CY-8070 Human NGAL/Lipocalin
Contents
1. 140318 Tii Human NGAL Lipocalin 2 ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Introduction Human neutrophil gelatinase associated lipocalin NGAL also called human lipocalin 2 vas originally identified as an associated protein to 92 kDa human neutrophil type IV collagenase also called gelatinase B or matrix metalloproteinase 9 MMP 9 1 The rat ortholog neu related lipocalin NRL or a2 microglobulin related protein was identified as a protein highly overexpressed ib mammary cancers The murine ortholog called 24p3 24 kDa superinducible protein Sip24 or uterocalin was identified as a protein induced in response to various proliferative signals and is highly expressed in uterine luminal fluids and epithelial cells Recently 24p3 has also been implicated in processes as diverse as apoptosis 2 and kidney cell differentiation 3 NGAL like most lipocalins is thought to modulate cellular processes by binding to ligand s and interacting with specific cell surface receptors Evidence for such a mammalian receptor for NGAL has recently been reported 2 One early hypothesis proposed that NGAL has immunomodulatory activity by binding and el aring lipophilic inflammatory mediators 4 such as the neutrophil tripeptide chemoattractant N formyl Met Leu Phe 5 6 It has recently been also demonstrated that the binding of NGAL to bacterial siderophores is pivotal in the inn2. Dilution factors need to be taken into consideration in calculating the human NGAL concentration Troubleshooting j The NGAL Standard should bemun i duplicate 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 by elevated values for wells containing no sample indicate insufficient washing If allansttuctions in the Detailed Protocol were followed accurately such results indicate a need for washes maintenance 3 Overall low signal May 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 aftergwash Cat CY 8070 10 Version 140318 Tii Human NGAL Lipocalin 2 ELISA Kit C 1rcu Lex 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 NGAL Lipocalin 2 ELISA Kit have been 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 NGAL Standard_must be stored at below 70 C Coated assay plates should be stored in the original foil bag sealed by the zip lock and containing a desiccant pack Assay Characteristics 1 Sensitivity The limit
3. Human NGAL Lipocalin 2 ELISA Kit is provided wath 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 NGAL 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 NGAL 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 ddH2O Mix well Store at 4 C for two weeks Of lt 20 C for long term storage 2 Reconstitute Hunam NGAL Standard with 0 8 mL of ddHL O he concentration of the human NGAL in vial should be 30 ng mL which is referred as a MastemStandard of human NGAL 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 serves as the high standard The Dilution Buffer serves as the zero standard Blank Volume of Standard Dilution Buffer Concentration Std 1 60 uL of Master Standard 540 uL 3 000
4. 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 Substrate Solution which contains hydrogen peroxide e Avoid contact with Stop Solution which contains Sulfuric Acid e Wear gloves and eye protection when handling immunodiagnostic materials and samples of human origin and these reagents In case of contact with the Stop Solution and the Substrate Solution wash skin thoroughly with water and seek medieal 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 i a Strong acid Wear disposable gloves and eye protection when handling Stop Solution Cat CY 8070 6 Version 140318 Human NGAL Lipocalin 2 ELISA Kit CircuLex Users Maita 4 For Research Use Only Not for use in diagnostic procedures Sample Collection and Storage Serum Use a serum separator tube and allow samp
5. cyelex cogp URL http www cyclex c0 jp CycLex CircuLex products are supplied for research 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 from CycLex Co Ltd To inquire about licensing for such commercial use please contact us via email Cat CY 8070 15 Version 140318
