预期应用
ELISA法定量测定大鼠血清、血浆、脑部提取物中CRH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CRH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CRH、生物素化的抗大鼠CRH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CRH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 rat CRH concentrations in serum, plasma and brain tissue extract samples.
Introduction
Corticotropin releasing factor (CRF, also CRH) was initially isolated from ovine hypothalamus by Vale et al., in 1981, and identified as a novel neuropeptide comprising 41 amino acid residues with molecular weight 4758. Later human CRF and rat CRF were also isolated and identified. The mouse CRF peptide is identical at amino acid level to the rat and human CRF peptides. CRF in anterior pituitary promotes the synthesis and secretion of ACTH, a main factor of hypothalamus-pituitary-adrenal (HPA) axis. In the rat and human, CRF distributes mainly in hypothalamus, but it was also found in spinal cord, stomach, spleen, duodenum, adrenal and placenta. In addition, immunochemical evidence supported the wide distribution of the peptide throughout the central nervous system (CNS) such as olfactory bulb, retina and central auditory system in the rat.
In mouse brain extracts, the highest concentrations of CRF-like immunoreactivity (CRF-LI) has been detected in the median eminence and hypothalamus and also existing in the amygdala, thalamus, frontal cortex, medulla/pons and cerebellum by radioimmunoassay. However because of the wide distribution, it is still disputing about CRF whether its blood level can reflect only the function of HPA axis.
The relationships between CRF and stress, CRF and Alzheimer disease (AD) were attracted much attention recently. In fact the peptide was also suggested to regulate endocrine, autonomic and behavioral responses to stress, based on an experiment with acute and chronic stress rat models that showed endocrine function changes similar to those seen in patients with depression CRF in serial cerebrospinal fluid（CSF）of patients with depression was strikingly reduced as compared to those of normal subjects. The mean CRF and ACTH levels in the CSF of AD patients were significantly lower than those of healthy controls. Only in the cortices of those with mild dementia, CRF was reduced significantly, thus CRF was proposed to serve as a potential neurochemical marker of early dementia and possible early AD.
A large proportion of the CRF in human brain was shown to be in the form of complex with its binding protein (CRF-BP). CRF molecule in the complex is unavailable for activation of the CRF receptor. Accordingly reductions in total CRF do not necessarily predict reductions of bioactive free CRF, and the levels of total CRF and CRF in the form of complex (CRF/CRF-BP) were suggested to be the main factors determining the quantity of bioactive free CRF in human brain. In AD there have been observed dramatic reductions in the content of free CRF in the brain and thus displacement of CRF from CRF-BP was proposed as a possible treatment for AD. In primary neuron culture, CRF exhibited protective effect against cell death induced by amyloid-beta peptide, suggesting that disturbances in HPA axis function can occur independently of alteration in CRF mRNA levels in AD brain and further suggesting an additional role for CRF in protecting neurons against cell death. On the other hand, Yanaihara et al., demonstrated immunoreative CRF in various neuroendocrine tumors, and suggested that the blood level of the peptide might be used as a tumor marker.
Test principle
This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for CRH has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any CRH present is bound by the immobilized antibody. An enzyme-linked monoclonal antibody specific for CRH 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 CRH bound in the initial step. The color development is stopped and the intensity of the color is measured.
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