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1. 5 6 86 13 36 21 40 6 7 20 13 61 22 47 MAX 7 39 14 42 22 90 MIN 6 86 12 78 20 85 MEAN 7 20 13 45 21 75 S D 0 197 0 591 0 786 C V 2 74 4 40 3 61 C CY 8051 12 Versions 120807 Mouse Adiponectin ELISA Kit x TM Circu 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 Mouse 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 98 4 101 0 104 2 93 6 108 7 100 4 89 4 99 7 A Recoveryrate 9836 10103 104 1577 jy L Nee 19ugml 5ug ml NIE Average ug ml 882 2286 1426 gmil33 Recoveryrate 5 9357 10874 FT w a po None 15 ug ml Sug ml Average ug ml 1405 2746 19987 1642 Recovery rate 8943 9 94 85 5 Linearity To assess the linearity of the assay samples c ng and or spiked with high concentrations of Adiponectin were serially diluted with the Dilution r to produce samples with values within the dynamic range of the assay 2 Linearity Serum A A Serum B Serum C gt mouse Adiponectin conc ng ml 0 1 2 3 4 5 6 Dilution ratio 10 000 C CY 8051 13 Versions 120807 Mouse Adiponectin ELISA Kit x TM Circu Lex2. For Research Use Only Not for use in diagnostic procedures Related Products CircuLex Rat Adiponectin ELISA Kit Cat CY 8049 CircuLex Human Adiponectin ELISA Kit Cat CY 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 CY P1031 PRODUCED BY Q CycLex Co Ltd 1063 103 Terasawaoka Ina Nagano 396 0002 Japan Fax 81 265 76 7618 e mail info 2 cyclex co jp URL http www cyclex co jp CycLex CircuLex products are supplied for use only CycLex CircuLex products and components thereof may not be resold modifie resale or used to manufacture commercial products without prior written approval from Cy Co Ltd To inquire about licensing for such commercial use please contact us via email Q e C CY 8051 15 Versions 120807
3. Measurement Range ample reading higher than the highest dilution and re assayed Dilution factors need iponectin concentration 1 The Mouse Adiponectin Standard sh in duplicate using the protocol described in the Detailed Protocol Incubation times emferatures significantly different from those specified may give erroneous results values for wells containing no sample indicate insufficient iled Protocol were followed accurately such results indicate a 2 Poor duplicates accompanied b washing If all instructions i need for washer maintenanc 3 Overall low signal ma icate that desiccation of the plate has occurred between the final wash and addition of Substrate Do not allow the plate to dry out Add Substrate Reagent immediately after wash included in the CycLex Research Product CircuLex Mouse Adiponectin ELISA for stability Reagents should not be used beyond the stated expiration date Upon 70 C Coated assay plates should be stored in the original foil bag sealed by the zip taining a desiccant pack CY 8051 Versions 120807 Mouse Adiponectin ELISA Kit TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures For Research Use Only Not For Use in Diagnostic or Therapeutic procedures Assay Characteristics 1 Sensitivity The limit of detection defined as such a concentration of Adiponectin giving absorban han mean absorbance of blank plus thr
4. Acrp30 AdipoQ and GBP 28 is a recently discovered 244 amino protein the product of the apM1 gene which is physiologically active and specifically and expressed in adipose cells adipokine The protein belongs to the soluble defense collagen it has a collagen like domain structurally homologous with collagen VIII and X and comple Clq like globular domain 1 2 Adiponectin forms homotrimers which are the build higher order complexes found circulating in serum 3 4 Circulating Adiponectin levels serum levels is accompanied by insulin resistance states such as obesity and ty 13 It is also reported in patients with coronary artery disease 13 Increasi associated with type 1 diabetes mellitus anorexia nervosa and chronicere es mellitus 12 nectin levels are ure Adiponectin ations and body mass lin stimulated glucose ease plasma glucose by erosclerosis suppressing the x index and positively with high density lipoprotein cholesterol levels a disposal Adiponectin has been shown to increase insulin sensitivit increasing tissue fat oxidation It inhibits the inflammatory processe expression of adhesion and cytokine molecules in vascular respectively This adipokine plays a role as a scaffold of remodeling after ischemic injury and also stimulates angiogen by AMP activated protein kinase and Akt signaling in endothe ial cells T4 Injection of Adiponectin into non obese diabetic ds t an insulin independent decrease in glu
