ab175820 – Estrogen BPA Environmental ELISA Kit
Contents
1. 10 4 Prepare Standard 2 by adding 100 uL of the Standard 1 to the microtube labeled Standard 2 Mix thoroughly and gently 10 5 Prepare Standard 3 by adding 100 uL of the Standard 2 to the microtube labeled Standard 3 Mix thoroughly and gently 10 6 Using the table below as a guide repeat for tubes 4 through 6 10 7 Standard B contains no protein and is blank control Volume Valme Starting Final fo E to Dilute Conc Conc Dilute md pg mL pg mL Standard Sample to 1 Step 10 3 1 000 000 2 Standard 1 100 900 1 000 000 100 000 3 Standard 2 100 900 100 000 10 000 4 Standard 3 100 900 10 000 1 000 5 Standard 4 100 900 1 000 100 6 Standard 5 100 900 100 10 Bo None 900 Discover more at www abcam com 9 P4464 ASSAY PREPARATION 11 11 SAMPLE COLLECTION AND STORAGE There are different protocols for isolating and purifying Estrogen BPA Environmental depending on the medium in which it is in Listed below are the different protocols for sample preparation For optimal results follow the appropriate protocol based on the biological sample present 11 1 Estrogen BPA Environmental measurement in urine 11 1 1 11 1 2 11 1 3 11 1 4 11 1 5 11 1 6 Make 8 0 mL of a 1 0 M citrate buffer solution pH 5 5 Dissolve the 8 mg of beta glucuronidase enzyme provided with the kit in 8 0 mL of the 1M citrate buffer Centrifuge t2. 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 15 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 wells and 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 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 96 Abs versus the concentration of Estrogen BPA Environmental 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 16 DATA ANALYSIS 15 TYPICAL DATA TYPICAL STANDARD CURVE Data provided for demonstration purposes only A new standard curve must be
3. 11 1 15 Follow the instructions in the Estrogen BPA Environmental instruction booklet to perform the ELISA 11 1 16 Subtract the value obtained with the zero time point from the 3 hour time point for a measurement of the Estrogen BPA Environmental level in the urine sample Make sure to include all dilution factors in your calculations 11 2 Estrogen BPA Environmental measurement in cells Discover more at www abcam com 11 ASSAY PREPARATION 11 2 1 Collect and homogenize and or sonicate the cells 11 2 2 Acidify the whole homogenized cells with acetic acid to a pH of approximately 3 4 Measure using standard pH paper 11 2 3 Extraction with ethyl acetate Add an equal volume of ethyl acetate to the homogenized cells and vortex very well Place the upper organic phase into a fresh clean tube after centrifugation Then add another equal volume of ethyl acetate to the homogenized cells to start the second time extraction It is strongly recommended that extraction is performed three times 11 2 4 Evaporate the pooled ethyl acetate from the extractions until all has dried up under argon or nitrogen gas 11 2 5 Add 10 uL to 20 uL ethanol or N N dimethyl formamide DMF to reconstitute the dried up residue from above step 11 2 4 Add 0 5 mL of 1x Sample Dilution Buffer provided in kit Load 100 pL in each well in triplicates on the ELISA plate Note We recommend measuring a different dilution of sample in attempt to fit th
4. 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 Estrogen BPA Environmental HRP conjugate 12 uL with 12 mL of HRP Buffer 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 8 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 Label 5 microtubes as Standard 2 6 10 2 Add 900 pL of the 1X Sample Dilution Buffer to the microtubes for Standards 2 to 6 10 3 Prepare a 1 ug mL Standard 1 by first spinning down the enclosed Estrogen BPA Environmental 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
5. years old with a mean total free glucuronidated Estrogen BPA Environmental level of 4 33 ng g of creatinine Urinary Estrogen BPA Environmental levels were correlated with cardiovascular diseases and diabetes A recent study revealed that a 12 ounce serving of canned soup for 5 days increased urinary Estrogen BPA Environmental level 12 fold due to Estrogen BPA Environmental containing epoxy resin lining of the cans The free Estrogen BPA Environmental level in urine or cell culture media can be measured using the Estrogen BPA Environmental ELISA without ethyl acetate extraction after 4 fold dilution of the sample The glucuronidated Estrogen BPA Environmental level can also be measured without extraction after beta glucuronidase treatment using our specific protocol Discover more at www abcam com 2 INTRODUCTION This competitive ELISA kit based on competition between the Estrogen BPA Environmental epitope and Estrogen BPA Environmental HRP conjugate for a limited number of binding sites available from the anti Estrogen BPA Environmental antibody which is coated on the bottom of the wells of the 96 well ELISA plate The conjugate concentration is held constant in each well while the concentration of the Estrogen BPA Environmental is variable based on the concentration of the sample or standard Thus the amount of the Estrogen BPA Environmental conjugate which is able to bind to each of the wells is inversely proportional to the conce
