基于抗体终点法观察亲和力在抗感染免疫中的作用及其与HLA-DQB1多态性的关联

发布时间：2018-03-07 02:38

  本文选题：抗体亲和力　切入点：ELISA　出处：《大连医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:基于酶联免疫检测(ELISA)技术,创建出一个具有适于临床标本抗体亲和力检测的新方法,并应用该方法探讨抗体亲和力在抗感染免疫中的作用;同时,以乙型肝炎病毒(HBV)抗体为例在分子水平上探讨HLA-DQB1基因的多态性与抗体亲和力的关联。方法:1.选取正常体检者的血清,基于ELISA检测方法,针对与抗体亲和力相关的影响因素分别进行单因素和多因素多水平定量分析,探讨其对ELISA检测结果的影响,并找出具有显著影响力的影响因素。2.基于传统的尿素洗脱法,选取两个对亲和力具有显著影响效果的因素开展抗体亲和力检测方法的设计,以乙型肝炎病毒表面抗体亲和力的检测为例,将待检抗体血清进行系列稀释,以检出限对应的抗血清浓度作为抗体终点浓度,并以此浓度作为基准进行分析。做批内及批间稳定性实验,通过分析变异系数验证方法的稳定性。同时为验证新创建的方法的适用性,用该方法对收集的已知抗体亲和力高低的家兔抗血清标本进行检测。3.分别采集抗-HBs抗体阳性的体检者血清(包括抗-HBe抗体阴性和抗-HBe抗体阳性)125例,和HCV抗体阳性的体检者血清(包含HCV-RNA阴性和HCV-RNA阳性)53例,并运用我们创建的检测抗体亲和力的抗体终点法分别来检测抗-HBs抗体和HCV抗体的亲和力,进而分析探讨抗体亲和力在临床检测中的应用。4.采集抗-HBs抗体阳性的体检者血清60例,运用我们创建的检测抗体亲和力的新方法筛选出高低亲和力抗血清,并分别用高低亲和力不同的抗血清中和乙型肝炎病毒表面抗原,检测中和指数进而探讨抗体亲和力在抗感染免疫中的作用。5.收集正常体检者的血清和全血标本70例,应用创建的抗体终点法筛选出高低亲和力抗体;同时采用PCR-SSP技术检测这70例体检者全血中HLA-DQB1等位基因的频率分布情况,进而分析不同高低的抗体亲和力与HLA-DQB1基因多态性的关联。结果:1.我们所探讨的与抗体亲和力有关的对ELISA技术具有显著影响效果的因素的影响程度由大到小依次为电解质强度作用时间孵育温度,其中以电解质强度的影响最为显著。2.新创建的抗体亲和力的检测方法能够良好的检测出高亲和力抗血清标本和低亲和力抗血清标本,并具有良好的准确度和稳定性。3.检测结果表明:抗-HBe抗体阳性组的血清标本的抗-HBs抗体的亲和力明显高于阴性组;HCV-RNA阴性血清组的HCV抗体的亲和力较阳性血清组高。4.在抗体总活性相同的条件下,抗体亲和力与抗体的蛋白量都与病毒抗原的中和指数成正相关关系。5.在抗体亲和力与HLA-DQB1基因多态性的相关性分析中,发现在高亲和力组和低亲和力组中,HLA-DQB1*02和HLA-DQB1*06两个位点的阳性率存在显著性差异(P0.05),其中,高亲和力组的HLA-DQB1*06位点阳性率明显高于低亲和力组,而HLA-DQB1*02位点的阳性率明显低于低亲和力组。结论:1.我们创建的抗体终点法具有良好的准确度和稳定性,将其应用于临床标本的检测,发现高亲和力抗体较低亲和力抗体可能具有更强的抗感染作用,适于临床检测应用。2.HLA-DQB1*02和HLA-DQB1*06基因的表达可能与HBV抗体亲和力的高低有关,提示HLA基因的多样性可能与HBV抗体亲和力有关。
[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.

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R446.6;R512.62
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本文编号：1577604