CY-8052 Dog Adiponectin ELISA Kit
Contents
1. e 4 m e 0 9 l e 9 0 0 25 5 0 75 10 0 125 15 0 17 5 Age year C CY 8052 14 Version 150119 Dog 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 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 ORS QN UA Ee CX pa r3 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 C2. 150119 Dog 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 Dog Adiponectin ELISA Kit 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 Dog 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 Characteristics 1 Sensitivity Twenty one assays were evaluated and the minimum detectable dose MDD of dog adiponectin The MDD defined as such a concentration of dog adiponectin giving absorbance higher than mean absorbance of blank plus three standard deviations of the absorbance of blank A blank 3SD blank is better than 163 4 pg ml of sample Dilution Buffer is pipetted into blank wells Typical Standard Curve Dog adiponectin standard curve SO er ee DI ee Cute i et ecuc ee 2 5 2 0 A450 c1 0 5 0 0 0 0 2 5 5 0 7 5 10 0 12 5 Dog adiponectin conc ng ml Cat CY 8052 11 Version 150119 TM Dog Adiponectin ELISA Kit L 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures 2 Precision Int
3. of the assay Linearity U Di cc a titu Serum 1 A Serum 2 7 5 Serum 3 ouf ooo X EE AE 2 5 Dog adiponectin conc ng ml 0 0 0 0 5 1 1 5 2 2 5 Serum Dilution Ratio 1 10 000 Example of Test Results Fig 1 Concentrations of adiponectin in female d g sera n 72 and male dog sera n 83 Dog serum adiponectin 70 e 60 T M S 50 x e H o 40 s O 5 o e o 30 t 8 c 8 H 20 o l 10 8 0 Male Female Cat CY 8052 13 Version 150119 Dog Adiponectin ELISA Kit L ircuLex User s Manual AN For Research Use Only Not for use in diagnostic procedures Fig 2 Relationship between age and serum adiponectin concentration in female dog sera n 72 male dog sera n 83 Relationship between Age and Adiponectin conc lt Female gt bt e M e 5 50 r e s 9 nO VRPPEPRO UE acd o e z e a e o e EPDE E E E ENEN E 9 25 P 0o e e e 2 s e Yaso e e e a ee ar C e e e e o o 9e e e L e j L e e 0 0 0 2 5 5 0 7 5 10 0 12 5 15 0 17 5 Age year Aw Relationship between Age and Adiponectin conc lt Male gt p ME E 2 e E e D m 2 c 0S p RRE c et ee Ee e e o c e a e o B ompegeee ete x t Esos 5 e e e lt e e ew s o 9 e e e e a oe gt 9 Co e e e 9e9 e e ee e
4. TM Dog Adiponectin ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures un N ELISA Kit for Measuring Dog Adiponectin CircuLex Dog Adiponectin ELISA Kit N Cat CY 8052 O Intended E Se eren vett n ka pax EDS 1 SIO ta T e eee ove cr T 1 FIAT TEC TOM PADS NOIL UR ENDE 2 Principle of the Assay 3 Materials Provided terrens 4 Materials Required but not Provided 5 Precautions and Recommendations 6 Sample Collection and Storage 7 Detailed Protocol seiss seose 8 9 Calculations seirer 10 Measurement Rates sceau mim retro etn 10 TroubleshO ting oeste tds r iat tRacis 10 Reagent Stab y unco etre eines tbitu prid eR ihn 11 Assay Characteristics c csss ccsscessssssseccetssces 11 Example of Test Results 1 Referens Me M 1 Related Products eere 15 Intended Use The CycLex Research Product CircuLex iponectin ELISA Kit is used for the quantitative measurement of dog adiponectin in seru asma tissue culture medium and other biological media This assay kit is for research use only z for use in diagnostic or therapeutic procedures Storage Upon receipt store all co Don t expose reagents to C CY 8052 1 Version 150119 Dog Adiponectin ELISA Kit L ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Introduction Adiponectin als
5. 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 fo bag with a desiccant pack Wells are coated with anti adiponectin antibody as a capture an 2 10X Wash Buffer One bottle containing 100 mL of 10X buffer containing 2 Tween 20 Dilution Buffer One bottle containing 50 mL of 1X buffer use for sample dilution to use Dog Adiponectin Standard One vial containing 48 ng of lyophilized dog adip ti HRP conjugated Detection Antibody One bottle containing 12 mL of HRP horseradish peroxidase conjugated anti adiponectin antibody Ready to use Substrate Reagent One bottle containing 20 mL of the chromo tetra methylbenzidine geni TMB Ready to use Stop Solution One bottle containing 20 mL of 1 N H2S014 Ready to C CY 8052 4 Version 150119 Dog Adiponectin ELISA Kit CircuLex Uses 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 pipettor Orbital microplate shaker Microcentrifuge and tubes for sample preparation Plate reader capable of measuring absorbance in 96 well plates at dual wa s of 450 nm 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be used n also be read at a single wavelength of 450 nm w
