Assessment of the IgA immunoassay diagnostic potential of the Mycobacterium tuberculosis MT10.3-MPT64 fusion protein in tuberculous pleural fluid

Abstract

Pleural tuberculosis (PL-TB) remains difficult to diagnose. An enzyme-linked immunosorbent assay (ELISA) was developed based on a construction containing the fusion of the Rv3019c (MT10.3) and Rv1980c (MPT64) gene sequences, and its performance was evaluated in an area where TB is endemic. A total of 92 pleural fluid (PF) samples at serial dilutions of 1:50 to 1:800 were included in the ELISA IgA MT10.3-MPT64 evaluation: 70 from TB patients and 22 from patients with other pleurisies. Confirmation of the expression and subsequent purification of the protein was made by SDS-PAGE and Western blot assays, resulting in a 36-kDa protein. ELISA IgA MT10.3-MPT64 showed sensitivities of 61.4%, 58.6%, 62.9%, 67.1%, and 70% at each PF dilution, respectively. The cumulative results of all dilutions increased sensitivity to 81.4% without jeopardizing specificity. Similar results were also obtained at the combined dilutions of 1:50, 1:200, and 1:800 or 1:50 plus 1:800 dilutions (80%). The overall sensitivity of the reference test, i.e., histopathological examination, was 74%. But, via the ELISA IgA MT10.3-MPT64 test, sensitivity was high for specimens with a negative culture (23/27; 85.2%) or nonspecific histopathology (17/18; 94.4%). Our findings demonstrated the promising use of this test as an adjunct in PL-TB diagnoses, particularly in cases with lower bacterial loads and false-negative results in the reference tests, since the new test includes such important features as quick and easy application, high sensitivity and, perhaps most importantly, affordability, which is so crucial for its widespread use in developing countries.

FIG. 1.

Cloning scheme of MT10.3-MPT64. (A) Digestion of the plasmid vector and amplicon of the Rv3019c gene (MT10.3) with the enzymes BamHI and SalI and subsequent ligation into the pQE80L vector. (B) Digestion of the pQE80L (Rv3019c) and Rv1980c (MPT-64) gene amplicon with SalI and HindIII, followed by enzyme linkage to generate MT10.3-MPT64-pQE80L.

FIG. 2.

(A) Coomassie-stained 12% SDS-PAGE gel of the purified fusion protein MT10.3-MPT64. Lanes 1 to 5, eluates collected at different times; lane 6, molecular mass marker. (B) Western blot of MT10.3-MPT64 fusion protein from a mixture of eluates 1 and 2 incubated with mouse IgG anti-His. Lane 1, prestained protein molecular ladder; lane 2, mixed eluates 1 and 2; lane 3, mixed eluates 3, 4, and 5.

FIG. 3.

Distribution of individual humoral responses of the IgA MT10.3-MPT64 fusion protein in patients with pleural tuberculosis (•) or other pleural diseases (○) tested in serial PF dilutions of 1:50, 1:100, 1:200, 1:400, and 1:800. Short bars, mean ODs; longer dashed bars, cutoffs.

FIG. 4.

Venn diagram showing the number of PF samples from patients with PL-TB in which IgA was detected via ELISA to single MT10.3 and MPT64 antigens and to the fusion protein MT10.3-MPT64 (combination of PF dilution results).