HMW and Total Adiponectin ELISA
Contents
1. papp 140 kDa gt Pa HMW ma MMW Hexamer lal Albumin binding LMW Trimer LMW Trimer da B C 11 Cross reactivity This ELISA does not cross react with mouse rat rabbit goat sheep pig horse or bovine samples No signal was detected when cross reactivity was tested for human Resistin Leptin TNF a and IL 6 at concentrations up to 100 ng ml 12 14 12 Reduction of Multimeric to Dimeric Adiponectin with Sample Pretreatment Buffer Boiling Human serum and plasma were incubated with Sample Pretreatment Buffer separated by SDS PAGE and analyzed by Western blot using the capture ab A or the detection antibody B The pretreatment conditions were as follows 1 50 mM Tris HCl buffer pH 6 8 containing 2 SDS without boiling 2 50 mM Tris HCl buffer pH 6 8 containing 2 SDS with boiling 3 Sample Pretreatment Buffer from the kit without boiling Serum Plasma Serum Plasma 1 2 3 C1 GB 1 2 3 Ad 2 3 A B 200 kDa 116 kDa gt 66 kDa ad _ a ee Dimer 42 kDa gt 30 kDa gt 13 14 Assay Procedure Short Form Pretreatment of samples Serum or plasma 10 ul Pretreatment for Pretreatment for Pretreatment for Total adiponectin assay combined MMW HMW adiponectin assay HMW adiponectin assay Protease Buffer Protease Protease Il 100 ul 100 ul 100 ul Protease Digestion gt Incubate 20 min at 37 C Sample Pretreatment Buffer 42. AAL HMW and Total Adiponectin ELISA For the quantitative determination of adiponectin in human serum and plasma EDTA citrate and heparin For Research Use Only Not for Use in Diagnostic Procedures Catalog Number 47 ADPHU E01 Size 96 wells Version 3 ALPCO October 14 2013 26 G Keewaydin Drive Salem NH 03079 P 800 592 5726 F 603 898 6854 ts alpco com www alpco com INTENDED USE The HMW amp Total Adiponectin ELISA is designed for the quantitative determination of High Molecular Weight HMW Mid Molecular Weight MMW Low Molecular Weight LMW and Total adiponectin in human serum and plasma EDTA citrate and heparin Upon request a supplemental protocol can be provided for use with cerebrospinal fluid Please note that all methodology described in this package insert for the HMW amp Total Adiponectin ELISA is protected by U S Patent No 7 608 405 B2 issued Oct 27 2009 INTRODUCTION Adiponectin is a 244 amino acid protein and one of several known adipocytokines secreted by the adipocyte Adiponectin circulates in the blood in various oligomeric complexes ranging from dimeric to bouquet structures of 9 or more proteins which include HMW adiponectin 12 mer or 18 mer Functions of adiponectin include protection against atherosclerosis improvement of insulin sensitivity and prevention of hepatic fibrosis In recent years the relationship between these physiological actions and the multimeri
3. Concentration of the sample from Pretreatment option 1 Total adiponectin minus the concentration of the sample from Pretreatment option 2 MMW HMW adiponectin PROCEDURAL NOTES 1 This kit has been validated using human serum and plasma EDTA citrate and heparin 2 Measurements of different adiponectin multimers in the same serum or plasma samples MUST be conducted on the same plate DO NOT calculate results between different plates 3 Acalibration curve must be run with each assay 4 Standards and samples should be assayed in duplicate 5 If the concentration of adiponectin in a sample exceeds the highest point of the calibration curve dilute the pretreated sample with Dilution Buffer and assay again 6 Observe all specified reaction times and temperatures outlined in this manual These parameters are especially important when pretreating samples with proteases 7 If the plate is not used entirely during the first run the remaining reagents may be stored as directed in the protocol and used once more before the expiration date 8 Samples must be promptly added to the MoAb Coated Plate following pretreatment and subsequent dilution 9 Remove all residual liquid completely after each step of the wash procedure 10 Do not allow the wells to dry out or be damaged during the wash procedure 11 Avoid carrying out this procedure in direct sunlight WARNINGS AND PRECAUTIONS 1 The human serum contained in the Calibrator was t
