Sample Data Sheet
Contents
1. 5 At what temperature should samples be stored both untreated and pre treated Samples should be stored at 70 C 6 How stable are the samples at 4 C and at room temperature RT Untreated serum and pre treated x5100 diluted serum can be stored at 4 C for up to 7 days Pre treated x100 diluted serum however cannot be stored refrigerated without a significant decrease in detectable adiponectin Figure 6 Figure 6 Measurement of Adiponectin After Storage at 4 C No 34 No 35 1 20 7 00 T 1 00 T809 6 A E 500 gt 0 80 e Serum ae aS gt 4 00 e Serum 0 60 AB 100 E ae E 100 D g D j 5 0 40 5100 5 ae 5100 a D Z 1 00 0 00 E 1 2 3 4 5 Days at 4C 1 2 3 4 5 Days at 4C Untreated serum and pre treated serum samples are stable for 8 hours at RT Pre treated x100 diluted serum samples cannot be stored at RT without a decrease in detectable Adiponectin Figure 7 Therefore samples should be diluted x5100 following pre treatment and heating Ideally all samples should be stored at 70 C Figure 7 Measurement of Adiponectin After Storage at RT Adiponectin ng mL Serum amp x 100 e 5100 0 1 2 3 4 5 6 7 8 9 Hours at RT Adiponectin ng mL 6 00 5 00 4 00 3 00 No 35 E Serum a x100 2 00 5100 1 002. Human Adiponectin ELISA Kit User Manual UM 100101 Published 01 October 2002 Catalog ADIP 1 and ADIP 2 Human Adiponectin ELISA Kit User Manual See List of Components for Storage Conditions FOR RESEARCH USE ONLY Table of Contents l Introduction and Protocol Overview 3 II List of Components 5 III Additional Materials Required 6 IV Reagent Preparation and Storage 7 V Adipocyte Treatment 8 9 VI Sample Pre Treatment VII Human Adiponectin ELISA Protocol 10 VIII Calculation of Results 12 IX Troubleshooting Guide and FAQs 13 X References 19 Notice to Purchaser This product is for research use only t is not intended for human veterinary or in vitro diagnostic use Proper precautions and biological containment should be taken when handling cells of human origin due to their potential biohazardous nature Always wear gloves and work behind a protective screen when handling primary human cells All media supplements and tissue culture ware used in this protocol should be sterile Human Adiponectin ELISA Kit User Manual l Introduction and Protocol Overview Obesity and obesity related disorders are reaching alarming proportions in the US and are on the increase in Europe and Asia A deeper understanding of the molecular and cellular dynamics of such disorders and their subsequent amelioration will have a far reaching impact on the quality of life of millions of people worldwide Adipocytes fat cells exp
3. the necessary volume is removed Next step is for treating samples from human cultured adipocytes only kit catalog ADIP 2 6 Sample Pre Treatment Solution Dilute sample pre treatment solution as follows Remove 2 ml sample pre treatment solution to clean conical tube Add 8 ml diH20 Invert to mix Prepare samples for pre treatment in this solution Note Do not mix reagents from different kits unless they have the same lot number Human Adiponectin ELISA Kit User Manual V Adipocyte Treatments applies only to ADIP 2 kit This kit contains one 96 well plate of cultured human subcutaneous adipocytes We recommend using no more than 84 wells for all treatments and controls in order to leave room on the ELISA plate for the standard curve duplicate wells We recommend the following procedure for treatment of the adipocytes On the day the cells arrive 1 Check the seal for each plate Please be aware that these cells are of human origin Please treat them as potentially infectious since we cannot test for all pathogens ALWAYS WEAR GLOVES AND USE PROTECTIVE MEASURES WHEN HANDLING HUMAN PRIMARY CELLS Place the package into a sterile environment THIS IS VERY IMPORTANT SINCE BREAKING THE VACUUM SEAL MAY POTENTIALLY INTRODUCE CONTAMINATION INTO THE PLATE Use scissors to snip open the bag at any end The vacuum seal should be released at this time You may notice some bubbling of the media in the plate at this time This is norm
