BPA (Bisphenol A) ELISA kit
Contents
1. 4 mL of H20 2 Acidify the homogenate by adding 8 uL of acetic acid to each homogenate 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 4 Dry the organic phase with argon or nitrogen gas 5 Dissolve the dried residue from above step 4 with ethanol or DMF Add approximately 20 uL of ethanol or DMF to reconstitute the dried up residue 6 Dilute further with 1x Sample Dilution Buffer Add approximately 0 5 mL of 1x Sample Dilution Buffer and centrifuge at 10 000 rpm for five minutes at room temperature The supernatant will be used for ELISA 7 Perform the ELISA for BPA according to the instructions of the manufacturer Samples Preperation Samples can be directly diluted into the 1X Sample Dilution Buffer if it is in solution For extracted and dried samples it is recommended to dissolve the dried up samples with a minimal amount of ethanol of N N dimethyl formanmide DMF 10 uL to 20 uL and vortex well Before ELISA assay add 100 uL of 1X Sample Dilution Buffer to make the stock sample solution ready for quantification with ELISA The stock sample solution can be further diluted to a proper range of concentration for ELISA test Plate Preparation Plate Setup Each plate must contain a minimum of three blank wells BL three maximum binding wells BO and a six point standard curve S1 S6 Each sa2. BPA HRP conjugate in the BO wells the standard wells and the sample wells Do NOT add HRP conjugate into the BL wells Step 5 Incubate the plate at room temperature for two hours Step 6 Wash the plate three times with 400 microliters of the diluted Wash Buffer per well Step 7 After the last of the three wash cycles pat the plate dry onto some paper toweling Step 8 Add 200 microliters of the TMB substrate to all of the wells including BL wells Step 9 Incubate the plate at room temperature for 15 30 minutes Step 10 Add 50 micoliters of 2N sulfuric acid to all of the wells Step 11 Read the plate at 450 nm Calculation Most plate readers provide data reduction software that can be used to plot the standard curve and determine the sample concentrations If your plate reader does not have this option then a data reduction program can be used 4 parameter of log log curve fit Creative Diagnostics All rights reserved 45 16 Ramsey Road Shirley NY 11967 USA Tel 631 624 4882 Fax 631 614 7828 E mail info creative diagnostics com www creative diagnostics com CD If you do not have these options the results can be obtained manually as follows 1 Average the absorbance readings from the blanks 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 2 Average the corrected absorbance readings fr
3. 631 614 7828 E mail info creative diagnostics com www creative diagnostics com CD Storage This kit will obtain optimal results if all of the components are stored at the proper temperature prior to use Items should be stored at the designated temperatures upon receipt of this kit All components are stored below 20 C and should not be re frozen and thawed more than necessary Specimen Collection And Handling There are different protocols for isolating BPA depending on the nature of the biological sample Listed below are the different protocols For optimal results follow the appropriate protocol based on the biological sample BPA measurement in urine preparations Make 8 0 mL of a 1 0 M citrate buffer solution pH 5 5 Dissolve the 8 mg of B glucuronidase enzyme provided with the kit in 8 0 mL of the 1M citrate buffer Centrifuge the urine sample to remove any solids Protocol 1 Homogenize 1 g of tissue 4 mL of H20 2 Acidify the homogenate by adding 8 uL of acetic acid to each homogenate 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 4 Dry the organic phase with argon or nitrogen gas 5 Dissolve the dried residue from above step 4 with ethanol or DMF Add approximately 20 uL of ethanol or DMF to reconstitute the dried up residue 6 Dilute further with 1x Sample Dilution Buffer
