Antigens in culture supernatant of Mycobacterium tuberculosis: epitopes defined by monoclonal and human antibodies

Abstract

~ ~~Antigens of Mycobacterium tuberculosis found in the supernatant of heat-treated cultures were characterized in order to explore whether antigens from this source could be used for the development of a serological test. Culture supernatants and sonicates of 12, 25 and 39 d cultures were analysed by SDS-PAGE. In culture supernatant, major protein bands of 65, 24, and 12 kDa were visible after Coomassie brilliant blue staining. Using murine monoclonal antibodies in Western blots, a pattern of protein bands distinct from that of the corresponding M. tuberculosis sonicates was found in all the culture supernatants. Gel permeation chromatography, in the presence of SDS, was used to separate the major protein bands in the culture supernatant. In ELISA, sera from 20 of 26 patients with tuberculosis reacted with fractions containing mainly 24 kDa or 12 kDa proteins, whereas none of the control sera reacted. In Western blots, each patient serum had its own characteristic banding pattern with culture Supernatant, but all the sera from tuberculosis patients and control subjects reacted with protein bands of 65,61,58,30 and 24 kDa. The 12 kDa protein was recognized only by sera from patients with tuberculosis in both Western blots and ELISA. This suggests that different kinds of epitopes on proteins of M. tuberculosis are detected by human antibodies in Western blots and ELISA. We assume that epitopes recognized in Western blots by patients with tuberculosis and control subjects are ubiquitous and are also present on normal commensal bacteria. Epitopes recognized by only some patients with tuberculosis in Western blots may be linear and M. tuberculosis specific. Epitopes recognized by tuberculosis patients but by none of the control subjects in ELISA may be conformation related and M. tuberculosis specific. The major protein bands found in supernatants of heat-treated cultures, 24 and 12 kDa, possess epitopes that may be M. tuberculosis specific and are potentially valuable for the development of a serological test.

Host Responses and Antigens Involved in Protective Immunity to Mycobacterium tuberculosis

“…In this study proteins were identified with a large range of secretion efficiencies, from 5 to 1000, as defined by the ratio of filtrate to sonicate concentrations. An alternative approach has been to study proteins released from M. tuberculosis at early culture timepoints [142,143]. The release of the metabolic enzyme isocitrate dehydrogenase (ICD) served in the study by Andersen and colleages [143] (Fig.…”

Characterization of a major polymorphic tandem repeat in Mycobacterium tuberculosis and its potential use in the epidemiology of Mycobacterium kansasii and Mycobacterium gordonae

“…The repetitive DNA-containing recombinant plasmid pPH7301 is a derivative of a lambda gt-11 recombinant that was originally selected because of its reactivity with the MAb F116-5 (14), Wvhich recognizes a 24-and a 30-kDa protein of M. tuberculosis (41). The M. tuberculosis DNA in pPH7301 located immediately downstream of the cro-lacZ gene has been used as a target sequence in PCR.…”

“…tpe 1 (41). In this study, we showed by epitope mapping that the epitope expressed by pPH7301 was artificially created because of the fusion of cro-lacZ with a M. tuberculosis DNA fragment, and presumably, this recombinant does not encode for the 24-or the 30-kDa polypeptide.…”

Lipoprotein Access to MHC Class I Presentation During Infection of Murine Macrophages with Live Mycobacteria

“…The 19-kDa protein has been implicated in a range of immunological responses. In addition to stimulation of innate immunity via toll-like receptors (21), it is recognized by humoral (22,23) and cell-mediated arms of the acquired immune system (24,25). It was one of the first Ags to be defined as the target of specific CD8 ϩ T cell responses during human infection (26).…”