Establishment of an indirect ELISA for Mycobacterium tuberculosis MTB39A protein antibody

ABSTRACT: The MTB39A protein is a member of the unique Mycobacterium tuberculosis (MTB) PE/PPE protein family and is the main candidate for tuberculosis (TB) diagnosis. The aim of this study was to establish a novel indirect ELISA (iELISA) method that uses antibodies against MTB. The MTB39A gene sequence was synthesized according to the MTB39A nucleotide sequence of the MTB H37Rv strain (GenBank accession number: NC_000962.3) and cloned into the pET28a( +) vector. After correct sequencing, it was transferred to Escherichia coli BL21 (DE3) receptor cells for expression and purification, and the purified recombinant protein was identified by SDS-PAGE and western blotting. The purified MTB39A protein was used as the capture antibody, and a rabbit polyclonal antibody against the MTB MTB39A protein was used as the detection antibody to establish an indirect ELISA method. The ELISA conditions were optimized, and the optimal coating concentration of the MTB39A antigen was determined to be 0.5 μg/mL. The optimal dilution of MTB39A rabbit polyclonal antibody was 1:4096, and the optimal dilution of HRP-goat anti-rabbit IgG was 1:4000. The results showed that this indirect ELISA method has high sensitivity, specificity and efficacy for MTB39A protein detection. Moreover, this indirect ELISA method has optimal stability and can be used for the initial detection of MTB antibodies in clinical human and bovine serum samples. The establishment of this assay provides a new method for the rapid diagnosis of MTB and technical support for the prevention and control of tuberculosis. KEY POINTS: • MTB MTB39A protein was expressed in a prokaryotic expression system. • Rabbit polyclonal antibody against MTB39A was prepared. • To establish an iELISA based on the MTB39A protein for the detection of MTB antibodies.