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1. A Roa CY 8058V2 2 Version 151118 o AO CircuLex Uer Manual For Research Use Only Not for use in diagnostic procedures Ss S100A12 EN RAGE ELISA Kit Ver 2 gt gt Summary of Procedure l Add 100 uL of diluted sample to the wells SS 4 Incubate for 1 hour at room temp K Wash the wells 74 Add 100 uL of HRP conjugated anti S100A12 antibody Q 4 Incubate for 1hour at room temp y Wash the wells aS gt Add 100 uL of Substrate Reagent gt S J Incubate for 10 20 mimes at room temp Add 100 uL of Stop Solution N Measure absorbance at 450 nm I Materials Provided X O All samples and standards should be assay MY duplicate The following components are supplied and are sufficient for the one 96 well mcroPlae Microplate One microplate supplied r Oio use with 96 wells 12 strips of 8 wells in a foil zip lock bag with a desiccant pack Wells ag pcs with anti S100A12 EN RAGE monoclonal antibody as a capture antibody H 10X Wash Buffer One 100 m amp Gpitle of 10X buffer containing Tween 20 Dilution Buffer One a ee 50 mL of 1X buffer use for standard and sample dilution Ready to use lt S Human S100A12 Efan One vial containing 25 6 ng of lyophilized recombinant human S100A12 EN RAGF HRP conjugat etection Antibody One bottle containing 12 mL of HRP horseradish peroxidase conjugated a 100A 12 EN RAGE monoclonal antibody Substrat agent One bottle contai2. A7 Low Endotoxin Cat CY R2457 N Human S100A8 Low Endotoxin Cat CY R2458 Human S100A9 Low Endotoxin Cat CY R2459 G Human S100411 Low Endotoxin Cat CY R2461 7 Human S100A12 Low Endotoxin Cat CY R2462 Qy Human S100414 Low Endotoxin Cat CRD Human S100P Low Endotoxin Cat CY RAA wW PRODUCED BY A N CycLex Co Ltd amp 1063 103 Terasawa Ina Nagano 396 000 Japan Fax 81 265 297618 E mail inf clex co j URL https w cyclex co j Cyc AdCircuLex products are supplied for research use only CycLex CircuLex products and si thereof may not be resold modified for resale or used to manufacture commercial prddfcts without prior written approval from CycLex Co Ltd To inquire about licensing for commercial use please contact us via email Roa CY 8058V2 15 Version 151118 o c
3. C Qu W Taguchi A Lu Y Avila C Kambham N i A Nawroth P Neurath M F Slattery T Beach D McClary J Nagashima M Stern D Schmidt A M RAGE mediates a novel proinflammatory axis a centra receptor for S100 calgranulin polypeptides Cell 97 889 901 1999 Q 2 Shishibori T Oyama Y Matsushita O Yamashita K Furuichi H Okabe A eta H Hata Y Kobayashi R Three distinct anti allergic drugs amlexanox cromolyn and tranifasF bind to S100A12 and S100A13 of the S100 protein family Biochem J 338 583 9 1999 AR 3 Foell D Wittkowski H Hammerschmidt I Wulffraat N Schmeling H F M Horneff G Kuis W Sorg C Roth J Monitoring neutrophil activation in juvenile aan hritis by S100A12 serum concentrations Arthritis Rheum 50 1286 95 2004 Q 4 Foell D Seeliger S Vogl T Koch HG Maschek H Harms BESorg C Roth J Expression of S100A12 EN RAGE in cystic fibrosis Thorax 58 613 7 2 Ry 5 Foell D Ichida F Vogl T Yu X Chen R Miyawaki T Sere C Roth J S100A12 EN RAGE in monitoring Kawasaki disease Lancet 361 1270 2 2003 amp 6 Neutrophil derived human S100A12 EN RAGE 49 strongly expressed during chronic active inflammatory bowel disease Gut 52 847 53 2003 Q Related Products CircuLex S100A13 ELISA Kit Cat CY 8057 CircuLex S100A12 ELISA Kit Cat 058V2 CircuLex S100P ELISA Kit Cat cgi CircuLex S100A8 MRP8 ELISA Kit at CY 8061 CircuLex S100A9 MRP14 ELISAQ
