Related Experiment Video
Updated: Oct 18, 2025
Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection
Published on: June 5, 2021
An evaluation of a FluoroSpot assay as a diagnostic tool to determine SARS-CoV-2 specific T cell responses
Sara M Mangsbo1, Sebastian Havervall2, Ida Laurén1
1Department of Pharmaceutical Biosciences, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
Insights
This study assessed SARS-CoV-2 T cell assays, finding an in-house peptide pool offered higher specificity (96.1%) than commercial options. While sensitivity varied, T cell responses currently have limited diagnostic value for COVID-19 infection.
Area of Science:
- Immunology
- Virology
- Diagnostic Assays
Background:
- Serological assays are cost-effective for characterizing SARS-CoV-2 immune responses.
- Cellular immune responses, like T cell assays, may diagnose past SARS-CoV-2 infection in seronegative individuals.
- Cross-reactivity with endemic human coronaviruses (HCoVs) can impact T cell assay specificity.
Purpose of the Study:
- To evaluate the specificity and sensitivity of a SARS-CoV-2 interferon-gamma (IFN-γ) Release Assay (IGRA) using FluoroSpot.
- To compare commercially available SARS-CoV-2 peptide pools with an in-house designed pool considering HCoV cross-reactivity.
- To assess the diagnostic utility of T cell responses for identifying previous SARS-CoV-2 infection.
Main Methods:
- Utilized a FluoroSpot-based IFN-γ Release Assay (IGRA).
- Employed commercially available and in-house designed SARS-CoV-2 peptide pools.
- Tested blood samples from SARS-CoV-2 seroconverted healthcare workers (HCW), seronegative HCW, and PCR-confirmed COVID-19 patients.
Main Results:
- The in-house peptide pool achieved 96.1% specificity, outperforming commercial pools (80.4% and 85.3%).
- Sensitivity was higher in hospitalized COVID-19 patients compared to the HCW cohort.
- Commercial pools showed higher sensitivity (92.0%) in seroconverted HCW than the in-house pool (76.0%).
Conclusions:
- The in-house peptide pool demonstrates superior specificity for SARS-CoV-2 T cell detection.
- The diagnostic value of T cell immune responses for individual COVID-19 diagnosis is currently limited.
- SARS-CoV-2 IGRA remains a valuable tool for research purposes.
Abstract:
Numerous assays evaluating serological and cellular responses have been developed to characterize immune responses against SARS-CoV-2. Serological assays are both cost- and time-effective compared to cellular assays, but cellular immune responses may provide a diagnostic value to determine previous SARS-CoV-2 infection in seronegative individuals. However, potential cross-reactive T cell responses stemming from prior encounters with human coronaviruses (HCoVs) may affect assay specificity. In this study, we evaluated the specificity and sensitivity of a SARS-CoV-2 IFN-γ Release Assay (IGRA) based on the FluoroSpot method employing commercially available SARS-CoV-2-specific peptide pools, as well as an in-house designed SARS-CoV-2 peptide pool restricted to 5 amino acid stretches or less aligning with endemic HCoVs. Blood samples were obtained from healthcare workers (HCW) 5-6 months post SARS-CoV-2 spike (S) IgG and nucleocapsid (N) IgG dual seroconversion (n = 187) and HCW who had been S IgG and N IgG dual seronegative at repeated occasions, including the current sampling time point (n = 102). In addition, samples were obtained 4 to 5 months post infection from 55 polymerase chain reaction (PCR)-confirmed COVID-19 patients. Assay specificity and sensitivity were calculated with serology as a reference standard for HCW. The in-house generated peptide pool displayed a specificity of 96.1%, while the commercially available peptide pools displayed specificities of 80.4% and 85.3%, respectively. Sensitivity was higher in a cohort of previously hospitalized COVID-19 patients (96.4% and 84.0% for the commercially available peptide pools and 92.7% for the in-house generated peptide pool) compared to the HCW cohort (92.0% and 66.8% for the commercially available peptide pools and 76.0% for the in-house generated peptide pool). Based on these findings, the individual diagnostic value of T cell immune responses against SARS-CoV-2 currently appears to be limited but remain an important research tool ahead.
More Related Videos
06:13Enzyme-linked Immunospot Assay ELISPOT: Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens
Published on: November 23, 2010
08:21Cell-Free Dot Blot as a Practical and Adaptable Immunoassay Platform for the Detection of Antibody Response in Human and Animal Sera
Published on: May 23, 2025