CY-8049 Rat Adiponectin ELISA Kit
Contents
1. HRP conjugated Detection Antibody One vial containing 12 mL of HRP conjugated anti Adiponectin antibody Ready to use Rat Adiponectin Standard One vial containing 144 ng of lyophilized recombinant A peroxidase Substrate Reagent One bottle containing 20 mL of the chromoge tetra methylbenzidine TMB Ready to use Q Stop Solution One bottle containing 20 mL of 1 N H2501 Rea C CY 8049 4 Versionft 150218 Rat Adiponectin ELISA Kit CircuLex Users Manual 4 For Research Use Only Not for use in diagnostic procedures Materials Required but not Provided Orbital microplate shaker e Pipettors 2 20 uL 20 200 uL and 200 1000 uL precision pipettors with disposable tips Precision repeating pipettor Microcentrifuge and tubes for sample preparation s of 450 nm 540 also be read at a Plate reader capable of measuring absorbance in 96 well plates at dual wa nm Dual wavelengths of 450 550 or 450 595 nm can also be used cy Vortex mixer Qy e Microplate washer optional Manual washing is possible but not preferable d n single wavelength of 450 nm which will give a somewhat higher readi Software package facilitating data generation and analysis opti 500 or 1000 mL graduated cylinder Reagent reservoirs Deionized water of the highest quality Disposable paper towels C CY 8049 5 Versions 150218 Rat Adiponectin ELISA Kit TM L 1rcu Lex User s Manual For Research Use Only Not2. USA 98 2005 Berg A H et al 2002 Trends Endocrinol Metab 13 84 Arita Y et al 1999 Biochem Biophys Res Commun 257 79 Yamauchi T et al 2003 Nature 423 762 10 Stefan N et al 2002 J Clin Endocrinol Metab 87 4652 11 Matsubara M et al 2002 Eur J Endocrinol 147 173 12 Weyer C et al 2001 J Clin Endocrinol Metab 86 1930 13 Hotta K et al 2000 Arterioscler Thromb Vasc Biol 20 1595 14 Tomas E et al 2002 Proc Natl Acad Sci USA 99 16309 15 Berg A H et al 2001 Nat Med 7 947 16 Fruebis J et al 2001 Proc Natl Acad Sci USA 98 2005 17 Yamauchi T et al 2001 Nat Med 7 941 Related Products CircuLex Rat Adiponectin ELISA Kit Cat CY 8049 CircuLex Human Adiponectin ELISA Kit Cat Y 8050 CircuLex Mouse Adiponectin ELISA Kit Cat CY 8051 CircuLex Dog Adiponectin ELISA Kit Cat CY 8052 Anti Human Adiponectin Cat CY P1017 Anti Mouse Adiponectin Cat CY P1018 Anti Human Adiponectin 17 36 aa Cat GY P1031 PRODUCED BY CycLex Co Ltd 1063 103 Terasawaoka Ina Nagano 396 0002 Japan Fax 81 265 76 7618 e mail info cyelex co jp URL http www yclex c jp CycLex CircuEex products are supplied for research use only CycLex CircuLex products and components thereof may not be resold modified for resale or used to manufacture commercial products without prior written approval from CycLex Co Ltd To inquire ab
3. C 2 Dilute samples with Dilution Buffer See Sample Preparation above o2 Pipette 100 uL of Standard Solutions Std1 Std7 Blank and diluted samples in duplicates into the appropriate wells 4 Incubate the plate at room temperature ca 25 C for 1 hour shaking at ca 300 rpm on an orbital microplate shaker Nn Wash 4 times by filling each well with Wash Buffer 350 uL using a sq irt4bottle multi channel pipette manifold dispenser or microplate washer 6 Add 100 uL of HRP conjugated Detection Antibody into each well 7 Incubate the plate at room temperature ca 25 C for 1 hour shaking at ca 300 rpm on an orbital microplate shaker 8 Wash 4 times by filling each well with Wash Buffer 350 BL using a squirt bottle multi channel pipette manifold dispenser or microplate washer 9 Add 100 uL of Substrate Reagent Avoid expositfig the microtiter plate to direct sunlight Covering the plate with e g aluminum foil is recommended Return Substrate Reagent to 4 C immediately after the necessary volume is removed 10 Incubate the plate at room temperature ca 25 C for 10 20 minutes shaking at ca 300 rpm on an orbital microplate shaker The incubation4fime may be extended up to 30 minutes if the reaction temperature is below than 20 C 11 Add 100 uL of Stop Solution to each well in the same order as the previously added Substrate Reagent 12 Measure absorbance in each w ll using a
