Optimization of a whole-blood gamma interferon assay for detection of Mycobacterium bovis-infected cattle

Irene Schiller, W. Ray Waters, H. Martin Vordermeier, Brian Nonnecke, Michael Welsh, Nicolas Keck, Adam Whelan, Teresa Sigafoose, Christoph Stamm, Mitchell Palmer, Tyler Thacker, Roland Hardegger, Beatrice Marg-Haufe, Alex Raeber, Bruno Oesch*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

41 Citations (SciVal)

Abstract

Antigens of Mycobacterium bovis elicit a cell-mediated immune response upon intradermal injection in cattle. In vitro, such antigens stimulate the production of gamma interferon (IFN-γ) by bovine T cells in whole-blood culture (IFN-γ assay). We have analyzed various parameters of the in vitro IFN-γ assay, ranging from blood sampling to execution of the IFN-γ test, in view of potential simplifications of the assay. Here, we show that IFN-γ responses may be reduced under certain animal handling/holding conditions and that a delayed time from blood collection to culture may lead to a reduced in vitro IFN-γ response. Delayed initiation of culture in a purified-protein-derivative-based assay (24 h compared to 8 h after blood collection), however, resulted in a significant improvement of specificity (97% compared to 85%), whereas there was only a modest reduction of sensitivity (from 96% to 90%), which was statistically not significant. Furthermore, we show that the stimulation temperature needs to be 33° C or higher; that carbon dioxide is not required for stimulation; and that various plate formats, ranging from 24 to 96 wells per plate, can be utilized. The produced IFN-γ is stable at 4° C for 28 days as well as after repeated freeze-thaw cycles. Thus, stimulation of samples may be initiated in the field without the need for a carbon dioxide source, and bovine IFN-γ is stable under various routine laboratory temperature scenarios. These findings demonstrate opportunities for improvements in the bovine IFN-γ test platform and flexibilities in test application.

Original languageEnglish
Pages (from-to)1196-1202
Number of pages7
JournalClinical and Vaccine Immunology
Volume16
Issue number8
Early online date29 Jul 2009
DOIs
Publication statusPublished - 01 Aug 2009

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