马波沙星单克隆抗体的制备及ELISA检测方法的初步建立
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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1.28×10~5,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,R~2=0.987 2,IC_(50)=1.029 pg/mL,LOD=45.13 pg/mL,LOQ=322.59 pg/mL。
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.
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.
引文
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[2] 魏雷雷.牛奶中氟喹诺酮类残留量测定新方法的研究与应用[D].衡阳:南华大学,2010.
[3] Mahmood A H,Medley G A,Grice J E,et al.Determination of trovafloxacin and marbofloxacin in sheep plasma samples by HPLC using UV detection[J].J Phamaceut Biomed Anal,2012,62(6):220-223.
[4] 谢军.荧光分光光度法对马波沙星含量的测定[J].分析试验室,2012,31(07):94-96.
[5] 张伟,王兴,刘金萍,等.动物性食品中头孢氨苄残留检测研究进展[J].安徽农业科学,2016,44(19):84-86+196.
[6] 刘秀梵.A单克隆抗体在农业上的应用[M].合肥:安徽科技出版社,1994.
[7] 汝晓飞,张伟,王兴,等.马波沙星人工抗原的合成及其免疫原性的鉴定[J].畜牧与兽医,2018,50(04):66-71.
[8] 宋春美,李青梅,刘庆堂,等.喹乙醇单克隆抗体的制备及其免疫学特性的鉴定[J].核农学报,2010,24(04):777-783.
[9] 刘宣兵,滕蔓,张改平,等.新霉素单克隆抗体的制备及其免疫学特性鉴定[J].华北农学报,2009,24(04):80-83.
[10] 张伟.头孢氨苄单克隆抗体的研制及免疫学检测方法的初步建立[D].扬州:扬州大学,2017.