Precision and linearity targets for validation of an IFNgamma ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptides.

Published

Journal Article

Single-cell assays of immune function are increasingly used to monitor T cell responses in immunotherapy clinical trials. Standardization and validation of such assays are therefore important to interpretation of the clinical trial data. Here we assess the levels of intra-assay, inter-assay, and inter-operator precision, as well as linearity, of CD8+ T cell IFNgamma-based ELISPOT and cytokine flow cytometry (CFC), as well as tetramer assays.Precision was measured in cryopreserved PBMC with a low, medium, or high response level to a CMV pp65 peptide or peptide mixture. Intra-assay precision was assessed using 6 replicates per assay; inter-assay precision was assessed by performing 8 assays on different days; and inter-operator precision was assessed using 3 different operators working on the same day. Percent CV values ranged from 4% to 133% depending upon the assay and response level. Linearity was measured by diluting PBMC from a high responder into PBMC from a non-responder, and yielded R2 values from 0.85 to 0.99 depending upon the assay and antigen.These data provide target values for precision and linearity of single-cell assays for those wishing to validate these assays in their own laboratories. They also allow for comparison of the precision and linearity of ELISPOT, CFC, and tetramer across a range of response levels. There was a trend toward tetramer assays showing the highest precision, followed closely by CFC, and then ELISPOT; while all three assays had similar linearity. These findings are contingent upon the use of optimized protocols for each assay.

Full Text

Duke Authors

Cited Authors

  • 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

Published Date

  • March 17, 2008

Published In

Volume / Issue

  • 9 /

Start / End Page

  • 9 -

PubMed ID

  • 18366814

Pubmed Central ID

  • 18366814

Electronic International Standard Serial Number (EISSN)

  • 1471-2172

International Standard Serial Number (ISSN)

  • 1471-2172

Digital Object Identifier (DOI)

  • 10.1186/1471-2172-9-9

Language

  • eng