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1. aa Human Chitotriosidase ELISA Kit CircuLex 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 fi 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 macrophages cDNA librar 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 ide e o date for the monitoring the efficacy of the extremely costly enzyme replacement 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 a2. The measurement range is 56 25 pg mL to 3 600 pe Any sample reading higher than the highest standard should be diluted with Dilution Buffer in iigher dilution and re assayed Dilution factors need to be taken into consideration in calculating the human Chitotriosidase concentration Troubleshooting 1 Human Chitotriosidase Standard shouldyb in duplicate using the protocol described in the Detailed Protocol Incubation times of temperatures 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 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 Q C CY 8074 9 Version 120710 Human Chitotriosidase 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 Chitotriosi ELISA Kit have been tested for stability Reagents should not be used beyond the stated expiratio Upon receipt kit reagents should be stored at 4 C except the reconstituted Human Chitots Standard must be stored at b
3. 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 e Software package facilitating data generation and analysis opti 500 or 1000 mL graduated cylinder e Reagent reservoirs e Deionized water of the highest quality e Disposable paper towels C CY 8074 4 Version 120710 Human Chitotriosidase 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 residues from glassware Qy e Use deionized water of the highest quality A e Do not mix reagents from different kits O 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 as
4. W E and Aerts Purification and characterization of human chitotriosidase a novel member of the chiti proteins J Biol Chem 270 2198 3 Boot R G Renkema G H Strijland A van Zonneveld A J and Aerts J M Cloning of a cDNA encoding chitotriosidase a human chitinase produced by macrophages Chem 270 26252 4 Hakala BE White C Recklies AD 1993 Human cartilage gp 39 a m etory product of articular chondrocytes and synovial cells is a mammalian member of chitinase ein family J Biol Chem 268 25803 Nn Renkema GH Boot RG Au FL Donker Koopman WE Strijland A JM 1998 Chitotriosidase a chitinase and the 39 kDa human ca lectin are homologues of family 18 glycosyl hydrolases se Biochem 251 504 AO Hrebicek M Aerts ycoprotein a chitin binding uman macrophages Eur J 6 Aerts J M and Hollak C E 1997 Plasma and metabohi normalities in Gaucher s disease Baillieres Clin Haematol 10 691 7 Guo Y He W Boer AM Wevers RA de Bruij van Diggelen OP 1995 Elevated plasma chi disorders J Inher Metab Dis 18 717 roener JE Hollak CE Aerts JM Galjaard H sidase activity in various lysosomal storage 8 Vedder A C Cox Brinkman J Hollak patients A marker for monitoring lipi replacement therapy Mol Genet Met al 2006 Plasma chitotriosidase in male Fabry macrophages and their correction by enzyme 9 Grosso S Margollicci M A Bar dis
5. for human chitotriosidase Standards and samples are pipetted into the wells and the an chitotriosidase present After washing away any unbound substances an HRP conjugat ibody specific for human chitotriosidase is added to the wells Following a wash to remo to react with the sub y unbound HRP conjugated antibody the remaining conjugate is allowed tetramethylbenzidine The reaction is stopped by addition of acidic he resulting yellow product is measured at 450 nm The absorbance is tion of human chitotriosidase A standard curve is constructed by plotting solution and ab a proportional to thefco absorbance value us human chitotriosidase concentrations of calibrators and concentrations of e unknown sam ermined using this standard curve The CycLe ch Product CircuLex Human Chitotriosidase ELISA Kit is designed to measure the co ti uman chitotriosidase in human serum plasma or cell culture conditioned medium C CY 8074 2 Version 120710 Human Chitotriosidase ELISA Kit n 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 wells Yy Incubate for 1 hour at room temp cA Wash the wells O v Add 100 uL of HRP conjugated anti human chitotriosidase antibody v Incubate for hour at room temp Wash the wells Add 100 uL of Substrate Reagent y v Add 100 uL of Stop Solution v Measure absorbance at 45