6. 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 14 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 f performing the test on an automatic ELISA system we recommend increasing the washing steps from three to five and the volume of 1X Wash Buffer from 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 of 1X Sample Dilution Buffer into maximum binding control wells 13 2 Add 100 pL 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 uL of 1X Wash Buffer per well 13 6 After the last of the 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
7. abcam discover more ab175820 Estrogen BPA Environmental ELISA Kit Instructions for Use A competitive immunoenzymatic assay for the quantitative measurement of Estrogen BPA Environmental in urine serum plasma cells and tissues This product is for research use only and is not intended for diagnostic use Table of Contents INTRODUCTION 1 BACKGROUND 2 2 ASSAY SUMMARY 4 GENERAL INFORMATION 3 PRECAUTIONS 5 4 STORAGE AND STABILITY 5 5 MATERIALS SUPPLIED 5 6 MATERIALS REQUIRED NOT SUPPLIED 6 7 LIMITATIONS 6 8 TECHNICAL HINTS 7 ASSAY PREPARATION 9 REAGENT PREPARATION 8 10 STANDARD PREPARATION 9 11 SAMPLE COLLECTION AND STORAGE 10 12 PLATE PREPARATION 14 ASSAY PROCEDURE 13 ASSAY PROCEDURE 15 DATA ANALYSIS 14 CALCULATIONS 16 15 TYPICAL DATA 17 16 ASSAY SPECIFICITY 18 RESOURCES 17 TROUBLESHOOTING 19 18 NOTES 21 Discover more at www abcam com 1 INTRODUCTION 1 BACKGROUND Abcam s Estrogen BPA Environmental competitive in vitro ELISA Enzyme Linked Immunosorbent Assay kit is designed for of Estrogen BPA Environmental in urine serum plasma cells and tissues following proper isolation and purification Estrogen BPA Environmental is a phenolic environmental estrogen which disrupts endocrine activity In human an Estrogen BPA Environmental glucuronide was a primary metabolite of BPA In a recent study the age group with highest Estrogen BPA Environmental exposure was 6 11
8. e results to the standard curve e g add 3 wells with 50 uL of the rest of sample plus 50 uL 1x Sample Dilution Buffer and 3 wells with 10 uL of the rest of sample plus 90 pL of 1x Sample Dilution Buffer 11 2 6 Perform the ELISA for Estrogen BPA Environmental according to the instructions of the manufacturer 11 3 Estrogen BPA Environmental measurement in tissues 11 3 1 Homogenize 1 g of tissue 4 mL of H20 11 3 2 Acidify the homogenate by adding 8 uL of acetic acid to each homogenate 11 3 3 Extract with an equal amount of ethyl acetate vortex thoroughly spin down and collect the organic phase Repeat this extraction twice more and combine all of the organic phases Discover more at www abcam com 12 ASSAY PREPARATION 11 3 4 11 3 5 11 3 6 11 3 7 Dry the organic phase with argon or nitrogen gas Dissolve the dried residue from above step with ethanol or DMF Add approximately 20 uL of ethanol or DMF to reconstitute the dried up residue Dilute further with 1X Sample Dilution Buffer Add approximately 500 uL of 1x Sample Dilution Buffer and centrifuge at 10 000 rpm for five minutes at room temperature The supernatant will be used for ELISA Perform the ELISA for Estrogen BPA Environmental according to the instructions of the manufacturer 11 4 Estrogen BPA Environmental measurement in plasma or serum 11 4 1 Combine 1 0 mL of plasma adjusted with acetic acid to pH 4 and 1 0 mL of ethyl aceta
9. ent in untested sample types Sample prepared Ensure proper sample incorrectly preparation dilution Ensure no bubbles present prior to Bubbles in wells reading plate Check that all ports of plate washer Mie area ae are unobstructed wash wells as qm goy recommended Incomplete reagent Ensure all reagents master mixes Large CV mixing are mixed thoroughly JT Use calibrated pipettes amp ensure Inconsistent pipetting accurate pipetting Ensure consistent sample Inconsistent sample preparation and optimal preparation or storage sample storage conditions e g minimize freeze thaws cycles Discover more at www abcam com RESOURCES Problem Cause Solution Wells are insufficiently Wash wells as per protocol washed recommendations High Contaminated wash Make fresh wash buffer background buffer Waiting too long to read plate after adding stop solution Read plate immediately after adding stop solution Store all reagents as Improper storage of recommended Please note all ELISA kit reagents may not have identical Low storage requirements sensitivity Using incompatible sample type e g Serum vs cell extract Detection may be reduced or absent in untested sample types Discover more at www abcam com 20 RESOURCES 18 NOTES Discover more at www abcam com 21 RESOURCES Discover more at www abcam com 22 abcam discover more UK EU and ROW Email tech
