CircuLex TM CircuLex TM
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1. e Upon receipt store aigeynponents at 4 C e Don t expose reagesits o excessive light L ircuLex User s Manual A S Human Calprotectin ELISA Kit For Research Use Only Not for use in diagnostic procedures 4 Introduction 3 S100A9 and S100A8 belong to the low molecular mass calcium binding S100 protein Ny 1 they are composed of two distinct helix loop helix motifs EF hands flanked by hytrophobic regions at either terminus and separated by a central hinge region In human S100A is usually co expressed with S100A8 and they form heterodimeric complexes which v named as calprotectin 2 Although a number of possible functions for calprotectin including antimicr hy l activity have been proposed the exact role of these proteins in cell metabolism is still uncffr In human they have been associated with several inflammatory diseases 3 phagoc expressing S100A9 belong to the early infiltrating cells and dominate acute inflammatory legieiis in addition elevated serum levels of calprotectin have been found in patients suffering fro umber of inflammatory inflammatory bowel disease IBD chronic bronchitis 3 some ignancies and autoimmune diseases 5 6 It could be demonstrated with human monocyt at both S100A9 protein and S100A8 proteins are secreted by an energy consuming pathway hich is dependent on an intact microtubule network and involves protein kinase C 7 Fecal calprotectin is a marke2. y Th Human Calprotectin ELISA Kit re CirculLlex User s Manual For Research Use Only Not for use in diagnostic procedures gt ELISA Kit for Measuring Human Calprotectin l CircuLex Human Calprotectin ELISA Rit Cat CY 8107 G Tine SING a coccatessucncsnmoieetocantatsecaacoannslesten 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 Troubles WO Otay sicccsiccesetsnsissse somanagavnnaunensses 9 Reagent SADLY sence scsesitcdspovsneecenenissssantessene 9 amp Assay Characteristics csssssescseeseeeeees Example of Test Results wissiseissscsscnssaavensnns 4 17 RETSLCHCES rss cacaatedepusetececdeaddivdaseumecsneoetauats Related Products cscs ciccescessssssieeteaseeaseace 4 19 amp Intended Use J The CycLex Research Product cine ex Human Calprotectin ELISA Kit is used for the quantitative measurement of human calprotectingy erum plasma and other biological media This assay kit is for research y only and not for use in diagnostic or therapeutic procedures 4 Storage
3. N 38 Human Calprotectin ELISA Kit CircuLex Users Manial For Research Use Only Not for use in diagnostic procedures i O D oO Q S Fig 2 Correlation between CycLex s CircuLex Human Calprotectin ELISA Kit and i counterpart in sera from Crohn s disease and CRP positive patients Correlation CycLex kit versus company B y 1 005x 0 6513 R 0 9243 z a 3 2 a a S measured by company B Kit 10 15 Calprotectin conc ug mL measured by CycLex Kit V w Fig 3 Human calprotectin concentration in tegf amp samples from healthy volunteers LA Human Calprotectin concentration in tear samples E oD 3 3 z 2 Ss S 5 t 7 CY 8107 16 Version 151027 Thi Human Calprotectin ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Fig 4 Human calprotectin concentration in milk samples from healthy volunteers Human Calprotectin concentration in milk samples gt in E E z S k 2 2 a 5 Fig 5 Human calprotectin concentration in saliva Eprples from healthy volunteers Human Calprotectin concentration in saliva samples H o S 3 Human Calprotectin conc ug ml bo saliva 1 saliva 2 saliva 3 saliva 4 saliva 5 saliva 6 Version 151027 P ini Human Calprotectin ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use
4. 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 different 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
5. Users Manial For Research Use Only Not for use in diagnostic procedures S Human Calprotectin ELISA Kit 8 OY Note 2 Reliable standard curves are obtained when either O D values do not exceed 0 25 units Ahe blank zero concentration or 3 0 units for the highest standard concentration We plate should be monitored at 5 minute intervals for approximately 30 minutes K A 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 c constructed using the values measured at 405 nm is used to determine human cal concentration of off scale samples The readings at 405 nm should not replace the ony e readings at 450 nm c Human Calprotectin ELISA Kit L ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 A Q O Q Calculations 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 nigel of the standards and draw the best curve The data can be linearized by using log log paper amg tegression analysis may be applied to the log transformation To determine the human calprotectin Qycentration 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 vert
