Extending the working range of immunoanalysis by exploitation of two monoclonal antibodies

A newly established rat monoclonal antibody (mAb) for isoproturon, namely, IOC 10G7, is described. This mAb shows a standard curve for isoproturon in phosphate-buffered saline with a test midpoint of 5.5 +/- 1.8 microg/L (n = 15). In combination with the formerly developed mAb IOC 7E1, IOC 10G7 can be exploited to extend the working range for the analysis of isoproturon. Both antibodies were formatted into a competitive enzyme-linked immunosorbent assay (ELISA), using the same enzyme-tracer. MAb IOC 7E1 and mAb IOC 10G7 have different affinities for the target compound, but the signal-response curves of the single antibodies overlap. Cross-reactivity (CR) patterns of both antibodies are comparable, showing the highest CR for the metabolite 1-(4-isopropylphenyl)-3-methylurea (IOC 10G7, 9%; IOC 7E1, 19%). The system described here includes the combined, but individual, usage of both assays on one microtiter plate, as well as the strategy for mixing the two antibodies for the utilization in one assay. When standards are performed in Milli-Q water, the working range for isoproturon with the individual ELISAs using mAb IOC 7E1 is from 0.01 to 1 microg/L (test midpoint = 0.11 +/- 0.03 microg/L; n = 17) and with IOC 10G7, it is 1-100 microg/L (test midpoint = 10.3 +/- 1.6 microg/L; n = 32). The working range with mixed antibodies is usually on the order of 0.03-30 microg/L (test midpoint = 0.5 +/- 0.2 microg/L; n = 17). These strategies (mAbs individually and mixed) cover a range of 4 and 3 orders of magnitude, respectively. As a demonstration, water samples of different origins and an extract of mixed sediment were analyzed. The advantages of these strategies are discussed.