Development Of A Biomimetic Enzyme-Linked Immunosorbent Assay Method For The Determination Of Methimazole Using Novel Molecularly Imprinted Films As Artificial Antibodies

Methimazole can promote the metabolism of protein, it has been applied illegally to animals’ feed to obtain more lean meat. Although MMZ has been used in the treatment of hyperthyroidism, the uncontrolled introduction of this and other thyreostats into the human’s food chain could have serious health implications.There was no report about using artificial antibody of ELISA to monitor methimazole.A novel molecularly imprinted polymer has been prepared using methimazole as template, methacrylic acid (MAA) as functional monomer, edma ethylene glycol dime-thacrylate (EGDMA) as cross-linker. The imprinted film was directly synthesized on the well surface of MaxiSorp polystyrene96-well plate in conjunction with molecular imprinting technology. The imprinted polymer was evaluated by static and kinetic adsorption experiments. The results showed that the imprinted sorbent exhibited good recognition, selective ability and offered a faster kinetics for the adsorption and desorption of the template methimazole. The saturated adsorption capacity (Qmax) of imprinted sorbent toward enrofloxacin was1.51mg well-1and the adsorption equilibrium time was40min.Based on the molecularly imprinted film as artificial antibody, we developed a sensitive direct competitive biomimetic enzyme-linked immunosorbent assay (BELISA) method. This BELISA method had a higher selectivity for methimazole, and competitive binding studies demonstrated various degrees of cross-reactivity with two chemically related compounds ranging from1.1%to1.3%. The IC50(calculated as the concentration giving50%inhibition of color development) and the detection limit values IC15under optimized experimental condition were70±4μg·L-1and0.9±0.04μg·L-1, respectively.To evaluate the usefulness of this method, the urine sample and pig sample, determined to be free of methimazole, were spiked with three levels of methimazole (50,100,150μg·L-1and10,20,μg·kg-1). At each concentration, good recoveries have ranged from90%to95%and77.0%-89.0%, respectively. It is proved that the detection of methimazole in animal-derived products is feasible in this method,. and a good agreement for methimazole in comparative analysis with that using HPLC.