人红细胞单链抗体的制备与血凝试验方法的初步建立
发布时间：2018-12-06 12:15
【摘要】： 自身红细胞凝集试验(Autologous red cell agglutination assay,AGEN)是一种简便、特异又不需要任何特殊仪器的快速诊断方法。其灵敏性和特异性不亚于ELISA,且可同时检测IgG和IgM,具有安全、快速、准确、价廉的特点,检测时无需分离血清,无需仪器设备,仅需一滴全血,在2 min内可得到检测结果,非常适合条件简陋的基层医院、义务献血前以及紧急输血前的使用,因而具有良好的应用前景。该方法依赖于一种双功能试剂,该试剂具有结合人红细胞和病人血液中的抗原或抗体的特性。当病人血液中有针对双功能试剂的抗原或抗体存在时,由于与双功能试剂的分子间交联而导致红细胞的凝集。因此本研究的目的是制备人红细胞非凝集型单克隆抗体,进而形成双功能抗体试剂,用于临床诊断。 本研究用三种免疫原免疫BALB/c小鼠来制备人红细胞非凝集型单克隆抗体,通过化学偶联法制备出双功能抗体,进而形成全血凝集检测试剂,并对血凝试验方法进行了初步探讨。主要研究内容如下: 1、分别用人O型红细胞、红细胞全膜和红细胞膜蛋白作为抗原,采用腹腔加尾部静脉注射免疫BALB/c小鼠,取免疫脾细胞和小鼠骨髓瘤细胞SP2/0进行细胞融合,细胞比例为5∶1,经间接ELISA、红细胞直接凝集试验和间接凝集试验检测,采用有限稀释法进行克隆,经过3次克隆后获得1株人红细胞非凝集型单克隆抗体杂交瘤细胞,命名为1C3。经过多次传代、冻存、复苏,杂交瘤细胞分泌抗体能力仍然稳定,其抗体亚类类型为IgG1。常规方法诱生腹水,辛酸-硫酸铵沉淀法和DEAE纤维素层析法对腹水进行纯化。腹水单抗ELISA效价在1:10~4～1:10~5之间,并与其它种属无交叉反应,亲和力高。 2、分别用胃蛋白酶消化人红细胞非凝集型单克隆抗体和乙肝表面抗体,经纯化得到人红细胞单抗和乙肝表面抗体的F(ab~')_2片段。通过戊二醛一步法和戊二醛两步法将非凝集型单抗和乙肝表面抗体的F(ab~')_2片段进行偶联来制备双功能抗体。经SDS-PAGE电泳鉴定,通过戊二醛一步法成功制备了双功能抗体,进而形成双功能检测试剂,对该试剂的活性进行了初步的鉴定,同时初步建立了一种检测血液中乙肝表面抗原的方法。
[Abstract]:Autologous hemagglutination test (Autologous red cell agglutination assay,AGEN) is a simple, specific and rapid diagnostic method without any special instrument. Its sensitivity and specificity are no less than that of ELISA, and it can detect IgG and IgM, simultaneously with the characteristics of safety, rapidity, accuracy and low price. The detection results can be obtained in 2 min without the need of separating serum, apparatus and equipment, and only a drop of whole blood. It is very suitable for basic hospitals with poor conditions. It is used before voluntary blood donation and before emergency blood transfusion, so it has a good application prospect. The method relies on a bifunctional reagent that binds human erythrocytes to antigens or antibodies in the patient's blood. When antigen or antibody against bifunctional reagents exists in the blood of patients, the agglutination of erythrocytes is caused by intermolecular crosslinking with bifunctional reagents. The aim of this study is to prepare non-agglutination monoclonal antibody of human red blood cell and to form bifunctional antibody reagent for clinical diagnosis. In this study, BALB/c mice were immunized with three kinds of immunogens to prepare non-agglutination monoclonal antibodies against human erythrocytes. Bifunctional antibodies were prepared by chemical coupling method, and then the whole blood agglutination assay was formed. The method of hemagglutination test was discussed. The main contents are as follows: 1. BALB/c mice were immunized with human type O erythrocytes, erythrocyte membrane proteins and erythrocyte membrane proteins respectively by intraperitoneal and tail intravenous injection. The immune spleen cells and mouse myeloma cells (SP2/0) were selected for cell fusion. The proportion of cells was 5: 1. Indirect ELISA, erythrocyte agglutination test and indirect agglutination test were used to clone the cells. After three clones, a human erythrocyte non-agglutinating monoclonal antibody hybridoma cell was obtained and named as 1C3. After repeated passage, cryopreservation and resuscitation, the ability of hybridoma cells to secrete antibodies is still stable, and its antibody subtype is IgG1.. Ascites were induced by routine methods. Ascites were purified by octanoic acid-ammonium sulfate precipitation and DEAE cellulose chromatography. The ELISA titer of ascitic monoclonal antibody was between 1: 10 and 4: 10 5, and had no cross reaction with other species and had high affinity. 2. Human erythrocyte non-agglutinating monoclonal antibody and hepatitis B surface antibody were digested with pepsin, and the F (ab~') _ 2 fragment of human erythrocyte monoclonal antibody and hepatitis B surface antibody were purified. The F (ab~') _ 2 fragment of non-agglutinating monoclonal antibody and hepatitis B surface antibody was coupled with glutaraldehyde one-step method and glutaraldehyde two-step method to prepare the bifunctional antibody. The bifunctional antibody was successfully prepared by one step method of glutaraldehyde by SDS-PAGE electrophoresis, and then the bifunctional detection reagent was formed. The activity of the reagent was preliminarily identified. At the same time, a method for the detection of hepatitis B surface antigen in blood was established.