4. CircuLex Uer Manual Ss S100A12 EN RAGE ELISA Kit Ver 2 gt For Research Use Only Not for use in diagnostic procedures gt ELISA Kit for Measuring Human S100A12 EN RAGE l Circulex S100A12 EN RAGE ELISA Bit Ver 2 Q Cat CY 8058V2 2 G Intended Use o nsoeneeseeeneeseeeessreesssesessresesee 1 ne Be at nsatsinanigayonenysareiocsciasiadsaenpiaaeeetsines 1 a ntroduction seeeeeeeeeseeeeeeeeereeserrrrsrrereseseee 2 O Principle Of the Assay 2 3 gt Materials Provided ccescccceeseeceeeseeeeees 3 N Materials Required but not Provided 4 Precautions and Recommendations 5 4 Sample Collection and Storage 6 e2 Detailed ProtoCOl ssisscrisrsicieirriririarsrrsrsanenana 7 8 4 Calculations ccccccccesessceeesereeeeeneeeesenees 9 gt Measurement Range 9 Troubleshooting eeseeeeeeeeeeeereereerrererresen RY Reagent Stability Assay Characteristics ccccsssssccsocarsansveencessgecs 0 12 Example of Test Results 13 References ccccccceesseceeeeeteeceseeeeeugeQecees 14 Related Products scsccsciscstoncosasecasece amp PR 14 15 amp Intended Use Ka The CycLex Research Prod irculex S100A12 EN RAGE ELISA Kit Ver 2 is used for the quantitative measurement of S100A12 EN RAGE in serum plasma and other biological media This assay kit is for reseafeh use only and not for use in diagnostic or therapeutic procedures Storage e Upon receipt KA component
5. EN RAGE ELISA Kit Ver 2 O For Research Use Only Not for use in diagnostic procedures 4 Spiking Recover l Serum samples were spiked with different amounts of S100A12 and assayed RA The recovery of S100A12 spiked to levels throughout the range of the assay was evaluated Ww Sample Average Recovery Range S Serum samples n 3 101 8 101 2 91 8 5 Linearity Ku To assess the linearity of the assay samples containing and or spiked wit Fed concentrations of S100A12 were serially diluted with the Dilution Buffer to produce me th values within the dynamic range of the assay SS The linearity of the assay gt X The linearity of the assay A sample 1 E sample 2 sample 3 3 3 _ g 80 kad a j lt S S bom n 5 w 0 3 7 5 06 07 08 09 Sample dilution ratio o gL eA Roa CY 8058V2 12 Version 151118 o AO A RS F I S100A12 EN RAGE ELISA Kit Ver 2 Circu Lex User s Manual A For Research Use Only Not for use in diagnostic procedures Example of Test Results 3 Fig l Human S100A12 concentrations in sera from Crohn s disease patients n 10 ang Qealthy volunteers n 8 RY s El B 2 600 S lt lt Zz S Healthy Crohn s volunteer disease o gL eA Roa CY 8058V2 13 Version 151118 amp lt C Human S100A5 Cat CY R2255 Roa CY 8058V2 14 Version 151118 ge References 1 Hofmann M A Drury S Fu
6. RAGE ELISA Kit Ver 2 L ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 A Q O Q Detailed Protocol 3 The CycLex Research Product Circulex S100A12 EN RAGE ELISA Kit Ver 2 is pro 2d with removable strips of wells so the assay can be carried out on separate occasions using only thesaimber of strips required for the particular determination Since experimental conditions may vary Daiguot of the S100A12 Standard within the kit should be included in each assay as a calibrator Digppsable pipette tips and reagent troughs should be used for all liquid transfers to avoid a a of reagents or samples Y Preparation of Working Solutions All reagents need to be brought to room temperature prior to the assay AS reagents are supplied ready to use with the exception of 10X Wash Buffer and Human S100A1QS andard deionized distilled water ddH20 Mix well Store at 4 C for weeks or 20 C for long term 1 Prepare a working solution of Wash Buffer by adding 100 mL of Ss Wash Buffer to 900 mL of storage C 2 Reconstitute Human S100A12 Standard with 1 mL of aafo The concentration of the human S100A12 in vial should be 25 6 ng mL which is referred asta Master Standard of human S100A12 Prepare Standard Solutions as follows R4 Use the Master Standard to produce a dilution serf s below Mix each tube thoroughly before the next transfer The 1 280 pg mL standard gO serves as the highest standa
