Mycobacterium tuberculosis expression of Antigen 85B (Ag85B) in different culture conditions

Abstract

Background: : Secretion of specific proteins by Mycobacterium tuberculosis contributes to pathogenesis and immune responses in tuberculosis disease yet the quantification of these secreted proteins in-vitro are poorly understood. Aim. To explore and find a culture system that provides a higher concentration in Ag85B expression. Methods. We cultured Mtb clinical isolates obtained from 6 different body compartments (lungs, CSF, blood, lymph nodes, pleura and MDR TB) in modified middlebrook 7H9 broth with tween 80 (treated media) and without tween 80 (untreated media). Culture supernatants were collected at 1 and 2 weeks of culture. We then used monoclonal antibodies that recognized the Mycobacterium tuberculosis secreted protein, Ag85B to establish and characterize a sensitive enzyme-linked immunosorbent assay (ELISA) to quantitate Ag85B in the supernatants. Data analysis: Collected data (concentration of Ag85B in ng/ml) was coded, entered and cleaned in Microsoft excel 2016 spread sheet and exported to SPSS software package for analysis. Data was represented as means and standard deviation. Pairwise comparison of means was done using t-test and statistical significance set at P<0.05. Results. We found that culture conditions (with Tween 80) significantly stimulated higher Ag85B expression in both Week 1 and Week 2 (p<0.05). In both culture conditions, there was significant difference in secreted Ag85B at Week 2 when compared with week 1 (p<0.05). Conclusion. These results indicate that bacterial protein secretion in vitro is affected by presence of Tween 80 and this can contribute to a better understanding of the culture conditions for the production of Ag85B. Recommendations. Further studies should be conducted to find the effect of tween 80 on other members of Ag85 complex (Ag85A and Ag85C).