AFB₁–AP Conjugate for Enzyme Immunoassay of Aflatoxin B₁ in Corn Samples

Abstract

This article describes the conjugation between aflatoxin B₁ (AFB₁), one of the major mycotoxins, and alkaline phosphatase (AP), one of the most used enzymes for immunoassays. In addition, an application of the ELISA method for aflatoxin B₁ determination in corn is presented. Three AFB₁–AP conjugates in different toxin–enzyme ratios were prepared and tested. The ELISA results, developed with the most effective conjugate obtained, showed a satisfactory working range between 2.4 and 4000 ng of toxin/g of corn. The detection limit was 2 ng/g in corn samples, and recoveries ranged from 105 to 120%.

Keywords:

• Aflatoxin B₁ (AFB₁)
• alkaline phosphatase (AP)
• conjugate preparation
• corn samples
• direct competitive ELISA

Acknowledgments

The authors gratefully acknowledge the projects AFLARID and EUROLAB for financial support and Prof. Mariano Venanzi (Physic Chemistry Laboratory of “Tor Vergata” University, Italy) for the fluorescence measurements.

Notes

^a Against 100 U/ml of native activity.

^a CR percentage is determined as IC₅₀(AFB₁)/IC₅₀ (test compound) × 100; IC₅₀ is the concentration of the test compound that inhibits 50% of color.

^a Three days, and corresponding four measurements for each day.

^a Each value is a mean of 12 measurements (3 replicates in 3 different days; each replicate is the mean of 4 measurements); the CRM is a mean of 4 measurements.