rev: January 18, 2000
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Cortisol (Salivary) ELISA Kit -made by IBL-Hamburg, Germany -sample data-see actual kit insert for batch specific information
Catalogue No : RDI-RE52261 Product group : Steroids; Gynaecology 12 x 8 Product name : Cortisol, Saliva ELISA 96 Method : ELISA Incubation time: 1h, 15 min Standard curve : 2 - 80 ng/ml Sample/Prep. : 50µl Saliva Isotope/Substr.: TMB, 450 nm -------------------------------------------------------------------------------- CONTENTS Introduction Contents of the Kit Principle of the Test Test Procedure Performance Characteristics Expected Values Alternative Applications (More) Clinical Background Sales Arguments Product Literature Miscellaneous
Introduction The IBL Cortisol Enzyme Immunoassay Kit provides materials for the quantitative determination of cortisol in saliva. This assay is intended for in vitro diagnostic use only. Cortisol (hydrocortisone, compound F) is the main corticosteroid secreted in humans by the adrenal cortex. This steroid hormone has a molecular weight of 363.5. In most physiological conditions, only about 10 % of plasma cortisol circulates unbound from transcortin and albumin. Among the products of the human adrenal cortex, only cortisol is involved in the regulation of ACTH secretion. As the level of free (non-protein bound) cortisol in blood rises, the release of ACTH is inhibited by the negative feedback effect. Conversely, if cortisol levels are subnormal, the negative feedback decreases, ACTH levels rise, and the adrenal cortex secretes cortisol until normal blood levels are restored. The release of ACTH is under the control of hypothalamic corticotrophin releasing hormone (CRH); the negative feedback system involving cortisol has been identified at both hypothalamic and pituitary levels. Normally during the day there is a fluctuation of cortisol achieving the highest level in the morning and the lowest in the night. Useful information is given when cortisol measurement is done in samples withdrawn at a fixed hour (8:00 a.m.). The main biological effects of cortisol are: Promotion of glucogenesis, deposition of liver glycogen, increase in blood glucose concentration when the carbohydrate utilisation is reduced, effect on fat metabolism and anti-inflammatory action. Cortisol measurement is a powerful tool for the evaluation of suspected abnormalities in glucocorticoid production: Cushing's Syndrome (hypercortisolism), Addison's disease or secondary adrenal insufficiency (hypocortisolism). In many cases, it is necessary to perform dynamic tests (suppression or stimulation) in order to localise the defect at one of the three main levels (i.e. adrenal, pituitary, hypothalamus). In saliva only free, biological active cortisol is present. Therefore the IBL salivary cortisol ELISA provides a reliable, non-invasive method for determination of free cortisol. Contents of the Kit Contents of the Testkit 1. Microtiter Strips 12 x 8 wells break apart strips coated with anti cortisol antibodies (mouse monoclonal) in foilbag with desiccant. 2. Standards (A - G) 7 vials 0.5 ml each, ready to use containing the below mentioned concentrations of cortisol in stripped human serum and preservative. Standard A B C D E F G Concentration in ng/ml 0 2 5 10 20 40 80 (conversion factor: 1 ng/ml = 2.76 nmol/l). 3. Enzyme Conjugate 1 vial 31 ml, ready to use cortisol conjugated to the enzyme horseradish peroxidase in buffer, supplemented by enzyme stabilisers and cortisol binding protein displacers. 4. TMB Substrate Solution 1 vial 11 ml, ready to use containing a solution of tetramethylbenzidine (TMB) in buffer supplemented by stabilisers. 5. TMB Stop Solution 1 vial 11 ml, ready to use 0.5 M sulphuric acid (H2SO4) Avoid contact with stop solution it may cause skin irritations and burns. 