Cortisol ELISA Kit. Catalog Number KA assays Version: 02. Intended for research use only.
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1 Cortisol ELISA Kit Catalog Number KA assays Version: 02 Intended for research use only
2 Table of Contents Introduction... 3 Intended Use... 3 Background... 3 Principle of the Assay... 3 General Information... 4 Materials Supplied... 4 Storage Instruction... 5 Materials Required but Not Supplied... 5 Precautions for Use... 5 Assay Protocol... 6 Reagent Preparation... 6 Sample Preparation... 6 Assay Procedure... 7 Data Analysis... 8 Calculation of Results... 8 Performance Characteristics... 9 Resources References Plate Layout KA / 11
3 Introduction Intended Use A solid-phase enzyme immunoassay for the quantitative determination of cortisol in saliva. This kit is designed for measurement of cortisol in saliva. The kit contains reagents sufficient for 96 determinations and allows to analyze 40 unknown samples in duplicates. Background Cortisol is a glucocorticoid with a MW of Dalton. Cortisol is the major hormone secreted by adrenals. Cortisol secretion undergoes circadian rhythms with maximal (up to 700 nmol/l) concentrations seen in the morning (6 9 am) and minimal (up to 55 nmol/l) in the midnight. In serum, cortisol is found both in free and bound form, the latter being a complex with transport proteins (transcortin, albumin). But only free form exerts biological activity, its proportion being ca. 10% of total cortisol. Currently, there are no reliable immunochemical method for quantitative determination of free cortisol in serum. At the same time, complicated reference laboratory methods (e.g., dialysis) are not suitable for everyday use. That is why the only method of direct determination of free cortisol is its determination in saliva. Follow-up of salivary cortisol level is a very informative and non-traumatic method widely used in endocrinology, physiology, sport medicine, veterinary, etc. for estimation of adrenal glands function, differential diagnostics of Cushing syndrome with its characteristic alteration of circadian rhythm of cortisol secretion being a good example. Elevated Cortisol concentrations are found in secreting tumors of adrenals, in virilizing hyperplasia of adrenals, in Cushing syndrome, in ACTH-producing tumors, during surgical stress, in cardiac insufficiency, diabetes, burns, pains, during pregnancy, during estrogen therapy, etc. Cortisol level may be increased by intake of ACTH, Cortisol, alcohol, nicotine, oral contraceptives. Decreased Cortisol levels are found in Addison syndrome, adrenogenital syndrome, hypopituitarism. Some drugs may decrease Cortisol level in blood, such as: L-DOPA, dexamethasone, etc. Decreased Cortisol level during pregnancy may indicate anencephaly of the fetus. Principle of the Assay This test is based on competition enzyme immunoassay principle. Tested specimen is placed into the microwells coated by specific murine monoclonal to cortisol-antibodies simultaneously with conjugated Cortisol-peroxidase. Salivary Cortisol from the specimen competes with the cortisol labelled with peroxidase enzyme for coating antibodies. After washing procedure, the remaining enzymatic activity bound to the microwell surface is detected and quantified by addition of chromogen-substrate mixture, stop solution and photometry at 450 nm. Optical density in the microwell is inversely related to the quantity of the measured analyte in the specimen. KA / 11
4 General Information Materials Supplied List of component Component Salivary Cortisol EIA strips: polystyrene microwells coated with murine monoclonal to cortisol Calibrator set (1-6): The set contains 6 calibrators: 0; 0.3; 1.5; 5, 15, 45 ng/ml. Human cortisol diluted in tris buffered BSA solution, preservative 0.01% Bronidox L, 0.01% 2-Methyl-4-isothiazolin-3-one-hydrochloride; also contains bright red dye Control sera: dilution of preselected human salivary, with high content of cortisol with preservative - 0,01% Bronidox L, 0,01% 2-Methyl-4-isothiazolin-3-one-hydrochloride; also contains red dye Conjugate: aqueous solution of cortisol coupled with horseradish peroxidase diluted on phosphate buffered solution preservative 0.01% Bronidox L, 0.01% 2-Methyl-4-isothiazolin-3-one-hydrochloride and red dye Substrate solution: ready-to-use, single-component tetramethylbenzidine (TMB) solution. Amount 96 (8x12) wells 1 ml x 6 1 ml x 2 14 ml 16 ml Washing solution concentrate 26x: aqueous solution of sodium chloride and detergent (Tween 20), contains proclin300 as a preservative Stop solution: 5.0% vol/vol solution of sulphuric acid 22 ml 14 ml Plate sealing tape 2 slides KA / 11
