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1. 13 Fax 908 233 0758 e E mail corp drg international com_e Web www drg international com DIRE DRG Adiponectin human EIA 4820 Revised 14 Nov 2011 rm Vers 7 1 RUO TROUBLESHOOTING POOR STANDARD 1 INACCURATE PIPETTING OR CHECK PIPETTES AND CALIBRATE CURVE PIPETTING ERROR REGULARLY IMPROPER STANDARD DILUTION VORTEX THE STOCK BEFORE USE AND DILUTE CAREFULLY IN AN EPPENDORF TUBE LOW SIGNAL SHORTER INCUBATION THAN ENSURE SUFFICIENT INCUBATION RECOMMENDED TIME INADEQUATE REAGENT VOLUMES OR IMPROPER CHECK PIPETTES AND ENSURE DILUTION OR PIPETTING ERROR CORRECT PERFORMANCE LARGE CV INACCURATE PIPETTING AND CHECK PIPETTES a n oe ee ao FILL THE WELLS PROMPTLY WITH WASH BUFFER AND REAGENTS HIGH BACKGROUND PLATE IS INSUFFICIENTLY REVIEW THE MANUAL FOR PROPER WASHED WASH IF USING A PLATE WASHER CONTAMINATED WASH BUFFER ee Oke UNOBSTRUCTED AND CLEAN WASH BUFFER VOLUME IS LESS THAN ADVISED MAKE A FRESH WASH BUFFER USE 300uL PER WELL LOW SENSITIVITY IMPROPER STORAGE OF THE STORE TEST KIT COMPONENTS AS ELISA KIT ADVISED IN THIS USER MANUAL KEEP SUBSTRATE SOLUTION PROTECTED FROM LIGHT STOP SOLUTION STOP SOLUTION SHOULD BE ADDED TO EACH WELL BEFORE MEASURE USE CLEAN STERILE TIPS DISCARD CONTAMINATED REAGENTS CONTAMINATION OF REAGENTS Version 2011 09 22 rm DRG International Inc USA 14 Fax 908 233 0758 e E mail corp ddrg international com e Web www drg internationa2. J Biol Chem 1996 271 10697 10703 Maeda K Okubo K Shimomura I Funahashi T Matsuzawa Y Matsubara K cDNA cloning and expression of a novel adipose specific collagen like factor apM1 adipose most abundant gene transcript 1 Biochem Biophys Res Commun 1996 221 286 289 Nakano Y Tobe T Choi Miura NH Mazda T Tomita M Isolation and characterization of GBP28 a novel gelatin binding protein purified from human plasma J Biochem 1996 120 803 812 Scherer PE Williams S Fogliano M Baldini G Lodish HF A novel serum protein similar to Clg produced exclusively in adipocytes J Biol Chem 1995 270 26746 26749 Saito K Tobe T Minoshima S et al Organization of the gene for gelatin binding protein GBP28 Gene 1999 229 67 73 Takahashi M Arita Y Yamagata K et al Genomic structure and mutations in adipose specific gene adiponectin Int J Obes Relat Metab Disord 2000 24 861 868 Kissebah AH Sonnenberg GE Myklebust J et al Quantitative trait loci on chromosomes 3 and 17 influence phenotypes of the metabolic syndrome Proc Natl Acad Sci USA 2000 97 14478 14483 Vionnet N Hani EH Dupont S et al Genomewide search for type 2 diabetes susceptibility genes in French whites evidence for a novel susceptibility locus for early onset diabetes on chromosome 3q27 qter and independent replication of a type 2 diabetes locus on chromosome 1q21 q24 Am J Hum Genet 2000 67 1470 1480 Matsuzawa Y Funahashi T Nakamura T Molecular m
3. Web www drg international com DIRE 6 DRG Adiponectin human EIA 4820 Revised 14 Nov 2011 rm Vers 7 1 RUO CONTENTS OF THE KIT TEST COMPONENTS AMOUNT VOLUME 96 WELL PLATE WITH 12 STRIPS BREAK APART MICROTITER TEST STRIPS EACH WITH 8 ADIPONECTIN ANTIBODY FRAME COATED SINGLE WELLS READY FOR USE ADIPONECTIN STANDARD 30 NG ML LYOPHILIZED amp STABILIZED RECOMBINANT HUMAN ADIPONECTIN 2 VIALS SEE LABEL FOR STOCK CONCENTRATION RECONSTITUTE WITH THE SAMPLE DILUENT VOLUME SHOWN ON THE LABEL BIOTINYLATED ADIPONECTIN ANTIBODY 10 ML READY FOR USE HRP CONJUGATED AVIDIN 12 ML READY FOR USE 20X WASH BUFFER CONCENTRATE SUFFICIENT FOR 1000 ML 50 ML DILUTE 1 20 SAMPLE DILUENT READY FOR USE misk k s STOP SOLUTION 0 9 N H SO 8 ML READY FOR USE ML TMB SUBSTRATE 2 READY FOR USE DRG International Inc USA 2 Fax 908 233 0758 e E mail corp ddrg international com e Web www drg international com DIRE Cc DRG Adiponectin human EIA 4820 Revised 14 Nov 2011 rm Vers 7 1 RUO STORAGE AND STABILITY Antibody coated 96 well plates Store at 2 8 C in closed aluminum pouch 3 months after opening with 12 strips with desiccant Strips which are not used must be stored in Break apart microtiter test strips each the re sealable aluminum pouch in humidity with 8 antibody coated single wells free and airtight conditions Adiponectin Standard Store at 2 8 C Until date of kit expiry in Lyophilized Avo