6. Wash Buffer, concentrate (20x) 1 vial 25 ml, concentrate containing buffer Material required but not provided * Automatic pipettes to dispense 50, 100 and 300 µl (a multichannel pipetting device such as Titertek is suitable for adding reagents to the wells) * Distilled water * Microtiter plate spectrophotometer (ELISA reader) with 450 nm filter Storage and Stability Store all reagents at 2 - 8 °C and use before expiry date. When stored at 2 - 8 °C unopened reagents will retain reactivity until expiration date stated on the reagent labels. Do not use reagents beyond this date. Once the foilbag of the coated microtiter strips has been broken, care should be taken to close it tightly again. The immuno reactivity of the coated microtiter strips is stable for approx. 8 weeks in the broken, but tightly closed bag when stored at 2 - 8 °C. Enzyme conjugate is stable for 8 weeks after first opening when stored at 2 - 8 °C. TMB Substrate Solution (-LS-) is stable for 8 weeks when stored at 2 - 8 °C or 2 weeks when stored at room temperature (18 - 24 °C). Allow all reagents and required number of strips to reach room temperature prior to use. Specimen Collection and Storage It is recommended to collect saliva samples with a wad of cotton and subsequently centrifugation of the wad of cotton. Equipment for this step of sample collection is commercially available. Saliva samples can be stored at 2 - 8 °C for one week. For a longer, the samples should be frozen at - 20 °C. Repeated freeze - thawing should be avoided. Thawed samples should be inverted several times prior to testing. Preparation of Samples and Reagents Wash Buffer Dilute the wash buffer concentrate with distilled water 1 to 20 (25 ml concentrate + 475 ml distilled water). Ready to use wash buffer is stable for 8 weeks when stored at room temperature. Principle of the Test This assay is based on the competition principle and the microtiter plate separation. An unknown amount of antigen present in the sample and a fixed amount of enzyme labelled antigen compete for the binding sites of the antibodies coated onto the wells. After an incubation the wells are washed to stop the competition reaction. Having added the TMB substrate solution the concentration of antigen is inversely proportional to the optical density measured. The measured ODs of the standards are used to construct a calibration curve against which the unknown samples are calculated. Test Procedure Assay Procedure GENERAL REMARKS: All reagents and specimens must be allowed to come to room temperature before use. All reagents must be mixed without foaming. Once the test has been started, all steps should be completed without interruption. The total dispensing time of standards, controls and sample should not exceed 15 minutes. Use new disposable plastic pipette tips for each reagent, standard or specimen in order to avoid cross contamination. For the dispensing of the TMB substrate solution and the TMB stop solution avoid pipettes with metal parts. Absorbance is a function of the incubation time and temperature. Before starting the assay, it is recommended that all reagents be ready, caps removed, all needed wells secured in holder, etc. This will ensure equal elapsed time for each pipetting step without interruption. 1. Leave sufficient microtiter strips in the strip holder to enable the running of standards, controls, and samples, plus one well for the chromogen blank. 2. Pipet 50 µl of standards, controls and samples into the appropriate wells of the strips. 3. Add 300 µl of enzyme conjugate to each well in sequence. 4. Incubate for 60 minutes without covering the plate at room temperature (18 - 24 °C). 5. Washing: discard the incubation solution, rinse the wells 3x with 300 µl wash buffer (dilute concentrate 1 to 20 with distilled water) and remove any residual. 6. Promptly pipet 100 µl of the TMB substrate solution into the rinsed wells. 7. Incubate for 15 minutes at room temperature (18 - 24 °C). 