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1. 2 5 2 0 gt 1 5 lt 1 0 0 5 0 0 0 500 1 000 1 500 2 000 2 500 Human YKL 39 conc pg ml Ww C CY 8087 10 Version 120710 Human YKL 39 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 Three samples of known concentration were tested sixteen times on one plate to assess intrazass precision e Intra assay Within Run n 16 CV 2 4 5 7 O Human YKL39 conc pg ml Sampel Sample 2 Sample 3 O WMOAN ANN PWN Se ee Se Ee RWNrF OO C CY 8087 11 Version 120710 Human YKL 39 ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Inter assay Precision Precision between assays Two samples of known concentration were tested in five separate assays to assess inter ass precision e Inter assay Run to Run n 5 CV 5 0 7 4 Human YKL39 conc pg ml O Sample 1 Sample 2 T 2 286 1 863 9 Qy 3 305 5 797 4 4 313 8 822 5 5 301 2 860 MAX 347 9 90 MIN 286 1 MEAN 310 9 3 Linearity To assess the linearity of the assay samples con human YKL 39 were serially diluted with the Dilut dynamic range of the assay ig and or spiked with high concentrations of ffer to produce samples with values within the Human YKL 39 conc pg mL 0 C 0 0 2 0 4 0 62. 2 Dilute serum sample 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 8 Wash 4 times by filling each well with Wash Buffer 350 u pipette manifold dispenser or microplate washer sin squirt bottle multi channel 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
3. Solutions as follows Use the Master Standard to produce a dilution series bel MiX each tube thoroughly before the next transfer The 2 400 pg mL standard Std 1 servestas 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 24 ng mL 540 uL 2 400 pg mL Std 2 300 uL of Std 1 2 400 pg mL 300 uL 1 200 pg mL Std 3 300 uL of Std 2 1 200 pg m 300 uL 600 pg mL Std 4 300 uL of Std 3 600 pg 300 uL 300 pg mL Std 5 300 uL of Std 4 300 p 300 uL 150 pg mL Std 6 300 uL of Std 5 15 300 uL 75 pg mL Std 7 300 uL of Std 6 7 300 uL 37 5 pg mL Blank 300 uL 0 pg mL Note Do not use a Repeat ipette Change tips for every dilution Wet tip with Dilution Buffer before dispensing Unu ortions of Master Standards should be aliquoted and stored at below 70 C immediatelysAyvoid multiple freeze and thaw cycles Sample Prep e Serum and mples require a 25 fold dilution e Oti amples require neat to appropriate dilution CY 8087 Version 120710 Human YKL 39 ELISA Kit n TM CircubLex User s Manual For Research Use Only Not for use in diagnostic procedures Assay Procedure 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
4. User s Manual For Research Use Only Not for use in diagnostic procedures Related Products CircuLex Human Chitotriosidase ELISA Kit Cat CY 8074 CycLex Chitotriosidase Fluorometric Assay Kit Cat CY 1249 CircuLex Human YKL 40 ELISA Kit Cat CY 8088 Coming soon CycLex Co Ltd 1063 103 Terasawaoka Ina Nagano 396 0002 Japan Fax 81 265 76 7618 e mail info cyclex co jp URL http www cyclex co jp PRODUCED BY a 2 CycLex CircuLex products are supplied for rese CycLex CircuLex products and components thereof may not be resold modifie le or used to manufacture commercial products without prior written approval from Cy Co Ltd To inquire about licensing for such commercial use please contact us via email C CY 8087 16 Version 120710
5. 0 8 1 Sample dilution ratio C CY 8087 12 Version 120710 Human YKL 39 ELISA Kit TM Circulex User s Manual For Research Use Only Not for use in diagnostic procedures Example of Test Results Fig 1 Concentrations of human YKL 39 in cell lysates of three cell lines 600 500 300 200 100 Human YKL 39 conc pg mg protein MCE7 DU145 U937 Cell lines AV dia of three cell lines Fig 2 Concentrations of human YKL 39 in conditii Human YLK 39 conc pg mL A S S 300 200 100 0 THP 1 Jarkat MCE7 Cell line C CY 8087 13 Version 120710 Human YKL 39 ELISA Kit TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Fig 3 Human YKL 39 levels in 15 sera of healthy volunteers 10 0 9 0 8 0 7 0 6 0 5 0 4 0 3 0 Human YKL 39 conc ng mL 2 0 C CY 8087 14 Version 120710 Human YKL 39 ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures References 1 Funkhouser J D and Aronson NN 2007 Chitinase family GH18 evolutionary insights fro genomic history of a diverse protein family BMC Evol Biol 7 96 3 Hu B Trinh K Figueira WF Price PA 1996 Isolation and sequence of a nov uman chondrocyte protein to mammalian members of the chi