4. 0 00 O 1 2 3 4 5 6 7 8 9 Hours at RT 15 Human Adiponectin ELISA Kit User Manual IX Troubleshooting Guide and FAQs continued 7 8 What if only heat the samples for 5 minutes in the pre treatment incubation step As shown in Figure 8 below samples were heated at 100 C in a heat block for 0 to 20 minutes then the ELISA was performed and the samples were tested For samples heated between 1 minute and 20 minutes there was no significant difference in the adiponectin concentrations recorded Figure 8 Effects of Pre Treatment Incubation Times Pre Treatment Incubation Time minutes Sample 0 1 3 5 7 10 20 No 34 0 083 0 985 0 918 0 901 0 931 0 899 0 976 No 35 0 270 5 192 5 443 5 104 5 202 4 405 4 744 Adiponectin ng mL What temperature range can use in the pre treatment incubation step When samples were heated for 5 minutes at 5 C intervals from 80 C to 100 C there was no significant difference in the adiponectin concentrations recorded Figure 9 Figure 9 Effects of Pre Treatment Incubation Temperature Pre Treaimeni Incubation Temp C Sample 80 85 90 95 100 Adiponectin ng mL How reproducible are the results Several experiments were performed to determine the reproducibility of data obtained using this kit In one experiment 8 control high and control low samples were assayed i e 16 samples total on one plate
5. 96 well plate that fits into a 96 well plate heat block and can withstand temperatures up to 100 C Human Adiponectin ELISA Kit User Manual IV Reagent Preparation and Storage 1 1X Wash Solution Prepare 1X Wash Solution by mixing all of the 25X Wash Solution 40mL with 960 mL of deionized water or equivalent If crystals are observed in the 25X Wash Solution bottle warm the bottle in a 37 C water bath until the crystals disappear After preparation store 1X Wash Solution at 2 8 C 2 1X Sample Diluent Prepare 1X Sample Diluent by mixing all of the 5X Sample Diluent 50mL with 200mL of deionized water or equivalent After preparation store 1X Sample Diluent at 2 8 C 3 Adiponectin Standard Solution Prepare each Adiponectin Standard 6 0 ng mL 3 0 ng mL 1 5 ng mL 0 75 ng mL 0 375 ng mL by serially diluting the supplied adiponectin standard solution 12 0 ng mL with 1X Sample Diluent Use undiluted adiponectin 12 0 ng mL and 1X Sample Diluent for 12 0 ng mL and 0 ng mL standard solutions respectively 4 Detection Antibody Solution Prepare the Detection Antibody Solution by adding one part Detection Antibody Concentrate to 200 parts Detection Antibody Diluent Prepare only as much Detection Antibody Solution as needed 5 Substrate Solution Prepare the Substrate Solution by adding one part Substrate A to one part Substrate B Prepare only as much Substrate Solution as needed Return Substrate A to 2 8 C immediately after
6. extracts or conditioned medium to obtain the correct result for undiluted samples Figure 3 Typical Standard Curve 5 D a 1 O 2 5 Z 0 1 0 1 1 10 20 Concentration of Adiponectin 12 Human Adiponectin ELISA Kit User Manual IX Troubleshooting Guide and FAQs Troubleshooting Guide 1 Lack of signal or weak signal in all wells Possible explanations e Omission of a reagent or a step e Improper preparation or storage of a reagent e Assay performed before reagents were allowed to come to 20 30 C e Plate reader did not perform well 2 High signal and background in all wells Possible explanations e Improper or inadequate washing be certain that all wash volumes and repetitions were correct e Improper dilution of detection antibody e Overdeveloping decrease the incubation time before the Stop Solution is added 3 High background in sample wells only Possible explanations e Sample concentration was too high e Improper dilution of detection antibody 4 Weak signal in sample wells only Possible explanations e Sample concentration was too low e Improper dilution of detection antibody FAQs Frequently Asked Questions 1 What is the shelf life of this kit ADIP 1 Currently all components of this kit have a shelf life of 9 months when stored at 2 8 C However it is fully anticipated that this will be extended in the future The expiration date appears on the top label of the product
