Serological expression cloning and immunological evaluation of MTB48, a novel Mycobacterium tuberculosis antigen

Improved diagnostics are needed for the detection of Mycobacterium tubercul osis, especially for patients with smear-negative disease. To address this problem, we have screened M. tuberculosis (H37Rv and Erdman strains) genomi c expression libraries with pooled sera from patients with extrapulmonary d isease and with sera from patients with elevated reactivity with M. tubercu losis lysate. Both serum pools were reactive,vith clones expressing a recom binant protein referred to here as MTB48. The genomic sequence of the resul ting clones was identical to that of the M. tuberculosis H37Rv isolate and showed 99% identity to the Mycobacterium bovis and M. bovis BCG isolate seq uences. The genomic location of this sequence is 826 bp upstream of a regio n containing the esat-6 gene that is deleted in the M. bovis BCG isolate. T he mtb48 1,380-bp open reading frame encodes a predicted 47.6-kDa polypepti de with no known function. Southern and Western blot analyses indicate that this sequence is present in a single copy and is conserved in the M. tuber culosis and M. bovis isolates tested but not in other mycobacterial species tested, including Mycobacterium leprae and Mycobacterium avium. In additio n, the native protein was detected in the cytoplasm, as was a processed for m that was also shed into the medium during culture. Serological analysis o f recombinant MTB48 and the M. tuberculosis 38-kDa antigen with a panel of patient and control sera indicates that the inclusion of recombinant MTB48 in a prototype serodiagnostic test increases assay sensitivity for M. tuber culosis infection when it is combined with other known immunodominant antig ens, such as the 38-kDa antigen.