Synthesis Of Artifical Antigen Of Cefalexin And Preparation Of Monoclone Antibody For Cefalexin

Cefhalexin (Cefalexin,CEX) is a semisynthetic first-generation oral cephalosporin antibiotics, was first synthesized in1967, similar to the antibacterial spectrum and Cefhalothin, Cefhaloridine, and other cefhalosporin antibiotics, isclinical and veterinary clinical application of one of the most antibiotics. Excessive abuse of such antibiotics lead to enhanced bacterial resistance to antibiotics, the widespread presence of drug residues in agricultural and livestock caused a great deal of potentially harmful allergy crowd and crowd NA drugs. Around cause severe allergy cases continue to occur due to cephalexin, how cases of anaphylactic shock and even deaths.The People’s Republic of China Ministry of Agriculture revised and released on December24,2002,235Notice MRLs of veterinary drugs in foods of animal provisions in the beef kidney in the CEX maximum residue limits for1000μg/kg and in the beef in the CEX maximum residue limits for200μg/kg. With the continuous development of science and technology and the attention of the national government the cephalexin detection method has evolved. Commonly used detection methods by UV spectrophotometry, HPLC, LC-MS usage, photochemical fluorescence,near-infrared diffuse reflectance spectroscopy, fluorescence spectrophotometry, flow injection chemiluminescence methods. However, these methods requires not only complex and expensive instruments, and need to go through the tedious pre-treatment, it is difficult to achieve rapid and simple field testing requirements, to carry out the complex matrix of animal trace residue analysis, specificity, sensitivity, accuracy,analysis of cost and efficiency are the most important assessment indicators. Immunochemical technology, especially enzyme-linked immunoassay technique with high selectivity, high sensitivity and high-throughput features, just to meet the above requirements, will play an important role in in CEX rapid detection.In order to find a rapid, sensitive cephalexin residual immunological analysis.This study based on the analysis of cephalexin immunological characteristics, using monoclonal antibodies prepared cephalexin monoclonal antibodies. The main results are as follows:1. Synthesis and identification of the artificial antigen for CefalexinTwo complete antigens of CEX (CEX-BSA, CEX-OVA) were prepared by glutaraldehyde method. The characterization of hapten-protein conjugates were examined by ultraviolet spectrophotometry(UV) and sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) respectively. BALB/c mice were immunized with CEX-BSA, the titre of polyclonal antibody (pAb) was detected by indirect ELISA, the sensitivity and specificity of pAb was identified by blocking ELISA. According to UV scanning, the conjugation ratio of CEX to BSA and OVA was about21.7:1and14.6:1.SDS-PAGE results show that the BSA’s swimming faster than CEX-BSA, the CEX-BSA molecular weight greater than BSA, This further shows that CEX-BSA has been coupled with success.The titer of pAb for2#mouse is the highest, its IC50o was769.588ng/mL, cross reaction rate to carbadox was1.283%, and no cross reaction to other antibiotics. The high-titer, sensitive and specific anti-CEX pAb had been produced.2. Preparation of monoclonal antibodies and immunological characterization of CefalexinThe titre and sensitivity of polyclonal antibody was detected by indirect ELISA and blocking ELISA, so as to select the mouse used in cell fusion. CEXmAb was prepared by hybridoma technology. The titer, affinity, sensitivity, specificity and subtype of the mAb were characterized. Massive CEXmAb were induced from in vivo method. There hybridoma cell lines of1B12、3E6、2G5were screened for specificity to CEX, all the isotypes of the mAb were IgG1. The indirect ELISA titer of the mAb were1:4.8×102～1:1.28×103in supernatant,1:2.56×105of3E6in ascites, and the affinity constant(Ka) was7.5×1010L/moL, the mAb of3E6showed good sensitivity with IC50of77.52ng/mL to CEX. The rate of cross reaction of CEX mAb with cefradine was0.969%, and there was no cross-reactivity to other compounds. CEX mAb of high-titer, sensitivity and specificity had been generated, it is possible to establish immunoassay of CEX residues in animal food.