ab175813 – 14,15 DHET ELISA Kit for Human Urine
Contents
1. ASSAY PREPARATION thoroughly and incubate for 1 h at 50 C This will yield an aqueous solution c Vortex and incubate for 1 h at 50 C This will yield an aqueous solution c 11 1 10 3 Dilute 2 mL of the aqueous solution c with 3 mL of H O Adjust the pH using 2096 formic acid 132 uL to pH 5 5 Add ethyl acetate 1 part aqueous solution c 1 part ethyl acetate vortex thoroughly and centrifuge at 2000 rpm for ten minutes at 22 C Repeat the procedure twice more using an equal volume of ethyl acetate per sample Collect the upper phase containing saponified lipids 11 1 10 4 Dry the pooled ethyl acetate upper phase d and dry in a Speedvac yielding the dried sample sediment e Store the sediment e at 20 C For ELISA assay dissolve the sediment e in 20 pL of DMF or ethanol then add 130 uL of 1X Sample Dilution Buffer 11 1 10 5 For the competitive 14 15 DHET ELISA the above 150 uL sample needs to be further diluted Dilute 1 4 e g 80 uL sample 320 pL 1x Sample Dilution Buffer Check the final pH should be pH 7 4 When calculating the final concentration consider all dilution factors 11 1 10 6 Perform the ELISA for 14 15 DHET 11 2 Measurement of Free and Glucuronidated 14 15 DHET without extraction 11 2 1 Measurement of free 14 15 DHET Dilute 1 mL urine 4 fold with 1X Sample Dilution Buffer and apply to ELISA plate 100 uL well A 4X dilution is recommended although other dilution factors may2. ICAL DATA TYPICAL STANDARD CURVE Data provided for demonstration purposes only A new standard curve must be generated for each assay performed The data shown here is an example of typical results obtained using the Abcam s14 15 DHET ELISA kit These results are only a guideline and should not be used to determine values from your 10000 100000 1000000 samples 100 0 90 0 80 0 70 0 60 0 g 50 0 oO 40 0 30 0 20 0 10 0 0 0 1 10 100 14 15 DHET pg ml Conc pg mL B Bo 10 85 4 100 68 2 1 000 47 3 10 000 27 9 100 000 11 0 1 000 000 5 4 Discover more at www abcam com DATA ANALYSIS 16 ASSAY SPECIFICITY The specificity of the 14 15 DHET ELISA was investigated using authentic 14 15 DHET and a panel of eiconsanoids Eiconsanoid Reactivity 14 15 DHET 100 00 8 9 DHET 3 30 11 12 DHET 3 30 14 15 EET 1 5 15 s HETE 1 00 8 9 EET 0 40 5 s 15 s DiHETE 0 2096 11 12 EET 0 0596 Arachidonic Acid 0 0596 5 6 DHET 0 0296 5 6 EET 0 02 Thromboxane B5 0 02 PGE2 0 01 96 PGF24 0 01 96 6 keto PGF 1 lt 0 01 SENSITIVITY The calculated minimal detectable MDD dose is 3 pg mL The MDD was determined by calculating the mean of zero standard replicates Discover more at www abcam com 17 RESOURCES 17 TROUBLESHOOTING Problem Cause Solution The HRP conjugate Redo the assay and
3. abcam discover more ab175813 14 15 DHET ELISA Kit for Human Urine Instructions for Use A competitive immunoenzymatic assay for the quantitative measurement of free and glucuronidated 14 15 DHET in urine This product is for research use only and is not intended for diagnostic use Version 2 Last Updated 2 March 2015 Table of Contents INTRODUCTION 1 BACKGROUND 2 2 ASSAY SUMMARY 3 GENERAL INFORMATION PRECAUTIONS STORAGE AND STABILITY MATERIALS SUPPLIED MATERIALS REQUIRED NOT SUPPLIED LIMITATIONS TECHNICAL HINTS PNP hw OaanharaA ASSAY PREPARATION 9 REAGENT PREPARATION 10 STANDARD PREPARATION 11 SAMPLE COLLECTION AND STORAGE 12 PLATE PREPARATION 1 oOo oN ASSAY PROCEDURE 13 ASSAY PROCEDURE 14 DATA ANALYSIS 14 CALCULATIONS 15 15 TYPICAL DATA 16 16 ASSAY SPECIFICITY 17 RESOURCES 17 TROUBLESHOOTING 18 18 NOTES 20 Discover more at www abcam com 1 INTRODUCTION 1 BACKGROUND Abcam s 14 15 DHET competitive in vitro ELISA Kit for Human Urine is designed for determination of free and glucuronidated 14 15 DHET levels in urine This competitive ELISA kit is based on competition between the 14 15 DHET epitope and the 14 15 DHET HRP conjugate for a limited number of binding sites available from the anti 14 15 DHET antibody which is coated to the wells of the 96 well ELISA plate The conjugate concentration is held as a constant in each well while the concentration o