4. bound by the immobilized antibody After washing awayg ny unbound substances an HRP conjugated antibody specific for Adiponectin is added to the wells E llowing a wash to remove any unbound antibody HRP conjugate the remaining conjugate is allowed to react with the substrate H2O2 tetramethylbenzidine The reaction is stopped by addition of acidic solution and absorbance of the resulting yellow product is measured at 450 nm The absorbanee is proportional to the concentration of Adiponectin A standard curve is constructed by plotting absorbance values versus Adiponectin concentrations of calibrators and concentrations of unknown samples are determined using this standard curve The Circulex Rat Adiponectin ELISA Kit is designed to measure the concentration of rat adiponectin from rat serum plasma adipocytes or conditioned medium The principle of the assay is shown in below Diluted samples and serially diluted standard recombinant rat adiponectin solutions are added to an appropri te number of wells of the microtiter plate and incubated Adiponectin in the sample will be bound by the primary anti adiponectin antibody immobilized in the well first reaction After washing the HRP conjugated anti adiponectin antibody is added to each well and allowed to incubate second reaction The HRP conjugated anti adiponectin antibody will bind to the adiponectin trapped in the well Cat CY 8049 2 Versionft 150218 Rat Adiponectin ELISA Kit C
5. the dynamic range of the assay 40 Serum 1 A Serum 2 Serum 3 30 20 rat Adiponectin conc ng ml 0 0 5 1 15 2 25 Dilution ratio 1 000 Cat CY 8049 13 Version 150218 ihi Rat Adiponectin ELISA Kit L 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Example of Test Results Fig 1 Relation between Rat Adiponectin concentration and age 25 0 Rat No 1 e W Rat No2 g 20 0 g Rat No 3 SS X Rat No 4 is O Rat No 5 2 15 0 o c T E g 8 gt 9 10 0 S 2 p o lt gt 90 amp N 0 0 0 5 10 15 20 25 30 35 age in week Fig 2 Relation between Rat Adiponectin concentration nd body weight 500 400 e e o we ao 300 M E oD D E gt 200 mo o B 100 0 0 0 5 0 10 0 15 0 20 0 25 0 rat Adiponectin conc ug ml Cat CY 8049 14 Version 150218 Rat Adiponectin ELISA Kit L ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures References Maeda K et al 1996 Biochem Biophys Res Commun 221 286 Kishore U and K B Reid 2000 Immunopharmacology 49 159 Shapiro L and P E Scherer 1998 Curr Biol 8 335 Nakano Y et al 1996 J Biochem Tokyo 120 803 Scherer P E et al 1995 J Biol Chem 270 26746 Fruebis J et al 2001 Proc Natl Acad Sci
6. 0 AdipoQ and GBP 28 is a recently discovered 244 aminoaeid protein the product of the apM1 gene which is physiologically active and specifically and highly expressed in adipose cells adipokine The protein belongs to the soluble defence collagen superfamily it has a collagen like domain structurally homologous with collagen VIII and X and complement factor Clq like globular domain 1 2 Adiponectin forms homotrimers which are the building blocks for higher order complexes found circulating in serum 3 4 Circulating Adiponectin levels are high 5 30 g mL accounting for approximately 0 01 of total plasma protein 5 8 Adiponectin receptors AdipoR1 and AdipoR2 have been recently cloned AdipoR1 is abundantly expressed in skeletal muscle whereas AdipoR2 is predominantly expressed in the liver 9 Paradoxically adipoSe tissue expressed adiponectin levels are inversely related to the degree of adiposity 10 11 A reduction in adiponectin serum levels is accompanied by insulin resistance states such as obesity and type diab tes mellitus 12 13 It is also reported in patients with coronary artery disease 13 Increas d adiponectin levels are associated with type 1 diabetes mellitus anorexia nervosa and chronic renal failure Adiponectin concentrations correlate negatively with glucose insulin triglyceride c neentr tions and body mass index and positively with high density lipoprotein cholesterol levels and insulin stimulated glucose dis