7. measured on a plate reader simultaneously data shown in Figure 10 first table In the second table are the results of measuring single control high and low samples from the same ELISA 6 times consecutively i e one sample of each measured on a plate reader 6 times in a row The third table shows the results of eight assays control high and low run by four different people 16 Human Adiponectin ELISA Kit User Manual IX Troubleshooting Guide and FAQs continued 10 11 Figure 10 Reproducibility 8 Samples From Same Plate Measured Simultaneously test 1 test 2 test 3 test 4 test 5 test 6 test 7 test 8 mean SD 2 Samples Measured 6 Times Consecutively test 1 test 2 test 3 test 4 test 5 test 6 mean _SD_ CV 8 Assays by 4 Different People test 1 test 2 test 3 test 4 mean SD_ Cv Adiponectin ng mL What is the range of adiponectin that can be detected by this kit We have established a minimum detectable limit as 23 4 pg mL of adiponectin unpublished data The ELISA is linear within the range of 0 375 ng mL to 12 0 ng mL What will the effect be if dilute my samples beyond what is recommended Three serum samples were pre treated as described in the protocol resulting in a final dilution of x5100 labeled in the table below as dilution 1 The samples were then further diluted x2 and x4 The data are linear Figure 11 Figure 11 Effects
8. of Dilution 6 wo A oa Adiponectin ng mL M Adiponectin ng mL ki 2309 4 985 0 943 1 A a aii 0 552 1 284 0 239 4 1 4 1 2 1 Dilution 17 12 Human Adiponectin ELISA Kit User Manual IX Troubleshooting Guide and FAQs continued Will the mouse anti human adiponectin monoclonal Ab detect adiponectin from other species Two experiments were conducted to examine the cross reactivity of the anti human monoclonal Ab In the first recombinant mouse adiponectin samples from a concentration of 0 313 to 320 ng mL were assayed see left table in Figure 12 below In the second experiment sera from different animals were pre treated according to protocol and assayed right table below The results from the adiponectin standards run simultaneously are shown in the bottom table of Figure 12 There was no cross reactivity observed Figure 12 Cross Reactivity Recombinant Mouse Adiponectin Pre Treated Sera From Different Species Antigen ng mL 320 0083 1 0 023 0 000 Mouse 0 025 0 027 0 000 a 022 g 028 0 022 0 000 9 0374 88 0 003 ice 021 one 037 mera pal 0 022 0 000 Goat ES use 0 035 0 005 0 022 022 0 0 037 er ues 0 022 0 000 Sheep pened pet 0 039 0 009 0 0 021 0 004 ers eee 0 022 0 000 Porcine oer Cal 0 024 0 000 0 0 021 0 0 027 E pes 0 022 0 000 Calf Soe pee 0 032 0 002 0 020 020 0 036 036 0 023 0 025 0 022 0 000 0 029 0 000 0 021 a ooze ooo oos He 0 0
9. 33 feces 0000 E A ues 0 022 0 000 Blank EA une 0 030 0 0 021 0 033 033 E eee 0 023 0 000 NUN 023 0 625 0 020 020 0 021 0 000 Recombinant Human Adiponectin 0 022 Antigen ng mL 0 313 EN del 0 021 0 000 12 000 e202 2 547 2 524 0 021 021 2 532 EE eet 0 023 6 000 ik A 1 746 1 723 0 021 021 1 1729 3 000 i ETA 1 105 1 082 T4140 1 500 EA 647 0657 0 634 0667 0 750 30 o 365 0 342 0 360 0 375 0 206 0 183 0199 E 024 0 023 0021 Human Adiponectin ELISA Kit User Manual X References 1 Nemet D et al 2002 Relationships among adiponectin and other adipose cytokines body composition and fasting insulin in lower socioeconomic middle school children American Physiological Society s APS Abstracts 2 Yamauchi T et al 2001 The fat derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity Nature Medicine Aug 7 8 941 6 3 Kubota N et al 2002 Disruption of adiponectin causes insulin resistance and neointimal formation J Biol Chem 277 29 25863 6 Epub 2002 May 24 4 Berg A H et al 2001 The adipocyte secreted protein Acrp30 enhances hepatic insulin action Nature Medicine Aug 7 8 947 53 UK amp Rest of World Switzerland North America AMIS Biotechnology Europe Ltd AMSS Biotechnology Europe Ltd amsbio LLC 184 Milton Park Abingdon Centro Nord Sud 2E 1035 Cambridge Street OX14
