Fish Corticotropin releasing hormone (CRH) ELISA Kit

This assay employs the competitive enzyme immunoassay technique. The microtiter plate provided in this kit has been pre-coated with goat-anti-rabbit antibody. Standards or samples are added to the appropriate microtiter plate wells with an antibody specific for CRH and Horseradish Peroxidase (HRP) conjugated CRH. The competitive inhibition reaction is launched between with HRP labeled CRH and unlabeled CRH with the antibody. A substrate solution is added to the wells and the color develops in opposite to the amount of CRH in the sample. The color development is stopped and the intensity of the color is measured.

Hormones are made up of proteins, which act on other sites or organs of the body. They are usually produced by organs of the endocrine system, which functions in the maintenance of normal metabolism inside the body. A corticotropin-releasing hormone (CRH), previously known as corticotropin- releasing factor, is produced by the hypothalamus of the brain. This hormone then stimulates the front region of the pituitary gland, a pea-shaped gland situated just below the hypothalamus, to secrete another hormone called the adrenocorticotropic hormone (ACTH). In short, a corticotropin-releasing hormone is often needed for the production of ACTH. The hormone ACTH, in turn, goes to the adrenal glands, small glands situated on top of each kidney, to trigger the secretion of three other substances. These substances are the mineralocorticoids, glucocorticoids, and androgens.

This assay has high sensitivity and excellent specificity for detection of Fish CRH. No significant cross-reactivity or interference between Fish CRH and analogues was observed.

Matrices listed below were spiked with certain level of recombinant Fish CRH and the recovery rates were calculated by comparing the measured value to the expected amount of Fish CRH in samples.

Intra-assay Precision (Precision within an assay) 

Three samples of known concentration were tested twenty times on one plate to assess intra-assay precision.

Inter-assay Precision (Precision between assays) 

Three samples of known concentration were tested in forty separate assays to assess inter-assay precision.

CV (%) = SD/meanX100

Intra-Assay: CV<8% 

Inter-Assay: CV<12%

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Fish CRH and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

The stability of ELISA kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within the expiration date under appropriate storage condition. 

The loss rate was determined by accelerated thermal degradation test. Keep the kit at 37°C for 4 and 7 days, and compare O.D.values of the kit kept at 37°C with that of at recommended temperature. (referring from China Biological Products Standard, which was calculated by the Arrhenius equation. For ELISA kit, 4 days storage at 37°C can be considered as 6 months at 2 - 8°C, which means 7 days at 37°C equaling 12 months at 2 - 8°C).

Serum: Use a serum separator tube (SST) and allow samples to clot for two hours at room temperature or overnight at 2 - 8°C before centrifugation for 15 minutes at 1000 × g. Remove serum and assay immediately or aliquot and store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles.

Plasma: Collect plasma using EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 × g at 2 - 8°C within 30 minutes of collection. Assay immediately or aliquot and store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles.

Other biological fluids: Centrifuge samples for 20 minutes at 1000 × g. Remove particulates and assay immediately or store samples in aliquot at -20°C or -80°C. Avoid repeated freeze/thaw cycles.

Store at 2-8°C. Please refer to Instruction Manual.