D-二聚体单克隆抗体制备及荧光胶乳免疫层析应用的研究

发布时间：2018-06-09 23:13

本文选题：D-二聚体 + 甲基纤维素半固体基　；参考：《华南理工大学》2012年硕士论文

【摘要】：D-二聚体是人体内纤维蛋白降解的特异性产物，血液中D-二聚体的含量反应了人体内血栓的形成及溶解状态，D-二聚体的含量测定对血栓性疾病的检测及监控有指导性作用。D-二聚体的检测试剂主要应用抗D-二聚体单克隆抗体与D-二聚体抗原的免疫反应，定性或定量检测D-二聚体。抗D-二聚体单克隆抗体是各种检测试剂的生物原材料，抗体的性质直接影响试剂检测结果的准确性和可靠性。故特异性强的抗D-二聚体单克隆抗体的制备是开发各类D-二聚体检测试剂的关键。国内外多利用传统杂交瘤细胞筛选方法或基因工程方法制备抗D-二聚体单克隆抗体，主要应用于D-二聚体酶联免疫检测试剂（其中包括微孔板ELISA、免疫荧光ELISA、化学发光ELISA）以及用于胶体金免疫层析试剂、半定量乳胶凝集试剂、全血凝集试剂和免疫比浊试剂。本试验以D-二聚体为免疫原，按常规方法免疫BALB/c小鼠；首先利用甲基纤维素半固体基改进的传统杂交瘤技术，筛选出了141株杂交瘤细胞株；接着应用D-二聚体、纤维蛋白原、纤维蛋白、及纤维蛋白降解物为筛选原料，利用间接ELISA筛选技术从141株杂交瘤细胞株中筛选出10株稳定的阳性杂交瘤细胞株；最后通过单克隆抗体体外生产法，，生产10株抗D-二聚体的单克隆抗体，分别命名为1-3C、2-1B、2-1D、2-4B、2-6D、4-1D、4-3C、4-3D、4-4C、5-3B。ELISA检测结果表明，10株单克隆抗体腹水效价可达1×10~6。抗体类—亚类—型鉴定结果显示，抗体型为IgG1的有2-1D，为IgG2a有1-3C、2-1B、2-4B、4-3C、4-3D，为IgG2b有2-6D、4-1D、4-4C、5-3B。Westernblot结果表明与纤维蛋白原有交叉抗体有4-1D、5-3B，与纤维蛋白原降解物有交叉的抗体有C1、C2，只与D-二聚体有反应而无交叉的抗体有1-3C、2-1B、2-6D、4-3C、4-3D、4-4C。通过双抗夹心ELISA方法进行单克隆抗体的配对，结果显示15对配对显示阳性结果。最后依次采用ELISA、胶体金及荧光胶乳定量法筛选出适用于荧光定量法的2-6D与C2抗体对。荧光胶乳免疫层析法是一种快速免疫学方法，具有简便快捷，敏感特异的优点。本试验利用配对的D-二聚体单克隆抗体，制备荧光胶乳免疫层析试纸条，并将其应用于荧光定量检测仪（广州万孚生物技术有限公司）。敏感性结果表明，本试验制备的荧光胶乳试纸条可以敏锐的检测出含量仅为100μg/L浓度的D-二聚体；在特异性试验中没有交叉，通过临床试验对医院样品进行检测，结果显示阳性符合率92.1%，阴性符合率更高达98%。本研究结果表明，改进的杂交瘤细胞筛选方法制备出的抗D-二聚体单克隆抗体亲和性和特异性好。通过双抗夹心ELISA法-胶体金法筛选出的抗体对应用于免疫荧光胶乳定量层析法具有高的敏感性和特异性，可达到与国外同类试剂盒相同的效果。该研究成果为我国血栓类疾病的检测、监控和治疗提供了有效的技术支持。
[Abstract]:D-dimer is a specific product of fibrin degradation in human body. The content of D- dimer in blood reflects the formation and dissolution of thrombus in human body. The determination of D- dimer content has instructive effect on the detection and monitoring of thrombotic diseases. The detection reagent of D- dimer is mainly used to resist D- dimer. Immune reaction of Monoclonal Antibody to Ddimer Antigen, Qualitative or quantitative detection of D-dimer. Anti-D-dimer monoclonal antibody is the biological raw material of various detection reagents. The nature of the antibody directly affects the accuracy and reliability of the detection results of the reagents. So the preparation of anti-D-dimer monoclonal antibody is the key to develop all kinds of D-dimer detection reagents. Traditional hybridoma cell screening methods or genetic engineering methods are used to prepare monoclonal antibodies against D- dimer at home and abroad. It is mainly used in D- dimer enzyme linked immunoassay (including microporous plate ELISA, immunofluorescence ELISA, chemiluminescence ELISA), colloidal gold immunochromatographic reagent, semi-quantitative latex agglutination reagent. BALB / c mice were immunized with D- dimer as immunogen, and 141 hybridoma cell lines were screened by methylcellulose semisolid modified hybridoma technique. Then 10 stable hybridoma cell lines were screened from 141 hybridoma cell lines by indirect Elisa using D- dimer fibrinogen fibrin and fibrin degradation as raw materials. Finally, 10 strains of monoclonal antibodies against D- dimer were produced by the method of monoclonal antibody production in vitro. The results showed that the ascites titers of 10 strains of McAbs were 1 脳 10 ~ (6). The results of antibody class-subclass-type identification showed that, The results of Western blot for IgG1, IgG2a, IgG2a and IgG2a were as follows: 1C 2a, 1-3CU 2-4BU 2-4BN 4-3D, and IgG2b, IgG2b, 2-6DX 4-4CU 4-3B Western blot. The results of Western blot showed that there were 4-1D5-3B with fibrinogen biodegradable antibody, C1C2with fibrinogen degradation, and 1-3CU 2-1B2-6B2-3C4-3D4-4C with fibrinogen biodegradable antibody. Monoclonal antibodies were paired by double antibody sandwich Elisa. The results showed that 15 pairs showed positive results. Finally, 2-6D and C2 antibodies suitable for fluorescence quantitative assay were screened by ELISA-colloidal gold and fluorescent latex quantitative method. Fluorescent latex immunochromatography was a rapid immunological method with the advantages of simplicity, rapidity, sensitivity and specificity. In this study, a fluorescent latex immunochromatographic strip was prepared by using the matched monoclonal antibody of D-dimer, and was applied to the fluorescence quantitative detector (Guangzhou Wanfu Biotechnology Co., Ltd.) The sensitivity results showed that the fluorescent latex test strip prepared in this experiment could be used to detect D- dimer with a concentration of only 100 渭 g / L, and there was no crossover in the specific test, and the hospital samples were detected by clinical trial. The results showed that the positive coincidence rate was 92.1%, and the negative coincidence rate was even higher. The results showed that the modified screening method of hybridoma cells had good affinity and specificity of anti-D-dimer monoclonal antibodies. The antibodies screened by double antibody sandwich Elisa and colloidal gold method have high sensitivity and specificity for immunofluorescence latex quantitative chromatography, and can achieve the same effect as foreign similar kits. The results provide effective technical support for the detection, monitoring and treatment of thrombotic diseases in China.
【学位授予单位】：华南理工大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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