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 mycolactonespecific 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.

An Antigen Capture Assay for the Detection of Mycolactone, the Polyketide Toxin of Mycobacterium ulcerans

“…1), were used to generate sets of mAbs with varying fine specificities (Table I). One of these sets of mAbs (JD5.1JD 5.12) has been previously described (11,12).…”

“…Fine specificity of subset 2b mAbs resembled subset 2a mAbs in that they recognized all mycolactone variants with an intact core, but in contrast to the latter, their binding to PG-183 was also strong. Subset 3 mAbs were obtained from two different PG-203immunized mice and include mAbs JD5.1JD5.12, which have been described before (11,12). This subset recognized PG-204 and MG-161 and, to varying degrees, the mycolactone derivatives MG-158 and MG-160 with more pronounced modifications at the upper side chain (Fig.…”

“…3), matched pairs comprising the subset 3 mAb LW7.11 as a capturing mAb and any of the (core-specific) subset 2b mAbs LW7.5LW7.9 as detecting mAb were prioritized for further tests. We have previously shown a TEA-based buffer to be beneficial for a mycolactonecompetitive ELISA, yielding improved assay sensitivity compared with a PBST-based buffer (12). Interestingly, in the current mycolactone capture ELISA, the TEA-based buffer was crucial for mycolactone detection as little or no signals were obtained with a PBST-based buffer.…”

“…We recently reported the generation of mAbs capable of specific binding to mycolactone and developed an Ag-competitive immunoassay based on these mAbs (11,12). Although this assay is highly specific and sensitive, competitive assays do have a few shortcomings compared with Ag capture assays.…”

“…For one, by using only one mAb, competitive assays have a higher probability of cross-reactivity (i.e., lower specificity) compared with Ag capture assays, which usually use two mAbs of different fine specificities (13,14). Also, the need to have a suitable reporter molecule for competitive assays, like a biotinylated mycolactone variant (12), increases the complexity of scale-up for such an assay. Mycolactone is a notoriously difficult molecule to synthesize, even though several total syntheses strategies have been reported (1518).…”