Corticosterone Enzyme Immunoassay Kit - B
Contents
1. do not use this data 1 2 0 8 0 6 Absorban 0 4 0 2 0 0 Corticosterone Conc Notes 100 pg ml of Corticosterone is equivalent to 288 6 pM Assay sensitivity is 18 6 pg ml Limit of detection is 16 9 pg ml 9 B Bridge International Inc www b bridge com Corticosterone ELISA 130806 customersupport b bridge com 1 408 252 62002. 0 and 590 nm Software for converting raw relative fluorescent unit FLU readings from the plate reader and carrying out four parameter logistic curve 4PLC fitting PRECAUTIONS For in vitro research use only As with all such products this kit should only be used by qualified personnel who are experienced in routine laboratory procedures and safety practices Read and understand the complete user manual before attempting to use the product The Stop Solution is 1N hydrochloric acid Appropriate precautions should be observed when handling this caustic solution Avoid contact with skin or eyes Wear gloves and laboratory coats when handling materials and in all cases please consult your institution s safety procedures for working with hazardous chemicals 4 B Bridge International Inc www b bridge com Corticosterone ELISA 130806 customersupport b bridge com 1 408 252 6200 REAGENT PREPARATION Allow the kit reagents to come to room temperature for 30 minutes We recommend that all standards and samples be run in for accurate determination of corticosterone concentrations Ensure that all samples have reached room temperature and have been diluted as appropriate prior to running them in the kit The corticosterone stock solution contains an organic solvent Pre rinse the pipet tip several times to ensure accurate delivery Wash Buffer Dilute 20X Wash Buffer 1 20 by adding one part of the concentrate to nineteen parts of de
3. B Bridge International Inc A Corticosterone Enzyme Immunoassay Kit User Manual Catalog K3014 1 1 Plate Kit K3014 5 5 Plate Kit 1 B Bridge International Inc www b bridge com Corticosterone ELISA 130806 customersupport b bridge com 1 408 252 6200 TABLE OF CONTENTS Intended Use Background Assay Principle Kit Components Materials Required Precautions Reagent Preparation Normal Range Standards Expanded Range Standards Sample Preparation Assay Protocol Calculations oO O AON OA a FP A BPW 0 0 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 2 B Bridge International Inc www b bridge com Corticosterone ELISA 130806 customersupport b bridge com 1 408 252 6200 B Bridge International In
4. ation in the kit assay buffer Spike one aliquot of your sample with a volume of the corticosterone solution Control spike and one aliquot of sample with the same volume of assay buffer Control sample Extract both the Control spike and the Control sample with ethanol as described above Urine Samples Urine Samples should be diluted gt 1 20 with the supplied Assay Buffer prior to running in the assay Please see our Urinary Creatinine Detection Kit Cat K3002 1 for assays to measure urine creatinine which can be used to allow normalization of corticosterone in a random urine specimen 7 B Bridge International Inc www b bridge com Corticosterone ELISA 130806 customersupport b bridge com 1 408 252 6200 ASSAY PROTOCOL Standards and samples should be run in duplicate Be sure to include duplicate Non Specific Binding NSB reaction and blank reactions The NSB reaction omits the Corticosterone antibody and provides a background signal level for the assay The blank only includes the Assay Buffer and provides the maximum signal level without corticosterone competition 1 Pipet 50 L of samples or standards into each well Also pipet 50 L of Assay Buffer into wells for blanks points Bo or 0 pg mL and 75 L of Assay Buffer into the non specific binding NSB wells 2 Add25 Lofthe Corticosterone Peroxidase Conjugate to each well using a repeater or multichannel pipet 3 Excluding the NSB wells add 25 L of the Corticosterone Anti
