Identification of Mycobacterium tuberculosis antigens of high serodiagnostic value

Abstract

Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis, with several million new cases detected each year. Current methods of diagnosis are time-consuming and/or expensive or have a low level of accuracy. Therefore, new diagnostics are urgently needed to address the global tuberculosis burden and to improve control programs. Serological assays remain attractive for use in resource-limited settings because they are simple, rapid, and inexpensive and offer the possibility of detecting cases often missed by routine sputum smear microscopy. The aim of this study was to identify M. tuberculosis seroreactive antigens from a panel of 103 recombinant proteins selected as diagnostic candidates. Initial library screening by protein array analysis and enzyme-linked immunosorbent assay (ELISA) identified 42 antigens with serodiagnostic potential. Among these, 25 were novel proteins. The reactive antigens demonstrated various individual sensitivities, ranging from 12% to 78% (specificities, 76 to 100%). When the antigens were analyzed in combinations, up to 93% of antibody responders could be identified among the TB patients. Selected seroreactive proteins were used to design 3 new polyepitope fusion proteins. Characterization of these antigens by multiantigen print immunoassay (MAPIA) revealed that the vast majority of the TB patients (90%) produced antibody responses. The results confirmed that due to the remarkable variation in immune recognition patterns, an optimal multiantigen cocktail should be designed to cover the heterogeneity of antibody responses and thus achieve the highest possible test sensitivity.

FIG. 1.

SDS-PAGE analysis of purified recombinant M. tuberculosis proteins. Individual M. tuberculosis proteins are listed by their H37Rv gene numbers. Two to 5 μg of each antigen was run in a 4 to 20% SDS-PAGE gel and stained with Coomassie blue to determine its relative purity. Lanes M, molecular size standards (160, 120, 80, 60, 40, 25, 20, 15, and 10 kDa).

FIG. 2.

Serum reactivity in recombinant M. tuberculosis protein arrays. Seventy-nine M. tuberculosis proteins were printed in duplicate, incubated with TB-positive or NEC sera, developed, and scanned. Representative images of 3 control sera (A) and 3 TB-positive sera (B) on protein arrays are shown.

FIG. 3.

ELISA results for recombinant M. tuberculosis proteins. TB+, confirmed sputum smear-positive pulmonary TB samples (n = 92) from Brazil; NEC, negative, nonendemic (U.S.) control sera (n = 46). Representative data for 24 recombinant M. tuberculosis antigens are shown. The median OD is represented by a horizontal line. Individual antigens are listed at the bottom of each graph, with positive samples determined as those samples giving ELISA readings 2-fold above the mean of the negative controls, with an OD₄₅₀ of >0.2.

FIG. 4.

Fusion protein serum ELISA. Results for DID90A, DID90B, DID104, TBF10, and the individual component antigens are displayed as box plots. TB+, confirmed pulmonary TB samples (n = 36) from India; NEC, negative, nonendemic (U.S.) control sera (n = 30); EC, negative, endemic (Indian) control sera (n = 20). Each box represents data from the 25th to the 75th percentile, the median is represented by a horizontal line, and the whiskers extend to the lowest and highest values.

FIG. 5.

M. tuberculosis antigen reactivity in MAPIA. The 4 fusion polyproteins and 6 single antigens were printed on nitrocellulose membranes (listed on the right), and the assay was performed as described in Materials and Methods. Each strip represents one serum sample and displays the antigen reactivity pattern for that sample. Results are shown for 10 negative-control sera (left) and 30 sera from TB patients, including 6 from India and 24 from Brazil (right).