New Hapten Synthesis, Antibody Production, and Indirect Competitive Enzyme-Linked Immnunosorbent Assay for Amantadine in Chicken Muscle

Two haptens were synthesized by introducing spacer arms of different lengths and structures on the free amino of amantadine (AMA). 4-(chlorosulfonyl) benzoic acid (CSBA) was reacted with AMA to produce hapten AMA-CSBA, and succinic anhydride was reacted with AMA to produce hapten AMA-HS. And then, the two haptens coupled with bovine serum albumin (BSA) were prepared as immunogens for the production of monoclonal antibody (mAb). High-sensitive mAbs were obtained and characterized by using indirect competitive enzyme-linked immnunosorbent assay (icELISA). The best antibody was obtained in a heterologous icELISA with an IC50 value of 0.84 and 0.41 ng/mL for AMA and rimantadine, respectively, and negligible cross-reactivity (CR) with memantine and ribavirin. An icELISA was developed to detect AMA residue in chicken muscle. The limit of detection (LOD) was 1 mu g/kg in spiked chicken muscle and the recoveries ranged from 93.6 to 116.9% with the coefficient of variations (CVs) below 9.5%. Good correlation between icELISA and HPLC-MS/MS results in the incurred tissues demonstrated the reliability of the developed icELISA. These results indicated that the new antibody was sufficiently sensitive and specific for the detection of AMA in real samples.