CircuLex Human Lacritin ELISA Kit
Contents
1. oo Wash 4 times by filling each well with Wash Buffer 350 tL using a squirt bottle multi channel pipette manifold dispenser or microplate washer 9 Add 100 uL of Substrate Reagent Avoid exposifig the microtiter plate to direct sunlight Covering the plate with e g aluminum foil is recommended Return Substrate Reagent to 4 C immediately after the necessary volume is removed 10 Incubate the plate at room temperature ca 25 C for 10 20 minutes shaking at ca 300 rpm on an orbital microplate shaker The incubation Aim may be extended up to 30 minutes if the reaction temperature is below 20 C 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 wellusing aspectrophotometric microplate reader at dual wavelengths of 450 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be used Read the microplate at 450 nm if only a single wayelength can be used Wells must be read within 30 minutes of adding the Stop Solution Note 1 Complete removal of liquid at each step is essential to good performance After the last wash remove any remaining Wash Buffer by aspirating or decanting Invert the plate and blot it against Clean pap r towels Note 2 Reliable standard curves are obtained when either O D values do not exceed 0 25 units for the blank zeto concentration or 3 0 units for the highest standard concentration The plate shouldbe2. E 4 Cat 8105 14 Version 150611 Human Lacritin ELISA Kit L ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures References Ma P Wang N McKown RL Raab RW Laurie GW Focus on molecules lacritin Exp Eye Res 2008 86 457 458 N McKown RL Wang N Raab RW et al Lacritin and other new proteins of the lacri al funetional unit Exp Eye Res 2009 88 848 858 w Morimoto Tochigi A Walkup RD Nakajima E Shearer TR Azuma M Mechanism for carbachol induced secretion of lacritin in cultured monkey lacrimal acinar cells Invest Ophthalmol Vis Sci 2010 51 4395 4406 4 Sanghi S Kumar R Lumsden A et al cDNA and genomic cloning of lacritifha novel secretion enhancing factor from the human lacrimal gland J Mol Biol 2001 310 127 139 n Nakajima T Walkup RD Tochigi A Shearer TR Azuma M Establishment of an appropriate animal model for lacritin studies cloning and characterization of lacritin in monkey eyes Exp Eye Res 2007 85 651 658 6 Tsai PS Evans JE Green KM et al Proteomic analysis of human meibomian gland secretions Br J Ophthalmol 2006 90 372 377 Weigelt B Bosma A J van t Veer L J Expression of anoyel lacrimal gland gene lacritin in human breast tissues J Cancer Res Clin Oncol 2003 129 735 6 8 Koo B Lee D Ha H Kim JC Kim CW Comparative analysis of the tear protein expression in blepharitis
3. 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 Deionized water of the highest quality e Disposable paper towels 8105 4 Version 150611 Human Lacritin ELISA Kit L 1rcu Lex User s Manual 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 e All microplate strips that are not immediately required should be returned to the zip lock pouch Which 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 e Use deionized water of the highest quality e Do not mix reagents from different kits e The buffers and reagents used in this kit contain NaN3 as preservatives Care 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 i argas where samples or reagents are handled e Dispose of tetra methylbenzidine TMB containifg solutions in compliance with local regulations e Avoid contact with the acidic Stop Solution and Substrate Solution which contains hydrogen peroxide e Wear gloves and ey
4. patients using two dimensional lectfophoresis J Proteome Res 2005 4 719 724 9 Nichols JJ Green Church KB Mass spectfometry based proteomic analyses in contact lens related dry eye Cornea 2009 28 1109 1117 10 Samudre S Lattanzio FA Jz Losse n V et al Lacritin a novel human tear glycoprotein promotes sustained basal tearing and 1s well tolerated Invest Ophthalmol Vis Sei 2011452 6265 6270 11 Wang J Wang N Xie J etal Restricted epithelial proliferation by lacritin via PKCalpha dependent NFAT and mTOR pathways J Cell Biol 2006 174 689 700 Cat OY 8105 15 Version 150611 Human Lacritin ELISA Kit CircuLex Users Manual For Research Use Only Not for use in diagnostic procedures Related Products CircuLex S100A4 ELISA Kit Ver 2 Cat CY 8086 CircuLex S100A6 ELISA Kit Cat CY 8097 CircuLex S100A10 ELISA Kit Cat CY 8095 CircuLex S100A11 ELISA Kit Cat CY 8063 CircuLex S100A14 ELISA Kit Cat CY 8064 CircuLex S100P ELISA Kit Cat CY 8060 PRODUCED BY G 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 CycLex CircuLex products are supplied for re components thereof may not be resold modifi fox products without prior written approval from C such commercial use please contact us via email y CycLex CircuLex products and esale or used to manufacture commercia
