Production of monoclonal antibodies against interferon gamma of asian elephant elephas maximus for tuberculosis diagnosis in elephants by interferon gamma release assay

Abstract

Tuberculosis is a zoonotic disease that can be transmitted from infected animals to humans. Wild animals such as elephants and non-human primate have been reported to be infected by M. tuberculosis (MTB) complex, which can cause tuberculosis in animals as well. The common TB diagnostic approaches in human such as chest X-ray and tuberculin skin test are not practical in the elephants. Moreover, the gold standard of the bacteria culture from trunk wash has low sensitivity, is time-consuming and can only detect tuberculosis in the active stage. Therefore, an accurate diagnosis covering all phases of the MTB infection is in need. Interferon gamma release assay (IGRA) is an alternative approach for tuberculosis diagnosis, which detects the interferon gamma (IFNγ) secreted from white blood cells stimulated with MTB antigens. The aim of this study is to develop IGRA for diagnosis of TB in elephants and possibly other wild animals. The peptides spanning the conserved region of IFNγ from ten mammalian species were identified and used as immunogen for stimulating specific antibody production in mice. Among twelve monoclonal antibodies that showed strong reactivity against recombinant elephant IFNγ (reIFNγ), monoclonal antibody from hybridoma No. nF1C3#15, which is an IgM, was chosen as a capture antibody in an IgM sandwich ELISA. This IgM sandwich ELISA was compared to the IgG sandwich ELISA developed previously using IgG from reIFNγ immunization. The sensitivity of IgM sandwich ELISA and IgG sandwich ELISA showed the limit of detection (LOD) at 190 ng/ml and 0.257 ng/ml, respectively. Therefore, the IgG sandwich ELISA was chosen for further development. Using the developed in-house IGRA for elephant TB diagnosis sixty-one elephant peripheral blood mononuclear cell (PBMC) samples were tested using PPD from M. bovis as stimulating antigen. We found that 37.7% of samples were diagnosed as infection negative, 4.9% were indeterminate, and 57.4% showed positive results for potential TB infection. In addition, by using ESAT-6 together with CFP10 as stimulating antigen, the response of the PBMCs was greater than using either ESAT-6 or CFP10 alone, accounting for 31.1%, 21.3%, and 8.2%, respectively. The comparison of the results by in-house IGRA and commercial DPP® VetTB assay using 32 samples, revealed that eleven samples showed positive results in both assays, while twenty-one samples were positive TB infection only with IGRA. In conclusion, the developed in-house IGRA has the potential for using as an alternative approach for elephant TB diagnosis with the same or better accuracy than using a commercial test kit.