Development and Validation of a Semi-quantitative Lateral Flow Device for Aflatoxin Detection in Corn and Nuts

Introduction: Aflatoxins are recognized as the most important group of mycotoxins. As secondary metabolites, they are synthesized by Aspergillus species (A. flavus and A. parasiticus) on agricultural produce grown under stressful conditions.  The mold is found in soil and on damaged or decaying grains, fruits or nuts.  There are several different types of aflatoxins, but B₁, B₂, G₁ and G₂ are the most common. Aflatoxin B₁ is considered the most toxic and carcinogenic substance produced in nature.  FDA has established action levels for the amount of aflatoxins allowed in food or feed to protect human and animal health. The lack of availability and simplicity of routine qualitative and semi-quantitative aflatoxin detection at storage/sorting facilities remains an essential problem in controlling aflatoxin contamination.

Purpose: To develop a semi-quantitative method for aflatoxin detection in different food matrices that is rapid, simple to use and does not require expensive equipment. 

Methods: A lateral flow device was developed for semi-quantitative aflatoxin detection based on a competitive inhibition immuno-assay.  For this purpose, high-affinity monoclonal antibodies against aflatoxin B₁ were obtained, conjugated to colloidal gold particles and used as a detection reagent in a lateral flow device.  For the capture reagent, aflatoxin-BSA was immobilized on a nitrocellulose membrane. To achieve semi-quantitative aflatoxin detection, different amounts of aflatoxin B₁-BSA were applied on several test lines. For the procedural control, goat anti-mouse IgG antibodies were immobilized downstream from test lines on the lateral flow device.

Results: :  A lateral flow test device was developed that allows users to determine aflatoxin concentration in a single step.  Levels of 5 ppb, 20 ppb and 100 ppb (B₁+B₂+G₁+G₂) are used as cut off levels. To validate the lateral flow method, 6 corn samples and 25 peanut samples spiked with different aflatoxin concentrations were tested and the results were compared with the gold standard method of quantitative HPLC.

Significance: A semi-quantitative method for aflatoxin detection was developed that allows, in a single step, determination aflatoxin levels at low (less then 5 ppb), middle (less then 20 ppb, but more than 5 ppb) or high range (more the 100 ppb) of contamination.