CY-8100 Human LOXL2 ELISA Kit
Contents
1. gt Human LOXL2 ELISA Kit CircuLex eer Manual For Research Use Only Not for use in diagnostic procedures 4 gt ELISA Kit for Measuring Human LOXL2 l CircuLex Human LOXL2 ELISA KiS Cat CY 8100 O Intended a coccatessucanssnmaieeiocanbatsecaacoannolesten 1 Q ENO trat A EPEE E E TE T 1 y DIAS OIN aiisis vesrodaddoeceseepansanaaoecanentaeeasess 2 ww Principle of the Assay 2 3 a Materials Provided sccscaccisccsavasnesocesencexsnes 3 Q Materials Required but not Provided 4 gt Precautions and Recommendation 5 Q Sample Collection and Storage 0 0 6 amp Detailed Prot sosateccssactanctsdasnsatacatenseaasecnees 7 8 Ca PATIOS lt a sarecicnccsiusiccenntenscheeatdueaseansecanes 9 oe Measurement Range 9 7 PrpmblesWO Ota scccsiccesetsnsissse somanaguuierccenaces 9 Reagent Stability sseeeseeseeeeeeeeeseseseeeses 9 amp Assay Characteristics eseeeeeeeeeeeeeeee seee RA Example of Test Results is ic c sccssessnree xQ RETSLCHCES eriseisund 4 Related Proguets cj ciccescassssssieeteasevasence 4 15 gO Intended Use K4 The CycLex Research Product CirgyLex Human LOXL2 ELISA Kit is used for the quantitative measurement of human LOXL2 in ulture supernatant and other biological media This assay kit is for research a only and not for use in diagnostic or therapeutic procedures 4 Storage e Upon receipt store aigeynponents at 4 C e Don t expose reagesits o ex2. S o A eA Reon CY 8100 11 Version 150612 S C Q Human LOXL2 ELISA Kit O Q TM C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures 4 3 Linearity N Three samples were diluted with Dilution Buffer and assayed after dilution The neat sa is set to 1 The results are summarized in the figure below Ww Sample Cell cultureg upernatant S Linearity 2 300 250 E 200 z 150 Z 5 100 50 0 0 0 25 0 5 0 75 1 1 Dilution Factor o gL eA Roa CY 8100 12 Version 150612 S C A CircuLex eer Manual For Research Use Only Not for use in diagnostic procedures 4 gt Example of Test Results gt oO Wh Fig 1 Human LOXL2 concentration in cell culture supernatants RY Qs Human LOXL2 concentration in culture supernatants of cancer cell lines Q Human LOXL2 ELISA Kit O Q Human LOXL2 ng mL eA Roa CY 8100 13 Version 150612 o c A CircuLex Uber Manual For Research Use Only Not for use in diagnostic procedures 4 References Csiszar K 2001 Lysyl oxidases a novel multifunctional amine oxidase family Prog Nuger Acid Res Mol Biol 70 1 32 RY gt Human LOXL2 ELISA Kit 2 Wang SX et al 1996 A crosslinked co factor in lysyl oxidase redox function for Mino acid side chains Science 273 1078 84 Molnar J et al 2003 Structural a
3. AA Werner JA 2003 Reduction of LOX and LOXL2 mRNA e sion in head and neck squamous cell carcinomas Anticancer Res 23 1565 73 13 Akiri G Sabo Dafni H et al 2003 Lysyl oxidase related protein 1 promotes tumor fibrosis and tumor progre ipn in vivo Cancer Res 63 1657 66 14 Kinhal DA Seftor EA Fong SF et al 2002 A molecular role for lysyl oxidase in breast cancer asion Cancer Res 62 4478 83 Q S 72 Roa CY 8100 14 Version 150612 S C RS P i Human LOXL2 ELISA Kit Circu Lex User s Manual A For Research Use Only Not for use in diagnostic procedures Related Products S CircuLex S100A4 ELISA Kit Ver 2 Cat CY 8086 CircuLex S100A6 ELISA Kit Cat CY 8097 RY CircuLex S100A10 ELISA Kit Cat CY 8095 CircuLex S100A11 ELISA Kit Cat CY 8063 Y CircuLex 100A14 ELISA Kit Cat CY 8064 N CircuLex S100P ELISA Kit Cat CY 8060 Y CycLex Co Ltd 1063 103 Terasawaoka Ina Nagano 396 0002 Japan amp Fax 81 265 76 7618 rA e mail info cyclex co jp URL http www cyclex co jp X gt RS PRODUCED BY gt gt c y s CycLex CircuLex products are supplied for gga use only CycLex CircuLex products and components thereof may not be resold modified for resale or used to manufacture commercial products without prior written approval fr ycLex Co Ltd To inquire about licensing for such commercial use please contact us via qua l amp o gL A R