7. Sample Preparation above amp Pipette 100 uL of Standard Solutions Std1 Std7 Blank and diluted sample duplicates into the appropriate wells O 4 Incubate the plate at room temperature ca 25 C for 1 hour shaking at EN rpm on an orbital microplate shaker aS Wash 4 times by filling each well with Wash Buffer 350 uL using squirt bottle multi channel pipette manifold dispenser or microplate washer 6 Add 100 uL of HRP conjugated Detection Antibody into each on Incubate the plate at room temperature ca 25 C for 1 ng skin at ca 300 rpm on an orbital microplate shaker 2 Wash 4 times by filling each well with Wash Bufeo uL using a squirt bottle multi channel pipette manifold dispenser or microplate washer 9 Add 100 uL of Substrate Reagent Avoid MNN the microtiter plate to direct sunlight Covering the plate with e g aluminum foil is recommeng y Return Substrate Reagent to 4 C immediately after the necessary volume is removed lt Incubate the plate at room temperatu 25 C for 10 20 minutes shaking at ca 300 rpm on an orbital microplate shaker The incubyon time may be extended up to 30 minutes if the reaction temperature is below than 20 C Add 100 uL of Stop Solution each well in the same order as the previously added Substrate Reagent Measure absorbance in caven using a spectrophotometric microplate reader at dual wavelengths of 450 540 nm Dual waveJtagths of 450 550 o
8. eins when working as anti alrgic drugs S100A12 serum concentrations indicate neutrophil activation in J 3 cystic fibrosis 4 Kawasaki disease 5 and Crohn s disease 6 Its function as a proin atory factor secreted by activated neutrophils makes this protein a potential target for future therpie Principle of the Assa 2 The Circulex S100A12 EN RAGE ELISA Kit Qe employs the quantitative sandwich enzyme immunoassay technique A monoclonal antibody ri for S100A12 EN RAGE has been pre coated onto a microplate Standards and samples are pipgtted into the wells and the immobilized antibody binds any S1O00A12 EN RAGE present After aa a any unbound substances an HRP conjugated monoclonal antibody specific for S100A124 N RAGE is added to the wells Following a wash to remove any unbound antibody HRP onge the remaining conjugate is allowed to react with the substrate H202 tetramethylbenzidine 2 eaction is stopped by addition of acidic solution and absorbance of the resulting yellow prodik 1s measured at 450 nm The absorbance is proportional to the concentration of S1O0A12 EN RAG standard curve is constructed by plotting absorbance values versus S100A12 EN RAGE concen 1ons of calibrators and concentrations of unknown samples are determined using this standard cure The Circulex S100A12 EN E ELISA Kit Ver 2 is designed to measure the concentration of human 100A 12 EN RAGE serum plasma and other biological media Ni O Q O RS A
9. fore use eS All microplate strips that are not immediately required should be returned to the zip lock Xa which must be carefully resealed to avoid moisture absorption amp e Do not use kit components beyond the indicated kit expiration date e Use only the microtiter wells provided with the kit Meg e Rinse all detergent residue from glassware Rd e Use deionized water of the highest quality gt e Do not mix reagents from different kits Ni The buffers and reagents in this kit may contain preservatives V chemicals Care should be taken to avoid direct contact with these reagents lt Q e Do not smoke eat or drink when performing the yay or in areas where samples or reagents are handled aS e Dispose of tetra methylbenzidine TMB contgiGhe solutions in compliance with local regulations e Do not mouth pipette or ingest any of the reagents e Avoid contact with the acidic Stop Song d Substrate Solution which contains hydrogen peroxide e Wear gloves and eye protection whey Fandling immunodiagnostic materials and samples of human origin and these reagents In case gf contact with the Stop Solution and the Substrate Solution wash skin thoroughly with water and se edical attention when necessary Biological samples may be EL minatead with infectious agents Do not ingest expose to open wounds or breathe aeros gt Wear protective gloves and dispose of biological samples properly e CAUTION Sulfu
