Synthesis Of Cefotaxime Immunogen And Study Of It’s Monoclonal Antibody

Objective: Because of the Obvious effect of cefotaxime sodium(CTX) on the treatmentof Escherichia coli, pseudomonas aeruginosa, poultry Salmonella and other gramnegativebacteria, more and more of the veterinary drug production enterprises and farmers use thecefotaxime sodium as animal treatment. It is necessary to establish a rapid and effectivedetection method for the supervision of reasonably application. This paper aims to synthesisthe immunogen of cefotaxime sodium and explore cefotaxime sodium monoclonal antibodiesto create the necessary conditions for the establishment of immunological detection methods.Synthesis and identification of cefotaxime immunogen: CTX was linked to bovine serumalbumin(BSA) and ovalbumin(OVA), by carbodiimide(EDC) and glutaraldehyde(GA) methodrespectively, and synthesized complete antigen CTX-BSA, CTX-OVA. Using ultraviolet abso-rption spectrum and gelelectrophoresis to identify bis-complete antigen. The displacement ofcharacteristic absorption peak obviously in compare with CTX-BSA,CTX-OVA and CTX,BS-A,OVA by using ultraviolet absorption spectrum,which indicate that is a Successful experime-nt.Compared the atlas of gel electrophoresis, CTX-BSA moved more slowly than BSAobviously，so does the CTX-OVA.It is Indicate that is a Successful experiment again.The immunogenicity evaluation of cefotaxime immunogen: Take the CTX-BSA whichsynthesised by EDC as immunogen on BALB/c mouse immunity. After4times immunization,detect the serum titer with Indirect ELISA method. Both of the two mous serum titer are over1:100,000. It is shown that the immunogen with good immunogenicity.Stduy of cefotaxime monoclonal antibody:1. Preparation of hybridoma cell:By the polyethylene glycol (PEG)4000mediated, takethe higher titre immune mouse spleen and SP-2myeloma cell fusion.2/3hybridoma cellcryopreservation and the other1/3fusion cells cultured by HAT-1640selective cell culturemedium. Observed the hybridoma cell after8days, with cell growth, suggesting that cellfusion is successful. Use indirect ELISA to detect, screened positive hybridoma cells.2. Screening of monoclonal cell lines: Take the screened positive cell amplificationculture, part reservation. At the same time clone the logarithmic growth phase of positive cellby limited dilution method(repeat3times). The indirect ELISA tests show, screened out threestrains of strong positive CTX monoclonal antibody cell. 3. Preparation of high titer antibody: Take12BALB/c mouse intraperitoneal injection ofparaffin oil, five days after intraperitoneal injection of monoclonal antibody cell.Through8~12days expanding culture in the mouse peritoneal cavity, according to the mouse livingstate take ascites at sterile condition.Each of the three monoclonal cell prepared about8mLascites successfully.