具有抗凝活性抗人组织因子单克隆抗体的制备与鉴定

Preparation and characterization of a monoclonal antibody against human tissue factor with anticoagulation activity

目的制备并鉴定具有抗凝血活性的抗人组织因子(h TF)单克隆抗体(m Ab)。方法以截短型重组h TF243蛋白免疫BALB/c小鼠,细胞融合后,采用间接ELISA联合血浆凝血酶原时间(PT)测定方法,筛选具有抗凝血活性的抗h TF m Ab的杂交瘤细胞株,并进行效价测定和特异性鉴定。结果成功建立1株可稳定分泌具有抗凝血活性的抗h TF m Ab的杂交瘤细胞株,该m Ab属于Ig G1亚型。间接ELISA测定腹水效价为1∶200 000,Western blot、PT测定结果表明,该m Ab可特异性结合重组h TF243,同时与SW620结肠癌细胞表面天然TF作用,显著延长血浆凝血酶原时间,具有良好的抗凝血活性。结论成功制备了具有抗凝血活性的抗h TF m Ab。

Objective To prepare and characterize a monoclonal antibody（ m Ab） against human tissue factor（ h TF） with anticoagulation activity. Methods BALB / c mice were immunized with truncated recombinant protein（ rh TF243）. Hybridoma cell lines were generated from cell fusion,and screened using indirect ELISA and prothrombin time（ PT）. After ascites was developed in BALB / c mice,antibody titers were determined using indirect ELISA. Western blotting was performed to study the antibody specificity. Anticoagulant activity of the antibody was detected by PT assay. Results A m Ab to h TF with excel ent anticoagulation activity was identified. Its immunoglobulin subclass belonged to Ig G1. Titer of ascites fluid was 1 ∶ 200 000.Western blotting and PT analysis confirmed the specificity and anticoagulant activity of the antibody. The m Ab reacted specifically to both recombinant h TF243 and natural TF on SW620 colon cancer cell surface. Conclusion A h TF m Ab with anticoagulation activity and high specificity has been successfully prepared.