检测NDV诱导特异性IFN-γ、IL-4的ELISPOT试验方法建立及应用
发布时间：2018-11-10 11:16
【摘要】： 在病毒免疫过程中,不仅需要体液免疫,而且需要细胞免疫。如在新城疫的免疫方面,细胞免疫与体液免疫协同发挥作用。Ghumman(1976)等发现,接种新城疫活苗和灭活苗后2-3天,就能检测到淋巴细胞内DNA合成增加。Agrawal(1991)等用白细胞游走抑制试验来评价接种ND疫苗后细胞免疫反应,发现接种后确实存在细胞免疫反应。机体针对新城疫的细胞免疫和体液免疫之间究竟是怎么变化的呢?为什么经常出现免疫失败呢?这些问题没有明确的答案。 酶联免疫斑点检测技术(ELISPOT)结合了细胞培养技术与酶联免疫吸附技术(ELISA)的长处,能够分析经特异抗原活化后分泌细胞因子(如IFN-γ、TNF-α等)的单个效应细胞的频数,具有敏感、特异、易于重复的优点。本研究将ELISPOT技术应用于新城疫细胞免疫的研究,通过对脾淋巴细胞浓度,培养时间,抗原刺激量进行选择,建立了检测NDV诱导特异性IFN-γ、IL-4的ELISPOT方法。结果显示,试验的特异性与培养时间和刺激抗原密切相关,在一定范围内,培养时间越长、刺激抗原越多,试验的特异性越低,当抗原量为5ug/孔,培养时间为7h时,试验的特异性最强。ELISPOT斑点数,随细胞浓度的增加而增加,当小鼠的脾淋巴细胞浓度为106/孔时,最有利于斑点的计数,试验误差最小。用建立的ELISPOT方法对NDV免疫小鼠的IFN-γ、Il-4水平进行检测发现,IFN-γ、Il-4与血清抗体的产生存在着一定的时差关系,在血清抗体阳性检出的前期,IFN-γ的分泌降至最低水平,Il-4升至最高水平。
[Abstract]:In the process of virus immunization, not only humoral immunity, but also cellular immunity is needed. For example, in the immunity aspect of Newcastle disease, the synergistic effect of cellular immunity and humoral immunity on. Ghumman (1976 was found, 2-3 days after inoculation of live and inactivated Newcastle disease vaccine, The leukocyte migration inhibition test was used to evaluate the cellular immune response after inoculation of ND vaccine. It was found that there was a cellular immune response after inoculation. How on earth does the cellular and humoral immunity of the body change in response to Newcastle disease? Why do you often fail to get immunized? There are no clear answers to these questions. (ELISPOT) combines the advantages of cell culture and enzyme linked immunosorbent assay (ELISA) to analyze cytokines (such as IFN- 纬) secreted by specific antigens. The frequency of single effector cells of TNF- 伪 is sensitive, specific and easy to repeat. In this study, ELISPOT technique was applied to the study of cellular immunity of Newcastle disease (NDV). By selecting the concentration of splenic lymphocytes, culture time and the amount of antigen stimulation, a ELISPOT method for detecting the specific IFN- 纬 and IL-4 induced by NDV was established. The results showed that the specificity of the test was closely related to the culture time and the stimulation antigen. In a certain range, the longer the culture time was, the more the stimulating antigen was, the lower the specificity of the test was. When the amount of antigen was 5ug/ pore, the culture time was 7 h. The specificity of the experiment was the strongest. The number of ELISPOT spots increased with the increase of cell concentration. When the concentration of spleen lymphocytes in mice was 106 / well, the number of spots was the most favorable, and the error of the experiment was the least. The levels of IFN- 纬 and Il-4 in mice immunized with NDV were detected by using the established ELISPOT method. It was found that there was a time-difference relationship between IFN- 纬, Il-4 and the production of serum antibodies. The secretion of IFN- 纬 decreased to the lowest level and Il-4 to the highest level.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392
本文编号：2322320
[Abstract]:In the process of virus immunization, not only humoral immunity, but also cellular immunity is needed. For example, in the immunity aspect of Newcastle disease, the synergistic effect of cellular immunity and humoral immunity on. Ghumman (1976 was found, 2-3 days after inoculation of live and inactivated Newcastle disease vaccine, The leukocyte migration inhibition test was used to evaluate the cellular immune response after inoculation of ND vaccine. It was found that there was a cellular immune response after inoculation. How on earth does the cellular and humoral immunity of the body change in response to Newcastle disease? Why do you often fail to get immunized? There are no clear answers to these questions. (ELISPOT) combines the advantages of cell culture and enzyme linked immunosorbent assay (ELISA) to analyze cytokines (such as IFN- 纬) secreted by specific antigens. The frequency of single effector cells of TNF- 伪 is sensitive, specific and easy to repeat. In this study, ELISPOT technique was applied to the study of cellular immunity of Newcastle disease (NDV). By selecting the concentration of splenic lymphocytes, culture time and the amount of antigen stimulation, a ELISPOT method for detecting the specific IFN- 纬 and IL-4 induced by NDV was established. The results showed that the specificity of the test was closely related to the culture time and the stimulation antigen. In a certain range, the longer the culture time was, the more the stimulating antigen was, the lower the specificity of the test was. When the amount of antigen was 5ug/ pore, the culture time was 7 h. The specificity of the experiment was the strongest. The number of ELISPOT spots increased with the increase of cell concentration. When the concentration of spleen lymphocytes in mice was 106 / well, the number of spots was the most favorable, and the error of the experiment was the least. The levels of IFN- 纬 and Il-4 in mice immunized with NDV were detected by using the established ELISPOT method. It was found that there was a time-difference relationship between IFN- 纬, Il-4 and the production of serum antibodies. The secretion of IFN- 纬 decreased to the lowest level and Il-4 to the highest level.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392
【参考文献】
相关期刊论文 前6条
1 张辉,潘志明,焦新安;一种细胞因子检测新方法——ELISPOT技术[J];动物医学进展;2004年06期
2 邵华斌,杨峻,陈守仁,王文清,黎秋华;鸡新城疫、传染性支气管炎二联油乳剂灭活疫苗试验研究报告(Ⅰ报)——NDV、IBV同胚培养繁毒效果试验[J];湖北畜牧兽医;1997年02期
3 杨秀文,边传周;鸡新城疫二价油乳剂灭活苗的研制及免疫效果观察[J];郑州牧业工程高等专科学校学报;2003年04期
4 栾怡,于修平,赵蔚明,周亚滨,齐眉;ELISPOT检测特异性抗体形成细胞功能[J];山东大学学报(医学版);2004年02期
5 李影;田波;;新城疫病毒F蛋白七肽重复序列(HR)三螺旋HR212蛋白的抗体抑制病毒和细胞融合[J];自然科学进展;2007年01期
6 黄金海,刘业兵,丁伯良,张健,王东;间接ELISA法检测鸡新城疫抗体[J];中国兽药杂志;2005年06期
相关博士学位论文 前1条
1 秦卓明;新城疫病毒流行株致病性和抗原性及其与F和HN基因变异的相关性[D];山东农业大学;2006年
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