while maintaining 98% (95% CI 97.6%-98.4%) specificity regardless of the combinations used. When AU/mL concentrations of IgG antibodies from the MSD assay were compared against the corresponding IgG signals acquired from the single target commercial assays, the following correlations were observed: Abbott (vs MSD N, R2 = 0.73), Siemens (vs MSD RBD, R2 = 0.92), and EUROIMMUN (vs MSD S, R2 = 0.82).ConclusionMSD assay offers an accurate and a comprehensive assessment of SARS-CoV-2 antibodies with higher sensitivity and equivalent specificity compared to the commercial IgG serology assays.
| S-EPMC8767881 | biostudies-literature
Project description:The rapid emergence of SARS-CoV-2 variants with high severity and transmutability adds further urgency for rapid and multiplex molecular testing to identify the variants. A nucleotide matrix-assisted laser-desorption-ionization time-of-flight mass spectrophotometry (MALDI-TOF MS)-based assay was developed (called point mutation array, PMA) to identify four major SARS-CoV-2 variants of concern (VOCs) including Alpha, Beta, Delta, and Omicron (namely PMA-ABDO) and differentiate Omicron subvariant (namely PMA-Omicron). PMA-ABDO and PMA-Omicron consist of 24 and 28 mutation sites of the spike gene. Both PMA panels specifically identified VOCs with as low as 10 viral copies/µl. The panel has shown a 100% concordant with the Next Generation Sequencing (NGS) results testing on 256 clinical specimens with real-time PCR cycle threshold (Ct) values less than 26. It showed a higher sensitivity over NGS; 25/28 samples were positive by PMA but not NGS in the clinical samples with PCR Ct higher than 26. Due to the mass of nucleotide used to differentiate between wild-type and mutation strains, the co-infection or recombination of multiple variants can be determined by the PMA method. This method is flexible in adding a new primer set to identify a new emerging mutation site among the current circulating VOCs and the turnaround time is less than 8 h. However, the spike gene sequencing or NGS retains the advantage of detecting newly emerged variants.
| S-EPMC9900542 | biostudies-literature
Project description:The humoral immune response to SARS-CoV-2 results in antibodies against spike (S) and nucleoprotein (N). However, whilst there are widely-available neutralization assays for S-antibodies, there is no assay for N-antibody activity. Here we present a simple in vitro method called EDNA (electroporated-antibody dependent neutralization assay) that provides a quantitative measure of N-antibody activity in unpurified serum from SARS-CoV-2 convalescents. We show that N-antibodies neutralize SARS-CoV-2 intracellularly and cell-autonomously but require the cytosolic Fc-receptor TRIM21. Using EDNA, we show that low N-antibody titres can be neutralizing, whilst some convalescents possess serum with high titres but weak activity. N-antibody and N-specific T cell activity correlates within individuals, suggesting N-antibodies may protect against SARS-CoV-2 by promoting antigen presentation. This work highlights the potential benefits of N-based vaccines and provides an in vitro assay to allow the antibodies they induce to be tested.
| S-SCDT-EMBOJ-2021-108588 | biostudies-other
Project description: Not available
| S-BSST379 | biostudies-other
Project description:Early diagnosis, early isolation, and early treatment are efficient solutions to control the COVID-19 pandemic. To achieve the accurate early diagnosis of SARS-CoV-2, a multiplex detection strategy is required for the cross-validation to solve the problem of "false negative" of the existing gold standard assay. Here, we present a multicomponent nucleic acid assay platform for SARS-CoV-2 detection based on lanthanide nanoparticle (LnNP)-tagging strategy. For targeting SARS-CoV-2's RNA fragments ORF1ab gene, RdRp gene, and E gene, three LnNP probes can be used simultaneously to identify three sites in one sample through elemental mass spectrometry detection with limits of detection of 1.2, 1.3, and 1.3 fmol, respectively. With the multisite cross-validation, we envision that this multiplex and sensitive detection platform may provide an effective strategy for SARS-CoV-2 fast screening with a high accuracy.
| S-EPMC8442555 | biostudies-literature
Project description:To combat the continued pandemic of COVID-19, multiplex serological assays have been developed to comprehensively monitor the humoral immune response and help to design new vaccination protocols to different SARS-CoV-2 variants. However, multiplex beads and stably transfected cell lines require stringent production and storage conditions, and assays based on flow cytometry is time-consuming and its application is therefore restricted. Here, we describe a phage display system to distinguish the differences of immune response to antigenic domains of multiple SARS-CoV-2 variants simultaneously. Compared with linear peptides, the recombinant antigens displayed on the phage surface have shown some function that requires the correct folding to form a stable structure, and the binding efficiency between the recombinant phage and existing antibodies is reduced by mutations on antigens known to be important for antigen-antibody interaction. By using Phage display mediated immuno-multiplex quantitative PCR (Pi-mqPCR), the binding efficiency between the antibody and antigens of different SARS-CoV-2 variants can be measured in one amplification reaction. Overall, these data show that this assay is a valuable tool to evaluate the humoral response to the same antigen of different SARS-CoV-2 variants or antigens of different pathogens. Combined with high-throughput DNA sequencing technology, this phage display system can be further applied in monitoring humoral immune response in a large population before and after vaccination.
| S-EPMC9441900 | biostudies-literature