Abstract: An indirect competitive ELISA (ic-ELISA) for the detection of amantadine residue in chicken muscle was mainly developed in this article, the hapten of amantadine was synthesized via reaction of N-(1-adamantylamino) carbamide and glyoxylic acid, and then conjugated to the carrier protein of keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) via active ester method to prepare immunogens and coating antigens. BALB/c mice were immunized with the immune antigen for antibody generation. subsequently, a specific amantadine monoclonal antibody was obtained. After optimized the ic-ELISA condition, the dilution of coating antigen and monoclonal antibody content were 1:8.00×104 and 122.50 μg/L, respectively, the IC50 of AMA was 0.69 μg/L, the limit of detection was 0.21 μg/L, the linear detection range was 0.07 μg/L～6.15 μg/L, the recoveries for chicken muscle were in the range from 101.69% to 108.71%, and the coefficients of variation of intra-assay and inter-assay were less than 10.57%. Furthermore, the results determined by ic-ELISA were very compatible with that of HPLC-MS (R2=0.97), which demonstrated that the established ic-ELISA has high accuracy and good reliability. Therefore, this ic-ELISA provided a valid detection method for amantadine in real chicken samples.

Key words: chicken muscle, amantadine, ic-ELISA, monoclonal antibody