4. add the No color was not added conjugate at the proper step present in A gag 1 APR sem Redo the assay and incubate for S the proper time the proper time The TMB substrate was not added Add substrate No color in RO Wes Tho TMB conjugate Continue incubation until desired was not incubated for Solor is teached the proper time Discover more at www abcam com 18 RESOURCES Problem Cause Solution E ware Redo the assay with the proper incubation times cut short i The TMB Substrate Redo the assay makin re all The coloris was not warmed up to y Mang Sure a faint room temperature reagents are at room temperature Be sure the lab temperature is The lab is too cold between 21 27 C and redo the assay The background Ee Ms subetale Redo the assay with a fresh bottle color is very contaminated of substrate high c Da Redo assay using an 8 channel obtained Incorrect loading of pipetman making sure the 8 F m samples channels are equal volume while sample loading Discover more at www abcam com RESOURCES 18 NOTES Discover more at www abcam com 20 RESOURCES Discover more at www abcam com 21 RESOURCES Discover more at www abcam com 22 abcam discover more UK EU and ROW Email technical abcam com Tel 44 0 1223 696000 Austria Email wissenschaftlicherdienst abcam com Tel 019 288 259 France Email supportscientifique abcam com Tel 01 46 94 62 96 Ge
5. be tried too Discover more at www abcam com 11 ASSAY PREPARATION 11 2 2 11 2 3 Measurement of glucuronidated 14 15 DHET This method is for determining the level of glucuronidated 14 15 DHET in urine after digestion of the molecule with glucuronidase Collect the first sample as soon as the Beta Glucuronidase is added to a reaction mixture 0 hour digestion and then a second sample at a time when digestion of the glucuronic acid moiety of the molecule is completed Subtract the level of the molecule in the first sample at 0 hour from the levels in the second sample after complete digestion usually 3 hrs to obtain the level of glucuronidated molecule Beta Glucuronidase digestion 11 2 3 1 Dilute 1 mL of urine 4 fold with 1X Sample Dilution Buffer 11 2 3 2 To 4 mL of urine add 1 mL of the Beta Glucuronidase solution pH 5 5 to each tube pH lt 6 0 11 2 3 3 Immediately transfer 2 mL of urine to a clean tube and flash freeze This is the Zero time point 11 2 3 4 Incubate the remaining 2 mL at 37 C for 3 hours This is the 3 hour time point 11 2 3 5 Follow the instructions for the ELISA kit 11 2 3 6 To calculate the amount of glucuronidated Discover more at www abcam com 12 14 15 DHET subtract the Zero time value from the 3 hour value ASSAY PREPARATION 12 PLATE PREPARATION e The 96 well plate included with this kit are supplied ready to use It is not necessary to rinse the plate
6. f the 14 15 DHET is variable based on the concentration of the sample or standard Thus the amount of the 14 15 DHET conjugate which is able to bind to each of the wells is inversely proportional to the concentration of 14 15 DHET in the standard or sample The amount of the conjugate which is bound to each well is then determined by the amount of color obtained when TMB is added The TMB reacts with the HRP available in the well With the addition of sulfuric acid the blue colored product is converted into a yellow colored product which can be read on a plate reader at 450 nm The 14 15 DHET is a representative metabolite of soluble epoxide hydrolase mediated metabolism of EETs which are generated by arachidonic acid epoxygenase activity of cytochromes P450 14 15 DHET level exhibited strong positive correlation with hypertension in rat and human and brain injury and stroke in rodents High levels of the glucuronidated form of 14 15 DHET have been found in human urine but not in urine collected from rodents Discover more at www abcam com 2 INTRODUCTION 2 ASSAY SUMMARY Capture Antibody YYY Sample Add standards and samples to o Y each well used Labeled HRP Conjugate Add prepared HRP conjugate Qo o to each well and incubate at Y Y Y room temp bac Substrate Colored Product Add TMB substrate to each well Incubate at room temperature Add Stop Solution Qo o to each well Read immediately YyY Prepare al