6. Citrate plasma has not been validated for use in this assay G Other biological samples Remove any particulates by centrifugation a eS immediately or aliquot and store samples at below 70 C Avoid repeated freeze thaw cycles C CY 8087 6 Version 120710 Human YKL 39 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 YKL 39 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 YKL 39 Standard within the kit should be included in each assay as a calibrate pipette tips and reagent troughs should be used for all liquid 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 YKL 39 Standa 1 Prepare a working solution of Wash Buffer by adding 100 mL of the 10X fer to 900 mL of deionized distilled water ddH O Mix well Store at 4 C for two fe C for long term storage 2 Reconstitute Human YKL 39 Standard with 0 5 mL of ddH O YKL 39 in vial should be 24 ng mL which is referred as a Mas centration of the human adard of human YKL 39 Prepare Standard
7. Human YKL 39 ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures ELISA Kit for Measuring Human YKL 39 CircuLex Human YKL 39 ELISA Kit cA Cat CY 8087 O Intended USe c cc eeeeccccceesseeeeeeseeeeseteeeeees 1 PUA I SEESE 1 Introduction eeccesesesseeeeseseeeeseteeeenenees 2 Principle Of the ASSAY cccsesscasectssassearsenssensvens 2 3 Materials Provided c ccccesccceserseeeeeeees 3 Materials Required but not Provided 4 Precautions and Recommendation 5 QO Sample Collection and Storage 6 Detailed Protocol ceesesceeeeseeeeseseeeeeeees 7 8 Calculations ccccecscecesesceeeeeeecesseeeeeeees 9 Measurement Range 9 Troubleshooting seeeeeeeeeeeeeeeeeereseereerssee 9 Reagent SCADIUY cccccassancaceasansaveicntessvsndiiadunsis 10 Assay Characteristics cesceesseeesseeeenneeees 10 Example of Test RESUl5S pcssssccasssscececassscevenes 14 ReferenC S ercssrriaenisestenssssi riss 15 Related Products ccccccesescceessreeeeeseneeees 16 Intended Use The CycLex Research Product CircuL YKL 39 ELISA Kit is used for the quantitative measurement of human YKL 39 in seru asma cell culture medium and other biological media This assay kit is for research use olfa t for use in diagnostic or therapeutic procedures Storage e Upon receipt store all co e Don t expose reagents toje C CY 8087 1 Versi
8. ate kit Microplate One microplate supplied ready t ith 96 wells 12 strips of 8 wells in a foil zip lock bag with a desiccant pack Wells are coated uman YKL 39 antibody as a capture antibody 10X Wash Buffer One bottle containing 100 4L of 10X buffer containing 2 Tween 20 Dilution Buffer One bottles contai of 1X buffer use for sample dilution Ready to use Human YKL 39 Standard O ntaining 12 ng of lyophilized recombinant human YKL 39 HRP conjugated Detectio conjugated anti human Y y One bottle containing 12 mL of HRP horseradish peroxidase ody Ready to use Substrate Reagent containing 20 mL of the chromogenic substrate tetra methylbenzidine TMB Ready t Stop Solution le containing 20 mL of 1 N H SOy Ready to use C CY 8087 3 Version 120710 Human YKL 39 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 wave s of 450 nm 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be used can also be read at a single wavelength of 450 n