4. Add approximately 0 5 mL of 1x Sample Dilution Buffer and centrifuge at 10 000 rpm for five minutes at room temperature The supernatant will be used for ELISA 7 Perform the ELISA for BPA according to the instructions of the manufacturer BPA measurement in plasma or serum 1 Combine 1 0 mL of plasma adjusted with acetic acid to pH 4 and 1 0 mL of ethyl acetate Vortex thoroughly Centrifuge at 2000 rpm for ten minutes at 22 C Three phases should result i Upper organic phase ethyl acetate phase lipoproteins ii Interphase proteins iii Lower phase aqueous phase 2 Collect the upper organic phase a and set aside 3 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 4 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 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 then add 130 uL of 1x Sample Dilution Buffer 6 For the competitive BPA ELISA the above 150 uL sample may need to be further diluted When calculating the concentration consider any dilution factors 7 Perform the ELISA for BPA according to the instructions of the manufacturer BPA measurement in cells 1 Collect and homogenize and or sonicate the ce
5. CD BPA Bisphenol A ELISA kit Cat No DEIA12664 Pkg Size 96T Intended use The BPA Bisphenol A ELISA kit can be used for the determination of BPA in urine serum plasma cells and tissues following proper isolation and purification The free BPA level in urine or cell culture media can be measured using the BPA ELISA without ethyl acetate extraction after 4 fold dilution of the sample The glucuronidated BPA level can also be measured without extraction after beta glucuronidase treatment using our specific protocol General Description BPA is a phenolic environmental estrogen which disrupts endocrine activity In human a BPA glucuronide was a primary metabolite of BPA In a recent study the age group with highest BPA exposure was 6 11 years old with a mean total free glucuronidated BPA level of 4 33 ng g of creatinine Urinary BPA 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 BPA level 12 fold due to BPA containing epoxy resin lining of the cans Principle Of The Test This competitive ELISA kit based on competition between the BPA epitope and BPA HRP conjugate for a limited number of binding sites available from the anti BPA 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 BPA is variable based
6. e must be used the same day and should not be stored for later use Standards Label 5 microtubes as Standard 1 through Standard 5 Dilute the entire contents of Sample Dilution Stock buffer 25 mL with 225 mL deionized water to yield a final volume of 250 mL of 1X Sample Dilution Buffer Add 0 9 mL of the Sample Dilution Buffer to the microtubes for Standards 1 to 5 Spin down the enclosed BPA standard vial 2 uL filled with inert gas and add 1 998 mL of Sample Dilution Buffer to obtain 2 mL of solution Label this Standard 6 Add 0 1 mL of the Standard 6 to the microtube labeled Standard 5 and mix thoroughly Next add 0 1 mL of Standard 5 into the microtube labeled Standard 4 and mix thoroughly Continue to serially dilute the standards using 1 10 dilutions for the remaining standards Assay Steps Note 1 Remove all of the reagents required including the TMB and allow them to equilibrate to room temperature before proceeding with the assay 2 It is necessary to thoroughly mix the concentrated buffer solutions A stir bar is contained within each buffer solution Assay Steps Step 1 Load 200 microliters of Sample Dilution Buffer into the blank BL wells and 100 microliters of Sample Dilution Buffer into the maximum binding BO wells Step 2 Load 100 microliters of each of the standards into the appropriate wells Step 3 Load 100 microliters of each of the samples into the appropriate wells Step 4 Load 100 microliters of the diluted
7. lls 2 Acidify the whole homogenized cells with acetic acid to a pH of approximately 3 4 Measure using standard pH paper 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 Creative Diagnostics All rights reserved 45 16 Ramsey Road Shirley NY 11967 USA Tel 631 624 4882 Fax 631 614 7828 E mail info creative diagnostics com www creative diagnostics com CD 4 Evaporate the pooled ethyl acetate from the extractions until all has dried up under argon or nitrogen gas 5 Add 10 uL to 20 uL ethanol or N N dimethyl formamide DMF to reconstitute the dried up residue from above step 4 Add 0 5 mL of 1x Sample Dilution Buffer provided in kit Load 100 uL in each well in triplicates on the ELISA plate Note We recommend measuring a different dilution of sample in attempt to fit the 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 uL of 1x Sample Dilution Buffer 6 Perform the ELISA for BPA according to the instructions of the manufacturer BPA measurement in tissues 1 Homogenize 1 g of tissue