6. 0 nm Materials Provided All samples and standards should be assayed in are sufficient for the one 96 well microplate kit e The following components are supplied and 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 coat anti human chitotriosidase antibody as a capture antibody 10X Wash Buffer One bottle oinn Kop of 10X buffer containing 2 Tween 20 Dilution Buffer One bottle co 50 mL of 1X buffer use for reconstitution of Human Chitotriosidase Standard and sample dilution Ready to use Human Chitotriosidase Sta chitotriosidase HRP conjugated Det conjugated anti iosidase antibody Ready to use One vial containing 18 ng of lyophilized recombinant human tibody One bottle containing 12 mL of HRP horseradish peroxidase Substrate Reag a ne bottle containing 20 mL of the chromogenic substrate tetra methylbenzidine TMB Ready e Stop S n ottle containing 20 mL of 1 N H SO Ready to use C CY 8074 3 Version 120710 Human Chitotriosidase 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
7. 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 chitotriosidase ti of each sample first find the absorbance value on the y axis and extend a horizontal line dard curve At the point of intersection extend a vertical line to the x axis and read the correspo man chitotriosidase concentration If the samples have been diluted the concentration read from ti andard 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 microplate readers perform automatic calculations of analyte curve is constructed by plotting the absorbance Y of calib concentration X of calibrators using the 4 parameter function can be used to linearize the calibration curve i e logit of absofba known concentration X of calibrators ration The calibration sus log of the known ively the logit log function is plotted versus log of the Measurement Range
8. ctivity 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 zyme in host defense ag her patients and sed by the abnormal 1 It has been shown erotic tissue showing a acrophages inside human th pathogen driven diseases ainst 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 In the blood stream tissue macrophages largely s about one third is directly routed to lysosomes and p 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 olytically processed to the 39 kDa unit Principle of the Assay The CycLex Research Produ quantitative sandwich enzyme imm has been pre coated onto a immobilized antibody bin ex Human Chitotriosidase ELISA Kit employs with e newly synthesized 50 kDa chitotriosidase but that iosidase gene polymorphism leads to a null allele and lations 30 to 40 of individuals are carriers of the ssay technique An antibody specific
9. ease activity and clinical stag al 2004 Serum levels of chitotriosidase as a marker of idosis Scand J Clin Lab Invest 64 57 10 Boot R G van Achterber chitinase family of protei Aken B E et al 1999 Strong induction of members of the erosclerosis chitotriosidase and human cartilage gp 39 expressed r Thromb Vasc Biol 19 687 in lesion macrophages 11 Artieda M Cen anan A et al 2003 Serum chitotriosidase activity is increased in subjects with W disease Arterioscler Thromb Vasc Biol 23 1645 12 Labadaridis g ou E Costalos C et al 1998 Serial chitotriosidase activity estimations in neonatal s 1 didiasis Acta Paediatr 87 605 13 i imitriou E Theodorakis M et al 2005 Chitotriosidase in neonates with fungal ections Arch Dis Child Fetal Neonatal Ed 90 F531 CY 8074 Version 120710 Human Chitotriosidase ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures chitotriosidase activity in acute Plasmodium falciparum malaria Clin Chim Acta 331 79 15 Di Rosa M Musumeci M Scuto A Musumeci S and Malaguarnera L 2005 Effe interferon gamma interleukin 10 lipopolysaccharide and tumor necrosis factor alp chitotriosidase synthesis in human macrophages Clin Chem Lab Med 43 499 16 Malaguarnera L Musumeci M Licata F Di Rosa M Messina A and Musum Prolactin induces chitotriosidase gene exp