5. Kit is designed to measure the concentration of mouse or rat adiponecti use or rat serum plasma adipocytes or conditioned medium The principle of the assay is in below Diluted samples and serially diluted standard recombinant mouse adipone solutions are added to an appropriate number of wells of the microtiter plate and incubated Adip cet in the sample will be bound by the primary anti adiponectin antibody immobilized in the eaction 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 Cati CY 8051 2 Version 120807 D Mouse Adiponectin ELISA Kit CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 the adiponectin trapped in the well in the first Reaction After washing the colorimetric substrate for enzyme is added to all wells and incubated The color development is terminated by the addition o stop solution The intensity of the color is measured at 450 nm The concentrations of the samples t are calculated using the absorbance values of the adiponectin standard solutions assayed at the time Summary of Procedure Q Add 100 uL of diluted sample to the wells Y Incubate for 1 hour at room temp Wash the wells Add 100 uL of HRP conjugated anti Adiponecti i Y Incubate for 1 hour at room t Wash the wells M Add 100 uL of Substrate Reagent M Add 100 u
6. User s Manual A For Research Use Only Not for use in diagnostic procedures Example of Test Results Fig 1 Secretion of mouse adiponectin into culture medium during OP9 differentiation into adipocyte Measurement of mouse Adioponectin in OP9 cell Culture Cup 125 ES on 100 o amp 7 8 5 amp 350 uo lt o 5 25 e E 0 0 2 4 6 8 10 12 14 Days of differentiation NJ References ommun 221 286 ology 49 159 335 Maeda K et al 1996 Biochem Biophys Kishore U and K B Reid 2000 Immuno Shapiro L and P E Scherer 1998 Curr Nakano Y et al 1996 J Biochem Scherer P E et al 1995 J Biol Che Fruebis J et al 2001 Proc Natl Berg A H et al 2002 Trends Arita Y et al 1999 Biochem Yamauchi T et al 2003 N 10 Stefan N et al 2002 J crinol Metab 87 4652 11 Matsubara M et al 2 Endocrinol 147 173 12 Weyer C et al 200 Clin Endocrinol Metab 86 1930 13 Hotta K et al 20 ioscler Thromb Vasc Biol 20 1595 14 Tomas E et al 2 c Natl Acad Sci USA 99 16309 15 Berg A H e t Med 7 947 16 Fruebis J et 4l 2001 Proc Natl Acad Sci USA 98 2005 17 DN O 001 Nat Med 7 941 0 7803 270 26746 USA 98 2005 1 Metab 13 84 l Res Commun 257 79 49 COO ect podes C CY 8051 14 Versions 120807 Mouse Adiponectin ELISA Kit TM CircuLex User s Manual
7. a mixture of EDTA Na and Futhan5 to stabilize the sample against spontaneous in vitro c ement activation Immediately centrifuge samples at 4 C for 15 minutes at 1 000 x g Assay immediately or store samples on ice for up to 6 hours before assaying Aliquots of plasma may als red 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 G5 Other biological samples Remove any particulates by centrifugation an 1mmediately or aliquot and store samples at below 70 C Avoid repeated freeze thaw cycles C CY 8051 7 Version 120807 Mouse Adiponectin ELISA Kit x TM Circu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol The CycLex Research Product CircuLex Mouse Adiponectin ELISA Kit is provided removable strips of wells so the assay can be carried out on separate occasions using only the nu strips required for the particular determination Since experimental conditions may vary angali the Mouse Adiponectin Standard within the kit should be included in each assay ag a Disposable pipette tips and reagent troughs should be used for all liquid trans 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 ready to use with the exception of 10X Wash Buffer and Mouse Adiponectin S are suppli
