Identification and characterization of mycobacterial antigens and enzymes : a methodological study / Ronny Öhman .

Tese (doutor) - University of Göteborg, 1995

Tuberculosis is a serious public health problem. The estimated global annual incidence of tuberculosis is about 8 million and the mortality 3 million. The causative agent of tuberculosis, Mycobacterium tuberculosis, is thus the single infectious agent with the highest mortality rate. Improved methods to control tuberculosis are therefore urgent. Genus Mycobacterium encompasses, in addition to M. tuberculosis, more than 50 other known species, many of which are common in the enviroment or in the normal flora of animals and humans. Some of them are well-known opportunists while other have not been recorded in connection with human disease. Knowledge concerning M. tuberculosis, M. bovis BCG (the vaccine against tuberculosis) and the other mycobacteria is comparatively limitid or absent. Only a small number of antigens has been identified and characterized and an even smaller number has been isolated. Knowledge about the function of mycobacterial antigens is also limited. Increased knowledge in this field is urgent, since it is a prerequisite for developing an improved vaccine and a serodiagnostic test, and for understanding the pathogenesis of these organisms. The aim of the studies has been to develop techniques for analysis of mycobacteria and to identify and characterize mycobacterial antigens, particularly antigens which are also enzymes. In addition the aim has been to compare antigens and enzymes of various mycobaterial taxa, to isolate antigens and to evaluate their potential in serodiagnosis. Faint or invisible mycobacterial precipitates of immunoelectrophoresis were made visible by an amplification technique using anti-IgG. Thus a considerably larger number of precipitation arcs was revealed. A technique was developed in which selective enzyme staining procedures follow immunoelectrophoresis. By means of this technique the antigens which are enzymes can be identified; e.g. several dehydrogenases were identified. Differences and similarities between the analysed mycobacteria were recorded. A large number of reactive dyes were investigated and some of these dyes proved useful in the purification on mycobacterial enzyme-antigens. Thus, two intracellular enzyme-antigens were purified, namely isocitrate dehydrogenase and malate dehydrogenase from Mycobacterium tuberculosis. Characterization of the two fractions containing these enzymes was made. An evaluation of their potential as antigens in serodiagnosis of tuberculosis showed that both fractions could distinguish patients with tuberculosis from healthy controls.