Cortisone Chemiluminescent Immunoassay Kit - B
Contents
1. 1 408 252 6200 SAMPLE PREPARATION Serum samples need to be treated with the supplied Dissociation Reagent Addition of this reagent will yield the total cortisone concentration in serum Dissociation Reagent is to be used only with Serum samples Free cortisone can be measured in saliva and urine samples as directed below Dried Fecal Samples Weigh out gt 0 2 gm of dried fecal solid into a tube Add 1 mL of Ethanol or Ethyl Acetate for every 0 1 gm of solid and shake vigorously for at least 30 minutes Centrifuge samples at 5 000 rpm for 15 minutes then transfer supernatant to a clean tube Evaporate supernatant to dryness in a SpeedVac or under nitrogen Store dried extracted samples at 20 C in a desiccator Just before assaying dissolve extracted sample with 100 L ethanol and add at least 400 L Assay Buffer Vortex well then allow to rest 5 minutes Do this 2 3 times to ensure complete resuspension The ethanol concentration in the final Assay Buffer dilution added to the well should be lt 5 Run reconstituted diluted samples in assay immediately according to insert directions Saliva Samples Saliva samples should be frozen and thawed then centrifuged at 14 000 rpm for 15 minutes The supernatant should be diluted 1 5 to 1 10 with the supplied Assay Buffer prior running in the assay Urine Samples Urine samples should be diluted 2 1 100 with the supplied Assay Buffer prior running in the assay Serum Samples Serum samples sh2. and cortisol concentrations exhibit a predictable diurnal pattern and can be measured in extracted dried feces or in serum plasma saliva and urine A recent publication8 has suggested that salivary cortisone is a good surrogate marker for serum cortisol O HO O OH ASSAY PRINCIPLE The B Bridge Cortisone Chemiluminescent Immunoassay kit is designed to quantitatively measure Cortisone present in extracted dried fecal samples urine saliva and serum samples Please read the complete kit insert before performing this assay 1 Sample or standard added to well in microtiter plate 2 Cortisone peroxidase conjugate is added to the standards and samples in the wells 3 A polyclonal antibody to cortisone is added to each well 4 Incubate for 2 hours wash plate and add substrate to each well 5 Read luminescence and calculate cortisone concentration from standard curve after short incubation 3 B Bridge International Inc www b bridge com Cortisone 150623 customersupport b bridge com 1 408 252 6200 KIT COMPONENTS Component Cat Coated White 96 Well Plates Cortisone Standard 1 000 ng mL Cortisone Antibody Rabbit Polyclonal Cortisone Conjugate Concentrate Conjugate Diluent Assay Buffer Dissociation Reagent use only with serum samples 20X Wash Buffer Concentrate Substrate Solution A Substrate Solution B Plate Sealer K3017 1 1 plate 50 I 3 ml 1 ml 3 ml 28 ml 1 ml 30 ml 6 ml 6 ml 1 each K3
3. 017 5 5 plates 125 13 ml 3 5 ml 13 ml 55 ml 5 ml 125 ml 28 ml 28 ml 5 each Once opened the kit can be stored at 4 C up to the expiration date on the kit label except for the PGE2 Conjugate which must be stored at 20 C MATERIALS REQUIRED BUT NOT SUPPLIED Distilled or deionized water Repeater pipet with disposable tips capable of dispensing 25 uL and 100 uL A microplate shaker 96 well microplate reader capable of reading glow chemiluminescence All luminometers read Relative Light Units RLU These RLU readings will vary with make or model of plate reader The number of RLUs obtained is dependant on the sensitivity and gain of the reader used If you are unsure of how to properly configure your reader contact your plate reader manufacturer or carry out the following protocol Dilute 5 L of the Cortisone Conjugate Concentrate into 995 L of deionized water Pipet 5 L of diluted conjugate into a white well and add 100 L of prepared substrate solution see page 5 for details This well will give you an intensity close to the maximum binding signal for the assay Adjust the gain integration time or sensitivity so that your reader is giving close to its maximum signal To properly analyze the data software will be required for converting raw RLU readings from the plate reader and carrying out four parameter logistic curve 4PLC fitting Contact your plate reader manufacturer for details 4 B Bridge I
