Introduction

2,4-D, a member of the phenoxy family of herbicides, was the first successful selective herbicide developed. Its major uses in agriculture are on wheat and small grains, sorghum, corn, rice, sugar cane, soybeans etc. It is also used on rights-of-way, roadsides, non-crop areas, forestry, lawn and turf care, and on aquatic weeds.

Principle of 2,4D Determination

The enzyme immunoassay for 2,4D is based on the competition between the 2,4D to be assayed and the 2,4D-Alkaline Phosphatase conjugate, for binding to rabbit antibody directed against 2,4D, coated onto microwells. The sample containing the 2,4D, and the 2,4D-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 2,4D 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-2,4D antibody.
• One vial of 0.25 ml of Positive Calibrator containing 1 μg/ml of 2,4D.
• One bottle containing 10.5 ml of 2,4D-Alkaline Phosphatase (24D-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 25 μl of standard or sample to the bottom of each well.
• Add 100 μl of 24D-ALP conjugate to each well. Incubate at room temperature for 40 minutes.
• 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 types of pesticides.

2,4D Inhibition Curve