Cortisol ELISA Kit

Catalog No. ABIN649057

Product Details Cortisol ELISA Kit

Target: Cortisol

Reactivity: Human

Detection Method: Colorimetric

Method Type: Competition ELISA

Application: ELISA

Purpose: Competitive Enzyme Immunoassay (TYPE 7): The essential reagents required for a enzyme immunoassay include antibody, enzyme-antigen conjugate and native antigen. Upon mixing biotinylated antibody, enzyme-antigen conjugate and a serum containing the native antigen, a competition reaction results between the native antigen and the enzyme-antigen conjugate for a limited number of antibody binding sites. The enzyme activity in the antibodybound fraction is inversely proportional to the native antigen concentration. By utilizing several different serum references of known antigen concentration, a dose response curve can be generated from which the antigen concentration of an unknown can be ascertained.

Analytical Method: Quantitative

Components: A. Cortisol Calibrators (1ml/vial). Six vials of serum reference for Cortisol at concentrations of 0 (A), 1. 0 (B), 4. 0 (C), 10. 0 (D), 20. 0 (E) and 50. 0 (F) µg/dl. Store at 2-8°C. A preservative has been added. B. Cortisol Enzyme Reagent (1. 0 ml/vial). One vial of Cortisol (Analog)-horseradish peroxides (HRP) conjugate in a protein stabilizing matrix with green dye. Store at 2-8°C. C. Steroid Conjugate Buffer (7. 0 ml/vial). One vial reagent containing buffer, red dye, preservative, and binding protein inhibitors. Store at 2-8°C. D. Cortisol Biotin Reagent (7. 0 ml). One bottle reagent containing anti-cortisol biotinylated mIgG conjugate in buffer, green dye and preservative. Store at 2-8°C. E. Streptavidin Coated Plate (96 wells). One 96-well microplate coated with 1. 0 µg/ml streptavidin and packaged in an aluminum bag with a drying agent. Store at 2-8°C. F. Wash Concentrate Solution (20ml). One vial containing a surfactant in buffered saline. A preservative has been added. Store at 2-30°C. G. Substrate A (7ml/vial). One vial containing tetramethylbenzidine (TMB) in buffer. Store at 2-8°C. H. Substrate B (7ml/vial). One vial containing hydrogen peroxide (H2O2) in buffer. Store at 2-8°C. I. Stop Solution (8ml/vial). One vial containing a strong acid (1N HCl). Store at 2-30°C. J. Product Instructions: Note 1: Do not use reagents beyond the kit expiration date. Note 2: Opened reagents are stable for 60 days when stored at 2-8°C. Note 3: Above reagents are for a single 96-well microplate.

Material not included: 1. Pipette capable of delivering 25µl, 50µl, and 100µl volumes with a precision of better than 1. 5%. 2. Dispenser(s) for repetitive deliveries of 0. 100ml and 0. 350ml volumes with a precision of better than 1. 5%. 3. Adjustable volume (200-1000µl) Dispenser(s) for conjugate. 4. Microplate washer or a squeeze bottle (optional). 5. Microplate Reader with 450nm and 620nm wavelength absorbance capability. 6. Absorbent Paper for blotting the microplate wells. 7. Plastic wrap or microplate cover for incubation steps. 8. Vacuum aspirator (optional) for wash steps. 9. Timer. 10. Quality control materials.

Application Details

Application Notes: Precautions: All products that contain human serum have been found to be non-reactive for Hepatitis B Surface Antigen, HIV 1&2 and HCV Antibodies by FDA required tests. Since no known test can offer complete assurance that infectious agents are absent, all human serum products should be handled as potentially hazardous and capable of transmitting disease. Good laboratory procedures for handling blood products can be found in the Center for Disease Control / National Institute of Health, Biosafety in Microbiological and Biomedical Laboratories, 2nd Edition, 1988, HHS Publication No. (CDC) 88-8395.