4. 5 ul to 350 ul 12 and 8 channel pipets Recommended for manual washings and reagent dispensing Adjustable 1 25 ml pipettes for reagent preparation 100 ml and 1 liter graduated cylinders Microplate washer or 12 well Multichannel pipet for washings Absorbent paper Distilled or de ionized water Log log graph paper or computer and software for ELISA data analysis Tubes to prepare standard or sample dilutions Timer AMOUNTS OF THE REAGENTS NEEDED TO PERFORM THE TEST REAGENTS NO OF STRIPS BIOTINYLATED AVIDIN TMB STOP WASH USED ANTIBODY HRP SUBSTRATE SOLUTION BUFFER WITH 8 WELL EACH 50 uL WELL 50 uL WELL 50 uL WELL 25 uL WELL 300 pL WELL 1 8 WELLS 500 pL 500 uL 500 uL 300 uL 30 ML REAGENT AND SAMPLE PREPARATION 1 Bring all reagents and samples to room temperature 18 25 C before use 2 Antibody coated plate Before opening the foil pouch determine the number of strips required to test the desired number of samples plus 16 wells needed for running standards and blanks in duplicate Remove non used strips from the plate frame and return them to the foil pouch containing the desiccant for up to 1 month at 2 8 C 3 Dilution of test standard Dissolve the lyophilised Adiponectin standard with Sample Diluent volume shown on the label Adiponectin standard is unstable after dissolving Use immediately or keep on ice if not used within 1 hr after dissolving To obtain a standard curve d
5. DIRE DRG Adiponectin human EIA 4820 Revised 14 Nov 2011 rm Vers 7 1 RUO This kit is intended for Research Use Only Not for use in diagnostic procedures Please use only the valid version of the package insert provided with the kit INTENDED USE The Human Adiponectin Acrp30 ELISA is an enzyme linked immunosorbent assay for measurement of human Adiponectin in cell culture supernatants plasma and human serum INTRODUCTION This Human Adiponectin ELISA test is a solid phase ELISA assay designed to measure the amount of total low middle and high molecular weight human adiponectin in cell culture supernates serum and plasma This assay employs an antibody specific for human adiponectin coated on a 96 well plate Standards samples and biotinylated anti human adiponectin are pipetted into the wells and adiponectin present in a sample is captured by the antibody immobilized to the wells and by the biotinylated adiponectin specific detection antibody After washing away unbound biotinylated antibody HRP conjugated streptavidin is pipetted to the wells The wells are again washed Following this second wash step TMB substrate solution is added to the wells resulting in color development proportional to the amount of Adiponectin bound The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm DRG International Inc USA 1 Fax 908 233 0758 e E mail corp drg international com_e
