Introduction

Aflatoxins are produced primarily by some strains of Aspergillus flavus and Aspergillus parasiticus. There are four principle aflatoxins B1, B2, G1 and G2. Aflatoxin B1 is the most frequently encountered and the most toxic of the group. The primary products contaminated with aflatoxins include peanuts, corn, wheat, rice, cottonseed, copra, and nuts. Because aflatoxins are naturally occurring compounds, precautions must be taken to control the quality of the food and feed in which they may occur. Aflatoxins are potent liver toxins in all animals in which they have been tested and carcinogens in some species. The United States Food and Drug Administration has set the guidelines of aflatoxin level in animal feeds and human foods at 20 parts per billion (20 ppb).

Principle of Aflatoxin Determination

The enzyme immunoassay for aflatoxin is based on the competition between the aflatoxin to be assayed and the Aflatoxin-Alkaline Phosphatase conjugate, for binding to rabbit antibody directed against aflatoxin, coated onto microwells. The sample containing the aflatoxin, and the Aflatoxin-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 aflatoxin 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-Aflatoxin antibody.
• One vial of 1.2 ml Negative Calibrator containing 0 ng/ml of aflatoxin.
• Three vials of Positive Calibrator (0.9 ml) containing 0.5, 2.0 and 8 ng/ml of aflatoxin.
• One bottle containing 10.5 ml of Aflatoxin-Alkaline Phosphatase (AFX-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 AFX-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

Cross react with aflatoxin B2. aflatoxin G1, Do not cross react with other mycotoxins.

Aflatoxin Inhibition Curve