Immunological and biochemical characterization of protein antigens of mycobacterium tuberculosis H 37 Ra, as recognised by sera from…

Abstract

Tuberculosis is a major health hazard in developing countries like India. Availability of new and powerful drug regimens has reduced the mortality rate, but the lack of a suitable diagnostic test and the absence of an effective vaccine still place tuberculosis as a major cause of human mortality. Many unique or novel substances reported from mycobacteria have been employed in attempts to overcome the above problems, but with more failure than success. Following a different approach, sera from tuberculosis patients were used in the present investigation to characterize the immunogenicity of the proteins of Mycobacterium tuberculosis. Sera from patients were analyzed using conventional immunological techniques as well as advanced methods such as ELISA and immunoblotting. Of the 75 sera tested, 45 showed very low antibody titres against the M. tuberculosis extract. Among the remaining sera with higher titres, five antigens (designated 1 to 5) were identified using immunoblot analysis. Two antigens (antigen 1 and antigen 2), recognized by the majority of sera, were further characterized. Molecularweight determination revealed antigen 1 to be 65 kDa, while antigen 2 was 36-38 kDa. Isoelectric focusing showed pI values of 6.8 and 7.2 for antigen 1 and antigen 2, respectively. Both antigens were found to contain none or very low amounts of carbohydrate. Mycobacterial extracts are known to contain high amounts of lipids and polysaccharides, complicating protein purification using standard techniques. Antigen 1 and antigen 2 were therefore bandcut and geleluted from nondenaturing polyacrylamide gels. Antibodies were raised against these proteins in rabbits. Immunochemical characterization of these antisera was performed using immunoelectrophoresis, tandem twodimensional immunoelectrophoresis, ELISA, and immunoblotting. Antiserum 1 (against antigen 1) recognized antigens 1, 2, and 5 in the M. tuberculosis (Ra) extract. Antiserum 2 (against antigen 2) recognized antigens 2, 3, and 5, but not antigen 1. On immunoblot analysis after SDSPAGE, antiserum 1 recognized 18 polypeptides (12-70 kDa), while antiserum 2 reacted with five polypeptides (18-50 kDa). The presence of crossreactive antigens in other mycobacteria was examined and equivalent antigens for antigens 1 and 2 were detected, but with lower crossreactivity than seen in the Ra extract. Displacement ELISA using these antisera with extracts from different mycobacterial species showed that crossreactivity was limited and confined to the respective related antigens among these species. The 65 kDa antigen has been reported to share epitopes with various molecules, including equivalent proteins from other microbes and the GroEL protein of E. coli. Displacement ELISA was used to evaluate crossreactivity of antigens 1 and 2 with E. coli extract and BSA, but the negligible binding observed was considered nonspecific. Antigen 1 could not be purified using an antiserum1 immunosorbent column. Surprisingly, antigen 2 and a few other proteins eluted from the column, suggesting that antigen 2 may be a degradation product of antigen 1. When the M. tuberculosis Ra extract was incubated for 48 hours at 37°C and electrophoresed, the amount of antigen 1 decreased markedly, while antigen 2 appeared in higher amounts. Incomplete peptide mapping of antigen 1 supported the possibility that antigen 2 is a degradation product of antigen 1. Circular dichroism analysis was attempted to determine the secondary structures of these proteins but was unsuccessful due to polyacrylamide contamination in the antigen preparations.