Development of a quantitative sandwich enzyme-linked immunosorbent assay for detecting the MPT64 antigen of Mycobacterium tuberculosis

Abstract

Purpose: Tuberculosis (TB) is a major infectious disease and is responsible for two million deaths annually. For the identification and quantitation of Mycobacterium tuberculosis (M. tuberculosis), a causative agent of TB, a sandwich enzyme-linked immunosorbent assay (ELISA) against the MPT64 protein of M. tuberculosis, an antigen marker of the M. tuberculosis complex, was developed.

Materials and methods: The MPT64 protein was expressed, and anti-MPT64 monoclonal antibodies were prepared. A sandwich ELISA was established using recombinant MPT64 protein and anti-MPT64 monoclonal antibodies. The sandwich MPT64 ELISA was evaluated using reference and clinical mycobacterial strains.

Results: The sandwich MPT64 ELISA detected MPT64 protein from 2.1 ng/mL to 250 ng/mL (equivalent to 1.7×10⁴ CFU/mL and 2.0×10⁶ CFU/mL). All 389 clinical M. tuberculosis isolates tested positive in the sandwich MPT64 ELISA (sensitivity, 100%), and the assay showed no cross reactivity to any tested nontuberculous mycobacterial strain (specificity, 100%).

Conclusion: The sandwich MPT64 ELISA is a highly sensitive and quantitative test for MPT64 protein, which can identify M. tuberculosis.

Keywords: MPT64 protein; Mycobacterium tuberculosis; sandwich ELISA.

Fig. 1

Analysis of expression of the MPT64 protein in TB-1 cells. TB-1 cells were inducted with 1 mM IPTG for 4 h, and the cell lysates were analysed by SDS-PAGE, after which the gel was stained with Coomassie blue (lanes 1 to 2) or analysed by Western blot with a mouse polyclonal anti-M. tuberculosis antibody (lane 3). Lane M: pre-stained protein molecular weight markers. Lane 1: cell lysate of TB-1 cells. Lane 2: purified recombinant MPT64. Lane 3: Western blot analysis with anti-MPB64 antibody. TB, tuberculosis; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; IPTG, isopropyl β-D-1-thiogalactopyranoside.

Fig. 2

Calibration curve of the developed sandwich ELISA for MPT64 protein (A), and detection limits of the assay (B). The calibration curve was obtained from the mean value of six sandwich ELISAs with the anti-MPT64 antibodies Mab-1A4 and Mab-2E9 under optimized conditions (A). Culture supernatants of M. tuberculosis H37Rv in Middlebrook 7H9 broth with enrichment were serially diluted and applied to the test. The cut-off of the MPT64 sandwich ELISA is indicated by a dotted line. ELISA, enzyme-linked immunosorbent assay; CFU, colony-forming unit; OD, optical density.