Introduction

Atrazine is a widely used agricultural pesticide in the world. It provides the control of broad-leaved weeds and grasses in asparagus, corn, fruit orchard, and citrus groves. The widespread application, stability and relatively high solubility of atrazine in water allow it to leach from soil and be a relatively persistent environmental contaminant. United States Environmental Protection Agency (EPA) has set an enforceable Maximum Contaminant Level for atrazine at 1 part per billion (ppb).

Principle of Atrazine Determination

The enzyme immunoassay for atrazine is based on the competition between the atrazine to be assayed and the Atrazine-Alkaline Phosphatase conjugate, for binding to rabbit antibody directed against atrazine, coated onto microwells. The sample containing the atrazine, and the Atrazine-Alkaline Phosphatase conjugate, when added to the microtiter wells, compete for binding to a limiting number of antibody sites. After incubation, each well is rinsed in order to remove non-bound components. The bound enzymatic activity is then measured by the addition of a chromogenic substrate. The intensity of the color developed is inversely proportional to the concentration of atrazine in the sample. The concentration is calculated on the basis of a standard curve.

Reagents

• 96-well microtiter plate. Twelve strips of 8 detachable wells, coated with rabbit anti-Atrazine antibody.
• One vial of 0.5 ml Negative Calibrator containing 0 ng/ml of atrazine.
• One vial of Positive Calibrator containing 0.25 ml of 1μg/ml atrazine.
• One bottle containing 10.5 ml of Atrazine-Alkaline Phosphatase (ATZ-ALP) conjugate.
• One bottle containing 10.5 ml of p-Nitrophenyl Phosphate (pNPP) substrate.
• One bottle containing 15 ml of 10xWash Buffer (PBS-Tween).
• One bottle containing 5.5 ml of Stop Solution, 3N NaOH.

Assay Procedure

• Add 50 μl of standard or sample to the bottom of each well.
• Add 100 μl of ATZ-ALP conjugate to each well. Incubate at room temperature for 40 min.
• Wash microtiter wells 3 times with wash buffer.
• Add 100 μl of pNPP substrate to each well. Incubate at room temperature for 20 min.
• Add 50 μl of stop solution to each well.
• Read absorbance at 405 nm using ELISA reader.

Cross Reactivity

Do not cross react with other pesticides such as 24 D, Paraquat, Parathion, Glyphosate.

Atrazine Inhibition Curve