9. hen hai iMmmunodiagnostic materials and samples of human origin and these reagents In case of co ith the Stop Solution and the Substrate Solution wash skin thoroughly with water and seek Ci when necessary Biological samples may be cont a ith infectious agents Do not ingest expose to open wounds or breathe aerosols Wear protective gloves and dispose of biological samples properly e CAUTION Sulfuric Aci ng acid Wear disposable gloves and eye protection when K amp Y C CY 8087 5 Version 120710 Human YKL 39 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 serum and assay immediately or store sam o 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 Na as the anticoagulant If possible collect the to a 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 Assaypimmediately or store samples on ice for up to 6 hours before assaying Aliquots of plasma may Gy at below 70 C for extended periods of time Avoid repeated freeze thaw cycles Note
10. ificantly 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 ndic 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 C CY 8087 9 Version 120710 Human YKL 39 ELISA Kit TM Circulex User s Manual For Research Use Only Not for use in diagnostic procedures Reagent Stability All of the reagents included in the CycLex Research Product CircuLex Human YKL 39 ELIS have been tested for stability Reagents should not be used beyond the stated expiration date receipt kit reagents should be stored at 4 C except the reconstituted YKL 39 Standard mus below 70 C Coated assay plates should be stored in the original foil bag sealed by the containing a desiccant pack Assay Characteristics 1 Sensitivity The limit of detection defined as such a concentration of human YKL 39 giyi ance higher than mean absorbance of blank plus three standard deviations of the absorban e o nk in 24 assay is better than 35 1 pg mL of sample Dilution Buffer is pipetted into blank wells Typical standard curve A A si Human YKL 39 Standard Curve
11. m which will give a somewhat higher readi Software package facilitating data generation and analysis opti 9 500 or 1000 mL graduated cylinder e Reagent reservoirs e Deionized water of the highest quality e Disposable paper towels C CY 8087 4 Version 120710 Human YKL 39 ELISA Kit n TM CircuLex User s Manual A 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 All microplate strips that are not immediately required should be returned to the zip lock W 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 detergent residue from glassware Qy e Use deionized water of the highest quality A e Do not mix reagents from different kits Q e The buffers and reagents used in this kit contain NaN as preserva e should 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 assay or eas where samples or reagents are handled e Dispose of tetra methylbenzidine TMB contain tions in compliance with local regulations e Avoid contact with the acidic Stop Solution and Substrate Solution which contains hydrogen peroxide e Wear gloves and eye protection w
12. nd extend a horizontal line t dard curve At the point of intersection extend a vertical line to the x axis and read the ding YKL 39 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 aving a 4 parameter logistic function It is important to make an appropriate mathematical adjus accommodate for the dilution factor 2 Most microtiter plate readers perform automatic calculations of analyte curve is constructed by plotting the absorbance Y of calib concentration X of calibrators using the four parameter f function can be used to linearize the calibration curve i e lo log of the known concentration X of calibrators ration The calibration sus log of the known tion Alternatively the logit log Ofyabsorbance Y is plotted versus Measurement Range The measurement range is 37 5 pg mL to 2 400 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 39 concentration Troubleshooting 2 1 The Human YKL 39 Standard should begun plicate using the protocol described in the Detailed Protocol Incubation times or temperatures sign
13. 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 icroplate 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 YKL 39 concentration of off scale amples The readings at 405 nm should not replace the on scale readings at 450 nm Note 1 Complete remoya CY 8087 Version 120710 Human YKL 39 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 YKL 39 co n each sample first find the absorbance value on the y axis a
