Serum is not required for ex vivo IFN-gamma ELISPOT: a collaborative study of different protocols from the European CIMT immunoguiding program
Serum is not required for ex vivo IFN-gamma ELISPOT: a collaborative study of different protocols from the European CIMT immunoguiding program
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.
elispot, serum free, harmonization, t cell enumeration, multicenter study
619-627
Mander, A.
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Gouttefangeas, C.
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Ottensmeier, C.
42b8a398-baac-4843-a3d6-056225675797
Welters, M.J.P.
c9970409-fd91-4bc3-bb3b-d7e4a1be4f17
Low, L.
a6686acc-764a-47c1-8fc5-f08d8a4617c3
van der Burg, S.H.
5962846d-001b-4e1c-980d-c3416911baea
Britten, C.M.
b1f904b6-e8b0-4ffc-a7b2-be91b4f2c1d6
6 January 2010
Mander, A.
2a92e8dd-4bb4-4f3b-b8ca-d62f5c4724bd
Gouttefangeas, C.
68075a2b-439d-4c6c-9d0e-2fd9e5c82b25
Ottensmeier, C.
42b8a398-baac-4843-a3d6-056225675797
Welters, M.J.P.
c9970409-fd91-4bc3-bb3b-d7e4a1be4f17
Low, L.
a6686acc-764a-47c1-8fc5-f08d8a4617c3
van der Burg, S.H.
5962846d-001b-4e1c-980d-c3416911baea
Britten, C.M.
b1f904b6-e8b0-4ffc-a7b2-be91b4f2c1d6
Mander, A., Gouttefangeas, C., Ottensmeier, C., Welters, M.J.P., Low, L., van der Burg, S.H. and Britten, C.M.
(2010)
Serum is not required for ex vivo IFN-gamma ELISPOT: a collaborative study of different protocols from the European CIMT immunoguiding program.
Cancer Immunology Immunotherapy, 59 (4), .
(doi:10.1007/s00262-009-0814-4).
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.
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Published date: 6 January 2010
Keywords:
elispot, serum free, harmonization, t cell enumeration, multicenter study
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Local EPrints ID: 160899
URI: http://eprints.soton.ac.uk/id/eprint/160899
ISSN: 0340-7004
PURE UUID: 819eb5b8-3971-4cc9-afda-31d5c81a652c
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Date deposited: 21 Jul 2010 10:22
Last modified: 14 Mar 2024 01:58
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Contributors
Author:
A. Mander
Author:
C. Gouttefangeas
Author:
M.J.P. Welters
Author:
L. Low
Author:
S.H. van der Burg
Author:
C.M. Britten
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