An immunochemical method for simultaneous analysis of cross-reacting analytes is presented. We demonstrate the general principle using triazine herbicides as the model system. The analysis is based on a combination of individual enzyme immunoassays (immunoarray) for triazine herbicides using antibodies with different cross-reactivity patterns towards the selected analytes. The assay signals obtained can be mathematically evaluated to estimate concentrations of each analyte out of a ternary or quaternary mixture. The mathematical model utilizes an extension of the empirical four parameter log-logistic fit. Using mono-and polyclonal antibodies it was possible to quantify the four analytes atrazine, simazine, cyanazine, and prometon in the low to sub-ppb range simultaneously.

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An antibody for simazine and atrazine has been developed that exhibits low cross-reactivity to propazine relative to most atrazine antibodies heretofore evaluated. The cross-reactivities obtained in an enzyme-linked immunosorbent assay were 100 ± 4% for simazine, 76 ± 9% for atrazine, and 12.6 ± 1.3% for propazine. This was achieved by immunizing rabbits with the hapten 6-[[[4-chloro-6-(methylamino)]-1,3,5-triazin-2-yl]amino]hexanoic acid coupled to keyhole limpet hemocyanin. The influence of tracer hapten structure on the assay sensitivity was investigated in two competitive formats. The performance of the assay with respect to pH differences and ionic strength was also examined. The lowest IC50 values achieved for simazine were in the 0.1 μg/L range, with the limit of quantitation being 50 ng/L. Spike−recovery studies in tap and ground water as well as analysis of crude ground water samples show the usefulness of this sensitive antibody for simazine detection. Keywords: Immunoassay; enzyme-linked immunosorbent assay (ELISA); simazine; atrazine; propazine; triazine herbicide

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We demonstrate the construction of an immunoarray to categorize, identify and quantitate different triazine herbicides or their environmental metabolites in water. In a first step cluster analysis is used to examine the performance of a small subset of antibodies out of a larger library, and hence to select a small array that is capable of categorizing the triazine herbicides into different groups. At the 1 ppb level it is possible to categorize an analyte as a chloro-s-triazine, a hydroxy metabolite, or as a methoxy /methylthio substituted triazine when using only 2-4 antibodies. At higher concentrations even the identity of the triazine can be determined with the same number of antibodies. A selected combination of antibodies is then used to identify and quantify Unknowns by comparing theii~unoassay responses to an array of calibration curves and usin~the maximum likelihood criterion.