6. postischemic rat kidney J Am Soc Nephrol 12 787A 10 Ohlsson S Wieslander J Segelniark M 2003 Increased circulating levels of proteinase 3 in patients with anti neutrophilic cytoplasmic autoantibodies associated systemic vasculitis in remission Clin Exp Immunol 131 528 535 11 Mishra J Ma Q Prada Ag Mitsnefes M Zahedi K Yang J Barasch J Devarajan P 2003 Identification of neutrophil gelatinase associated lipocalin as a novel early urinary biomarker for ischemic renal injury Am Soc Nephrol 14 2534 2543 12 Amin RP Vickers AF Sistare F Thompson KL Roman RJ Lawton M Kramer J Hamadeh HK Collins J Grissom S Bennett L Tucker CJ Wild S Kind C Oreffo V Davis JW 2nd Curtiss S Naciff JM Cunningham M Tennant R Stevens J Car B Bertram TA Afshari CA 2004 Identification 6f putative gene based markers of renal toxicity Environ Health Perspect 112 465 479 13 Mishra JgMori K Ma Q Kelly C Barasch J Devarajan P 2004 Neutrophil gelatinase associated lipocalin a novel early urinary biomarker for cisplatin nephrotoxicity Am J Nephrol 24 307 315 Cat CY 8070 14 Version 140318 Human NGAL Lipocalin 2 ELISA Kit C ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures 14 Mishra J Dent C Tarabishi R Mitsnefes MM Ma Q Kelly C Ruff SM Zahedi K Shao M Bean J Mori K Barasch J Devarajan P 2005 Neutrophil gelatinase associated lipocalin NGAL as_a bioma
7. 5 MAX 19 23 66 98 110 95 MIN 16 70 61 40 96 81 MEAN 17 99 63 59 104 93 S D 1 087 2 108 6 151 C V 6 0 3 3 5 9 Cat CY 8070 12 Version 140318 Human NGAL Lipocalin 2 ELISA Kit C ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures 3 Spiking Recover Serum samples were spiked with different amounts of NGAL and assayed The recovery of NGAL spiked to levels throughout the range of the assay was evaluated Sample Average Recovery Range Cell culture media n 4 92 87 98 109 4 Linearity To assess the linearity of the assay samples containing and or spiked with high co NGAL were serially diluted with the Dilution Buffer to produce samples with values within range of the assay The linearity of the assay Qy a of amic 20 Serum A A Serum B Serum C human NGAL cone ng ml Dilution ratio 100 C CY 8070 13 Version 140318 Tii Human NGAL Lipocalin 2 ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures References j Kjeldsen L Johnsen AH Sengelov H Borregaard N 1993 Isolation and primary structure of NGAH a novel protein associated with human neutrophil gelatinase J Biol Chem 268 10425 32 2 Devireddy L R Teodoro J G Richard F A and Green M R 2001 Induction of apoptosis by a secreted lipocalin that is transcriptionally regulated by IL 3 deprivati
8. Human NGAL Lipocalin 2 ELISA Kit C ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures ELISA Kit for Measuring Human NGAL Lipocalin 2 Y CircuLex Human NGAL Lipocalin 2 ELISA Kit oO Cat CY 8070 Intended US 0 cssscscsscesceosnivsssasadarcaadacveanienatands 1 NEG SETE 1 TOU OU OI os iejccascOeceaiatesilenttaaiatoteciacinmesantcaud 2 Principle of the Assay 2 3 Materials Provided 4 Materials Required but not Provided 5 Precautions and Recommendations 6 Sample Collection and Storage 6 7 Detailed Protocol ssiiscccsasastecssadasreccsasvseancans 8 10 CACM LAUIOIS cccnscacsmenesascesaasetesianeenVeusnisaiaads 10 Measurement Range eeeeeeeeeeeeeeeeee 10 Troubleshooting icscccesssatsesessasnscersenvosssvene 10 Reagent StaDiLty vecicesessasdecdesseesacaconsnssaasoas 1 Assay CharacterisStics ccccssssccsecareavsveancassanes 1 Referens sssissisisrrscsssssraicassraiossssecisrsstsas 14 Related PRODUCES a sivesscencsssasassanateaanonnmatseinys 15 Intended Use The CycLex Research Product Circu Lipocalin 2 ELISA Kit is used for the quantitative measurement of human NGAL Lipocali i serum plasma urine tissue culture medium and other biological media This assay kit is for research use only and not for use in diagnostic or therapeutic procedures Storage e Upon receipt store all gt e Don t expose reagents cessive light C CY 8070 1 Version
9. ate immune response to bacterial infection Upon encountering imyading bacteria the Toll like receptors on immune cells stimulate the transcription translation and secretion of NGAL Secreted NGAL then limits bacterial growth by sequestrating the iron laden siderophore 7 NGAL is released from the secondary granules of activated neutrophils 1 and plasma levels rise in inflammatory or infective conditions especially in bacterial infections 8 Thus the level of NGAL in plasma or serum has been proposed as a marker of infection However as levels of NGAL may also be raised in neoplastic conditions and renal disorders independently of any infective