6. bstrate 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 incubation Aim 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 wellUsing aspectrophotometric microplate reader at dual wavelengths of 450 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be used Read the microplate at 450 nm if only a single wayelength 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 remaining 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 zetoyconcentration or 2 5 units for the highest standard concentration The plate shouldbe monitored at 5 minute intervals for approximately 30 minutes Note 3 1f the miicfoplate 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 sing the values measured at 405 nm is used to determine A
7. diponectin concentration of off scale samples The readings at 405 nm should not replace the on scale readings at 450 nm Cat CY 8052 9 Versionft 150119 Dog 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 versus the concentration of the standards and draw the best curve The data can be linearized by using log log paper and regression analysis may be applied to the log transformation 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 point 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 1 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 programghaving a 5 parameter logistic function It is important to make an appropriate mathematical adjustinent to accommodate for the dilution factor 2 Most microtiter plate readers perform automatic calculations of analyte concentration The calibration curv
8. e Solution which contains hydrogen peroxide Avoid contact with Stop Solution which contains Sulfuric Acid Wear gloves and eye protection when handling immunodiagnostic materials and samples of dog origin and these reagents In case of contact with he 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 aerosols Wear protective gloves and dispose of biological samples properly CAUTION Sulfuric Acid i a Strong acid Wear disposable gloves and eye protection when handling Stop Solution Cat CY 8052 6 Version 150119 Dog Adiponectin ELISA Kit CircuLex Uses Mobil 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 tume repeated freeze thaw cycles Plasma Collect plasma using EDTA Naz as the anticoagulant If possible collect the T mixture of EDTA Naz and Futhan5 to stabilize the sample against spontaneous in vitro ple activation Immediately centrifuge samples at 4 C for 15 minutes at 1 000 x g Assay immediately or st
9. e 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 3 Dilution factors need to be taken into consideration in calculating tbe Adiponectin concentration Measurement Range The measurement range is 0 19 ng mL to 12 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 into consideration in calculating the dog adiponectin concentration Troubleshooting 1 The Dog Adiponectin Standard should bejrun in duplicate using the protocol described in the Detailed Protocol Incubation tim S 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 inthe Detailed Protocol were followed accurately such results indicate a need for washer maintenance 3 Overall low signal n y indicate 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 8052 10 Version
10. for two weeks or lt 20 C for long term storage 2 Reconstitute Dog Adiponectin Standard with 1 0 mL of ddHzOg The concentration of the dog Adiponectin in vial should be 48 ng mL which is referred as ae Master Standard of dog 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 12 ng mL standard Std serves as the highest standard The Dilution Buffer serves as the zero standard Blank Volume of Standard Dilution Buffer Concentration Std 1 150 uL of Master Standard 450 uL 12 00 ng mL Std 2 300 uL of Std 1 12 00 ng ml 300 uL 6 00 ng mL Std 3 300 uL of Std 2 6 00 ng ml 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 4 1 50 ng ml 300 uL 0 75 ng mL Std 6 300 uL of Std 0 75 ng ml 300 uL 0 38 ng mL Std 7 300 uL of Std 6 0 38 ng ml 300 uL 0 19 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 Unused portions of Master Standards should be aliquoted and stored at below 70 C immediately Avoid multiple freeze and thaw cycles Sample Preparation e Serum and plasma samples require a 5 000 fold dilution e g step Orte Dilute samples 1 100 with Dilution Buffer 2 uL of sample 198 uL of Dilution Buffer Step Ewo Dilute S
11. hich will give a somewhat higher readi Vortex mixer Qy e Microplate washer optional Manual washing is possible but not preferable f 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 8052 5 Version 150119 Dog Adiponectin ELISA Kit TM L 1rcu Lex User s Manual 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 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 Substrat
12. ircuLex 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 cyclex co jp URL http www cyclexieo jp CycLex CircuLexyproducts 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 witho t prior written approval from CycLex Co Ltd To inquire about licensing for such commercial use please contact us via email Cat CY 8052 15 Version 150119
13. o referred as Acrp30 AdipoQ and GBP 28 is a recently discovered 244 amino aeid 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 defense 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 re 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 s
