Data Sheet
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1. Chitotriosidase Fluorometric Assay Kit P cLex User s Manual For Research Use Only Not for use in diagnostic procedures Chitotriosidase Activity Assay Procedure Following Table 1 below first add Test Sample or 3 X10 diluted Recombinant Chitotriosidase or 1 1X Assay Buffer to each well of a microplate Table 1 Reaction mixture o Test Positive e Assay Reagents Assay Control ol Test Sample 5 uL Qy 3 X10 diluted Recombinant Chitotriosidase gw 1 1X Assay Buffer e a PI s 2 Reaction Buffer 95 uL L 95 uL Test sample Human serum plasma sample or culture supernat ted macrophages If sample might contain high concentration of chitotriosidase such as seru sma from Gaucher s disease patients samples should be diluted 25 50 times in 1 1X A er before incubation See page 5 section Preparation of Reagents within 2 Finally initiate the reaction by adding 95 pL 4 thoroughly See page 5 section Preparation of Reagents within the kit 3 Read fluorescence intensity for 30 60 min Qi length of time at 2 to 5 minute intervals using a microplate fluorometer with excitatio 4 nm and emission at 440 460 nm at 30 C Any assay temperature from roo Cy to 37 C may be used c tion while the reaction velocity remains constant eaction Buffer to each well and mixing 4 Measure and calculate the rate o 3 After incubatio2. 4 Chitotriosidase Fluorometric Assay Kit cLex User s Manual For Research Use Only Not for use in diagnostic procedures Quantitative test kit for chitotriosidase activity CycLex Chitotriosidase Fluorometric cA Assay Kit Fo ays Cat CY 1249 Intended MISE occsscicea snccnaconioasmauteaeenaecteanies 1 of SLOT tat e gt eee ee ere eee eee ere eee ens err re 1 TIAA OM case orensuancaoutsincaaacdandeotoanearaeadectese 2 Principle Of the ASSAY isscccenssecascescssesnesaesaces 3 Materials Provided sscssisissscsesasassstevenssseiaceesees 3 Materials Required but not Provided 4 PEE CAUIIONS soscccaacesdhaconacacetacdsivsidaratasyehunauenes 4 Detailed Protocol aia ccsssisiiss seisactssestieseecesensses 5 8 Evaluation of RESults cccccccssessearscessareeee 9 10 TrombleshoOun gs sisccccecssinsisncatencdssioveaereatves 11 Reagent Stability siissssicarcccececessavevcasanessss sees 11 Referents is cciviesritaceussasedspesceeancsanvinsarencdeass 11 Related Proguets wi is sscavaccseusssnncasecssovevadeneass 1 Intended Use The CycLex Research Product CycLex Chitetriosidase Fluorometric Assay Kit is used for the quantitative measurement of chitotriosidase in serum plasma culture supernatant of activated macrophages and other biological samples Applications for this kit include 1 Measuring chitotriosidase activitypi plasma and other biological samples 2 Evaluating the effects of ph c ical compounds on chitotriosidase activi
3. al Dose Dependency Curve Qy y 4 Chitotriosidase Dose Dependency 20min IO e cen eg ect ng pect ag tena ae epey matinee mets ict cea geo a 450 300 counts F340 F460 x10 a 0 2 4 6 8 10 12 Chitotriosidase amount ng w Analysis of Inhibitor Effect Assay Vehicle Control r Control 3 Calculate the Intensi Reaction velocity of Test Assay ity X 100 Reaction velocity of Vehicle Control Intensity 1 Run reactions with test comp ehicle as described in the Detailed Protocol 2 Subtract fluorescence inte No Enzyme Control from all other experimental samples Test NOTE This tensity is a rough value of enzyme activity or inhibition For greater accuracy plot a anid ve of Chitotriosidase for each new set of reactions and estimate the Activity see section Chitotriosidase Standard Curve and Activity Catt CY 1249 10 Version 121011 Chitotriosidase Fluorometric Assay Kit Pa ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Troubleshooting 1 All assays should be run in duplicate using the protocol described in the Detailed Protocol Incub times or temperatures significantly different from those specified may give erroneous results 2 The reaction curve is nearly a straight line if the kinetics of the assay is of the first order the protocol can lead to non linearity of the curve as can assay kinet
