DEVELOPMENT OF ELISA TECHNIQUE FOR THE ANALYSIS OF ATRAZINE RESIDUES IN WATER

Abstract

A highly sensitive enzyme immunoassay is described for the detection of atrazine residues in water. Atrazine derivative was conjugated to Bovine Serum Albumin (BSA) to obtain an immunizing antigen and to Horseradish Peroxidase enzyme (POD) to obtain a marker for immunoassay. The formation of these conjugations was confirmed by UV spectroscopy as well as by gel-electrophoresis. Polyclonal antibodies were raised in rabbits by immunization with an atrazine–BSA conjugate containing 29 atrazine residues per BSA molecule. An ELISA on microtitration plates was optimized with peroxidase–atrazine conjugate. The middle of the test (50% B/Bo) was found to be at 90 ng/l, which is well below the maximum concentration permitted by the EC guidelines for drinking water.

Detection limits for atrazine of about 1 ng/l could be reached. The assay did not require concentration or cleanup steps for drinking or ground water samples. Validation experiments showed good accuracy and precision. No cross-reactivities were shown by other s-triazines like terbutryn, ametryn, terbuthylazine, des-isopropylatrazine, and de-ethylatrazine except hydroxyatrazine. The latter was present at very low levels that can be calibrated/standardized before analysis or it may be considered as leftover residues of atrazine. Based on these results, it is suggested that this test can be applied to obtain fairly accurate results for atrazine concentration in water samples from different sources.

Keywords:

• ELISA
• Development
• Standardization
• Triazines
• Cross-reactivity
• Matrix effect

ACKNOWLEDGMENTS

We thank the International Atomic Energy (IAEA) for the grant. We thank NOVARTIS (Basel) for providing the relevant literature and standards for atrazine and its metabolites. We are grateful to the Plant Protection Institute, AARI, Faisalabad, for providing s-triazine standards.