Sample Volume: 10 μL

Plate: Pre-coated

Reagent Preparation:

1. Working Enzyme Reagent - Stable for 1 year: Measure 0. 7 ml of Cortisol Enzyme Reagent and add to the vial containing Steroid Conjugate Buffer. Store at 2-8°C. 2. Wash Buffer: Dilute contents of wash solution to 1000ml with distilled or deionized water in a suitable storage container. Store at room temperature 20-27 °C for up to 60 days. 3. Working Substrate Solution: Pour the contents of the amber vial labeled Solution A into the clear vial labeled Solution B. Place the yellow cap on the clear vial for easy identification. Mix and label accordingly. Store at 2 - 8 °C. Note: Do not use the working substrate if it looks blue.

Sample Collection: The specimens shall be blood, serum or plasma in type and the usual precautions in the collection of venipuncture samples should be observed. For accurate comparison to established normal values, a fasting morning serum sample should be obtained. The blood should be collected in a plain redtop venipuncture tube without additives or anti-coagulants (for serum) or evacuated tube(s) containing EDTA or heparin. Allow the blood to clot for serum samples. Centrifuge the specimen to separate the serum or plasma from the cells. Samples may be refrigerated at 2-8°C for a maximum period of five days. If the specimen(s) cannot be assayed within this time, the sample(s) may be stored at temperatures of -20 °C for up to 30 days. Avoid repetitive freezing and thawing. When assayed in duplicate, 0. 050ml of the specimen is required.

Calculation of Results:

A dose response curve is used to ascertain the concentration of cortisol in unknown specimens. 1. Record the absorbance obtained from the printout of the microplate reader. 2. Plot the absorbance for each duplicate serum reference versus the corresponding cortisol concentration in µg/dl on linear graph paper (do not average the duplicates of the serum references before plotting). 3. Connect the points with a best-fit curve. 4. To determine the concentration of cortisol for an unknown, locate the average absorbance of the duplicates for each unknown on the vertical axis of the graph, find the intersecting point on the curve, and read the concentration (in µg/dl) from the horizontal axis of the graph (the duplicates of the unknown may be averaged as indicated). In the following example, the average absorbance (1. 071) intersects the dose response curve at (10. 2 µg/dl) cortisol concentration.

Restrictions: For Research Use only

Handling

Handling Advice: Before proceeding with the assay, bring all reagents, serum references and controls to room temperature (20-27°C). 1. Format the microplates' wells for each serum reference, control and patient specimen to be assayed in duplicate. Replace any unused microwell strips back into the aluminum bag, seal and store at 2-8°C. 2. Pipette 0. 025 ml (25µl) of the appropriate serum reference, control or specimen into the assigned well. 3. Add 0. 050 ml (50µl) of the working Cortisol Enzyme Reagent to all wells (see Reagent Preparation Section). 4. Swirl the microplate gently for 20-30 seconds to mix. 5. Add 0. 050 ml (50µl) of Cortisol Biotin Reagent to all wells. 6. Swirl the microplate gently for 20-30 seconds to mix. 7. Cover and incubate for 60 minutes at room temperature. 8. Discard the contents of the microplate by decantation or aspiration. If decanting, blot the plate dry with absorbent paper. 9. Add 350µl of wash buffer (see Reagent Preparation Section), decant (tap and blot) or aspirate. Repeat two additional times for a total of three washes. An automatic or manual plate washer can be used. Follow the manufacturer's instruction for proper usage. If a squeeze bottle is employed, fill each well by depressing the container (avoiding air bubbles) to dispense the wash. Decant the wash and Repeat two additional times. 10. Add 0. 100 ml (100µl) of working substrate solution to all wells (see Reagent Preparation Section). Always add reagents in the same order to minimize reaction time differences between wells. DO NOT SHAKE THE PLATE AFTER SUBSTRATE ADDITION. 11. Incubate at room temperature for 15 minutes. 12. Add 0. 050ml (50µl) of stop solution to each well and gently mix for 15-20 seconds. Always add reagents in the same order to minimize reaction time differences between wells. 13. Read the absorbance in each well at 450nm (using a reference wavelength of 620-630nm to minimize well imperfections) in a microplate reader. The results should be read within 30 minutes of adding the stop solution. Note: Dilute the samples suspected of concentrations higher than 50 µg/dl 1:5 and 1:10 with cortisol 0 µg/dl patient serum.

