Identification and monitoring of Mycobacterium tuberculosis growth in liquid culture by Antigen 85 detection

Abstract

Background: Mycobacterium tuberculosis is the most common causative agent of tuberculosis. It releases secretory proteins, especially the Ag85 complex, from actively growing mycobacteria, which can be detected in mycobacterial liquid culture. Ag85B is the most abundant in the Ag85 complex and is an interesting target for the detection of tuberculosis. In addition, measuring Ag85 level is beneficial for comparing growing and non-growing mycobacteria in liquid culture. Objectives: To detect Ag85B protein from active growing M. tuberculosis in liquid culture for the diagnosis of tuberculosis and compare the level of Ag85B between growing and non-growing mycobacteria. Materials and methods: A sandwich ELISA assay using anti-Ag85B monoclonal antibody was performed to detect Ag85B protein. Drug-susceptible mycobacteria, M. tuberculosis H37Ra and M. tuberculosis H37Rv, as well as 12 other M. tuberculosis complex strains isolated from clinical specimens were cultured in liquid media. In addition, mycobacterial culture was separated into two conditions: untreated and treated with streptomycin. Then the liquid media were collected, filtered for sterility, and used for the detection of Ag85B. The levels of Ag85B between treated and untreated conditions were analyzed. Results: In untreated mycobacterial culture, Ag85B protein was detected and continuously increased each day. However, Ag85B in treated mycobacterial culture increased slightly in the early days and then stabilized. These results demonstrate that the growth of mycobacteria was inhibited after culturing with streptomycin. Thus, the increase of Ag85B was not observed because this protein cannot be secreted by dead mycobacteria. On the other hand, living mycobacteria can secrete the Ag85B protein continuously. Moreover, Ag85B accumulated and increased with each passing day. Conclusion: M. tuberculosis can produce and release Ag85B protein. In this study, detection of Ag85B in liquid culture by a sandwich ELISA assay was used to identify M. tuberculosis. When M. tuberculosis was suppressed or killed by anti-TB medications, it stopped growing and the increase in Ag85B proteins disappeared. As a result, the levels of Ag85B could be used to detect living mycobacteria. Furthermore, this knowledge can be further applied to monitor mycobacterial growth affected by testing mycobacteria with anti-TB drugs.