【学位授予单位】：河南工业大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
本文编号：2366008
[Abstract]:Autologous hemagglutination test (Autologous red cell agglutination assay,AGEN) is a simple, specific and rapid diagnostic method without any special instrument. Its sensitivity and specificity are no less than that of ELISA, and it can detect IgG and IgM, simultaneously with the characteristics of safety, rapidity, accuracy and low price. The detection results can be obtained in 2 min without the need of separating serum, apparatus and equipment, and only a drop of whole blood. It is very suitable for basic hospitals with poor conditions. It is used before voluntary blood donation and before emergency blood transfusion, so it has a good application prospect. The method relies on a bifunctional reagent that binds human erythrocytes to antigens or antibodies in the patient's blood. When antigen or antibody against bifunctional reagents exists in the blood of patients, the agglutination of erythrocytes is caused by intermolecular crosslinking with bifunctional reagents. The aim of this study is to prepare non-agglutination monoclonal antibody of human red blood cell and to form bifunctional antibody reagent for clinical diagnosis. In this study, BALB/c mice were immunized with three kinds of immunogens to prepare non-agglutination monoclonal antibodies against human erythrocytes. Bifunctional antibodies were prepared by chemical coupling method, and then the whole blood agglutination assay was formed. The method of hemagglutination test was discussed. The main contents are as follows: 1. BALB/c mice were immunized with human type O erythrocytes, erythrocyte membrane proteins and erythrocyte membrane proteins respectively by intraperitoneal and tail intravenous injection. The immune spleen cells and mouse myeloma cells (SP2/0) were selected for cell fusion. The proportion of cells was 5: 1. Indirect ELISA, erythrocyte agglutination test and indirect agglutination test were used to clone the cells. After three clones, a human erythrocyte non-agglutinating monoclonal antibody hybridoma cell was obtained and named as 1C3. After repeated passage, cryopreservation and resuscitation, the ability of hybridoma cells to secrete antibodies is still stable, and its antibody subtype is IgG1.. Ascites were induced by routine methods. Ascites were purified by octanoic acid-ammonium sulfate precipitation and DEAE cellulose chromatography. The ELISA titer of ascitic monoclonal antibody was between 1: 10 and 4: 10 5, and had no cross reaction with other species and had high affinity. 2. Human erythrocyte non-agglutinating monoclonal antibody and hepatitis B surface antibody were digested with pepsin, and the F (ab~') _ 2 fragment of human erythrocyte monoclonal antibody and hepatitis B surface antibody were purified. The F (ab~') _ 2 fragment of non-agglutinating monoclonal antibody and hepatitis B surface antibody was coupled with glutaraldehyde one-step method and glutaraldehyde two-step method to prepare the bifunctional antibody. The bifunctional antibody was successfully prepared by one step method of glutaraldehyde by SDS-PAGE electrophoresis, and then the bifunctional detection reagent was formed. The activity of the reagent was preliminarily identified. At the same time, a method for the detection of hepatitis B surface antigen in blood was established.
【学位授予单位】：河南工业大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
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