Corticotropin-releasing hormone (CRH) is a 41-amino acid peptide derived from a 191-amino acid preprohormone. CRH is secreted by the paraventricular nucleus (PVN) of the hypothalamus in response to stress. Marked reduction in CRH has been observed in association with Alzheimer's disease, and autosomal recessive hypothalamic corticotropin deficiency has multiple and potentially-fatal metabolic consequences including hypoglycemia and hepatitis. In addition to being produced in the hypothalamus, CRH is also synthesized in peripheral tissues, such as T lymphocytes, and is highly expressed in the placenta. In the placenta, CRH is a marker that determines the length of gestation and the timing of parturition and delivery. A rapid increase in circulating levels of CRH occurs at the onset of parturition, suggesting that, in addition to its metabolic functions, CRH may act as a trigger for parturition.
Human corticotropin releasing hormone[CRH] ELISA Kit
<PRINCIPLE OF THE ASSAY>
The microtiter plate provided in this kit has been pre-coated with an antibody specific to CRH.Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for CRH and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB (3,3'5, 5' tetramethyl-benzidine) substrate solution is added to each well. Only those wells that contain CRH, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of CRH in the samples is then determined by comparing the O.D. of the samples to the standard curve.
This assay recognizes recombinant and natural humanCRH. No significant cross-reactivity or interference was observed.
0.16 ng/ml-10 ng/ml. The standard curve concentrations used for the ELISA’s were10 ng/ml, 5 ng/ml, 2.5 ng/ml, 1.25 ng/ml, 0.62 ng/ml, 0.32 ng/mlC0.16 ng/ml.
The minimum detectable dose of human CRH is typically less than 0.04 ng/ml. The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero.
|3.||Sample Diluent||1 x 20 ml|
|4.||Biotin-antibody Diluent||1 x 10 ml|
|5.||HRP-avidin Diluent||1 x 10 ml|
|6.||Biotin-antibodyt||1 x 120μl|
|7.||HRP-avidin||1 x 120μl|
|8.||Wash Buffer||1 x 20 ml (25×concentrate)|
|9.||TMB Substrate||1 x 10 ml|
|10.||Stop Solution||1 x 10 ml|