金标免疫渗滤法检测类风湿性关节炎自身抗体的研究

发布时间：2018-06-09 17:34

本文选题：金标免疫渗滤法 + 类风湿性关节炎　；参考：《东南大学》2015年硕士论文

【摘要】：胶体金标记技术的建立催生了两种经典的免疫学检测方法,斑点金免疫渗滤(DIGFA)和胶体金免疫层析技术(GICA),其中DIGFA由于具有快速、结果可直接判读、可单人份检测、结果稳定等优点逐渐成为开发各类快速诊断试剂的技术基础,一系列针对临床、食品等多个领域的DIGFA被建立和应用。类风湿性关节炎(RA)是一种自身免疫性疾病,晚期关节多发生畸形难以治愈,早期诊断对疾病的控制尤为重要,大量研究已经表明在临床症状出现好几年前患者血清中就会出现特异的自身抗体,对这些自身抗体的检测对RA的早期诊断非常有利。本文拟建立检测RA两种自身抗体的DIGFA,旨在简化检测操作、降低检测成本、在样本量少的情况下可快速开展检测。为此,首先通过与欧蒙试剂盒对比及检测临床确定的血清对待检抗体特异的抗原进行了筛选制备,采用链酶亲和素-生物素结合方式将环瓜氨酸肽(CCP)与载体蛋白进行偶联,优化了新的含18个氨基酸的环肽序列；确定了较稳定的类风湿因子(RF)特异抗原的原料来源,同时优化了特异性高的HRP标记F(ab’)2片段作为酶标二抗,并比较了兔IgG、人IgG、Fc片段几种常用于RF检测的抗原性能。接着,本文制备了抗自身抗体的胶体金探针,对各反应参数进行了优化,确定了较好的偶联pH在8.2-8.5之间(7μL K2CO3 (5%)/mL Au),最小偶联蛋白量为22μg/mLAu,最适大分子稳定剂是BSA(免疫级)和PEG-20K,较优洗涤纯化缓冲液是Tris-HCl(pH 8.2),最后优化了探针冻干的最佳保护剂为甘露醇。最后,将前两章制备的抗原和探针应用于DIGFA的建立,对影响检测的多个因素进行了优化,确定了RIgG最适稀释液是Tris-HCl (pH 8.2),样品下渗速度是1.4-3.3虬/s,大分子封闭剂是BSA,洗涤条件是PBST(pH7.4,1%Tween-20),同时发现PEG和Tween对斑点显色有一定增强作用,用该试剂检测部分临床血清,显示了良好的检测效果。
[Abstract]:The establishment of colloidal gold labeling technique gave birth to two classical immunological detection methods, DIGFA and GICA, in which DIGFA can be read directly and can be detected by single person because of its rapidity. Results Stability has gradually become the technical basis for the development of various rapid diagnostic reagents, and a series of DIGFA for clinical, food and other fields have been established and applied. Rheumatoid arthritis (RA) is a kind of autoimmune disease. A large number of studies have shown that specific autoantibodies are present in patients' serum several years before the onset of clinical symptoms. The detection of these autoantibodies is very helpful for the early diagnosis of RA. The purpose of this paper is to establish a DIGFA for detecting two kinds of autoantibodies in RA. The aim is to simplify the detection operation and reduce the detection cost, so that the detection can be carried out quickly under the condition of small sample size. For this reason, the specific antigens of antibodies were screened and prepared by comparing and detecting the serum specific antigens determined by Ohm kit. The cyclic citrullinated peptide (CCPP) was coupled with the carrier protein by the way of chain enzyme affinity and biotin binding. The new cyclic peptide sequence containing 18 amino acids was optimized, the source of the stable RFRFs specific antigen was determined, and the HRP labeled FGBA 2 fragment was optimized as the enzyme labeled second antibody. The antigenicity of rabbit IgG, human IgG Fc fragment for RF detection was compared. Then, the colloidal gold probe of anti-autoantibody was prepared, and the reaction parameters were optimized. The optimum coupling pH was between 8.2-8.5 渭 L K2CO3 and 5 渭 L / mL au, the minimum coupling protein content was 22 渭 g / mL au, the optimal macromolecular stabilizers were BSA (immune level) and PEG-20K, the optimal washing and purification buffer was Tris-HCl pH 8.2, and the best protection agent for the lyophilization of the probe was mannitol. Finally, the antigens and probes prepared in the first two chapters are applied to the establishment of DIGFA, and several factors affecting the detection are optimized. The optimal diluent of RIgG was Tris-HCl (pH 8.2), the osmotic rate of the sample was 1.4-3.3 / s, the macromolecular sealant was BSA, and the washing condition was PBST-pH7.4 ~ (-1) Tween-20. At the same time, it was found that PEG and Tween had a certain enhancement effect on spot color, and some clinical sera were detected with this reagent. Good detection effect is shown.
【学位授予单位】：东南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R593.22

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