7. Rat Adiponectin ELISA Kit CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures ELISA Kit for Measuring Rat Adiponectin NN CircuLex Rat Adiponectin ELISA Kit Catit CY 8049 C Intended Nr T 1 AOI aT EEEE 1 TPO CUO NNNM 2 3 Principle of the Assay 3 4 Materials Provided cereo rtis 4 Materials Required but not Provided 5 Precautions and Recommendations 6 Sample Collection and Storage 7 Detailed Protocoles ense rrr matte tnots 8 0 CACM LAGOS T 10 Measurement Rative cssecsccccarsnsvssaaceass 10 Troubleshooting vis scsacsesseicecacctnnsasasanayeevente 10 Reagent Stability esteso tenants 10 Assay CharacteriStics ccccsasnccacecersnnvvnancns seve 11 Example of Test Results 1 icio up I Related PtOd uctsa er eri rires 15 Intended Use The CycLex Research Product Circulex ponectin ELISA Kit is used for the quantitative gt pl measurement of rat or rat adiponectin G asma tissue culture medium and other biological media This assay kit is for research use o ih d not for use in diagnostic or therapeutic procedures Storage Upon receipt store all co e Don t expose reagents A C CY 8049 1 Versionft 150218 Rat Adiponectin ELISA Kit L ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Introduction Adiponectin also referred as Acrp3
8. User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol The CycLex Research Product Circulex Rat Adiponectin ELISA Kit is provided with removable strips of wells so the assay can be carried out on separate occasions using only the number of strips required for the particular determination Since experimental conditions may vary an aliquot of the Rat Adiponectin Standard within the kit should be included in each assay as a calibrator Disp sable pipette tips and reagent troughs should be used for all liquid transfers to avoid cross contamination of reagents or samples Preparation of Working Solutions All reagents need to be brought to room temperature prior to the assay Assay r agents are supplied ready to use with the exception of 10X Wash Buffer and Rat Adiponectin Standard 1 Prepare a working solution of Wash Buffer by adding 100 mL of the 10X Wash Buffer to 900 mL of deionized distilled water ddH2O Mix well Store at 4 C for two weeks or 20 C for long term storage 2 Reconstitute Rat Adiponectin Standard with 1 mL of ddH2 The concentration of the rat adiponectin in vial should be 144 ng mL which is referred as a Master Standard of rat adiponectin Prepare Standard Solutions as follows Use the Master Standard to produce a dilution series below Mix each tube thoroughly before the next transfer The 36 ng mL standard Std 1 serves as the highest standard The Dilution Buffer s
9. erosols Wear protective gloves and dispose of biological samples properly CAUTION Sulfuric Acid is a Strong acid Wear disposable gloves and eye protection when handling Stop Solution Cat CY 8049 6 Versions 150218 Rat Adiponectin ELISA Kit CircuLex Users Manual For Research Use Only Not for use in diagnostic procedures Sample Collection and Storage Serum Allow blood samples to clot for 60 30 minutes Centrifuge the samples at 4 C for 10 mi at 1 000 x g Remove serum and assay immediately or store samples on ice for up to 6 hours befo assaying Aliquots of serum may also be stored at below 70 C for extended periods of time id repeated freeze thaw cycles Plasma Collect plasma using EDTA Naz as the anticoagulant If possible collect the amp oa mixture of EDTA Na and Futhan5 to stabilize the sample against spontaneous in vitro lement store samples on ice for up to 6 hours before assaying Aliquots of plasma may als ored at below 70 C for extended periods of time Avoid repeated freeze thaw cycles Note Citrate plasma has not been validated for use in this assay G Other biological samples Remove any particulates by centrifugation an immediately or aliquot and store samples at below 70 C Avoid repeated freeze thaw cycles activation Immediately centrifuge samples at 4 C for 15 minutes at 1 000 x g i immediately or C CY 8049 7 Version 150218 CircuLex Rat Adiponectin ELISA Kit