5. 6 hours before assaying Aliquots of serum may also be stored at below 70 C for extended periods of time Avoid repeated freeze thaw cycles Plasma Collect plasma using EDTA Naz as the anticoagulant Ifjpossible collect the plasma into a mixture of EDTA Na2 and Futhan to stabilize the sample 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 before assaying Aliquots o plasma may also be stored at below 70 C for extended periods of time Avoid repeated freeze thaw eycles Note Citrate plasma has not been validated for us im this assay Preparation of tear extract from Schirmer s test strip 1 Place one free end of the Schirmer s test strip within your lower eyelid Both eyes are tested at the same time 2 Keep your eyes gently closed for 5 minuf s w9 Remove the wet test strips from each lower eyelid 4 Cut the wet test strip 5 mm length with seissors or cutter 6 Transfer 5 mm piece of the wetest strip to microcentrifuge tube N Add 300 ul PBS with protease inhibitor cocktail to the microcentrifuge tube incubated at 4 C for 3 hours with intermittent mixing 8 Centrifuge at 15 000 xpm for 5 minutes at 4 C 9 Transfer the supernatant to new microcentrifuge tube store at 80 C until processing e This teamextract from Schirmer s test strip requires 100 to 300 fold dilutio
6. Human Lacritin ELISA Kit CircuLex Uers Manual For Research Use Only Not for use in diagnostic procedures ELISA Kit for Measuring Human Lacritin Y CircuLex Human Lacritin ELISA Kit Cat CY 8105 oO Intended Use 1 gt trat A eee E ene een arr ern eee 1 Introduction ccccceceesssessssesessseeeeees 2 Principle of the Assay 2 3 Materials Provided cc cccccceeseeeseeeeeees 3 Materials Required but not Provided 4 Precautions and Recommendation 5 Sample Collection and Storage 00 6 Detailed Protocol ccccccceseseeeeeeeeeees 8 9 Calculations ccccccccccccccccceeeseee es esssseeeeees 10 Measurement Range 10 Troubleshooting isssccssecsczacsssccesacasssssoevaccacas 10 Reagent SCAB cccctsisssitatonsevovecatadacsamaaseores 10 Assay Characteristics ereere 11 Example of Test Results ssissisacccsissecescesseseeene 1 Referen oE Se eere E ESAE Related Prod ctS s sscessssisssssiossssssersaressses 16 Intended Use 2s The CycLex Research Product CircuLex an Lacritin ELISA Kit is used for the quantitative re supernatant and other biological media measurement of human lacritin in tear fluidgi e This assay kit is for research use only a for use in diagnostic or therapeutic procedures Storage e Upon receipt store all co e Don t expose reagents to Cat 8105 1 Version 150611 Human Lacritin ELISA Kit L ircuLex User s Manual For Research Use Only N
7. cat ithat 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 Reagent Stability All of the r agents included in the CycLex Research Product CircuLex Human Lacritin ELISA Kit have beenstested for stability Reagents should not be used beyond the stated expiration date Upon receipt kit reagents should be stored at 4 C except the reconstituted Human Lacritin Standard must be stored at belows 70 C Coated assay plates should be stored in the original foil bag sealed by the zip lock and containing a desiccant pack Cat OY 8105 10 Version 150611 Human Lacritin ELISA Kit CircuLex Users Manual For Research Use Only Not for use in diagnostic procedures Assay Characteristics 1 Sensitivity The limit of detection defined as such a concentration of human lacritin giving absorbance high an mean absorbance of blank plus three standard deviations of the absorbance of blank blank is better than 32 4 pg mL of sample Dilution Buffer is pipetted into blank wells Typical Standard Curve Qy Human Lacritin Standard Curve Version 150611 Human Lacritin ELISA Kit CircuLex Users 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 four times on one plate to as
