Standardization of natural mycolic acid antigen composition and production for use in biomarker antibody detection to diagnose active tuberculosis

This research article published by Elsevier B.V., 2016Mycobacterium tuberculosis, the causative agent of tuberculosis, is characterized by the abundance of species specific, antigenic cell wall lipids called mycolic acids. These wax-like molecules all share an identical, amphiphilic mycolic motif, but have different functional groups in a long hydrophobic hydrocarbon mero-chain that divide them into three main classes: alpha-, keto- and methoxy-mycolic acids. Whereas alpha-mycolic acids constitutively maintain an abundance of around 50%, the ratio of methoxy- to keto-mycolic acid types may vary depending on, among other things, the growth stage of M. tuberculosis. In human patients, antibodies to mycolic acids have shown potential as diagnostic serum biomarkers for active TB. Variations in mycolic acid composition affect the antigenic properties and can potentially compromise the precision of detection of anti-mycolic acids antibodies in patient sera to natural mixtures. We demonstrate this here with combinations of synthetic mycolic acid antigens, tested against TB patient and control sera. Combinations of methoxy- and α-mycolic acids are more antigenic than combinations of keto- and α-mycolic acids, showing the former to give a more sensitive test for TB biomarker antibodies. Natural mixtures of mycolic acids isolated from mature cultures of M. tuberculosis H37Rv give the same sensitivity as that with synthetic methoxy- and α-mycolic acids in combination, in a surface plasmon resonance inhibition biosensor test. To ensure that the antigenic activity of isolates of natural mycolic acids is reproducible, we cultured M. tuberculosis H37Rv on Middlebrook 7H10 solid agar plates to stationary growth phase in a standardized, optimal way. The proportions of mycolic acid classes in various batches of the isolates prepared from these cultures were compared to a commercially available natural mycolic acid isolate. LC-MS/MS and NMR data for quantitation of mycolic acids class compositions show that the variation in batches is small, suggesting that the quality of the results for anti-mycolic acid antibody detection in the TB patients should not be affected by different batches of natural mycolic acid antigens if prepared in a standard way

PEER REVIEWED ARTICLE No 5 - EFFECT OF PROCESSING AND PRESERVATION METHODS ON VITAMIN C AND TOTAL CAROTENOID LEVELS OF SOME VERNONIA (BITTER LEAF) SPECIES

Vernonia is one of the leafy vegetables that can be used in an attempt to alleviate the problem of micronutrient malnutrition, prominent in tropical Africa. Commonly known as Ndole or Bitterleaf in most Central and West African countries, it is one of the most widely consumed leafy vegetable in Cameroon. Though eaten by a large proportion of the population, cultivation is limited to the southern parts of the country and mostly in the rainy season. In order to ensure availability in nongrowing areas or seasons, the vegetable is usually processed and preserved. Processing and preservation methods used are thought to influence the nutrient content of these vegetables. The study was aimed at determining the effects of processing and preservation methods on vitamin C and total carotenoid levels of some species of Vernonia (V.amygdalina , V.calvoana var. bitter , V.colorata and V.calvoana var. non bitter ) consumed in Cameroon. The processing methods were squeeze-washing, boiling and squeeze-washing with 0N, 1N and 2N concentrations of natron. The methods of preservation were sun drying, oven drying at different temperatures (45, 60 and 75°C) and freezing for 0, 10, 30 60 and 120 days. Results show that these leafy vegetables were good sources of vitamin C and total carotenoid. Vitamin C values vary from 137.5±3.3 in V. calvoana non bitter to 197.5 ± 3.5mg/100g in V. colorata, while total carotenoid levels range from 30.0 ± 1.0 in V. amygdalina to 41.5±0.9mg/100g in V. colorata for the raw samples. Greater losses of both vitamins were observed for the bitter species. Drying caused significant losses, especially in Vitamin C. The best temperature for drying the vegetables to preserve carotenoids and vitamin C was at 45°C, whereas sun drying and oven drying at 75°C caused the highest losses. Freezing for up to ten days had no significant influence on total carotenoids and vitamin C levels. Comparatively, squeezewashing proved to be the best processing method that ensured minimum loss of both vitamins