4. B Bridge International Inc kA Cortisone Chemiluminescent Immunoassay Kit User Manual Catalog K3017 1 1 Plate Kit K3017 5 5 Plate Kit 1 B Bridge International Inc www b bridge com Cortisone 150623 customersupport b bridge com 1 408 252 6200 TABLE OF CONTENTS Intended Use Background Assay Principle Kit Components Materials Required Precautions Reagent Preparation Sample Preparation Assay Protocol Calculations N N O Aa dl dl sa FW dd W Typical Standard Curve Example Notice to Purchaser This product is to be used for Research Purposes Only It is not to be used for Drug or Diagnostic Purposes nor is it intended for Human Use B Bridge products may not be resold modified for resale or used to manufacture commercial products without the express written consent of B Bridge International Inc EXCEPT AS OTHERWISE EXPRESSLY SET FORTH IN THIS USER MANUAL B BRIDGE DOES NOT MAKE ANY REPRESENTATION OR WARRANTIES OR CONDITIONS OF ANY KIND EITHER EXPRESSED OR IMPLIED WITH RESPECT TO THE PRODUCTS OR INFORMATION DISCLOSED HEREUNDER INCLUDING BUT NOT LIMITED TO THE IMPLIED WARRANTIES OF MERCHANTABILITY FIT FOR A PARTICULAR PURPOSE OR NONINFRINGEMENT OF THE INTELLECTUAL PROPERTY RIGHTS OF THIRD PARTIES B Bridge International Inc All Rights Reserved 2 B Bridge International Inc www b bridge com Cortisone 150623 customersupport b bridge com 1 408 252 6200 INTENDED USE The B Bridge Cortisone Chemi
5. d the Cortisone conjugate is stable for one month when stored at 4 C Chemiluminescent Substrate Mix one part of the Substrate Solution A with one part of Substrate Solution B in a brown bottle Once mixed the substrate is stable for one month when stored at 4 C Standard Preparation 1 Label six test tubes as 1 through 9 2 Pipet 490 Lof Assay Buffer into tube 1 and 250 L of Assay Buffer into tubes 2 9 3 Carefully add 10 L of the cortisone stock solution to tube 1 and vortex completely 4 Take 250 L of the solution in tube 1 and add it to tube 2 and vortex completely 5 Repeat the serial dilutions for tubes 3 through 9 The concentration of cortisone in tubes 1 through 5 will be 20 000 10 000 5 000 2 500 1 250 625 312 5 156 3 and 78 1 pg mL Use all Standards within 2 hours of preparation Reagent Sid 1 Sid 2 Std 3 Std 4 Std 5 Std 6 Std 7 Std 8 Std 9 Assay Buffer 490 pl 250ul 250ul 250ul 250p 25041 250ul 25041 250 pil Cortisone Stock 10 pl Standard 1 250 pl Standard 2 250 pl Standard 3 250 pl Standard 4 250 pl Standard 5 250 pl Standard 6 250 pl Standard 7 250 pl Standard 8 250 pl Standard 9 Final Concentration 20 000 10 000 5 000 2 500 1 250 625 312 5 156 25 78 1 pg mL 5 B Bridge International Inc www b bridge com Cortisone 150623 customersupport b bridge com
6. luminescent Immunoassay Kit quantitatively measures Cortisone present in extracted dried fecal samples urine saliva and serum samples This assay is species independent Please read the complete kit insert before performing this assay BACKGROUND Cortisone C21H2805 Kendall s Compound E was identified by Mason Myers and Kendall in 1936 as Compound E extracted from bovine suprarenal gland tissue that had the qualitative but not quantitative activity of cortin The presence of multiple cortin like compounds led the authors to speculate that the study of Compound E would reveal the nature of cortin Compound E is now called cortisone and the more active Compound F cortisol and the concentrations of these two glucocorticoids vary due to the activity of two 11B hydroxysteroid dehydrogenases 11 HSD While most tissues have the ability to express either enzyme 11B HSD1 is found primarily in the liver where it converts cortisone to cortisol while 118 HSD2 is found in tissues such as the kidney where cortisol receptor binding is required 118 HSD2 deactivates cortisol to cortisone prohibiting receptor activation This glucocorticoid shuttle helps to initiate and regulate the anti inflammatory response making cortisone one of the modern wonder drugs Monitoring the ratio of cortisone cortisol has applications in diabetes obesity metabolic syndrome osteoporosis and chronic fatigue syndrome in addition to adrenal diseases Cortisone