4. The Dilution Buffer serves as the zero standard Blank Volume of Standard Dilution Buffer Concentration Std 1 60 uL of Master Standard 2 mL 540 uL 25 ng mL Std 2 300 uL of Std 1 25 ng m 300 uL 12 5 ng mL Std 3 300 uL of Std 2 12 5 n 300 uL 6 250 ng mL Std 4 300 uL of Std 3 6 25 yg mL 300 uL 3 125 ng mL Std 5 300 uL of Std 4 3 ng mL 300 uL 1 563 ng mL Std 6 300 uL of Std 63 ng mL 300 uL 0 781 ng mL Std 7 300 uL of Sta 781 ng mL 300 uL 0 391 ng mL Blank a 300 uL 0 ng mL 2 Q Note Do not use Repeating pipette Change tips for every dilution Unused portions of Master Standard shi be aliquoted and stored at below 70 C immediately Avoid multiple freeze and thaw cycles Sample Dilun e Cell cule supernatant require 5 to 30 fold dilution Q S 72 Roa CY 8100 T Version 150612 e ee Standard Assay Procedure for Human LOXL2 j N w D n N oo 10 12 Human LOXL2 ELISA Kit 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 Dilute samples with Dilution Buffer See Sample Dilution above Pipette 100 uL of Standard Solutions Std1 Std7 Blank and diluted samplen duplicates int
5. 00 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 y e Microplate washer optional Manual washing is possible but not prefett e Plate reader capable of measuring absorbance in 96 well plates toe wavelengths of 450 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be used THEYlate can also be read at a single wavelength of 450 nm which will give a somewhat higher Ey e Software package facilitating data generation and analysig o tional 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 8100 4 Version 150612 o c y Human LOXL2 ELISA Kit gt O CircuLex Cpe Manual For Research Use Only Not for use in diagnostic procedures amp Precautions and Recommendations 3 e Allow all the components to come to room temperature before use we e 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 e Rinse all detergent residues from glassware e Use deionized water of the highest quality e Do not mix reagents from di
6. 612 o c Human LOXL2 ELISA Kit 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 CireuLex Human LOXL2 ELISA Kit is provided with vable strips of wells so the assay can be carried out on separate occasions using only the numb Of strips required for the particular determination Since experimental conditions may vary an xe of the Human LOXL2 Standard within the kit should be included in each assay as a calibragpy Disposable pipette tips and reagent troughs should be used for all liquid transfers to avoid a Aao of reagents or samples Y Preparation of Working Solutions RU All reagents need to be brought to room temperature prior to the assay reagents are supplied ready to use with the exception of 10X Wash Buffer and Human LOXL ndard 1 Prepare a working solution of Wash Buffer by adding 100 mL of the Wash Buffer to 900 mL of deionized distilled water Mix well Store at 4 C for two weeks QS C for long term storage 2 Reconstitute Human LOXL2 Standard with 0 4 mL of Dil Buffer The concentration of the human LOXL2 in vial should be 250 ng mL which is ref amp fed as a Master Standard of human LOXL2 amp G Prepare Standard Solutions as follows Q Use the Master Standard to produce a dilution N below Mix each tube thoroughly before the next transfer The 25 ng mL standard St Q serves as the highest standard
7. cessive light o gL A Roa CY 8100 1 Version 150612 o AO Human LOXL2 ELISA Kit L ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 A Q O Q Introduction 3 Lysyl oxidase like 2 LOXL2 is a member of the lysyl oxidase LOX protein family th nsists of five members 1 All the members of the family contain a highly conserved carboxyeterminal copper binding domain and a lysyl tyrosyl quinone cofactor which are necessary fi e amine oxidase activity of these extracellular matrix enzymes 1 2 and the N terminal re divergent scavenger receptor cysteine rich domain which is thought to determine the role and tissue distribution of each isoenzyme 3 In addition to its biological role in ffpper elastic fiber homeostasis and cardiovascular system development 4 5 the LOX family fypteins has recently been implicated in tumorigenesis and metastasis 6 7 Up regulation of LOXL2 has been reported in breast colon 8 esop 1 8 head and neck 9 and oral squamous cell carcinomas 10 and pancreatic er 11 although its down regulation of LOXL2 mRNA has been reported in head and A cell carcinomas 12 It was also reported that LOXL2 overexpression promotes th asiveness of tumor cells in vivo and in vitro 13 14 In addition LOXL2 protein was detect the serum of 83 of patients with chronic HCV infection but was not detected in serum fro rmal healthy donors This result
