Cortisol Human ELISA Kit

Transcription

1 ab Cortisol Human ELISA Kit Instructions for Use For the quantitative measurement of Human Cortisol in saliva This product is for research use only and is not intended for diagnostic use.

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3 Table of Contents 1. Introduction 3 2. Assay Summary 4 3. Kit Contents 5 4. Storage and Handling 6 5. Additional Materials Required 6 6. Preparation of Reagents 7 7. Preparation and Collection of Specimen 8 8. Assay Method 9 9. Data Analysis Limitations Specificity Troubleshooting 16 2

4 1. Introduction ab154996, Cortisol Human ELISA Kit is a Competitive immunoenzymatic colorimetric method for quantitative determination of Cortisol in saliva. 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. The majority of cortisol in saliva is not-bound and enters the saliva via intracellular mechanisms. Salivary cortisol levels are unaffected by salivary flow rate or salivary enzymes. It is a high correlations between serum and saliva cortisol levels. These normal endogenous functions are the 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 longterm use of glucocorticoid drugs. 3

5 2. Assay Summary ab is based on the principle of a solid phase enzyme-linked immunosorbent assay. Microtiter strip wells are precoated with anti-corisol antibodies (solidphase). Cortisol in the sample competes with added horseradish peroxidase labelled Estriol (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 free Estriol in the sample. Sulphuric acid is added to stop the reaction. This produces a yellow endpoint color. Absorption at 450 nm is read using an ELISA microwell plate reader. 4

6 3. Kit Contents Anti-Cortisol IgG Coated Wells: 12 breakapart 8-well snap-off strips coated with anti-cortisol IgG; in resealable aluminium foil. Stop Solution: 1 bottle containing 15 ml sulphuric acid, 0.15 mol/l (avoid any skin contact). Cortisol-HRP Conjugate conc: 1 bottle containing 1 ml of horseradish peroxidase labelled Cortisol. TMB Substrate Solution: 1 bottle containing 15 ml 3, 3, 5, 5 tetramethylbenzidine (H 2 O 2 -TMB 0.25 g/l) (avoid any skin contact). Incubation buffer: 1 bottle containing 30 ml phosphate buffer 50 mm, ph 7.4, BSA 1 g/l. Cortisol Standards: 7 bottles, 1 ml each Standard 0: 0 ng/ml Standard 1: 0.5 ng/ml Standard 2: 1.0 ng/ml Standard 3: 5.0 ng/ml Standard 4: 10.0 ng/ml Standard 5: 20.0 ng/ml Standard 6: ng/ml For SI units: ng/ml x 2.76 = nmol/l 1 Strip holder 2 Cover foils 5

7 4. Storage and Handling The reagents are stable up to the expiry date stated on the label when stored at 2-8 C in the dark. Keep microtiter plate in a sealed bag with desiccant to minimize exposure to damp air. 5. Additional Materials Required ELISA microwell plate reader, equipped for the measurement of absorbance at 450 nm Incubator 37 C Manual or automatic equipment for rinsing wells Pipettes to deliver volumes between 10 and 1000 μl Vortex tube mixer Distilled water Disposable tubes Glass Centrifuge tubes Timer 6

8 6. Preparation of Reagents It is very important to bring all reagents, samples and standards to room temperature (22-28 C) before starting the assay. 1. Coated snap-off Strips: The ready to use break apart snap-off strips are coated with anti-cortisol IgG antibodies. Store at 2-8 C. Open the bag only when it is at room temperature. Immediately after removal of strips, the remaining strips should be resealed in the aluminium foil along with the desiccant supplied and stored at 2-8 C; stability until expiry date. Do not remove the adhesive sheets on the unused strips. 2. Cortisol-HRP Conjugate: Prepare immediately before use. Add 10 μl stock solution to 1 ml Incubation buffer. Mix gently for 5 min with a rotating mixer. Stable for 3 hours at room temperature. 3. Cortisol Standards: The standards are ready to use. Incubation buffer is used as standard 0. After first use the standards are still stable for another 6 months at 2-8 C. 4. TMB Substrate Solution: The bottle contains 12 ml of a tetramethylbenzidine/hydrogen peroxide system. The reagent is ready to use and has to be stored at 2-8 C in the dark. The solution should be colorless or could have a slight blue tinge. If the substrate turns into blue, it may have become contaminated and should be thrown away. 7

