本文選題：抗體親和力　切入點(diǎn)：ELISA　出處：《大連醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:基于酶聯(lián)免疫檢測(ELISA)技術(shù),創(chuàng)建出一個具有適于臨床標(biāo)本抗體親和力檢測的新方法,并應(yīng)用該方法探討抗體親和力在抗感染免疫中的作用;同時,以乙型肝炎病毒(HBV)抗體為例在分子水平上探討HLA-DQB1基因的多態(tài)性與抗體親和力的關(guān)聯(lián)。方法:1.選取正常體檢者的血清,基于ELISA檢測方法,針對與抗體親和力相關(guān)的影響因素分別進(jìn)行單因素和多因素多水平定量分析,探討其對ELISA檢測結(jié)果的影響,并找出具有顯著影響力的影響因素。2.基于傳統(tǒng)的尿素洗脫法,選取兩個對親和力具有顯著影響效果的因素開展抗體親和力檢測方法的設(shè)計,以乙型肝炎病毒表面抗體親和力的檢測為例,將待檢抗體血清進(jìn)行系列稀釋,以檢出限對應(yīng)的抗血清濃度作為抗體終點(diǎn)濃度,并以此濃度作為基準(zhǔn)進(jìn)行分析。做批內(nèi)及批間穩(wěn)定性實(shí)驗(yàn),通過分析變異系數(shù)驗(yàn)證方法的穩(wěn)定性。同時為驗(yàn)證新創(chuàng)建的方法的適用性,用該方法對收集的已知抗體親和力高低的家兔抗血清標(biāo)本進(jìn)行檢測。3.分別采集抗-HBs抗體陽性的體檢者血清(包括抗-HBe抗體陰性和抗-HBe抗體陽性)125例,和HCV抗體陽性的體檢者血清(包含HCV-RNA陰性和HCV-RNA陽性)53例,并運(yùn)用我們創(chuàng)建的檢測抗體親和力的抗體終點(diǎn)法分別來檢測抗-HBs抗體和HCV抗體的親和力,進(jìn)而分析探討抗體親和力在臨床檢測中的應(yīng)用。4.采集抗-HBs抗體陽性的體檢者血清60例,運(yùn)用我們創(chuàng)建的檢測抗體親和力的新方法篩選出高低親和力抗血清,并分別用高低親和力不同的抗血清中和乙型肝炎病毒表面抗原,檢測中和指數(shù)進(jìn)而探討抗體親和力在抗感染免疫中的作用。5.收集正常體檢者的血清和全血標(biāo)本70例,應(yīng)用創(chuàng)建的抗體終點(diǎn)法篩選出高低親和力抗體;同時采用PCR-SSP技術(shù)檢測這70例體檢者全血中HLA-DQB1等位基因的頻率分布情況,進(jìn)而分析不同高低的抗體親和力與HLA-DQB1基因多態(tài)性的關(guān)聯(lián)。結(jié)果:1.我們所探討的與抗體親和力有關(guān)的對ELISA技術(shù)具有顯著影響效果的因素的影響程度由大到小依次為電解質(zhì)強(qiáng)度作用時間孵育溫度,其中以電解質(zhì)強(qiáng)度的影響最為顯著。2.新創(chuàng)建的抗體親和力的檢測方法能夠良好的檢測出高親和力抗血清標(biāo)本和低親和力抗血清標(biāo)本,并具有良好的準(zhǔn)確度和穩(wěn)定性。3.檢測結(jié)果表明:抗-HBe抗體陽性組的血清標(biāo)本的抗-HBs抗體的親和力明顯高于陰性組;HCV-RNA陰性血清組的HCV抗體的親和力較陽性血清組高。4.在抗體總活性相同的條件下,抗體親和力與抗體的蛋白量都與病毒抗原的中和指數(shù)成正相關(guān)關(guān)系。5.在抗體親和力與HLA-DQB1基因多態(tài)性的相關(guān)性分析中,發(fā)現(xiàn)在高親和力組和低親和力組中,HLA-DQB1*02和HLA-DQB1*06兩個位點(diǎn)的陽性率存在顯著性差異(P0.05),其中,高親和力組的HLA-DQB1*06位點(diǎn)陽性率明顯高于低親和力組,而HLA-DQB1*02位點(diǎn)的陽性率明顯低于低親和力組。結(jié)論:1.我們創(chuàng)建的抗體終點(diǎn)法具有良好的準(zhǔn)確度和穩(wěn)定性,將其應(yīng)用于臨床標(biāo)本的檢測,發(fā)現(xiàn)高親和力抗體較低親和力抗體可能具有更強(qiáng)的抗感染作用,適于臨床檢測應(yīng)用。2.HLA-DQB1*02和HLA-DQB1*06基因的表達(dá)可能與HBV抗體親和力的高低有關(guān),提示HLA基因的多樣性可能與HBV抗體親和力有關(guān)。
[Abstract]:Objective: Based on the enzyme-linked immunosorbent assay (ELISA) technology, to create a new method for clinical samples with antibody affinity detection, and the application of the method of antibody affinity in anti infectionimmunity; at the same time, the association of hepatitis B virus (HBV) antibody to investigate the polymorphism of HLA-DQB1 gene and antibody affinity cases at the molecular level. Methods: 1. normal subjects were selected in serum, ELISA detection method based on the related factors of antibody affinity respectively for single and multi factors analysis of multi level quantitative, evaluate its effect on the ELISA test results, and find out the influence factors have a significant influence to traditional.2. based on urea elution the design method, selecting two has significant effect on the affinity factors of antibody affinity detection method, the hepatitis B virus surface antibody affinity detection Cases to be detected serum antibody was diluted serially, with the detection limit of the corresponding antiserum concentration as the antibody concentration and the concentration of end point, as the benchmark for analysis. Do the intra batch and inter batch stability experiment, through stability analysis and verification methods of coefficient of variation. At the same time as the method to verify the applicability of the newly