4. Protease II is stable for 2 days at 2 10 C Reconstituted Protease II is stable below 20 C for the life of the kit Low High and Serum Controls Reconstitute each Control with deionized water according to the Certificate of Analysis Replace the rubber stopper and cap Gently swirl the vial and let stand for 10 minutes prior to use The contents of the vial should be in solution with no visible particulate matter Store Controls in aliquots at 20 C Reconstituted Low and High Controls are ready for use Reconstituted Serum Control should be pretreated and diluted before use in the same manner as the serum samples Refer to the Control Set Certificate of Analysis for lot specific reference ranges Sample Collection Serum or plasma samples can be used with this assay Because the measurement of adiponectin multimers requires samples be pretreated with a protease it is recommended that serine protease inhibitors such as aprotinin not be used when collecting samples ASSAY PROCEDURE It is critical that all reagents be allowed to reach room temperature prior to use 1 Pretreatment of samples The Adiponectin Multimeric ELISA allows for the independent quantification of Total and HMW adiponectin respectively and the indirect measurement of MMW and LMW adiponectin Separate aliquots of sample and Serum Control must be used for each option For example if HMW and Total adiponectin concentrations are being quantified for the same sample
5. then digested to dimers using Sample Pretreatment Buffer which also stops the protease digestion Subsequent measurement in the ELISA quantifies the amount of reduced MMW HMW adiponectin but does not detect the digested LMW 4 To measure MMW adiponectin MMW hexamer concentration is calculated by subtracting the concentration of HMW from the concentration of combined MMW HMW adiponectin 5 To measure LMW_adiponectin LMW trimers albumin bound trimers concentration is calculated by subtracting the concentration of combined MMW HMW from Total adiponectin 2 14 This ELISA utilizes an antibody sandwich for detection of adiponectin The microplate wells have been coated with an antihuman adiponectin monoclonal antibody and adiponectin in the standards and pretreated samples is captured during the first incubation Washing removes all unbound material and a biotinconjugated anti human adiponectin monoclonal antibody binds to the immobilized adiponectin in the wells After the second incubation and subsequent wash step HRP Labeled streptavidin is added Following a third incubation and subsequent wash step O phenylenediamine OPD is added as substrate and the colorimetric reaction is terminated with the addition of diluted H2S04 The intensity of the color development i e absorbance value is proportional to the adiponectin concentration in the sample Please note that all methodology described in this package insert for the A
