Preparation of antisera and development of a direct enzyme-linked immunosorbent assay for the determination of the antifouling agent Irgarol 1051

The development of an immunoassay to determine the antifouling agent Irgarol 1051 (2-methylthio-4-tert-butylamino-6-cyclopropylamino-s-triazine) in seawater samples is described. Three polyclonal antisera were obtained by immunizing with a hapten preserving important antigenic determinants such as the t-butyl and the methylthio groups of Irgarol. Protein conjugates were characterized by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). Several usable competitive immunoassays have been obtained by screening a battery of 11 enzyme tracers. The optimized immunoassay presents an I50 of 0.64 nM (0.163 μg/l) and a detection limit of 0.062 nM (0.016 μg/l). Cross-reactivity studies have demonstrated that the immunoassay is specific for Irgarol 1051. Other triazine compounds are only detected to a minor extent. Performance of the immunoassay is not affected by the high salinity content of seawater.