Data Sheet
Contents
1. 9 Add 100 uL of Substrate Reagent Avoid expos the plate with e g aluminum foil is recommende etutn Substrate Reagent to 4 C immediately after the necessary volume is removed 10 Incubate 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 25 units for the oncentration or 3 0 units for the highest standard concentration The plate nitored at 5 minute intervals for approximately 30 minutes plate 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 e values measured at 405 nm is used to determine sRAGE concentration of off scale amples The readings at 405 nm should not replace the on sca2. TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Summary of Procedure Add 100 uL of diluted samples to the wells Yy Incubate for 1 hour at room temp cA Wash the wells O Add 100 uL of HRP conjugated anti human sRAGE antibody v Incubate for 1 hour at room temp Wash the wells Add 100 uL of Substrate Reagent y Add 100 uL of Stop Solution v Measure absorbance at 450 nm Materials Provided All samples and standards should be assayed in e The following components are supplied and are sufficient for the one 96 well microplate kit Microplate One microplate supplied ready to with 96 wells 12 strips of 8 wells in a foil zip lock bag with a desiccant pack Wells are coated i human sRAGE monoclonal antibody Y K 2B4 as a capture antibody 10X Wash Buffer One bottle containing 100 of 10X buffer containing 2 Tween 20 Dilution Buffer One bottle contain Standard and sample dilution Rea use Human sRAGE Standard Peers 8 ng of lyophilized recombinant human sRAGE A mL of 1X buffer use for reconstitution of Human sRAGE HRP conjugated Detect tibody One bottle containing 12 mL of HRP horseradish peroxidase conjugated anti huma ntibody Ready to use TMB Ready to Rp containing 20 mL of 1 N H2SOx Ready to use Substrate sas tle containing 20 mL of the chromogenic substrate tetra methylbenzidine Stop Solutio C CY 8083 3 Version 120710 Human
3. a central t 100 calgranulin polypeptides Cell 1999 97 889 901 an SF and Schmidt AM Receptor for advanced glycation endproducts RAGE ations of diabetes Ageing Res Rev 2002 1 1 15 12 Hofmann M cell surface 13 Stern DM and the ca C CY 8083 15 Version 120710 Human sRAGE ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures 14 Schlueter C Hauke S Flohr A M Rogalla P and Bullerdiek J Biochim Biophys Acta 2 1630 1 6 15 Yonekura H Yamamoto Y Sakurai S Petrova R G Abedin M J Li H Yasui K T M Makita Z Takasawa S Okamoto H Watanabe T and Yamamoto H Bioche 370 1097 1109 16 Malherbe P Richards J G Gaillard H Thompson A Diener C Schuler A G Brain Res Mol Brain Res 1999 71 159 170 Related Products CircuLex S100A12 EN RAGE ELISA Kit Cat CY 8058 Human S100A12 Cat CY R2262 G Human S100A12 Cat CY R2262 H O Human S100A12 Low Endotoxin Cat CY R2462 G CML BSA Ne carboxymethyl Lysine BSA Cat CY R2052 CML OVA Ne carboxymethyl Lysine OVA Cat CY R2053 CEL BSA Ne carboxyethyl Lysine BSA Cat CY R2054 CEL OVA Ne carboxyethyl Lysine OVA Cat CY R2055 Glucose AGE BSA Cat CY R2056 Glucose AGE OVA Cat CY R2057 Glyceraldehyde AGE BSA Cat CY R2058 Glyceraldehyde AGE OVA Cat CY R2059 Glycolaldehyde AGE BSA Cat CY R2060 Gl
4. Buffer before dispensi sed portions of Master Standard should be aliquoted and stored at below 70 C immedi id multiple freeze and thaw cycles Sample Prepa n e Serum an samples require a 8 fold dilution logical samples require neat to appropriate dilution C CY 8083 7 Version 120710 Human sRAGE ELISA Kit n TM Circulex User s Manual For Research Use Only Not for use in diagnostic procedures Standard Assay Procedure for Human sRAGE 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 N Incubate the plate at room temperature ca 25 C for 1 hour at ca 300 rpm on an orbital microplate shaker oo Wash 4 times by filling each well with Wash Buffer 350 u pipette manifold dispenser or microplate washer sin squirt bottle multi channel
