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。
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 aseites. 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-rhTFPI-1 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. Westem 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 effectively inhibits the activities of rhTFPI-1.
出处
《基础医学与临床》
CSCD
北大核心
2006年第11期1186-1190,共5页
Basic and Clinical Medicine
基金
国家十五科技攻关项目(2004BA720A06-02)
上海市重大科技攻关项目(04DZ19104)
上海市曙光计划基金(02SG05)
