tetano
Editor, Senior Moderator
Hum Vaccin Immunother
. 2024 Dec 31;20(1):2428516.
doi: 10.1080/21645515.2024.2428516. Epub 2024 Nov 28. Validation of an interferon-gamma enzyme-linked immunosorbent spot assay to evaluate cell-mediated immunity against severe acute respiratory syndrome coronavirus 2
Yunhwa Kim[SUP] 1 [/SUP], Eun-Jeong Jang[SUP] 1 [/SUP], Ji-Young Hwang[SUP] 1 [/SUP], Kyung-Min Lee[SUP] 1 [/SUP], Sivilay Xayaheuang[SUP] 1 [/SUP], Seok-Tae Choi[SUP] 1 [/SUP], Hosun Park[SUP] 1 2 [/SUP]
Affiliations
The COVID-19 pandemic forced the rapid development of methods to measure humoral and cellular immunity against SARS-CoV-2. The lack of a global standardized protocol and the high variability of intra- and inter-assay precision of the T-cell response made it difficult to compare T-cell assay results with those of other laboratories. The interferon-gamma enzyme-linked immunosorbent spot (IFN-γ ELISpot) assay for immunogenicity evaluation was validated using naturally infected donor peripheral blood mononuclear cells, a commercially available IFN-γ ELISpot kit, and a SARS-CoV-2 specific peptide pool. Depending on anti-CD3 and peptide pool stimulation, the mean coefficients of variation (CVs) of the intra-assay precision were 19.0% and 13.4%, respectively. The mean CVs of the inter-assay precision were 26.1% and 25.4%, and the mean CVs for reproducibility were 6.7% and 15.9%, respectively. Linearity with an R-squared value between 0.98 and 0.99 was established, and the mean CVs between the lots were 17.6% and 6.6%, depending on the anti-CD3 and peptide pool stimulation, respectively. The limit of detection was 11 spot-forming counts per well. Taken together, we demonstrated that the IFN-γ ELISpot assay is feasible for evaluating SARS-CoV-2-specific cell-mediated immune function through validation based on standard operating procedures.
Keywords: COVID-19; ELISpot assay; SARS-CoV-2; cell-mediated immunity; validation.
. 2024 Dec 31;20(1):2428516.
doi: 10.1080/21645515.2024.2428516. Epub 2024 Nov 28. Validation of an interferon-gamma enzyme-linked immunosorbent spot assay to evaluate cell-mediated immunity against severe acute respiratory syndrome coronavirus 2
Yunhwa Kim[SUP] 1 [/SUP], Eun-Jeong Jang[SUP] 1 [/SUP], Ji-Young Hwang[SUP] 1 [/SUP], Kyung-Min Lee[SUP] 1 [/SUP], Sivilay Xayaheuang[SUP] 1 [/SUP], Seok-Tae Choi[SUP] 1 [/SUP], Hosun Park[SUP] 1 2 [/SUP]
Affiliations
- PMID: 39610025
- PMCID: PMC11610554
- DOI: 10.1080/21645515.2024.2428516
The COVID-19 pandemic forced the rapid development of methods to measure humoral and cellular immunity against SARS-CoV-2. The lack of a global standardized protocol and the high variability of intra- and inter-assay precision of the T-cell response made it difficult to compare T-cell assay results with those of other laboratories. The interferon-gamma enzyme-linked immunosorbent spot (IFN-γ ELISpot) assay for immunogenicity evaluation was validated using naturally infected donor peripheral blood mononuclear cells, a commercially available IFN-γ ELISpot kit, and a SARS-CoV-2 specific peptide pool. Depending on anti-CD3 and peptide pool stimulation, the mean coefficients of variation (CVs) of the intra-assay precision were 19.0% and 13.4%, respectively. The mean CVs of the inter-assay precision were 26.1% and 25.4%, and the mean CVs for reproducibility were 6.7% and 15.9%, respectively. Linearity with an R-squared value between 0.98 and 0.99 was established, and the mean CVs between the lots were 17.6% and 6.6%, depending on the anti-CD3 and peptide pool stimulation, respectively. The limit of detection was 11 spot-forming counts per well. Taken together, we demonstrated that the IFN-γ ELISpot assay is feasible for evaluating SARS-CoV-2-specific cell-mediated immune function through validation based on standard operating procedures.
Keywords: COVID-19; ELISpot assay; SARS-CoV-2; cell-mediated immunity; validation.