10. it Cat CY 8062 CircuLex S100A11 ELISA Kit Ggt CY 8063 CircuLex S100A14 ELISA Kit at CY 8064 CircuLex S100A7 Psoriasi SA Kit Cat CY 8073 CircuLex S100A4 ELISAGgit Ver 2 Cat CY 8086 Anti Human S100A ne YK 3E3 Cat CY M1039 Anti Human S100 a Cat CY P1026 Anti Human S10 at CY P1028 Anti Human S100A10 Cat CY P1033 Anti Human A16 Cat CY P1034 0A3 Cat CY P1039 100A2 Cat CY P1040 Hum Sion Cat CY R2250 Hunfah S100A1 Cat CY R2251 Hugpan S100A2 Cat CY R2252 HGman S100A3 Cat CY R2253 fuman S100A4 Cat CY R2254 A ni S 100A12 EN RAGE ELISA Kit Ver 2 C 1rcu Lex User s Manual AS For Research Use Only Not for use in diagnostic procedures y ra S100A12 EN RAGE ELISA Kit Ver 2 re L 1rcu Lex User s Manual rey For Research Use Only Not for use in diagnostic procedures s gt Human S100A6 Cat CY R2256 re Human 100A7 Cat CY R2257 aN Human S100A8 Cat CY R2258 lt Human S100A9 Cat CY R2259 G Ww Human S100A9 Cat CY R2259 H K Human S100410 Cat CY R2260 Human S100A12 Cat CY R2262 G Human S100412 Cat CY R2262 H N Human 100A13 Cat CY R2263 y Human S100414 Cat CY R2264 Human S100A16 Cat CY R2266 Human S100P Cat CY R2267 Human S100A11 Cat CY R2269 Human S100A1 Low Endotoxin Cat CY R2451 Human 100A3 Low Endotoxin Cat CY R2453 Human S100A4 Low Endotoxin Cat CY R2454 oY Human S100
11. linearized by using log log paper sio analysis may be applied to the log transformation To determine the human S100A12 aQycentration of each sample first find the absorbance value on the y axis and extend a horizontal li the standard curve At the point of intersection extend a vertical line to the x axis and read the c ponding human 100A 12 concentration If the samples have been diluted the concentration read fr he standard curve must be multiplied by the dilution factor AR 1 The dose response curve of this assay fits best to a sigmoidal 5 paragr logistic equation The results of unknown samples can be calculated with any computer progfam having a 5 parameter logistic function It is important to make an appropriate mathematical ANustment to accommodate for the dilution factor 2 Most microtiter plate readers perform automatic calculations of flyte concentration The calibration curve is constructed by plotting the absorbance Y of Mbravors 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 logit of absorbance Y is plotted versus log of the known concentration X of calibrators Measurement Range amp Y The measurement range is 20 pg mL to 1 28 nL Any sample reading higher than the highest standard should be diluted with Dilution Buff igher dilution and re assayed Dilution factors need to be taken into c
12. ning 20 mL of the chromogenic substrate tetra methylbenzidine TMB Ready to use Sto lution One bottle containing 20 mL of 1 N H2S014 Ready to use e Roa CY 8058V2 3 Version 151118 amp lt C A CircuLex Uie Manual Ss S100A12 EN RAGE ELISA Kit Ver 2 gt For Research Use Only Not for use in diagnostic procedures 4 Materials Required but not Provided 3 e Pipettors 2 20 uL 20 200 uL and 200 1000 uL precision pipettors with disposable tips RS N Precision repeating pipettor N Orbital microplate shaker g e Microcentrifuge and tubes for sample preparation 2 e Vortex mixer oO e Optional Microplate washer Manual washing is possible but not proggie e Plate reader capable of measuring absorbance in 96 well plates Gi wavelengths of 450 nm 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be us The plate can also be read at a single wavelength of 450 nm which will give a somewhat en Optional Software package facilitating data generation q analysis 500 or 1000 mL graduated cylinder I e Reagent reservoirs amp Y e Deionized water of the highest quality wW O e Disposable paper towels Roa CY 8058V2 4 Version 151118 o c Ss S100A12 EN RAGE ELISA Kit Ver 2 gt CircuLex Uer Manual For Research Use Only Not for use in diagnostic procedures F Precautions and Recommendations 3 e Allow all the components to come to room temperature be