8. etermine the human LOXL2 oQycentration 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 capone human LOXL2 concentration If the samples have been diluted the concentration read fry e standard curve must be multiplied by the dilution factor R 1 The dose response curve of this assay fits best to a sigmoidal t paragr logistic equation The results of unknown samples can be calculated with any computer prog am having a 4 parameter logistic function It is important to make an appropriate mathematical Austment to accommodate for the dilution factor wy 2 Most microtiter plate readers perform automatic calculations of flyte concentration The calibration curve is constructed by plotting the absorbance Y of Moras versus log of the known concentration X of calibrators using the 4 parameter fungtion Alternatively the logit log function can be used to linearize the calibration curve i e logit ofghtorbance Y is plotted versus log of the known concentration X of calibrators Ss Measurement Range O The measurement range is 0 391 ng mL to 2 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 consideration in calculating thg human LOXL2 concentration Troubleshooting 2 1 The Human LOXL2 Sta
9. fferent kits RU S Y A RG N amp The buffers and reagents used in this kit contain NaN3 as presgttives Care should be taken to avoid direct contact with these reagents lt Do not mouth pipette or ingest any of the reagents 4 gt e Do not smoke eat or drink when performing the yay or in areas where samples or reagents are handled andle aS e Dispose of tetra methylbenzidine TMB contgi he solutions in compliance with local regulations e Avoid contact with the acidic Stop Solutiggspnd Substrate Solution which contains hydrogen peroxide e Wear gloves and eye protection whey Analing immunodiagnostic materials and samples of human origin and these reagents In case ay ntact with the Stop Solution and the Substrate Solution wash skin thoroughly with water and Shy edical attention when necessary e Biological samples may be P with infectious agents Do not ingest expose to open wounds or breathe aeros gt Wear protective gloves and dispose of biological samples properly handli p Solution O nw a Qe amp eo Roa CY 8100 5 Version 150612 e CAUTION Sulfuric is a strong acid Wear disposable gloves and eye protection when o AO Human LOXL2 ELISA Kit CircuLex De Manual For Research Use Only Not for use in diagnostic procedures 4 A Q O Q Sample Collection and Storage 3 Cell culture supernatant Remove any particulates by centrifugation and assay immediatel
10. indicates that LOXL2 is a promising candidate biomarker foe non invasive assessment of liver fibrosis lt A Principle of the Assay The CycLex Research Product CircuLex Hg LOXL2 ELISA Kit employs the quantitative sandwich enzyme immunoassay technique An antibddy specific for human LOXL2 is pre coated onto a microplate Standards and samples are pipette the wells and the immobilized antibody binds any human LOXL2 present After washing away any unbound substances an HRP conjugated monoclonal antibody specific for human LOXL2 is a ago the wells Following a wash to remove any unbound antibody HRP conjugate the regen is allowed to react with the substrate H202 tetramethylbenzidine The reaction is ped by addition of acidic solution and absorbance of the resulting yellow product is measured 450 nm The absorbance is proportional to the concentration of human LOXL2 A standard curve nstructed by plotting absorbance values versus human LOXL2 concentrations of calibrators and Ppcentrations of unknown samples are determined using this standard curve Ss o oe Roa CY 8100 2 Version 150612 o AO CircuLex eer Manual For Research Use Only Not for use in diagnostic procedures gt Human LOXL2 ELISA Kit O Summary of Procedure Add 100 uL of diluted samples to the wells we 4 Incubate for 1 hour at room temp RY Wash the wells amp Add 100 uL of HRP conjugated anti human LOXL2 anito 4 Incubate for 1 hour at r