8. L of Stop Soluti M Measure absorbance at 450 C CY 8051 3 Versions 120807 Mouse Adiponectin ELISA Kit x TM Circu Lex User s Manual For Research Use Only Not for use in diagnostic procedures 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 bag with a desiccant pack Wells are coated with anti mouse Adiponectin antibody as a cat 10X Wash Buffer One 100 mL bottle of 10X buffer containing 2 Tween 20 Dilution Buffer One bottle containing 50 mL each of 1X buffer use for sample dil Zi eady to use Mouse Adiponectin Standard One vial containing 52 8 ng of lyophili ombinant mouse Adiponectin HRP conjugated Detection Antibody One vial containing 12 mL of orseradish peroxidase conjugated anti mouse Adiponectin antibody Ready to use Substrate Reagent One bottle containing 20 mL of the chromo trate tetra methylbenzidine TMB Ready to use Stop Solution One bottle containing 20 mL of 1 N H5SO se C CY 8051 4 Version 120807 Mouse Adiponectin ELISA Kit x TM Circu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Materials Required but not Provided Pipettors 2 20 uL 20 200 uL and 200 1000 uL precision pipettors with disposable tips Precision repeating
9. Mouse Adiponectin ELISA Kit x TM Circu Lex User s Manual For Research Use Only Not for use in diagnostic procedures ELISA Kit for Measuring Mouse Adiponectin CircuLex Mouse Adiponectin ELISA KIN Cat CY 8051 Q 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 Q Sample Collection and Storage 7 Detailed Protocoles ense rrr matte tnots 8 0 CAC Euri T 10 Measurement Rative cssecscctcersnsvssaecenss 10 Troubleshooting eerta rto mte 10 Reagent Stability eese errans 10 11 Assay Characteristics us cree ener saree 11 Example of Test Results 1 icio up 1 Related PROGUCtS cariissesncasessatsciectaanesenoesesanss 15 Intended Use The CycLex Research Product CircuLe diponectin ELISA Kit is used for the quantitative measurement of mouse or rat adiponecti serum plasma tissue culture medium and other biological media This assay kit is for research use only and not for use in diagnostic or therapeutic procedures Storage e Upon receipt store all coim Don t expose reagent C CY 8051 1 Version 120807 Mouse Adiponectin ELISA Kit x TM Circu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Introduction Adiponectin also referred as
10. cose levels 15 This is likely due to insulin se s involving Adiponectin regulation of triglyceride metabolism 15 A truncated form onectin gAdiponectin containing only the C terminal globular domain has been identified in lood and recombinant gAdiponectin has been shown to regulate weight reduction as well as free fat id oxidation in mouse muscle and liver 16 med collagen in myocardial promoting cross talk between 17 The full length recombinant Adiponectin in is apparently less potent at mediating these effects 16 17 The CircuLex Mouse Adipon immunoassay technique An anti microplate Standards and samples the immobilized antibody Af specific for Adiponectin is conjugate the remaining A Kit employs the quantitative sandwich enzyme cific for mouse Adiponectin has been pre coated onto a dh tted into the wells and any Adiponectin present is bound by away any unbound substances an HRP conjugated antibody e Eu Following a wash to remove any unbound antibody HRP is allowed to react with the substrate H5O tetramethylbenzidine The reaction is stopped b dom of acidic solution and absorbance of the resulting yellow product is measured at 450 nm bance is proportional to the concentration of Adiponectin A standard curve is constru tting absorbance values versus Adiponectin concentrations of calibrators and concentrations o mples are determined using this standard curve The CircuLex e Adiponectin ELISA