10. elow 70 C Coated assay plates should be stored in the ori sealed by the zip lock and containing a desiccant pack Assay Characteristics 1 Sensitivity The limit of detection defined as such a concentration of human chitotriosi ing absorbance higher than mean absorbance of blank plus three standard deviations of th ance of blank A blank 3SD blank is better than 48 3 pg ml of sample Dilution Buffer is pipetted into blank wells Typical Standard Curve O Human Chitotriosidase Standard Curve J paaa aaa EEES A450 0 0 1 0 2 0 3 0 4 0 Human Chitotriosidase conc ng ml amp C CY 8074 10 Version 120710 Human Chitotriosidase 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 1 2 6 O Human Chitotriosidase conc ng ml Sewmi Serum f Serum3 1 2 3 4 5 6 7 8 9 C CY 8074 11 Version 120710 Human Chitotriosidase ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Inter assay Precision Precision between assays Three samples of known concentration were tested in five separate assays to assess inter precision e Inter assay R
11. g 100 mL of the 10X fer to 900 mL of deionized distilled water Mix well Store at 4 C for one month or 20 erm storage 2 Reconstitute Human Chitotriosidase Standard with 0 5 mL of Dilu er The concentration of the reconstituted human Chitotriosidase should be 36 ng m I s referred as a Master Standard of human chitotriosidase Prepare Standard Solutions as follows Use the Master Standard to produce a dilution series AS each tube thoroughly before the next transfer The 3 600 pg mL standard Std 1 ser s the highest standard The Dilution Buffer serves as the zero standard Blank Volume of Standard ilution Buffer Concentration Std 1 60 uL of Master Standard 540 uL 3 600 pg mL Std 2 300 uL of Std 1 4 000 pg mL 300 uL 1 800 pg mL Std 3 300 uL of Std 2 2 000 300 uL 900 pg mL Std 4 300 uL of Std 3 1 000 300 uL 450 pg mL Std 5 300 uL of Std 4 S00 pg 300 uL 225 pg mL Std 6 300 uL of Std 5 25 300 uL 112 5 pg mL Std 7 300 uL of Std g mL 300 uL 56 25 pg mL Blank 300 uL 0 ng mL Note Do not use a Repeat ipette Change tips for every dilution Wet tip with Dilution Buffer before dispensing rtions of Master Standard should be aliquoted and stored at below 70 C immediate void multiple freezes and thaw cycles Sample Prep A e Serum and pl amples require a 50 fold dilution S e g 6 Q ple 294 uL Dilution Buffer e ia of cultured cells require neat to appropriate dil
12. moved 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 ard curves are obtained when either O D values do not exceed 0 25 units for the mcentration or 3 0 units for the highest standard concentration plate reader is not capable of reading absorbance greater than the absorbance of the dard perform a second reading at 405 nm A new standard curve constructed e values measured at 405 nm is used to determine human chitotriosidase tration of off scale samples The readings at 405 nm should not replace the on scale Note 1 Complete remoya Note 2 Reliabl bla Note 3 If t CY 8074 Version 120710 Human Chitotriosidase ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Calculations
13. ontaneous in vitro c ement activation Immediately centrifuge samples at 4 C for 15 minutes at 1 000 x g Assay immediately or store samples on ice for up to 6 hours before assaying Aliquots of plasma may als ed at bolow 70 C for extended periods of time Avoid repeated freeze thaw cycles Note Citrate plasma has not been validated for use in this assay Conditioned medium of cultured cells Remove any particulates trifugation and assay immediately or aliquot and store samples at below 70 C Avoid repeated aw cycles C CY 8074 6 Version 120710 Human Chitotriosidase 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 Chitotriosidase ELISA Kit is provided removable strips of wells so the assay can be carried out on separate occasions using only the nu strips required for the particular determination Since experimental conditions may vary angali the Human Chitotriosidase Standard within the kit should be included in each assay a Disposable pipette tips and reagent troughs should be used for all liquid trans cross contamination of reagents or samples Preparation of Working Solutions All reagents need to be brought to room temperature prior to the assay Assay Gy supplied ready to use with the exception of 10X Wash Buffer and Human Chitotriosidase d 1 Prepare a working solution of Wash Buffer by addin
