Purification of serum IgM in Acanthopagrus latus and preparation of rabbit sera anti-IgM--《Journal of Fishery Sciences of China》2008年01期
Purification of serum IgM in Acanthopagrus latus and preparation of rabbit sera anti-IgM
LIU Zhen-xing1,2,ZHANG Dian-chang1,SU Tian-feng1,GONG Shi-yuan2,JIANG Shi-gui1 (1.South China Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Guangzhou .Huazhong Agricultural University,Wuhan 430070,China)
The serum IgM of Acanthopagrus latus was purified by using Sepharose-6B gel filtration chromatography,Phenyl Sepharose FF hydrophobic interaction chromatography and rProtein A Sepharose affinity chromatography. The purified IgM obtained through the three methods was then compared. By the methods of Sepharose-6B gel filtration chromatography and Phenyl Sepharose FF hydrophobic interaction chromatography the IgM was obtained at the purity of only 56%. While through combination of Sepharose-6B gel filtration chromatography and Phenyl Sepharose FF hydrophobic interaction chromatography,we obtained a bit higher purity at about 69%. The purity reached approximately 89% when using rProtein A Sepharose affinity chromatography. However,the rProtein A Sepharose affinity chromatography suffered relatively high cost. Sepharose-6B gel filtration chromatography and Phenyl Sepharose FF hydrophobic interaction chromatography could be good choices for the purification of the IgM in terms of cost and performance. It was revealed that molecular weights of the heavy- and light- chains of IgM were 73.6 and 26.5kD,respectively. The IgM purified by rProtein A Sepharose affinity,chromatography was used to prepare rabbit anti-IgM sera. The antibody activity of anti-sera was tested by Western Blot and double agar diffusion.Indirect ELISA revealed that the anti-serum titer reached 1∶25 600. It is considered that the purified IgM and developed anti-sera provide a foundation for further studies in relation to fish immune response.
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(C)2006 Tsinghua Tongfang Knowledge Network Technology Co., Ltd.(Beijing)(TTKN) All rights reserved鲫鱼IgM单克隆抗体的制备及嗜水气单胞菌抗体ELISA检测方法的建立--《南京农业大学》2011年硕士论文
鲫鱼IgM单克隆抗体的制备及嗜水气单胞菌抗体ELISA检测方法的建立
