抗细胞间粘附分子1单克隆抗体的制备、鉴定以及初步应用

发布时间：2018-01-21 16:02

本文关键词： 细胞间粘附分子-1 单克隆抗体杂交瘤细胞格雷夫斯病　出处：《天津医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 细胞间粘附分子1 (intercellular adhesion molecule-1 ICAM-1)是一种细胞表面单链糖蛋白,参与抗原识别、补体结合、细胞粘附功能。可溶性细胞间粘附分子1 (soluble intercellular adhesion molecule-1 sICAM-1)为细胞表面ICAM-1脱落所形成,其表达是导致Graves'病发生的重要因素之一,并且其血清水平对于判断Graves'病的停药和复发具有重要意义。利用纯品ICAM-1免疫小鼠制备得到抗ICAM-1的单克隆抗体,为进一步治疗Graves'病奠定了基础,将会有一定的经济意义和临床应用前景。目的：1.利用纯品ICAM-1免疫小鼠制备得到具有生物活性的抗ICAM-1的单克隆抗体。 2.利用抗ICAM-1的单克隆抗体,治疗Graves'病模型小鼠。方法：1.用纯品ICAM-1来免疫动物。将纯品ICAM-1蛋白与佐剂混合腹腔注射BALB/C小鼠三次,每次间隔大约3周,取小鼠内眦静脉血测免疫效价,取效价高的小鼠尾静脉注射加强免疫一次,三天后取小鼠脾细胞,与骨髓瘤细胞以5：1的数量混合,在PEG(polyethylene glycol,聚乙二醇)作用下进行细胞融合。一周后取细胞生长孔上清经ELISA检测抗体分泌情况,将阳性分泌孔扩大培养并进行亚克隆。三次亚克隆后,将单克隆孔细胞扩大培养,进行杂交瘤细胞及单克隆抗体的鉴定,同时采用动物体内诱生的方法大量制备单克隆抗体。最后用Protein G SepharoseTM 4 Fast Flow(简称protein G)纯化腹水得到抗ICAM-1的单克隆抗体。 2.腹腔注射抗ICAM-1的单克隆抗体治疗Graves'病模型小鼠：6只,10ug/只,一共注射3次,每次间隔24小时,3天后检测,比较治疗前后小鼠血清T4和TRAb(以刺激性抗体TSAb为主)含量的变化,评价治疗效果。结果：1.利用纯品ICAM-1免疫小鼠三次,经三次基础免疫后小鼠血清中的抗体效价可达到1：10000。融合后一周左右,杂交瘤细胞可生长至1/3-1/2培养孔面积时,取上清液经ELISA检测抗体分泌情况,筛选出4个阳性孔,阳性OD405值均约2.0。将阳性孔扩大培养后进行三次亚克隆化,获得单克隆,扩大培养后进行杂交瘤细胞及单克隆抗体的鉴定。杂交瘤细胞染色体分析发现染色体数目众数为98-104。数目上接近两种亲代细胞染色体数目的总和,结构上多数为端着丝点染色体,还有少数亚中部着丝点染色体。4株细胞分泌的单克隆抗体的免疫球蛋白类别均为IgG1型。动物体内诱生的腹水中抗体效价均可达1:200000, Protein G纯化腹水上清效价均可达1：200000,纯化后的单抗蛋白浓度为1.2 mg/ml,并均可与ICAM-1特异性结合。 2.利用抗ICAM-1的单克隆抗体治疗Graves'病模型小鼠6只,比较治疗前后小鼠血清：1)T4治疗前为36.63±3.64,治疗后为20.12±2.15,两者比较为t=7.945,p=0.001,差别具有统计学意义；2)TRAb以刺激性抗体TSAb为主,治疗前为2.02±0.27,治疗后为0.54±0.14,两者比较为t=11.388,p=0,差别具有统计学意义。结论：1.成功地制备出4株抗ICAM-1的单克隆抗体,分别为：B9,F1,C11,D6. 2利用抗ICAM-1的单克隆抗体治疗Graves'病模型小鼠,有一定的治疗效果,为进一步的研究奠定了基础。
[Abstract]:Intercellular adhesion molecule 1 (intercellular adhesion molecule-1 ICAM-1) is a cell surface glycoprotein scFv, involved in antigen recognition, complement binding, cell adhesion function. Soluble intercellular adhesion molecule 1 (soluble intercellular adhesion molecule-1 sICAM-1) for cell surface ICAM-1 shedding formed, its expression is one of the important factors leading to Graves'disease, and the serum level of Graves' has an important significance for the judgment of disease withdrawal and relapse. Preparation of monoclonal antibodies against ICAM-1 using purified ICAM-1 immunized mice, which laid the foundation for the further treatment of Graves'disease, there will be some economic significance and Prospect of clinical application.