process this proposed application should be treated with caution An early and dramatic upregulation was later observed in rat proximal tubule cells after ischemia reperfusion injury 9 and raised plasmadevels of NGAL were found to be strongly correlated with decreased renal function in patients with renal damage due to systemic vasculitis 10 The results for renal ischemia reperfusion injury were subsequentlyiconfirmed and extended to nephrotoxic agents 11 13 It has been suggested that urinary NGAL levels may serve as an early marker for ischemic renal injury in children after cardiopulmonary bypass 14 In addition Wang Y et al reported that both clinical and experimental evidence demonstrating that circulating NGAL is a marker for obesity and its associated pathologies Although many tissues e
10. e best curve To determine the NGAL concentration 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 NGAL onec ntration If the samples have been diluted the concentration read from the standard curve must bemultiplied by the dilution factor 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 program having a 4 parameter logistic function It is important to make an appropriate mathematical adjustment to 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 scalibrators versus log of the known concentration X of calibrators using the four parameter function Alternatively the logit log function can be used to linearize the calibration curve i e logit of absorbance Y is plotted versus log of the known concentration X of calibrators 3 Dilution factors need to be taken into consideration in calculating the NGAL concentration Measurement Range The measurement range is 46 9 pg mL to 3 000 pg mL Any sample reading higher than the highest standard should be diluted with Dilution Bufferin higher dilution and re assayed
11. gths 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 singlegwavelength 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 paper towels Note 2 Reliable standard curves are obtained when either O D values do not exceed 0 2 units for the blank Zero concentration or 2 5 units for the highest standard concentration The plate Should be monitored at 5 minute intervals for approximately 30 minutes Note 3 Ithe microplate reader is not capable of reading absorbance greater than the absorbance of the Cat CY 8070 9 Version 140318 Tii Human NGAL Lipocalin 2 ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures highest standard perform a second reading at 405 nm A new standard curve constructed using the values measured at 405 nm is used to determine NGAL concentration of off scale samples The readings at 405 nm should not replace the on scale readings at 450 nm 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 th
12. ion Buffer See Sample Preparation above 3 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 cae300 mpm on an orbital microplate shaker 5 Wash 4 times by filling each well with Wash Buffer 350 uL using aisquirt bottle multi channel pipette manifold dispenser or microplate washer 6 Add 100 uL of HRP conjugated Detection Antibody into each well 7 Incubate the plate at room temperature ca 25 C for 1 hour shaking at ca 300 rpm on an orbital microplate shaker 8 Wash 4 times by filling each well with Wash Buffer 50 uD using a squirt bottle multi channel pipette manifold dispenser or microplate washer 9 Add 100 uL of Substrate Reagent Avoid exposing 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 time may be extended up 30 minutes if the reaction temperature is below than 20 C 11 Add 100 uL of Stop Solution t each well in the same order as the previously added Substrate Reagent 12 Measure absorbance in each well using a spectrophotometric microplate reader at dual wavelen
13. les 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 Na or heparin as the anticoagulant If possible colle sma into a mixture of EDTA Na and Futhan5 to stabilize the sample against spontaneous vitro complement activation Immediately centrifuge samples at 4 C for 15 minutes at 1000 x g Assay immediately or store samples on ice for up to 6 hours before assaying Aliquots of may also be stored at below 70 C for extended periods of time Avoid repeated freeze thaw cycl Note Citrate plasma has not been validated for use in this assay Urine Immediately centrifuge samples at 4 C for 15 minutes at 15 000 supernatant or store on ice for up to 24 hours before assaying Aliquots below 70 C for extended periods of time Avoid repeated freeze tha Assay immediately the may also be stored at Other biological samples Remove any particulates by centrifug assay immediately or aliquot and store samples at below 70 C Avoid repeated freeze thaw c C CY 8070 7 Version 140318 Tii Human NGAL Lipocalin 2 ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol The CycLex Research Product CircuLex