11. ed 1 Prepare a working solution of Wash Buffer by adding 100 mL of the 10X deionized distilled water ddH O Mix well Store at 4 C for two fer to 900 mL of 0 C for long term storage 2 Reconstitute Mouse Adiponectin Standard with 1 1 mL of ddH he Concentration of the mouse Adiponectin in vial should be 48 ng mL which is referred aster Standard of mouse Adiponectin Prepare Standard Solutions as follows Use the Master Standard to produce a dilution series b Mix each tube thoroughly before the next transfer The 12 ng mL standard S the highest standard The Dilution Buffer serves as the zero standard Blank Volume of Standard Dilution Buffer Concentration Std 1 150 uL of Master Standar 450 uL 12 00 ng mL Std 2 300 uL of Std 1 12 00 300 uL 6 00 ng mL Std 3 300 uL of Std 2 6 0 300 uL 3 00 ng mL Std 4 300 uL of Std 3 3 00 ng ml 300 uL 1 50 ng mL Std 5 300 uL of Std 1 300 uL 0 75 ng mL Std 6 300 uL of St ng ml 300 uL 0 38 ng mL Std 7 300 uL of Std ng ml 300 uL 0 19 ng mL Blank J 300 uL 0 ng mL Note Do not use a R before dispensing immediately pette Change tips for every dilution Wet tip with Dilution Buffer ed portions of Standards should be aliquoted and stored at below 70 C tiple freeze and thaw cycles tin S Sample Prepa n Serum an samples require a 2 500 fold dilution e g fold dilution 2 uL of sample 98 uL of Dilution Bu
12. ee standard deviations of the absorbance of blank A bla 3 SD blank is better than 0 173 ng ml of sample Dilution Buffer is pipetted into blank wells and the microtiter plate is blanked on Eighty assays were evaluated and the minimum detectable dose MDD of n ranged from 0 116 0 197 ng mL The mean MDD was 0 173 ng mL The MDD was de by adding three standard deviations to the mean optical density value of twenty zero stand eplieates and calculating the corresponding concentration ili 25 20 A450 0 5 0 0 i 0 2 4 6 8 10 12 14 mouse Adiponectin conc ng ml v C CY 8051 11 Version 120807 Mouse Adiponectin ELISA Kit x TM Circu Lex User s Manual A For Research Use Only Not for use in diagnostic procedures 2 Specificity The antibodies in the CircuLex Mouse Adiponectin ELISA Kit are highly specific of mouse and r Adiponectin with no detectable cross reactivity to other cytokines that may be present in mouse andmrat serum 3 Precision Intra assay Precision Precision within an assay Three samples of known concentration were tested twelve times on one plate to asses assay precision Intra assay Within Run n 12 mouse Adiponectin conc ug Inter assay Precision Precision between Three samples of known concentra precision e tested in six separate assays to assess inter assay Inter assay Run to Run n 6 mouse Adiponectin conc ug ml
13. ffer d Make 2 500 fold dilution 5 uL of 50 fold diluted sample 245 uL Dilution Buffer Cati CY 8051 8 Versions 120807 Mouse Adiponectin ELISA Kit x TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Assay Procedure Remove the appropriate number of microtiter wells from the foil pouch and place them into the holder Return any unused wells to the foil pouch refold seal with tape and store at 4 C 2 Dilute samples with Dilution Buffer See Sample Preparation above w Pipette 100 uL of Standard Solutions Std1 Std7 Blank and diluted samples in dup the appropriate wells 4 Incubate the plate at room temperature ca 25 C for 1 hour shaking at ca 3 on an orbital microplate shaker 5 Wash 4 times by filling each well with Wash Buffer 350 uL using a s le multi channel pipette manifold dispenser or microplate washer 6 Add 100 uL of HRP conjugated Detection Antibody into each well Incubate the plate at room temperature ca 25 C for 1 hour at ca 300 rpm on an orbital microplate shaker 8 Wash 4 times by filling each well with Wash Buffer 350 u pipette manifold dispenser or microplate washer sin squirt bottle multi channel 9 Add 100 uL of Substrate Reagent Avoid expos the plate with e g aluminum foil is recommende eturn Substrate Reagent to 4 C immediately after the necessary volume is removed 10 Inc
14. in diagnostic procedures Calculations Average the duplicate readings for each standard control and sample and subtract the average standard optical density Plot the optical density for the standards versus the concentration th standards and draw the best curve The data can be linearized by using log log paper an analysis may be applied to the log transformation To determine the Adiponectin concen sample first find the absorbance value on the y axis and extend a horizontal line to the s At the point of intersection extend a vertical line to the x axis and read the corresponding 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 uation The results of unknown samples can be calculated with any computer progr ing a 5 parameter logistic function It is important to make an appropriate mathematical adjust accommodate for the dilution factor tration The calibration sus log of the known Alternatively the logit log ance Y is plotted versus log B Most microtiter plate readers perform automatic calculations of analyt curve is constructed by plotting the absorbance Y of calib concentration X of calibrators using the four parameter f function can be used to linearize the calibration curve i e logit of the known concentration X of calibrators