14. ore 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 o Other biological samples Remove any particulates by centrifugation an ediately or aliquot and store samples at below 70 C Avoid repeated freeze thaw cycles C CY 8052 7 Version 150119 CircuLex Dog Adiponectin ELISA Kit User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol The CycLex Research Product CircuLex Dog 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 Dog Adiponectin Standard within the kit should be included in each assay as a calibrator Disposable 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 Dog Adiponectin Standard 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
15. ra assay Precision Precision within an assay Three samples of known concentration were tested fourteen times on one plate to assess intra assay precision Intra assay Within Run n 14 CV 3 1 5 1 96 Dog adiponectin conc ug ml Serum No Serum 1 Serum 2 Serum 3 1 13 9 20 0 36 7 2 14 0 18 9 35 3 3 14 0 18 7 34 0 4 14 5 17 9 33 1 5 14 3 18 4 34 1 6 14 1 20 0 35 4 7 14 2 20 2 38 8 13 4 20 3 37 3 9 13 3 19 2 36 2 10 13 4 19 0 34 3 11 13 4 17 8 33 3 12 13 7 17 5 34 2 13 14 3 18 0 35 6 14 14 5 19 6 38 3 Max 14 55 20 28 38 53 Min 13 29 17 46 33 07 Mean 13 93 18 97 35 44 S D 0 428 0 960 1 747 C V 3 1 Dl 4 9 Inter assay Precision Precision between assays Three samples of known concentration were tested in four separate assays to assess inter assay precision Inter assay Run to Run n24 V 26 0 7 1 Dog adiponectin conc ug ml Serum 1 Serum 2 Serum 3 1 14 0 19 4 35 3 2 14 4 21 3 40 0 3 14 0 19 9 36 1 4 12 2 18 4 39 5 Max 14 4 21 3 40 0 Min 12 2 18 4 35 3 Mean 13 7 19 7 37 7 S D 1 0 1 2 2 8 C V 7 1 6 0 6 2 Cat CY 8052 12 Version 150119 TM Dog Adiponectin ELISA Kit L 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures 3 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 the dynamic range
16. technique An antibody specific for adiponectin has been pre coated onto a microplate Standar samples are pipetted into the wells and the immobilized antibody binds any adiponectin present After washing away any unbound substances an HRP conjugated antibody specific for adiponec the wells Following a wash to remove any unbound antibody HRP conjugate the remainit allowed to react with the substrate H2O tetramethylbenzidine The reaction is stopped b acidic solution and absorbance of the resulting yellow product is measured at 450 nm The absorba proportional to the concentration of adiponectin A standard curve is constructed by plotting absorbance values versus adiponectin concentrations of calibrators and concentrations of u samples are determined using this standard curve The CircuLex Dog Adiponectin ELISA Kit is designed to measure G tion of dog adiponectin from dog serum plasma adipocytes or conditioned medium Summary of Procedure Q Add 100 uL of diluted sample to the wells 4 Incubate for 1 hour p Wash the wells Add 100 uL of HRP conju diponectin antibody 4 Incu hour at room temp Wash the wells t Add 100 uL of s ye t Add 100 uL of lution Measure a bance at 450 nm Q 2 C CY 8052 3 Version 150119 Dog Adiponectin ELISA Kit CircuLex Uses Mobil MM For Research Use Only Not for use in diagnostic procedures Materials Provided All samples and standards should be assayed in duplicate
17. tep one samples 1 50 with Dilution Buffer Cagk CY 8052 5 uL of Step One samples 245 uL of Dilution Buffer 8 Version 150119 Dog Adiponectin ELISA Kit TM L 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Assay Procedure 1 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 C 2 Dilute samples with Dilution Buffer See Sample Preparation above 3 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 5 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 Su
18. timulated glucose disposal 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 remodeling after ischemic injury and also stimulates angiogenesis by promoting cross talk between AMP activated protein kinase and Akt signaling in endothelial cells 4 Injection of adiponectin into non obese diabetic mice leads to an insulin independent decrease in glucose levels 15 This is likely due to insulin sen itizing effects involving adiponectin regulation of triglyceride metabolism 15 A truncated form of adiponectin gAdiponectin containing only the C terminal globular domain has been identified in the blood and recombinant gAdiponectin has been shown to regulate weight reduction as well as free fatty acid oxidation in mouse muscle and liver 16 17 The full length recombinant adiponectin protein is apparently less potent at mediating these effects 16 17 Cat CY 8052 2 Version 150119 Dog Adiponectin ELISA Kit CircuLex Uses Mobil 4 For Research Use Only Not for use in diagnostic procedures Principle of the Assay The CircuLex Dog Adiponectin ELISA Kit employs the quantitative sandwich enzyme immuno
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