Introduction

Zearalenone is a Mycotoxin produced by Fusarium graminearum on corn, wheat and barley. Zearalenone induced hyperestrogenism in animals and can exert estrogenic effects on the mammalian reproductive system. The extensive biological effects of zearalenone require that its presence be routinely monitored in human foods and animal feeds. The zearalenone EIA is intended for the quantitative detection of zearalenone levels in grains and milk.

Principle of zearalenone Determination

The enzyme immunoassay for zearalenone is based on the competition between the zearalenone to be assayed and the zearalenone-alkaline phosphatase conjugate, for binding to rabbit antibody directed against zearalenone, coated onto microwells. The sample containing the zearalenone, and the zearalenone-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 zearalenone 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-zearalenone antibody.
• One vial of 0.9 ml Negative Calibrator containing 0 ng/ml of zearalenone.
• Three vials of Positive Calibrators containing 0.75 ml each of 1, 3 and 10 ng/ml of zearalenone.
• One bottle (#3) containing 10.5 ml of Zearalenone-Alkaline phosphatase conjugate (ZLN-ALP).
• 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 ZLN-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 mycotoxins.

Zearalenone Inhibition Curve