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Cancer Immunology, Immunotherapy
Published in: Cancer Immunology, Immunotherapy 4/2010

Open Access 01-04-2010 | Original Article

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

Authors: A. Mander, C. Gouttefangeas, C. Ottensmeier, M. J. P. Welters, L. Low, S. H. van der Burg, C. M. Britten

Published in: Cancer Immunology, Immunotherapy | Issue 4/2010

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Abstract

The Cancer Immunotherapy Immunoguiding Program has conducted an IFN-γ 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-γ 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.
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Literature
1.
van der Burg SH (2008) Therapeutic vaccines in cancer: moving from immunomonitoring to immunoguiding. Expert Rev Vaccines 7:1–5CrossRefPubMed
2.
Hoos A, Parmiani G, Hege K, Sznol M, Loibner H, Eggermont A, Urba W, Blumenstein B, Sacks N, Keilholz U, Nichol G (1997) A clinical development paradigm for cancer vaccines and related biologics. J Immunother 30:1–15CrossRef
3.
Copier J, Dalgleish AG, Britten CM, Finke LH, Gaudernack G, Gnjatic S, Kallen K, Kiessling R, Schuessler-Lenz M, Singh H, Talmadge J, Zwierzina H, Hakansson L (2009) Improving the efficacy of cancer immunotherapy. Eur J Cancer 45:1424–1431CrossRefPubMed
4.
Britten CM, Janetzki S, van der Burg SH, Gouttefangeas C, Hoos A (2007) Toward the harmonization of immune monitoring in clinical trials: Quo vadis? Cancer Immunol Immunother 57:285–288CrossRefPubMed
5.
Cox JH, Ferrari G, Kalams SA, Lopaczynski W, Oden N, D’souza MP (2005) Results of an ELISPOT proficiency panel conducted in 11 laboratories participating in international human immunodeficiency virus type 1 vaccine trials. AIDS Res Hum Retroviruses 21:68–81CrossRefPubMed
6.
Hanekom WA, Dockrell HM, Ottenhoff TH, Doherty TM, Fletcher H, McShane H, Weichold FF, Hoft DF, Parida SK, and Fruth UJ (2008) Immunological outcomes of new tuberculosis vaccine trials: WHO panel recommendations. PLoS Med 5:e145
7.
Butterfield LH, Disis ML, Fox BA, Lee PP, Khleif SN, Thurin M, Trinchieri G, Wang E, Wigginton J, Chaussabel D, Coukos G, Dhodapkar M, Hakansson L, Janetzki S, Kleen TO, Kirkwood JM, Maccalli C, Maecker H, Maio M, Malyguine A, Masucci G, Palucka AK, Potter DM, Ribas A, Rivoltini L, Schendel D, Seliger B, Selvan S, Slingluff CL Jr, Stroncek DF, Streicher H, Wu X, Zeskind B, Zhao Y, Zocca MB, Zwierzina H, Marincola FM (2008) A systematic approach to biomarker discovery; preamble to “the iSBTc-FDA taskforce on immunotherapy biomarkers”. J Transl Med 6:81CrossRefPubMed
8.
Janetzki S, Panageas KS, Ben-Porat L, Boyer J, Britten CM, Clay TM, Kalos M, Maecker HT, Romero P, Yuan J, Kast WM, Hoos A (2008) Results and harmonization guidelines from two large-scale international Elispot proficiency panels conducted by the Cancer Vaccine Consortium (CVC/SVI). Cancer Immunol Immunother 57:303–315CrossRefPubMed
9.
Britten CM, Janetzki S, Ben Porat L, Clay TM, Kalos M, Maecker H, Odunsi K, Pride M, Old L, Hoos A, Romero P (2009) Harmonization guidelines for HLA-peptide multimer assays derived from results of a large scale international proficiency panel of the Cancer Vaccine Consortium. Cancer Immunol Immunother 58:1701–1713CrossRefPubMed
10.
Britten CM, Gouttefangeas C, Welters MJ, Pawelec G, Koch S, Ottensmeier C, Mander A, Walter S, Paschen A, Muller-Berghaus J, Haas I, Mackensen A, Kollgaard T, Thor SP, Schmitt M, Giannopoulos K, Maier R, Veelken H, Bertinetti C, Konur A, Huber C, Stevanovic S, Wolfel T, van der Burg SH (2008) The CIMT-monitoring panel: a two-step approach to harmonize the enumeration of antigen-specific CD8+ T lymphocytes by structural and functional assays. Cancer Immunol Immunother 57:289–302CrossRefPubMed
