Two-step sandwich enzyme immunoassay using monoclonal antibodies for detection of soluble and membrane-associated human membrane type 1-matrix metalloproteinase
Two-step sandwich enzyme immunoassay using monoclonal antibodies for detection of soluble and membrane-associated human membrane type 1-matrix metalloproteinase
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DOI：
10.1081/ias-120002274
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发表时间：
2002-02-01
影响因子：
--
通讯作者：
Seiki, M
中科院分区：
文献类型：
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作者：
Aoki, T;Yonezawa, K;Seiki, M
应用两种抗重组人膜基质金属蛋白酶(MT1-MMPs)的单抗，建立了检测MT1-MMP二步夹心酶免疫分析(EIA)体系。标本与固相抗体反应后，用过氧化物酶标记的第二抗体进行检测。该系统的检出限为1.25 ng/m L，线性范围为1.25-160 ng/m L。该EIA系统是MT1-MMP特异性的，与其他几种检测的MMPs没有交叉反应。该系统还检测到一些癌细胞株将可溶性MT1-MMPs释放到培养液中。而正常人和癌症患者血清中可溶性MT1-MMP值均低于检测限。用含有洗涤剂的提取缓冲液增溶癌细胞后，也检测到膜相关的MT1-MMPs。此外，还检测了临床标本中的MT1-MMPs。癌组织匀浆中MT1-MMP值高于正常组织，且肿瘤组织中MT1-MMP值与区域淋巴结转移率相关。因此，我们证明了该EIA系统是第一个在临床标本中检测MT1-MMPs的方法，从而表明它对癌症的诊断或恶性肿瘤的预测是有用的。
A two-step sandwich enzyme immunoassay (EIA) system for the detection of human membrane Type 1-matrix metalloproteinase (MT1-MMP) was established by using two monoclonal antibodies against recombinant MT1-MMP. MT1-MMP in which samples were reacted with solid-phase antibody and then detected with peroxidase-labeled second antibody. At least 1.25 ng/mL was detected by the EIA system, and linearity was obtained between 1.25 and 160 ng/mL. This EIA system is specific for MT1-MMP and did not show cross-reactivity against several other MMP's examined. Shedding of soluble MT1-MMP into the medium by some cancer cell lines was also detected by this system. However, soluble MT1-MMP in serum from normal and cancer patients was under the detection limit. Membrane-associated MT1-MMP of cancer cell lines was also detected after solubilization of the membranes with extraction buffer containing detergent. Additionally, MT1-MMP in clinical samples was examined. Elevated levels of MT1-MMP were detected in homogenate of cancer tissue compared with the levels for normal tissue and the level of MT1-MMP in tumors correlated with the rate of metastasis to the regional lymph nodes. Thus, we demonstrated that this EIA system is the first to measure MT1-MMP in clinical specimens, thus suggesting its useful for diagnosis of cancer or prediction of malignancy.