A ONE-STEP SANDWICH ENZYME-IMMUNOASSAY FOR TISSUE INHIBITOR OF METALLOPROTEINASES-2 USING MONOCLONAL-ANTIBODIES
A ONE-STEP SANDWICH ENZYME-IMMUNOASSAY FOR TISSUE INHIBITOR OF METALLOPROTEINASES-2 USING MONOCLONAL-ANTIBODIES
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DOI：
10.1016/0009-8981(93)90004-n
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发表时间：
1993-10-29
影响因子：
5
通讯作者：
HAYAKAWA, T
中科院分区：
文献类型：
--
作者：
FUJIMOTO, N;ZHANG, J;HAYAKAWA, T
使用一对单克隆抗体开发了一步夹心酶免疫测定系统，该抗体针对由人类组织金属蛋白酶抑制剂 2 (TIMP-2) 的氨基酸序列制备的两个单独的寡肽。该测定系统由两个同时进行的免疫反应组成，使用固相单克隆抗体和辣根过氧化物酶标记的单克隆抗体。该系统检测到游离形式的 TIMP-2 以及与活性形式的基质金属蛋白酶 (MMP) 复合的 TIMP-2，这对每种 MMP 具有不同的敏感性，但未检测到与 72 kDa 明胶酶/IV 型胶原酶 (MMP-2) 前体复合的 TIMP-2。系统的灵敏度为 1.6 mug/l pg/测定），线性度在 6.3 至 50 mug/l（63-500 pg/测定）之间。 20 名类风湿性关节炎患者（68 +/- 25 mugl，平均值+/- S.D.）和 13 名肝细胞癌患者（76 +/- 46 mug/l）血清中 TIMP-2 水平显着高于（P < 0.05）18 名正常受试者（5.6 +/- 7.4 mug/1）。相比之下，10名胃癌患者（45±18μg/l）和7名子宫癌患者（36±13μg/l）的血清中该水平显着低于正常受试者（P<0.05或P<0.01）。 免疫反应性分析表明，正常血清中MMP-2的前体通过与TIMP-2的C端结构域相互作用，以与TIMP-2复合的形式存在。
A one-step sandwich enzyme immunoassay system was developed with a pair of monoclonal antibodies against two individual oligopeptides prepared from the amino acid sequence of the human tissue inhibitor of metalloproteinases-2 (TIMP-2). The assay system consisting of two simultaneous immunoreactions used a solid phase monoclonal antibody and a horseradish peroxidase-labeled monoclonal antibody. The system detected a free form of TIMP-2 and that complexed with active forms of matrix metalloproteinases (MMPs) giving a different sensitivity for each MMP but not TIMP-2 complexed with the precursor of 72 kDa gelatinase/type IV collagenase (MMP-2). The sensitivity of the system was 1.6 mug/l pg/assay) and linearity was obtained between 6.3 and 50 mug/l (63-500 pg/assay). TIMP-2 levels in the sera of 20 patients with rheumatoid arthritis (68 +/- 25 mugl, mean +/- S.D.) and 13 patients with hepatocellular carcinoma (76 +/- 46 mug/l) were significantly higher (P < 0.05) than those of 18 normal subjects (5.6 +/- 7.4 mug/1). In contrast, the levels in the sera of 10 patients with gastric cancer (45 +/- 18 mug/l) and 7 patients with cancer of the uterus (36 +/- 13 mug/l) were significantly lower (P < 0.05 or P < 0.01) than those of normal subjects. Immunoreactivity analyses suggested that the precursor of MMP-2 in normal sera exists in a complexed form with TIMP-2 by interacting with the C-terminal domain of TIMP-2.