7. nternational Inc customersupport b bridge com www b bridge com Cortisone 150623 1 408 252 6200 PRECAUTIONS As with all such products this kit should only be used by qualified personnel who have had laboratory safety instruction The complete insert should be read and understood before attempting to use the product This kit utilizes a peroxidase based readout system Buffers including other manufacturers Wash Buffers containing sodium azide will inhibit color production from the enzyme Make sure all buffers used for samples are azide free Ensure that any plate washing system is rinsed well with deionized water prior to using the supplied Wash Buffer In all cases please consult your institution s safety procedures for working with hazardous chemicals REAGENT PREPARATION Allow the kit reagents to thaw and come to room temperature for 30 60 minutes We recommend that all standards and samples be run in duplicate to allow the end user to accurately determine prostaglandin E2 concentrations Ensure that all samples have reached room temperature and have been diluted as appropriate prior to running them in the kit Wash Buffer Dilute Wash Buffer Concentrate 1 20 by adding one part of the concentrate to nineteen parts of de ionized water Once diluted this is stable at room temperature for 3 months Cortisone Conjugate The supplied Cortisone Conjugate Concentrate should be diluted 1 4 with the Conjugate Diluent Once dilute
8. ould be diluted with an equal volume of the supplied Dissociation Reagent The diluted samples should then be further diluted 1 50 with the supplied Assay Buffer prior running in the assay NOTE Dissociation Reagent is to be used only with Serum samples Tissue Culture Media For measuring cortisone in tissue culture media TCM samples should be read off a standard curve generated in TCM Samples may need to be diluted further in TCM Use all samples within 2 hours of preparation ASSAY PROTOCOL 1 Determine the number of wells to be used and return unused wells to the foil pouch with desiccant Seal the ziploc plate bag and store at 4 C 2 Pipet 50 L of samples or standards into wells in the plate 3 Pipet 75 L of Assay Buffer into the non specific binding NSB wells 4 Pipet 50 L of Assay Buffer into wells to act as maximum binding wells B or 0 pg mL 5 Add 25 L of the Cortisone Conjugate to each well using a repeater pipet 6 Add 25 L of the Cortisone Antibody to each well except the NSB wells using a repeater pipet 7 Gently tap the sides of the plate to ensure adequate mixing of the reagents Cover the plate with the plate sealer and shake at room temperature for 2 hours If the plate is not shaken signals will be approximately 45 lower 8 Aspirate the plate and wash each well 4 times with 300 L wash buffer Tap the plate dry on clean absorbent towels 9 Add 100 L ofthe mixed Chemiluminescent Substrate to each
9. well using a repeater pipet 10 Immediately read the luminescence generated from each well in a mutimode or chemiluminescent plate reader using a 0 1 second read time per well The chemiluminescent signal will decrease about 40 over 60 minutes 6 B Bridge International Inc www b bridge com Cortisone 150623 customersupport b bridge com 1 408 252 6200 11 Use the plate reader s built in 4PLC software capabilities to calculate Cortisone concentration for each sample CALCULATIONS All luminometers read Relative Light Units RLU These RLU readings will vary with make or model of plate reader Average the duplicate RLU readings for each standard and sample Create a standard curve by reducing the data using the 4PLC fitting routine on the plate reader after subtracting the mean RLU s for the NSB The sample concentrations obtained calculated from the B By curve should be multiplied by the dilution factor to obtain neat sample values TYPICAL STANDARD CURVE EXAMPLE ONLY 100 TT Io OSCE CT HOSS TE IN 70 E 50 B BO N Ih 10 000 100 000 NU TR ail anil T 20 i m o 1 Cortisone ne ve fo Always run your own standard curves for calculation of results Do not use this data 7 B Bridge International Inc www b bridge com Cortisone 150623 customersupport b bridge com 1 408 252 6200
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