6. two aliquots of the sample are needed and require independent pretreatments The pretreatment options are listed below Pretreatment option 1 For Total adiponectin assay Add 100 ul of Protease Buffer and 400 pl of Sample Pretreatment Buffer to 10 pl of sample sample dilution 1 51 Stir thoroughly or vortex Pretreatment option 2 For combined MMW HMW adiponectin assay Add 100 pl of Protease I to 10 ul of sample and incubate for 20 min at 37 C Immediately add 400 ul of Sample Pretreatment Buffer sample dilution 1 51 Stir thoroughly or vortex e Pretreatment option 3 For HMW adiponectin assay Add 100 ul of the Protease II to 10 pl of sample and incubate for 20 min at 37 C Immediately add 400 pl of the Sample Pretreatment Buffer sample dilution 1 51 Stir thoroughly or vortex Pretreated samples are stable at 4 or 25 C for up to 2 days Precipitate may form but will dissolve when brought to room temperature stir thoroughly or vortex 5 14 2 Dilution of pretreated samples Dilute pretreated samples 1 101 as follows Add 10 ul of the pretreated sample from Pretreatment Options 1 2 and or 3 to 1 0 ml of Dilution Buffer Since precipitation may occur when a pretreated samples are added to Dilution Buffer stir thoroughly or vortex FINAL Sample dilution 1 5 151 Samples must be used within 2 hours of dilution at room temperature 3 Assay Method 1 Plan your plate configuration Determine h
7. 00 ul Dilute pretreated samples 1 101 with Dilution Buffer Proceed to Assay Method within 2 hours of dilution Assay Method Standards and diluted pretreated samples 50 ul Monoclonal Ab Coated Plate Incubate 1 hour at RT Wash 3 times 350 400 ul per well Biotin Labeled Monoclonal Ab 50 ul Incubate 1 hour at RT Wash 3 times 350 400 ul per well Enzyme Labeled Streptavidin 50 ul Incubate 30 min at RT Wash 3 times 350 400 ul per well Substrate Solution 50 ul Incubate 10 min at RT Stop Reagent 50 ul Measure absorbance at 492 nm within 30 min after adding Stop Reagent 14 14
8. W MMW LMW Adiponectin Adiponectin Adiponectin Adiponectin Adiponectin Mean ug ml 3 98 2 16 1 08 1 08 1 82 SD ug ml 0 21 0 11 0 05 0 11 0 13 CV 5 4 5 0 5 0 10 2 7 3 Serum Direct Calculated Sample 2 Total MMW HMW HMW MMW LMW Adiponectin Adiponectin Adiponectin Adiponectin Adiponectin Mean ug ml 9 22 6 60 4 53 2 08 2 62 SD ug ml 0 48 0 27 0 15 0 29 0 34 CV 5 3 4 1 3 3 13 7 13 0 Inter assay n 8 Serum Total MMW HMW HMW Sample 3 Adiponectin Adiponectin Adiponectin Mean ug ml 7 66 5 35 3 45 SD ug ml 0 38 0 32 0 20 CV 5 0 6 0 5 7 8 14 4 Dilution Linearity Pretreated human serum and plasma samples diluted per the Assay Procedure 1 5 151 were serially diluted with Dilution Buffer and assayed for Total I diponectin 3 0 Human Serum 1 g 2 514 O Human Serum 2 BB A Human Plasma 1 c 2 0 Human Plasma 2 Cc 15t o 5 o 1 0 To lt 0 5 0 0 1 8 1 4 1 2 1 5 Spike and Recovery Varying amounts of purified human HMW adiponectin were added to a human serum samples and assayed for Total HMW and MMW tdiponectin HMW RAPySGiy Observed Expected Recovery O E Added ug ml ug ml ug ml Total Adiponectin 0 00 7 33 7 31 100 1 14 8 51 8 46 101 2 29 9 88 9 60 103 4 57 11 8 11 9 99 9 14 16 9 16 5 103 MMW HMW 0 00 5 69 5 69 100 Adiponectin 1 14 7 02 6 83 103 Protea
9. c structure of circulating adiponectin has attracted wide attention and data has shown that the ratio of circulating HMW adiponectin to total adiponectin is more informative than total adiponectin levels alone PRINCIPLE OF THE ASSAY In the HMW amp Total Adiponectin ELISA both Total and HMW adiponectin can be measured independently on the same plate Samples are pretreated with or without proteases diluted and then assayed for the different multimers of adiponectin as described below The principle of the sample pretreatment procedure is outlined briefly here and explained in greater detail in the ASSAY PROCEDURE 1 To measure Total adiponectin Samples are pretreated with Sample Pretreatment Buffer Citrate buffer SDS which reduces multimeric adiponectin to dimers Subsequent measurement in the ELISA quantifies the amount of all multimers of adiponectin in the sample 2 To measure HMW adiponectin Samples are pretreated with Protease II which selectively digests LMW and MMW The remaining HMW fraction is treated with Sample Pretreatment Buffer which reduces these multimers to dimers while also stopping the protease digestion Subsequent measurement in the ELISA quantifies the amount of reduced HMW adiponectin but does not detect the digested LWW and MMW adiponectin 3 To measure combined MMW HMW adiponectin Samples are pretreated with Protease which selectively digests only LMW adiponectin The remaining MMW and HMW are
