In an article in the October issue of the Journal of Lipid Research, researchers in Singapore report the development of a new antibody-based method for detecting Mycobacterium tuberculosis, the causative agent of tuberculosis infection. Mycolic acid, whose long fatty acids are the main component of M. tuberculosis’ bacterial cell wall, is found in infected patients’ sputum and appears to be the perfect target for such an assay.
According to the Centers for Disease Control and Prevention, one-third of the world’s population is infected with M. tuberculosis. The World Health Organization reports that more than 95 percent of deaths caused by tuberculosis worldwide occur in low- and middle-income countries. Treatment for TB is most effective when the infection is diagnosed quickly, but developing countries are likely to lack the resources that allow for such quick diagnosis.
Current TB diagnosis methods using blood cultures, mucosal sputum smears, polymerase chain reaction and chest X-rays in such settings are not ideal because of the poor sensitivity and high expense of the tests, the length of time it takes to receive results, and the lack of infrastructure in which to conduct the tests. While assays to detect protein and glycolipid antigens of the bacterium exist, none represents a true improvement over standard diagnostics, and too few studies have been done to confirm their usefulness.
In the study reported in the JLR, Conrad E. Chan of the National University of Singapore and colleagues there and at DSO National Laboratories in Singapore first screened a phage display library to isolate four specific antibodies for mycolic acid. Then they optimized a rapid lipid-extraction protocol to use these antibodies in detecting mycolic acid in mycobacterial culture. Sensitivity of each antibody to detect mycolic acid was tested against serially diluted concentrations of M. tuberculosis-derived mycolic acid as well as against synthetic mycolic acid subclasses. To free mycolic acid’s covalent bond with a bacterium’s inner cell membrane, alkaline hydrolysis proved to be a quicker method than treatment with hexane, which required overnight incubation. The most sensitive of the four isolated antibodies detected 4.5 nanograms of mycolic acid or methoxy mycolic acid by ELISA, a result similar to other currently used, antibody-based diagnostic assays.
|Mycobacterium_tuberculosis, Centers for Disease Control and Prevention. CREDIT: Ray Butler and Janice Carr of Wikimedia Commons.jpg.
The researchers used high-resolution tandem mass spectrometry to analyze the alkaline hydrolysis products to confirm that the isolated antibodies were indeed detecting mycolic acid accurately. While MS analysis is powerful and practical in clinical use, it is not likely feasible for use in a point-of-care test in developing countries. While Chan et al. admit that their assay is less sensitive than using MS, they note that it is able to detect dead bacterial fragments, whereas conventional assays require that live, intact bacteria be present for positive results. The researchers conclude that further testing on clinical sputum samples will help validate the clinical utility of these isolated antibodies for use in assays.
Overall, Chan and colleagues have proved it is possible to extract lipids efficiently and quickly and detect them in a rapid-format assay if needed and without expensive equipment and extensive training. This gives hope that future assays can be developed to diagnose deadly infections like TB more quickly, even in resource-poor settings.
Mary L. Chang (firstname.lastname@example.org) is publications manager for the Journal of Lipid Research and Molecular & Cellular Proteomics.