11.
Martinuzzi E, Scotto M, Enee E, Brezar V, Ribeil JA, van Endert P, Mallone R (2008) Serum-free culture medium and IL-7 costimulation increase the sensitivity of ELISpot detection. J Immunol Methods 333:61–70CrossRefPubMed
12.
Ahmad S, Sundaramoorthy E, Arora R, Sen S, Karthikeyan G, Sengupta S (2009) Progressive degradation of serum samples limits proteomic biomarker discovery. Anal Biochem 394:237–242CrossRefPubMed
13.
Gislefoss RE, Grimsrud TK, Morkrid L (2008) Long-term stability of serum components in the Janus Serum Bank. Scand J Clin Lab Invest 68:402–409CrossRefPubMed
14.
Zhang W, Caspell R, Karulin AY, Ahmad M, Haicheur N, Abdelsalam A, Johannesen K, Vignard V, Dudzik P, Georgakopoulou K, Mihaylova A, Silina K, Aptsiauri N, Adams V, Lehmann PV, McArdle S (2009) ELISPOT assays provide reproducible results among different laboratories for T-cell immune monitoring- even in the hands of inexperienced investigators. J Immunotoxicol 6:227–234CrossRefPubMed
15.
Janetzki S, Britten CM, Kalos M, Levitsky HI, Maecker HT, Melief CJM, Old LJ, Romero P, Hoos A, Davis MM (2009) ‘‘MIATA’’—minimal information about T cell assays. Immunity 31:527–528CrossRefPubMed
16.
Maecker HT, Hassler J, Payne JK, Summers A, Comatas K, Ghanayem M, Morse MA, Clay TM, Lyerly HK, Bhatia S, Ghanekar SA, Maino VC, Delarosa C, Disis ML (2008) Precision and linearity targets for validation of an IFNgamma ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptides. BMC Immunol 9:1–9CrossRef
17.
Janetzki S, Cox JH, Oden N, Ferrari G (2005) Standardization and validation issues of the ELISPOT assay. Methods Mol Biol 302:51–86PubMed
18.
Mander A, Chowdhury F, Low L, Ottensmeier CH (2009) Fit for purpose? A case study: validation of immunological endpoint assays for the detection of cellular and humoral responses to anti-tumour DNA fusion vaccines. Cancer Immunol Immunother 58:789–800CrossRefPubMed
19.
Janetzki S, Price L, Britten CM, van der Burg SH, Caterini J, Currier JR, Ferrari G, Gouttefangeas C, Hayes P, Kaempgen E, Lennerz V, Nihlmark K, Souza V, Hoos A (2009) Performance of serum-supplemented and serum-free media in IFNgamma Elispot Assays for human T cells. Cancer Immunol Immunother (ePub ahead of print)
20.
Weinberg A, Song LY, Wilkening C, Sevin A, Blais B, Louzao R, Stein D, Defechereux P, Durand D, Riedel E, Raftery N, Jesser R, Brown B, Keller MF, Dickover R, McFarland E, Fenton T (2009) Optimization and limitations of use of cryopreserved peripheral blood mononuclear cells for functional and phenotypic T-cell characterization. Clin Vaccine Immunol 16:1176–1186CrossRefPubMed
21.
Smith JG, Joseph HR, Green T, Field JA, Wooters M, Kaufhold RM, Antonello J, Caulfield MJ (2007) Establishing acceptance criteria for cell-mediated-immunity assays using frozen peripheral blood mononuclear cells stored under optimal and suboptimal conditions. Clin Vaccine Immunol 14:527–537CrossRefPubMed
22.
Elliott P, Peakman TC (2008) The UK Biobank sample handling and storage protocol for the collection, processing and archiving of human blood and urine. Int J Epidemiol 37:234–244CrossRefPubMed
Metadata
Title
Serum is not required for ex vivo IFN-γ ELISPOT: a collaborative study of different protocols from the European CIMT Immunoguiding Program
Authors
A. Mander
C. Gouttefangeas
C. Ottensmeier
M. J. P. Welters
L. Low
S. H. van der Burg
C. M. Britten
Publication date
01-04-2010
Publisher
Springer-Verlag
Published in
Cancer Immunology, Immunotherapy / Issue 4/2010
Print ISSN: 0340-7004
Electronic ISSN: 1432-0851
DOI
https://doi.org/10.1007/s00262-009-0814-4

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