14. mmary of Procedure QO Add 100 uL of diluted sample to the wells Incubate for 1 hour p Wash the wells t Add 100 uL of HRP conju 4 Incu Wash the wells t Add 100 uL of s yer Add 100 pL iG gunn Measure a bance at 450 nm L antibody hour at room temp C CY 8070 3 Version 140318 Human NGAL Lipocalin 2 ELISA Kit C ircuLex User s Manual 4 For Research Use Only Not for use in diagnostic procedures Materials Provided All samples and standards should be assayed in duplicate The following components are supplie 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 bag with a desiccant pack Wells are coated with anti NGAL antibody as a capture antibo 10X Wash Buffer One bottle containing 100 mL of 10X buffer containing 2 Tween 20 Dilution Buffer One bottle containing 50 mL of 1X buffer use for sample OF use Human NGAL Standard One vials containing 24 ng each of lyophilized reco a an NGAL HRP conjugated Detection Antibody One bottle containing 12 mL of HRP horseradish peroxidase conjugated anti human NGAL antibody Ready to use Substrate Reagent One bottle containing 20 mL of the chromogeni tetra methylbenzidine TMB Ready to use o Stop Solution One bottle containing 20 mL of 1 N H2SO4 Rea C CY 8070 4 Version 140318 Human NGAL Lipocalin 2 ELISA Kit C ircuLex User
15. of detection defined as such a concentration of NGAL giving absorbanee higher than mean absorbance of blank plus three standard deviations of the absorbance of blank blank 3SD blank is better than 26 7 pg ml of sample Dilution Buffer is pipetted into blank wells Eighty assays were evaluated and the minimum detectable dose MDD of NGAL ranged from 23 7 31 6 pg mL The mean MDD was 26 7 pg mL The MDD was determined by adding three standard deviations to the mean optical density value of twenty zero standard geplicates and calculating the corresponding concentration Typical standard curve 1 1 5 2 2 5 human NGAL cone ng ml Cat CY 8070 11 Version 140318 Human NGAL Lipocalin 2 ELISA Kit C ircuLex 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 twelve times on one plate to assess intra assay precision e Intra assay Within Run n 12 CV 6 2 3 4 5 1 human NGAL conc ng ml Inter assay Precision Precision between assays Three samples of known concentration wef ptested in five separate assays to assess inter assay precision e Inter assay Run to Run n 4 CV 6 0 3 3 5 9 human NGAL conc ng ml B C Day A 1 17 54 66 98 96 81 2 16 70 63 68 110 65 3 17 47 61 40 110 95 4 19 02 63 51 100 89 5 19 23 62 38 105 3
16. on Science 293 829 834 3 Yang J Goetz D Li J Y Wang W Mori K Setlik D Du T Erdjument Bromage H Tempst P Strong R and Barasch J 2002 Iron delivery pathway mediated by a lipocalin Mol Cell 10 1045 1056 4 Bundgaard J R Sengelov H Borregaard N and Kjeldsen L 1994 Molecular cloning and expression of a cDNA encoding NGAL a lipocalin expressed in human Meutfophils BBRC 202 1468 1475 5 Chu S T Lin H J and Chen Y H 1997 Complex formation between a formyl peptide and 24p3 protein with a blocked N terminus of pyroglutamate J Pept Res 4955822585 6 Sengelov H Boulay F Kjeldsen L and Borregaard N4 1994 Subcellular localization and translocation of the receptor for N formylmethionyl leucylphenylalanine in human neutrophils Biochem J 299 473 479 7 Flo T H Smith K D Sato S Rodriguez D J Holmes M A 2004 Strong R K Akira S Aderem A Lipocalin 2 mediates an innate immune fesponse to bacterial infection by sequestrating iron Nature 432 917 921 8 Xu SY Pauksen K Venge P 1995 Serum measurements of human neutrophil lipocalin HNL discriminate between acute bacterial and viral infections Scand J Clin Lab Invest 55 125 131 9 Matthaeus T Schulze Lohoff E Ichimura Ty Weber M Andreucci M Park KM Alessandrini A Bonventre JV 2001 Co regulation of netifrophil gelatinase associated lipocalin and matrix metalloproteinase 9 in the
