Development of Antigen Detection Assay for Diagnosis of Tuberculosis Using Sputum Samples

Abstract

The rising incidence of tuberculosis worldwide means an increasing burden on diagnostic facilities, so tests simpler than Ziehl-Neelsen staining are needed. Such tests should be objective, reproducible, and have at least as good a detection limit as 10 4 bacteria/ml. A capture enzyme-linked immunosorbent assay (ELISA) was developed for detection of lipoarabinomannan (LAM) in human sputum samples. As a capture antibody, we used a murine monoclonal antibody against LAM, with rabbit antiserum against Mycobacterium tuberculosis as a source of detector antibodies. The sensitivity of the capture ELISA was evaluated by using purified LAM and M. tuberculosis whole cells. We were able to detect 1 ng of purified LAM/ml and 10 4 M. tuberculosis whole cells/ml. LAM could also be detected in culture filtrate of a 3-week-old culture of M. tuberculosis. The culture filtrate contained approximately 100 g of LAM/ml. The detection limit in sputum pretreated with N-acetyl-L-cysteine and proteinase K was 10 4 M. tuberculosis whole cells per ml. Thirty-one (91%) of 34 sputum samples from 18 Vietnamese patients with tuberculosis (32 smear positive and 2 smear negative) were positive in the LAM detection assay. In contrast, none of the 25 sputum samples from 21 nontuberculous patients was positive. This specific and sensitive assay for the detection of LAM in sputum is potentially useful for the diagnosis of tuberculosis.

Identification and characterization of Mycobacterium tuberculosis antigens in urine of patients with active pulmonary tuberculosis: an innovative and alternative approach of antigen discovery of useful microbial molecules

“…Indeed, a test that is based on detection of M. tuberculosis lipoarabinomannan (LAM) in the urine of tuberculosis patients is under clinical validation. Despite some conflicting results regarding the sensitivity and specificity of this test, a significant correlation between the mycobacterial burden in sputum and LAM antigen concentrations in the patients' urines has been found [26][27][28][29]. Therefore, the proteins identified in the present studies are interesting candidates for the development of a similar antigen detection assay for the diagnosis of active tuberculosis.…”

Clinical Utility of a Commercial LAM-ELISA Assay for TB Diagnosis in HIV-Infected Patients Using Urine and Sputum Samples

“…The standardized Clearview® LAM ELISA has not previously been evaluated using sputum samples. We hypothesised, based on a prior study of LAM antigen in sputum [28] , that detection of LAM antigen in smear negative patients would be diagnostically useful. However, although the Clearview® TB ELISA had a high sensitivity in sputum samples, the specificity was dismally poor.…”

Low sensitivity of a urine LAM-ELISA in the diagnosis of pulmonary tuberculosis

“…LAM is immunogenic and suspected to be an important virulence factor and therefore a potential drug target [ 12 , 13 ]. Various tests have been developed to detect LAM in serum [ 14 , 15 ], pleural effusion [ 16 ] or sputum [ 17 - 19 ] of tuberculosis patients. None of the tests is, however, widely used.…”