【摘要】 目的研制抗重组人组织因子途径抑制物1单克隆抗体(rhTFPI-1 M cAb),了解其对rhTFPI-1抗凝活性的抑制作用。方法将分泌rhTFPI-1 M cAb的杂交瘤细胞注入经降植烷预处理的Balb/C小鼠腹腔中,诱生腹水。腹水先经硫酸铵沉淀处理,再用rprote in A亲和层析柱进一步纯化。对纯化后的单克隆抗体进行SDS-PAGE检测和W estern b lot分析。并用发色底物法测定rhTFPI-1 M cAb对rhTFPI-1的活性抑制。结果纯化后的抗rhTFPI-1 M cAb经SDS-PAGE检测,其纯度为98%;W estern b lot分析显示其能特异识别rhTFPI-1抗原。发色底物法检测结果表明,其能有效阻断rhTFPI-1的抗FVIIa/TF活性,M cAb浓度为8 mg/L时FVIIa/TF剩余活性达到86%;但对rhTFPI-1的抗FXa活性作用不明显,M cAb浓度为80 mg/L时FXa的剩余活性为48.4%。结论rhTFPI-1 M cAb可明显抑制rhT-FPI-1活性,阻碍rhTFPI-1对TF/FVIIa的抑制活性。纯化后的rhTFPI-1 M cAb的各项鉴定指标均较为理想,rprote inA亲和层析柱纯化方法简便易行值得推广。更多还原

【Abstract】 Objective To develop the monoclonal antibody for the anti-recombinant human tissue factor pathway inhibitor 1(rhTFPI-1) and to investigate its anticoagulation inhibition effects on rhTFPI.Methods We injected the hybridoma cells that secrete anti-rhTFPI-1 McAb to the Balb/C mouse pretreated with pristane to induce ascites.The ascites were purified with rprotein A affinity chromatograghy.The purified anti-rhTFPI-1 McAb was analyzed by SDS-PAGE and Western blot.The inhibition effects of anti-rhTFPI1 McAb on rhTFPI-1 were measured by chromophoric substrate assay.Results The purity of the anti-rhTFPI-1 McAb was 98% by SDS-PAGE analysis.Western blot demonstrated that the anti-rhTFPI-1 McAb specifically reacted with(rhTFPI-1.) The chromophoric substrate assay showed that the anti-rhTFPI-1 McAb could effectively block the(anti-TF/FVIIa) activity,but not the anti-FXa activity,of rhTFPI-1.The residual TF/FVIIa activity was up to(86% at (8 mg/L)) anti-rhTFPI-1 McAb,but the residual FXa activity was 48.4% at (80 mg/L) anti-rhTFPI-1 McAb.(Conclusion) The anti-rhTFPI-1 McAb effectivelyinhibits the activities of rhTFPI-1.更多还原