8. e protection when handlingjimmunodiagnostic materials and samples of human origin and these reagents In case of contaetywith the Stop Solution and the Substrate Solution wash skin thoroughly with water and seek medical attention when necessary Biological samples may be contaminated with infectious agents Do not ingest expose to open wounds or breathe aerosols Wear protective gloves and dispose of biological samples properly e CAUTION Sulfuric Acid is a strong acid Wear disposable gloves and eye protection when handling Stop Solution Cat OY 8105 5 Version 150611 Human Lacritin ELISA Kit L ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Sample Collection and Storage Tear fluid Remove any particulates by centrifugation and assay immediately or aliquot and store samples at below 70 C Avoid repeated freeze thaw cycles Cell culture supernatant Remove any particulates by centrifugation and assay immediately or aliquot and store samples at below 70 C Avoid repeated freeze thaw cycles Other biological samples Remove any particulates by centrifugation and assay immediately or aliquot and store samples at below 70 C Avoid repeated freeze thaw cycles For reference Serum Use a serum separator tube and allow samples to clot for 60 30 mifutes Centrifuge the samples at 4 C for 10 minutes at 1 000 x g Remove serum and assay immediately or store samples on ice for up to
9. ied ready t ith 96 wells 12 strips of 8 wells in a foil zip lock bag with a desiccant pack Wells are coated uman lacritin antibody as a capture antibody 10X Wash Buffer One bottle containing 100 mL of 10X buffer containing Tween 20 Dilution Buffer One bottle contai of 1X buffer use for reconstitution of human lacritin Standard and sample dilution Rea Human Lacritin Standard ntaining 44 ng of lyophilized recombinant human lacritin HRP conjugated Detection A dy One bottle containing 12 mL of HRP horseradish peroxidase conjugated anti human lac antibody Ready to use Substrate Rea TMB Ready to Qj e containing 20 mL of 1 N H2S01 Ready to use le containing 20 mL of the chromogenic substrate tetra methylbenzidine Stop Solutio Cat 8105 3 Version 150611 Human Lacritin ELISA Kit For Research Use Only Not for use in diagnostic procedures CircuLex Waers Manual 4 Materials Required but not Provided e Pipettors 2 20 uL 20 200 uL and 200 1000 uL precision pipettors with disposable tips e Precision repeating pipettor e Orbital microplate shaker e Microcentrifuge and tubes for sample preparation e Vortex mixer s of 450 540 nm e read at a single e Plate reader capable of measuring absorbance in 96 well plates at dual wa e Microplate washer optional Manual washing is possible but not preferable i Dual wavelengths of 450 550 or 450 595 nm can also be used The O b wavelength
10. ips for every dilution Unused portions of Master Standard should be aliquofed and stored at below 70 C immediately Avoid multiple freeze and thaw cycles Sample Preparation Tear fluid requires 5 000 to 10 000 fold dilution e Tear extra et from Schirmer s test strip requires 100 to 300 fold dilution e Othembiological samples require neat to appropriate dilution Cat CY 8105 8 Version 150611 Human Lacritin ELISA Kit L ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Standard Assay Procedure for Human Lacritin j Remove the appropriate number of microtiter wells from the foil pouch and place them into the well holder Return any unused wells to the foil pouch refold seal with tape and store at 4 C 2 Dilute samples with Dilution Buffer See Sample Preparation above W Pipette 100 uL of Standard Solutions Std1 Std7 Blank and diluted samples in duplicates into the appropriate wells 4 Incubate the plate at room temperature ca 25 C for 1 hour shaking at ca 300frpim on an orbital microplate shaker n Wash 4 times by filling each well with Wash Buffer 350 uL using a squtirtebottle 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 shaking at ca 300 rpm on an orbital microplate shaker
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12. monitored at 5 minute intervals for approximately 30 minutes Note 3 Jf the micfoplate reader is not capable of reading absorbance greater than the absorbance of the highestystandard perform a second reading at 405 nm A new standard curve constructed using the values measured at 405 nm is used to determine human lacritin concentration of off scale samples The readings at 405 nm should not replace the on scale readings at 450 nm Cat CY 8105 9 Version 150611 Ti Human Lacritin ELISA Kit L 1rcu Lex 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 zero standard optical density Plot the optical density for the standards versus the concentration of the standards and draw the best curve The data can be linearized by using log log paper and_regression analysis may be applied to the log transformation To determine the human lacritin concentfatton of each sample first find the absorbance value on the y axis and extend a horizontal line to the standard curve At the point of intersection extend a vertical line to the x axis and read the corresponding human lacritin 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 logistic equation The
