Fecha de publicación: 11/10/2024 Fuente: Biomolecules - Revista científica (MDPI) Biomolecules, Vol. 14, Pages 1286: Validation of the Enzyme-Linked ImmunoSpot Analytic Method for the Detection of Human IFN-γ from Peripheral Blood Mononuclear Cells in Response to the SARS-CoV-2 Spike Protein
Biomolecules doi: 10.3390/biom14101286
Authors:
Laura E. Carreto-Binaghi
Milton Nieto-Ponce
Andrea Palencia-Reyes
Rodolfo L. Chávez-Domínguez
Jessica Blancas-Zaragoza
Pablo Franco-Mendoza
Montserrat A. García-Ramos
Claudia I. Hernández-Lázaro
Martha Torres
Claudia Carranza

COVID-19 vaccine evaluations are mainly focused on antibody analyses, but there is growing interest in measuring the cellular immune responses from the researchers evaluating these vaccines. The cellular responses to several COVID-19 vaccines have been studied using the enzyme-linked immunospot (ELISPOT) assay for IFN-γ. However, the ELISPOT assay is no longer used only for research purpose and so the performance of this assay must be validated. Since the bioanalytical validation of ELISPOT-IFN-γ is essential for evaluating the method’s effectiveness and establishing confidence in a vaccine’s immunogenicity, the present work validates the ELISPOT-IFN-γ assay’s performance in determining the frequency of IFN-γ-producing cells after stimulation with the SARS-CoV-2 spike protein. The validation was performed in peripheral blood mononuclear cells from volunteers immunized with anti-COVID-19 vaccines. According to the findings, the LOD was 17 SFU and the LLOQ was 22 SFU, which makes the method highly sensitive and suitable for evaluating low levels of cellular responses. The procedure’s accuracy is confirmed by the correlation coefficients for the spike protein and anti-CD3+, being 0.98 and 0.95, respectively. The repeatability and intermediate precision tests were confirmed to be reliable by obtaining a coefficient of variation of ≤25%. The results obtained in this validation enable the assay to be employed for studying antigen-specific cells and evaluating cellular responses to vaccines.