Purpose: The purpose of this work was an alternative way to improve the recovery levels of OTA in overparticular matrices avoiding the pre-column treatment.
Methods: Fifty grams of the above samples were extracted with 70% methanol and after filtration the OTA 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 OTA-HRP for 5 minutes followed by washing steps and the addition of chromogen for 5 minutes followed by the addition of acidic solution. The OTA-HRP and the immunogens were produced by active ester biochemical conjugation, optimization of conjugates molarity and gel filtration purification.
Results: Although the immunoassay requires only 20% of extract, the sensitivity was increased 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 treatment.
Significance: This immunoassay is a very valuable tool in the OTA analysis without pre-column treatment, which optimizes the recovery percentage and is an effortless and rapid procedure.