13. n for Assay The tear extfactpfrom Schirmer s test strip can be stored at below 70 C Avoid multiple freeze thaw cycles After thaw the tear extract Centrifuge at 15 000 rpm for 5 minutes at 4 C again since the cell Cat CY 8105 6 Version 150611 Human Lacritin ELISA Kit CircuLex Waers Manual For Research Use Only Not for use in diagnostic procedures lysates should be clear of any sediments or particulate matter Typical data using this protocol are shown in Fig 1 of the section Example of Test Results below NOTE THE ABOVE PROCEDURES ARE INTENDED ONLY AS A GUIDELINE OPTIMAL EXPERIMENTAL CONDITIONS WILL VARY DEPENDING PARAMETERS BEING INVESTIGATED AND MUST BE DETERMIN INDIVIDUAL USER NO WARRANTY OR GUARANTEE OF PER USING THESE PROCEDURES IS MADE OR IMPLIED Cat 8105 7 Version 150611 Human Lacritin ELISA Kit L ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol The CycLex Research Product CircuLex Human Lacritin ELISA Kit is provided with removable strips of wells so the assay can be carried out on separate occasions using only the number of strips required for the particular determination Since experimental conditions may vary an aliquot of the Human Lacritin Standard within the kit should be included in each assay as a calibrator Disposable pipette tips and reagent troughs should be used for all liquid transfers to avoid cross contaminatio
14. n of reagents or samples Preparation of Working Solutions All reagents need to be brought to room temperature prior to the assay Assay r agents are supplied ready to use with the exception of 10X Wash Buffer and Human Lacritin Standard 1 Prepare a working solution of Wash Buffer by adding 100 mL of the 10X Wash Buffer to 900 mL of deionized distilled water Mix well Store at 4 C for two weeks or 20 C for long term storage 2 Reconstitute Human Lacritin Standard with 1 mL of Dilution Buffer The concentration of the human lacritin in vial should be 44 ng mL which is referred as a Master Standard of human lacritin Prepare Standard Solutions as follows Use the Master Standard to produce a dilution series below Mix each tube thoroughly before the next transfer The 3 000 pg mL standard Std 1 servesyas the highest standard The Dilution Buffer serves as the zero standard Blank Volume of Standard Dilution Buffer Concentration Std 1 50 uL of Master Standard 44 ng mL 683 uL 3 000 pg mL Std 2 300 uL of Std 1 3 000 pg mL 300 uL 1 500 pg mL Std 3 300 uL of Std 2 1 500 pg mL 300 uL 750 pg mL Std 4 300 uL of Std 3 750 pg mL 300 uL 375 pg mL Std 5 300 uL of Std 4 375 pg mL 300 uL 187 5 pg mL Std 6 300 uL of Std 5 187 5 pg mL 300 uL 93 8 pg mL Std 7 300 uL of Std 6 93 8 pg mL 300 uL 46 9 pg mL Blank 3 300 uL 0 pg mL Note Do not use a repeating pipette Change t
15. ndards and samples are pipetted into the wells and the immobilized antibody binds any human lacritin present After washing away any unbound substanees an HRP conjugated antibody specific for human lacritin is added to the wells Followifg a Wash to remove any unbound antibody HRP conjugate the remaining conjugate is fallowed bto react with the substrate H202 tetramethylbenzidine 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 concentration of human lacritin A standard curve is constructed by plotting absorbance values versus human lacritin concentrations of calibrators and concentrations of unknown samples are determined using this standard curve Cat OY 8105 2 Version 150611 Human Lacritin ELISA Kit L ircuLex User s Manual A For Research Use Only Not for use in diagnostic procedures Summary of Procedure Add 100 uL of diluted samples to the wells 4 Incubate for 1 hour at room temp Wash the wells Add 100 uL of HRP conjugated anti human lacritin antibody 4 Incubate for 1 hour at room temp Wash the wells Add 100 uL of Substrate Reagent G Add 100 uL of Stop Solution O t Measure absorbance at 450 nm Materials Provided All samples and standards should be assayed in e The following components are supplied and are sufficient for the one 96 well microplate kit Microplate One microplate suppl
