Abstract: An indirect competitive enzyme linked immunoassay (ic-ELISA) for the detection of amantadine residue in chicken muscle was developed in this paper. The hapten was synthesized via the reaction of N-(1-adamantylamino) carbamide with glyoxylic acid, and then conjugated to the carrier proteins keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) by the active ester method to generate an immunogen and a coating antigen, respectively. BALB/c mice were immunized with the immunoantigen for antibody generation. Subsequently, a specific monoclonal antibody against amantadine was obtained. The optimal dilution of coating antigen and antibody concentration were 80 000 and 122.50 μg/L, respectively. Under the optimized experimental conditions, the ic-ELISA showed an IC50 of 0.69 μg/L with a limit of detection (LOD) of 0.21 μg/L. The linear range was 0.07–6.15 μg/L, and the recoveries for spiked chicken muscle were in the range from 101.69% to 108.71% with coefficients of variation less than 15%. Furthermore, the results of ic-ELISA were highly correlated with those of high performance liquid chromatography-mass spectrometry (HPLC-MS) (R2 = 0.97), which demonstrated that the established ic-ELISA is of high accuracy and good reliability. Therefore, the ic-ELISA proved to be a suitable method for the rapid detection of amantadine in real chicken samples.

Key words: chicken muscle, amantadine, indirect competitive enzyme linked immunosorbent assay, monoclonal antibody