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.

Detection of BCG bacteria using a magnetoresistive biosensor: A step towards a fully electronic platform for tuberculosis point-of-care detection

“…Mechanical biosensors in attenuated Mtb cells achieved LOD of 2 × 10 3 (He et al, 2003) and 10 5 (He and Zhang, 2002), respectively, with acoustic wave and quartz crystal microbalance. In optical biochips, LOD was 10 3 and 10 4 cells/mL with Mtb sputum samples, respectively, when using isothermal loop mediated amplification (Creecy et al, 2015) and capture ELISA of lipoarabinomannan antigen (Arias-Bouda et al, 2000). Similar LOD results, 10 3 and 10 4 , were achieved by magnetic methods, respectively, by using a magnetic barcode in sputum (Liong et al, 2013) and a magnetoelastic biosensor in Mtb H37Ra strain cells (Pang et al, 2008).…”

“…In these settings, direct Ziehl-Neelsen (ZN) sputum smear microscopy is an important diagnostic technique, although the method is laborious and prone to human error and to infection exposure (Yeo et al, 2009;Matu and Juma, 2013;Creecy et al, 2015). ZN only identifies TB pulmonary cases in sputum samples, with a limit of detection (LOD) of 10 4 cells/mL (Arias-Bouda et al, 2000;Yeo et al, 2009;Creecy et al, 2015). Therefore, active TB cases with lower number of cells, patients with negative sputum results, children, HIV patients and extrapulmonary TB, need a different diagnostic approach (Moreno-Pérez et al, 2010;Parsons et al, 2011;Veigas et al, 2012;WHO, 2012).…”

“…Therefore, active TB cases with lower number of cells, patients with negative sputum results, children, HIV patients and extrapulmonary TB, need a different diagnostic approach (Moreno-Pérez et al, 2010;Parsons et al, 2011;Veigas et al, 2012;WHO, 2012). Inexpensive point-of-care (POC) tests that can be used with blood or urine, are extremely necessary (Arias-Bouda et al, 2000;Yeo et al, 2009;Lange and Mori, 2010).…”

Detection of Mycobacterium Tuberculosis With Tb Antigen Rapid Test in Pulmonary Tuberculosis Patients With Four Types of Spuctum Sample Preparation

“…This process will cause damage to the cells of M.tuberculosis, so many proteins will be secreted. 8,18 In addition, the reading results of TB Ag Rapid Test Device using routine procedures (Tube 1) were 43.3% for the positive one and 56.7% for the negative one. The low results were possibly caused by Ag of M.tuberculosis still trapped in the viscous mucous.…”

New tools and emerging technologies for the diagnosis of tuberculosis: Part II. Active tuberculosis and drug resistance

“…In addition to these approaches, efforts are also underway to develop immune-based assays that focus on antigen rather than antibody detection. For example, antigen-capture enzymelinked immunosorbent assays for the detection of lipoarabinomannan in sputum [23] and urine [24] samples have demonstrated promise in early trials. Further work is necessary to determine their utility.…”