10. erves as the zero standard Blank Volume of Standard Dilution Buffer Concentration Std 1 150 uL of Master Standard 450 uL 36 00 ng mL Std 2 300 uL of Std 1 36 00 ng ml 300 uL 18 00 ng mL Std 3 300 uL of Std 2 18 00 ng ml 300 uL 9 00 ng mL Std 4 300 uL of Std 3 9 00 ng ml 300 uL 4 50 ng mL Std 5 300 uL of Std 4 4 50ng ml 300 uL 2 25 ng mL Std 6 300 uL of Std 5 2 25 ng ml 300 uL 1 13 ng mL Std 7 300 uL of Std 6 1 13 ng ml 300 uL 0 56 ng mL Blank 300 uL 0 ng mL Note Do not use a Repeating pipette Change tips for every dilution Wet tip with Dilution Buffer before dispensing nused portions of Standards should be aliquoted and stored at below 70 C immediately Avoid multiple freeze and thaw cycles Sample Preparation Serum and plasina samples require a 1 000 fold dilution e g First Make 20 fold dilution 5 uL of sample 95 uL of Dilution Buffer Seconidz Make 1 000 fold dilution 6 uL of 20 fold diluted sample 294 uL Dilution Buffer Oth r biological samples require neat 10 and 100 fold dilution Capt CY 8049 Version 150218 Rat Adiponectin ELISA Kit L ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Assay Procedure paa Remove the appropriate number of microtiter wells from the foil pouch and place them into the well holder Return any unused wells to the foil pouch refold seal with tape and store at 4
11. for use in diagnostic procedures Precautions and Recommendations Allow all the components to come to room temperature before use All microplate strips that are not immediately required should be returned to the zip lock pouch which must be carefully resealed to avoid moisture absorption Do not use kit components beyond the indicated kit expiration date Use only the microtiter wells provided with the kit Rinse all detergent residue from glassware Use deionized water of the highest quality Do not mix reagents from different kits The buffers and reagents in this kit may contain preservatives or other chemicals Care should be taken to avoid direct contact with these reagents Do not mouth pipette or ingest any of the reagents Do not smoke eat or drink when performing the assay or in areas where samples or reagents are handled Dispose of tetra methylbenzidine TMB containifig solutions in compliance with local regulations Avoid contact with the acidic Stop Solution and Substrate Solution which contains hydrogen peroxide Wear gloves and eye protection when handling 3mmunodiagnostic materials and samples of rat origin and these reagents In case of contact with the Stop Solution and the Substrate Solution wash skin thoroughly with water and seek medical attention when necessary Biological samples might be contaminated with infectious agents Do not ingest expose to open wounds or breathe a
12. haracteristics 1 Sensitivity The limit of detection defined as such a concentration of Adiponectin giving absorbance higher than mean absorbance of blank plus three standard deviations of the absorban ee of blank A blank 3 SD blank is better than 0 249 ng ml of sample Dilution Buffer is pipetted into blank wells Eighty assays were evaluated and the minimum detectable dose MDD of Adiponectin ranged from 0 134 0 325 ng mL The mean MDD was 0 249 ng mL The MDD was determined by adding three standard deviations to the mean optical density value of twenty zero standard replicates and calculating the corresponding concentration 2 5 2 0 A450 0 5 0 0 0 10 20 30 40 rat Adiponectin conc ng ml Cat CY 8049 11 Version 150218 ihi Rat Adiponectin ELISA Kit L 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures 2 Specificity The antibodies in the Circulex Rat Adiponectin ELISA Kit are highly specific of rat and_rat Adiponectin with no detectable cross reactivity to other cytokines that may be present in rat and rat serum 3 Precision Intra assay Precision Precision within an assay Three samples of known concentration were tested twelve times on one plate to assess intra assay precision Intra assay Within Run n 12 rat Adiponectin conc ug ml 1 2 3 4 5 6 7 8 Inter assay Precision Precision between assays T
