Bode C, Runge M S, Schönermark S, Eberle T, Newell J B, Kübler W, Haber E
Medizinische Klinik III (Kardiologie), Universität Heidelberg, FRG.
Circulation. 1990 Jun;81(6):1974-80. doi: 10.1161/01.cir.81.6.1974.
Single-chain urokinase plasminogen activator (scu-PA) that had been modified with N-succinimidyl-3-(2-pyridyldithio)propionate was covalently linked by disulfide bonds to the Fab' of a monoclonal antibody specific for the beta-chain of fibrin (antibody 59D8). scu-PA-59D8 Fab' conjugate was separated from free scu-PA and two-chain urokinase coupled to 59D8 Fab' by two-step affinity chromatography. First, the reaction mixture was chromatographed on a column containing Sepharose linked to the peptide that had been used as immunogen for antibody 59D8; scu-PA-59D8 Fab' conjugate and unconjugated 59D8 Fab' were retained but not unconjugated scu-PA. Then, the eluate from the peptide-Sepharose column was chromatographed on a column containing Sepharose linked to benzamidine, which acts as a ligand for two-chain urokinase. The molecular weight of the scu-PA-59D8 Fab' conjugate was approximately 100 kDa when electrophoresed on a nonreducing sodium dodecylsulfate-polyacrylamide gel. Enzymatic assay after purification revealed that more than 97% of the scu-PA present in the conjugate retained the single-chain form. The Fab' portion of the conjugate functioned in a manner indistinguishable from that of native antibody 59D8. In an in vitro assay for lysis of fibrin monomer, the fibrinolytic potency of scu-PA-59D8 Fab' was 33-fold more than that of tissue plasminogen activator (p less than 0.001), 230-fold more than that of unconjugated scu-PA (p less than 0.0001), and 420-fold more than that of urokinase (p less than 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)
用N-琥珀酰亚胺基-3-(2-吡啶二硫基)丙酸修饰的单链尿激酶型纤溶酶原激活剂(scu-PA)通过二硫键与针对纤维蛋白β链的单克隆抗体的Fab'共价连接(抗体59D8)。通过两步亲和层析将scu-PA-59D8 Fab'缀合物与游离scu-PA以及与59D8 Fab'偶联的双链尿激酶分离。首先，将反应混合物在含有与用作抗体59D8免疫原的肽连接的琼脂糖的柱上进行层析；scu-PA-59D8 Fab'缀合物和未缀合的59D8 Fab'被保留，但未缀合的scu-PA没有。然后，将来自肽-琼脂糖柱的洗脱液在含有与苯甲脒连接的琼脂糖的柱上进行层析，苯甲脒作为双链尿激酶的配体。在非还原十二烷基硫酸钠-聚丙烯酰胺凝胶上电泳时，scu-PA-59D8 Fab'缀合物的分子量约为100 kDa。纯化后的酶活性测定表明，缀合物中存在的scu-PA超过97%保持单链形式。缀合物的Fab'部分的功能与天然抗体59D8的功能没有区别。在纤维蛋白单体溶解的体外测定中，scu-PA-59D8 Fab'的纤溶活性比组织纤溶酶原激活剂高33倍(p<0.001)，比未缀合的scu-PA高230倍(p<0.0001)，比尿激酶高420倍(p<0.0001)。(摘要截断于250字)
