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Position: Home > Articles > 马波沙星单克隆抗体的制备及ELISA检测方法的初步建立 Animal Husbandry & Veterinary Medicine 2019 (7) 100-106

马波沙星单克隆抗体的制备及ELISA检测方法的初步建立

作  者：
赵贵山;林梦舟;李辛洁;宋炎博;侯珂;汝晓飞;王捍东
单  位：
扬州大学兽医学院
关键词：
马波沙星;人工抗原;ELISA;单克隆抗体
摘  要：
马波沙星(MBF)是一种氟喹诺酮类抗菌药.本试验利用碳二亚胺法合成马波沙星免疫原和包被原,经紫外分光光度法、SDS-PAGE和免疫学方法,鉴定人工抗原合成成功.通过免疫小鼠和单克隆抗体技术,获得2株可分泌马波沙星抗体的杂交瘤细胞株(5G2和7H5),并将建筑细胞注入小鼠的腹腔内,获得腹水型单克隆抗体.经过纯化和ELISA鉴定,结果表明5G2株单克隆抗体的效价较高,达1.28*105,其单抗亚型是IgG2b,轻链是Kappa.经ELISA方法优化了最佳封闭液和抗原抗体的最佳工作浓度,当MBF药物浓度在100~5 000 pg/mL时,有良好的线性关系,线性方程y=0.379 8x-0.661 3,R2=0.987 2,IC50=1.029 pg/mL,LOD=45.13 pg/mL,LOQ=322.59 pg/mL.
作  者：
ZHAO Guishan;LIN Mengzhou;LI Xinhao;Song Yanbo;HOU Ke;RU Xiaofei;WANG Handong;College of Veterinary Medicine, Yangzhou University;
关键词：
Marbofloxacin;;artificial antigen;;ELISA;;monoclonal antibody
摘  要：
Marbofloxacin(MBF) is a kind of fluoroquinolone antibiotic with a carboxyl. In the present experiment, the immunogen and coated antigen of MBF were synthesized using the carbodiimide method; and the synthesis was proved successful ussing ultraviolet spectrophotometry, SDS-PAGE analysis and the immunological method. Two strains of hybridoma cell lines(5 G2 and7 H5) secreting MBF monoclonal antibodies were obtained by immunization in mice and monoclonal antibody technology. The cells were then injected into the abdominal cavity of mice to obtain ascitic monoclonal antibodies which ascites were purified and determined by ELISA. The results showed that the monoclonal antibody of the strain of 5 G2 had a higher titer of 1.28 x10~5, and its subtype monoclonal antibody was IgG2 b with a light chain being Kappa. The best blocking liquid and the best working concentration of the antigen antibody optimized by ELISA showed that, when the MBF drug concentration was between 100 and 5000 pg/mL, a good linear relationship was present with a linear equation being y=0.379 8x-0.661 3, R~2=0.987 2, IC_(50)=1.029 pg/mL, LOD=45.13 pg/mL and LOQ=322.59 pg/mL.

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