8-Isoprostane ELISA kit Urinary 8
Contents
1. 8 Isoprostane ELISA kit Urinary 8 Isoprostane ELISA kit D etroit R amp D Inc Catalog Number 8iso1 8iso11 8iso21 8iso101 8isoU1 8sioU11 8isoU21 8isoU101 Store at 20 C Introduction This competitive ELISA kit is for determination of 8 isoprostane levels in biological samples The isoprostanes are a family of eicosanoids of non enzymatic origin produced by the random oxidation of tissues phospholipids by oxygen radicals A recent NIH sponsored study on Biomarkers of Oxidative Stress has indicated that 8 isoprostane is the best index of oxidative injury in a well accepted oxidant stress rat model 1 2 In addition plasma 8 isoprostane levels were found to be elevated in elderly subjects with severe hypertension 3 and in urine from subjects with high fat diet induced liver steatosis 4 This kit can be used for the determination of 8 isoprostane in diluted urine serum plasma cells and tissues following proper isolation and purification of the eicosanoid from the isoprostane containing sample Instructions are provided as to the proper isolation and purification in the following pages Storage and Stability 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 Materials Provided2. Part Item Description Quantity Number 1 8 isoprostane ELISA Solid 96 well plate coated with anti 8 isoprostane antibody in 1 Plate each well 2 8 isoprotane Standard 1 10 ng in 2 uL Stock standard at a concentration of 5 ug mL 8 isoprostane HRP 3 Conjugates 1 12 uL 1000 X concentrated solution 4 Sampie Dae 10 X solution of Tris buffered saline with preservatives 1 25 mL 5 E a 1 X solution of Tris buffered saline with preservatives 1 Wash Buffer Solution 10 X solution of Tris buffered saline with detergents and 6 1 25 mL preservatives TMB Substrate 7 24 mL A solution of TMB tetra methyl benzadine 1 2727 Second Ave Suite 4113 Detroit MI 48201 tel 313 961 1606 fax 313 963 7130 info detroitrandd com www detroitRandD com Page 2 of 9 Additional Required Materials Not Provided An 8 channel adjustable pipetter and an adjustable pipetter Storage bottles Costar cluster tubes 1 2 mL and microcentrifuge tubes Speed vac optional or argon or nitrogen gas Precautions Please read all instructions carefully before beginning the assay The reagents in this kit have been tested and formulated to perform optimally This kit may not perform correctly if any of the reagents are replaced or any of the procedures are modified This kit is intended for research use only and is not to be used as a diagnostic Procedural Notes Remove all of the reagents required including the TMB a
3. isoprostane ELISA was investigated using authentic 8 isoprostane and a panel of eicosanoids structurally similar to the 8 isoprostane 8 isoprostane 2 3 dinor 8 isoPGF2a 2 3 dinor 11B PGF2a PGF la 8 iso PGE2 8 iso PGEI Troubleshooting No color present in standard wells 100 00 lt 0 01 lt 0 01 lt 0 01 lt 0 01 lt 0 01 The HRP conjugate was not added Redo the assay and add the conjugate at the proper step 2727 Second Ave Suite 4113 Detroit MI 48201 tel 313 961 1606 fax 313 963 7130 info detroitrandd com www detroitRandD com Page 8 of9 The HRP conjugate was not incubated for the proper time Redo the assay and incubate for the proper time No color in any wells including the TA wells The TMB substrate was not added Add substrate The TMB substrate was not incubated for the proper time Continue incubation until desired color is reached The color is faint One or all of the incubation times were cut short Redo the assay with the proper incubation times The TMB substrate was not warmed up to room temperature Redo the assay making sure all reagents are at room temperature The lab is too cold Be sure the lab temperature is between 21 27 C and redo the assay The background color is very high The TMB substrate has been contaminated Redo the assay with a fresh bottle of substrate Scattered O D obtained from the sample Redo assay using an 8 channel pipetm
4. an making sure that 8 channels are equal volume while loading References Morrow JD Quantification of isoprostanes as indices of oxidant stress and the risk of atherosclerosis in humans Arterioscler Thromb Vasc Biol 2005 25 279 86 Gross M et al Plasma F2 isoprostanes and coronary artery calcification the CARDIA Study Clin Chem 2005 51 125 31 Hozawa A et al Increased plasma 8 isoprostane levels in hypertensive subjects the Tsurugaya Project Hypertens Res 2004 27 557 61 Carabelli J et al High fat diet induced liver steatosis promotes an increase in liver mitochondrial biogenesis in response to hypoxia J Cell Mol Med 2010 Warranty Detroit R amp D Inc makes no warranty of any kind expressed or implied including but not limited to the warranties of fitness for a particular purpose and merchantability 2727 Second Ave Suite 4113 Detroit MI 48201 tel 313 961 1606 fax 313 963 7130 info detroitrandd com www detroitRandD com Page 9 of9 Detroit R amp D Inc Detroit R amp D Inc Metro Center For High Technology Bldg MCHT 2727 Second Ave Suite 4113 Detroit MI 48201 Phone 313 961 1606 Fax 313 963 7130 E mail info detroitrandd com www DetroitRandD com 2727 Second Ave Suite 4113 Detroit MI 48201 tel 313 961 1606 fax 313 963 7130 info detroitrandd com www detroitRandD com