4. d Recombinant Chitotriosidase 5 uL 1 1X Assay Buffer MQ VT 5L Test Compound Vehicle of Test Compound A sup ed Sul 10X allosamidin 500 uM 2 Reaction Buffer See page 5 section Preparation of Reagen the kit Not provided in this kit See page 4 s n Materials Required but not Provided 2 Initiate reactions by adding 90 pL oft action Buffer to each well and mixing thoroughly See page 5 section Preparati agents within the kit 3 Read fluorescence intensit 60 min or desired length of time at 2 to 5 minute intervals using microtiter plate fluoro T xcitation at 340 380 nm and emission at 440 460 nm at 30 C Any assay temper room temperature to 37 C may be used 4 Measure and ate of reaction while the reaction velocity remains constant Caution an Qi ance S be assayed in duplicate Alls off microplate shaker is recommended for complete mixing sst Compounds or samples themselves emit fluorescence at excitation wavelength 340 380 nm and fl orescence wavelength 440 460 nm the test assay cannot be evaluated correctly Cat CY 1249 7 Version 121011 Chitotriosidase Fluorometric Assay Kit G ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Determine microplate reader conversion factorfor 4 Methylumbelliferone fluoroph The exact 4 Methylumbelliferone concentratio
5. diagnostic procedures Principle of the Assay The CycLex Chitotriosidase Fluorometric Assay Kit is based on an exclusive fluores substrate 4 Methylumbelliferyl B D N N N triacetylchitotriose This homogenous assay 1 sensitive and convenient Summary of Procedure Dispense 5 uL of sample or Recombinant chitotriosidase in the well Add 95 uL of 1X Fluoro Substrate solution Q Measure velocity of fluorescence intensity with excitation O nm and emission at 440 460 nm for 30 60 min at 30 C Materials Provided Each kit contains Materials Da Quantity Storage 10X Chitotriosidase Assay Buffer x r 1 0mL x 2 Below 20 C 50X Fluoro Substrate 1 mM 4 MU chitotrioside 220 uL x1 Below 20 C Recombinant Chitotriosidase ca 5 pg ml 50uLx1 70 C 4 Methylumbelliferone standard 1 MM Jooo 200 uLx1 Below 20 C lnstruction manual room temp C CY 1249 3 Version 121011 Chitotriosidase Fluorometric Assay Kit G ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Materials Required but not Provided e Allosamidin Allosamidin is available from 2A PharmaChem cat 2A 300198 CAS nu 103782 08 7 e Microplate suitable for use with a fluorometric plate reader black microplates et signal to noise ratio e Microplate reading fluorometer capable of excitation at a wavelength in the range 0 nm and detection of
6. dures The CycLex Research Product CycLex Chitotriosidase Fluorometric Assay Kit is provided concentrated reagents Disposable pipette tips and reagent troughs should be used for all liquid tr e Thaw the reagents at room temperature except Recombinant Chitotriosidase and keep all reagents on ice until use Use them only after they are completely thawed and mixed deionized water Mix well Unused buffer should be stored at 20 C 1 Prepare 1X Assay Buffer by diluting the 10X Assay Buffer ee 2 Prepare Reaction Buffer according to the table below Mix well in distilled y unused Reaction Buffer Reaction Buffer Assay reagents lassay 8assa 1 ays 32 assays 48 assays 10X Assay Buffer 10 uL 160uL 320pL 480 uL 50X Fluoro Substrate 2 uL 32 uL 64 uL 96 uL Distilled water 88 u 1408 uL 2816 uL 4224 uL Total volume 100 u 800 uL 1600 uL 3200uL 4800 uL 3 Prepare X10 diluted Recombinant Chitotriosidase provided 1 10 in 1 unused X10 diluted Recombinant Chitotsi after diluted itotriosidase by diluting say Buffer Mix well Put it on ice Discard any the Recombinant Assay reagents say 8 assays 16 assays 32 assays 48 assays 1 1X Assay Buffer 9 uL 45 uL 81 uL 153 uL 225 uL Recombinant Chitotriosid 1 uL 5 uL 9 uL 17 uL 25 uL Total volume O 10 uL 50 uL 90 uL 170 uL 250 uL CY 1249 Version 121011
