Background: An NDV-based vector has been used as a veterinary vaccine and, recently, as a human COVID-19 vaccine. However, data for the potential immune response against the vector in humans are scarce; therefore, it is important to evaluate the levels of antibodies produced. The HI assay is the gold standard for assessing the humoral response against NDV in poultry serum. Objective: Here, the objective was to validate the HI assay against the NDV-vectored vaccine to analyze antibodies in human serum. Methods: First, we standardized the conditions in human sera before validation. Results: The results for analytical performance in terms of selectivity, sensitivity, specificity, and positive and negative predictive values, as well as positive and negative diagnostic reliability, indicate that the assay is highly selective, allowing clear discrimination between positive and negative samples. Regarding repeatability and intermediate precision, we demonstrated that the assay has the precision to obtain consistent results, guaranteeing their reliability and truthfulness. Finally, the results regarding accuracy, linearity, and robustness indicate that the assay is accurate across the evaluated concentration intervals, with a linear correlation between low and high levels, and demonstrate that it is robust and consistent when serum–antigen interaction times are changed. Conclusions: We conclude that the suitability of the analytical method for its intended use is confirmed, guaranteeing the reliability of the results obtained under the established operating conditions.

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.

NDV-based vector has been used as a veterinary vaccine and, recently also for human COVID-19 vaccine; however, data about the potential immune response against vector in humans is scarce, therefore, it is important evaluate levels of antibodies produced. The HI assay is the gold standard for assessing the humoral response against NDV in poultry serum. Here the objective of was validate the HI assay against the NDV-vectored vaccine to analyze antibodies in human serum. First, we standardized conditions in human sera before validation. The results from analytical performance: selectivity, sensitivity, specificity, positive and negative predictive value, as well as positive and negative diagnostic reliability, indicate that the assay is highly selective, as it allows clear discrimination between positive and negative samples. Regarding repeatability and inter-mediate precision, we demonstrated that the assay has precision to obtain consistent results and guarantees their reliability and truthfulness. Finally, the results of accuracy, linearity and robustness, indicate that the assay is accurate at the concentration intervals evaluated with a linear correlation between the low and high level and demonstrated that it is robust and consistent when the serum-antigen interaction times changed. We conclude that the suitability of the analytical method for its intended use is confirmed and guarantees the reliability of the results obtained under the established operating conditions.