10. diponectin Multimeric ELISA is protected by U S Patent No 7 608 405 B2 issued Oct 27 2009 KIT COMPONENTS Reagent Composition Quantity Preparation Wash Buffer Concentrate Phosphate buffer pH 7 2 100 ml x 1 vial 10X Sample Pretreatment Buffer Citrate buffer pH 3 0 containing SDS 50 ml x 1 vial Ready to use Dilution Buffer Phosphate buffer pH 7 2 containing BSA 100 ml x 1 vial Ready to use Monoclonal Ab Coated Plate Anti human adiponectin mouse monoclonal Ab coated plate 1 plate 96 wells Ready to use Calibrator stock solution Human serum stabilized in sample pretreatment buffer 0 25 ml x 1 vial Concentration indicated on vial Biotin conjugated anti human Biotin Labeled Monoclonal Ab i A i 6 ml x 1 vial Ready to use adiponectin monoclonal antibody Enzyme Labeled Streptavidin Nar eraun peroxidase ARE 6 ml x 1 vial Ready to use labeled streptavidin Substrate O phenylenediamine OPD 2 vials Lyophilized Substrate Buffer Citrate buffer pH 5 0 containing H202 15 ml x 1 vial Ready to use Stop Reagent 7 7 H2SO04 15 ml x 1 vial Ready to use Protease Protease 1 vial Lyophilized Protease II Protease 1 vial Lyophilized Protease Buffer Tris buffer pH 8 0 50 ml x 1 vial Ready to use Phosphate buffer containing High and Low Controls pr cessed h man serm k fa Lyophil
11. e Precipitation may occur when the Calibrator is added to Dilution Buffer therefore stir thoroughly EE of Fra concentration i 10 ul of Calibrator 1 000 ul fe stock 2 150 ul of Standard 1 150 ul 2 4 3 150 ul of Standard 2 150 ul 1 2 4 150 ul of Standard 3 150 ul 0 6 5 150 ul of Standard 4 150 ul 0 3 6 150 ul of Standard 5 150 ul 0 15 7 150 ul of Standard 6 150 ul 0 075 8 0 150 ul 0 Approximate concentrations are listed actual concentration of the Calibrator stock is indicated on the vial label Unused Calibrator should be stored at 2 10 C until subsequent use The Standards should be prepared just prior to loading the plate e Diluted Standards are not stable and should be discarded 4 14 Substrate Just prior to use reconstitute the Substrate lyophilized by adding 6 ml of Substrate Buffer to the Substrate vial The Substrate solution should be used immediately after reconstitution and any remaining solution should be discarded Protease Reconstitute Protease lyophilized by adding 10 ml of Protease Buffer to the vial and dissolve completely by mixing at room temperature for 15 30 min Protease is stable for 2 days at 2 10 C Reconstituted Protease is stable below 20 C for the life of the kit Protease Il Reconstitute Protease II lyophilized by adding 10 ml of Protease Buffer to the vial and dissolve completely by mixing at room temperature for 15 30 min
12. ested and found negative for presence of the HBs antigen HIV antibody and HCV antibody however all samples should be handled carefully as though capable of transmitting infection 2 Stop Reagent 7 7 H2SOa is hazardous and can cause severe burns In case of eye contact rinse immediately with plenty of water and seek medical advice In case of contact with skin or clothing rinse immediately with plenty of water STORAGE OF REAGENTS The kit reagents should be stored at 2 10 C DO NOT FREEZE EXPIRATION DATE Indicated on the package REFERENCES 1 Waki H et al 2003 J Biol Chem 278 40352 63 2 Pajvani UB et al 2003 J Biol Chem 278 9073 85 3 Tsao TS et al 2003 J Biol Chem 278 508 10 7 4 