5. in patients with various ati pgical conditions Decreased level of sRAGE is a biomarker for deficient and or altered inflammatory control in humans It was shown that reduced level of sRAGE is associated with higher pronary disease In Alzheimer disease there is a decrease in serum sRAGE in comparison controls On the other hand an increased level of serum sRA renal disease and acute lung injury 1s found in patients with end stage Principle of the Assay GE ELISA Kit employs the quantitative onoclonal antibody specific for human sRAGE is pre coated onto a microplate Standards and an are pipetted into the wells and the immobilized antibody binds any human sRAGE pres et washing away any unbound substances an HRP conjugated antibody specific for human GE s added to the wells Following a wash to remove any The CycLex Research Product CircuLex Human sandwich enzyme immunoassay technique unbound antibody HRP conjugate the ing conjugate is allowed to react with the substrate H O gt tetramethylbenzidine The rea 1 ped by addition of acidic solution and absorbance of the resulting yellow product is measur d at nm The absorbance is proportional to the concentration of human sRAGE A standard curve nstructed by plotting absorbance values versus human sRAGE concentrations of calibrators centrations of unknown samples are determined using this standard curve N C CY 8083 2 Version 120710 Human sRAGE ELISA Kit
6. lock and desiccant pack CY 8083 Version 120710 Human sRAGE ELISA Kit TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Assay Characteristics 1 Sensitivity The limit of detection defined as such a concentration of human sRAGE giving absorbance ighe than mean absorbance of blank plus three standard deviations of the absorbance of blank lanky 3SD blank is better than 1 6 pg ml of sample Standard Sample Dilution Buffer is pipetted into blank wells Typical Standard Curve Qj sRAGE Standard Curve 3 0 A450 0 200 400 600 800 sRAGE conc pg ml C CY 8083 10 Version 120710 Human sRAGE ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures 2 Precision Intra assay Precision Precision within an assay Four samples of known concentration were tested eight times on one plate to assess zA precision l e Intra assay Within Run n 8 CV 2 5 5 3 Human sRAGE concentration pg mL Sample 1 Sample 2 Sample 3 Sample 4 1 222 9 587 0 644 1 923 7 2 218 6 564 0 628 8 819 1 3 196 5 534 7 614 8 898 6 4 224 0 555 6 624 4 807 1 5 213 7 551 0 610 5 855 5 6 197 3 605 6 647 0 909 4 7 215 1 562 2 624 5 842 3 8 214 3 530 3 602 1 818 0 max 224 0 605 6 647 0 923 7 min 196 5 530 3 602 1 807 1 mean 212 8 561 3 624 5 859 2 SD 10 5 25 2 15 6 45 6 CV 4 9 i ZE 2 5 5 3
7. sRAGE ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Materials Required but not Provided e Pipettors 2 20 uL 20 200 uL and 200 1000 uL precision pipettors with disposable tips Precision repeating pipettor e Orbital microplate shaker e Microcentrifuge and tubes for sample preparation e 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 e s of 450 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be used The pl so be read at a single wavelength of 450 nm which will give a somewhat higher reading Software package facilitating data generation and analysis opti pal e 500 or 1000 mL graduated cylinder e Reagent reservoirs Deionized water of the highest quality e Disposable paper towels C CY 8083 4 Version 120710 Human sRAGE ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Precautions and Recommendations e Allow all the components to come to room temperature before use e All microplate strips that are not immediately required should be returned to the zip lock h must be carefully resealed to avoid moisture absorption e Do not use kit components beyond the indicated kit expiration date e Use only the microtiter wells provided with the kit e Rinse all deterg
8. C CY 8083 11 Version 120710 Human sRAGE ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Inter assay Precision Precision between assays Four samples of known concentration were tested in four separate assays to assess inter ass precision e Inter assay Run to Run n 4 CV 4 9 8 0 Human sRAGE concentration ng mL O Sample 1 Sample 2 Sample 3 Sample 4 208 3 451 5 669 3 1 022 0 192 2 405 5 694 3 868 3 196 1 430 5 625 2 910 7 179 5 411 0 634 5 865 max 208 3 451 5 694 3 1 022 0 min 179 5 405 5 625 2 865 5 mean 194 0 424 6 655 8 916 6 SD 11 9 20 9 31 9 73 2 CV 6 1 4 9 4 9 8 0 BWN Fe 3 Linearity Four serum specimens were diluted with Standard Sam The neat sample is set to 1 4 4 Please note that all s as stated in the Assay Procedure The results are s bon Buffer and assayed after dilution ing the neat sample are 8 fold diluted ed in the figure below Linearity 600 Sample 1 Sample 2 500 Sample 3 Sample 4 400 g Sb 2 m 300 amp 3 200 100 0 lf 2 3 4 Sample dilution ratio 4 C CY 8083 12 Version 120710 Human sRAGE ELISA Kit TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Example of Test Results Fig 1 Correlation between the human sRAGE concentrations measured by CircuLex ELISA kit a competitor ELISA kit Correlati
