Original Article

Cancer Immunology, Immunotherapy

, Volume 59, Issue 4, pp 609-618

First online:

Open Access This content is freely available online to anyone, anywhere at any time.

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

  • Sylvia JanetzkiAffiliated withCancer Vaccine Consortium of the Cancer Research InstituteZellNet Consulting, Inc Email author 
  • , L. PriceAffiliated withDepartment of Biostatistics, New York University Medical Center
  • , C. M. BrittenAffiliated withDivision of Experimental and Translational Oncology, Department of Internal Medicine III, Johannes Gutenberg-University
  • , S. H. van der BurgAffiliated withDepartment of Clinical Oncology, Leiden University Medical Center
  • , J. CateriniAffiliated withsanofi pasteur
  • , J. R. CurrierAffiliated withHenry M. Jackson Foundation
  • , G. FerrariAffiliated withDepartment of Surgical Sciences, Duke University Medical Center
  • , C. GouttefangeasAffiliated withDepartment of Immunology, Institute for Cell Biology, Eberhard-Karls University
  • , P. HayesAffiliated withIAVI Human Immunology Laboratory, Imperial College
    • , E. KaempgenAffiliated withDepartment of Dermatology, University Hospital Erlangen
    • , V. LennerzAffiliated withDepartment of Internal Medicine III, Johannes Gutenberg-University
    • , K. NihlmarkAffiliated withMabtech AB
    • , V. SouzaAffiliated withWyeth Research
    • , A. HoosAffiliated withCancer Vaccine Consortium of the Cancer Research InstituteBristol-Myers Squibb


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.


Elispot Serum Immune monitoring Harmonization