Preparation And Estblished Primary Immunoassay Of Monoclonal Antibodies Against Amantadine

Amantadine(AMT)belongs to tricyclic amines and is the first antiviral to be used to suppress influenza virus.In livestock and poultry breeding,mainly for the early treatment and prevention of poultry and influenza,as well as the prevention of swine gastroenteritis,but the side effects caused by mental disorders and the increasing resistance of livestock consumers greater security risks.Therefore,China's Ministry of Agriculture 560 Bulletin has been banned amantadine and rimantadine and other antiviral drugs for livestock and poultry breeding industry,the United States FAD has also explicitly prohibit the use of such drugs in livestock and poultry breeding.Detection of amantadine residues can be used the traditional instrument detection method.Mainly by liquid chromatography,gas chromatography,liquid chromatography-tandem mass spectrometry,ultra-high liquid chromatography-tandem mass spectrometry,hydrophilic chromatography-tandem mass spectrometry,potentiometric titration.However,traditional instrumentation methods require not only expensive equipment,but also complex sample processing processes that require professional technical personnel to operate,and these detrimental factors make these detection methods unsuitable for on-site high-throughput sample testing.ELISA detection method based on antigen-specific antibody binding,with a high degree of selectivity and sensitivity,detection method is low cost,simple,efficient,flexible and easy to carry,suitable for rapid screening in the field of large quantities of samples.In this study,glutaraldehyde method was used to synthesize amantadine complete antigen.Connect bovine serum albumin(BSA)to the synthetic immunogen,link the ovalbumin(OVA)synthetic coating antigen.Balb/c mice were immunized with immunogen.A monoclonal antibody with strong specificity and high titer against amantadine was prepared by monoclonal antibody technique,enzyme-linked immunosorbent assay(ELISA)an d mouse ascites preparation.And an indirect competitive ELISA method for the detection of amantadine was established.1.Synthesis and identification of AMT complete antigen.AMT belongs to the tricyclic amine and is an amino derivative of saturated tricyclodecane.As the homobifunctional cross-linker,the two aldehyde groups of glutaraldehyde can form Schiff s base with the primary amino groups on the hapten and the carrier protein.The two substances were linked by a five-stranded bridge to synthesize the complete antigen.Artificial antigens were initially judged successfully by UV-scanning method.Finally,complete antigen by immunization,fusion and ELISA assay,indicating that the preparation of two antigens was successful.The protein concentrations of AMT-BSA and AMT-OVA were 3.2mg/mL and 3.4mg/mL measured by BCA method.2.Preparation of monoclonal antibodies against AMTImmunization of female BALB/c mice using conventional immunization regimens,AMT-BSA as immunogen,AMT-OVA as a coating antigen.On the seventh day after the fifth immunization,the blood titer of blood collected from mouse tail vein was as high as 1:16000.Using cell fusion techniques,ELISA screening methods and limited dilution methods,two strains of hybridoma cell lines that were able to secrete anti-AMT monoclonal antibodies were obtained.They were named 1D11 and 2F9 respectively.Hybridoma cells were injected into the mouse peritoneum to prepare ascites containing monoclonal antibodies.Ascites protein concentrations were 20.7mg/mL and 25.3 mg/mL,respectively.Titers of the monoclonal antibodies were 32000,128000.Purification of ascites was performed using a HiTrap Protein G HP purification column.The results of the cross-experiments showed that the antibody had almost no cross-reaction with adamantane,ribavirin,morpholine guanidine,amoxicillin and ceftiofur,the cross-reactivity with rimantadine hydrochloride was 15%and the inhibition rate for AMT was 100%.3.The establishment of ELISA detection methodEstablishment of Indirect Competitive ELISA method for detection of Amantadine residues in chicken based on purified 2F9 monoclonal antibodies.The best dilution concentration is 1:4000 and the best working concentration of purified 2F9 antibody is 1:64000.The standard curve of R to antibody-antigen reaction was prepared with the linear equation y=0.3785x-0.2179(R2=0.9925),the linear range was 10-500ng/mL.IC50=77.83ng/mL,LOD = 5.98ng/mL,LOQ=54.78ng/mL.4.The addition and reclamation test of AMT in the chickenThe addition and reclamation test of AMT was carried on in the chicken.Chicken samples need to be pretreated before they can be used for experimental determination.The experimental results show that the matrix interference of the sample disappears when the treatment sample of chicken was diluted more than 16 times.In the range of 10?1000 ng/mL,the standard equation was y = 0.3377x-0.1658(R2=0.9901),IC50=92.67ng/mL,LOD=6.74ng/mL and LOQ=132.82ng/mL.When adding 10,500,900ng/mL AMT to the chicken,the recovery ratio was between 83.4%to 102.3%.