CY-8089 Human Lactoferrin ELISA Kit
Contents
1. C CY 8089 1 Version 140318 T Human Lactoferrin ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Introduction Lactoferrin is an iron binding glycoprotein of the transferrin family that is expressed in most biol gic fluids and is a major component of mammals innate immune system 1 It is a glycoprotein consisting of a single polypeptide chain of about 80 kDa with two globular lobes each containing an iron binding site It is found in most exocrine secretions including milk particularly the colostrunis and other secretions such as tears saliva intestinal mucus and genital secretions and in the specific granules of neutrophils The physiological roles that have been proposed for lactoferrin include anti inflammatory immunomodulatory antimicrobial antiviral and antitumoral functions For this reason lactoferrin is regarded as a host defense mediator Specific lactoferrin receptors exist in a variety of Cells including monocytes lymphocytes adipocytes hepatocytes and endothelial cells 2 Therefore determination of the lactoferrin concentration in various body fluids can be a marker of inflammation 3 6 It was reported that fecal lactoferrin is useful as a sensitive and specific marker ing d ntifying intestinal inflammation such as Crohn s disease and chronic inflammatory bowel disease ABD 7 Combination of several markers such as S100A8 A9 complex defensin elastase M2. microplate shaker n Wash 4 times by filling each well with Wash Buffer 350 uL using a sqtirt bottle 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 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 4im may be extended up to 30 minutes if the reaction temperature is below than 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 welljusing 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 eac
3. ug ml ng ml ng ml ug g 1 1514 5 1420 1 37 8 324 8 8 0 2 1574 0 1413 6 40 6 377 0 8 9 3 1725 3 1366 9 36 7 323 5 9 7 max 1725 3 1420 1 40 6 377 0 9 7 min 1514 5 1366 9 36 7 323 5 8 0 Sample 1 Milk mean 1604 6 1400 2 38 4 341 8 8 9 Sample 2 Tear SD 108 7 29 0 2 0 30 5 0 8 Sample 3 Urine GV 6 8 2 1 5 2 8 9 9 5 Sample 4 Serum Sample 5 Stool Cat CY 8089 11 Version 140318 Human Lactoferrin ELISA Kit C ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures 3 Linearity Two biological samples were diluted with Dilution Buffer and assayed after dilution The n sample is set to 1 4 4 Please note all samples including the neat sample are 200 000 fold diluted_as stated in the Assay Procedure The results are summarized in the figure below Linearity 2800 r 2600 A 2400 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 Human Lactoferrin ug ml 0 1 2 3 4 Sample Dilutin Ratio 4 Sample 1 Milk Sample 2 Tear C CY 8089 12 Version 140318 Human Lactoferrin ELISA Kit C ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures MA Example of Test Results Fig 1 Lactoferrin levels in many biological samples a In milk of healthy volunteers hLF conc sample mg ml 1 2 4 2 2 2 3 6 2 4 27 b In tear of healthy volunteers G ALF cone sample mg ml QO or 53 2 1 8 3 5 3 4 4
4. 7 5 c In urine of healthy volunteers l conc sample 1 5 0 3 3 28 8 9 9 4 5 d In saliva of healthy T hLF conc sample ug ml Qy 1 3 0 4 8 20 6 5 0 9 9 3 7 AU A UN C CY 8089 13 Version 140318 Human Lactoferrin ELISA Kit C ircuLex User s Manual A For Research Use Only Not for use in diagnostic procedures e In serum of healthy volunteers hLF conc sample ng ml 1 474 5 cA 2 260 7 3 296 8 O f In stool of healthy baby volunteers hLF conc sample ug g 1 776 4 90 4 33 4 QO 55 2 18 6 nA A UN C CY 8089 14 Version 140318 Human Lactoferrin ELISA Kit CircuLex User s Manual 4 For Research Use Only Not for use in diagnostic procedures References Legrand D Pierce A Elass E Carpentier M Mariller C Mazurier J Lactoferrin structure functions Adv Exp Med Biol 606 163 194 2008 2 Ward PP Paz E Conneely OM Multifunctional roles of lactoferrin a critical overview Sci 62 2540 8 2005 3 Martins CA Fonteles MG Barrett LJ Guerrant RL Correlation of lactoferrin with hilic inflammation in body fluids Clin Diagn Lab Immunol 2 763 5 1995 4 Rado T A Bollekens J St Laurent G Parker L Benz E J Jr Lactoferrin esis during granulocytopoiesis Blood 64 1103 9 1984 5 Guerrant RL Araujo V Soares E Kotloff K Lima AA Cooper WH Lee AG lactoferrin as a marker of fecal leukocytes J Clin Microbiol 30 1238 sure