9. Human sRAGE ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures ELISA Kit for Measuring Human sRAGE CircuLex Human sRAGE ELISA Kit cA Cat CY 8083 O Intended USse cceeccecccccccecceessesssseeesseeecees 1 SON AE A occe tale eeteae cose een 1 FINNS C tiO Mei sgcctbacoespeonasnysooenamcareananicind 2 Principle of the Assay 2 3 Materials Provided caiccciscocccssssnserecaisdaessnneoecds 3 Materials Required but not Provided 4 Precautions and Recommendation 5 Sample Collection and Storage 00 6 Detailed Prod siccetszesatanareusnieccarateazadenass Calculations oninia Measurement Range WrowbleshO Oi visa ctccsicadeesansceacsentiosisooriatares Reagent Stabity ccccscccsdinccessesssnntesceesaaccecne Assay Characteristics ccceessceseceesneeeees Example of Test Results References cccccccccccccccceesssescceseeeesseeuseseees Related Products cccccccccccccsssseessseeeeseeees Intended Use The CycLex Research Product CircuLex sRAGE ELISA Kit is used for the quantitative measurement of human soluble RAGE s serum plasma and other biological media This assay kit is for research use onlypz for use in diagnostic or therapeutic procedures Storage e Upon receipt store all co e Don t expose reagents to C CY 8083 1 Version 120710 Human sRAGE ELISA Kit n TM CircuLex User s Manual For Research Use On
10. ent residues from glassware Qy e Use deionized water of the highest quality e 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 mouth pipette or ingest any of the reagents e Do not smoke eat or drink when performing the N where samples or reagents are handled e Dispose of tetra methylbenzidine TMB containi ions in compliance with local regulations e Avoid contact with the acidic Stop Solution and Subs Solution which contains hydrogen peroxide e Wear gloves and eye protection when han unodiagnostic materials and samples of human origin and these reagents In case of conta th the Stop Solution and the Substrate Solution wash skin thoroughly with water and seek m l attention when necessary Biological samples may be cont ith infectious agents Do not ingest expose to open wounds or breathe aerosols W ective gloves and dispose of biological samples properly handling Sto Q e CAUTION Sulfuric See g acid Wear disposable gloves and eye protection when n C CY 8083 5 Version 120710 Human sRAGE ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Sample Collection and Storage Serum Use a serum separator tube and allow samples to clot for 60 30 minutes Centrifug samples at 4 C for 10 minutes at 1 000 x g Remove s
11. erum and assay immediately or store sam 0 ice for up to 6 hours before assaying Aliquots of serum may also be stored at below 70 C e periods of time Avoid repeated freeze thaw cycles Plasma Collect plasma using EDTA Nay as the anticoagulant If possible collect the toa mixture of EDTA Na and Futhan5 to stabilize the sample against spontaneous in vitro complement activation Immediately centrifuge samples at 4 C for 15 minutes at 1 000 x g Assayeimmediately 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 Other biological samples Remove any particulates by centrifugation a ess immediately or aliquot and store samples at below 70 C Avoid repeated freeze thaw cycles C CY 8083 6 Version 120710 Human sRAGE ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol The CycLex Research Product CircuLex Human sRAGE ELISA Kit is provided with remo strips of wells so the assay can be carried out on separate occasions using only the number o required for the particular determination Since experimental conditions may vary an alique human sRAGE Standard within the kit should be included in each assay as a calibrat pipette tips and reagent troughs should be used for all liq