5. body to each reaction 4 Gently tap the sides of the plate to ensure adequate mixing then cover the plate with the plate sealer and shake at room temperature for 1 hour Note shaking is necessary to ensure maximum sensitivity 5 Aspirate the plate and wash 4 times with 300 LWash Buffer Tap the plate dry on absorbent towels 6 Add 100 L ofthe TMB Substrate to each well 7 Incubate the plate at room temperature for 30 minutes without shaking 8 Add 50 L ofthe Stop Solution to each well 9 Read the absorbance at 450 nm using a plate reader the correction should be set at approximate 580 nm B Bridge International Inc www b bridge com Corticosterone ELISA 130806 customersupport b bridge com 1 408 252 6200 CALCULATIONS After averaging duplicate absorbance readings subtract the signals for the NSB to obtain the signal value for each sample and standard Create a standard curve by fitting the standards to a 4 parameter logistic curve 4PLC The concentration of corticosterone in each sample is then using the B BO from the curve then multiply by the dilution value to obtain original concentrations amounts TypicaL DATA Mean OD B BO Corticosterone Conc EE Standard 1 0 270 0 223 as Standard 2 0 380 0 333 2 500 Standard 3 0 529 0 482 1 250 es es mo __ Sample 2 TYPICAL STANDARD CURVE EXAMPLE ONLY Standard curves vary with each assay Always run your own standard curves for calculation of results
6. c All Rights Reserved INTENDED USE The B Bridge Corticosterone ELISA Kit quantitatively measures Corticosterone present in dried fecal extracts serum EDTA and heparin plasma samples and tissue culture medium Please read the complete kit insert before performing this assay This kit is species independent BACKGROUND Corticosterone is a glucocorticoid secreted by the cortex of the adrenal gland and is produced in response to stimulation of the adrenal cortex by ACTH and the precursor of aldosterone Corticosterone is a major stress steroid Since the chemical structure of corticosterone appears identical across all known species the assay provides consistent results regardless of the species source of the samples Corticosterone ASSAY PRINCIPLE The B Bridge Corticosterone ELISA Kit uses an antibody sandwich to link corticosterone to the microtiter plate Levels of corticosterone are measured by competition with known amounts of a detectable corticosterone peroxidase conjugate The procedure is straightforward and involves only two incubations a 1 hour incubation to capture the corticosterone complexes followed by a second 30 minute incubation to develop the detection reagent The microtiter plate has been coated with antibodies that recognize sheep antibodies Sheep anti corticosterone antibodies are then used to capture corticosterone and bind it to the plate A corticosterone level in the samples is quantified using a corticoster
7. ionized water Once diluted this is stable at room temperature for 3 months at room temperature Normal Range Standard Preparation Using the Corticosterone standard stock at 100 000 pg ml prepare a series of 7 standards by serial dilution as follows referring to the table below Label seven glass test tubes as 1 through 7 Briefly spin vial of standard in a microcentrifuge to ensure contents are at the bottom of the vial Pipet475 Lof Assay Buffer into tube 1 and 250 L into tubes 2 to 7 Add25 Lofthe corticosterone stock solution to tube 1 and vortex completely Take 250 Lofthe corticosterone solution in tube 1 and add it to tube 2 and vortex completely Repeat the serial dilutions for tubes 3 through 7 The concentration of corticosterone in tubes 1 through 7 will be 5 000 2 500 1 250 625 312 5 156 25 and 78 125 pg mL Standard Standard Standard Standard Standard Standard Standard Reagent 1 2 3 4 5 6 7 Assay Buffer Volume 475 ul 250 ul 250 ul 250 ul 250 ul 250 ul 250 ul Corticosterone Stock 25 pl Standard 1 250 pl Standard 2 250 pl Standard 3 250 ul Standard 4 250 ul Standard 5 250 ul Standard 6 250 pl Final Concentration pg mL 5 000 2 500 1 250 625 312 5 156 25 78 125 Use all Standards within 2 hours of preparation B Bridge International Inc www b bridge com Corticosterone ELISA 130806 customersupport b bridge com 1 408 252 6200 Ex
