Serum is not required for ex vivo IFN-gamma ELISPOT: a collaborative study of different protocols from the European CIMT Immunoguiding Program

Abstract

The Cancer Immunotherapy Immunoguiding Program has conducted an IFN-gamma ELISPOT proficiency panel to examine the influence of serum supplementation of test media on assay performance. Sixteen European laboratories analyzed the same PBMC samples using different locally established protocols. Participants generated two simultaneous data sets-one using medium supplemented with serum and one without serum. Performances of the two test conditions were compared by quantifying: (1) the number of viable cells, (2) background spot formation induced in the medium only control and (3) the ability to detect antigen-specific T cell responses. The study demonstrated that the number of viable cells recovered and the overall background spot production were not significantly different between the two conditions. Furthermore, overall laboratory performance was equivalent for the two test conditions; 11 out of 16 laboratories reported equal or greater detection rates using serum-free medium, while 5 laboratories reported decreased detections rates under serum-free conditions. These results show that good performance of the IFN-gamma ELISPOT assay can be achieved under serum-free conditions. Optimization of the protocol for serum-free conditions should result in excellent detection rates and eliminate the requirement of serum batch and stability testing, allowing further harmonization of the assay.

Fig. 1

Overall results from sixteen IFNγ-ELISPOT protocols using serum-supplemented and serum-free media. a Recovery of viable PBMC using trypan blue and a manual haemocytometer, following resting for 2–20 h. b Background spot production (spots per 100,000 PBMC) in the cells plus medium only control. c Detection rates, shown as % of responses detected among the seven possible donor–antigen combinations. The mean values for all samples (D1–D5) and all labs (n = 16) were used to plot minimum and maximum value, the range (error bars), inter-quartile range (boxes), median (horizontal line) and mean (black triangle) for the whole group under serum supplemented or serum-free conditions

Fig. 2

Inverse correlation between background and detection rates. Mean detection rates for each lab expressed, as the % positive responses as defined in “Materials and methods”, are plotted against the mean cells plus medium only background (spots per 100,000 PBMC) using serum supplemented (open triangles) and serum-free (filled circles) media. Under both conditions, detection rate decreases as background increases. Correlation co-efficients: serum, −0.66; serum-free, −0.72