Performance of serum-supplemented and serum-free media in IFNγ Elispot Assays for human T cells

Abstract

The choice of serum for supplementation of media for T cell assays and in particular, Elispot has been a major challenge for assay performance, standardization, optimization, and reproducibility. The Assay Working Group of the Cancer Vaccine Consortium (CVC-CRI) has recently identified the choice of serum to be the leading cause for variability and suboptimal performance in large international Elispot proficiency panels. Therefore, a serum task force was initiated to compare the performance of commercially available serum-free media to laboratories’ own medium/serum combinations. The objective of this project was to investigate whether a serum-free medium exists that performs as well as lab-own serum/media combinations with regard to antigen-specific responses and background reactivity in Elispot. In this way, a straightforward solution could be provided to address the serum challenge. Eleven laboratories tested peripheral blood mononuclear cells (PBMC) from four donors for their reactivity against two peptide pools, following their own Standard Operating Procedure (SOP). Each laboratory performed five simultaneous experiments with the same SOP, the only difference between the experiments was the medium used. The five media were lab-own serum-supplemented medium, AIM-V, CTL, Optmizer, and X-Vivo. The serum task force results demonstrate compellingly that serum-free media perform as well as qualified medium/serum combinations, independent of the applied SOP. Recovery and viability of cells are largely unaffected by serum-free conditions even after overnight resting. Furthermore, one serum-free medium was identified that appears to enhance antigen-specific IFNγ-secretion.

Enzyme-Linked Immunospot Assay for Detection of Human Respiratory Syncytial Virus F Protein-Specific Gamma Interferon-Producing T Cells

“…Additionally, samples frozen in SFM maintain stable responses for at least 60 days when stored in LN 2 . These results are consistent with other reports that suggest it is beneficial to freeze freshly isolated PBMC in SFM rather than SCM (22)(23)(24)(25).…”

“…Minimization of nonspecific background responses is key to ensuring a high signal-to-noise ratio in an assay and is especially important in a setting where a low signal is expected. Avoidance of serum components in cellular processing has been recommended for T cell-based assays, as antigenspecific responses to serum components have been observed (22)(23)(24)(25). The performances of serum-free culture media (X-Vivo, AIM-V, and OpTmizer), previously described by Mander et al (26) and Janetzki et al (24), were evaluated against CTL-Test medium in the RSV F-specific IFN-␥ ELISPOT assay using PBMC from 6 individual donors.…”

Merkel Cell Polyomavirus-Specific CD8+ and CD4+ T-cell Responses Identified in Merkel Cell Carcinomas and Blood

“…PBMC availability prevented proteome-wide screens for some subjects, therefore we focused on the oncogenic portions of T-Ag persistently expressed in tumors. Cultured ELISPOT may sometimes generate false positive reactivity via in vitro priming (46). In order to eliminate such artifactual results, we employed a stringent ELISPOT interpretation criteria (SUPPLEMENTARY methods).…”

White Paper on Adoptive Cell Therapy for Cancer with Tumor-Infiltrating Lymphocytes: A Report of the CTEP Subcommittee on Adoptive Cell Therapy

“…A series of validation panels have been run to assess the reliability, robustness, sensitivity, specificity, and predictive value of a number of immune assays. The investigators that were part of the proficiency panels determined whether ELISPOT, tetramer, and intracellular cytokine assays could achieve a high level of reliability for use as immune assays in clinical trials (68,69). Only the ELISPOT assay, a type of assessment also employed by the AIDS Clinical Trials Group, achieved the threshold wherein it was felt to be a practical, reliable, reproducible, and robust assay for clinical use.…”