Objective: 1. the monoclonal antibodies with bioactive anti ICAM-1 were obtained from mice immunized with pure ICAM-1.
2. the anti ICAM-1 monoclonal antibody was used to treat the Graves'disease model mice.
Methods: 1. with pure ICAM-1. The purified ICAM-1 immunized animal protein and adjuvant intraperitoneal injection of BALB/C mice three times, each time interval of about 3 weeks, the mice medial venous blood samples showed high titers, were injected into tail vein of mice three days after booster immunization, spleen cells and bone marrow. The number of tumor cells in mixed 5:1, PEG (polyethylene glycol, polyethylene glycol) under the action of cell fusion. The cell growth by Kong Shangqing ELISA for detecting antibody secretion after a week, the positive hole secretion expanding culture and subcloned. Three sub cloning, cell culture expansion monoclonal antibody hybridoma, were identified. Cells and monoclonal, while using the method of animal in vivo induced a large number of monoclonal antibody. Finally Protein SepharoseTM 4 Fast G Flow (protein G) was purified from ascites monoclonal anti ICAM-1 Antibodies.
2. intraperitoneal injection of anti ICAM-1 monoclonal antibody was used to treat Graves'disease model mice: 6, 10ug/, only 3 times, 3 hours interval, 24 hours after interval. After 3 days, the changes of serum T4 and TRAb levels were evaluated before and after treatment.
Results: 1. using three pure ICAM-1 immunized mice, the antibody titer was three times of immunization in serum can be a week to reach 1:10000. after fusion, hybridoma cells can grow to 1/3-1/2 medium pore area, the supernatant was detected by ELISA antibody secretion, screened 4 positive holes, positive OD405 value all about 2.0. will be positive hole after culture expansion subcloning three times, obtained after identification of monoclonal expansion culture and monoclonal antibody hybridoma cell. Chromosome analysis of hybridoma cells showed the number of number of number of 98-104. close to two kinds of parent cell chromosome number sum, most of them were telocentric and there are a few submetacentric with monoclonal antibody acrocentric chromosome.4 strain cells of the immunoglobulin class was IgG1. The animal in vivo induced ascites antibody titer can reach 1 200000, the titer of purified ascites supernatant of Protein G can reach 1:200000, and the purified McAb protein concentration is 1.2 mg/ml, and it can be specifically combined with ICAM-1.
2. the use of anti ICAM-1 monoclonal antibody in the treatment of Graves'disease model of 6 mice were compared before and after treatment in serum of mice: 1) T4 before treatment was 36.63 + 3.64, 20.12 + 2.15 after treatment, compared to t=7.945, p=0.001, the difference was statistically significant; 2 TRAb) to stimulate antibody TSAb, before treatment was 2.02 after treatment was 0.54 + 0.27, + 0.14, compared to t=11.388, p=0, the difference was statistically significant.
Conclusion: 1. 4 monoclonal antibodies against ICAM-1 were successfully prepared, which were B9, F1, C11, D6., respectively.
2 the treatment of Graves'disease model mice by using anti ICAM-1 monoclonal antibody has certain therapeutic effect, which lays a foundation for further research.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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