5 Storage Instruction Store the whole kit at C upon receipt until the expiration date. After opening the pouch keep unused microtiter wells TIGHTLY SEALED BY ADHESIVE TAPE (INCLUDED) to minimize exposure to moisture. Materials Required but Not Supplied Distilled or deionized water; Automatic or semiautomatic multichannel micropipettes, µl, is useful but not essential; Calibrated micropipettes with variable volume, range volume µl; Dry thermostat for +37 С ± 2 С; Calibrated microplate photometer with 450 nm wavelength and OD measuring range Precautions for Use For professional use only. This kit is intended for research only. INFECTION HAZARD: There is no available test methods that can absolutely assure that Hepatitis B and C viruses, HIV-1/2, or other infectious agents are not present in the reagents of this kit. All individual products, including human samples, should be considered potentially infectious. Handling and disposal should be in accordance with the procedures defined by an appropriate national biohazard safety guidelines or regulations. Avoid contact with stop solution containing 5.0% H 2 SO 4. It may cause skin irritation and burns. Wear disposable latex gloves when handling specimens and reagents. Microbial contamination of reagents may give false results. Do not use the kit beyond the expiration date. All indicated volumes have to be performed according to the protocol. Optimal test results are only obtained when using calibrated pipettes and microplate readers. Do not smoke, eat, drink or apply cosmetics in areas where specimens or kit reagents are handled. Chemicals and prepared or used reagents have to be treated as hazardous waste according to the national biohazard safety guidelines or regulations. Do not mix reagents from different lots. Replace caps on reagents immediately. Do not swap caps. Do not pipette reagents by mouth. Specimens must not contain any AZIDE compounds they inhibit activity of peroxidase. The Material Safety Data Sheet for this product is available upon request directly from Abnova. The Material Safety Data Sheet fits the requirements of EU Guideline 91/155 EC. KA / 11
6 Assay Protocol Reagent Preparation All reagents (including unsealed microstrips) should be allowed to reach room temperature ( C) before use. All reagents should be mixed by gentle inversion or vortexing prior to use. Avoid foam formation. It is recommended to spin down shortly the tubes with calibrators on low speed centrifuge. Prepare washing solution from the Washing solution concentrate 26x by 26 dilutions in distilled water. Sample Preparation Specimen type Saliva Sample collection, storage and preparation Salivary Sampler collection Salivary samples may be taken not earlier than 30 minutes after tooth brushing, having meals or use of a chewing-gum. Polystyrene vials are recommended to be used for sample collection. Salivary samples containing traces of blood i.e., with rose or yellowish coloration should not be used. Sample storage Salivary samples may be stored for up to 2 weeks at 2-8 С or for up to 6 months at -20 С. Sample preparation. Before assaying, salivary samples should be stored at -20 С for at least 3 hours, then thawed at room temperature and centrifuged at 3000 rpm. To detect a morning peak of Cortisol, it is recommended to obtain 5 serial salivary samples the 1st one should be taken just after awakening, the other 4 samples should be taken consecutively with 30 minutes intervals. To investigate the whole day Cortisol secretion profile, it is recommended to assay 5 morning samples, 1 sample taken in the midday (ca ), 1 sample obtained at ca and 1 evening sample taken at ca samples in total. A simplified scheme includes only 2 samples obtained at 8.00 and at Sample in to Calculation the well, µl factor 50 1 KA / 11