13. hree samples of known concentration were tested in five separate assays to assess inter assay precision nter assay Run to Run n 5 rat Adiponectin conc ug ml Day Serum 1 Serum 2 Serum 3 1 16 90 11 14 3 31 2 18 92 12 22 3 33 3 17 95 12 42 3 28 4 18 25 12 20 3 46 5 17 48 11 98 3 30 MAX 18 92 12 42 3 46 MIN 16 90 11 14 3 28 MEAN 17 90 11 99 3 34 S D 0 764 0 502 0 072 C V 4 27 4 19 2 15 Cat CY 8049 12 Version 150218 ihi Rat Adiponectin ELISA Kit L 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures 4 Spiking Recover Serum samples were spiked with different amounts of Rat Adiponectin and assayed The recovery of Adiponectin spiked to levels throughout the range of the assay was evaluated Sample Average Recovery Range Cell culture media n 4 114 6 104 9 100 8 96 99 5 102 6 106 4 96 99 5 96 87 0 9230 Serum 1 Nene 10 ug ml Average ug ml 13 51 24 97 16 66 14 52 Recovery rate 11457 104 93 100 80 Serum 2 PCE Nore 10 ug ml Average ug ml 17 42 10 55 Recoveryrate 99 54 102 64 106 40 Serum 3 Oo None 10ug ml Average ug ml 13 17 Recovery rate 99 50 87 01 92 03 5 Linearity To assess the linearity of the assay samples containing and or spiked with high concentrations of Adiponectin were serially diluted with the Dilution Buffer to produce samples with values within
14. ircuLex Users Manual For Research Use Only Not for use in diagnostic procedures in the first Reaction After washing the colorimetric substrate for the enzyme is added to all wells incubated The color development is terminated by the addition of a stop solution The intensity of color is measured at 450 nm The concentrations of the samples tested are calculated using the absorbance values of the adiponectin standard solutions assayed at the same time Summary of Procedure Add 100 uL of diluted sample to the wells 3 Incubate for 1 hour at room temp Wash the wells Add 100 uL of HRP conjugated anti Adiponectin vin 4 Incubate for 1hour at room te Wash the wells i Add 100 uL of Substrate Reagent t Add 100 uL of Stop Solution Measure absorbance at Q C CY 8049 3 Versions 150218 TM Rat Adiponectin ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures d Materials Provided All samples and standards should be assayed in duplicate The following components are supplie are sufficient for the one 96 well microplate kit Microplate One microplate supplied ready to use with 96 wells 12 strips of 8 wells in a bag with a desiccant pack Wells are coated with anti Adiponectin antibody as a capture a 10X Wash Buffer One 100 mL bottle of 10X buffer containing 2 Tween 20 Dilution Buffer One bottle containing 50 mL of 1X buffer use for sample Q2 use 1ponectin
15. o consideration in calculating the rat adiponectin concentration Troubleshooting 1 The Rat Adiponectin Standard should be sun in d plicate using the protocol described in the Detailed Protocol Incubation times or temperatures significantly different from those specified may give erroneous results 2 Poor duplicates accompanied by elevated values for wells containing no sample indicate insufficient washing If all instructions in the Detailed Protocol were followed accurately such results indicate a need for washer maintenance 3 Overall low signal may Andicate that desiccation of the plate has occurred between the final wash and addition of Substrate Reagent Do not allow the plate to dry out Add Substrate Reagent immediately after wash Cat CY 8049 10 Version 150218 Rat Adiponectin ELISA Kit L ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Reagent Stability All of the reagents included in the CycLex Research Product Circulex Rat Adiponectin ELISA dit have been tested for stability Reagents should not be used beyond the stated expiration date Upon receipt kit reagents should be stored at 4 C except the reconstituted Rat Adiponectin Standard musb be stored at below 70 C Coated assay plates should be stored in the original foil bag sealed by the zip lock and containing a desiccant pack For Research Use Only Not For Use in Diagnostic or Therapeutic procedures Assay C
16. out licensing for such commercial use please contact us via email Cat CY 8049 15 Version 150218