7. emitted light in the range 440 460 nm Pipettors 2 20 uL 20 200 uL and 200 1000 uL precision pipettors with dispos tips Multi channel pipette G e Microplate shaker QO Deionized water of the highest quality e Reagent reservoirs Stop Solution Optional Add 23 6 ml of deionized e 2 gof sodium carbonate Cat 2127 and mix well until completely dissolved Store Solution c a 1 M Na2CO3 at room temperature Precautions and Recommendation e Please avoid repeated freezing and thawing Recombinant chitotriosidase in this kit There is a possibility that the enzyme activity may be i d Aliquot to 10 uL and store at 70 C e Do not use kit components beyond the i it expiration date e Rinse all detergent residue from gl e Use deionized water of the highes lity ddH 0 e Do not mix reagents from di t Kits e Do not mouth pipette or 1 t any of the reagents e Do not smoke ik when performing the assay or in areas where samples or reagents are handled Biological s S may be contaminated with infectious agents Do not ingest expose to open wounds or aerosols Wear protective gloves and dispose of biological samples properly C CY 1249 4 Version 121011 yclex Detailed Protocol User s Manual to avoid cross contamination of reagents or samples Preparation of Reagents within the kit Chitotriosidase Fluorometric Assay Kit For Research Use Only Not for use in diagnostic proce
8. f chitinase protein family J Biol Chem 268 25803 Zonneveld A J and Aerts J M 1995 Cloning of a itinase produced by macrophages J Biol Chem 270 5 Renkema GH Boot Donker Koopman WE Strijland A Muijsers AO Hrebicek M Aerts JM 1998 Chitotri a chitinase and the 39 kDa human cartilage glycoprotein a chitin binding lectin are ho family 18 glycosyl hydrolases secreted by human macrophages Eur J ollak C E 1997 Plasma and metabolic abnormalities in Gaucher s disease matol 10 691 oer AM Wevers RA de Bruijn AM Groener JE Hollak CE Aerts JM Galjaard H elen OP 1995 Elevated plasma chitotriosidase activity in various lysosomal storage Version 121011 CyY 1249 Chitotriosidase Fluorometric Assay Kit P cLex User s Manual For Research Use Only Not for use in diagnostic procedures disorders J Inher Metab Dis 18 717 8 Vedder A C Cox Brinkman J Hollak C E et al 2006 Plasma chitotriosidase in male F patients A marker for monitoring lipid laden macrophages and their correction by e replacement therapy Mol Genet Metab 89 239 9 Grosso S Margollicci M A Bargagli E et al 2004 Serum levels of chitotriosidase disease activity and clinical stage in sarcoidosis Scand J Clin Lab Invest 64 57 10 Boot R G van Achterberg T A van Aken B E et al 1999 Strong induction of members of the chitinase family of proteins in atheroscler
9. ics of other than fir non linear curve point to point or quadratic curve fit methods should be used or a 3 Poor duplicates accompanied by elevated values for wells containing no sample indicate inaccurate dispensing of assay reagents If all instructions in the Detailed Protocol were accurately such results indicate a need for multi channel pipette maintenance y Kit have been tested receipt all kit reagents mbinant Chitotriosidase All of the reagents included in the CycLex Chitotriosidase Fluoromet for stability Reagents should not be used beyond the stated expiration should be stored at 70 C Avoid repeated freeze thaw cycles of After use return kit reagents to 70 C as soon as possible For research use only not for use in human diagnostic or utic procedures References 1 Hollak C E van Weely S van Oers M H chitotriosidase activity A novel hallmark of Gauchi s J M 1994 Marked elevation of plasma sease J Clin Invest 93 1288 2 Renkema G H Boot R G Muijsers A Purification and characterization of human proteins J Biol Chem 270 2198 Donker Koopman W E and Aerts J M 1995 iosidase a novel member of the chitinase family of 3 Boot R G Renkema G H Strijland cDNA encoding chitotriosidase 26252 4 Hakala BE White C Rec 1993 Human cartilage gp 39 a major secretory product of articular chondrocytes and 1 l cells is a mammalian member o