11. nd functional diversity of lysyl oxidase and POX ike proteins Biochim Biophys Acta 1647 220 24 R w 4 Maki JM Rasanen J Tikkanen H et al 2002 Inactivation of the lys idase gene Lox leads to aortic aneurysms cardiovascular dysfunction and perinatal death in mige Circulation 106 2503 9 5 Liu X Zhao Y Gao J et al 2004 Elastic fiber homeostasis requires tysyl oxidase like 1 protein Nat Genet 36 178 82 6 Erler JT Bennewith KL Nicolau M et al 2006 Lysyl og ise is essential for hypoxia induced metastasis Nature 440 1222 6 lt 7 Payne SL Hendrix MJ Kirschmann DA 2007 Par Ggxical roles for lysyl oxidases in cancer a prospect J Cell Biochem 101 1338 54 S 8 Fong SF Dietzsch E Fong KS et al 2007 Lys wxidase like 2 expression is increased in colon and esophageal tumors and associated with less gi rentiated colon tumors Genes Chromosomes Cancer 46 644 55 4 9 Chung CH Parker JS Karaca G et al 04 Molecular classification of head and neck squamous cell carcinomas using patterns of geng ression Cancer Cell 5 489 500 10 Ziober AF Patel KR Alawi F 2006 Identification of a gene signature for rapid screening of oral squamous cell carcinoma Cancer Res 12 5960 71 11 Gronborg M Kristanse yg Iwahori A et al 2006 Biomarker discovery from pancreatic cancer secretome using a differgn 1 proteomic approach Mol Cell Proteomics 5 157 71 12 Rost T Pyritz V x IO Gorogh T Dunne
12. ndard shouldbe run in duplicate using the protocol described in the Detailed Protocol Incubation times or peratures significantly different from those specified may give erroneous results Q 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 sign ay 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 Reagei Stability All of ae included in the CycLex Research Product CircuLex Human LOXL2 ELISA Kit have be n tested for stability Reagents should not be used beyond the stated expiration date Upon recip kit reagents should be stored at 4 C except the reconstituted Human LOXL2 Standard must be S at below 70 C Coated assay plates should be stored in the original foil bag sealed by the zip lock 3 containing a desiccant pack A y P Thi Human LOXL2 ELISA Kit Circu Lex User s Manual A For Research Use Only Not for use in diagnostic procedures amp Assay Characteristics 3 1 Sensitivity ay The limit of detection defined as such a concentration of human LOXL2 giving ee er than mean absorbance of blank plus three standard deviations of the absorbance of blank A 3SD blank is bette
13. o the appropriate wells O 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 20 C Add 100 uL of Stop Solution each well in the same order as the previously added Substrate Reagent We Measure absorbance in caven using a spectrophotometric microplate reader at dual wavelengths of 450 540 nm Dual wav amp f ngths of 450 550 or 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 minu
14. oa CY 8100 15 Version 150612 o c
15. oom temp y Wash the wells RA bo r gt Add 100 uL of Substrate Reagent wy t ON Add 100 uL of Stop Solution N o 4 A G Materials Provided L Measure absorbance at 450 nm All samples and standards should be assayed fwduplicate The following components are supplied and are sufficient for the one 96 well microplate Microplate One microplate supplied re to use with 96 wells 12 strips of 8 wells in a foil zip lock bag with a desiccant pack Wells are coat d with anti human LOXL2 antibody as a capture antibody 10X Wash Buffer One bottle coping 100 mL of 10X buffer containing 1 3 Tween 20 Dilution Buffer One bate ining 50 mL of 1X buffer use for reconstitution of Human LOXL2 Standard and sample dilution Weady to use Human LOXL2 Standa None vial containing 100 ng of lyophilized recombinant human LOXL2 HRP conjugated D tXtion Antibody One bottle containing 12 mL of HRP horseradish peroxidase conjugated anti huggan LOXL2 monoclonal antibody KH 2A7 Ready to use Substrate Rent One bottle containing 20 mL of the chromogenic substrate tetra methylbenzidine TMB Rege use Stop Sgn One bottle containing 20 mL of 1 N H2S014 Ready to use S 72 Roa CY 8100 3 Version 150612 S C A CircuLex De Mai gt Human LOXL2 ELISA Kit 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 2