7. ffer One vial makes enough conjugate for one plate The conjugate must be used the same day and should not be stored for later use 9 3 1X Sample Dilution Buffer Prepare 1X Sample Dilution Buffer by adding 25 mL of 10X Sample Dilution Buffer to 225 mL of dH20 Mix gently and thoroughly Discover more at www abcam com 7 ASSAY PREPARATION 10 STANDARD PREPARATION Prepare serially diluted standards immediately prior to use Always prepare a fresh set of positive controls for every use 10 1 10 2 10 3 10 4 10 5 10 6 10 7 Standard Label 5 microtubes as Standard 2 6 Add 900uL of the 1X Sample Dilution Buffer to the microtubes for Standards 2 to 6 Prepare a 1 ug mL Standard 1 by first spinning down the enclosed 14 15 DHET standard vial 2 uL filled with inert gas and then adding 1 998 mL of 1X Sample Dilution Buffer to obtain 2 mL of solution Prepare Standard 2 by adding 100uL of the Standard 1 to the microtube labeled Standard 2 Mix thoroughly and gently Prepare Standard 3 by adding 100uL of the Standard 2 to the microtube labeled Standard 3 Mix thoroughly and gently Using the table below as a guide repeat for tubes 4 through 6 Standard B contains no protein and is blank control Sample to Dilute Volume to Dilute Volume of Diluent 22 Starting Conc pg mL Final Conc pg mL Step 10 3 1 000 000 Standard 1 100 900 1 000 000 100 000 S
8. g wells e Storage bottles e Rotating mixer e Deionised or freshly distilled water e Disposable tubes e Timer e 2N Sulfuric acid e 1M Citric acid 7 LIMITATIONS e ELISA kit intended for research use only Not for use in diagnostic procedures e Use only clean pipette tips dispensers and lab ware e Donotinterchange screw caps of reagent vials to avoid cross contamination e Close reagent vials tightly immediately after use to avoid evaporation and microbial contamination e After first opening and subsequent storage check conjugate and control vials for microbial contamination prior to further use e To avoid cross contamination and falsely elevated results pipette patient samples and dispense conjugate without splashing accurately to the bottom of wells Discover more at www abcam com 5 GENERAL INFORMATION 8 TECHNICAL HINTS e Avoid foaming or bubbles when mixing or reconstituting components e Avoid cross contamination of samples or reagents by changing tips between sample standard and reagent additions e Ensure plates are properly sealed or covered during incubation steps e Complete removal of all solutions and buffers during wash steps is necessary for accurate measurement readings e Addition of the TMB Substrate solution initiates a kinetic reaction which is terminated by the addition of the Stop Solution Therefore the TMB Substrate and the Stop Solution should be added in the same sequence to eli
9. he three wash cycles pat the inverted plate dry onto some paper towels 13 7 Add 200 uL of the TMB substrate to all of the wells 13 8 Incubate the plate at room temperature for 15 30 minutes 13 9 Add 50 uL of 2 N sulfuric acid to all of the wells 13 10 Read the plate at 450 nm Discover more at www abcam com 14 DATA ANALYSIS 14 CALCULATIONS If data redaction software is not available on your plate reader then the results can be obtained manually as follows 14 1 Average the absorbance Abs readings from the blank wellsand subtract that value from each well of the plate to obtain the corrected readings Note Some plate readers do this automatically Consult the user manual of your plate reader 14 2 Average the corrected absorbance readings from the maximum binding control wells This is your maximum binding 14 3 Calculate the 96 Abs for Standard 1 by averaging the corrected absorbance of the two wells divide the average by the Maximum Binding Control well average absorbance then multiply by 100 Repeat this formula for the remaining standards 14 4 Plot the Abs versus the concentration of 14 15 DHET from the standards using semi log paper 14 5 Calculate the 96 Abs for the samples and determine the concentrations utilizing the standard curve 14 6 Multiply the concentrations obtained for each of the samples by their corresponding dilution factor Discover more at www abcam com 15 DATA ANALYSIS 15 TYP