【摘要】：嗜水气单胞菌(Aeromonas hydrophila,Ah)分布于世界各地,广泛存在于淡水、污水、淤泥、土壤和人类粪便中,对水产养殖及野生鱼、虾、蛙、甲鱼等生物有致病性,并可进一步通过水产动物和水产品感染人畜,造成人畜腹泻和败血症等。它们所致的鱼类等嗜水气单胞菌病具有流行广、发病快、死亡率高等特点,常常造成养殖渔业的巨大经济损失。因此,建立快速、特异、有效的检测和鉴定嗜水气单胞菌的方法显得尤为重要。
研究证实,鱼类在受到微生物、寄生虫等病原体感染或接受人工免疫后,均能产生由免疫球蛋白(Immunoglobulin,Ig)介导的特异性体液免疫反应。这些免疫球蛋白在鱼体的免疫防御中起着十分重要的作用。因此,制备高纯度的鱼类免疫球蛋白对于研究鱼类体液免疫应答规律、建立以免疫球蛋白为核心的病原快速诊断和鱼类血清学分析方法具有重要的意义。
采用饱和硫酸铵分步盐析结合Macro-prep high Q Cartridge阴离子交换柱以及SephacrylTM S-300凝胶柱层析提取纯化了非免疫鲫鱼血清IgM,免疫BALB/c鼠,制备免疫脾细胞,与SP2/0骨髓瘤细胞融合,建立了6株分泌抗鲫鱼血清IgM单克隆抗体(mAbs)的杂交瘤细胞株,分别命名为A1、C2、C3、F3a、F3b和F3c，Ig亚类分别是IgG1、IgG1、IgG2a、IgG1、IgG1和IgM。用间接ELISA检测,C3和F3a的细胞培养上清液的效价均为1×103,腹水效价为1×108～1×1011；A1、C2、F3b和F3c的细胞培养上清液的效价为1×101～1×102,腹水效价为1×103～4×106。用间接ELISA分析特异性,6株mAbs均为抗鲫鱼血清IgM特异性单克隆抗体。Western blotting表明,6株mAbs在蛋白分子量约79.2kDa处有特异性条带。运用HisTrap Protein G HP亲和层析柱纯化腹水并进行了HRP标记；同时将纯化好的腹水与CNBr活化的琼脂糖凝胶偶联,制备亲和层析柱,纯化鲫鱼血清IgM,SDS.PAGE检测表明,提纯的鲫鱼血清IgM纯度较高,其分子量为837kDa左右,重链分子量为79.2kDa左右,轻链分子量为25.5kDa左右。
为了监测免疫动物的抗体水平以及流行病学调查的需要,本试验以灭活Ah J-1为包被抗原,抗鲫鱼血清IgM单克隆抗体作为酶标二抗,建立检测鲫鱼IgM抗体水平的间接ELISA方法。经筛选确定,抗原最适工作浓度为107cfu/mL;血清最佳稀释度为1：160；抗原抗体最佳作用条件为37℃1.5h；酶标二抗最佳工作浓度和反应时间分别为1：200,37℃1.5h；底物的最佳反应时间为15min；血清样品OD450值≥0.208,且P/N≥2.1判为阳性,OD450值≤0.178为阴性；建立的间接ELISA检出下限为1：1280,高于玻片凝集试验的1：320；重复性试验和稳定性试验表明：血清的批内重复试验和批间重复试验的变异系数均小于10%,表明建立的间接ELISA方法具有良好的可重复性和稳定性。运用已建立的间接ELISA方法测定了鲫鱼不同免疫及感染时期血清中IgM抗体水平的动态变化,结果显示：免疫组首免7d未在鲫鱼血清中检测到IgM抗体,8-10d开始出现IgM抗体并呈上升趋势；30-40d血清中IgM抗体水平达到高峰；感染组感染后3d未在鲫鱼血清中检测到IgM抗体,8-10d开始出现IgM抗体并呈上升趋势；20-30d血清中IgM抗体水平达到高峰。
以灭活Ah J-1作为检测抗原,兔抗Ah J-1作为竞争抗体,利用竞争原理针对淡水鱼感染嗜水气单胞菌建立了一种快速的间接竞争ELISA检测方法,并对工作条件进行了优化。结果表明：抗原最适工作浓度为107cfu/mL;兔抗血清最佳稀释度为1：5000；酶标二抗最佳工作浓度和反应时间分别为1：.0h；检测血清的最佳稀释度为1：2；最佳竞争反应时间为1.0h；底物的最佳反应时间为10min。该方法对60份健康鲫鱼血清样品进行检测,并用统计学方法对检测数据进行了分析,确定了阳性值判定标准为：在阴性对照PI30%情况下,抑制率大于36%为阳性。对实验室感染的60份鲫鱼血清分别用间接竞争ELISA.间接ELISA和凝集试验进行病原体检测,结果表明,建立的间接竞争ELISA检出率为56.7%,比传统的凝集试验(30%)要敏感,虽然没有间接ELISA(66.67%)敏感,但是它能用一种兔的抗体检测不同的血清样品,而不需要针对不同鱼种的二抗,为病原的检测提供方便。同时对临床的93份血清进行检测,阳性率为35.48%,与凝集试验的符合率为89.24%。
【学位授予单位】：南京农业大学【学位级别】：硕士【学位授予年份】：2011【分类号】：S943
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李永芹;[D];中国海洋大学;2010年
黎满香;[D];湖南农业大学;2010年
闫振贵;[D];山东农业大学;2011年
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罗晶晶;[D];华中农业大学;2010年
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