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11. ARY ANTIBODY COATED PLATE 1 Plate One plate holds 12x8 well strips 96 wells with adsorbed mouse anti human adiponectin monoclonal antibody Plate is provided in a resealable foil pouch with desiccant 5 ADIPONECTIN STANDARD 1 Vial 2mL Recombinant human adiponectin 12 0 ng mL 6 SECONDARY ANTIBODY SOLUTION Rabbit anti human adiponectin polyclonal antibody Bottle 12mL 7 DETECTION ANTIBODY CONCENTRATE Horseradish peroxidase HRP conjugated goat anti rabbit IgG Vial 0 1mL 8 DETECTION ANTIBODY DILUENT Bottle 15mL 9 SUBSTRATE A Bottle 7 5mL 10 SUBSTRATE B Bottle 7 5mL 11 STOP SOLUTION Bottle 15mL Components 12 15 for ADIP 2 kit only 12 ZEN BIO PPARy AGONIST 1 vial 500ul 13 BASAL MEDIUM BM 1 1 Bottle 830mL 14 ADIPOCYTE MEDIUM AM 1 1 Bottle 50ml 15 HUMAN SUBCUTANEOUS ADIPOCYTES 96 well plate PLATE SEALERS 1 Package Six sealers per package Human Adiponectin ELISA Kit User Manual Additional Materials Required The following materials are required but not supplied Graduated cylinder Micropipettor s and disposable pipette tips Null strips for 96 well plate 96 well plate or manual strip washer Paper towels or absorbent paper Plate reader capable of measuring absorbance at a wavelength of 450nm reference filter at 650 nm optional Heat block or equivalent Well closed containers such as microtubes 1 5 mL or more in capacity or
12. Dispense 100 uL of Stop Solution into each well 16 Read the plate at 450 nm using a plate reader If using a dual filter instrument the recommended reference wavelength is 650nm Figure 2 Flow Chart of Assay Procedure Mouse Anti Human Adiponectin Monoclonal Ab Coated Plate Sample Pre Treatment Wash 350 pL x 1 Standards and Pre Treated Samples 100 uL 1st Reaction 20 30 C 60min Wash 350 uL x 3 Secondary Rabbit Anti Adiponectin Ab 100 uL 2nd Reaction 20 30 C 60min Prepare Detection Ab Solution Wash 350 uL x 3 Detection Ab Solution 100 uL Prepare Substrate Solution Substrate Solution 100 uL Color Development 20 30 C 15min 3rd Reaction 20 30 C 60min Wash 350 uL x 3 Stop Solution Read Absorbance at 450nm Reference at 650nm 11 Human Adiponectin ELISA Kit User Manual VIII Calculation of Results Subtract the mean absorbance value of the 0 ng mL blank from each mean absorbance value of the standard series and samples tested Net Absorbance Plot the concentrations of each standard and the calculated Net Absorbances on the X axis and Y axis respectively Fit an appropriate regression curve on the plots Determine the adiponectin concentrations of the samples by interpolation of the regression curve formula The adiponectin concentrations calculated must be multiplied by the appropriate dilution factor x5100 for serum or plasma samples and x25 for adipocyte cellular
13. VI gt Human Adiponectin ELISA Kit User Manual VI Sample Pre Treatment Human Serum or Plasma Samples 1 Allow all the reagents needed for sample pre treatment including serum or plasma EDTA or heparin containing to come to room temperature 20 30 C prior to the start of the sample pre treatment Mix 10 uL of serum or plasma samples with 90 uL of Sample Pre treatment Solution and heat the mixture for 5 minutes at 100 C using a heat block To heat the samples use well closed containers 1 5 mL or more in capacity such as microtubes NOTE Make sure to eliminate the precipitate in the Sample Pre treatment Solution by warming to 37 C Add 900 uL of 1X Sample Diluent to each container after heating 1 100 diluted samples at final volume Transfer 20 uL of each diluted sample to a clean container and then add 1 0 mL of 1X Sample Diluent to the container 1 5100 dilution at final volume Repeat for each sample Human Adipocyte Cellular Extracts or Conditioned Media From Human Adipocytes ADIP 2 kit 1 Allow all the reagents needed for sample pre treatment including cellular extracts or conditioned media to come to room temperature 20 30 C prior to the start of the sample pre treatment Mix 20 uL of the adipocyte cellular extract or conditioned media with 80 uL of Diluted Sample Pre treatment Solution see step 6 in Reagent Preparation and Storage on page 7 and heat the mixture for 5 minutes at 100 C using a he