6. ant Centrifuge samples at 2000 x g for 10 minutes and assay Dilute plasma samples 1 1000 with Sample Diluent We recommend to perform the dilution in two steps e g first diluting 1 100 5 ul sample 495 ul Sample Diluent and further 1 10 100 ul diluted sample 900ul Sample Diluent Do not use grossly haemolyzed or lipemic specimens Undiluted samples can be stored at 20 C or below Avoid repeated freeze thaw cycles Serum Samples should be collected into a serum separator tube After clot formation centrifuge samples at 2000 x g for 10 minutes Remove serum and assay Dilute serum samples 1 1000 with Sample Diluent We recommend to perform the dilution in two steps e g first diluting 1 100 Sul sample 49Sul Sample Diluent and further 1 10 100ul diluted sample 900ul Sample Diluent Undiluted samples can be stored at 20 C or below for up to 3 months Avoid repeated freeze thaw cycles Cell Culture Supernatants Centrifuge cell culture media at 2000 x g for 10 minutes to remove debris Collect supernatants and assay Dilute Cell Culture Supernatants 1 10 with Sample Diluent e g 10 uL Sample 90 uL diluent Store samples at 20 C or below Avoid repeated freeze thaw cycles Samples with high absorbance values Samples that exceed the measuring range should be diluted 1 2 or 1 5 with Sample Diluent and measured again Samples with absorbance values gt 1 900 can be serially diluted 1 1000 1 2000 and 1 4000 The dilution fac
7. bstrate to test well TME substrate DRG International Inc USA Fax 908 233 0758 e E mail corp drg international com_e Web www drg international com DIRE DRG Adiponectin human EIA 4820 Revised 14 Nov 2011 rm Vers 7 1 RUO ASSAY PROCEDURE 1 Bring all reagents and samples to room temperature 18 25 C before use It is recommended that all standards and samples be run at least in duplicate Leave some wells as a reagent blank 2 to 4 wells FIRST STEP STANDARD SAMPLES AND BLANK BIOTINYLATED ANTIBODY Pipette 50 ul of sample and 50 ul of each diluted standard starting from 15 ng mL into appropriate wells see chapter 7 Pipette 50 ul of sample diluent to the wells which will be used as a blank Incubate for one hour wash 5 times with 1x Wash Solution 300 ul each Add 50 ul of green colored biotinylated detection antibody to all wells containing standards and samples Incubate for one hour Wash 5 times with 1x Wash Solution 300 ul each To wash Empty plate contents Use a multi channel pipette to fill each well with 300 ul of wash buffer then empty plate contents again Repeat procedure 4 additional times for a total of FIVE washes Gently blot plate onto paper towels or other absorbent material Note For automated washing aspirate all wells and wash 5 times with Wash Buffer Blot plate onto paper towels or other absorbent material Never let reaction wells dry Continue to the next step wi
8. e run in duplicate o Samples that contain gt 15 ng mL Adiponectin should be diluted serially with Sample Diluent o When pipetting reagents maintain a consistent order of addition from well to well This ensures equal incubation times for all wells Cover or cap all reagents when not in use Do not use reagents after the kit expiration date Read absorbances within 15 minutes of assay completion In house controls should be run with every assay O O O All residual wash liquid must be drained from the wells by efficient aspiration or by decantation followed by tapping the plate forcefully on absorbent paper Never insert absorbent paper directly into the wells o Because TMB Chromogen is light sensitive avoid prolonged exposure to light Also avoid contact between Stabilized Chromogen and metal or color may develop DRG International Inc USA 8 Fax 908 233 0758 e E mail corp ddrg international com e Web www drg international com PRE les DRG Adiponectin human EIA 4820 Revised 14 Nov 2011 rm Vers 7 1 RUO Test Principle Yr Adiponectin Antibody coated test well m Add 5OuL of diluted Adiponectin D standards and samples to test wells aj O TIT e STEP 2 Add 50 uL of Biotinylated Adiponectin i j antibody to test well STEP 3 Adiponectin Add 50 uL of HRP Streptavidin to test Biotinylated Adiponectin Adiponectin Y Antibody li HRP streptavidin Add 50 uL of TMB su
