Cortisol ELISA. For the quantitative determination of Cortisol in human serum. Research Use Only. Not for Use in Diagnostic Procedures.

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Cortisol ELISA For the quantitative determination of Cortisol in human serum. Research Use Only. Not for Use in Diagnostic Procedures. Catalog Number: 11CORHUE01 Size: 96 Wells Version: V 6.0 ALPCO June
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Cortisol ELISA For the quantitative determination of Cortisol in human serum. Research Use Only. Not for Use in Diagnostic Procedures. Catalog Number: 11CORHUE01 Size: 96 Wells Version: V 6.0 ALPCO June 5, G Keewaydin Drive, Salem, NH P: (800) F: (603) Page 1 of 6 INTENDED USE For the direct quantitative determination of Cortisol by an enzyme immunoassay in human serum. For Research Use Only. Not for Use in Diagnostic Procedures. PRINCIPLE OF THE TEST The principle of the following enzyme immunoassay test follows the typical competitive binding scenario. Competition occurs between an unlabeled antigen (present in standards, controls and samples) and an enzymelabelled antigen (conjugate) for a limited number of antibody binding sites on the microplate. The washing and decanting procedures remove unbound materials. After the washing step, the enzyme substrate is added. The enzymatic reaction is terminated by addition of the stop solution. The absorbance is measured on a microtiter plate reader. The intensity of the color formed is inversely proportional to the concentration of cortisol in the sample. A set of standards is used to plot a standard curve from which the amount of cortisol in samples and controls can be directly read. Introduction Cortisol is the most abundant circulating steroid and the major glucocorticoid secreted by the adrenal cortex. Cortisol is physiologically effective in blood pressure maintenance and antiinflammatory activity. It is also involved in calcium absorption, gluconeogenesis as well as the secretion of gastric acid and pepsin. Measurement of blood cortisol levels can be used to research adrenal function and investigate the differential diagnosis of Addison s and Cushing s diseases as well as adrenal hyperplasia and carcinoma. Most circulating cortisol is bound to cortisol binding globulin or transcortin. Therefore, the free cortisol concentration excreted in the urine is very small, and the 24hour collection of urine is a must in order to obtain an accurate measurement of urinary cortisol. Cortisol in blood shows a diurnal rhythm with the highest levels in the morning and the lowest levels at night. PROCEDURAL CAUTIONS AND WARNINGS 1. Users should have a thorough understanding of this protocol for the successful use of this kit. Reliable performance will only be attained by strict and careful adherence to the instructions provided. 2. Control materials should be included in every run at a high and low level for assessing the reliability of results. 3. When the use of water is specified for dilution or reconstitution, use deionized or distilled water. 4. In order to reduce exposure to potentially harmful substances, gloves should be worn when handling kit reagents and human samples. 5. All kit reagents and samples should be brought to room temperature and mixed gently but thoroughly before use. Avoid repeated freezing and thawing of reagents and samples. 6. A calibrator curve must be established for every run. 7. The controls should be included in every run and fall within established confidence limits. 8. Improper procedural techniques, imprecise pipetting, incomplete washing as well as improper reagent storage may be indicated when assay values for the controls do not reflect established ranges. 9. When reading the microplate, the presence of bubbles in the wells will affect the optical densities (ODs). Carefully remove any bubbles before performing the reading step. 10. The substrate solution (TMB) is sensitive to light and should remain colorless if properly stored. Instability or contamination may be indicated by the development of a blue color, in which case it should not be used. 11. When dispensing the substrate and stop solution, do not use pipettes in which these liquids will come into contact with any metal parts. 12. To prevent contamination of reagents, use a new disposable pipette tip for dispensing each reagent, sample, standard and control. 13. Do not mix various lot numbers of kit components within a test and do not use any component beyond the expiration date printed on the label. 14. Kit reagents must be regarded as hazardous waste and disposed of according to national regulations. LIMITATIONS 1. All the reagents within the kit are calibrated for the direct determination of cortisol in human serum. The kit is not calibrated for the determination of cortisol in saliva, plasma or other samples of human or animal origin. 2. Do not use grossly hemolyzed, grossly lipemic, icteric or improperly stored serum. 3. Any samples or control sera containing azide or thimerosal are not compatible with this kit, as they may lead to false results. 