Introduction

Ochratoxin-A is a toxic metabolite produced by several molds of the Aspergillus flavus and Penicillium genera, including Aspergillus ochraceus. The fungal species has the potential to produce ochratoxin-A, a known nephrotoxin and carcinogen. It has been frequently detected in human foods and animal feed, mainly in cereal products, although a range of commodities has been reported to contain the toxin. In humans, exposure to ochratoxin-A has been linked with Balken endemic nephropathy (BEN), a chronic kidney disease associated with tumors of the renal system. In animals, impairment of renal function has been reported in swine. In turkeys and chickens symptoms included retarded growth, decreased feed conversion, nephropathy and mortality. Feed refusal has also been observed in turkeys. A decrease in egg production and shell quality was reported in both turkeys and chickens. The ochratoxin-A EIA is intended for the quantitative detection of ochratoxin-A levels in grains, cereals, coffee, and other commodities including animal feeds.

Principle of Ochratoxin-A Determination

The enzyme immunoassay for ochratoxin-A is based on the competition between the ochratoxin-A in the sample and the Ochratoxin-A-Horseradish Peroxidase conjugate, for binding to antibody directed against ochratoxin-A, coated onto microwells. The sample containing the ochratoxin-A, and the Ochratoxin-A-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. If no or small amount of ochratoxin-A is present in the sample more enzyme labeled ochratoxin-A will bind the antibody on the solid surface. On the other hand, if large or significant amount of ochratoxin- A is present in urine sample, less enzyme labeled ochratoxin-A will bind to the antibody, producing less color signal. Therefore, the intensity of the color developed is inversely proportional to the concentration of ochratoxin-A 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-Ochratoxin-A antibody.
• One vial of 0.9 ml containing 0 ng/ml of ochratoxin-A.
• Three vials of Positive Calibrators containing 0.75 ml each of 0.4, 1.0 and 4.0 ng/ml of ochratoxin-A.
• One bottle containing 10.5 ml of Ochratoxin-A-Horseradish Peroxidase (ORT-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

• Carefully add 25 μl of standard or samples to the bottom of each well.
• Add 100 μl of ORT-HRP conjugate to each well. Incubate at room temperature for 30 minutes.
• Wash microtiter wells 3 times with wash buffer.
• 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

Cross react with Ochratoxin-B (9%). Do not cross react with other toxins.

Ochratoxin A Inhibition Curve