14. adiponectin polyclonal antibody will bind to the adiponectin trapped in the well in the 12 Reaction After washing a conjugate of horseradish peroxidase and goat anti rabbit IgG is added to each well and allowed to incubate 3 Reaction The detection antibody will recognize and bind to the rabbit anti adiponectin antibody trapped in the well in the 2 Reaction After washing the colorimetric substrate for the enzyme is added to all wells and incubated The color development is terminated by the addition of a stop solution The intensity of the 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 Human Adiponectin ELISA Kit User Manual Figure 1 Assay Principle Rabbit HRP conjugated Adiponectin A as goat anti rabbit IgG Substrate A OOO En A AA MAA 000 AAA 1st Reaction 2nd Reaction 3rd Reaction Color Development gt e OO 6 WA O KH tt iit ma ki Anti Adiponectin monoclonal antibody coated plate o gt o gt H O oe Human Adiponectin ELISA Kit User Manual ll List of Components Store components 1 13 at 2 8 C DO NOT FREEZE Store component 14 in a humidified CO incubator at 37 C 5 COs 1 25X WASH SOLUTION wk Bottle 40mL 2 SAMPLE PRE TREATMENT SOLUTION ys Bottle 10mL _h 3 5X SAMPLE DILUENT Bottle 50mL 4 PRIM
15. al and will not affect cell performance In a sterile environment remove the plate from the bag taking care to not disturb the cover top from the plate Open the lid and remove the white liner using sterile forceps or a hemostat and discard Carefully remove the clear adhesive seal by grabbing the edge with sterile forceps or hemostat and lifting the film slowly towards the other end Discard adhesive film in appropriate biohazard waste container Replace lid on plate Warm the BM 1 included with the cells to 37 C in a water bath When the medium is warm remove 250 ul of the medium from all wells of the plate and replace with 150 ul BM 1 Remove 150 ul of the medium and replace with a fresh 150 ul aliquot of BM 1 This process removes all serum and hormone from the cells Place cells in a 37 C humidified incubator at 5 COs Allow cells to rest for 3 days On the day of the assay 1 Prepare all treatments in AM 1 500 uL PPARy agonist in AM 1 has been provided with this kit and is ready for use 2 Remove medium from treatment wells and replace with appropriate treatment medium AMSBIO recommends treating in triplicate wells 4 No more than 89 of the wells can be used for treatment to allow room on the ELISA plate for the standard curve 5 After plate is treated place cells in a 37 C humidified incubator at 5 CO Suggested treatment time is 72 hours 6 Atthe end of the treatment period prepare the samples as described in section
16. at block To heat the samples use well closed containers 1 5 mL or more in capacity such as microtubes Alternatively samples can be prepared in a 96 well plate suitable for PCR work and heated in a PCR machine or other multi well plate warmers Add 400 uL of 1X Sample Diluent to each container after heating 1 25 diluted samples at final volume Alternatively transfer 50 uL of pretreated solution from step 2 to a new 96 well plate and add 200 uL 1X Sample Diluent to each well Pipet up and down gently to mix Human Adiponectin ELISA Kit User Manual Vil Human Adiponectin ELISA Protocol 1 Allow all reagents to come to room temperature 20 30 C prior to the start of the assay Prepare 1X Wash Solution 1X Sample Diluent and Adiponectin Standards according to Reagent Preparation Steps 1 2 and 3 2 Remove Primary Antibody Coated Plate from its foil pouch Remove any unneeded strips from the plate frame reseal them in the foil pouch and return the foil pouch to 2 8 C If a 96 well plate washer is used the plate frame should be completely filled with wells by adding as many null strips as necessary Identify well position s for each sample on a data sheet or plate map 3 Fill the wells with 1X Wash Solution 350 uL and immediately aspirate using a plate washer If wells are washed manually invert the plate s and gently tap on a clean absorbent towel 4 Add 100 uL of adiponectin standard or pre treated sample to the appropriat