8. one peroxidase conjugate Corticosterone in the samples competes with the corticosterone peroxidase conjugate After the reaction components equilibrate the level of corticosterone in the samples negatively correlates with the level of detectable corticosterone peroxidase conjugate After addition of the substrate the assay signal decreases with increasing amounts of corticosterone A corticosterone standard is provided to generate a standard curve for the assay and all samples should be read off the standard curve 3 B Bridge International Inc www b bridge com Corticosterone ELISA 130806 customersupport b bridge com 1 408 252 6200 KIT COMPONENTS Component K3014 1 K3014 5 Donkey Anti Sheep IgG Coated 96 Well Plate 1 plate 5 plates Corticosterone Standard 100 000 pg mL 125 625 Corticosterone Antibody 3ml 13 ml Corticosterone Conjugate 3 ml 13 ml 1X Assay Buffer 28 ml 55 ml Dissociation Reagent 1 ml 5 ml To be used only with serum and plasma samples 20X Wash Buffer Concentrate 30 ml 125 ml TMB Substrate 11 ml 55 ml Stop Solution 5 ml 25 ml 1N hydrochloric acid solution HCI CAUSTIC Plate Sealer 1 each 5 each Store above components at 4 C MATERIALS REQUIRED BUT NOT SUPPLIED Supply of distilled or deionized water Microplate washer Repeater pipet with disposable tips capable of dispensing 25ul 50ul and 100ul Colorimetric 96 well microplate reader capable of reading OD at 450 nm with correction between 57
9. panded Range Standard Preparation Using the Corticosterone standard stock at 100 000 pg ml prepare a series of 8 standards by serial dilution as follows referring to the table below Label eight glass test tubes as 1 through 8 Briefly spin vial of standard in a microcentrifuge to ensure contents are at the bottom of the vial Pipet 450 Lof Assay Buffer into tube 1 and 250 L into tubes 2 to 7 Add50 Lofthe corticosterone stock solution to tube 1 and vortex completely Take 250 Lofthe corticosterone solution in tube 1 and add it to tube 2 and vortex completely Repeat the serial dilutions for tubes 3 through 7 The concentration of corticosterone in tubes 1 through 8 will be 10 000 5 000 2 500 1 250 625 312 5 156 25 and 78 125 pg mL Standard Standard Standard Standard Standard Standard a IN 1 E 2 3 4 5 6 450u 250i 250u 250ui l asou 2504 2604 2500 z 50 ul Standard 1 250 ul Loo CC Standard 2 250 ul _ __ ee Standard 3 250 ul Doo Oooo Standard 4 250 Ul a ION Standard 5 250 Standard 6 250ul _250u Standard 7 250ul Final Concentration pg mL 10 000 5 000 2 500 1 250 312 5 156 25 78 125 Use all Standards within 2 hours of preparation B Bridge International Inc www b bridge com Corticosterone ELISA 130806 customersupport b bridge com 1 408 252 6200 SAMPLE PREPARATION This as
10. say has been validated with serum plasma EDTA or heparin cell extract and dried fecal extract samples Samples containing visible particulate should be centrifuged prior to using Moderate to severely hemolyzed samples should not be used in this kit Use samples within 2 hours of dilution Serum and Plasma Samples Serum and plasma samples should be diluted with an equal volume of Dissociation Reagent Diluted samples should then be further diluted at least 1 50 with Assay Buffer Tissue Culture Media Corticosterone can be directly measured in Cell Culture supernatant This assay has been validated with RPMI 1640 medium The standard curve should be generated using the culture media instead of the Assay Buffer and samples with a high concentration of Corticosterone should be diluted in medium Dried Fecal Samples Weigh out 0 2 g of dried fecal solid Add 1 mL of Ethanol or Ethyl Acetate for every 0 1 gm of solid and vortex for at least 30 minutes Centrifuge samples at 5 000 rpm for 15 minutes then transfer supernatant to a clean tube Evaporate supernatant 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 Repeat vortex and rest 5 minutes 2 3 times to ensure complete resuspension Recommendation Determine the extraction efficiency by preparing a corticosterone solution of know concentr
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