7 Quality Control It is recommended to use control samples according to state and federal regulations. The use of control samples is advised to assure the day to day validity of results. The test must be performed exactly as per the manufacturer s instructions for use. Moreover the user must strictly adhere to the rules of GLP (Good Laboratory Practice) or other applicable federal, state, and local standards and/or laws. This is especially relevant for the use of control reagents. It is important to always include, within the test procedure, a sufficient number of controls for validating the accuracy and precision of the test. The test results are valid only if all controls are within the specified ranges and if all other test parameters are also within the given assay specifications. Assay Procedure Procedure Note It is recommended that pipetting of all calibrators and samples should be completed within 3 minutes. 1. Put the desired number of microstrips into the frame; allocate 16 wells for the calibrators (1-6) and two wells for each unknown sample. DO NOT REMOVE ADHESIVE SEALING TAPE FROM UNUSED STRIPS. 2. Pipet 50 µl of calibrators (1 6) and unknown samples into the wells. 3. Dispense 100 µl of Conjugate into the wells. Cover the wells by plate adhesive tape (included into the kit). 4. Incubate 60 minutes at 37 C. 5. Prepare washing solution by 26x dilution of washing solution concentrate 26x by distilled water. Wash the strips 5 times. 6. Dispense 150 µl of Substrate solution into the wells 7. Incubate minutes at C 8. Dispense 100 µl of Stop solution into the wells. 9. Measure OD (optical density) at 450 nm. 10. Set photometer blank on air 11. Apply lin-log method for data reduction. KA / 11
8 Data Analysis Calculation of Results Calculate the mean absorbance values (OD450) for each pair of calibrators and samples. Plot a calibration curve on graph paper: OD versus cortisol concentration. Determine the corresponding concentration of cortisol in unknown samples from the calibration curve. Manual or computerized data reduction is applicable on this stage. Point-by-point or linear data reduction is recommended due to non-linear shape of curve. Below is presented a typical example of a standard curve. Not for calculations! Calibrators Value Absorbance Units (450 nm) 1 0 ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml no ng/ml 0.25 Expected Values Each laboratory should establish its own normal range for Salivary Cortisol. The following normal range is recommended (see below). Timing/patient group Units, ng/ml Units alternative, nmol/l Lower limit Upper limit Lower limit Upper limit Night Morning peak (ca. 1.5 hr after awakening) hrs after the morning peak hrs after the morning peak KA / 11
9 Performance Characteristics Analytical specificity / Cross reactivity Analyte Cross-reactivity, % wt/wt Cortisol Deoxycortisol 0,9 Prednisolone 5,6 Corticosterone 0,6 11-Deoxycorticosterone <0,1 Progesterone <0,1 17-Hydroxyprogesterone <0,1 Testosterone, Estradiol, Estriol <0,1 Danazol <0,01 Analytical sensitivity Sensitivity of the assay was assessed as being ng/ml. Linearity Linearity was checked by assaying dilution series of 5 samples with different cortisol concentrations. Linearity percentages obtained ranged within 90 to 110%. Recovery Recovery was estimated by assaying 5 mixed samples with known cortisol concentrations. The recovery percentages ranged from 90 to 110%. KA / 11
10 Resources References 1. L. Thomas, Labor und Diagnose, 4. Auflage, Tietz, N.W., Textbook of Clinical Chemistry, Saunders, Foster, L. B. and Dunn, R.T. Clin. Chem: 20/3, 365 (1974) 4. De Lacerda, L., Kowarski, A., and Migeon, C.J. J. Clin. Endocr. and Metab: 36, 227 (1973) 5. Rolleri, E., Zannino, M., Orlandini, S., Malvano,R. Clin chim Acta (1976) 6. Kobayashi, Y., et al Steroids, 32 no. 1 (1978) 7. Arakawa, H., Maeda, M., Tsuji, A. Anal. Biochem (1979) KA / 11
11 Plate Layout A B C D E F G H KA / 11
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