Development of an ELISA for the quantification of mycolactone, the cytotoxic macrolide toxin of Mycobacterium ulcerans

Abstract

Mycolactones, macrolide cytotoxins, are key virulence factors of Mycobacterium ulcerans, the etiological agent of the chronic necrotizing skin disease Buruli ulcer. There is urgent need for a simple point-of-care laboratory test for Buruli ulcer and mycolactone represents a promising target for the development of an immunological assay. However, for a long time, all efforts to generate mycolactone-specific antibodies have failed. By using a protein conjugate of a truncated non-toxic synthetic mycolactone derivative, we recently described generation of a set of mycolactone-specific monoclonal antibodies. Using the first mycolactone-specific monoclonal antibodies that we have described before, we were able to develop an antigen competition assay that detects mycolactones. By the systematic selection of a capturing antibody and a reporter molecule, and the optimization of assay conditions, we developed an ELISA that detects common natural variants of mycolactone with a limit of detection in the low nanomolar range. The mycolactone-specific ELISA described here will be a very useful tool for research on the biology of this macrolide toxin. After conversion into a simple point-of-care test format, the competition assay may have great potential as laboratory assay for both the diagnosis of Buruli ulcer and for the monitoring of treatment efficacy. The macrolide toxin mycolactone is the key virulence factor of Mycobacterium ulcerans, the causative agent of the chronic necrotizing skin disease Buruli ulcer. Mycolactone has cytotoxic activity and is secreted by the mycobacteria, causing tissue necrosis and immunosuppression. For research on Buruli ulcer, there is urgent need for a simple tool to quantify mycolactone. Using the first mycolactone-specific monoclonal antibodies that we have described previously, we have optimized here an antigen competition ELISA that detects common natural variants of mycolactone at a low nanomolar scale. Sensitivity of the assay is sufficient to detect the toxin in M. ulcerans culture supernatants and in the tissue of experimentally infected animals. Converted into a simple point-of-care test format, the competition assay may in future also be suitable as a diagnostic laboratory test for Buruli ulcer.