13. onsideration in calculating thg human S100A12 concentration Troubleshooting Pi 1 All samples and standards should b assayed in duplicate using the protocol described in the Detailed Protocol Incubation times or peratures significantly different from those specified may give erroneous results CA 2 Poor duplicates accompafeed by elevated values for wells containing no sample indicate insufficient washing If all instructi is in the Detailed Protocol were followed accurately such results indicate a need for washer mai ce 3 Overall low signa may 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 7 ReagQt Stability All N reagents included in the CycLex Research Product Circulex 100A12 EN RAGE ELISA Kit Ver have been tested for stability Reagents should not be used beyond the stated expiration date Up ceipt kit reagents should be stored at 4 C except the reconstituted Human S100A12 Standard e stored at below 70 C Coated assay plates should be stored in the original foil bag sealed by the lock and containing a desiccant pack kA at CY 8058V2 9 Version 151118 o AO RS I S100A12 EN RAGE ELISA Kit Ver 2 Circu Lex User s Manual A For Research Use Only Not for use in diagnostic procedures amp Assay Characteristics 1 Sensitivity N The limit of detec
14. r 450 595 nm can also be used Read the microplate at 450 nm if only a sin velength can be used Wells must be read within 30 minutes of adding the Stop Solution iS Note 1 Compleferemoval of liquid at each step is essential to good performance After the last wash remo ny remaining Wash Buffer by aspirating or decanting Invert the plate and blot it agai clean paper towels Note 2 R le standard curves are obtained when either O D values do not exceed 0 2 units for the zero concentration or 2 5 units for the highest standard concentration The plate ould be monitored at 5 minute intervals for approximately 30 minutes Note 3wIf the microplate reader is not capable of reading absorbance greater than the absorbance of the highest standard perform a second reading at 405 nm A new standard curve constructed using the values measured at 405 nm is used to determine S100A12 EN RAGE concentration PN of off scale samples The readings at 405 nm should not replace the on scale readings at 450 nm Roa CY 8058V2 8 Version 151118 o AO S100A12 EN RAGE ELISA Kit Ver 2 L ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 A Q O Q Calculations Average the duplicate readings for each standard control and sample and subtract the a Zero standard optical density Plot the optical density for the standards versus the concentr of the standards and draw the best curve The data can be
15. rd The Dilution Buffer serves as the zero standard Blank wO Volume of sanae Dilution Buffer Concentration Std 1 50 uL of Master Standard ng mL 950 uL 1 280 pg mL Std 2 300 uL of Std 1 1 280 p 300 uL 640 pg mL Std 3 300 uL of Std 2 640 p 300 uL 320 pg mL Std 4 300 uL of Std 3 320 g mL 300 uL 160 pg mL Std 5 300 uL of Std 4 g mL 300 uL 80 pg mL Std 6 300 uL of Std pg mL 300 uL 40 pg mL Std 7 300 uL of SQ 40 pg mL 300 uL 20 pg mL Blank A 300 uL 0 pg mL 2 lt S Note Do not use Keating pipette Change tips for every dilution Wet tip with Dilution Buffer before ipl Unused portions of Master Standard should be aliquoted and stored at below 70 C D we Avoid multiple freeze and thaw cycles Sample Prefaration Dilute s with Dilution Buffer e Ther mended dilution for serum and plasma samples is 100 to 1 000 fold UsQ ould determine appropriate dilution ratio of other biological samples amp A Roa CY 8058V2 7 Version 151118 o c Assay Procedure paa w n N oo 10 12 S100A12 EN RAGE ELISA Kit Ver 2 L ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Remove the appropriate number of microtiter wells from the foil pouch and place them ingee well holder Return any unused wells to the foil pouch refold seal with tape and store at 4 C Q 2 Dilute sample with Dilution Buffer See