16. ot for use in diagnostic procedures Introduction Lacritin is a 12 3 kDa tear glycoprotein that is apically released from human lacrimal acinar cells during reflex tearing 1 3 and can be detected in mixed reflex and basal human tears 4 5 Lacritin is produced by corneal conjunctival 5 meibomian 6 as one of the most eye restricted genes However it has been detected in a subpopulation of ductal cells in salivary glands thyroid 4 and mammary glands 7 Lacritin modulates lacrimal acinar cell secretion promotes ductal cell proliferation and stimulates signaling through tyrosine phosphorylation and release of calcium Since a decrease of tear lacritin was reported in patients with blepharitis 8 and in Contact lens related dry eye 9 lacritin might play an important role in maintaining tear fluid and the ocular surface In fact ocular instillation of recombinant human lacritin stimulated basal tear flow in rabbits 10 Lacritin also promotes basal tear secretion by cultured rat 4 and monkey 3 lacrimal acinar cells and stimulates human corneal epithelial cell growth 11 Thus Jacritin appears to be a multifunctional eye specific factor with a potential role in tear secretion fand corneal epithelial renewal Principle of the Assay The CycLex Research Product CircuLex Human Lacritin ELISA Kit employs the quantitative sandwich enzyme immunoassay technique An antibody specifiafor human lacritin is pre coated onto a microplate Sta
17. results of unknown samples can be calculated with any computer programghaving a 4 parameter logistic function It is important to make an appropriate mathematical adjustment t0 accommodate for the dilution factor 2 Most microtiter plate readers perform automatic calculations of analyte concentration The calibration curve is constructed by plotting the absorbance Y of calibrators versus log of the known concentration X of calibrators using the 4 parameter function Alternatively the logit log function can be used to linearize the calibration curve i e logit of absorbance X is plotted versus log of the known concentration X of calibrators Measurement Range The measurement range is 46 9 pg mL to 3 000 pg mb Any sample reading higher than the highest standard should be diluted with Dilution Buffer in higher dilution and re assayed Dilution factors need to be taken into consideration in calculating the human lacritin concentration Troubleshooting 1 The Human Lacritin Standard should be run induplicate using the protocol described in the Detailed Protocol Incubation times or temperatures significantly different from those specified may give erroneous results 2 Poor duplicates accompanied bypelevated values for wells containing no sample indicate insufficient washing If all instructions in the Detailed Protocol were followed accurately such results indicate a need for washer maintenang 3 Overall low signal may sndi
18. sess intra precision e Intra assay Within Run n 4 CV 4 6 5 4 Sample tear Human lacritin conc ng mL Sample No Sample 1 Sample 2 Sample 3 1 266 656 7 967 2 265 679 7 628 3 251 628 8 370 4 242 713 8 471 MAX 266 713 8 471 MIN 242 628 7 628 MEAN 256 669 8 109 S D 12 36 387 CY 4 6 5 4 4 8 Ww Inter assay Precision Precision between assays Three samples of known concentration wer in separate assays to assess inter assay precision e Inter assay Run to Run n 4 CV 2 4 8 5 Sample Human tear Human lacritin conc ng mL Q le No le 1 le2 le3 Assay 1 669 7 292 8 109 Assay 2 619 7419 8 207 Assay 3 589 8 015 8 560 4 547 7 057 8 241 MAX 669 8 015 8 560 MIN 547 7 057 8 109 MEAN 606 7 446 8 279 SD 51 408 196 CV 8 5 5 5 2 4 Cat 8105 12 Version 150611 Ti Human Lacritin ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures 3 Linearity Three samples were diluted with Dilution Buffer and assayed after dilution The neat sample is s et to 1 The results are summarized in the figure below Sample Human te 3 2 S E z 5 Version 150611 Human Lacritin ELISA Kit CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Example of Test Results Fig 1 Human lacritin concentration in tear extracts from Schirmer s test strips Homan Lacritin Conc ng mL g S
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