Antigen Analysis of Mycobacterium tuberculosis H37Rv Culture Filtrate Proteins

Abstract

Culture filtrates of Mycobacterium tuberculosis H37Rv highly enriched with secreted proteins were used to identify antigens recognized by a serum pool from tuberculosis patients. Two different approaches were used to separate the culture filtrate protein mixture: (i) proteins were fractionated according to their hydrophobicity using an HPLC‐C18 chromatography column followed by separation based on their molecular mass by SDS‐PAGE and subsequent immunoblotting or (ii) proteins were separated by two‐dimensional gel electrophoresis, based on their isoelectric point and their molecular mass. Twenty serologically reactive proteins were ultimately identified by both methods, including four novel antigens. Further, to estimate the immunogenicity of the identified culture filtrate proteins, the relative antibody quantities were measured using Image master software. Our results show that the antibodies against proteins belonging to the antigen 85 complex were the most abundant in the serum of patients with active tuberculosis. The most immunogenic proteins in terms of high antibody‐to‐protein‐ratio were Rv3881c and three lipoproteins Rv0934 (the 38 kDa antigen), Rv0932c (pstS2), and Rv3006 (LppZ). Rv3881c is located in the region of difference 1 (RD1) which is deleted from Mycobacterium bovis BCG, and is therefore a particularly promising candidate for development of serodiagnostic assays to detect active tuberculosis. The proteins from the M. tuberculosis H37Rv culture filtrate are strong candidates to be evaluated for improvement of the serodiagnostic tests of tuberculosis.

Nonclassical Transpeptidases of Mycobacterium tuberculosis Alter Cell Size, Morphology, the Cytosolic Matrix, Protein Localization, Virulence, and Resistance to β-Lactams

“…4; also, see Table S1 in the supplemental material). These proteins range from major secreted antigens encoded by Rv1174c (37), Rv1810 (38), Rv1926c (39), Rv1980c (38)(39)(40), Rv3004 (37), and Rv3312A (41) to integral membrane proteins (see Table S1). Notably, an L,D-transpeptidase, Ldt Mt4 , encoded by Rv0192 (24), is 4.4-fold more abundant in the cytoplasm of M12 than in that of the wild-type strain.…”

Diagnostic Potential of Ag85C in Comparison to Various Secretory Antigens for Childhood Tuberculosis

“…ESAT-6 and CFP-10 are the secretory antigens encoded by RD1 region genes which are deleted in M. bovis BCG but present in M. tuberculosis [18]. Humoral response to different components of a major secretory protein Ag85complex in tuberculosis has been reported [19][20][21]. Hence utility of these antigens in the diagnosis of TB has been envisaged.…”

Serodiagnosis of tuberculous lymphadenitis using a combination of antigens

“…Previous studies have reported that Ag85A, Rv3881c, LAM, Ara 6-BSA and ESAT-6 may be useful for the serodiagnosis of pulmonary and bovine TB respectively [7][8][9][10][11]. Ag85A, ESAT-6 and TB10.4 are strongly recognized by T-cells isolated from TB patients and have been seen to induce a robust cell mediated immune response [7,12,13].…”