Extending the Working Range of Immunoanalysis by Exploitation of Two Monoclonal Antibodies

Abstract

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.

A membrane-based immunosensor for the analysis of the herbicide isoproturon

“…However, noticeable cross-reactivities were noted for both assays with various isopropylphenyl amino compounds. Combination of this rat monoclonal antibody with a less sensitive antibody (test-midpoint of 5.5 g L −1 ) allowed development of a test with an extended working range [10]. The same group also developed a series of ELISA tests for common herbicides for application within the limits given by the Water Framework Directive [11].…”

Improving an Immunoassay Response to Related Polychlorinated Biphenyl Analytes by Mixing Antibodies

“…We also describe in detail the models we developed and employed to predict assay response curves for mixed antibodies and to predict optimum antibody mix ratios. We are aware of other work describing use of mixed antibodies to increase the working range of an immunoassay , but believe this is the first description of controlled mixing of antibodies to create a generic immunoassay able to respond to a class of analytes.…”