Microbiol

Microbiol. lipid biomarkers. Keywords: phage display, mycolic acid, biomarker, antibody-based assay Tuberculosis (TB) is one of the world’s most significant causes of mortality due to infectious disease, with an estimated 1.45 million deaths in 2010 2010 (1). Rapid identification of new cases is key to the proper treatment of TB, the majority of which occur in resource-poor settings. However, current detection methods such as sputum smears, culture, PCR, and X-ray are unsuitable in such areas due to poor sensitivity, slow turnaround time, high operating costs, and infrastructure requirements, respectively (2C4). An alternative point-of-care diagnostic test is antibody-based assays, which rely on detecting ((BCG) strain. We therefore demonstrate that insoluble lipids previously inaccessible to antibody-based assays, such as mycolic acid, can be detected using novel recombinant anti-lipid antibodies in combination with rapid and simple lipid extraction techniques and thus such antibodies are useful for the detection of novel lipid biomarkers. Material and methods Reagents and bacterial strains was a kind contribution from Dr Ayi Teck Choon, DSO National Laboratories and ((ATCC BAA-1517) and (ATCC 8368) were purchased from American Type Culture Collection. Phage panning and IgG expression A nonimmune human Fab phage display library (Humanyx Pte Gliotoxin Ltd. was panned based on a previously developed protocol for generating anti-lipid antibodies (17). Briefly, a MaxiSorp immunotube (Nunc) was coated with ST6GAL1 200 g/ml of was cultured in Middlebrook 7H9 broth (BD Biosciences) while and were cultured in Brain-Heart Infusion Broth (BD Biosciences) at 37C in flasks in a shaking incubator. (BCG) was cultured in 100 ml of Middlebrook 7H9 broth at 37C with 5% CO2 in T175 Gliotoxin cell culture flasks for 2 weeks. Cultures were grown until log-phase growth (optical density, OD600 0.5) was reached. Cultures were then pelleted by centrifugation at 4, 000 and lipids extracted by adapting a procedure from Minnikin et al (21). Briefly, pelleted bacteria was resuspended in 30 ml of n-hexane per gram of wet cell mass, vortexed and Gliotoxin incubated at room temperature on rollers overnight for initial extraction based on wet cell mass. The next day, the mixtures were centrifuged at 16,000 for 5 min and the organic layer containing lipids liberated from bacterial surfaces was recovered. Various methods of lipid extraction, as described below, were subsequently performed on in order to derive at an optimal method for lipid extraction. Optimization of lipid extraction One milliliter of OD 0.1 (equivalent to 5.8 107 cfu/ml) was centrifuged from pure culture grown as above and the pellet was resuspended in 1 ml of n-hexane. The mixture was vortexed for 15 min, incubated on a rotator at room temperature overnight, then centrifuged and the organic layer was recovered. To enhance the effectiveness of solvent extraction, a second methodology was tried where the sample was heated for 2 h at 70C before vortexing. To lyse the bacteria to improve extraction, a third protocol was carried out where the same amount of mycobacteria was resuspended in 1ml of Milli-Q water and incubated at 99C for 30 min using a Thermomixer heating block (Eppendorf). One milliliter of n-hexane was then added and processed as above without heating. A fourth protocol was also performed where the bacteria was pelleted after incubation in boiling water and then resuspended in 1 ml of n-hexane as above. Centrifugations were carried out at 16,000 for.