16. r than 0 286 ng mL of sample 2 Dilution Buffer is pipetted into blank wells A S Typical Standard Curve aS gt Standard curve wy 0 0 5 0 10 0 15 0 20 0 25 0 Human LOXL2 ng ml A Roa CY 8100 10 Version 150612 o AO A ni Human LOXL2 ELISA Kit C 1rcu Lex User s Manual AS For Research Use Only Not for use in diagnostic procedures 2 Precision N Intra assay Precision Precision within an assay Ww Three samples of known concentration were tested six times on one plate to asseg intra assay precision e Intra assay Within Run n 6 CV 3 2 5 2 Sample Cep Mure supernatant Human LOXL2 conc ng ml bw No Sample1 Sample2 Sample 3 Y 1 117 8 107 8 261 7 SS 2 112 1 98 7 238 0 gt 3 108 5 109 7 2646 4 1173 1080 2629 o 5 113 2 105 9 235 1 6 117 1 107 9 2524 amp max 1178 109 7 264 6 Pg min 108 5 98 7 2351 mean 1143 106 3 2524 amp SD 3 7 3 9 30 X CV 3 2 3 7 5 2 Inter assay Precision Precision between ass Three samples of known concentratiOr were tested in six separate assays to assess inter assay precision 4 e Inter assay Run to Run n 6 2 8 8 6 2 Sample Cell culture supernatant Human LOXL2 conc ng ml Assay Sample 1 Sample 2 Sample 3 1 17 0 49 4 258 2 2 pA By 57 6 267 9 3 18 9 48 5 265 0 4 21 2 52 2 264 9 5 19 1 50 6 271 8 6 19 5 50 4 251 1 max 21 7 57 6 271 8 min 17 0 48 5 251 1 mean 19 6 51 4 263 2 sD i7 33 7 4 CV 8 6 6 4 2 8
17. tes 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 25 units for the zero concentration or 3 0 units for the highest standard concentration The plate ould be monitored at 5 minute intervals for approximately 30 minutes Note 3 If 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 gL using the values measured at 405 nm is used to determine human LOXL2 concentration of off scale samples The readings at 405 nm should not replace the on scale readings at 450 nm Roa CY 8100 8 Version 150612 o AO kA at CY 8100 9 Version 150612 o AO L ircuLex User s Manual A gt Human LOXL2 ELISA Kit For Research Use Only Not for use in diagnostic procedures 4 Calculations 3 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 linearized by using log log paper asio analysis may be applied to the log transformation To d
18. yp fealiquot and store samples at below 70 C Avoid repeated freeze thaw cycles RY Other biological samples Remove any particulates by centrifugation and assay immed tgly or aliquot and store samples at below 70 C Avoid repeated freeze thaw cycles For reference Ww Serum Use a serum separator tube and allow samples to clot for 60 SP ninutes Centrifuge the samples at 4 C for 10 minutes at 1 000 x g Remove serum and assay imately or store samples on ice for up to 6 hours before assaying Aliquots of serum may also be sto at below 70 C for extended periods of time Avoid repeated freeze thaw cycles Plasma Collect plasma using EDTA Naz as the anticoagulant ossible collect the plasma into a mixture of EDTA Na2 and Futhan FUT175 to stabilize th ple against spontaneous in vitro complement 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 befor ssaying Aliquots of plasma may also be stored at below 70 C for extended periods of time Avoi leated freeze thaw cycles Note Citrate plasma has not been validated for use jhis assay wW NOTE Although we suggest to conduct ae as outlined above the optimal experimental conditions will vary depending on the parameters being investigated and must be determined by the individual user NO NTY OR GUARANTEE OF PERFORMANCE USING THESE PROCEDUR MADE OR IMPLIED Roa CY 8100 6 Version 150
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