Devepolment of a high sensitivity assay for activated coagulation fadctor-inhibitor complex and its diagnostic application to thrombosis

活化凝血因子-抑制剂复合物高灵敏度检测方法的开发及其在血栓形成中的诊断应用

• 批准号: 60480280
• 负责人: SAITO Hidehiko
• 金额: $ 3.39万
• 依托单位: Nagoya University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1985
• 资助国家: 日本
• 起止时间: 1985 至 1987
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-60480280/
• 关键词: Hypercoagulability; Activated factor IX; Antithrombin III; 活性【X】因子-アンチトロンビン【III】複合体; 活性【IX】因子-アンチトロンビン【III】複合体

An enzyme-linked immunosorbent Assay (ELISA) using activated Thiol-Sepharose has been developed for the quantitation of activated human factor IX-antithrombin III (IXa-AT III) complex. IXa-AT III complex is bound to a mini-column of thiol-Sepharose to whixh monospecific rabbit antibody against AT III was coupled. IXa-AT III complex is then quantified by a second antibody, rabbit anti-human factor IX Fab', labeled with <beta>-d-galactosidase. IXa-AT III complex was detected, when a final concentration of 5.0 ng[1.0 ug/ml of purified IXa-AT III complex was added to buffer or plasma. The minimum detectable amount of the complex (5 ng/ml) corresponds to activation of as little as 0.05% of plasma factor IX. Experiments with purified F.IXa, AT III, normal plasma and AT III-depleted plasma demonstrated the specificity of the assay. We measured Ixa-At III complex generation in clotting normal blood in glass tubes, and found that IXa-AT III complex appears within 60 sec after venipuncture. No detectable amount of the complex was observed in native whole blood from patients complex in 10 normal individuals were less than 5.0 ng/ml, wheresa those in 3 patients with disseminated in travascular coagulation (DIC) were more than 20 ng/ml. The ELISA using activated thiol-Sepharose described in this paper seems to provide a useful tool to detect activation of coagulation system in vitro as well as in vivo.

已开发出一种使用活化硫醇-琼脂糖的酶联免疫吸附测定 (ELISA)，用于定量活化人因子 IX-抗凝血酶 III (IXa-AT III) 复合物。 IXa-AT III 复合物与硫醇-Sepharose 微型柱结合，偶联抗 AT III 的单特异性兔抗体。然后通过用β-d-半乳糖苷酶标记的第二抗体、兔抗人因子IX Fab'来定量IXa-AT III复合物。当将终浓度为 5.0 ng[1.0 ug/ml 的纯化 IXa-AT III 复合物添加到缓冲液或血浆中时，检测到 IXa-AT III 复合物。复合物的最小可检测量 (5 ng/ml) 相当于激活低至 0.05% 的血浆因子 IX。使用纯化的 F.IXa、AT III、正常血浆和去除 AT III 的血浆进行的实验证明了该测定的特异性。我们测量了玻璃管中正常血液凝固过程中 Ixa-At III 复合物的生成，发现 IXa-AT III 复合物在静脉穿刺后 60 秒内出现。在10名正常个体的患者天然全血中未观察到可检测到的复合物含量，复合物含量低于5.0 ng/ml，而3名弥散性血管内凝血（DIC）患者的复合物含量超过20 ng/ml。本文中描述的使用活化硫醇-琼脂糖的 ELISA 似乎提供了一种有用的工具来检测体外和体内凝血系统的激活。

项目成果

Mukaiyama, H. ;Shionoya, S. ;Tkezawa, T. ;Kamiya, T. ;Hamaguchi, M. and Saito, H.: J. Vascular. Surg.6. 600-604 (1987)

Mukaiyama，H.；Shionoya，S.；Tkezawa，T.；Kamiya，T.；Hamaguchi，M. 和 Saito，H.：J. 血管。

Furui,H. Taniguchi,N. Yamauchi,K. Sotobata,I. Saito,H. and Inagaki,H.: "Effects of treadmill exercise on platelet with atrrial fibrillation." Jap. Heart. J.28. 177-184 (1987)

古井，H.

Kamiya, T. ;Sugihara, T. ;Ogata, K. ;Saito, H. ;Suzuki, K. ;Nishioka, J. ;Hashimoto, S. and Yamagata, K.: Blood. 67. 496-410 (1986)

Kamiya, T.；Sugihara, T.；Ogata, K.；Saito, H.；Suzuki, K.；Nishioka, J.；Hashimoto, S. 和 Yamagata, K.：血液。

Mukaiyama,H. Shionoya,S. Isezawa,T. Kamiya,T. Hamaguchi,M. and Saito,H.: "Abdominal aortic aneurysma complicated with chronic disseminated intravascular coagulopathy: a case of surgical treatment." J. Vascular. Surg.6. 600-604 (1987)

向山，H.

Ogura, M. ;Tanabe, N. ;Nishioka, J. ;Suzuki, K. and Saito, H.: Blood. 70. 301-306 (1987)

Ogura, M.；Tanabe, N.；Nishioka, J.；Suzuki, K. 和 Saito, H.：血液。