Description of Target: Product Name: Cortisol
# of Samples: 1 x 96 Assays
Intended Use: Competitive immunoenzymatic colorimetric method for quantitative determination of Cortisol in human serum or plasma.
Introduction: Cortisol is a steroid hormone released from the adrenal cortex in response to a hormone called ACTH (produced by the pituitary gland), it is involved in the response to stress; it increases blood pressure, blood sugar levels, may cause infertility in women, and suppresses the immune system.
Cortisol acts through specific intracellular receptors and has effects in numerous physiologic systems, including immune function, glucose-counter regulation, vascular tone, substrate utilization and bone metabolism. Cortisol is excreted primarily in urine in an unbound (free) form.
Cortisol is bound with high affinity in plasma from corticosteroid-binding globulin (CBG, transcotin) and from albumin. Only free cortisol is available to most receptors.
The amount of cortisol present in the serum undergoes diurnal variation, with the highest levels present in the early morning, and lower levels in the evening, several hours after the onset of sleep. Highest levels are at about 6 – 8 a.m. and lowest levels are at about midnight. These normal endogenous functions are basis for the physiological consequences of chronic stress- prolonged cortisol secretion causes muscle wastage, hyperglycaemia, and suppresses immune/ inflammatory responses. The same consequences arise from long-term use of glucocorticoid drugs.
Principles of the assay: Microtiter strip wells are precoated with anti-Cortisol antibodies (solid-phase). Cortisol in the sample competes with added horseradish peroxidase labelled Cortisol (enzyme-labelled antigen) for antibody binding. After incubation a bound/free separation is performed by solid-phase washing. The immune complex formed by enzyme-labelled antigen is visualized by adding Tetramethylbenzidine (TMB) substrate which gives a blue reaction product. The intensity of this product is inversely proportional to the amount of Cortisol in the sample. Sulphuric acid is added to stop the reaction. This produces a yellow endpoint colour. Absorption at 450 nm is read using an ELISA microwell plate reader.
Storage and Stability: The reagents are stable up to the expiry date stated on the label when stored at 2...8 C in the dark.
Limitations of the Test: Sample(s), which are contaminated microbiologically, should not be used in the assay. Highly lipemic or haemolysed specimen(s) should similarly not be used. It is important that the time of reaction in each well is held constant for reproducible results. Pipetting of samples should not extend beyond ten minutes to avoid assay drift. If more than one plate is used, it is recommended to repeat the dose response curve. Addition of the substrate solution initiates a kinetic reaction, which is terminated by the addition of the stop solution. Therefore, the addition of the substrate and the stopping solution should be added in the same sequence to eliminate any time deviation during reaction. Plate readers measure vertically. Do not touch the bottom of the wells. Failure to remove adhering solution adequately in the aspiration or decantation wash step(s) may result in poor replication and spurious results.
References Foster, L. B. and Dunn, R.T. (1974) Clin. Chem 20 (3), 365.De Lacerda, L., Kowarski, A., and Migeon, C.J. (1973) J. Clin. Endocr. and Metab 36, 227.Rolleri, E., Zannino, M., Orlandini, S. and Malvano, R. (1976) Clin chim Acta 66, 319.Kobayashi, Y. et al. (1978) Steroids 32 (1), 137 – 44.Arakawa, H., Maeda, M., Tsuji, A. (1979) Anal. Biochem. 97, 248.
English
Deutsch