created, using the method of collect the known antibody affinity of rabbit antiserum was detected.3. examination of anti -HBs antibody positive serum were collected (including negative anti -HBe antibody and anti -HBe antibody) in 125 cases, and HCV antibody positive serum examination (including HCV-RNA negative and HCV-RNA positive) in 53 cases, the detection of anti -HBs antibody and HCV antibody and application of detection of antibody to end point antibody affinity we create the affinity, and then using.4. analysis to study antibody affinity in clinical detection of anti -HBs antibody positive acquisition Physical examination of serum of 60 cases, using the new methods for detection of antibody affinity we create the selected high affinity antiserum, and respectively with high affinity neutralizing antiserum of different hepatitis B virus surface antigen detection and discuss the neutralization index of antibody affinity in anti infection effect of.5. immunization in normal subjects collected serum and whole blood samples of 70 cases, end point method is used to create antibodies screened high affinity antibodies; at the same time using PCR-SSP technology to detect the frequency distribution of the 70 cases of HLA-DQB1 in whole blood. The gene and association analysis of antibody affinity and HLA-DQB1 gene polymorphism in different level. Results: 1. we discuss the related antibody with affinity the significant effect of factors influence degree from high to low is the strength of the electrolyte time incubation temperature on the ELISA technology. The effect of electrolyte strength is the most significant method for detection of antibody affinity of the newly created.2. can detect the antiserum with high affinity and low affinity were good with antiserum samples, the accuracy and stability of.3. good results showed that serum anti -HBe antibody positive group and anti -HBs antibody affinity was significantly higher than that of negative group; HCV HCV-RNA antibody negative serum group affinity than positive serum.4. antibody in the same group of high activity under the condition of protein and antibody affinity antibody and virus antigen neutralization index has positive correlation with.5. in analysis of the relationship between antibody affinity and HLA-DQB1 gene polymorphism, found in the high and low affinity group the affinity group, there was significant difference between the positive rate of HLA-DQB1*02 and HLA-DQB1*06 two loci (P0.05, HLA-DQB1*06), a high affinity group positive point The rate was significantly higher than that of the low affinity group, while the positive rate of HLA-DQB1*02 was significantly lower than that of the low affinity site group. Conclusion: with accuracy and good stability of the 1. antibodies we create end point method, its application in the detection of clinical specimens, found that high affinity antibodies of low affinity antibody has stronger anti infection effect, suitable for expression detection and clinical application of.2.HLA-DQB1*02 and HLA-DQB1*06 gene may be associated with HBV antibody level, suggesting that the diversity of HLA gene may be related with HBV antibody affinity.

【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R446.6;R512.62
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本文編號：1577604