本试剂盒应用双抗体夹心酶标免疫分析法测定标本中H-ras水平。用纯化的抗体包被微孔板，制成固相抗体，往包被抗体的微孔中依次加入H-ras、生物素化的抗人H-ras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H-ras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 H-ras concentrations in cell culture supernates, serum, and plasma.
Introduction
RAS is a G protein (specifically a small GTPase): a regulatory GTP hydrolase that cycles between two conformations–an activated or inactivated form, respectively RAS-GTP and RAS-GDP.
H-Ras is well known as one of the essential components of Ras/Raf/MEK/ERK cascade, which is a critical prosurvival signaling mechanism in most eukaryotic cells. Ras targets Raf/MEK/ERK cascade by integrating and transmitting extracellular signals from growth factor receptors to Raf, leading to the propagation of signals to modulate a serious of cellular survival events. Apoptosis signal-regulating kinasel (ASK1) serves as a general mediator of cell death because it is responsive to a variety of death signals.
It is activated by guanine exchange factors (GEFs, eg. CDC25, SOS1 and SOS2, SDC25 in yeast), which are themselves activated by mitogenic signals and through feedback from Ras itself. A GEF usually heightens the dissociation rate of the nucleotide – while not changing the association rate (effectively lower the affinity of the nucleotide)–thereby promoting its exchange. The cellular concentration of GTP is much higher than that of GDP so the exchange is usually GDP vs. GTP.It is inactivated by GTPase-activating proteins (GAPs, the most frequently cited one being RasGAP), which increase the rate of GTP hydrolysis, returning RAS to its GDP-bound form, simultaneously releasing an inorganic phosphate.
Test principle
This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal
antibody specific for H-ras has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any H-ras present is bound by the immobilized antibody. An enzyme-linked polyclonal antibody specific for H-ras 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 H-ras bound in the initial step. The color development is stopped and the intensity of the color is measured.