Pajvani UB et al 2004 J Biol Chem 279 12 152 62 5 Tonelli J et al 2004 Diabetes 53 162 19 6 Kobayashi H et al 2004 Circ Res 94 27 31 7 Ebinuma H et al 2006 Clin Chim Acta 372 47 53 8 U S Patent No 7 608 405 B2 issued Oct 27 2009 7 14 Performance Characteristics 1 Typical Calibration Curve bh pt ttn a 0 1 Absorbance at 492 nm 0 01 0 001 0 1 1 Concentration of Adiponectin ng ml 2 Sensitivity The analytical sensitivity was determined by calculating the mean 3 standard deviations for 20 replicates of the Zero Standard The sensitivity of the assay is 0 019 ng ml 3 Reproducibility Intra assay n 8 Serum Calculated Sample 1 Total MMW HMW HM
13. ized Serum Control Processed human serum 1 f Lyophilized Plate Sealers n a 3 each Ready to use MATERIALS REQUIRED BUT NOT INCLUDED e Microfuge tubes 1 ml e Precision pipettes with disposable tips capable of dispensing 10 ul 100 ul 500 ul e Repeating or multi channel pipette e Volumetric container e Volumetric pipettes 3 14 Distilled deionized water Incubator or water bath 37 C Microplate washer or wash bottle Microplate reader with 492 and optional 600 700 nm filter REAGENT PREPARATION AND STORAGE It is critical that all reagents be allowed to reach room temperature prior to use All components are stable at 2 10 C until the expiration date of the kit unless otherwise indicated Wash Buffer Dilute Wash Buffer Concentrate with 900 ml of distilled water Working Wash Buffer should be stored at 2 10 C and is stable for the life of the kit Sample Pretreatment Buffer To dissolve any visible precipitate warm the solution to room temperature and stir thoroughly before use Monoclonal Antibody Coated Plate Unused strips should be returned to the laminate bag sealed and stored at 2 10 C Calibrator e The Calibrator is comprised of normal human serum stabilized in Sample Pretreatment Buffer e To dissolve any visible precipitate warm the solution to room temperature and stir thoroughly before use Avoid foaming e Create a standard curve by serial dilution as indicated in the table below
14. o 492 nm with a reference wavelength of 600 Ljj j nm if desired 4 Calculations Construct a calibration curve from the Standards The Zero Standard should be used as a blank with its average value subtracted from each well Plot the calibration curve using a log log scale It is preferable to use a software program to calculate the calibration curve and to determine the concentration of the samples The preferred curvefit method is quadratic If a manual calculation is to be utilized the Zero Standard should be used as a blank with its average value subtracted from each well The standard concentrations are plotted on the X axis and the absorbance values are plotted on the Y axis The concentrations of the unknowns are determined by plotting the absorbance of the unknown against the calibration curve The corresponding value on the X axis is the concentration of the unknown sample Read the concentrations for the diluted samples from the calibration curve Calculate the final concentration for the samples by multiplying by the dilution factor 1 5 151 6 14 Calculation of each adiponectin multimer fraction Total adiponectin Concentration of the sample from Pretreatment option 1 HMW adiponectin Concentration of the sample from Pretreatment option 3 MMW adiponectin Concentration of the ssample from Pretreatment option 2 MMW HMW adiponectin minus the concentration of the sample from Pretreatment option 3 HMW LMW adiponectin