10. generated for each assay performed The data shown here is an example of typical results obtained using the Abcam s Estrogen BPA Environmental ELISA kit These results are only a guideline and should not be used to determine values from your samples 0 1 10 100 1 000 10 000 100 000 BPA pgimL Conc a ImL O D Abs Blank Pg Abs Sample 1 2 834 98 7 10 2 555 89 0 100 2 077 72 3 1 000 1 172 40 8 10 000 0 388 13 5 100 000 0 209 7 3 Blank well mean abs 0 052 Maximum Control Well mean abs 2 870 Discover more at www abcam com 17 DATA ANALYSIS 16 ASSAY SPECIFICITY The specificity of the Estrogen BPA Environmental ELISA was investigated using authentic Estrogen BPA Environmental and a panel of bisphenols and related chemicals Chemicals Reactivity Estrogen BPA Environmental 100 00 BPF lt 0 01 BPS lt 0 01 Resveratrol 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 18 17 TROUBLESHOOTING RESOURCES Problem Cause Solution MM time to Try overnight incubation at 4 C Precipitate can form in wells upon substrate addition Increase dilution factor of sample when concentration of Low signal target is too high Reds wide Detection may be reduced 2 Im igs rise or abs
11. he urine sample to remove any solids Dilute 1 0 mL of human urine 4 fold with 1X Sample Dilution Buffer You must dilute the 10X Sample Dilution Buffer provided in the kit Divide the diluted urine sample into two equal aliquots of 2 0 mL Add 0 5 mL of the beta glucuronidase enzyme solution you prepared ahead of time to both of the diluted 2 0 mL urine aliquots Discover more at www abcam com 10 ASSAY PREPARATION 11 1 7 To one of the aliquots quickly add 2 0 mL of ethyl acetate vortex vigorously and centrifuge for 3 minutes at gt 5 000 g to separate the aqueous and organic layers 11 1 8 Remove and save the ethyl acetate layer in a clean tube and place on ice This is the zero time point sample 11 1 9 Incubate the other 2 0 mL aliquot at 37 C for 3 hours A shaking water bath is recommended This is the 3 hour time point sample 11 1 10 After the three hour incubation extract with ethyl acetate as you did with the zero time point sample 11 1 11 Evaporate the ethyl acetate using a speed vac or a gentle stream of nitrogen or argon 11 1 12 Resuspend the dried extract with 20 uL of ethanol Vortex sample to ensure the extract is completely dissolved 11 1 13 Add 1 0 mL of the 1X Sample Dilution Buffer to the tube 11 1 14 Add 100 uL to the sample wells of the ELISA plate If necessary the sample can be diluted to ensure that the absorbance reading of the well falls within the limits of the standard curve
12. ming 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 eliminate 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 T7
13. nents in section 9 Reagent Preparation 5 MATERIALS SUPPLIED Storage Condition After Preparation BPA ELISA Plate 96 Wells 20 C BPA Standard 2 uL 20 C BPA HRP Conjugates 12 uL 20 C 10X Sample Dilution Buffer 25 mL 20 C HRP Buffer 15 mL 20 C 10X Wash Buffer Solution 25 mL 20 C TMB Substrate 24 mL 20 C Discover more at www abcam com 5 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 Deionized water e An 8 channel adjustable pipette and an adjustable pipette e Storage bottles e Costar cluster tubes 1 2 mL and microcentrifuge tubes e 2N Sulfuric 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 Do not interchange 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 6 GENERAL INFORMATION 8 TECHNICAL HINTS e Avoid foa
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15. ntration of Estrogen BPA Environmental 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 Discover more at www abcam com 3 INTRODUCTION 2 ASSAY SUMMARY Capture Antibody Y YY 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 Substrate Colored Product Prepare all reagents and samples as instructed Add TMB substrate to each E well Incubate at room temperature Add Stop Solution Qo o to each well Read immediately YyYY j Discover more at www abcam com 4 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 compo
16. te Vortex thoroughly Centrifuge at 2000 rpm for ten minutes at 22 C Three phases should result 11 4 2 11 4 3 11 4 4 Discover more at www abcam com 13 11 4 1 1 Upper organic phase ethyl acetate phase lipoproteins 11 4 1 2 Interphase proteins 11 4 1 3 Lower phase aqueous phase Collect the upper organic phase a and set aside Discard the interphase Transfer the lower phase with a glass pipette to a new tube and repeat the ethyl acetate extraction step 2 more times Evaporation of pooled organic phase There should be approximately 3 mL of the ethyl acetate phase a Dry the pooled organic phase in a Speedvac to get the extracted sediment b ASSAY PREPARATION 11 4 5 Store the sediment e at 20 C if performing assay later For ELISA assay dissolve the sediment e in 20 uL of ethanol or DMF and then add 130 uL of 1x Sample Dilution Buffer 11 4 6 For the competitive Estrogen BPA Environmental ELISA the above 150 uL sample needs to be further diluted When calculating the concentration consider any dilution factors 11 4 7 Perform the ELISA for Estrogen BPA Environmental according to the instructions of the manufacturer 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 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
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