8. mple should be assayed in triplicate A suggested plate format is shown below Figure 1 E e080 Y Oe O08 J IID 00000000 JJJ OO O O88 O IIIO 00000000 23939000000000 IIIO 00000000 IIIO 00000000 Standard Dilutions Table B amp E H S S Samples Standard Dilutions Table Table 1 Creative Diagnostics All rights reserved 45 16 Ramsey Road Shirley NY 11967 USA Tel 631 624 4882 Fax 631 614 7828 E mail info creative diagnostics com www creative diagnostics com CD Standards Final Concentration pg mL Add Sample Dilution Buffer mL Serial Dilutions Procedure No 6 100 000 1 998 2 uL of stock solution No 5 10 000 0 9 Add 0 1 mL of No 6 No 4 1 000 0 9 Add 0 1 mL of No 5 No 3 100 0 9 Add 0 1 mL of No 4 No 2 10 0 9 Add 0 1 mL of No 3 No 1 l 0 9 Add 0 1 mL of No 2 Reagent Preparation The solid 96 well plate and TMB solution are provided ready to use The preparations of other assay reagents are detailed below Wash Buffer Mix the solution with a stir bar applying low heat until a clear colorless solution is obtained Dilute the entire contents of the Wash Buffer Concentrate 25 mL with 225 mL of deionized water to yield a final volume of 250 mL of 1X Wash Buffer This can then be refrigerated for the entire life of the kit HRP Conjugate Dilute 1 vial of the BPA HRP conjugate 0 012 mL with 12 00 mL of 1X HRP buffer One vial makes enough conjugate for one plate The conjugat
9. om the BO wells This is your maximum binding 3 Calculate the B BO for Standard 1 by averaging the corrected absorbance of the two S1 wells divide the average by the maximum binding then multiply by 100 Repeat this formula for the remaining standards 4 Plot the B BO versus the concentration of BPA from the standards using semi log paper 5 Calculate the B BO for the samples and determine the concentrations utilizing the standard curve 6 Multiply the concentrations obtained for each of the samples by their corresponding dilution factor Typical Standard Curve Tipical Standard Curve Figure 2 BIBO 1 10 100 1000 10000 100000 BPA pg mi The data shown here is an example of typical results obtained using the BPA ELISA kit These results are only a guideline and should not be used to determine values from your samples The user must run their own standard curve every time Specificity The specificity of the BPA ELISA was investigated using authentic BPA and a panel of bisphenols and related chemicals BPA 100 BPF lt 0 01 BPS lt 0 01 Resveratrol lt 0 01 Precautions 1 Please read all instructions carefully before beginning the assay 2 The reagents in this kit have been tested and formulated to perform optimally This kit may not perform corretly if any of the reagents are replaced or any of the procedures are modified REFERENCES 1 Zhao M Zhou S Yan J Li L Immunochemical analysis of endogeno
10. on the concentration of the sample or standard Thus the amount of the BPA conjugate which is able to bind to each of the wells is inversely proportional to the concentration of BPA 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 Reagents And Materials Provided BPA ELISA Plate Solid 96 well plate coated with anti BPA antibody in each well BPA Standard 2 uL Stock standard at a concentration of 1 mg mL BPA HRP Conjugates 12 uL 1000 X concentrated solution Sample Dilution Buffer 25 mL 10 X solution of Tris buffered saline with preservatives HRP Buffer 15 mL 1 X solution of Tris buffered saline with preservatives Wash Buffer Solution 25 mL 10 X solution of Tris buffered saline with detergents and preservatives TMB Substrate 24 mL A solution of TMB tetra methyl benzadine NOOO PR 0 Na Materials Required But Not Supplied 1 An 8 channel adjustable pipetter and an adjustable pipetter 2 Storage bottles 3 Cluster tubes 1 2 mL and microcentrifuge tubes 4 Deionized water 5 2N Sulfuric acid Creative Diagnostics All rights reserved 45 16 Ramsey Road Shirley NY 11967 USA Tel 631 624 4882 Fax
11. us and exogenous estrogens Current Pharmaceutical Analysis 2007 3 25 38 2 V lkel W Colnot T Csanady GA Filser JG Dekant W Metabolism and kinetics of bisphenol a in humans at low doses following oral administration Chem Res Toxicol 15 1281 1287 2002 3 Fourth National Report on human exposure to environmental chemicals Centers for Disease Control and Prevention 2009 www cdc gov exposurereport Creative Diagnostics All rights reserved 45 16 Ramsey Road Shirley NY 11967 USA Tel 631 624 4882 Fax 631 614 7828 E mail info creative diagnostics com www creative diagnostics com CD CREAT DIAGNOS Creative Diagnostics All rights reserved 45 16 Ramsey Road Shirley NY 11967 USA Tel 631 624 4882 Fax 631 614 7828 E mail info creative diagnostics com www creative diagnostics com
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