Evaluation of Heating & Irradiation Methods for Production of Purified Protein Derivative (PPD) of Mycobacterium Tuberculosis

Document Type: Original Articles

Authors

1 Reference Laboratory of Bovine Tuberculosis, Razi Vaccine and Serum Research Institute, Agricultural Research Education and Extension Organization (AREEO), Tehran, Iran

2 Department of Molecular Medicine, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran

3 Nuclear Agriculture Research School, Nuclear Science and Technology Research Institute, Karaj, Iran

4 Animal Science Research Institute of Iran, Alborz, Iran

5 Young Researchers and Elites Club, Science and Research Branch, Islamic Azad University, Tehran, Iran

Abstract

Tuberculin skin test, also known as the tuberculin or purified protein derivative (PPD) test, is a widely used diagnostic test for detection of primary infection with Mycobacterium tuberculosis (Mtb). The production of PPD is accompanied by some difficulties that require a series of modifications in the production and purification processes. In this study, we aimed to determine the facilitation level of the manufacturing process by modifying methods to evaluate optimal methods for production of bovine PPD tuberculin. Mtb strains were cultured on Lowenstein-Jensen media. Cultured strains were inoculated to the Dorset-Henley liquid medium by biphasic medium of potato-Dorset-Henley. After incubation, flasks containing cultured strain were subjected for bacterial inactivation and the optimal gamma radiation dose(s) was determined. Tuberculoprotein were precipitated by ammonium sulfate (AS) and Trichloroacetic acid (TCA). Protein concentrations were determined by using the Bradford and Kjeldahl protein assay. In finally, Lymphocyte Transformation Test and potency test was done. Results showed that, liquid Dorset Henley liquid medium is suitable for massive growth of the bacterium. Transferring of Mtb from solid to liquid medium was directly carried out without intermediate culture. We found that during tuberculoprotein production, for killing mycobacterium, heating at 100° C for 3 hr is safe. Simultaneous use of heating and gamma irradiation in 8 kGgy killed all of the mycobacteria, while 1, 1.5 and 7 kGy decreased a significant number of bacterial cells. The results also showed that concentration of extracted tuberculoprotein by AS precipitation method was higher than TCA method. There was no significant difference between the potency of produced tuberculoproteins by these two methods in the lymphocyte transformation test (P>0.05). Moreover, the protein measurement was more efficiently carried out by the Kjeldahl method, compared to the Bradford method, due to the high volume of produced protein. Finally the results of this study showed that optimum methods besides a novel approach of gamma irradiation in production of bovine PPD tuberculin are suitable and applicable.

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