DEVELOPMENT AND VALIDATION OF AN INDIRECT ENZYME-IMMUNOASSAY FOR THE DETECTION OF THE HERBICIDE ISOPROTURON IN WATER MATRICES

An indirect enzyme immunoassay (EIA) for the detection of the phenylur ea herbicide isoproturon is described. The specific antibodies did not cross-react with other structurally related compounds. The concentrat ion 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. M atrix effects were observed when river water samples were analyzed sho wing recoveries as high as 150%. The IC50 was increased to 0.81 ng/mL. To overcome these difficulties, a novel method of antibody purificati on was developed to reduce the heterogeneity of the medium when the te st was performed with complex surface water matrices. This technique i nvolved the extraction of the specific anti-isoproturon antibodies fro m the crude anti-serum. The refined fraction gave an IC50 not higher t han 0.29 ng/mL and an IC90 of 0.01 ng/mL, when assayed with river wate r samples. The method was validated using a HPLC procedure with a clea n up step involving an immune-affinity column using the same antibodie s. 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.