17. 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 6 988 pg mL 300 uL 46 9 pg mL Blank 300 uL 0 pg mL Note Do not use a Repeating pipette Change tips for every dilution Wet tip with Dilution Buffer before dispensing Unused portions of Standards should be aliquoted and stored at below 70 C immediately Avoid multiple freeze and thaw cycles Sample Preparation e Serum and plasmasamples require a 100 fold dilution with Dilution Buffer e g 3 uL sample 297 uL Dilution Buffer e Unne samplesifequire a 25 fold and 50 fold dilution with Dilution Buffer Note Dilutions lower than 10 fold should not be used Cat CY 8070 8 Version 140318 Tii Human NGAL Lipocalin 2 ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Assay Procedure 1 Prepare the assay protocol assigning the appropriate wells for setting up Standard solutions diluted patient specimens and any internal laboratory controls in duplicate 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 Dilut
18. rker for acute renal injury after cardiac surgery Lancet 365 1231 1238 15 Wang Y Lam KS Kraegen EW Sweeney G Zhang J Tso AW Chow WS Wat NM XudY Hoo RE Xu A 2007 Lipocalin 2 is an inflammatory marker closely associated with obesity insulin resistance and hyperglycemia in humans Clin Chem 53 34 41 Related Products CircuLex Rat NGAL ELISA Kit Cat CY 8053 CircuLex Human NGAL ELISA Kit Cat CY 8070 CircuLex Mouse FABP1 L FABP ELISA Kit Cat CY 8054 CircuLex Mouse FABP3 H FABP ELISA Kit Cat CY 8055 CircuLex Mouse FABP5 E FABP mall ELISA Kit Cat CY 8056 CircuLex Mouse FABP4 A FABP ELISA Kit Cat CY 8077 CircuLex Rat FABP4 A FABP ELISA Kit Cat CY 8076 CircuLex Rat Adiponectin ELISA Kit Cat CY 8049 CircuLex Human Adiponectin ELISA Kit Cat CY 8050 CircuLex Mouse Adiponectin ELISA Kit Cat CY 8051 CircuLex Dog Adiponectin ELISA Kit Cat CY 8052 CircuLex S100A13 ELISA Kit Cat CY 8057 CircuLex 100A12 EN RAGE ELISA Kit Cat CY 8058 CircuLex S100A4 ELISA Kit Cat CY 8059 CircuLex S100P ELISA Kit Cat CY 8060 CircuLex S100A8 MRP8 ELISA Kit Cat CY 8061 CircuLex S100A9 MRP14 ELISA Kit Cat CY 8062 CircuLex S100A7 Psoriasin ELISA Kit Cat Y 8073 CircuLex CML N carboxymethyl Lysine ELISA Kit Cat CY 8066 CircuLex High Sesitivity CRP ELISA Kit C at CY 8071 PRODUCED BY CycLex Co Ltd 1063 103 Terasawaoka Ina Nagano 396 0002 Japan Fax 81 265 76 7618 e mail info
19. 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 Plate reader capable of measuring absorbance in 96 well plates at dual wa s of 450 nm 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be used n also be read at a single wavelength of 450 nm which will give a somewhat higher readi e Vortex mixer Qy e Microplate washer optional Manual washing is possible but not preferable f 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 8070 5 Version 140318 Tii Human NGAL Lipocalin 2 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 detergent residue from glassware e
20. xpress NGAL their results suggest that adipose tissue and liver are probably the two principal sources that contribute to the increased circulating concentrations of this protein in obesity states 15 Cat CY 8070 2 Version 140318 Human NGAL Lipocalin 2 ELISA Kit C ircuLex User s Manual A 4 For Research Use Only Not for use in diagnostic procedures Principle of the Assay The CycLex Research Product CireuLex Human NGAL Lippocalin 2 ELISA Kit employ quantitative sandwich enzyme immunoassay technique An antibody specific for human NGAL has pe pre coated onto a microplate Standards and samples are pipetted into the wells and the immobilized antibody binds any NGAL present After washing away any unbound substances an HR antibody specific for NGAL is added to the wells Following a wash to remove any unb ody HRP conjugate the remaining conjugate is allowed to react with t rate H O tetramethylbenzidine The reaction is stopped by addition of acidic solution and absorb of the resulting yellow product is measured at 450 nm The absorbance is proportional to the concentration of NGAL A standard curve is constructed by plotting absorbance values versus NG centrations of calibrators and concentrations of unknown samples are determined using this stand The CircuLex Human NGAL Lipocalin 2 ELISA Kit is designed to measurey t centration of human NGAL Lipocalin 2 from serum plasma urine cultured macrophages o toned medium Su
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