Purpose: The Elispot (Enzyme-linked immunosorbent spot) assay provides a powerful tool in the development of new vaccines and novel immunotherapy agents. An expansion in recent years of treatments using immune system modulators, such as PD-1 inhibitors, as well as increased interest in vaccines with emergence of global viral outbreaks. Monitoring the immune system is key to understanding a patient’s response to such treatments. Elispot is an effective technique to determine a patient’s immune response by quantifying antigen specific T-Cell reactivity as measured by secretion of INF-gamma, other cytokines, or secreted molecules such as granzyme B. Elispot and other immune monitoring assays such as intracellular cytokine staining (ICS) provides unique challenges as no reference material or gold standard can be utilized, and FDA Bioanalytical Method Validation guidance is not always applicable. Global harmonization studies have been carried out, creating optimized protocols and guidelines (Janetzki et al., 2008,2015), as well as targets for precision and linearity (Maecker et al., 2008). IFN-gamma is the most common analyte measured with the Elispot assay in clinical studies. Utilizing optimized protocols and guidelines in established literature, a qualification plan was developed for an IFN-gamma Elispot.
Methods: Important parameters in qualifying an Elispot assay for use in the bioanalytical context include accuracy, specificity, precision, limit of detection (LOD), and the linearity and range of the assay. We address each of these terms that are commonly used in bioanalytical assays, and the unique challenges of applying them in a complex immune response assays such as Elispot. Experiments were run under optimal conditions, established prior to carrying out the qualification plan. For qualification, peptide pools (CEF and CMVpp65) corresponding to proteins in Cytomegalovirus, Epstein-Bar, and Influenza were utilized along with mitogen (PHA-L) to determine the number of INF-gamma producing cells. Target criteria were developed based on Maecker et al. Experiments addressing each component of the qualification plan were carried out on cryopreserved PBMCs. Precision was evaluating by determining spot counts from PBMCs treated with CEF or CMVpp65 for 3 donors over two days with two operators.
Results: The mean percent CV for donors with spot count >100 was 12.5%. The range of the inter-batch CV was from 10.4% up to 13.9%
Conclusion: The challenges of qualifying an immune biomarker assay in the bioanalytical environment can be addressed by a comprehensive qualification plan carried out within the framework of the bioanalytical environment in a GLP facility. We will present a detailed qualification plan developed based on global harmonization guidelines and peer reviewed literature references, along with the experimental data resulting from the qualification study.
Janetzki, S., Panageas, K.S., Ben-Porat, L. et al. (2008) Results and harmonization guidelines from two large-scale international Elispot proficiency panels conducted by the Cancer Vaccine Consortium (CVC/SVI) Cancer Immunol Immunother 57: 303.
Janetzki, S., Price, L., Schroeder, H., Britten, C. M., Welters, M. J. P., & Hoos, A. (2015). Guidelines for the automated evaluation of Elispot assays. Nature Protocols, 10(7), 1098–1115.
Maecker, H. T., Hassler, J., Payne, J. K., Summers, A., Comatas, K., Ghanayem, M., … Disis, M. L. (2008). Precision and linearity targets for validation of an IFNγ ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptides. BMC Immunology, 9, 1–9.