预期应用

ELISA法定量测定人血清、血浆或其它相关液体中eNOS含量。

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eNOS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eNOS抗原、生物素化的抗人eNOS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eNOS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 human eNOS concentrations in cell culture supernates, serum and plasma.

Introduction

Nitric oxide (NO) is produced by a group of enzymes called nitric oxide synthases (NOS). These enzymes catalyze the production of NO and L-citrulline from L-arginine, O2, and NADPH derived electrons. Mammalian systems contain three well-characterized isoforms of the enzyme: neuronal NOS (nNOS, also called NOS1), inducible NOS (iNOS or NOS2), and endothelial NOS (eNOS or NOS3).

The cell types that express eNOS include vascular endothelial cells, cardiomyocytes and others. In blood vessels, NO produced by the eNOS in endothelial cells functions as a vasodilator thereby regulating blood flow and pressure. Mutant eNOS knockout mice have blood pressure that is 30% higher than wild-type littermates. Within cardiomyocytes, eNOS affects Ca2+ currents and contractility. Expression of eNOS is usually reported to be constitutive, though modest degrees of regulation occur in response to factors such as shear stress, exercise, chronic hypoxia, and heart failure.The unique N-terminal sequence of eNOS is about 70 residues long and functions to localize the enzyme to membranes. Upon myristoylation at one site and palmitoylation at two other sites within this segment, the enzyme is exclusively membrane-bound. Palmitoylation is a reversible process that is influenced by some agonists and is essential for membrane localization. Within the membrane, eNOS is targeted to the caveolae, small invaginations characterized by the presence of proteins called caveolins. These regions serve as sites for the sequestration of signaling molecules such as receptors, G proteins and protein kinases. The oxygenase domain of eNOS contains a motif that binds to caveolin-1, and calmodulin is believed to competitively displace caveolin resulting in eNOS activation. Bound calmodulin is required for activity of eNOS, and this binding occurs in response to transient increases in intracellular Ca2+. Thus, eNOS occurs at sites of signal transduction and produces short pulses of NO in response to agonists that elicit Ca2+ transients. Physiological concentrations of eNOS-derived NO are in the picomolar range .Within the cardiovascular system, eNOS generally has protective effects. Studies with nNOS and eNOS knockout mice clearly indicate that eNOS plays a protective role in cerebral is chemia by preserving cerebral blood flow . During inflammation and atherosclerosis, low concentrations of NO prevent apoptotic death of endothelial cells and preserve the integrity of the endothelial cell monolayer. NO also acts as an inhibitor of platelet aggregation, adhesion molecule expression, and vascular smooth muscle cell proliferation.

 

Test principle

This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for eNOS has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any eNOS present is bound by the immobilized antibody. An enzyme-linked monoclonal antibody specific for eNOS 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 eNOS bound in the initial step. The color development is stopped and the intensity of the color is measured.