Preparation Of Monoclone Antibody And Establishment Of Immunology Rapid Test Method Of Olaquindox

Olaquindox is a chemosynthetic broad-spectrum antibacterial drug. As a derivative of quinoxaline-1, 4-dioxide with protein assimilation, it can be used to improve feed conversion rate,lean meat percentage so as to promote the growth of animals and prevent and treat bacterial infection. Not standardized use of olaquindox is not only directly against the body of the animal health, but also on human health through the food chain causing tremendous harm in the production practic. Along with technological development and national attention, its detection method has also developed continuously. Diferent analytical methods are currently used: electrochemical analysis, Spectrometry, Chromatography, LC-MS, IAS, etc. In order to find a rapid and sensitive means of immunological analysis to detect the Olaquindox residue, in this study, based on the analysis of molecular structure and immunogenicity of Olaquindox, the technology of monoclonal antibodies production was applied to prepare the monoclone antibody against Olaquindox and then assemble rapid ELISA kit and strip for OLA residual detection. The main contents and resuLts of this study were as follows:1. Synthesis and identification of the artificial antigen for OlaquindoxThe derivative of olaquindox, called as OLA-SH, was synthesized by succinic anhydride method. Two complete antigens of olaquindox (OLA-BSA, OLA-OVA) were prepared by active ester method (AE). The characterization of hapten-protein conjugates were examined by two methods, ultraviolet spectrophotometry(UV), sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) respectively. BALB/c mice were immunized with OLA-BSA, the titre of polyclonal antibody(pAb)was detected by indirect ELISA, the sensitivity and specificity of pAb was identified by blocking ELISA. The result of UV spectrum show that the largest artificial antigen absorption peak migration occurred, the conjugation ratio of OLA-HS to BSA and OVA was about 17.1:1and 12.4:1. SDS-PAGE results show that the BSA's swimming faster than OLA-BSA, the OLA-BSA molecular weight greater than BSA, This further shows that OLA-BSA has been coupled with success. The titer of pAb for 3# mouse is the highest, its IC50 was 58.923ng/mL, cross reaction rate to carbadox was 1.841%, and no cross reaction to other antibiotics. The high-titer, sensitive and specific anti-OLA pAb had been produced.2. Preparation of monoclonal antibodies and immunological characterization of OlaquindoxThe titre and sensitivity of polyclonal antibody was detected by indirect ELISA and blocking ELISA, so as to select the mouse used in cell fusion. OLAmAb was prepared by hybridoma technology. The titer, affinity, sensitivity, specificity and subtype of the mAb were characterized. Massive OLAmAb were induced from in vivo method. There hybridoma cell lines of 2B1,4F5,4E12,5B5 and 5E5 were screened for specificity to OLA, all the isotypes of the mAb were IgG1. The indirect ELISA titer of the mAb were 1:3.0×10~2 ~1:1.28×10~3 in supernatant, 1:5.12×10~5 of 4E12 in ascites, and the affinity constant(Ka) was 3.75×1010L/moL, the mAb of 4E12 showed good sensitivity with IC50 of 1.66ng/mL to OLA. The rate of cross reaction of OLAmAb with carbadox was 5.19%, and there was no cross-reactivity to other compounds. OLAmAb of high-titer, sensitivity and specificity had been generated, it is possible to establish immunoassay of OLA residues in animal food.3. Development of rapid determination of Difloxacin residue methodBased on the enzyme-linked immunosorbent assay principle, a competitive ELISA kit for determination of OLA (OLA-Kit) was developed with OLAmAb. The calibration curve of OLA-Kit with standard OLA inhibitor was typical sigmoid curve fitted to the four parameters logistic equation, the detection limit for OLA was 1ng/mL and IC50 was1.66ng/mL. The precision and accuracy of the assay as determined by inter-assay and intra-assay coefficient variation were below 15%. The recoveries from pig liver and pork were 77.6% and 79.68%, respectively, when 2, 10, 50, 100ng/mL OLA were spiked. The validity of OLA-Kit in 4℃was above six months.