Preparation Of A Monoclonal Antibodie And Establishment Of An Enzyme-Linked Immunosorbent Assay For The Determination Of Amantadines In Edible Chicken Tissues

Amantadines and ribavirin commonly used as human medicine are widely applied to the prevention and treatment of viral diseases in poultry.Based on a large number use of antivirals in food-producing animals may result in virus resistance,enabling the lose of medicine resources for human viral diseases,thereby the US FDA and the Chinese Ministry of Agriculture have issued the ban of these two drugs in animals in 2005,especially in food animals.Therefore,it will be of great significance to strengthen the research of these two types of drugs residue detection methods in edible animal tissues.Currently,the main method for detection of these drugs residues in animal edible tissues is instrument methods.Although instrument methods have high accuracy and precision,but they are expensive,often require complex sample cleanp and sophisticated laboratory equipment as well as highly trained operators.Then instrument methods are unsuitable for high sampling frequency or rapid assessment of results.Enzyme-linked immunosorbent assay(ELISA)methods,with high sensitivity,simplicity,and cost-effectiveness,are suit for screening large numbers of samples.ELISA for detecting residues of amantadines and ribavirin have not been reported.In this study,through designing and synthesizing the haptens,synthesizing and identifying the antigens and utilizing monoclonal antibody preparation technology,we obtained one monoclonal antibody which can recognize amantadine and rimantadine.By optimizing sample pretreatment methods,ensuring the performance parameter of limit detection,accuracy and precision,preliminarily established an indirect competitive ELISA that could detect amantadine drugs residue in edible chicken tissues.The main results were as follows:1.The synthesis of hapten and complete antigen: Adamantane,rimantadine,1-Adamantoic Acid and 5-carboxyl-2-ketone of adamantane as a raw material synthesized four haptens with different connection arms or sites,and they were identified successfully by mass spectrometry.Then linked to keyhole limpet(KLH),albumin human serum(HSA)and bovine serum albumin(BSA)by carbodiimide method,N,N dicyclohexyl carbon imine method and glutaraldehyde method,and finally six kinds of complete antigens were prepared successfully through UV spectrum.2.Screening of mice serum: Female Balb/c mice were immunized with the antigens,the antisera titers and specificity of mice were monitored by ELISA through the heterologous and homologous assay format.It was best in both of sensitivity and cross-reactivity when using AMA-HS-DCC-KLH and RMA-EDC-BSA,the antisera colud identify amantadine and rimantadine.By monoclonal antibody technology,we obtained one hybridomas line named AMA/2G3.3.Preparation of monoclonal antibody AMA/2G3: The average chromosome number of the monoclonal hybridoma line AMA/2G3 is 102.1.By ascites tumor,we prepared monoclonal antibody,and the MAb had an Ig G1 subclass.A ci ELISA for detecting the residue of AMA was established by optimizing conditions.The results showed that the best coating concentration of AMA/2G3 MAb was 1 μg/m L,and the best dilution was1:38000.A good linearity was achieved over a concentration range of 5 to 80 μg/L,and IC50 value was 15.83±0.44 μg/L.The inter-assay and intra-assay coefficient of variation were below 15%.The sensitivity of ci ELISA was 2.479 μg/L.The cross-reactivity with RMA was 70.67%.4.Determination of tissue samples: For a spiking study of chicken and chicken liver,samples were spiked with AMA at three different levels(5,10 and 20 μg/kg).We developed rapid extraction methods with acetonitrile and PBS,and the recoveries of spiked samples of AMA were 68.85%-105.26%.The inter-assay and intra-assay coefficient of variation were less than 15%.The results indicated the ELISA method had good accuracy and reproducibility.5.Compared with LC-MS/MS,the ELISA results correlated well,indicating the reliability of the test we developed.Besides,two new RIB haptens with different connection sites were prepared by alkaline hydrolysis and succinic anhydride.Then analogue adenosine and the new haptens were linked to KLH,HSA,BSA,finally we prepared three kinds of complete antigens.The antisera from groups of ribavirin had high titers but no specificity,so we did not obtaine monoclonal antibody of ribavirin.In this study we obtained one antibody,which could detect amantadine and rimantadine and established a simple and rapid extraction method for AMA in chicken and chicken liver.This study provided a theoretical basis for new determination technology research in detecting antiviral agents and had well academic value and wide application prospect.