预期应用
ELISA法定量测定大鼠血清、血浆、唾液、细胞培养物上清或其它相关液体中MMP-9含量。
概述
MMP-9基因位于染色体20q11.1~13.1，26~27kbp，具有13个外显子和9个内含子。哺乳类动物MMP-9mRNA的同源性约为80%。MMP-9的启动子区内含有活化蛋白(AP)-1、AP-2和刺激蛋白(SP)-1因子结合位点，大鼠的MMP-9启动子区还有核因子(NF)κB、ETS结合位点和转化生长因子(TGF)-β抑制元件MMPs主要由3个独特的、保守的结构域构成，包括前肽区(氨基末端区)、催化区和羧基末端区(类血红素结合蛋白酶区)。MMP-9是明胶酶中的一种，除了有MMPs的原型结构外，MMP-9的催化区还包括3个重复的型纤维连接蛋白结构域，这个结构域与明胶或弹性蛋白有高度的亲和力。MMP-9包含一个V型的胶原蛋白结构域,这个结构域有高度的糖基化作用，,它影响底物的特异性以及有抗衰变的作用。MMP-9有许多作用底物，如Ⅳ、Ⅴ、Ⅶ、Ⅹ、Ⅺ型胶原、蛋白聚糖的核心蛋白、明胶、纤维粘连蛋白、层粘连蛋白、弹性蛋白等，细胞因子及其受体也是MMP-9作用的底物。
MMP-9是以酶原的形式从胞内分泌到胞外，在体外，MMP-9通过有机汞制剂反应才有活性，在体内则可经一系列蛋白酶级联而激活。这个胶原蛋白结构域可被MMP-3、MMP-2或者次氯酸裂解，而不是纤维蛋白溶酶、凝血酶或者MMP-1。MMP-3可能是MMP-9最有效的激活剂。MMP-9主要的循环抑制剂是а2巨球蛋白，活化的MMP-9被а2巨球蛋白捕获并通过清除受体被清除出循环系统。金属蛋白酶组织抑制因子(TIMPs)是MMPs家族的组织抑制剂，广泛分布于组织和体液中，共价结合MMPs而抑制其活性。TIMP-1作为MMP-9的抑制剂，与MMP-9的酶原或活化后酶的催化区的羧基末端特异性结合，形成复合物，,从而特异性抑制MMP-9的活性。另外，血小板反应蛋白和蛋白酶组织抑制因子2(TFPI-2)也能抑制MMP-9的活性。
MMP-9的生理作用广泛。MMP能分解呼吸道和肺内的结构复合物如ECM和基底膜,因此能参与呼吸道和肺的重建，它还能调节其他蛋白酶及细胞因子的活性，它能降解α1抗胰蛋白酶，保护中性粒细胞弹性蛋白酶活性,能加强胶原质胶体中胶原细胞和MMP-13的溶胶原活动，并且MMP-9还能从白细胞介素8(CXCL8/CL8)上分解一个62氨基酸肽,使其向中性粒细胞的趋化活性增加10倍，但它也抑制其他中性粒细胞的趋化因子。此外，MMP-9结合CD44可释放储存的TGF-β1，另外，MMP-9可通过释放血管内皮生长因子(VEGF)以参与血管生成。
实验原理
本试剂盒应用双抗体夹心酶标免疫分析法测定标本中MMP-9水平。用纯化的抗体包被微孔板，制成固相抗体，往包被单抗的微孔中依次加入MMP-9抗原、生物素化的抗大鼠MMP-9抗体、HRP标记的亲和素，经过彻底洗涤后用底物TMB显色。TMB在过氧化物酶的催化下转化成蓝色，并在酸的作用下转化成最终的黄色。颜色的深浅和样品中的MMP-9呈正相关。用酶标仪在450nm波长下测定吸光度（OD值），计算样品浓度。
Intended use
This immunoassay kit allows for the specific measurement of total rat active and pro-Matrix Metalloproteinase 9 (total MMP-9) concentrations in cell culture supernates, serum, and plasma.
Introduction
Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that degrade extracellular matrix proteins. They are secreted as zymogens (pro-MMPs) that are activated by a variety of proteinases or by reaction with organic mercurials. They are inhibited by specific tissue inhibitors of metalloproteinases (TIMPs) and by α2-macroglobulin. The regulation of MMP activity is important in tissue remodeling, inflammation, tumor growth and metastasis.
Rat MMP-9 (also known as gelatinase B) is secreted as a 92 kDa zymogen. Cleavage of pro-MMP-9 at or near residue 87 results in the active enzyme with a mass of approximately 82 kDa. MMP-9 has three fibronectin type II domains, a hemopexin-like domain and a proline-rich type V collagen-homologous domain. Pro-MMP-9 can be activated by MMP-3 or by certain bacterial proteinases. MMP-9 is inhibited byα2-macroglobulin or by TIMP-1, which binds to pro-MMP-9 as well as to active MMP-9.
Pro-MMP-9 is secreted by monocytes, macrophages, neutrophils, keratinocytes, fibroblasts, osteoclasts, chondrocytes, skeletal muscle satellite cells, endothelial cells, and various tumor cells. Pro-MMP-9 expression is upregulated by TGF-β1, IL-1β, TGF-α, PDGF-AB, TNF-α and IL-1α. Substrates for MMP-9 include denatured collagen type I (gelatin), native collagens type IV, V, VII, X and XI, fibrinogen, vitronectin, IL-1β and entactin, a molecule that bridges laminin and type IV collagen.
MMP-9 is involved in inflammation, tissue remodeling, wound healing, mobilization of matrix-bound growth factors and processing of cytokines. Its expression correlates with the desmoplasia (abnormal collagen deposition) that accompanies pancreatic cancer, with the metastasis to lymph nodes by rat breast carcinoma cells and with the invasion of regional vessels in giant cell tumors of bones. MMP-9 may be elevated in gingival crevicular fluid and saliva in patients with gingivitis and periodontal diseases.
Test principle
This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for MMP-9 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any MMP-9 present is bound by the immobilized antibody. An enzyme-linked monoclonal antibody specific for MMP-9 is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of total MMP-9 bound in the initial step. The color development is stopped and the intensity of the color is measured. | 