9. echanism of metabolic syndrome X contribution of adipocytokines adipocyte derived bioactive substances Ann N Y Acad Sci 1999 892 146 54 Hotta K Funahashi T Arita Y et al Plasma concentrations of a novel adipose specific protein adiponectin in type 2 diabetic patients Arterioscler Thromb Vasc Biol 2000 20 1595 9 Ouchi N Kihara S Arita Y et al Novel modulator for endothelial adhesion molecules adipocyte derived plasma protein adiponectin Circulation 1999 100 2473 6 Kojima S Funahashi T Sakamoto T Miyamoto S Soejima H Hokamaki J Kajiwara I Sugiyama S Yoshimura M Fujimoto K Miyao Y Suefuji H Kitagawa A Ouchi N Kihara S Matsuzawa Y Ogawa H The variation of plasma concentrations of a novel adipocyte derived protein adiponectin in patients with acute myocardial infarction Heart 2003 89 667 Watanabe S Okura T Kurata M Irita J Manabe S Miyoshi K Fukuoka T Murakami K Higaki J The effect of losartan and amlodipine on serum adiponectin in Japanese adults with essential hypertension Clin Ther 2006 28 1677 85 Engeli S Feldpausch M Gorzelniak K Hartwig F Heintze U Janke J Mohlig M Pfeiffer AF Luft FC Sharma AM Association between adiponectin and mediators of inflammation in obese women Diabetes 2003352 942 7 Zoccali C Mallamaci F Panuccio V Tripepi G Cutrupi S Parlongo S Catalano F Tanaka S Ouchi N Kihara S Funahashi T Matsuzawa Y Adiponectin is markedly increased in patients with nephr
10. ermine the amount of adiponectin in an unknown sample The standard curve is generated by plotting the average O D 450 nm obtained for each of the standard concentrations on the vertical Y axis versus the corresponding adiponectin concentration ng mL on the horizontal X axis Construct the standard curve using graph paper or statistical software If samples generate values higher than the highest standard dilute the samples with sample diluent and repeat the assay Note that the concentration read from the standard curve must be multiplied by the dilution factor Multiply the plasma or serum value by the dilution factor of 1000 and the cell culture value by the dilution factor of 10 TYPICAL DATA The following graph was obtained for the various Adiponectin standards over the range of 0 to 15 ng mL Please note This graph is an example only A standard curve should be generated each time the assay is performed Do not use these values in your calculations Adiponectin 3 3 2 4 A 1 5 O 1 0 5 0 0 2 4 6 8 10 12 14 16 ng ml DRG International Inc USA 11 Fax 908 233 0758 e E mail corp ddrg international com e Web www drg international com DIRE DRG Adiponectin human EIA 4820 Revised 14 Nov 2011 rm Vers 7 1 RUO REFERENCES LITERATURE l 10 11 12 13 14 15 Hu E Liang P Spiegelman BM AdipoQ is a novel adipose specific gene dysregulated in obesity
11. id contamination lyophilized format Use clean sterile tips Unstable Use immediately after dissolving Keep on ice if not used within 1 hr after dissolving Biotinylated antibody Store at 2 8 C 3 months after opening Ready for use Avoid contamination Use clean sterile tips HRP Conjugated Avidin Store at 2 8 C 3 months after opening Ready for use Avoid contamination Use clean sterile tips Sample Diluent Store at 2 8 C 3 months after opening Avoid contamination use clean sterile tips or pipettes 20x Concentrated Wash Buffer Store at room temperature Until expiry date at room temperature Diluted Wash Buffer Ix working dilution 3 working days at room Bottles used for the working dilution should temperature or 2 weeks at be cleaned regularly discard cloudy 44 C solutions TMB Substrate Solution Ready for use solution at 2 8 C protected Until expiry date from light written on the bottle Avoid contamination Use clean sterile tips Stop Solution Store at 2 8 C May also be stored at Room Until expiry date Temperature DRG International Inc USA 3 Fax 908 233 0758 e E mail corp drg international com_e Web www drg international com DIRE DRG Adiponectin human EIA 4820 Revised 14 Nov 2011 rm Vers 7 1 RUO ADDITIONAL MATERIALS REQUIRED Microplate reader capable of measuring absorbance at 450 nm Precision pipettes 2 ul to 1 ml volumes Multi channel pipette 2