4. Only calibrator A may be used to dilute any high serum samples. The use of any other reagent may lead to false results. SAFETY CAUTIONS AND WARNINGS POTENTIAL BIOHAZARDOUS MATERIAL Human serum that may be used in the preparation of the standards and controls has been tested and found to be nonreactive for Hepatitis B surface antigen and has also been tested for the presence of antibodies to HCV and Human Immunodeficiency Virus (HIV) and found to be negative. No test method however, can offer complete assurance that HIV, HCV and Hepatitis B virus or any infectious agents are absent. The reagents should be considered a potential biohazard and handled with the same precautions as applied to any blood sample. CHEMICAL HAZARDS Avoid contact with reagents containing TMB, hydrogen peroxide and sulfuric acid. If contacted with any of these reagents, wash with plenty of water. TMB is a suspected carcinogen. 26G Keewaydin Drive, Salem, NH P: (800) F: (603) Page 2 of 6 SPECIMEN COLLECTION AND STORAGE Approximately 0.1 ml of serum is required per duplicate determination. Collect 4 5 ml of blood into an appropriately labelled tube and allow it to clot. Centrifuge and carefully remove the serum layer. Store at 4 C for up to 24 hours or at 10 C or lower if the analyses are to be done at a later date. Consider all human samples as possible biohazardous materials and take appropriate precautions when handling. SAMPLE PRETREATMENT This assay is a direct system; no specimen pretreatment is necessary. REAGENTS AND EQUIPMENT NEEDED BUT NOT PROVIDED 1. Precision pipettes to dispense 20, 50, 100, 150 and 300 μl 2. Disposable pipette tips 3. Distilled or deionized water 4. Plate shaker 5. Microplate reader with a filter set at 450 nm and an upper OD limit of 3.0 or greater* (see assay procedure step 10) 6. Timer REAGENTS PROVIDED 1. Rabbit AntiCortisol AntibodyCoated BreakApart Well Microplate Ready To Use Contents: One 96well (12x8) polyclonal antibodycoated microplate in a resealable pouch with desiccant. 2. CortisolHorseradish Peroxidase (HRP) Conjugate Concentrate Requires Preparation X100 Contents: CortisolHRP conjugate in a proteinbased buffer with a nonmercury preservative. Volume: 300 μl/vial Preparation: Dilute 1:100 in assay buffer before use (eg. 20 μl of concentrate in 2 ml of assay buffer). If the whole plate is to be used, dilute 120 µl of concentrate in 12 ml of assay buffer. Discard any that is left over. 3. Cortisol Calibrators Ready To Use Contents: Seven vials containing cortisol in a human serum based buffer with a nonmercury preservative. Prepared by spiking serum with a defined quantity of cortisol. * Listed below are approximate concentrations, please refer to bottle labels for exact concentrations. Calibrator Concentration Volume/Vial Calibrator A 0 µg/dl 1.0 ml Calibrator B 0.5 µg/dl 0.3 ml Calibrator C 2 µg/dl 0.3 ml Calibrator D 5 µg/dl 0.3 ml Calibrator E 10 µg/dl 0.3 ml Calibrator F 30 µg/dl 0.3 ml Calibrator G 60 µg/dl 0.3 ml Stability: 12 months in unopened vials or as indicated on label. Once opened, the standards should be used within 14 days or aliquoted and stored frozen. Avoid multiple freezing and thawing cycles. 4. Controls Ready To Use Contents: Two vials containing cortisol in a human serum based buffer with a nonmercury preservative. Prepared by spiking serum with defined quantities of cortisol. Refer to vial labels for the acceptable range. Volume: 0.3 ml/vial Stability: 12 months in unopened vial or as indicated on label. Once opened, the controls should be used within 14 days or aliquoted and stored frozen. Avoid multiple freezing and thawing cycles. 5. Wash Buffer Concentrate Requires Preparation X10 Contents: One bottle containing buffer with a nonionic detergent and a nonmercury preservative. Volume: 50 ml/bottle Preparation: Dilute 1:10 in distilled or deionized water before use. If the whole plate is to be used dilute 50 ml of the wash buffer concentrate in 450 ml of water. 6. Assay Buffer Ready To Use Contents: One bottle containing a proteinbased buffer with a nonmercury preservative. 26G Keewaydin Drive, Salem, NH P: (800) F: (603) Page 3 of 6 Volume: 15 ml/bottle 7. TMB Substrate Ready To Use Contents: One bottle containing tetramethylbenzidine and hydrogen peroxide in a nondmf or DMSO containing buffer. Volume: 16 ml/bottle 8. Stop Solution Ready To Use. Contents: One bottle containing 1M sulfuric acid. Volume: 6 ml/bottle ASSAY PROCEDURE Sample Pretreatment: None. All reagents must reach room temperature before use. Calibrators, controls and specimen samples should be assayed in duplicate. Once the procedure has been started, all steps should be completed without interruption. 1. Prepare working solutions of the cortisolhrp conjugate and wash buffer. 2. Remove the required number of well strips. Reseal the bag and return any unused strips to the refrigerator. 3. Pipette 20 μl of each calibrator, control and sample into correspondingly labelled wells in duplicate. 4. Pipette 100 μl of the conjugate working solution into each well. (It is recommended to use a multichannel pipette.) 