Introduction

The herbicide paraquat (1,1’-dimethyl-4,4’-bipuridinium ion) is a potent herbicide, useful for the control of both terrestrial and aquatic weeds. It was also used as preharvest desiccant and defoliant. The use for these purposes requires a sensitive method for the estimation of residues in various crops, soil and water. Estimation of the concentrations of paraquat in biological fluids is a useful diagnostic and in cases of poisoning. It has been shown that paraquat concentrations in human plasma can be estimated by enzyme immunoassay and that the results so obtained correlate well with those of other methods.

Principle of Paraquat Determination

The enzyme immunoassay for Paraquat is based on the competition between the paraquat to be assayed and the Paraquat-Horseradish Peroxidase conjugate, for binding to rabbit antibody directed against paraquat, coated onto microwells. The sample containing the paraquat, and the Paraquat-Horseradish Peroxidase 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 paraquat 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 anti-Paraquat antibody.
• One vial of 0.9 ml Negative Calibrator containing 0 ng/ml of paraquat.
• Three vials of Positive Calibrator (0.75ml) containing 0.1, 1.0 and 4.0 ng/ml of paraquat.
• One bottle containing 10.5 ml of Paraquat- Horseradish Peroxidase (PRQ-HRP) Conjugate.
• One bottle containing 10.5 ml of Tetramethylbenzidine (TMB) substrate
• One bottle containing 15 ml of 10xWash Buffer (PBS-Tween).
• One bottle containing 10.5 ml of Stop Solution, 3N HCl

Assay Procedure

• Add 25 μl of standard or sample to the bottom of each well.
• Add 100 μl of PRQ-HRP conjugate to each well. Incubate at room temperature for 30 minutes.
• Wash microtiter wells 3 times with wash buffer to remove the non-bound conjugate.
• Add 100 μl of TMB substrate to each well and incubate at room temperature for 15 min.
• Add 100 μl of stop solution to each well.
• Read absorbance at 450 nm using an ELISA reader.

Cross Reactivity

Do not cross react with aflatoxin, ochratoxin, zearalenone, deoxynivalenol and T2 toxin.

Paraquat Inhibition Curve