Establishment of a new immunological method for direct detection of Mycobacterium in solution

Tuberculosis (TB) is a crucial health problem. Prevention of the disease requires rapid diagnosis. Rapid liquid culture systems, nucleic acid amplification tests, and high-performance liquid chromatography (HPLC) are among the rapid tests used for detecting Mycobacterium species. However, these tests are expensive and require extensive equipment and expertise, which is hardly affordable in resource-poor countries. Although direct microscopy is performed routinely as an initial step for detection of the bacteria, it is not sufficiently sensitive. As a result, we thought of establishing a low-cost immunological test that can potentially replace direct microscopy with higher sensitivity and specificity.

The assay is based on pre-incubation of biotinylated rabbit antibody against Antigen 60 (A60) with a solution containing Bacillus Calmette-Guérin (BCG) or Mycobacterium tuberculosis (MTB) followed by incubation with a streptavidin-alkaline phosphatase (STA-ALP) conjugate. The test is devised in enzyme-linked immunosorbent assay (ELISA) and non-ELISA formats, therefore it does not require extensive facilities and expertise.

The ELISA format showed a 100-fold improvement in the lower detection limit of BCG compared with direct microscopy. With the non-ELISA formats, there was a 2- and 16-fold improvement for the cartridge assay and the microfuge tube assay, respectively.

In conclusion, we successfully detected BCG and MTB in solution using the new immunological method. Our results are very promising and the new immunological method could potentially replace direct microscopy with higher sensitivity and specificity.