9 5. Stop Solution: The bottle contains 12 ml 0.15 M sulphuric acid solution. This ready to use solution has to be stored at 2-8 C. 7. Preparation and Collection of Specimen The determination of Cortisol can be performed in saliva. It is recommended to collect saliva samples with a centrifuge glass tube and a plastic straw: let the saliva flow down through the strawn into the centrifuge glass tube; centrifuge at 3000 rpm for 5 min. SALIVETTE collector device can be used in Cortisol Saliva assay. Other equipment of sample collection commercially available has not been tested. Samples can be stored for one week at 2-8 C. For longer periods samples should be stored at -20 C. Repeated freeze- thawing should be avoided. Thawed samples should be inverted several times prior to use. 8

10 Precaution: 1. Maximum precision is required for dispensation of the reagents. 2. Avoid the exposure of reagent TMB substrate to direct sunlight, metals or oxidants. 3. This method allows the determination of Cortisol from ng/ml. 4. Treatment of the patient with corticosteroids, natural or synthetic steroids can impair Cortisol determination. 8. Assay Method Test Preparation: Please read the test protocol carefully before performing the assay. Result reliability depends on strict adherence to the test protocol as described. Prior to commencing the assay, the distribution and identification plan for all specimens and standards should be carefully established. Select the required number of microtiter strips or wells and insert them into the holder. 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. 9

11 Please allocate at least: 1 well (e.g. A1) for the substrate blank 2 wells (e.g. B1+C1) for standard 0 2 wells (e.g. D1+E1) for standard 1 2 wells (e.g. F1+G1) for standard 2 2 wells (e.g. H1+A2) for standard 3 2 wells (e.g. B2+C2) for standard 4 2 wells (e.g. D2+E2) for standard 5 2 wells (e.g. F2+G2) for standard 6 It is recommended to determine standards and samples in duplicate. Perform all assay steps in the order given and without any appreciable delays between the steps. A clean, disposable tip should be used for dispensing each standard and each sample. Assay Procedure: 1. Dispense 25 μl incubation buffer, standards and samples into their respective wells. 2. Add 200 μl diluted Conjugate to each well. Leave well A1 for substrate blank. 3. Cover wells with the foil supplied in the kit. 4. Incubate for 1 hour at 37 C. 10

12 5. When incubation has been completed, remove the foil, aspirate the content of the wells and wash each well twice with 300 μl distilled water. Avoid overflows from the reaction wells. The soak time between each wash cycle should be >5sec. At the end carefully remove remaining fluid by tapping strips on tissue paper prior to the next step! Note: Washing is critical! Insufficient washing results in poor precision and falsely elevated absorbance values. 6. Dispense 100 μl TMB Substrate Solution into all wells. 7. Incubate for exactly 15 min at room temperature (22-28 C) in the dark. 8. Dispense 100 μl Stop Solution into all wells in the same order and at the same rate as for the TMB Substrate Solution. Any blue colour developed during the incubation turns into yellow. 9. Measure the absorbance (E) of the specimen at 450 nm within 30 min after addition of the Stop Solution. Measurement: Adjust the ELISA Microwell Plate Reader to zero using the substrate blank in well A1. If - due to technical reasons - the ELISA reader cannot be adjusted to zero using the substrate blank in well A1, subtract the absorbance value of well A1 from all other absorbance values measured in order to obtain reliable results! 11