Storage: 4 °C/-20 °C

References for Cortisol ELISA Kit (ABIN649057)

Akbari, Dalir-Naghadeh, Asri-Rezaei, Hadian, Boston: "Experimental hyperlipidemia induces insulin resistance in sheep." in: Domestic animal endocrinology, Vol. 53, pp. 95-102, (2015) (PubMed).

Target Details for Cortisol

Target: Cortisol

Abstract: Cortisol Products

Target Type: Hormone

Background: Summary and Explanation of the test: Cortisol (hydrocortisone, compound F) is the most potent glucocorticoid produced by the human adrenal cortex. As with other adrenal steroids, cortisol is synthesized from cholesterol, through a series of enzymatically mediated steps, by the adrenal cortex [reviewed in 1, 2]. The first and rate-limiting step in adrenal steroidogenesis, conversion of cholesterol to pregnenolone, is stimulated by pituitary adrenocorticotropic hormone (ACTH) which is, in turn, regulated by hypothalamic corticotropin releasing factor (CRF). ACTH and CRF secretion are inhibited by high cortisol levels. In plasma, the major portion of cortisol is bound with high affinity to corticosteroid-binding globulin (CBG, transcortin), with most of the remainder loosely bound to albumin. Physiologically effective in anti-inflammatory activity and blood pressure maintenance, cortisol is also involved in gluconeogenesis. Cortisol acts through specific intracellular receptors and has effects in numerous other physiologic systems, including immune function, glucose-counter regulation, vascular tone, substrate utilization and bone metabolism [1-3]. Cortisol is excreted primarily in urine in an unbound (free) form. Cortisol production has an ACTH-dependent circadian rhythm with peak levels in the early morning and a nadir at night. The factors controlling this circadian rhythm are not completely defined. The circadian rhythm of ACTH/cortisol secretion matures gradually during early infancy, and is disrupted in a number of physical and psychological conditions [4]. Furthermore, increased amounts of ACTH and cortisol are secreted independently of the circadian rhythm in response to physical and psychological stress [4, 5]. Elevated cortisol levels and lack of diurnal variation have been identified in patients with Cushing's disease (ACTH hyper secretion) [2, 6]. Elevated circulating cortisol levels have also been identified in patients with adrenal tumors [7]. Low cortisol levels are found in primary adrenal insufficiency (e. G. adrenal hypoplasia, congenital adrenal hyperplasia, Addison's disease) and in ACTH deficiency [1, 2, 8, and 9]. Due to the normal circadian variation of cortisol levels, distinguishing normal and abnormally low cortisol levels can be difficult. Therefore, various tests to evaluate the pituitary-adrenal (ACTH-cortisol) axis, including insulin-induced hypoglycemia, short- and long-term ACTH stimulation, CRF stimulation and artificial blockage of cortisol synthesis with metronome have been performed [8-10]. Cortisol response characteristics for each of these procedures have been reported. Cross-reactivity to other naturally-occurring steroids is low. The employment of several serum references of known cortisol concentration permits construction of a graph of activity and concentration. From comparison to the dose response curve, an unknown specimen's activity can be correlated with cortisol concentration. Intended Use: The Quantitative Determination of Total Cortisol Concentration in Human Serum or Plasma by a Microplate Enzyme Immunoassay. Q. C. Parameters: In order for the assay results to be considered valid the following criteria should be met: 1. The absorbance (OD) of calibrator 0 µg/dl should be greater than 1.3. 2. Four out of six quality control pools should be within the established ranges.