抗体的非对应性激发及其与H2-Eb等位基因多态性的关联

发布时间：2018-02-25 05:34

本文关键词： 非对应性激发 H2-Eb等位基因多态性 ELISA 天然自身抗体　出处：《大连医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的：天然自身抗体(natural autoantibody, NAA)指的是没有经过任何抗原的主动免疫,机体所产生的针对一种或者多种自身和(或)外来抗原的抗体,广泛存在于几乎所有的脊椎动物体内。本文通过对免疫小鼠得到的血清进行四种抗体(抗牛血清白蛋白抗体、抗人血清白蛋白抗体、抗卵清蛋白抗体和抗鱼精蛋白抗体)的检测,观察其非对应性激发现象,并用上述四种抗原进行交叉检测；应用PCR-SSP技术等,分析其与H2-Eb等位基因的多态性的关联；探索天然自身抗体的来源。方法： 1.用已知浓度的牛血清白蛋白标准品和待测血清,做酶联免疫吸附试验(ELISA),检测待测血清中抗体的相对含量,建立一个基于ELISA法来测定抗体含量的精密方法。 2.采用回收试验法对以上方法进行验证。 3.选出与小鼠亲缘关系较近的两种白蛋白(牛血清白蛋白和人血清白蛋白)和两种与小鼠亲缘关系较远的两种蛋白(卵清蛋白和鱼精蛋白)作为抗原物质,采用间隔免疫的方法建立非对应性激发的动物模型,制备实验用的待测血清。 4.分别用以上四种抗原包被酶标板,并测量待测血清中各种抗体的水平。将加强免疫两次得到的小鼠血清按照抗原种类分别混合后作为标准品。 5.取用加强免疫组的小鼠的肝脏,提取基因组DNA,采用PCR-SSP技术检测小鼠H2-Eb等位基因中MudoEb5和MudoEb7位点的基因多态性,并分析H2-Eb等位基因多态性与小鼠体内抗体的变化情况之间的关系。结果： 1.回收试验的回归系数R2=0.994,回归方程为Y=1.097X-11.021,回归曲线(1ine)的线性良好,抗体相对含量与血清浓度呈正相关,回收试验成功,ELISA实验方法准确可靠。 2.间断免疫法制备待检血清的方法有效。小鼠体内抗体被相关抗原自身激发后,加强免疫组与基础免疫组的抗体含量有显著性差异,P0.05,差异有统计学意义。 3.四种抗体中,抗人血清白蛋白抗体在受到鱼精蛋白激发后,明显增多,P0.05,差异有统计学意义；另外3种抗体受到非对应性抗原激发后变化不明显,P0.05,差异没有统计学意义。 4.45只小鼠中MudoEb5检测到阳性的有36只,阳性率为80%,MudoEb7检测到阳性的有40只,阳性率为88.89%；抗卵清蛋白抗体在受到人血清白蛋白激发后,没有MudoEb5等位基因的,加强免疫组比基础免疫组的抗体相对含量明显增多,P0.05,差异有统计学意义。其余的非对应性激发反应,无论有无MudoEb5等位基因和(或)MudoEb7等位基因,加强免疫组与基础免疫组之间的抗体相对含量均无明显差异,P0.05,差异没有统计学意义。结论： 1.应用ELISA检测抗体含量的方法精密有效。 2.某些抗体(如小鼠抗人血清白蛋白抗体)可能被无关的非对应性抗原(如鱼精蛋白)非对应性激发。 3.非对应性激发与某些基因位点可能有一定关系(如人血清白蛋白非对应激发小鼠抗卵清蛋白抗体可能与MudoEb5相关)。
[Abstract]:Objective: natural autoantibody (NAA) refers to antibodies produced by the body against one or more of its own and / or foreign antigens without active immunization with any antigen. In this paper, four kinds of antibodies (anti-bovine serum albumin antibody, anti-human serum albumin antibody) were carried out in the sera of immunized mice. The detection of anti-ovalbumin antibody and anti-protamine antibody was used to observe the non-corresponding excitation phenomenon, and to cross-detect the above four antigens, and to analyze the association between H2-Eb allele polymorphism and anti-ovalbumin antibody by PCR-SSP technique, and to analyze the relationship between H2-Eb allele polymorphism and anti-ovalbumin antibody. To explore the source of natural autoantibodies. Methods:. 1. Using the known concentration of bovine serum albumin (BSA) standard and serum to be tested, Elisa was performed to detect the relative content of antibodies in the serum to be tested, and a precise method based on ELISA method was established for the determination of antibodies. 2. The method of recovery test is used to verify the above method. 3. Two kinds of albumin (bovine serum albumin and human serum albumin) and two proteins (ovalbumin and protamine) closely related to mice were selected as antigens. The animal model of non-corresponding excitation was established by interval immunity, and the experimental serum was prepared. 4. The four kinds of antigens were coated with the enzyme labeled plates, and the levels of various antibodies in the serum were measured. The sera of mice obtained from the two times of enhanced immunization were mixed as standard samples according to the kinds of antigens. 5. The MudoEb5 and MudoEb7 loci of H2-Eb allele in mice were detected by PCR-SSP technique, and the relationship between H2-Eb allele polymorphism and antibody in mice was analyzed. 5. The liver of mice immunized with H2-Eb was extracted and genomic DNA was extracted. The polymorphism of MudoEb5 and MudoEb7 in H2-Eb allele was detected by PCR-SSP technique. Results:. 1. The regression coefficient of recovery test was 0.994, and the regression equation was YC1.097X-11.021. The regression curve was linear. The relative content of antibody was positively correlated with serum concentration. The method of Elisa was accurate and reliable. 2. The method of preparing serum to be tested by intermittent immunization was effective. After the antibody was excited by the autoantigen in mice, there was significant difference in the antibody content between the immunized group and the basic immunization group (P 0.05), and the difference was statistically significant. 3. Among the four antibodies, the level of anti-human serum albumin antibody increased significantly (P 0.05) after being stimulated by protamine, the difference was statistically significant, but the change of the other three antibodies was not significant (P 0.05) after the challenge of non-corresponding antigens, the difference was not statistically significant. Among the 45 mice, 36 were positive for MudoEb5, 40 were positive for Mudo Eb7, and 88.89 were positive for anti-ovalbumin antibody, and there was no MudoEb5 allele after stimulation by human serum albumin. The relative content of antibody in the enhanced immunization group was significantly higher than that in the basic immunization group (P 0.05), the difference was statistically significant. The other non-corresponding excitation responses, whether or not there were MudoEb5 alleles and (or MudoEb7) alleles, There was no significant difference in the relative content of antibody between the two groups (P 0.05), and there was no significant difference between the two groups. Conclusion:. 1. The method of detecting antibody content by ELISA is accurate and effective. 2. Some antibodies (such as mouse anti-human serum albumin antibodies) may be excited by unrelated non-corresponding antigens (e.g. protamine). 3. There may be some relationship between non-corresponding excitation and some gene loci (for example, the anti-ovalbumin antibody against ovalbumin in mice stimulated by human serum albumin may be related to MudoEb5).
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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