5. 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 1 X Sample Dilution Buffer Spin down the enclosed 8 isoprostane 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 1 6 mL of 1 X Sample Dilution Buffer to Standard 5 1 mL of Sample Dilution Buffer to Standard 4 1 6 mL of 1 X Sample Dilution Buffer into Standard 3 1 mL of 1 X Sample Dilution Buffer into Standard 2 and 1 mL of Sample Dilution Buffer into Standard 1 Take 400 uL of Standard 6 5 ng mL into Standard 5 1 ng mL Take 1 mL of the Standard 5 solution into Standard 4 500 pg mL From Standard 4 take 400 uL into Standard 3 100 pg mL Take 1 mL of Standard 3 into Standard 2 50 pg mL The last step is to add 400 uL of Standard 2 to Standard 1 10 pg mL The serially diluted standards for 1 to 6 are ready for analysis from concentration 10 pg mL to 5000 pg mL Samples Samples can be directly diluted into the 1 X 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 1 X Sample Dilution Buffe
6. nd allow them to equilibrate to room temperature before proceeding with the assay Itis necessary to thoroughly mix the concentrated buffer solutions A stir bar is contained within each buffer solution Sample Preparations There are different protocols for isolating and purifying 8 isoprostane depending on the medium it is in Listed below are the different protocols For optimal results follow the appropriate protocol based on the biological sample present 8 isoprostane measurement in urine 1 Dilute urine 4 fold with sample dilution buffer containing a final concentration of 0 1 mM TPP triphenylphosphine 0 03 0 05 mg mL TPP is an antioxidant which looks like a precipitate in samples because it does not easily dissolve Before using the stored samples containing TPP spin samples to separate the precipitated TPP from sample solution 2 Perform the ELISA for 8 isoprostane according to the instructions of the manufacturer 8 isoprostane measurement in cells 1 Collect and homogenize and or sonicate the cells using a solution containing a final concentration of 0 1 mM TPP triphenylphosphine 0 03 0 05 mg mL TPP is an antioxidant which looks like a precipitate in samples because it does not easily dissolve Before using the stored samples containing TPP spin samples to separate the precipitated TPP from sample solution 2 Acidify the whole homogenized cells with acetic acid to a pH of approximately 3 4 Measure using standard
7. orking solution 1 mL of 2 M KOH 4 mL methanol so that the final concentration of KOH 0 4 N Vortex and incubate for 1 h at 50 C Add 1 5X H O to the solution and adjust pH with 20 formic acid to pH 5 Re extract the solution with ethyl acetate 1 part aqueous solution 1 part ethyl acetate and dry 8 isoprostane measurement in tissues 1 2 3 4 S Homogenize 1 g of tissue 4 mL of H20 and 0 01 mg TPP Acidify the homogenate by adding 8 uL of acetic acid to each homogenate 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 Dry the organic substance with argon or nitrogen gas Saponification if needed see 8 isoprostane measurement in cells 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 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 measuring Perform the ELISA for 8 isoprostane according to the instructions of the manufacturer 8 isoprostane measurement in plasma or serum 3 Combine 1 8 mL of plasma adjusted with approximately 20 uL of acetic acid to pH 4 and 1 8 mL of ethyl acetate Vortex thoroughly Centrifuge at 2000
8. 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 4 Evaporate the pooled ethyl acetate from the extractions until all has dried up under argon or nitrogen gas 5 Saponification if needed see below 2727 Second Ave Suite 4113 Detroit MI 48201 tel 313 961 1606 fax 313 963 7130 info detroitrandd com www detroitRandD com 7 Page 3 of9 Add 10 uL to 20 uL ethanol or N N dimethyl formamide DMF to dissolve 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 of 10 uL of the rest of sample and 90 uL of 1x Sample Dilution Buffer Perform the ELISA for 8 isoprstane according to the instructions of the manufacturer Saponification to cleave fatty acid from glycerol backbone 1 Ow Dissolve dried fatty acids after 3X ethyl acetate extractions in 20 KOH solution make w
