本文關(guān)鍵詞： 非對(duì)應(yīng)性激發(fā) H2-Eb等位基因多態(tài)性 ELISA 天然自身抗體　出處：《大連醫(yī)科大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：天然自身抗體(natural autoantibody, NAA)指的是沒有經(jīng)過任何抗原的主動(dòng)免疫,機(jī)體所產(chǎn)生的針對(duì)一種或者多種自身和(或)外來抗原的抗體,廣泛存在于幾乎所有的脊椎動(dòng)物體內(nèi)。本文通過對(duì)免疫小鼠得到的血清進(jìn)行四種抗體(抗牛血清白蛋白抗體、抗人血清白蛋白抗體、抗卵清蛋白抗體和抗魚精蛋白抗體)的檢測(cè),觀察其非對(duì)應(yīng)性激發(fā)現(xiàn)象,并用上述四種抗原進(jìn)行交叉檢測(cè)；應(yīng)用PCR-SSP技術(shù)等,分析其與H2-Eb等位基因的多態(tài)性的關(guān)聯(lián)；探索天然自身抗體的來源。 方法： 1.用已知濃度的牛血清白蛋白標(biāo)準(zhǔn)品和待測(cè)血清,做酶聯(lián)免疫吸附試驗(yàn)(ELISA),檢測(cè)待測(cè)血清中抗體的相對(duì)含量,建立一個(gè)基于ELISA法來測(cè)定抗體含量的精密方法。 2.采用回收試驗(yàn)法對(duì)以上方法進(jìn)行驗(yàn)證。 3.選出與小鼠親緣關(guān)系較近的兩種白蛋白(牛血清白蛋白和人血清白蛋白)和兩種與小鼠親緣關(guān)系較遠(yuǎn)的兩種蛋白(卵清蛋白和魚精蛋白)作為抗原物質(zhì),采用間隔免疫的方法建立非對(duì)應(yīng)性激發(fā)的動(dòng)物模型,制備實(shí)驗(yàn)用的待測(cè)血清。 4.分別用以上四種抗原包被酶標(biāo)板,并測(cè)量待測(cè)血清中各種抗體的水平。將加強(qiáng)免疫兩次得到的小鼠血清按照抗原種類分別混合后作為標(biāo)準(zhǔn)品。 5.取用加強(qiáng)免疫組的小鼠的肝臟,提取基因組DNA,采用PCR-SSP技術(shù)檢測(cè)小鼠H2-Eb等位基因中MudoEb5和MudoEb7位點(diǎn)的基因多態(tài)性,并分析H2-Eb等位基因多態(tài)性與小鼠體內(nèi)抗體的變化情況之間的關(guān)系。 結(jié)果： 1.回收試驗(yàn)的回歸系數(shù)R2=0.994,回歸方程為Y=1.097X-11.021,回歸曲線(1ine)的線性良好,抗體相對(duì)含量與血清濃度呈正相關(guān),回收試驗(yàn)成功,ELISA實(shí)驗(yàn)方法準(zhǔn)確可靠。 2.間斷免疫法制備待檢血清的方法有效。小鼠體內(nèi)抗體被相關(guān)抗原自身激發(fā)后,加強(qiáng)免疫組與基礎(chǔ)免疫組的抗體含量有顯著性差異,P0.05,差異有統(tǒng)計(jì)學(xué)意義。 3.四種抗體中,抗人血清白蛋白抗體在受到魚精蛋白激發(fā)后,明顯增多,P0.05,差異有統(tǒng)計(jì)學(xué)意義；另外3種抗體受到非對(duì)應(yīng)性抗原激發(fā)后變化不明顯,P0.05,差異沒有統(tǒng)計(jì)學(xué)意義。 4.45只小鼠中MudoEb5檢測(cè)到陽性的有36只,陽性率為80%,MudoEb7檢測(cè)到陽性的有40只,陽性率為88.89%；抗卵清蛋白抗體在受到人血清白蛋白激發(fā)后,沒有MudoEb5等位基因的,加強(qiáng)免疫組比基礎(chǔ)免疫組的抗體相對(duì)含量明顯增多,P0.05,差異有統(tǒng)計(jì)學(xué)意義。其余的非對(duì)應(yīng)性激發(fā)反應(yīng),無論有無MudoEb5等位基因和(或)MudoEb7等位基因,加強(qiáng)免疫組與基礎(chǔ)免疫組之間的抗體相對(duì)含量均無明顯差異,P0.05,差異沒有統(tǒng)計(jì)學(xué)意義。 結(jié)論： 1.應(yīng)用ELISA檢測(cè)抗體含量的方法精密有效。 2.某些抗體(如小鼠抗人血清白蛋白抗體)可能被無關(guān)的非對(duì)應(yīng)性抗原(如魚精蛋白)非對(duì)應(yīng)性激發(fā)。 3.非對(duì)應(yīng)性激發(fā)與某些基因位點(diǎn)可能有一定關(guān)系(如人血清白蛋白非對(duì)應(yīng)激發(fā)小鼠抗卵清蛋白抗體可能與MudoEb5相關(guān))。
[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).
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392

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