环丙沙星人工完全抗原的制备与鉴定

分别采用碳二亚胺法和氯甲酸异丁酯法制备环丙沙星的牛血清白蛋白与卵清白蛋白载体的人工完全抗原CPFX-BSA和CPFX-OVA.人工完全抗原经SDS-PAGE凝胶电泳初步鉴定后,考马斯亮蓝染色法测定蛋白质含量分别为1.24和0.58 mg.mL-1,将偶联抗原稀释至一定浓度,紫外分光光度法测其波形,根据偶联前后波形及吸收峰的变化鉴定偶联物分子量大于载体蛋白,证实CPFX与BSA和OVA偶联成功,根据游离半抗原、载体蛋白和完全抗原偶联物各自的紫外吸光度、摩尔吸光系数,初步定量计算出偶联结合比分别为7.2∶1和6.4∶1.质谱法测定CPFX-BSA和CPFX-OVA分子量分别为6.895×104和4.586×104,推算偶联比分别为5.6∶1和4.2∶1,进一步证实了偶联比和人工抗原制备成功.制备的人工完全抗原CPFX-BSA和CPFX-OVA可分别作为免疫抗原和包被抗原,为环丙沙星单克隆抗体的制备以及环丙沙星快速检测试剂盒的研制奠定基础.

The synthesis and identification of the artificial ciprofloxacin antigen

The artificial antigen ciprofloxacin-bovine serum albumin(CPFX-BSA) and ciprofloxacin ovalbumin(CPFX-OVA) were synthesized by carbodiimide(EDC) method and isobutyl chloroformate method,respectively.The conjugations were preliminary identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE),then measured the protein concentration by Coomassie brilliant blue determination.The result showed that the protein concentration of CPFX-BSA was 1.24 and 0.58 mg·mL-1 for CPFX-OVA.According to the waveforms and the absorption peak changes of the conjugations by UV spectrophotometry,the molecular weight was larger than the carrier protein,which proved CPFX successfully conjugating with BSA and OVA.It was calculated that the conjugate ratio of CPFX-BSA was 7.2∶ 1 and 6.4∶ 1 for CPFX-OVA.The results of mass spectroscopy showed that the molecular weight of CPFX-BSA was 6.895×104 and 4.586×104 for CPFX-OVA.It was calculated that the conjugate ratio of them were 5.6∶ 1 and 4.2∶ 1,respectively.The mass spectroscopy analysis assured the conjugate ratio and the success in artificial antigens.This experiment took the CPFX-BSA as the immunizing antigen and the CPFX-OVA is served as the coating antigen,which built a solid material base for the establishment of monclone antibody and the rapid test kit of ciprofloxacin.