12. le readings at 450 nm Note 1 Complete remoya CY 8083 Version 120710 Human sRAGE ELISA Kit n TM CircuLex 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 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 and n analysis may be applied to the log transformation To determine the human sRAGE co n each sample first find the absorbance value on the y axis and extend a horizontal line t dard curve At the point of intersection extend a vertical line to the x axis and read the correspo man SRAGE 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 4 parameter lo c equation The results of unknown samples can be calculated with any computer progr av a 4 parameter logistic function It is important to make an appropriate mathematical adjus accommodate for the dilution factor ration The calibration sus log of the known ively the logit log function is plotted versus log of the 2 Most microtiter plate readers perform automatic calculations of analyte curve is constructed by plotting the absorba
13. ly Not for use in diagnostic procedures Introduction RAGE is a multi ligand member of the immunoglobulin superfamily of cell surface molecules t expressed in a variety of cell lines including endothelial cells smooth muscle cells mono e phagocytes pericytes neurons cardiac myocytes mesangial cells and hepatocytes 1 interacts with different structures to transmit a signal into the cell and recognizes thre structures rather than specific amino acid sequences Therefore RAGE seems to fulfill th of a pattern recognition receptor As a member of the immunoglobulin superfamily it int diverse class of ligands including AGEs 3 4 HMGB1 also known as Amphoterin B peptide 6 amyloid A 7 leukocyte adhesion receptors 8 prions 9 Escherichia coli curli operons 10 B sheet fibrils 11 and several members of the S100 protein sup l including S100 calgranulins 12 Thus RAGE may have potential involvement in several pa processes including inflammation diabetes Alzheimer s disease AD systemic amyloidosis and tumor growth 13 Soluble version of RAGE termed soluble RAGE sRAGE created by pro tical cleavage by matrix metalloproteases can be detected in sera In addition to sRAGE er soluble RAGE as endogenous secretory RAGE esRAGE derives from alternative splicing e RAGE mRNA 14 16 was discovered Because of the possible neutralization effect of sk studies have examined the significance of sRAGE serum concentration
14. nce Y of calib concentration X of calibrators using the 4 parameter function can be used to linearize the calibration curve i e logit of abs known concentration X of calibrators Measurement Range The measurement range is 12 5 pg mL to 800 standard should be diluted with Dilution Buffer in to be taken into consideration in calculating the hum ample reading higher than the highest dilution and re assayed Dilution factors need AGE concentration Troubleshooting 1 The Human sRAGE Standard should b licate using the protocol described in the Detailed Protocol Incubation times or temp fatureg significantly different from those specified may give erroneous results 2 Poor duplicates accompanied by elev values for wells containing no sample indicate insufficient washing If all instructions i iled Protocol were followed accurately such results indicate a need for washer maintenan 3 Overall low signal may mdic at desiccation of the plate has occurred between the final wash and addition of Substrate Re t Do not allow the plate to dry out Add Substrate Reagent immediately after wash included in the CycLex Research Product CircuLex Human sRAGE ELISA Kit stability Reagents should not be used beyond the stated expiration date Upon agents should be stored at 4 C except the reconstituted sRAGE Standard must be stored at ted assay plates should be stored in the original foil bag sealed by the zip
