| Ampicillin (AMP), a kind of β-lactam antibiotics, is a kind of hapten and a broad-spectrumantibiotic. AMP also has a role in promoting growth of animals, so in the process of breeding,ampicillin is often used as feed additives or used in the control of animal mastitis and other diseases,which causes ampicillin antibiotics residues in secretion of fluid and it would lead to do harm tohuman beings. Therefore, it is essential that to establish an accurate, rapid and convenient ampicillinresidue detection method.The first study is to prepare efficient ampicillin whole antigen, and to obtain a high affinity,high specificity of the monoclonal antibodies in mice immunized, and then to establish ampicillinELISA detecting techniques.(1)Firstly, ampicillin was coupled with carrier proteins bovine serum albumin (BSA),ovalbumin (OVA) and keyhole limpet hemocyanin (KLH) by method of physiology, EDC method,glutaraldehyde, NHS+EDC method, BS3method and SMCC method to prepare whole antigen. UsingUV scan method and SDS-PAGE gel electrophoresis to identify the couplings effect.(2)Artificial antigens were used to analysis of immunogenicity, among which the mouse③serum titer up to2x105. The best mouser is③of inhibition to standard AMP with100ng/ml whoseinhibition rate reached86.2%. The mouse③was selected to prepare monoclonal antibodiesaccording to Hybridoma Technology. And the last, three hybridoma cell line were screened,whichnamed2C3,4E5and4C7. The antibody of2C3hubridoma cell secrete had stable nature and highertiter, so2C3hybridoma cell was elected for prepareing ascites and monoclonal antibody. The pure2C3antibody’s protein concentration was9.51mg/ml, the titer of ascites was4×105, subtype wasIgG1,IC50value was9.3ng/ml and had no cross-reaction with the carrier protein BSA, KLH andantibiotics except penicillins.(3)The condition of indirect ELISA experiment were preliminary builded, and the sensitivityand detection limit were9.37ng/ml and0.82ng/ml. |