5. Human Lactoferrin ELISA Kit CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures ELISA Kit for Measuring Human Lactoferrin Y CircuLex Human Lactoferrin ELISA aw Cat CY 8089 O Intended USe i cccccceeccccceccccceesssssseeseseseseees 1 EOI tat A A nee ee ene ee eee ee 1 ist hae 6 076140 6 9 een nen een een Serene nee irer err aren 2 Qy Principle of the Assay 2 3 Materials Provided cisccccsnsccsssctsnnsacsstcsannensseeceee 3 Materials Required but not Provided 4 Precautions and Recommendationg a Sample Collection Storage and Preparation 6 Detailed Pros Colac pcsinsatcntawersothiectarcicanenennenstes Coda PAO INS asa cicarecoxssccosuncdicaeesxeantan dd teacinteseard Measurement Range Troubleshootiie siicecatssisvatsncteanesesasccsatossvnccasens Reagent Stability icccazssssssccedvesteaveceatassoeaemsvavee Assay Characteristics cicsscsccssesssesseteccesssneesacs Example of Test Results RETSRCHICES j i zsseesivsvacaamecuaasesboncadeasasaiocmenevseeses Related Proqucts isssssscvesetace sssssncesacnssasssevaeaasers Intended Use The CycLex Research Product CircuLex actoferrin ELISA Kit is used for the quantitative 1 a measurement of human lactoferrin in se nd other biological media 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
6. PO and PFABP may be useful for classifying IBD Lactoferrin also directly interacts with modified LDL to prevent its interaction with scavenger receptors 8 9 A region rich in basic amino acid residues near the lactoferrin N terminus is responsible for the interaction with acetylated or oxidized LDL Principle of the Assay The CycLex Research Product CireuLex Human Lactoferrin ELISA Kit employs the quantitative sandwich enzyme immunoassay technique A monoclonal antibody specific for human lactoferrin is pre coated onto a microplate Standards and samples are pipetted into the wells and the immobilized antibody binds any human lactoferrin present After Washing away any unbound substances an HRP conjugated monoclonal antibody specific for human lactoferrin is added to the wells Following a wash to remove any unbound monoclonal antibody HRP conjugate the remaining conjugate is allowed to react with the substrate H O 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 lactoferrin A standard curve is constructed by plotting absorbance values versus human lactOferrif concentrations of calibrators and concentrations of unknown samples are determined usingsthis standard curve Cat CY 8089 2 Version 140318 Human Lactoferrin ELISA Kit C ircuLex User s Manual A For Research Use On
7. ailed 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 sndicat 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 reagents included in the CycLex Research Product CircuLex Human Lactoferrin ELISA Kit havesbeen tested 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 Lactoferrin Standard must be stored at below 70 G Coated assay plates should be stored in the original foil bag sealed by the zip lock and containing a desiccant pack Cat CY 8089 9 Version 140318 Human Lactoferrin ELISA Kit C ircuLex User s Manual A 4 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 lactoferrin giving absorbance than mean absorbance of blank plus three standard deviations of the absorbance of blank A b 3SD blank is better than 259 pg
8. for use in diagnostic procedures Detailed Protocol The CycLex Research Product CircuLex Human Lactoferrin 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 Lactoferrin 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 contamination 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 Lactoferrin 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 Cfor long term storage 2 Reconstitute Human Lactoferrin Standard with 1 0 mL of Dilution Buffer The concentration of the human lactoferrin in vial should be 200 ng mL which is ref wed as a Master Standard of human lactoferrin Prepare Standard Solutions as follows Use the Master Standard to produce a dilution series below Mix each tube thoroughly before the next transfer The 20 ng mL standard Std 1 serves as the highest standard The Dilution Buffer serves as the zero standard Bla