9. r 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 2727 Second Ave Suite 4113 Detroit MI 48201 tel 313 961 1606 fax 313 963 7130 info detroitrandd com www detroitRandD com Page 5 of9 Performing the Assay Plate Setup Each plate must contain a minimum of three blank wells B1 three maximum binding wells Bo and a six point standard curve S S Each sample should be assayed in triplicate A suggested plate format is shown below aeaooo000000 OO RI O BL En Bo CI S Se Samples Standard Dilutions Table Standards Final Concentration pg mL Add Sample Dilution Buffer mL Serial Dilutions Procedure No 6 5 000 1 998 2 uL of stock solution No 5 1 000 1 600 Add 0 4 mL of No 6 No 4 500 1 000 Add 1 0 mL of No 5 No 3 100 1 600 Add 0 4 mL of No 4 No 2 50 1 000 Add 1 0 mL of No 3 No 1 10 1 600 Add 0 4 mL of No 2 Assay Procedure Step 1 Load 200 microliters of Sample Dilution Buffer into the blank B1 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 8 isoprostane HRP conjugate in the Bo wells the standard wells and the
10. 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 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 5 6 mL of the ethyl acetate phase a Dry the pooled organic phase in a Speedvac or under argon or nitrogen gas to get the extracted sediment b Saponification to cleave fatty acid from glycerol backbone Dissolve the dried residues b in 2 mL of 20 KOH solution for preparation see 8 isoprostane measurement in cells Vortex thoroughly and incubate for 1 h at 50 C This will yield an aqueous solution c Dilute 2 mL of the aqueous solution c with 3 mL of H O Adjust the pH using 20 formic acid 132 uL to pH 5 5 Add 2 mL 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 with saponified lipids Dry the pooled ethyl acetate upper phase d in a Speedvac or under argon or nitrogen gas yielding the dried sample sediment e Store the sediment e at 20 C For ELISA assay dissolve the sediment e in 20 uL of e
11. sample wells Do NOT add HRP conjugate into the B 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 By wells 2727 Second Ave Suite 4113 Detroit MI 48201 tel 313 961 1606 fax 313 963 7130 info detroitrandd com www detroitRandD com Page 6 of 9 Step 9 Incubate the plate at room temperature for 15 30 minutes Step 10 Add 50 micoliters of 2 N sulfuric acid to all of the wells Step 11 Read the plate at 450 nm Calculating the Results 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 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 from the Bo wells This is your maximum binding 3 Calculate the B Bo for Standard 1 by a
12. thanol then add 130 uL of 1x Sample Dilution Buffer 10 For the competitive 8 isoprostane ELISA the above 150 uL sample needs to be further diluted Dilute 1 4 e g 80 uL sample 320 uL Sample Dilution Buffer Check the final pH should be pH 7 4 2727 Second Ave Suite 4113 Detroit MI 48201 tel 313 961 1606 fax 313 963 7130 info detroitrandd com www detroitRandD com Page 4 of 9 When calculating the concentration consider the dilution factor In this case 150 uL total sample volume from 1 8 mL plasma 12 fold concentration and then 80 uL sample in 400 uL SDB 5 fold dilution Since the samples are concentrated 2 4 fold to get the actual concentration you must divide by 2 4 11 Perform the ELISA for 8 isoprostane according to the instructions of the manufacturer Assay Preparations 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 1 X Wash Buffer This can then be refrigerated for the entire life of the kit HRP Conjugate Dilute 1 vial of the 8 isoprostane HRP conjugate 0 012 mL with 12 00 mL of 1 X HRP buffer One vial makes enough conjugate for one plate The conjugate must
13. veraging the corrected absorbance of the two S wells divide the average by the maximum binding then multiply by 100 Repeat this formula for the remaining standards Plot the B Bo versus the concentration of 8 isoprostane from the standards using semi log paper Calculate the B Bo for the samples and determine the concentrations utilizing the standard curve Multiply the concentrations obtained for each of the samples by their corresponding dilution factor Oe 2727 Second Ave Suite 4113 Detroit MI 48201 tel 313 961 1606 fax 313 963 7130 info detroitrandd com www detroitRandD com Typical Results B B0O Net OD at 450 nm of Bo 1 394 30 min color development 110 0 100 0 90 0 80 0 70 0 60 0 50 0 40 0 30 0 20 0 10 0 0 0 LL rrm rm 100 1000 8 Isoprostane pg ml 1 10 Page 7 of 9 10000 The data shown here is an example of typical results obtained using the Detroit R amp D 8 isoprostane 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 B wells Bo wells Standard Concentration No No No No No No 1 2 3 4 5 6 0 14 1 387 10 pg mL 50 pg mL 100 pg mL 500 pg mL 1000 pg mL 5000 pg mL Specificity of anti 8 isoprostane IgG O D 1 394 1 174 0 985 0 504 0 328 0 090 BIBo 100 0 73 4 70 7 36 2 23 5 64 The specificity of the 8
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