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
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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。
Intercellular adhesion molecule-1 (ICAM-1) is a kind of glucoprotein on cell surface and concerned with recognition of antigen combination with complement and cell adhesion. ICAM-1 sheds from the cell surface to form the soluble intercellular adhesion molecule-1 (sICAM-1). The abnormal expression of sICAM-1 is one of the important factors in Graves' disease(GD) and the level of serumal sICAM-1 has significant value in judging the relapse of GD and stopping drug administration. If we could acquire the pure protein of monoclonal antibody against Intracellular adhesion molecule-1, we can use it to treat GD.It will have economics significance and clinical application prospect.
Objective:1.To prepare the monoclonal antibody against Intracellular adhesion molecule-1 with biological activity.
2. The use of monoclonal antibody against ICAM-1 to treat the mice model of Graves'.
Methods:1. BALB/C mice were immunized with ICAM-1 protein. After 4 times of immunization, the mice's spleen was removed and fused with myeloma cells. The secration wells were selected with ELISA and sub-clone was done. Hybridoma cells and monoclonal antibody were identified after 3 times of sub-clone. Monoclonal antibody was prepared in quantity after hybridoma cells was injected into the abdominal cavity of BALB/C mice.The ascites was purified by Protein G SepharoseTM 4 Fast Flow(Protein G).
2. The 6 mice of Graves'were treated with intraperitoneal injection of anti ICAM-1 mAb for 3 times, lOug per mouse.Compare the content of T4 and TRAb (mostly measures TSAb) to evaluate the therapeutic effect.
Results:l.The serum antibody titre can reach 1:10000 after three times of immunization.4 secration wells(OD405≈2.0) were obtained after fusion. Monoclonal antibody was prepared after 3 times of sub-clone and identified as IgGl. Chromosome analysis of hybridoma cells showed the mean number was 98-104, most of them were telocentric chromosome and a few were submetacentric chromosome. The titre of monoclonal antibody induced in the peritoneal fluid reached 1:200000 and reached a protein content of 1.2mg/ml, which bound to ICAM-1 specifically.
2.After the use of anti ICAM-1 mAb on mice model of Graves',we comepare the content of T4(prior-treatment is 36.63±3.64, post-treatment is 20.12±2.15,t=7.945, p=0.001) and TRAb which mostly measures TSAb (prior-treatment is 2.02±0.27, post-treatment is 0.54±0.14, t=11.388, p=0).The data suggested that the use of anti ICAM-1mAb could be of value in treatment on mice model of Graves'.
Conclusion:1.This study successfully prepared the monoclonal antibody against ICAM-1, which were B9, F1, C11 and D6.
2. The use of anti ICAM-1mAb could be of value in treatment on mice model of Graves', which laid a foundation of further study on its application.
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。
Intercellular adhesion molecule-1 (ICAM-1) is a kind of glucoprotein on cell surface and concerned with recognition of antigen combination with complement and cell adhesion. ICAM-1 sheds from the cell surface to form the soluble intercellular adhesion molecule-1 (sICAM-1). The abnormal expression of sICAM-1 is one of the important factors in Graves' disease(GD) and the level of serumal sICAM-1 has significant value in judging the relapse of GD and stopping drug administration. If we could acquire the pure protein of monoclonal antibody against Intracellular adhesion molecule-1, we can use it to treat GD.It will have economics significance and clinical application prospect.
Objective:1.To prepare the monoclonal antibody against Intracellular adhesion molecule-1 with biological activity.
2. The use of monoclonal antibody against ICAM-1 to treat the mice model of Graves'.
Methods:1. BALB/C mice were immunized with ICAM-1 protein. After 4 times of immunization, the mice's spleen was removed and fused with myeloma cells. The secration wells were selected with ELISA and sub-clone was done. Hybridoma cells and monoclonal antibody were identified after 3 times of sub-clone. Monoclonal antibody was prepared in quantity after hybridoma cells was injected into the abdominal cavity of BALB/C mice.The ascites was purified by Protein G SepharoseTM 4 Fast Flow(Protein G).
2. The 6 mice of Graves'were treated with intraperitoneal injection of anti ICAM-1 mAb for 3 times, lOug per mouse.Compare the content of T4 and TRAb (mostly measures TSAb) to evaluate the therapeutic effect.
Results:l.The serum antibody titre can reach 1:10000 after three times of immunization.4 secration wells(OD405≈2.0) were obtained after fusion. Monoclonal antibody was prepared after 3 times of sub-clone and identified as IgGl. Chromosome analysis of hybridoma cells showed the mean number was 98-104, most of them were telocentric chromosome and a few were submetacentric chromosome. The titre of monoclonal antibody induced in the peritoneal fluid reached 1:200000 and reached a protein content of 1.2mg/ml, which bound to ICAM-1 specifically.
2.After the use of anti ICAM-1 mAb on mice model of Graves',we comepare the content of T4(prior-treatment is 36.63±3.64, post-treatment is 20.12±2.15,t=7.945, p=0.001) and TRAb which mostly measures TSAb (prior-treatment is 2.02±0.27, post-treatment is 0.54±0.14, t=11.388, p=0).The data suggested that the use of anti ICAM-1mAb could be of value in treatment on mice model of Graves'.
Conclusion:1.This study successfully prepared the monoclonal antibody against ICAM-1, which were B9, F1, C11 and D6.
2. The use of anti ICAM-1mAb could be of value in treatment on mice model of Graves', which laid a foundation of further study on its application.
引文
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