14. ression in human monocyte derived Immunol Lett 94 57 17 Renkema G H Boot R G Strijland A Donker koopman W E van den Be Muijsers A O and Aerts J M F G 1997 Synthesis sorting and processing into distin s of human macrophage chitotriosidase Eur J Biochem 244 279 18 Eiberg H den Tandt WR 1997 Assignment of human pla ethylumbelliferyl tetra N acetylchitotetraoside or chitinase to chromosome 19 by a li tady Hum Genet 101 205 Related Products CircuLex Human Chitotriosidase ELISA Kit Cat CY 80 CycLex Chitotriosidase Fluorometric Assay Kit Cat C Chitotriosidase Cat CY E1249 PRODUCED BY 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 ed for research use only CycLex CircuLex products and components thereof may old modified for resale or used to manufacture commercial products without prior writt roval from CycLex Co Ltd To inquire about licensing for such commercial use pleas contact us via email N CycLex CircuLex products C CY 8074 15 Version 120710
15. rr Human Chitotriosidase ELISA Kit Circ uL ex User s Manual For Research Use Only Not for use in diagnostic procedures ELISA Kit for Measuring Human Chitotriosidase CircuLex Human Chitotriosidase ELISA Cat CY 8074 Intended USE cos ccacesancsaccepaioheuacaceeencediont 1 OPA E E E T 1 ntroduction sesseeeesseeeesesesssesessressessesssees 2 Principle of the Assay 2 3 Materials Provided iss sscsiacesssdsacnccescnevsenssesses 3 Materials Required but not Provided 4 Precautions and Recommendation 5 Sample Collection and Storage 6 Detailed Protocol aiscisissacscerccsvasescerseesossensavect 7 8 KALCALACIONS 55 sccsacesssusssusseneenvsasvansca teresina 9 Measurement Range 9 Troubleshooting ss isssicccecceedsssssccaccessontervecsnes 9 Reagent SCAB Y scasenssassscesstotuseccuadiorsvectnndeas 10 Assay Characteristics csscsessreesreeeeee 1 Example of Test ReSults ssissisciccsscscecccesvasevenee Referents nuncii rossii neries 1 Related Products sssvscescsescssssasacesaneoavencvensess 15 Intended Use The CycLex Research Product Circu an Chitotriosidase ELISA Kit is used for the quantitative measurement of human chitoffiosidase in human serum plasma cell extract cell conditioned medium and other biological samples This assay kit is for research use Only and not for use in diagnostic or therapeutic procedures Storage e Upon receipt store all co C CY 8074 1 Version 120710
16. say 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 when hai iMmunodiagnostic 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 i when necessary e 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 8074 5 Version 120710 Human Chitotriosidase 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 t d 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 sp
17. un to Run n 5 CV 3 07 5 02 Hu Chitotriosidase conc ng ml Serum Serum 2 Serum 3 32 7 20 4 1 1 2 3 4 5 3 Linearity To assess the linearity of the assay samples conta r spiked with high concentrations of chitotriosidase were serially diluted with the Dilut er to produce samples with values within the dynamic range of the assay 1 0 0 8 0 5 0 3 Human Chitotriosidase conc ng ml 0 0 C 0 0 5 1 1 5 2 2 5 Serum Dilution Ratio 1 100 C CY 8074 12 Version 120710 Human Chitotriosidase ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Example of Test Results Fig 1 Chitotriosidase concentrations in healthy Japanese volunteers sera n 36 and CRP positive n 35 45 p e 9 pons amp Y e 35 cl e a ee an 3 lt So Oe ee 2 a b w OO Lorne e 8 15 j A P a x 8 ae ee 8 EEEE e 5 0 8 C CY 8074 13 Version 120710 Human Chitotriosidase ELISA Kit n TM Circulex User s Manual For Research Use Only Not for use in diagnostic procedures References Hollak C E van Weely S van Oers M H and Aerts J M 1994 Marked elevation of pl chitotriosidase activity A novel hallmark of Gaucher disease J Clin Invest 93 1288 2 Renkema G H Boot R G Muijsers A O Donker Koopman
18. ution CY 8074 Version 120710 Human Chitotriosidase ELISA Kit n TM Circulex User s Manual For Research Use Only Not for use in diagnostic procedures Standard Assay Procedure ja 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 Dilute samples with Dilution Buffer See Sample Preparation above Pipette 100 uL of Standard Solutions Std1 Std7 Blank and conditioned media of A ells or diluted samples in duplicates into the appropriate wells 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 N w D 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 sing squirt bottle multi channel pipette manifold dispenser or microplate washer 9 Add 100 uL of Substrate Reagent Avoid exp e IMieroplate to direct sunlight Covering the plate with e g aluminum foil is recommended bstrate Reagent to 4 C immediately after the necessary volume is re
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