12. ilute as follows DRG International Inc USA 4 Fax 908 233 0758 e E mail corp drg international com_e Web www drg international com DIRE 6 DRG Adiponectin human EIA 4820 Revised 14 Nov 2011 rm Vers 7 1 RUO a Add 125 ul of Adiponectin standard and 125 ul of sample diluent in the first tube to obtain 15 ng ml Adiponectin standard b Add 200 ul of sample diluent to other 4 wells and start 3 fold serial dilutions in dilution tubes as described in the figure c Add only sample diluent to tube no 6 using a clean tip 100 ul 100 ul 100 pl 100 ul 200 pi Sample Diluent as blank 125 ul of Adiponectin a a ul Sample Diluent 15 5 1 67 0 56 0 185 0 ng ml ng ml ng ml ng ml ng ml ng ml 4 Sample preparation and dilution Sample diluent is used for dilution of all samples serum plasma samples culture supernatants and urine requiring dilution Store and dilute all samples in tubes or plates made of material with low binding surface such as polypropylene Prior to the assay frozen samples should be thawed as quickly as possible in tap water 18 25 C Do not use 37 C or 56 C water bath for this purpose DRG International Inc USA Fax 908 233 0758 e E mail corp drg international com_e Web www drg international com DIRE DRG Adiponectin human EIA 4820 Revised 14 Nov 2011 rm Vers 7 1 RUO 5 Plasma Collect plasma using one tenth volume of 0 1 M sodium citrate as an anticoagul
13. l com
14. otic syndrome and is related to metabolic risk factors Kidney Int Suppl 2003 84 S98 102 DRG International Inc USA 12 Fax 908 233 0758 e E mail corp ddrg international com e Web www drg international com DIRE DRG Adiponectin human EIA 4820 Revised 14 Nov 2011 rm Vers 7 1 RUO 16 Ryan AS Berman DM Nicklas BJ Sinha M Gingerich RL Meneilly GS Egan JM Elahi D Plasma adiponectin and leptin levels body composition and glucose utilization in adult women with wide ranges of age and obesity Diabetes Care 2003 26 2383 8 17 Steffes MW Gross MD Schreiner PJ Yu X Hilner JE Gingerich R Jacobs DR Jr Serum adiponectin in young adults interactions with central adiposity circulating levels of glucose and insulin resistance the CARDIA study Ann Epidemiol 2004 14 492 8 LIABILITY This kit 1s intended for research use only If the recipient of this kit passes it on in any way to a third party this instruction must be enclosed and said recipient shall at own risk secure in favor of DRG all limitations of liability herein DRG shall not be responsible for any damages or losses due to using the kit in any way other than as expressly stated in these Instructions The liability of DRG shall in no event exceed the commercial value of the kit DRG shall under no circumstances be liable for indirect special or consequential damages including but not limited to loss of profit DRG International Inc USA