13 Measure the absorbance of all wells at 450 nm and record the absorbance values for each standard and sample in the distribution and identification plan. Where applicable calculate the mean absorbance values of all duplicates. Quality Control: Each laboratory should assay controls at normal, high and low levels range of Cortisol for monitoring assay performance. These controls should be treated as unknowns and values determined in every test procedure performed. Quality control charts should be maintained to follow the performance of the supplied reagents. Pertinent statistical methods should be employed to ascertain trends. The individual laboratory should set acceptable assay performance limits. Other parameters that should be monitored include the 80, 50 and 20% intercepts of the standard curve for run-to-run reproducibility. In addition, maximum absorbance should be consistent with past experience. Significant deviation from established performance can indicate unnoticed change in experimental conditions or degradation of kit reagents. Fresh reagents should be used to determine the reason for the variations. 12

14 9. Data Analysis 1. Calculate the mean absorbance for each point of the standard curve and each sample. 2. Plot the mean value of absorbance of the standards against concentration. Draw the best-fit curve through the plotted points. (Four Parameter Logistic). 3. Interpolate the values of the samples on the standard curve to obtain the corresponding values of the concentrations expressed in ng/ml. A. Sensitivity The lowest detectable concentration of Estriol that can be distinguished from the zero standard is 0.05 ng/ml at the 95 % confidence limit. B. Recovery The recovery of ng/ml of Cortisoll added to sample gave an average value (±SD) of 100% ± 6.3 % with reference to the original concentrations. 13

15 C. Reproducibility Intra-Assay: Within-run variation was determined by replicate determinations (16x) of two different control sera in one assay. The within-assay variability is 7%. Inter-Assay: Between-run variation was determined by replicate measurements of three different control sera in a two different lots. The between-assay variability is 9.3%. 10. Limitations Bacterial contamination or repeated freeze-thaw cycles of the specimen may affect the absorbance values. 14

16 11. Specificity The cross reaction of the antibody calculated at 50% according to Abraham is: Material Tested Cross reactivity Cortisol 100% Cortisone 10.8% 11 α-deoxicortisol 18.7% Corticosterone 2.4% Progesterone 0.1 % Aldosterone 0.01 % 11 α-oh-progesterone 0.01 % Cholesterol < 1x10-6 % 15

17 12. Troubleshooting Problem Cause Solution Poor standard curve Low signal High background Improper standard dilution Standard improperly reconstituted (if applicable) Standard degraded Curve doesn't fit scale Incubation time too short Target present below detection limits of assay Precipitate can form in wells upon substrate addition when concentration of target is too high Using incompatible sample type (e.g. serum vs. cell extract) Sample prepared incorrectly Wells are insufficiently washed Contaminated wash buffer Waiting too long to read plate after adding STOP solution Confirm dilutions made correctly Briefly spin vial before opening; thoroughly resuspend powder (if applicable) Store sample as recommended Try plotting using different scale Try overnight incubation at 4 C Decrease dilution factor; concentrate samples Increase dilution factor of sample Detection may be reduced or absent in untested sample types Ensure proper sample preparation/dilution Wash wells as per protocol recommendations Make fresh wash buffer Read plate immediately after adding STOP solution 16

18 Large CV Low sensitivity Bubbles in wells All wells not washed equally/thoroughly Incomplete reagent mixing Inconsistent pipetting Inconsistent sample preparation or storage Improper storage of ELISA kit Using incompatible sample type (e.g. Serum vs. cell extract) Ensure no bubbles present prior to reading plate Check that all ports of plate washer are unobstructed/wash wells as recommended Ensure all reagents/master mixes are mixed thoroughly Use calibrated pipettes and ensure accurate pipetting Ensure consistent sample preparation and optimal sample storage conditions (e.g. minimize freeze/thaws cycles) Store all reagents as recommended. Please note all reagents may not have identical storage requirements. Detection may be reduced or absent in untested sample types For further technical questions please do not hesitate to contact us by or phone (select contact us on for the phone number for your region). 17

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20 UK, EU and ROW Tel: +44 (0) US, Canada and Latin America Tel: ABCAM (22226) China and Asia Pacific Tel: ( 中國聯通 ) Japan technical@abcam.co.jp Tel: +81-(0) Copyright 2012 Abcam, All Rights Reserved. The Abcam logo is a registered trademark. All information / detail is correct at time of going to print. 19