9. h 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 zetoyconcentration or 3 0 units for the highest standard concentration The plate shouldbe 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 lactoferrin concentration of off scale samples The readings at 405 nm should not replace the on scale readings at 450 nm Cat CY 8089 8 Version 140318 Human Lactoferrin ELISA Kit C ircuLex 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 lactoferrin concentration of each sample first find the absorbance value on the y axis and extend a h
10. llow 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 0 ice for up to 6 hours before assaying Aliquots of serum may also be stored at below 70 C forme d periods of time Avoid repeated freeze thaw cycles Plasma Collect plasma using EDTA Nay as the anticoagulant If possible collect the toa mixture of EDTA Na and Futhan5 to stabilize the sample against spontaneous in vitro ment 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 red at below 70 C for extended periods of time Avoid repeated freeze thaw cycles Note Citrate plasma has not been validated for use in this assay 7 Other biological samples Remove any particulates by centrifugation an ediately or aliquot and store samples at below 70 C Avoid repeated freeze thaw cycles Sample Preparation e Serum and plasma samples require a 100 fold dilution e Other biological samples require neat to appropriate diluti For example Milk 1 0 x 10 to 1 0 x 10 fold dilution Tear 1 0 x 10 to 1 0 x 10 fold dilution Saliva 1 000 to 5 000 fold dilution Serum 100 to 500 fold dilution Urine 1 to 10 fold dilution C CY 8089 6 Version 140318 T Human Lactoferrin ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not
11. ly Not for use in diagnostic procedures Summary of Procedure Add 100 uL of diluted samples to the wells J Incubate for 1 hour at room temp Wash the wells Add 100 uL of HRP conjugated anti human lactoferrin monoclonal ody 4 Incubate for 1 hour at room temp Wash the wells Add 100 uL of Substrate Reagent 4 Add 100 uL of Stop Solution O t Measure absorbance at 450 nm Materials Provided All samples and standards should be assayed in The following components are supplied and are sufficient for the one 96 well microplate kit Microplate One microplate supplied ready to use with 96 wells 12 strips of 8 wells in a foil zip lock bag with a desiccant pack Wells are c ith anti human lactoferrin monoclonal antibody AF 2D12 as a capture antibody 10X Wash Buffer One bottle containing 100 of 10X buffer containing 2 Tween 20 Dilution Buffer One bottle contai mL of 1X buffer use for reconstitution of Human Lactoferrin Standard and sample dilution Rea u Human Lactoferrin Standar ial containing 200 ng of lyophilized human lactoferrin HRP conjugated Detecti ntibody One bottle containing 12 mL of HRP horseradish peroxidase conjugated anti human in monoclonal antibody AF 1E4 Ready to use Substrate Rea bottle containing 20 mL of the chromogenic substrate tetra methylbenzidine TMB Ready to CG le containing 20 mL of 1 N H2SOxz Ready to use Stop Solutio C CY 8089 3 Version 140318 Human Lact
12. ment of fecal 6 Parsi MA Saadeh SN Zein NN Davis GL Lopez R Boone J diagnosis of spontaneous bacterial peritonitis Gastroenterology 1 tic fluid lactoferrin for 7 2008 7 Buderus S Boone J Lyerly D Lentze MJ Fecal lactoferrin ga ne ameter to monitor infliximab therapy Dig Dis Sci 49 1036 9 2004 8 Kajikawa M Ohta T Takase M Kawase K Shimamur da I Lactoferrin inhibits cholesterol accumulation in macrophages mediated by acety r Oxidized low density lipoproteins Biochim Biophys Acta 1213 82 90 1994 9 Suginohara Y Miyazaki A Hakamata H Sak o Y Ohta T Matsuda I Horiuchi S The heparin bound fraction of human lipoprotein deficient serum inhibits endocytic uptake of oxidized low density lipoprotein by macrophages A lerosis 120 167 79 1996 C CY 8089 15 Version 140318 Human Lactoferrin ELISA Kit C ircuLex User s Manual 4 For Research Use Only Not for use in diagnostic procedures Related Products CircuLex S100A12 EN RAGE ELISA Kit Cat CY 8058 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 CycLex CircuLex products are supplied for research use onl components thereof may not be resold modified for resale products without prior written approval from CycLex Co ditd such commercial use please contact us via email x CircuLex products and manufacture commercial i