10. l reagents and samples as instructed Discover more at www abcam com 3 GENERAL INFORMATION 3 PRECAUTIONS Please read these instructions carefully prior to beginning the assay All kit components have been formulated and quality control tested to function successfully as a kit Modifications to the kit components or procedures may result in loss of performance 4 STORAGE AND STABILITY Store kit at 2 8 C or 20 C immediately upon receipt Refer to list of materials supplied for storage conditions of individual components Observe the storage conditions for individual prepared components in section 9 Reagent Preparation 5 MATERIALS SUPPLIED Storage Amount Condition After Preparation 14 15 DHET ELISA plate 96 Wells 2 8 C 14 15 DHET Standard 1 mg mL 2 uL 2 8 C 1 000X 14 15 DHET HRP Conjugates 12 uL 2 8 C 10X Sample Dilution Buffer 25 mL 2 8 C HRP Buffer 15 mL 2 8 C 10X Wash Buffer Solution 25 mL 2 8 C TMB Substrate 24 mL 2 8 C Beta Glucuronidase enzyme 8 mg 2 8 C Discover more at www abcam com 4 GENERAL INFORMATION 6 MATERIALS REQUIRED NOT SUPPLIED These materials are not included in the kit but will be required to successfully utilize this assay e Microplate reader capable of measuring absorbance at 450 nm e Incubator at 37 C e Multi and single channel pipettes to deliver volumes between 10 and 1 000 uL e Optional Automaticplate washer for rinsin
11. minate any time deviation during the reaction e It is important that the time of reaction in each well is held constant for reproducible results Pipetting of samples should not extend beyond ten minutes to avoid assay drift If more than 10 minutes are needed follow the same order of dispensation If more than one plate is used it is recommended to repeat the dose response curve in each plate e The incomplete or inaccurate liquid removal from the wells could influence the assay precision and or increase the background e This kit is sold based on number of tests A test simply refers to a single assay well The number of wells that contain sample control or standard will vary by product Review the protocol completely to confirm this kit meets your requirements Please contact our Technical Support staff with any questions Discover more at www abcam com 6 ASSAY PREPARATION 9 REAGENT PREPARATION Equilibrate all reagents samples and controls to room temperature 18 25 C prior to use 9 1 1X Wash Buffer Mix the 10X Wash Buffer Solution with a stir bar applying low gentle heat until a clear colorless solution is obtained Dilute the entire contents of the 10X Wash Buffer Solution 25 mL with 225 mL of deionized water to yield a final volume of 250 mL of 1 X Wash Buffer This can then be refrigerated for the entire life of the kit 9 2 1X HRP Conjugate Dilute 1 vial of the 14 15 DHET HRP conjugate 12 uL with 12mL of HRP Bu
12. prior to adding reagents e Unused well strips should be returned to the plate packet and stored at 4 C e For each assay performed a minimum of 2 wells must be used as a blank omitting sample and conjugate from well addition Another 2 wells must be used for a maximum binding control e For statistical reasons we recommend each standard and sample should be assayed with a minimum of two replicates duplicates Discover more at www abcam com 13 ASSAY PROCEDURE 13 ASSAY PROCEDURE e Equilibrate all materials and prepared reagents to room temperature prior to use e Please read the test protocol carefully before performing the assay Result reliability depends on strict adherence to the test protocol as described e If performing the test on anautomatic ELISA system we recommend increasing the washing steps from three to five and the volume of 1X Wash Bufferfrom 300 pL to 350 uL to avoid washing effects e Assay all standards controls and samples in duplicate 13 1 Add 200 uL of 1X Sample Dilution Buffer into the blank wells and 100 uL of1XSample Dilution Buffer into maximum binding control wells 13 2 Add 100 uL of each of the standards or samples into the appropriate wells 13 3 Add 100 uL of the 1X HRP conjugate in the all wells except the blank control wells 13 4 Incubate the plate at room temperature for two hours 13 5 Wash the plate three times with 400 uLof 1X Wash Buffer per well 13 6 After the last of t
