Optimisation of the interferon - gamma assay to maximise responses to M. ptb. antigen in sheep

摘要

The Bovigam~R assay (CSL, Australia) detects IFN-gamma released in response to M. paratuberculosis antigen in a whole blood assay. While there are published data on optimisation of the assay for cattle, there is little information for sheep. Investigations were conducted in sheep on anticoagulant, sample transit temperature and time delay before incubation and duration of incubation. Blood from vaccinated (Gudair~R) Merino sheep was collected into tubes containing different anticoagulants and incubated at 37 degC for 24, 48 or 72 hours in either media alone or media containing M. ptb. antigen. Plates containing blood and media +- M. ptb. antigen (added either at plate setup or just prior to incubation) were held for 4, 8 or 24 hours at both room temperature and 4degC prior to incubation. Maximal IFN-gamma responses were obtained when blood was collected into lithium heparin and incubated with antigen for at least 48 hours. When the blood was stored for 24 hours prior to incubation, storage at 4degC resulted in lower IFN-gamma responses than storage at room temperature; in this case adding the antigen at the time of blood collection resulted in greater IFN- gamma responses. While the findings of maximal IFN-gamma response with lithium heparin and storage of blood at room temperature are consistent with previous studies in cattle, maximal IFN-gamma responses in sheep were obtained by harvesting plasma at 48 hours rather than 16 to 24 hours as is recommended by the manufacturer. The results also suggest that if incubation is to be delayed, collection tubes containing antigen and media supplementation may improve IFN-gamma responses.