金葡萄5型荚膜多糖单克隆抗体制备及ELISA方法建立
发布时间：2019-07-04 17:37
【摘要】： 荚膜多糖(Capsular polysaccharide,CP)是存在于细菌表面的一类特殊物质,是细菌的致病因子之一。对于金黄色葡萄球菌(Staphylococcus aureus,S.aureus)来说,荚膜多糖有11种血清型,其中5型荚膜多糖(CP5)是主要的血清型,它可以使细菌长期定居在粘膜和内皮表面,一旦发生感染,荚膜多糖通过抗吞噬作用,增强了金葡菌的毒力。金葡菌可以引起包括奶牛乳腺炎在内的多种疾病,金葡菌感染一旦发生,抗生素治疗效果并不明显,因此建立一种能快速、准确的区分金葡菌荚膜多糖血清型的诊断方法对预防和治疗金葡菌性疾病是十分重要的。 本实验利用化学试剂十六烷基三甲基溴化铵(CTAB),提取、纯化出CP5157.47mg,纯度为72.48%,并且用CP5与胎牛血清白蛋白偶联,制备了人工完全抗原。采用常规免疫方法,免疫BalB/C小鼠。将免疫后的BalB/C小鼠脾细胞与SP2/0细胞进行融合,进而筛选、克隆,共获得能稳定分泌抗CP5的单克隆抗体杂交瘤细胞2株,分别命名为2F3和5E8。其中2F3分泌抗体亚类为IgG1,5E8分泌的抗体为IgG2a。在对单抗的效价、相对亲和力等进行分析的基础上,选择单抗2F3用于建立CP5的间接竞争ELISA检测方法,对间接竞争ELISA方法进行优化后,建立了标准曲线,其标准曲线的回归方程为y=-1.6629x+4.9697,相关系数R2=0.9901,检测范围为62.5～4000ng/ml,最低检测限为62.5ng/mL。CP5间接竞争ELISA的建立为金葡菌性疾病的快速诊断以及金葡菌荚膜多糖的流行病学调查奠定了物质基础。
文内图片：
图片说明：电镜下的血清5型金葡菌Figure1.1Type5CapsularPolysaccharidesofStaphylococcusAureusbytransmissionelectronmicroscope
[Abstract]:Capsule polysaccharide (Capsular polysaccharide,CP) is a kind of special substance which exists on the surface of bacteria and is one of the pathogenic factors of bacteria. For Staphylococcus aureus (Staphylococcus aureus,S.aureus), there are 11 serotypes of capsula polysaccharide, among which type 5 capsula polysaccharide (CP5) is the main serotype, which can make bacteria settle on the surface of mucous membrane and endothelial for a long time. Once infection occurs, capsule polysaccharide enhances the virulence of Staphylococcus aureus by anti-phagocytosis. Staphylococcus aureus can cause a variety of diseases, including mastitis in dairy cows. Once Staphylococcus aureus infection occurs, the effect of antibiotics is not obvious. Therefore, it is very important to establish a rapid and accurate diagnostic method to distinguish Staphylococcus aureus capsule polysaccharide serotypes for the prevention and treatment of Staphylococcus aureus. In this experiment, the purity of CP5157.47mg, was 72.48% by (CTAB), extraction with cetyltrimethylammonium bromide, and the artificial complete antigen was prepared by coupling CP5 with fetal bovine serum albumin (FSA). BalB/C mice were immunized by routine immunization. After fusion of immunized BalB/C mouse spleen cells with SP2/0 cells, two hybridoma cells, named 2F3 and 5E8, were obtained, which could stably secrete anti-CP5 monoclonal antibodies. Among them, the antibody secreted by 2F3 is IgG2a., which is secreted by IgG1,5E8. On the basis of analyzing the titer and relative affinity of monoclonal antibody, the monoclonal antibody 2F3 was selected to establish the indirect competitive ELISA detection method of CP5. After optimizing the indirect competitive ELISA method, the standard curve was established. The regression equation of the standard curve was y 鈮，
本文编号：2510089
文内图片：
图片说明：电镜下的血清5型金葡菌Figure1.1Type5CapsularPolysaccharidesofStaphylococcusAureusbytransmissionelectronmicroscope
[Abstract]:Capsule polysaccharide (Capsular polysaccharide,CP) is a kind of special substance which exists on the surface of bacteria and is one of the pathogenic factors of bacteria. For Staphylococcus aureus (Staphylococcus aureus,S.aureus), there are 11 serotypes of capsula polysaccharide, among which type 5 capsula polysaccharide (CP5) is the main serotype, which can make bacteria settle on the surface of mucous membrane and endothelial for a long time. Once infection occurs, capsule polysaccharide enhances the virulence of Staphylococcus aureus by anti-phagocytosis. Staphylococcus aureus can cause a variety of diseases, including mastitis in dairy cows. Once Staphylococcus aureus infection occurs, the effect of antibiotics is not obvious. Therefore, it is very important to establish a rapid and accurate diagnostic method to distinguish Staphylococcus aureus capsule polysaccharide serotypes for the prevention and treatment of Staphylococcus aureus. In this experiment, the purity of CP5157.47mg, was 72.48% by (CTAB), extraction with cetyltrimethylammonium bromide, and the artificial complete antigen was prepared by coupling CP5 with fetal bovine serum albumin (FSA). BalB/C mice were immunized by routine immunization. After fusion of immunized BalB/C mouse spleen cells with SP2/0 cells, two hybridoma cells, named 2F3 and 5E8, were obtained, which could stably secrete anti-CP5 monoclonal antibodies. Among them, the antibody secreted by 2F3 is IgG2a., which is secreted by IgG1,5E8. On the basis of analyzing the titer and relative affinity of monoclonal antibody, the monoclonal antibody 2F3 was selected to establish the indirect competitive ELISA detection method of CP5. After optimizing the indirect competitive ELISA method, the standard curve was established. The regression equation of the standard curve was y 鈮，
本文编号：2510089
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