10. ment therap male Fabry patients 8 Elevated levels of serum chitotriosidase were also found in disor activation of immune system including sarcoidosis 9 and atherosclero that chitotriosidase activity was elevated up to 55 fold in extracts offfathe clear connection between chitotriosidase expression and lipid atherosclerotic vessel wall 10 Human chitotriosidase also ass and in particular with fungal infections suggesting the role chitin containing pathogens 12 13 Other clinical data for i her patients and sed by the abnormal 1 It has been shown erotic tissue showing a acrophages inside human th pathogen driven diseases zyme in host defense against at chitotriosidase activity is alciparum malaria 14 Additional evidence for a role of chitotriosidase during immunologi s is the observation that the enzyme is shortly and acutely up regulated both at the lev d activity following stimulation about one third is directly routed to lysosomes and proteolytically processed to the 39 kDa unit remains catalytically active 17 A common chi therefore a defective enzyme activity In whi this abnormal chitotriosidase allele and appro tely 6 are homozygous 1 18 C CY 1249 2 Version with iosidase gene polymorphism leads to a null allele and lations 30 to 40 of individuals are carriers of 121011 Chitotriosidase Fluorometric Assay Kit c3 ycLex User s Manual For Research Use Only Not for use in
11. n a Solution Fluoresce re as Tead on a microplate fluorometer with excitation at 340 380 nm and emission at 440 460 Alternatively See page 4 Materials Required but not Provided C CY 1249 6 Version 121011 4 Chitotriosidase Fluorometric Assay Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Inhibitor Screening Assay Procedure In order to estimate the inhibitory effect on chitotriosidase activity by test compounds correctly dt necessary to conduct the control experiment of Vehicle Control at least once for every experi and Inhibitor Control at least once for the first experiment in addition to Test Assay assi in the Table 1 below When test chemicals cause an inhibitory effect on Chitotriosidas level of increase of fluorescence intensity is weakened as compared with Vehicle increase in fluorescence intensity is not observed in Inhibitor Control 1 Following Table 1 below first add 3 X10 diluted Recombinant Chitotriosi Assay Buffer to microplate wells Second add Test Compound or Compound or 10X allosamidin to each well of the microplate and mix well se or 1 1X icle of Test Optional For best accuracy it is advisable to pre incubate the plat 0 min at assay temperature Table 1 Reaction mixture Assay Reagents Raa nhibitor No Enzyme ssay Control Control 3 X10 dilute
12. n range that will be useful for preparing a sta curve will vary depending on the fluorometer model the gain setting and the exact excitation jan emission wavelengths used Please dilute the 100X 4 Methylumbelliferone standard prowide uM to 1 0 uM as the highest standard and make 4 fold serial dilution with Assay B measuring the fluorescence of 50 ul in a microtiter plate fluorometer with excitation at 340 380 nm and emission at 440 460 nm The estimate of uM RFU obtained with this measurement toge observed range of values obtained in the enzyme assays can then be used to plan an appropriate se dilutions for a standard curve The slope of the standard curve can then be use the uM RFU conversion factor 4 Methylumbelliferone Standard Curve RE ye eee ee Typical 4 Methylumbelliferone Standard Curve ee i eae al y 20338x 548 55 2 S000 EEEE e EE 7 nob Ghelatebinacs a RFU F340 F460 LOU Steet a teen iment Rene 0 0 0 5 1 0 1 5 2 0 2 5 4 Methylumbelliferone conc uM C CY 1249 8 Version 121011 p Chitotriosidase Fluorometric Assay Kit f ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Evaluation of Results Analysis of Kinetics Time course curve 1 Run reactions as described in the Detailed Protocol 2 Subtract fluorescence intensity at time 0 from all reaction time points 3 Plot fluorescence intensity at 440 460 nm versus reaction
