Validation of advanced immunoassays under ISO/IEC 17025:2017: a strategic framework for immunogenicity assays

Validation of analytical methods is a mandatory requirement for testing laboratories accredited under ISO/IEC 17025:2017. Immune response to vaccination is inherently heterogeneous, multidimensional, and distributed along continua rather than binary outcomes. ISO/IEC 17025:2017 provides valuable principles and a structured framework to support method validation, but the unique performance characteristics and biological variability of immunoassays often require adapting ISO guidance to assay-specific validation criteria. Its general requirements (impartiality, competence, method validation, measurement uncertainty, and result interpretation) can be meaningfully applied to complex immune-response assays to establish regulatory credibility, but compliance often requires innovation. This study presents a process framework for validating advanced immunological methods, including ELISA cutoff-based assays, multiplex cytokine profiling, functional IFN-γ T cell readouts, and microneutralization assays, in accordance with international guidelines, and provides clearly defined acceptance criteria for each parameter. The validation parameters included evaluation of analytical performance (precision, accuracy, specificity) and uncertainty estimation for the analytical methods we specifically used to assess the immunogenicity of the AVX-COVID-12 anti-SARS-CoV-2 vaccine. Here, we discuss the main challenges encountered, propose methodological strategies, and present validation outcomes to meet stringent accreditation requirements, using robust experimental designs and acceptance criteria informed by the literature. This process framework, based on lessons learned, ensures the reliability and traceability of critical results needed to approve newly developed biotechnological products targeting the immune response in similar conditions.