Optimization of Total Aflatoxin Recovery Levels in Overparticular Matrices Using a Twenty-Minute ELISA by Dilution Normalization and Two Incubation Steps

Introduction: There have been many problems with total aflatoxin recovery levels using most commercial competitive ELISAs for grains. Especially, corn germ, corn silage, cottonseed, ginger, paprika and walnuts require clean-up pre-columns because when extracted, overparticular matrices are formed. Extracts usually constitute one-third of the incubated mixture affecting negatively the HRP and competitive step. Although most immunossays run better after pre-column treatment, unsatisfactory recovery levels are avoidable.

Purpose: The purpose of this work was an alternative way to improve the recovery levels of total aflatoxin in overparticular matrices avoiding the pre-column treatment.

Methods: Fifty grams of the above samples were extracted with 70% methanol and after filtration the total aflatoxin was determined by a direct competitive ELISA with or without pre-column treatment. Micoplates were coated with a high affinity monoclonal antibody produced after mice immunisation, preparation of hybridomas, clones screening and protein G purification. The immunoassay had two incubation steps (in contrast with other tests), the first incubation of 20% extract and 80% HRP-free buffer mixture for 10 minutes followed by washing steps, the second incubation of aflatoxin-HRP for 5 minutes followed by washing steps and the addition of chromogen for 5 minutes followed by the addition acidic solution. The aflatoxin-HRP and the immunogens were produced by organic modification of hapten, active ester biochemical conjugation, optimization of conjugates molarity and gel filtration purification.

Results: The immunoassay requires only 20% of extract and the sensitivity remains in increased levels giving the maximum range of quantification. Due to increased dilution normalization of matrix and enzyme protection from methanol by using two incubation steps, the recovery levels were more acceptable without pre-column step.

Significance: This ELISA constitutes a very valuable tool in the total aflatoxin analysis that optimizes the recovery levels and is an effortless and rapid immunoassay without pre-column treatment.