A novel small molecule immunoassay to detect the mycobacterial siderophore carboxymycobactin

Abstract

Background: To diagnose tuberculosis (TB), it is necessary to demonstrate the presence of Mycobacterium tuberculosis in a clinical specimen such as sputum. A simple, low-cost rapid test for blood or urine is urgently needed but remains elusive. Tests for host response-derived biomarkers or secreted bacterial compounds have so far failed to provide sufficient sensitivity or specificity and alternative approaches are needed. Carboxymycobactins are amphiphilic siderophores unique to the mycobacteria that have the ability to transverse mammalian cell walls. They have insufficient mass to induce an antibody response and there are currently no simple sensitive tests for their detection. Methods: We report the development of an enzyme-linked immunosorbent assay (ELISA) for carboxymycobactin. Polyclonal antibodies were raised in rabbits following conjugation to a carrier protein, bovine serum albumin allowing development of a sensitive indirect double antibody ELISA. A second keyhole limpet hemocyanin conjugate was manufactured to adhere the hapten to the wells of the microplate. We established a limit of detection for the assay of 1 pg/ml. Carboxymycobactin was detected in culture supernatant from mycobacteria including clinical isolates of M. tuberculosis , but not from cultures of Rhodococcus, Nocardia, Streptomyces, Bacillus cereus, Klebsiella oxytoca, Staphylococcus aureus, Escherichia coli , and Pseudomonas aeruginosa . Sample concentration was achieved following extraction with chloroform. Results: We have demonstrated for the first time the direct detection of carboxymycobactin in culture supernatant by immunoassay. Conclusions: We recommend testing samples from humans and animals to establish the potential utility of carboxymycobactin as a diagnostic marker of active mycobacterial disease.