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Corticotropin-Releasing Hormone (CRH) Release Kit Instructions – CRH RIA Kit
Corticotropin-releasing hormone (CRH) is a key neuropeptide produced by hypothalamic neurons that plays a crucial role in the regulation of the stress response. It stimulates the release of proopiomelanocortin (POMC), which leads to the production of adrenocorticotropic hormone (ACTH) in the pituitary gland, ultimately influencing adrenal cortex activity. Beyond its central nervous system function, CRH is also synthesized and secreted by various peripheral tissues, including the placenta, amnion, chorion, decidua, rat thymus, spleen, and even human T-lymphocytes when stimulated by hemagglutinin. Human CRH shares 100% homology with its rat counterpart, both consisting of 41 amino acids, while differing from ovine CRH by seven residues.
The CRH RIA Kit includes the following components:
1. CRH Standards: Six vials containing concentrations of 0, 1.5, 5.0, 15.0, 45.0, and 135.0 ng/ml.
2. 125I-CRH: One vial for radiolabeling.
3. CRH Antibody: One vial for specific binding.
4. Buffer: One vial for dilution and sample preparation.
5. Separating Agent (Second Antibody): One vial for precipitation of immune complexes.
Sample Preparation:
For plasma collection: Draw 3 ml of venous blood into a tube containing 0.5 M EDTA-Na2 (30 μl), mix thoroughly, centrifuge, and collect the plasma for measurement. If stored at -20°C, samples can remain stable for up to 2–3 months without affecting results.
For tissue extraction: Take an appropriate amount of tissue, weigh it immediately, and boil in 1 ml of saline for 3 minutes. Add 0.5 ml of 1N glacial acetic acid, homogenize, neutralize with 0.5 ml of 1N NaOH, and centrifuge at 3000 rpm for 30 minutes (preferably using a cryogenic centrifuge). Store the supernatant at -20°C, and samples can be kept for 2–3 months before testing.
Assay Procedure:
- Add 200 μl of buffer to each well.
- Add 100 μl of standards, control, or samples.
- Add 100 μl of CRH antibody to each well.
- Add 100 μl of 125I-CRH.
- Incubate overnight at 4°C.
- Add 500 μl of separating agent and incubate at room temperature for 15 minutes.
- Centrifuge at 3500 rpm for 15 minutes.
- Remove the supernatant and measure the radioactivity in the pellet (cpm).
Data Analysis:
Calculate the percentage of bound radioactivity relative to total (B/T) and non-specific binding (NSB). Plot the standard curve on semi-logarithmic paper using B/B0 values. Determine the concentration of CRH in the samples based on the standard curve or directly via automated gamma counter.
Notes:
- Each CRH standard should be reconstituted with 0.8 ml of buffer.
- The CRH antibody should be diluted with 5.0 ml of buffer.
- The 125I-CRH should be reconstituted with 5.5 ml of buffer.
Technical Specifications:
- Sensitivity: ≤ 3 pg/ml
- Intra-assay Coefficient of Variation (CV): < 10%
This kit is ideal for research purposes in endocrinology, neurobiology, and stress-related studies. For more information, visit the booth of Shanghai Xinfan Biotechnology Co., Ltd. or download the full instructions in PDF format.
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