5. Incubate on a plate shaker (approximately 200 rpm) for 45 minutes at room temperature. 6. Wash the wells 3 times with 300 μl of diluted wash buffer per well and tap the plate firmly against absorbent paper to ensure that it is dry. (The use of a washer is recommended.) 7. Pipette 150 μl of TMB substrate into each well at timed intervals. 8. Incubate on a plate shaker for minutes at room temperature (or until calibrator A attains dark blue color for desired OD). 9. Pipette 50 μl of stop solution into each well at the same timed intervals as in step Read the plate on a microplate reader at 450 nm within 20 minutes after addition of the stop solution. * If the OD exceeds the upper limit of detection or if a 450 nm filter is unavailable, a 405 or 415 nm filter may be substituted. The optical densities will be lower; however, this will not affect the results of sample//controls.. CALCULATIONS 1. Calculate the mean optical density of each calibrator duplicate. 2. Draw a calibrator curve on semilog paper with the mean optical densities on the Yaxis and the calibrator concentrations on the Xaxis. If immunoassay software is being used, a 4parameter or 5parameter curve is recommended. 3. Calculate the mean optical density of each unknown duplicate. 4. Read the values of the unknowns directly off the calibrator curve. 5. If a sample reads more than 60 µg/dl, then dilute it with calibrator A at a dilution of no more than 1:8. The result obtained should be multiplied by the dilution factor. TYPICAL TABULATED DATA Sample data only. Do not use to calculate results. Calibrator OD 1 OD 2 Mean OD Value (μg/dl) A B C G Keewaydin Drive, Salem, NH P: (800) F: (603) Page 4 of 6 D E F G Unknown TYPICAL CALIBRATOR CURVE Sample curve only. Do not use to calculate results. Cortisol (μg/dl) PERFORMANCE CHARACTERISTICS SENSITIVITY The lower detection limit is calculated from the standard curve by determining the resulting concentration of the mean OD of Calibrator A (based on 10 replicate analyses) minus 2 SD. Therefore, the sensitivity of the Cortisol ELISA kit is 0.4 μg/dl. SPECIFICITY (CROSSREACTIVITY) The following compounds were tested for crossreactivity with the Direct Cortisol ELISA kit with cortisol crossreacting at 100%. Steroid % Cross Reactivity Cortisol 100 Prednisolone 13.6 Corticosterone 7.6 Deoxycorticosterone 7.2 Progesterone 7.2 Cortisone 6.2 Deoxycortisol 5.6 Prednisone 5.6 Dexamethasone 1.6 No cross reaction was detected with DHEAS and Tetrahydrocortisone. Please note that there is an observed crossreactivity of 13.6% with prednisolone. Since prednisone is converted to prednisolone in vivo, caution must be exercised when assaying the cortisol levels of subjects undergoing either therapy. INTRAASSAY PRECISION Three samples were assayed ten times each on the same calibrator curve. The results (in µg/dl) are tabulated below: Sample Mean SD CV % INTERASSAY PRECISION Three samples were assayed ten times over a period of four weeks. The results (in µg/dl) are tabulated below: Sample Mean SD CV % G Keewaydin Drive, Salem, NH P: (800) F: (603) Page 5 of 6 RECOVERY Spiked samples were prepared by adding defined amounts of cortisol to three serum samples (1:1). The results (in µg/dl) are tabulated below: Sample Obs. Result Exp. Result Recovery % 1 Unspiked Unspiked Unspiked LINEARITY Three serum samples were diluted with calibrator A. The results (in µg/dl) are tabulated below: 1 1:2 1:4 1:8 2 1:2 1:4 1:8 3 1:2 1:4 1:8 Sample Obs. Result Exp. Result Recovery % REFERENCES Brock P, et al. Direct SolidPhase 125I Radioimmunoassay of Serum Cortisol. Clin Chem. 1978; 24(9): Morris R. A Simple and Economical Method for the Radioimmunoassay of Cortisol in Serum. Ann Clin Biochem. 1978; 15(3): Silver AC, et al. Radioimmunoassay of Cortisol in Saliva with the GammaCoat Kit. Clin Chem. 1983; 29(10): Vecsei P, et al. Radioimmunological Determination of Plasma Cortisol. Experimentia. 1972; 28(9): Abraham GE, et al. Radioimmunoassay of Plasma Cortisol. Anal Lett. 1972; 5(11): GomezSanchez C, et al. Radioiodinated Derivatives for Steroid Radioimmunoassay. Application to the Radioimmunoassay of Cortisol. J Lab Clin Med. 1977; 89(4): Demers LM, Derck DD. Comparison of Competitive Protein Binding Analysis and Radioimmunoassay for the Determination of Cortisol in Serum and Urine. Clin Biochem. 1977; 10(3): Poland RE, Rubin RT. Saliva Cortisol Levels Following Dexamethasone Administration in Endogenously Depressed Patients. Life Sci. 1982; 30(2): Peters JR, et al. Salivary Cortisol Assays for Assessing PituitaryAdrenal Reserve. Clin Endocrinol (Oxf). 1982; 17(6): Papanicolaou DA, et al. Nighttime Salivary Cortisol: A Useful Test for the Diagnosis of Cushing s Syndrome. J Clin Endocrinol Metab. 2002; 87(10): Check JH, et al. Falsely Elevated Steroidal Assay LevelsRelated to Heterophile Antibodies Against Various Animal Species. Gynecol Obstet Invest. 1995; 40(2): G Keewaydin Drive, Salem, NH P: (800) F: (603) Page 6 of 6
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