A method of determining the presence or absence in a sample of a biomarker indicative of exposure to mycobacteria, the method comprising: (a) providing a substrate carrying a mycolic acid derived antigen; (b) contacting the substrate with the sample; (c) contacting the substrate with a fluorophore species; (d) creating an evanescent wave at the boundary of the substrate and the sample; (e) detecting the presence or absence of fluorescence.