13. osis chitotriosidase and human cartil 39 expressed in lesion macrophages Arterioscler Thromb Vasc Biol 19 687 11 Artieda M Cenarro A Ganan A et al 2003 Serum chitotriosidas is increased in subjects with atherosclerosis disease Arterioscler Thromb Vasc Biol 23 12 Labadaridis J Dimitriou E Costalos C et al 1998 Serial chitot gidas activity estimations in neonatal systemic candidiasis Acta Paediatr 87 605 13 Labadaridis I Dimitriou E Theodorakis M et al 2005 14 Barone R Simpore J Malaguarnera L Pignatelli chitotriosidase activity in acute Plasmodium falciparu Clin Chim Acta 331 79 15 Di Rosa M Musumeci M Scuto A interferon gamma interleukin 10 lipopolys chitotriosidase synthesis in human macrophages S and Malaguarnera L 2005 Effect of ide and tumor necrosis factor alpha on Chem Lab Med 43 499 Prolactin induces chitotriosidase gene r ssion in human monocyte derived macrophages 16 Malaguarnera L Musumeci M Licata i Rosa M Messina A and Musumeci S 2004 Immunol Lett 94 57 17 Renkema G H Boot R G Strijlan onker koopman W E van den Berg M Muijsers A O and Aerts J M F G 1997 is sorting and processing into distinct isoforms of human macrophage chitotriosidase Earn J hem 244 279 18 Eiberg H den T 1997 Assignment of human plasma methylumbelliferyl tetra N acetylchitot Si chitinase to ch
14. romosome 19 by a linkage study Hum Genet 101 205 amp C CY 1249 12 Version 121011 wn Chitotriosidase Fluorometric Assay Kit e ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Related Products CircuLex Human Chitotriosidase ELISA Kit Cat CY 8074 Human Chitotriosidase Cat CY E1249 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 CycLex CircuLex products are supplied for rese CycLex CircuLex products and components thereof may not be resold modifie es r 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 1249 13 Version 121011
15. time 4 Determine the reaction time range in which the increase in fluorescence intensit 440 460 nm is linear 5 Calculate activity Fluorescence Intensity of Test amp Activity reaction velocity Reaction time mi NOTE Usually the linear range is from O to 30 min This val able depending on reaction conditions and storage handling of the Recombinant Chi Decreasing the amount of Recombinant Chitotriosidase in the assay may help to lefgthen the time range Fig l Typical Time Course Curve A Chitotriosidase Assay Time Course 250 N j nn RFU F340 F460 x10 _ S on Time min C CY 1249 9 Version 121011 Chitotriosidase Fluorometric Assay Kit ca ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Chitotriosidase Standard Curve and Activity Make serial dilutions of 3 X10 diluted Recombinant Chitotriosidase with 1 1X Assay Buffer 100 50 25 12 5 6 25 3 13 and 0 2 Run reactions with Vehicle and serial dilutions of Recombinant Chitotriosidase as describe Detailed Protocol 3 Plot standard curve data as fluorescence intensity at 460 nm versus dose of Chitotriosid ay 4 Obtain a line fit to the data using appropriate calculations 5 Use the slope and Y intercept to calculate the amount of Chitotriosidase activity for the e ental data Fig 2 Typic
16. ty 3 Screening inhibitors or activatorsof chitotriosidase y and not for use in diagnostic or therapeutic procedures Storage e Upon receipt C CY 1249 1 Version 121011 Chitotriosidase Fluorometric Assay Kit Pa ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Introduction Chitotriosidase was discovered in plasma of patients suffering from Gaucher s disease it was f that the 1 000 fold elevated enzyme originates from lipid laden macrophages that accumulate in tissues of Gaucher s patients 1 Chitotriosidase has subsequently been purified from thegsplee Gaucher s patient and its cDNA was cloned from a human macrophage cDNA library enzyme is a human chitinase member of family 18 glycosyl hydrolases 3 5 and has the capabi hydrolyze chitin This enzyme is selectively expressed in activated tissue macrophages tha in various tissues of several lysosomal diseases 6 Therefore its activity has been proposed biochemical marker of macrophage accumulation in Gaucher s disease 1 7 In so lysosomal storage disorders especially sphingolipidoses such as Niemann Pick and Krabbe disease which involve accumulation of different lipids more modest ou other inherited ngliosidosis s in plasma chitotriosidase have been noted 7 Chitotriosidase is the only biomarker identifie o date for the monitoring the efficacy of the extremely costly enzyme replace
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