Preparation and application of an immunoaffinity column based on an antibody with strong affinity and packing material with good stability

Abstract

This article describes the production of an anti-citrinin antibody that showed a high affinity constant (Ka) of 6.28 × 10⁹ and good tolerance to organic solvent and low pH, the synthesis of a Cu (II)-embedded polymer that showed strong binding with this antibody and the preparation of packing material for an immunoaffinity column (IAC) that show good stability. Most of the IACs reported either use harsh elution conditions and are used only once or use gentle elution conditions and are reused many times. Here, through the combined use of a strong-affinity antibody and packing material with good stability, high recoveries during clean-up and yet simultaneously good stability of the IAC were successfully achieved. Under optimised conditions of 80% methanol (pH 3), the IACs were used to clean-up the extracts of Monascus colour and red yeast rice samples, followed by HPLC detection. The recoveries of citrinin from spiked samples at levels of 50-200 μg kg⁻¹ were in the range of 84-97%.

Aflatoxin-specific Monoclonal Antibody Selection for Immunoaffinity Column Development

“…The initial criteria for selection of the antibody-producing hybridoma clones is specificity, where the produced antibodies should be able to interact with free AF isotypes but not with proteins used in immunization or other mycotoxins. Antibodies were evaluated for solvent tolerance and extract cross-reactivity where high solvent tolerance may also facilitate further studies leading to reusable IACs by the optimization of elution conditions [33]. Suitable candidates were tested for their toxin-binding performance in food extracts and their affinity towards AFs.…”