Preparation,Identification And Preliminary Application Of Monoclonal Antibody Against Intercellular Adhesion Molecule-1

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.