15. temperature Wash 5 x with 1x wash buffer 5 Add 50 ul TMB One Step Substrate Reagent to each well DRG International Inc USA Fax 908 233 0758 e E mail corp drg international com_e Web www drg international com DIRE DRG Adiponectin human EIA 4820 Revised 14 Nov 2011 rm Vers 7 1 RUO Incubate 20 minutes at room temperature L 6 Add 25 ul Stop Solution to each well Read at 450 nm against 630 nm immediately Correcting for optical imperfections in the microplates by subtracting A630 nm is recommended but not an essential procedure PROCEDURAL NOTES LAB QUALITY CONTROL o When not in use kit components should be refrigerated All reagents should be warmed to room temperature before use o Microtiter plates should be allowed to come to room temperature before opening the aluminum pouches o Once the desired number of strips has been removed immediately reseal the pouch and store at 2 8 C to maintain plate integrity Protect from humidity o Samples should be collected in pyrogen endotoxin free tubes o Samples should be frozen if not analyzed shortly after collection Avoid multiple freeze thaw cycles of frozen samples Thaw completely and mix well prior to analysis o When possible avoid use of badly hemolyzed or lipemic sera If large amounts of particulate matter are present centrifuge or filter prior to analysis o Itis recommended that all standards controls and samples b
16. thout delay or interruption SECOND STEP STREPTA VIDIN HRP Add 50 ul of prepared Streptavidin HRP solution Ready to use to each well Incubate for 30 minutes at room temperature Wash 5 times with 1x wash solution 300 ul each THIRD STEP TMB SUBSTRATE Add 50 ul of TMB ready to use substrate reagent to each well Incubate for 20 minutes at room temperature in the dark FOURTH STEP STOP REACTION ANDREAD THE PLATE Add 25 ul of Stop Solution to each well Read at 450 nm within 15 minutes Correcting for optical imperfections in the microplates by subtracting A630 nm is recommended but not an essential procedure FIFTH STEP READING AND CALCULATION Calculate the mean of reagent blank absorbance values and subtract it from all test well values standard and test samples Mean reagent blank absorbance value at 450 nm should be less than 0 200 Calculate your results against standard DRG International Inc USA 10 Fax 908 233 0758 e E mail corp ddrg international com e Web www drg international com DIRE DRG Adiponectin human EIA 4820 Revised 14 Nov 2011 rm Vers 7 1 RUO CALCULATION OF RESULTS The standard curve must be determined individually for each experiment Correct each absorbance value of all standards by subtracting from it the O D value of the reagent blank BI only sample diluent Calculate the mean absorbance value for each standard from the duplicates The standard curve is used to det
17. tor must be taken in account when calculating the results 6 Wash Buffer If the 20x concentrated Wash Buffer contains visible crystals warm it at 37 C and mix gently until dissolved Dilute 25 ml of Wash Buffer Concentrate with de ionized or distilled water to yield 500 ml of 1x Wash Buffer 7 Vortex mix Biotinylated antibody solution gently before use 8 Vortex mix peroxidase HRP labeled avidin gently before use Caution TMB substrate Tetramethylbenzidine and the Stop solution H SO are toxic or corrosive and should be handled with care Use gloves during handling DRG International Inc USA 6 Fax 908 233 0758 e E mail corp drg international com e Web www drg international com DIRE 6 DRG Adiponectin human EIA 4820 Revised 14 Nov 2011 rm Vers 7 1 RUO TEST PROCEDURE SUMMARY 1 Prepare all reagents samples and standards Dilute samples as follows Serum amp Plasma 1 1000 diluted samples Cell Culture Supernatants 1 10 diluted samples L 2 Add 50 ul of each diluted sample diluted standards starting from 15 ng mL and sample diluent as a blank into the appropriate wells of the strips Incubate hour at room temperature Wash 5 x with 1x wash buffer 3 Add 50 ul ready for use biotin antibody promptly to each well Incubate 1 hour at room temperature L Wash 5 x with 1x wash buffer 4 Add 50 ul ready for use HRP Streptavidin solution Incubate 30 minutes at room
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