8. Stop the reaction by adding 100 µl of TMB stop solution to each well. 9. Shake gently the microtiter strips being careful not to let the content come from the wells and read at 450 nm within 60 minutes from the stopping. Calculation of Results Any ELISA reader capable of determining the absorbance at 450 ñ 10 nm may be used. The antigen concentration of each sample is obtained as follows: Using linear-linear or semi log graph paper, construct an standard curve by plotting the absorbance (Y) of each reference standard (OD) against its corresponding concentration (X) in ng/ml or calculate %B/Bmax for each standard as follows: Use the absorbance or B/Bmax of each sample to determine the corresponding antigen value by simple interpolation from this standard curve, multiplying by the initial sample dilution, if necessary. Alternatively the use of electronic device is possible. The results can also be calculated with normal programs for automatic data processing, i.e. 4 parameter, spline, logit-log. Any sample reading greater than the highest standard should be diluted appropriately with zero standard and reassayed. The result has to be multiplied by the corresponding dilution factor. Do not use this calibration curve. In the laboratory the standard curve should be established in each assay run. Standards B/Bmax ng/ml (%) ------------------------------------------- 0 100.00 2 91.76 5 79.21 10 64.52 20 47.31 40 30.47 80 19.00 Performance Characteristics Back to Contents 1. Expected values The following values can be used as preliminary guidelines until each laboratory establishes its own normal ranges. As indicated, 95.5 % of normal samples yield results within the below mentioned ranges. ng/ml µg/dl A.M. 4 - 10 0.4 - 1.0 P.M. 0.8 - 1.3 0.08 - 0.13 Conversion factor: 1 ng/ml = 2.76 nmol/l 2. Specificity The specificity of the cortisol assay was assessed according to Abraham's method. The percentage indicate cross-reactivity at 50 % displacement compared to cortisol. Component % Cross-Reactivity Cortisol 100.0 Dexamethazone < 0.1 Prednisolone 60.0 Deoxycorticosterone < 0.1 Corticosterone 29.0 Dehydroepiandrosterone sulphate < 0.1 Cortisone 3.0 Estradiol < 0.1 11-Deoxycortisol < 1.0 Estriol < 0.1 17-OH Progesterone < 0.5 Estrone < 0.1 Prednisone < 0.1 Testosterone < 0.1 Progesterone < 0.1 3. Sensitivity The minimal detectable concentration of cortisol by this assay is estimated to be 1.14 ng/ml (defined as 2x standard deviation to the zero standard). 4. Precision 4.1. Intra Assay Variation Within run variation was determined by replicate determination of 3 different control sera in one assay. The within assay variability is shown below: Sample n Mean Standard Deviation CV (%) 1 18 3.45 0.18 5.14 2 20 18.07 0.83 4.58 3 20 29.13 1.06 3.65 4.2. Inter Assay Variation Between run variation was determined by replicate measurements of 3 different control sera in several different assay. The between assay variability is shown below: Sample n Mean Standard Deviation CV (%) 1 19 3.48 0.20 5.88 2 21 19.78 1.25 6.33 3 21 31.25 1.48 4.73 5. Linearity 3 patient samples were serially diluted with zero standard in a linearity study. Sample Dilution Measured Expected Recovery conc.(ng/ml) conc.(ng/ml) (%) neat 40.80 -- 1:2 21.50 20.40 105.4 1 1:4 9.50 10.20 93.1 1:8 4.80 5.10 94.1 1:16 2.75 2.55 107.8 neat 45.80 -- 1:2 22.53 22.90 98.4 2 1:4 10.44 11.45 91.2 1:8 5.77 5.73 100.8 1:16 2.75 2.86 96.1 neat 10.20 -- 1:2 5.77 5.10 113.1 3 1:4 2.37 2.55 92.9 1:8 1.30 1.28 101.7 6. Recovery Spiked serum samples were prepared by adding varying levels of cortisol to 3 different saliva samples. Sample Added Cortisol Measured Cortisol Expected Cortisol Recovery ng/ml ng/ml ng/ml (%) 40 40.84 41.60 98.2 1 20 21.76 21.60 100.7 10 11.97 11.60 103.2 40 55.06 54.90 100.3 2 20 34.22 34.90 98.1 10 22.30 4.90 89.6 40 58.59 56.65 103.4 3 20 37.98 36.65 103.6 10 28.70 26.65 107.7 Expected Values Back to Contents The following values can be used as preliminary guidelines until each laboratory establishes its own normal ranges. As indicated, 95.5 % of normal samples yield results within the below mentioned ranges. ng/ml µg/dl A.M. 4 - 10 0.4 - 1.0 P.M. 0.8 - 1.3 0.08 - 0.13 Conversion factor: 1 ng/ml = 2.76 nmol/l Product Literature References 1. Tietz, N.W. Testbook of Clinical Chemistry, Saunders, 1986. 2. Kirschbaum, C; Hellhammer, DH. Salivary Cortisol in Psychoneuro-endocrine Research: Recent Developments and Applications. Psychoneuroendocrinology, 19: 313-333 (1994). 3 Thomas, L: Labor und Diagnose, 5.th edition, Frankfurt/Main, 1998.
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