尿激酶抗人交联纤维蛋白-D-二聚体单抗化学偶合体的制备

Preparation of Conjugate of Urokinase With Monoclonal Antibody against Fragment D dimer of Human Cross linked Fibrin

利用双功能试剂Ｎ－琥珀酰亚胺－３（２－二硫吡啶）丙酸酯（ＳＰＤＰ）作交联剂，合成了尿激酶（ＵＫ）－抗人交联纤维蛋白降解物Ｄ－二聚体单抗（ＭＡ－ＨＩＤ１）化学偶合体（ＵＫＭＡ－ＨＩＤ１），并用苯甲脒－Ｓｅｐｈａｒｏｓｅ６Ｂ及人交联纤维蛋白降解物Ｄ－二聚体－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和柱纯化，获得偶合体产物．ＳＤＳ－ＰＡＧＥ呈现一条带，其分子量约为２０００００．纤维蛋白平板法测活结果显示，偶合体中酶比活为５３０００ＩＵ／ｍｇ尿激酶蛋白，与偶联前的５４３００ＩＵ／ｍｇ蛋白相仿．ＥＬＩＳＡ测试显示，偶合体对人交联纤维蛋白降解物Ｄ－二聚体有免疫反应性。

Urokinase (UK) and a murine monoclonal antibody(MA HID 1) directed against fragment D dimer of human cross linked fibrin were conjugated,using the cross linked agent N Succinimidyl 3(2 pyridylidithio)propionate(SPDP).The conjugate(UK·MA HID 1)was purified by affinity chromatography on Benzamidine Sepharose 6B and fragment D dimer Sepharose 4B columns.On SDS PAGE,it migrated as a main band with apparent M r of 200 000 .Specific activity on fibrin plates was 53 000 IU/mg urokinase protein as compared to 54 000 IU/mg protein for urokinase protein before conjugation.The results of the ELISA showed that the conjugate had immuno reactivity for fragment D dimer,and similar to reactivity of MA HID 1 for this antigen.