16. ric N is a strong acid Wear disposable gloves and eye protection when handli p Solution O nw a Qe amp A Roa CY 8058V2 5 Version 151118 o c S100A12 EN RAGE ELISA Kit Ver 2 CircuLex Uie Manual For Research Use Only Not for use in diagnostic procedures 4 A Q O Q Sample Collection and Storage 3 Serum Use a serum separator tube and allow samples to clot for 60 30 minutes Copuatfase the samples at 4 C for 10 minutes at 1 000 x g Remove serum and assay immediately or stor simples on ice for up to 6 hours before assaying Aliquots of serum may also be stored at below 70 r extended periods of time Avoid repeated freeze thaw cycles Y Plasma Collect plasma using EDTA Naz as the anticoagulant If possible colle e plasma into a mixture of EDTA Naz and Futhan5 to stabilize the sample against spontaneous vitro complement activation Immediately centrifuge samples at 4 C for 15 minutes at 1 000 x ssay immediately or store samples on ice for up to 6 hours before assaying Aliquots of plasma mgyjalso be stored 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 Yon gt Other biological media Remove any particulates by centrifugation hd assay immediately or aliquot and store samples at below 70 C Avoid repeated freeze thaw cy Xi ate CY 8058V2 6 Version 151118 o c S100A12 EN
17. s at 4 C e Don t expose sents to excessive light o gL eA Roa CY 8058V2 1 Version 151118 o c S100A12 EN RAGE ELISA Kit Ver 2 L ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 A Q O Q Introduction 3 Members of the S100 protein family are low molecular mass acidic proteins chara d by cell type specific expression and the presence of 2 EF hand calcium binding domains The anulins are S100 proteins that are expressed in neutrophils and are abundant in infiltrating me amp Ocytes and granulocytes under conditions of chronic inflammation Hofmann et al 1999 reported that RAGE is a central cell surface receptor for S1 2 which they referred to as EN RAGE Extracellular Newly identified RAGE binding protein elated members of the S100 calgranulin superfamily Interaction of EN RAGE S100A12 witf gellular RAGE on endothelium mononuclear phagocytes and lymphocytes triggered cellular activation with generation of key proinflammatory mediators In murine models blockade of EN E RAGE quenched delayed type hypersensitivity and inflammatory colitis by arresting acti n of central signaling pathways and expression of inflammatory gene mediators 1 S100A12 was also isolated as proteins binding to three different allergic drugs amlexanox cromolyn and tranilast by drug affinity chromatography 2 This ing implies that these three compounds might interact with these prot
18. tion defined as such a concentration of S100A12 EN RAGE givin sorbance higher than mean absorbance of blank plus three standard deviations of the absorban y of blank A blank 3SD blank is better than 8 2 pg mL of sample Dilution Buffer was pipetted into blank wells 2 y Typical standard curve RA lt Standard Curve 400 600 800 1 000 1 200 1 400 100A12 conc pg mL Roa CY 8058V2 10 Version 151118 o c CircuLex Uer Manual For Research Use Only Not for use in diagnostic procedures gt S100A12 EN RAGE ELISA Kit Ver 2 O 2 Specificity l The antibodies in the Circulex S100A12 EN RAGE ELISA Kit Ver 2 react with human S1 2 and without detectable cross reactivities to other human S100 proteins as indicated below Reactivity to various recombinant S100 proteins eA Reactivity of 100A12 EN RAGE ELISA kit to various recombinant 100 proteins 3 Precision ge Intra assay Precision Preci within an assay Three samples of knowf oncentration were tested eight times on one plate to assess intra assay precision x e Intra assay WitGm Run n 8 CV 3 4 5 3 Inter assay Precip Precision between assays Three samples of known concentration were tested in four separate assays to assess inter assay precision amp iggy Run to Run n 4 CV 5 3 6 2 amp N at CY 8058V2 11 Version 151118 o c CircuLex Wace Manual gt S100A12
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