15. on between the concentrations measured by CircuLex ELISA kit and by competitor ELISA kit i v y 1 0099x 138 27 2 000 R 0 8682 1 500 f 1 000 Ff 500 p Competitor Human RAGE ELISA pg ml 0 500 1 000 1 500 2 000 CircuLex Human sRAGE ELISA pg ml O lt amp Y C CY 8083 13 Version 120710 Human sRAGE ELISA Kit TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Fig 2 Serum sRAGE level in 40 healthy volunteers Human sRAGE n 40 Max 1783 4 Min 302 9 Mean 676 3 sRAGE pg ml C CY 8083 14 Version 120710 Human sRAGE ELISA Kit n TM Circulex User s Manual For Research Use Only Not for use in diagnostic procedures References Neeper M Schmidt AM Brett J et al Cloning and expression of a cell surface receptor for adv glycosylation end products of proteins J Biol Chem 1992 267 14998 5004 2 Brett J Schmidt AM Yan SD et al Survey of the distribution of a newly characterize advanced glycation end products in tissues Am J Pathol 1993 143 1699 712 W Neeper M Schmidt AM Brett J et al Cloning and expression of a cell surface receptor for advanced glycosylation end products of proteins J Biol Chem 1992 267 14998 15004 4 Schmidt AM Vianna M Gerlach M et al Isolation and characterization of two ing proteins for advanced glycosylati
16. on end products from bovine lung which are presen dothelial cell surface J Biol Chem 1992 267 14987 14997 5 Hori O Brett J Slattery T Cao R et al The receptor for advanced gly products RAGE is expression of rage and 5761 6 Yan SD Zhu H Fu J Yan SF Roher A et al Amyloidbeta p endproduct interaction elicits neuronal expression of m proinflammatory pathway in Alzheimer disease Proc Natl ptor for advanced glycation olony stimulating factor a A 1997 94 5296 5301 7 Yan SD Zhu H Zhu A Golabek A Du H Roh Receptor dependent cell stress and amyloid accu 643 651 to C Schmidt AM Stern D et al stemic amyloidosis Nat Med 2000 6 8 Chavakis T Bierhaus A Al Fakhri N et al pattern recognition receptor RAGE is a counterreceptor for leukocyte integrins a 1 pathway for inflammatory cell recruitment J Exp Med 2003 198 1507 1515 9 Sasaki N Takeuchi M Chowei H Ki S Advanced glycation end products AGE and their receptor RAGE in the brain of patients with Creutzfeldt Jakob disease with prion plaques Neurosci Lett 2002 326 117 120 10 Chapman MR Robinson LS efJS et al Role of Escherichia coli curli operons in directing amyloid fiber formation Sci 295 851 855 11 Bierhaus A Humpert glycation end product os M et al Understanding RAGE the receptor for advanced ed 2005 83 876 886 U C Qu W et al RAGE mediates a novel proinflammatory axis
17. uid transfers to avoid cross co reagents or samples Preparation of Working Solutions All reagents need to be brought to room temperature prior to the assay Assay r are supplied ready to use with the exception of 10X Wash Buffer and Human sRAGE Stan 1 Prepare a working solution of Wash Buffer by adding 100 mL of the 10X ffer provided to 900 mL of deionized distilled water Mix well Store at 4 C for two 0 C for long term storage 2 Reconstitute Human sRAGE Standard with 1 0 mL of Dilutio Buffe he concentration of the human sRAGE in vial should be 8 ng mL which is referred sa aster Standard of human sRAGE Prepare Standard Solutions as follows Use the Master Standard to produce a dilution series b Mix each tube thoroughly before the next transfer The 800 pg mL standard S s the highest standard The Dilution Buffer serves as the zero standard Blank Volume of Standard Dilution Buffer Concentration Std 1 60 uL of Master Standard 8 n 540 uL 800 pg mL Std 2 300 uL of Std 1 800 pg mL 300 uL 400 pg mL Std 3 300 uL of Std 2 400 pg 300 uL 200 pg mL Std 4 300 uL of Std 3 200 pg mL 300 uL 100 pg mL Std 5 300 uL of Std 4 100 300 uL 50 pg mL Std 6 300 uL of Std 5 5 300 uL 25 pg mL Std 7 300 uL of Std 6 2 m 300 uL 12 5 pg mL Blank 300 uL 0 pg mL Note Do not use a R ting pipette Change tips for every dilution Wet tip with Dilution
18. ycolaldehyde AGE OVA Cat CY R2061 Methylglyoxal AGE BSA Cat CY R2062 Methylglyoxal AGE OVA Cat CY R2063 Glyoxal AGE BSA Cat CY R2064 Glyoxal AGE OVA Cat CY R2065 CML HSA Ne carboxymethy Lysine t CY R2066 CEL HSA Ne carboxyethyl m CY R2067 PRODUCED BY cA CycLex Co Ltd 1063 103 Terasawaoka Ina Nagano 396 0002 Japan Fax 81 265 7 e mail info c CycLex Circ oducts are supplied for research use only CycLex CircuLex products and compo may not be resold modified for resale or used to manufacture commercial pro hout prior written approval from CycLex Co Ltd To inquire about licensing for such use please contact us via email C CY 8083 16 Version 120710
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