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

• Published in: Yonsei medical journal Vol. 55; no. 3; pp. 746 - 752
• Main Authors: Ji, Mijung, Cho, Byungki, Cho, Young Shik, Park, Song-Yong, Cho, Sang-Nae, Jeon, Bo-Young, Yoon, Byoung-Su
• Format: Journal Article
• Language: English
• Published: Korea (South) Yonsei University College of Medicine 01.05.2014; 연세대학교의과대학
• Subjects: Antigens, Bacterial - analysis; Antigens, Bacterial - immunology; Enzyme-Linked Immunosorbent Assay - methods; Mycobacterium tuberculosis - immunology; Original; 의학일반; MPT64 protein; Mycobacterium tuberculosis; sandwich ELISA
• ISSN: 0513-5796; 1976-2437
• DOI: 10.3349/ymj.2014.55.3.746

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