17. e number of antibody coated wells Every plate must include the standard series to properly correlate the sample readings 5 Cover plate s securely with a plate sealer and incubate at 20 30 C for 60 minutes 6 Wash the plate s as follows a At the end of the incubation carefully remove the plate sealer avoiding splashing and discard appropriately b Completely aspirate the liquid from the wells using a plate washer c Fill each well with 1X Wash Solution 350 uL well and immediately aspirate Avoid Wash Solution overflow d Repeat Step 6c two more times for a total of three washes e Invert the plate s and gently tap on a clean absorbent towel 7 Dispense 100 uL of the Secondary Antibody Solution into each well 8 Cover plate s securely with a new clean plate sealer and incubate at 20 30 C for 60 minutes 9 Repeat the wash procedure described in step 6 Prepare Detection Antibody Solution according to Reagent Preparation Step 4 10 Dispense 100 uL of Detection Antibody Solution into each well 11 Cover plate s securely with a plate sealer and incubate at 20 30 C for 60 minutes Prepare Substrate Solution according to Reagent Preparation Step 5 12 Repeat the wash procedure described in step 6 13 Dispense 100 uL of Substrate Solution into each well 14 Incubate the plates at 20 30 C for 15 minutes 10 Human Adiponectin ELISA Kit User Manual VII Human Adiponectin ELISA Protocol continued 15
18. package ADIP 2 Must be used within 1 week of arrival 2 Can pool reagents Yes as long as the reagents are from the same lot 3 What is the effect of freezing thawing the standard and samples No significant effect was observed when adiponectin standards untreated samples pre treated samples and diluted samples were frozen and thawed five times Figure 4 13 Human Adiponectin ELISA Kit User Manual IX Troubleshooting Guide and FAQs continued Figure 4 Effects of Freeze Thaw Untreated Adiponectin ng mL Std ng mL NF F Tx3 F Tx5 NF F T x3 F T x5 2 Diluted x5100 Diluted Adiponectin ng mL x100 F T x3 F T x5 NF Not Frozen F T Freeze Thaw Does the method of separation of serum plasma affect the measurement of adiponectin There is no significant difference in measurements of adiponectin from separated serum samples However neither whole blood nor whole blood treated with citrate can be used Samples from three healthy individuals were taken and serum plasma were treated by several different methods results shown below Figure 5 Figure 5 Effects of Separation Method Serum Plasma Coagulated Coagulated Sep n Gel Heparin Citrate EDTA Na 2 3 234 3 422 3 136 2 766 3 304 1 941 2 157 2 036 1 539 1 744 1 275 1 289 1 206 0 990 1 127 Adiponectin ng mL 14 Human Adiponectin ELISA Kit User Manual IX Troubleshooting Guide and FAQs continued
19. ress a variety of proteins that function in the homeostatic control of glucose and lipid metabolism Insulin regulates the translocation and secretion of many of these proteins in response to changes in energy balance Adipocyte complement related protein of 30 kDa Acrp30 now known as adiponectin is a protein whose secretion from adipocytes is enhanced by insulin stimulation It has been suggested that the development of non insulin dependent Type diabetes may involve dysregulation of adiponectin secretion 1 In support of the link between obesity and Type diabetes it has been shown that decreased expression of adiponectin correlates with insulin resistance 2 3 and that adiponectin appears to be a potent insulin enhancer linking adipose tissue and whole body glucose metabolism 4 This Human Adiponectin ELISA Kit is designed to measure the concentration of human adiponectin from human serum plasma human adipocytes or conditioned medium The principle of the assay is shown in Figure 1 Pre treated samples and serially diluted standard recombinant human adiponectin solutions are added to an appropriate number of wells of the microtiter plate and incubated Adiponectin in the sample will be bound by the primary anti adiponectin monoclonal antibody immobilized in the well 1 Reaction After washing the secondary rabbit anti adiponectin antibody is added to each well and allowed to incubate 2 Reaction The secondary rabbit anti
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