BOVIGAM™ Mycobacterium bovis Interferon-gamma Test System for Cattle

The BOVIGAM™ Mycobacterium bovis Interferon-gamma Test System for Cattle is a rapid, in vitro, blood-based assay of cell-mediated response to M. bovis tuberculin purified protein derivitive (PPD) for the diagnosis of bovine tuberculosis infection in cattle. Tuberculin PPD antigens are presented to lymphocytes in whole-blood culture. IFN-gamma production by the cells is detected using a monoclonal antibody-based sandwich enzyme immunoassay (EIA). Lymphocytes from cattle not infected with M. bovis do not produce IFN-gamma. Therefore, detection of IFN-gamma correlates to M. bovis infection. The BOVIGAM M. bovis IFN-gamma Test System for Cattle consists of a BOVIGAM kit and the following stimulation agents: Bovine Tuberculin PPD 3000, Avian Tuberculin PPD 2500, as well as Pokeweed Mitogen, a viability control of the whole blood cells.

The test procedure

Stimulation
Tuberculin PPD antigens are presented to a whole-blood sample containing lymphocytes. Viable lymphocytes from M. bovis-infected animals react by producing IFN-gamma, whereas lymphocytes from uninfected animals do not. Thus, the detection of IFN-gamma correlates with an infection with M. bovis. IFN-gamma production is detected by conducting an IFN-gamma-specific sandwich enzyme-linked immunosorbent assay (EIA). Lymphocyte viability is measured by incubating the whole-blood sample with Pokeweed Mitogen. Pokeweed is a lectin from Phytolacca americana that acts as a non-specific mitogen on T- and B-cells. Viable cells produce IFN-gamma upon stimulation with pokeweed.

ELISA
1. Reconstitute freeze dried components of BOVIGAM kit.
2. Add Green Diluent to required wells.3. Add test plasmas (simultaneously for each animal) and controls to wells.
4. Incubate for 60 minutes and wash wells.
6. Prepare Conjugate Reagent and add to wells.
7. Incubate for 60 minutes and wash wells.
10. Add Substrate to wells and incubate for 30 minutes.
12. Stop reaction.
13. Read results with 450/620-650 nm filters.
14. Validate and interpret results.