Preparation Of Monoclonal Antibody Against Amantadine And Its Application In Rapid Detection Of Poultry Meat

Amantadine（AMD）,a type of tricyclic amines,and is a human antiviral drug that was used for the prevention and treatment of influenza.AMD has also been used to prevent and treat bird flu,swine flu and gastroenteritis by farmers.However,AMD may cause severe viral resistance and do harm to humans by accumulating in the human body.Therefore,AMD has been forbidden as an antiviral drug in the veterinary drug by the former Ministry of Agriculture of China.In the second chapter,the immunogen of AMD-BSA and the coating antigen of AMD-OVA were synthesized by EDC/NHS mediated method.The mice were immunized with AMD-BSA,and the splenocytes from the mouse with high titer and inhibition ratio were fused with SP2/0 myeloma cells.After the fusion,five kinds of monoclonal cell lines that produced specific antibodies against AMD were obtained.The anti-AMD monoclonal antibodies were named J1C6,J2B3,J2C3,J4E5,and J5E2,respectively.The sensitivity and specificity of these antibodies were evaluated by Enzyme-linked immunosorbent assay（ELISA）.The results showed that the half maximal inhibitory concentration(IC₅₀)of J1C6 J2B3,J2C3,J4E5,and J5E2 were 1.28ng/mL,0.2 ng/mL,2.5 ng/mL,0.32 ng/mL,and 0.61 ng/mL,respectively.The antibody J2B3 in most sensitivity was selected for specificity experiments.Due to no cross-reactivity with rimantadine,1-adamantanic acid,ribavirin,acetaminophen,olaquindox,and danofloxacin,the antibody J2B3 had good specificity.In the third chapter,a method for the quantitative detection of AMD residue in chicken with colloidal gold immunochromatographic assay（CG-ICA）was developed based on antibody J2B3.Some key parameters were optimized and the results showed that the pH of CG solution,the labeling amount of antibody J2B3,the concentration of AMD-BSA,and the detection time were 7.0,0.5μg/mL,0.3 mg/mL,and 16 min,respectively.Under optimal conditions,the standard curve of CG-ICA was Y=0.592lg X+0.749（R²=0.975）with the linear range at 0.1-2.5 ng/mL.The limit of detection（LOD）was 0.077 ng/mL.The recovery rate of the method in chicken was 96.4%-123%while the CVs of intra and inter were 2.4%-5.8%and 5.9%-9.2%,respectively.In the fourth chapter,colloidal gold immunochromatographic test strip was prepared for the qualitative and rapid detection of AMD residues in poultry meat.Some key parameters were optimized and the results showed that the gold-labeled antibody concentration,the spray volume of gold-labeled antibody,the detection environment temperature,the detection time,and the sample volume of the analyte were 1.0μg/mL,5μL/cm,25°C,10 min,and 120μL,respectively.Combining pretreatment,the sensitivity of colloidal gold immunochromatographic test strip was 1μg/kg for the detection of chicken,duck,and goose.The method was rapid（1 h for 12 samples）,accurate,and could be used for on-site detection after detection of a lot of chicken,duck,and goose samples.The method has practical significance for the rapid detection of AMD residues in poultry meat.In summary,the quantitative and qualitative colloidal gold immunochromatographic assay were developed after preparing the highly sensitive and specific anti-AMD monoclonal antibodies for sensitive,accurate,and rapid detection of AMD residues in chicken,duck,and goose samples.It provides effective technical support for the rapid detection of AMD residues in poultry meat.