15. outh pipette or ingest any of the reagents Do not smoke eat or drink when performing the assay o as where samples or reagents are handled Dispose of tetra methylbenzidine TMB containi ions in compliance with local regulations Avoid contact with the acidic Stop Solution and Subs Solution which contains hydrogen peroxide Wear gloves and eye protection when ha unodiagnostic materials and samples of mouse origin and these reagents In case of conta th the Stop Solution and the Substrate Solution wash skin thoroughly with water and seek m la ion when necessary Biological samples might be contami ith infectious agents Do not ingest expose to open wounds or breathe aerosols W ective gloves and dispose of biological samples properly CAUTION Sulfuric Acid i g acid Wear disposable gloves and eye protection when handling voile Q C CY 8051 6 Versions 120807 Mouse Adiponectin ELISA Kit x TM Circu Lex User s 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 efor assaying Aliquots of serum may also be stored at below 70 C for extended periods of ti id repeated freeze thaw cycles Plasma Collect plasma using EDTA Na as the anticoagulant If possible collect the to
16. pipettor Orbital microplate shaker Microcentrifuge and tubes for sample preparation Vortex mixer Qy e Microplate washer optional Manual washing is possible but not preferable e Plate reader capable of measuring absorbance in 96 well plates at dual wave s of 450 nm 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be used can also be read at a single wavelength of 450 nm which will give a somewhat higher readi Software package facilitating data generation and analysis opti 9 500 or 1000 mL graduated cylinder Reagent reservoirs Deionized water of the highest quality Disposable paper towels C CY 8051 5 Versions 120807 Mouse Adiponectin ELISA Kit x TM Circu Lex User s Manual A For Research Use Only Not 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 h 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 Qy Use deionized water of the highest quality Do not mix reagents from different kits The buffers and reagents used in this kit contain NaN as preservati hould be taken to avoid direct contact with these reagents Do not m
17. ubate the plate at room temperature ca orbital microplate shaker The incubation temperature is below than 20 C ay be extended up to 30 minutes if the reaction 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 well usi pectrophotometric microplate reader at dual wavelengths of 450 540 nm Dual wavelen 0 550 or 450 595 nm can also be used Read the microplate at 450 nm if only a single wa an be used Wells must be read within 30 minutes of adding the Stop Solution liquid at each step is essential to good performance After the last wash g Wash Buffer by aspirating or decanting Invert the plate and blot it towels Note 2 Reliabl ard curves are obtained when either O D values do not exceed 0 2 units for the oncentration or 2 5 units for the highest standard concentration The plate nitored at 5 minute intervals for approximately 30 minutes icroplate reader is not capable of reading absorbance greater than the absorbance of the andard perform a second reading at 405 nm A new standard curve constructed he values measured at 405 nm is used to determine Adiponectin concentration of ff scale samples The readings at 405 nm should not replace the on scale readings at 450 nm Note 1 Complete remoya CY 8051 Versions 120807 Mouse Adiponectin ELISA Kit x TM Circu Lex User s Manual For Research Use Only Not for use
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