17. posal Adiponectin has been shown to increase insulin sensitivity and decrease plasma glucose by increasing tissue fat oxidation It inhibits the inflammatory processes of atherosclerosis suppressing the expression of adhesion and cytokine molecules in vascular endothelial cells and macrophages respectively This adipokine plays a role as a scaffold of newly formed collagen in myocardial remodelling after ischaemic injury and also stimulates angiogenesis by promoting cross talk between AMP activated protein kinase and Akt signalling in endothelial Cells 14 Injection of Adiponectin into non obese diabetic micefleads to an insulin independent decrease in glucose levels 15 This is likely due to insulin sensitizing effects involving Adiponectin regulation of triglyceride metabolism 15 A truncated form of Adiponectin gAdiponectin containing only the C terminal globular domain has been identified inthe blood and recombinant gAdiponectin has been shown to regulate weight reduction as well as free fattyyacid oxidation in mouse muscle and liver 16 17 The full length recombinant Adiponectin protein is apparently less potent at mediating these effects 16 17 Principle of the Assay The Circulex Rat Adiponectin ELISA Kityemploys the quantitative sandwich enzyme immunoassay technique An antibody specific for rodent Adiponectin has been pre coated onto a microplate Standards and samples are pipetted into the wells and any Adiponectin present is
18. rsus the concentration of the standards and draw the best curve To determine the Adiponectin concentration of each sample first find the absorbance value on the y axis and extend a horizontal line to the standard curve At the p int of intersection extend a vertical line to the x axis and read the corresponding Adiponectin concentration If the samples have been diluted the concentration read from the standard curve must be multiplied by the dilution factor A The dose response curve of this assay fits best to a sigmoidal 5 parameter logistic equation The results of unknown samples can be calculated with any computer program having 5 parameter logistic function It is important to make an appropriate mathematical adjustment to a eommodate for the dilution factor B Most microtiter plate readers perform automatic calculations of analyte once ntration The calibration curve is constructed by plotting the absorbance Y of calibrators versus log of the known concentration X of calibrators using the four parameter function Alternatively the logit log function can be used to linearize the calibration curve i e logit of absorbance Y is plotted versus log of the known concentration X of calibrators Measurement Range The measurement range is 0 56 ng mL to 36 ng ml Any Sample reading higher than the highest standard should be diluted with Dilution Buffer in higher dilution and re assayed Dilution factors need to be taken int
19. spectrophotometric microplate reader at dual wavelengths of 450 540 nm Dual wavelengthsjof 450 550 or 450 595 nm can also be used Read the microplate at 450 nm if only a singleAwavelength can be used Wells must be read within 30 minutes of adding the Stop Solution Note 1 Complete removal of liquid at each step is essential to good performance After the last wash remove any femaining Wash Buffer by aspirating or decanting Invert the plate and blot it against Clean pap r towels Note 2 Reliable standard curves are obtained when either O D values do not exceed 0 2 units for the blank Zero concentration or 2 8 units for the highest standard concentration The plate shouldbe monitored at 5 minute intervals for approximately 30 minutes Note 3 If themicfoplate reader is not capable of reading absorbance greater than the absorbance of the highestystandard perform a second reading at 405 nm A new standard curve constructed using the values measured at 405 nm is used to determine Adiponectin concentration of off scale samples The readings at 405 nm should not replace the on scale readings at 450 nm Cat CY 8049 9 Versions 150218 Rat Adiponectin ELISA Kit L ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Calculations Average the duplicate readings for each standard control and sample and subtract the average zero standard optical density Plot the optical density for the standards ve
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