Ecotoxicity program turns to xMAP® Technology for multi-biomarker detection
A vital tool for assessing the toxicity of freshwater ecosystems is the tiny Gammarus fossarum, an amphipod crustacean often used as model organisms and bioindicators for ecotoxicology programs. In a 30-minute webinar presentation, Associate Professor Stéphane Azoulay, from the University of Côte d’Azur, described the design and development of a multiplexed immunoassay to detect essential proteins for these efforts.
Proteogenomic Approach
To develop the assay, Azoulay and his team used xMAP® Technology for the first time. There are many assay techniques for ecotoxicology applications, he said in the presentation, but none of them are capable of being multiplexed.
The scientists began with a search for novel biomarkers that could be used as a diagnostic tool for ecotoxicology needs, with a specific focus on assessing endocrine disruptors. A proteogenomic approach led to a list of 204 proteins with significant sexual dimorphism; further filtering identified 44 proteins sensitive to all three toxicants of interest. The team selected five of those proteins for a multiplex assay and developed polyclonal antibodies that were immunized in rabbits.
Assay Tips and Optimization
Since this was his first time using xMAP Technology, Azoulay and his team carefully followed the recommended protocols. In the webinar, he did offer one additional suggestion for fellow users of the platform: the use of TCEP (Tris-(carboxyethyl) phosphine hydrochloride) as a reducing agent was helpful for breaking disulfide bonds in the protein conjugation process. The scientists found that maleimide-modified beads led to reaction problems, so they incorporated a thiol molecule to deactivate the maleimide function and provide better signal patterns.
Assay optimization steps included testing chemical spacers and improving the buffer. Individual assays were tested before developing the multiplex assay, which required a study of cross-reactivity of the antibodies after individual results proved they were successful for the proteins of interest. Validation of the multiplex assay showed no matrix effects and strong recovery of the proteins.
With the assay in hand, Azoulay has been testing biomarkers throughout the life cycle of the gammarids and studying the impact of toxin exposure on endocrine disruptors. His team is also looking at expanding the number of biomarkers used, as well as rolling out multiplex assays for other applications they study.
Resources
- Getting Started with xMAP® Technology [Video]
- Browse 1,200+ Partner Kits with xMAP® Kit Finder [Online Tool]
- xMAP® Cookbook to Design Your Own Assays [Download]
- View the xMAP® Webinar Blog Series [Video]