13. ml of sample Dilution Buffer is pipetted into blank wells Typical Standard Curve Human Lactoferrin Standard Curve 3 0 p A450 0 5 10 15 20 Human Lactoferrin Conc ng ml C CY 8089 10 Version 140318 T Human Lactoferrin ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures 2 Precision Intra assay Precision Precision within an assay Five samples of known concentration were tested seven times on one plate to assess intra assay precision e Intra assay Within Run n 7 CV 4 2 7 3 Human lactoferrin concentration Sample 1 Sample 2 Sample3 Sample 4 Sample 5 ug ml ug m ng ml ng ml __ ug g 1 1454 6 1375 8 39 9 328 7 9 0 2 1483 9 1447 7 39 1 338 4 7 6 3 1597 5 1477 5 38 2 330 0 8 4 4 1701 3 1308 3 38 9 343 1 8 9 5 1423 6 1333 9 35 5 330 0 75 6 1464 2 1271 1 36 4 340 0 8a 7 1509 7 1356 1 37 1 383 4 84 max 1701 3 1477 5 39 9 383 4 9 0 min 1423 6 1271 1 35 5 328 7 7 5 Sample 1 Milk mean 1519 3 1367 2 37 9 341 9 8 3 Sample 2 Tear SD 97 4 73 8 16 19 2 0 6 e o ample 4 Serum CV 6 4 5 4 4 2 5 6 7 3 Sample 5 Stool Inter assay Precision Precision between assays Five samples of known concentration werejjtested in three separate assays to assess inter assay precision e Inter assay Run to Run n 3 CV 21 9 5 Human lactoferrin concentration Sample ijSample 2 Sample 3 Sample 4 Sample 5 Assay _ ug ml 4
14. nk Volume of Standard Dilution Buffer Concentration Std 1 100 uL of Master Standard 200 ng mL 900 uL 20 ng mL Std 2 300 uL of Std 1 20 ng mL 300 uL 10 ng mL Std 3 300 uL of Std 2 10 ng mL 300 uL 5 ng mL Std 4 300 uL of Std 3 5 ng mL 300 uL 2 5 ng mL Std 5 300 uL of Std 4 2 5 ng mL 300 uL 1 25 ng mL Std 6 300 uL of Std 5 1 25 ng mL 300 uL 0 63 ng mL Std 7 300 uL of Std 6 0 625 ng mL 300 uL 0 31 ng mL Blank 300 uL 0 ng mL Note Do not use a Repeating pipette Change tips for every dilution Wet tip with Dilution Buffer before dispensingsUnusedportions of Master Standard should be aliquoted and stored at below 70 C immediately Avoid multiple freeze and thaw cycles Cat CY 8089 T Version 140318 T Human Lactoferrin ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Standard Assay Procedure for Human Lactoferrin 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 serum sample 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
15. nquire about licensing for C CY 8089 16 Version 140318
16. oferrin ELISA Kit C ircuLex User s Manual 4 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 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 of 450 nm which will give a somewhat higher reading Software package facilitating data generation and analysis opti e 500 or 1000 mL graduated cylinder e Reagent reservoirs Deionized water of the highest quality e Disposable paper towels C CY 8089 4 Version 140318 T Human Lactoferrin ELISA Kit C 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 de
17. orizontal line to the standard curve At the point of intersection extend a vertical line to the x axis and read the corresponding human lactoferrin 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 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 Wersus 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 0 31 ng mL to 20 ng ml 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 lactoferrin concentration Troubleshooting 1 The Human Lactoferrin Standard should be un in duplicate using the protocol described in the Det
18. tergent residues from glassware e Use deionized water of the highest quality e Do not mix reagents from different kits The buffers and reagents in this kit may contain preservatives or othetchemiu als Care should be taken to avoid direct contact with these reagents e Do not mouth pipette or ingest any of the reagents e Do not smoke eat or drink when performing the assay or in 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 eye 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 medieal att ntion 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 CY 8089 5 Version 140318 Human Lactoferrin ELISA Kit CircuLex User s Manual 4 For Research Use Only Not for use in diagnostic procedures Sample Collection Storage and Preparation Serum Use a serum separator tube and a
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