Proteinase K-pretreated ConA-based ELISA assay: a novel urine LAM detection strategy for TB diagnosis

Abstract

ObjectivesLipoarabinomannan (LAM), an abundant cell wall glycolipid of mycobacteria including Mycobacterium tuberculosis (Mtb), is a promising TB diagnostic marker. The current commercially available urine LAM assays are not sufficiently sensitive, and more novel detection strategies are urgently needed to fill the current diagnostic gap.MethodsA proteinase K-pretreated Concanavalin A (ConA)-based ELISA assay was developed. Diagnostic performance was assessed by several bacterial strains and clinical urine samples.ResultsThe limit of detection (LoD) of the assay against ManLAM was 6 ng/ml. The assay reacted strongly to Mtb H37Rv and M. bovis BCG, intermediately to M. smegmatis mc2155, and weakly to four non-mycobacteria pathogens. This method could distinguish TB patients from healthy controls (HCs) and close contacts (CCs) in 71 urine samples treated with proteinase K, which increases urine LAM antibody reactiveness. In TB+HIV+ and TB+HIV− patients, the sensitivity was 43.8 and 37.5%, respectively, while the specificity was 100.0%. The areas under ROC curves (AUCs) were 0.74 and 0.82, respectively.ConclusionThis study implies that ConA can be paired with antibodies to detect LAM. Proteinase K treatment could effectively enhance the sensitivity by restoring the reactiveness of antibodies to LAM.