ELISPOT assays provide reproducible results among different laboratories for T-cell immune monitoring—even in hands of ELISPOT-inexperienced investigators

Abstract

Measurements of antibodies in bodily fluids (e.g., by ELISA) have provided robust and reproducible results for decades and such assays have been validated for monitoring of B-cell immunity. In contrast, measuring T-cell immunity has proven to be a challenge due to the need to test live cells in functional assays ex vivo. Several previous efforts looking into the reproducibility of ex vivo T-cell assays between different laboratories, or even within the same laboratory, have provided rather discouraging results. The hypothesis we tested in this study is that those poor results are due to the lack of assay and data analysis standardization, rather than the inherent complexity of T-cell assays. In this study, 11 laboratories across Europe and the United States were provided identical reagents and were asked to follow the same protocol while testing aliquots of the same three cryopreserved peripheral blood mononuclear cells (PBMC) in an interferon-γ (IFNγ) ELISPOT assay measuring the antigen-specific T-cell response to a CMV peptide. All individuals performing the assays were ELISPOT novices. At their first attempt, while three of these individuals failed with the basic logistics of the trial, eight detected the peptide-specific CD8+ T-cells in frequencies approximating the values established by the Reference Laboratory. The data show that ELISPOT assays provide reproducible results among different laboratories when the assay procedure and data analysis is standardized. Since ELISPOT assays have been qualified and validated for regulated studies, they are ideal candidates for robust and reproducible monitoring of T-cell activity in vivo.

Keywords::

• Immune monitoring
• CD8 cell immunity
• immune assay standardization

Acknowledgments

We thank Cellular Technology Ltd. (CTL) for donating their reference PBMC, CMV pp65 peptide, and the serum-free wash- (CTL Anti-Aggregate-Wash) and test-media (CTL-Test). We also thank BD Biosciences for donating their human IFNγ ELISPOT kits. Most of all, we thank the directors of the laboratories who supported this project by assigning an ELISPOT inexperienced member of their staff to this project, namely Drs. C. Baxevanis, A. DeLeo, G. Gaudernack, F. Garrido, F. Jotereau, P. Laidler, A. Line, E. Tartour, E. Naumova, and R. Rees.

Declaration of interest: Dr. Lehmann is employed by CTL. CTL is the manufacturer of ELISPOT readers, of the PBMC, and serum-free media used in this study. Independent evaluation of the study was conducted by Dr. McArdle. The Authors alone are responsible for the content and writing of this paper. The Authors did not receive funding for this study.