13. rmany Email wissenschaftlicherdienst abcam com Tel 030 896 779 154 Spain Email soportecientifico abcam com Tel 911 146 554 Switzerland Email technical abcam com Tel Deutsch 0435 016 424 Tel Fran ais 0615 000 530 US and Latin America Email us technical abcam com Tel 888 77 ABCAM 22226 Canada Email ca technical abcam com Tel 877 749 8807 China and Asia Pacific Email hk technical abcam com Tel 108008523689 t piim Japan Email technical abcam co jp Tel 81 0 3 6231 0940 www abcam com www abcam cn www abcam co jp Copyright 2013 Abcam All Rights Reserved The Abcam logo is a registered trademark All information detail is correct at time of going to print
14. ta Glucuronidase digestion 11 1 3 1 To 4 mL of urine add 1 mL of the Beta Glucuronidase solution pH 5 5 to each tube pH lt 6 0 11 1 3 2 Immediately transfer 2 mL of urine to a clean tube and flash freeze This is the zero time point 11 1 3 3 Incubate the remaining 2 mL at 37 C for 3 hours This is the 3 hour time point 11 1 3 4 Extract 11 1 4 Extraction protocol 11 1 5 Combine an equal amount of urine sample from steps 11 1 3 2 Zero time point and 11 1 3 3 3 hour time point above adjusted with approximately 20 uL of acetic acid to pH 4 and ethyl acetate Vortex thoroughly Centrifuge at 2000 rpm for ten minutes at 22 C Three phases should result 11 1 6 Upper organic phase ethyl acetate phase lipoproteins 11 1 7 Interphase proteins 11 1 8 Lower phase aqueous phase 11 1 9 Collect the upper organic phase a and set aside 11 1 10 Discard the interphase Transfer the lower phase with a glass pipette to a new tube and repeat the ethyl acetate extraction step two more times 11 1 10 1 Evaporation of pooled organic phase There should be approximately 5 6 mL of the ethyl acetate phase a Dry the pooled organic phase in a Speedvac to get the extracted sediment b 11 1 10 2 Saponification to cleave fatty acid from glycerol backbone Dissolve the dried residues b in 2 mL of 20 KOH solution for preparation see 14 15 DHET measurement in cells Vortex Discover more at www abcam com 10
15. tandard 2 100 900 100 000 10 000 Standard 3 100 900 10 000 1 000 Standard 4 Standard 5 1 000 100 100 10 None Discover more at www abcam com 8 ASSAY PREPARATION 11 SAMPLE COLLECTION AND STORAGE There are different protocols for isolating and purifying 14 15 DHET depending on the medium in which it is in For optimal results follow the appropriate protocol based on the biological sample present Dissolve 8 mg of Beta Glucuronidase in 8 mL of 1 M Citric acid and adjust to pH 5 5 400 U mL 11 1 Measurement of free and glucuronidated 14 15 DHET in sample extraction with ethyl acetate 11 1 1 Measurement of free 14 15 DHET Extract 4 mL of urine with ethyl acetate using the Extraction Protocol described in 11 1 4 below 11 1 2 Measurement of free and 14 15 DHET glucuronide This method is for determining the level of glucuronidated 14 15 DHET in urine after digestion of the molecule with glucuronidase Collect the first sample as soon as the Beta Glucuronidase is added to a reaction mixture 0 hour digestion and then a second sample at a time when digestion of the glucuronic acid moiety of the molecule is completed Subtract the level of the molecule in the first sample at 0 hour from the levels in the second sample after complete digestion usually 3 hrs to obtain the level of glucuronidated molecule Discover more at www abcam com 9 ASSAY PREPARATION 11 1 3 Be
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