14. on 120710 Human YKL 39 ELISA Kit n TM Circulex User s Manual For Research Use Only Not for use in diagnostic procedures Introduction YKL 39 also known as chitinase 3 like 2 is secreted chitinase like protein which contains Glyc domain with no chitinase catalytic activity but with putative lectin properties 1 2 In contras closest homologue YKL 40 YKL 39 is not a glycoprotein YKL 39 is secreted a chondrocytes also synoviocytes 3 and activates signal regulated kinases ERK1 ERE embryonic kidney cells 4 Increased levels of YKL 39 have been demonstrated in syn patients with osteoarthritis OA or rheumatoid arthritis RA 5 6 as well as in malig particularly in glioblastomas 7 It is currently recognized as a biomarker for the activation of chondrocytes and the progress of the osteoarthritis in human 5 In addition autoantibodies to YKL 39 were detected in 8 11 8 of patients with RA 8 9 and 11 1 of patients with O ile only 1 of patients with RA had autoantibodies to YKL 40 8 Maturation of monocyte derived macrophages in the presence of Th2 cyto and TGF beta observed during chronic inflammations can not be attributed solely to the activity of chondrogyt s In perspective YKL 39 is also proposed as a useful biomarker to detect macrophag fic response in tumor atherosclerosis and neurodegenerative diseases including Alzheimer in addition to the chondrocyte specific activation The Ci
15. rcuLex Human YKL 39 ELISA Kit employs the q technique An antibody specific for human YKL 39 has oated onto a microplate Standards and samples are pipetted into the wells and the imm i After washing away any unbound substances an is added to the wells Following a wash to remove conjugate is allowed to react with the substrate H202 ethylbenzidine The reaction is stopped by addition of acidic solution and absorbance of the resulting yellow product is measured at 450 nm The absorbance is proportional to the inn TA uman YKL 39 A standard curve is constructed by plotting absorbance values versus human Y ncentrations of calibrators and concentrations of unknown samples are determined using oa curve Q jugated antibody specific for human YKL 39 bound antibody HRP conjugate the remaining C CY 8087 2 Version 120710 Human YKL 39 ELISA Kit TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Summary of Procedure Add 100 uL of diluted sample to the wells Yy Incubate for 1 hour at room temp Wash the wells O Add 100 uL of HRP conjugated anti human YKL 39 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 te The following components are supplied and are sufficient for the one 96 well micropl
16. tinase protein family J Biol Chem 271 2 Kzhyshkowska J Gratchev A and Goerdt S 2007 Human Chitinases and Proteins as Indicators for Inflammation and Cancer Biomarker Insights 2 128 19415 4 Areshkov PA and Kavsan VM 2010 Chitinase 3 like protein 2 CH 39 activates phosphorylation of extracellular signal regulated kinases ERK1 E man embryonic kidney HEK293 and human glioblastoma U87 MG cells Tsitol Ge 5 Knorr T Obermayr F Bartnik E Zien A Aigner T 2003 YKL but not YKL 40 chitinase 3 like protein 1 is up regulated i Rheum Dis 62 995 nase 3 like protein 2 itic chondrocytes Ann 6 Steck E Breit S Breusch S J et al 2002 Enhanced ex sio the human chitinase 3 like 2 gene YKL 39 but not chitinase 3 like 1 gene YK ost arthritic cartilage Biochem Biophys Res Commun 299 109 7 Kavsan V Dmitrenko V Boyko O et al 2 e ssion of YKL 39 Gene in Glial Brain Tumors Scholarly Research Exchange 200 ID 814849 doi 10 3814 2008 814849 8 Sekine T Masuko Hongo K Matsui T et al Recognition of YKL 39 a human cartilage related protein as a target antigen in Q rheumatoid arthritis Ann Rheum Dis 60 49 h i 9 Tsuruha J Masuko Hongo K Kato t al 2002 Autoimmunity against YKL 39 a human cartilage derived protein in O Oarthritis J Rheumatol 29 1459 C CY 8087 15 Version 120710 Human YKL 39 ELISA Kit TM CircuLex
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