Assessment of critical parameters in blood processing for the bovine interferon-gamma ELISPOT assay to detect Mycobacterium bovis infected cattle in India

作者：Veerasami Maroudam^*; Moorthy Loganathan; Das Dipankar; Parthasarathy Sugumar; Chandran Dev; Lingala Rajendra; Srinivasan Villuppanoor Alwar

来源：Veterinaria Italiana, 2011, 47(1): 25-34.

• Bovine tuberculosis
• ELISPOT assay
• Enzyme-linked immunosorbent spot
• IFN-gamma
• India
• Interferon gamma
• Mycobacterium bovis
• Peripheral blood mono-nuclear cells
• Tuberculosis

In vitro production of bovine interferon gamma (BoIFN-gamma) cyokine from bovine peripheral blood mononuclear cells (PBMCs) can be detected using the most sensitive enzyme-linked immunosorbent spot (ELISPOT) assay. ELISPOT assays are dependent on the quantity and quality of PBMC preparations and hence contribute significantly to the performance of this assay. In order to standardise the methods for isolation of PBMCs, we compared two methods for the processing of bovine blood which included aliquots of blood that were stored in a horizontal position without dilution or agitation and aliquots of blood that were immediately diluted 1:1 with complete Rosewell Park Memorial Institute (RPMI) 1640 medium and stored in a horizontal position with gentle agitation. PBMCs were isolated at 2, 4, 6, 8 and 24 h and at 4 degrees C and at 22 degrees C+/-2 degrees C. They were stimulated using tuberculosis-specific antigens, after which the ELISPOT assay was performed. Quantities of spot-forming cells (SFC) created by the release of BoIFN-gamma in ELISPOT assays were significantly greater in the samples stored at 22 degrees C+/-2 degrees C than those held +4 degrees C and the intensity of the signals dropped following processing after 6 h. A further drop in SFC was observed in those samples that had been stored undiluted and without agitation. These findings demonstrated that optimisation of PBMC isolation procedures can lead to increased sensitivity in the detection of BoIFN-gamma using the ELISPOT assay, thus contributing to an enhanced diagnosis of bovine tuberculosis.

• 出版日期2011-3