15. ow many strips will be needed and store the remaining strips in the sealed laminate bag at 2 10 C 2 Add 50 ul each of the Standards and diluted pretreated samples to the appropriate wells according to the plate configuration 3 Cover the plate with a plate sealer and incubate for 1 hour at room temperature 20 30 C 4 Decant the plate and strike the plate against absorbent towels to remove any excess liquid Do not introduce absorbent materials into the wells Wash by adding 350 400 ul of Wash Buffer to each well using a laboratory squeeze bottle wash manifold or automated plate washer decant Wash Buffer and strike plate against absorbent towels to remove residual liquid Repeat this cycle twice for a total of 3 washes 5 Add 50 ul of Biotin Labeled Monoclonal Ab to each well Cover the plate with a plate sealer and incubate for 1 hour at room temperature 20 30 C 6 Repeat wash step as described in Step 4 7 Add 50 ul of the Enzyme Labeled Streptavidin to each well Cover the plate with a plate sealer and incubate for 30 min at room temperature 20 30 C 8 Repeat wash step as described in Step 4 9 Add 50 ul of the Substrate Solution to each well Protect the plate from light and incubate for 10 min at room temperature 20 30 C 10 Add 50 ul of the Stop Reagent to each well 11 The absorbance of each well should be measured within 30 min after the addition of the PL Reagent using a microplate reader set t
16. se 2 29 8 14 7 98 102 treatment 4 57 10 8 10 3 105 9 14 16 2 14 8 109 HMW Adiponectin 0 00 3 88 3 89 100 Protease II 1 14 5 01 5 03 100 treatment 2 29 6 26 6 17 101 4 57 8 89 8 46 105 9 14 12 6 13 0 97 9 14 6 Comparison of Serum vs Plasma Samples EDTA heparin and citrate plasma samples were compared with serum samples obtained from four healthy volunteers and assayed for Total HMW and MMW adiponectin Sample Mean Adiponectin ug ml Mean Plasma Serum Total Adiponectin Serum 5 84 100 EDTA plasma 5 37 93 Heparin 5 37 94 Citrate plasma 4 45 76 MMW HMW Serum 3 69 100 Adiponectin Protease EDTA plasma 3 72 102 treatment Heparin 3 77 101 Citrate plasma 3 12 83 HMW Adiponectin Serum 2 77 100 Protease Il treatment EDTA plasma 2 56 94 Heparin 2 63 94 Citrate plasma 2 20 79 7 Freeze Thaw Stability Four human serum samples were frozen and thawed three times and assayed for Total HMW and MMW adiponectin Recovery Sample n 4 Mean ug ml F T 1X F T 2X F T 3X Total Adiponectin 8 34 99 92 95 MMW HMW Adiponectin 6 60 92 91 92 HMW Adiponectin 4 41 97 100 98 10 14 8 Specificity of Protease Digestion Human serum samples were pretreated per the Assay Procedure separated by native PAGE and analyzed by Western Blot using goat anti adiponectin antibody 1 2 3 Lane 1 Pretreatment Option 1 no protease shows detec
17. tion Q9 of HMW MMW Alb LMW and LMW tdiponectin 669 kDA HMW QR Lane 2 Pretreatment Option 2 Protease shows specific 443 kDA MMW QP digestion of Alb LMW and LMW tdiponectin Alb LMW A Lane 3 Pretreatment Option 3 Protease II shows specific os digestion of all multimers except HMW I diponectin 140 kDa LMW x oof 9 Reference Range Forty seven serum samples from healthy donors were assayed for l RALPYSGIVY d ers Data are presented as Total HMW MMW and LMW Idiponectin and as ratios of the individual multimers to Total I diponectin HMWR MMWR and LMWR Male n 27 Total HMW MMW LMW Mean ug ml 4 30 1 62 1 15 1 54 SD 1 76 1 02 0 38 0 50 HMWR MMWR LMWR Mean 34 5 27 9 37 6 SD 11 1 5 41 7 78 Female n 21 Total HMW MMW LMW Mean ug ml 6 62 3 24 1 70 1 68 SD 3 04 Holt jy j Ky HMWR MMWR LMWR Mean 44 5 26 7 28 8 SD 12 3 4 47 10 3 11 14 10 Specificity of Antibodies In order to demonstrate specific reactivity to the intact multimeric forms of adiponectin human serum or plasma was separated by native PAGE and analyzed by Western blot using the ELISA capture ab A detection ab B or agoat anti human adiponectin polyclonal ab C Human Serum Human Plasma Human Serum i Human Plasma Human Serum 4 tid vd A Human Plasma m eet 669 kDa MEE i CI 443 kDa SM Si MaR a E
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