Development and validation of a bullfrog-immunoaffinity column clean-up for citrinin determination in red yeast rice

The effects of serum protein and muscle protein of bullfrog (rana earesbeiana) on citrinin (CIT) adsorption were evaluated by analyzing high-performance liquid chromatography-fluorescence detection (HPLC-FLD), fluorescence emission spectra and fluorescence microscopy. A specific immunoaffinity column (IAC) for determination of the adsorption capacity of CIT was prepared, and the adsorption capacity was evaluated. From the results, groups of immunized supernatant and muscle protein binding CIT revealed strong binding capacities for CIT. Immunofluorescence images indicated that the antibodies exhibited stronger binding capacities for CIT in serum supernatant, and it was identified as an immunoglobulin M (IgM) pentamer. CIT treatment promoted light chain detachment of Ig and strengthened the binding of CIT to antibody. The recoveries of bullfrog-IAC ranged from 75.5% to 80.1% for CIT-spiked samples indicating that bullfrog serum proteins were specific to CIT with high affinity. Thus, bullfrog has a special immunity response to CIT, and antibodies could be obtained by amphibian immunization with hapten. The proposed bullfrog-IAC column would provide a new alternative approach for CIT clean-up from food samples.