Development and Validation of an Indirect Enzyme Immunoassay for the Detection of the Herbicide Isoproturon in Water Matrices

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Abstract
An indirect enzyme immunoassay (EIA) for the detection of the phenylurea herbicide isoproturon is described. The specific antibodies did not cross-react with other structurally related compounds. The concentration of isoproturon that inhibits 50% of antibody-antigen binding (IC50) was 0.64 ng/mL. The sensitivities were 0.07 ng/mL (IC80) and 0.02 ng/mL (IC90) respectively, when the crude serum was used in the assay. Matrix effects were observed when river water samples were analyzed showing recoveries as high as 150%. The IC50 was increased to 0.81 ng/mL. To overcome these difficulties, a novel method of anatibody purification was developed to reduce the heterogeneity of the medium when the test was performed with complex surface water matrices. This technique involved the extraction of the specific anti-isoproturon antibodies from the crude anti-serum. The refined fraction gave an IC50 not higher than 0.29 ng/mL and an IC90 of 0.01 ng/mL, when assayed with river water samples. The method was validated using a HPLC procedure with a clean up step involving an immuno-affinity column using the same antibodies. Excellent correlation (r = 0.998) was obtained between HPLC and the EIA results when the refined antibody was used in the assay. The use of affinity purified antibodies as an effective procedure in reducing matrix effects was demonstrated.