6. in diagnostic procedures Fig 6 Human calprotectin concentration in sweat samples from healthy volunteers Human Calprotectin concentration in sweat samples E wo oh Z E 3 3 E S S wy Fig 7 Human calprotectin concentration in urine epples from healthy volunteers Human Calprotectin concentration in urine samples Human Calprotectin cone ug ml 0 0 urine 1 urine2 urine3 urine4 urine5 urine6 urine7 S CY 8107 18 Version 151027 CircuLex Users Manial A y Human Calprotectin ELISA Kit For Research Use Only Not for use in diagnostic procedures 4 References X 1 Odink K Cerletti N Br ggen J Clerc RG Tarcsay L Zwadlo G Gerhards G Schlegel Re C3 Two calcium binding proteins in infiltrate macrophages of rheumatoid arthritis 1987 re 330 80 82 2 Steinbakk M Naess Andresen C F Lingaas E Dale I Brandtzaeg P Fagerhol Q niani actions of calcium binding leucocyte L1 protein calprotectin 1990 Lancet 336 76 5 3 Sorg C The calcium binding proteins MRP8 and MRP14 in acute and chronig inflammation 1990 Behring Inst Mitt 91 126 137 Yon 4 Foell D Hernandez Rodriguez J Sanchez M Vogl T Cid MC ba J Early recruitment of phagocytes contributes to the vascular inflammation of giant cell arteritis 04 J Pathol 204 311 316 5 Nacken W Roth J Sorg C amp Kerkhoff C S100A9 S100A8 Jeloid representatives of the S100 pro
7. infectious agents Do not ingest expose to open wounds or breathe aeros gt Wear protective gloves and dispose of biological samples properly e CAUTION Sulfuric Nid is a strong acid Wear disposable gloves and eye protection when handli p Solution Human Calprotectin ELISA Kit CircuLex Users Manual For Research Use Only Not for use in diagnostic procedures A Q O Q lt Sample Collection and Storage 3 Sample Collecion arid Store Serum Use a serum separator tube and allow samples to clot for 60 30 minutes Copuatfnse the samples at 4 C for 10 minutes at 1 000 x g Remove serum and assay immediately or stor simples on ice for up to 6 hours before assaying Aliquots of serum may also be stored at below 70 r extended periods of time Avoid repeated freeze thaw cycles Y Plasma Collect plasma using EDTA Naz as the anticoagulant If possible colle e plasma into a mixture of EDTA Naz and FUT 175 Futhan to stabilize the sample against Gpontaneous in vitro complement activation Immediately centrifuge samples at 4 C for 15 minufgg at 1 000 x g Assay immediately or store samples on ice for up to 6 hours before assaying Aliqu f plasma may also be stored at below 70 C for extended periods of time Avoid repeated freeze t cycles Note Citrate plasma has not been validated for use in this assay Yon N Cell culture supernatant Remove any particulates by centrifug and assay immediately or aliquot and store sampl
8. wells 2 Typical Standard Curve Yon Standard curve 20 30 40 60 Humann calprotectin conc ng mL Human Calprotectin ELISA Kit CircuLex Users Manial For Research Use Only Not for use in diagnostic procedures 2 Precision O Intra assay Precision Precision within an assay A Four samples of known concentration were tested sixteen times on one plate to assess in assay precision Ww e Intra assay Within Run n 8 CV 3 2 5 7 samge uman serum Human calprotectin conc ug mL le 1 le 2 le 3 le 4 2 1 1 63 8 86 11 79 16 15 RU 2 1 67 8 59 11 67 16 65 gt 3 1 63 8 88 11 92 20 68 4 1 58 9 41 122 17 66 5 171 9 04 12 06 17 39 6 1 54 8 73 11 81 17 22 7 1 61 8 75 12 23 17 87 8 1 63 28 12 33 18 43 9 1 42 8 39 12 43 17 80 10 1 49 8 53 12 78 18 07 11 1 52 9 05 12 06 18 23 2 1 60 9 19 12 98 17 17 13 1 64 8 97 12 48 18 15 14 1 54 8 95 11 79 16 75 15 1 63 8 63 12 45 18 37 16 1 63 9 10 12 92 18 17 MAX 171 9 41 12 98 20 68 MIN 1 42 8 39 11 67 16 15 MEAN 1 59 8 90 12 24 17 80 sD 0 07 0 28 0 41 1 02 Cv 4 6 3 2 3 4 5 7 ge A S Ti Human Calprotectin ELISA Kit Circu Lex User s Manual A For Research Use Only Not for use in diagnostic procedures Inter assay Precision Precision between assays g Four samples of known concentration were tested in four separate assays to assess inter gt asay precision lt Wh e Inter assay Run to Run n 4 CV 4 6 8 7 X a le serum Human calprotectin con
9. Wash Buffer by adding 100 mL of tl X Wash Buffer to 900 mL of deionized distilled water Mix well Store at 4 C for two weeks 0 C for long term storage 2 Prepare HRP conjugated Detection Antibody by 100 fold dilg 100X HRP conjugated Detection Antibody with Conjugate Dilution Buffer at the time of ass ly Prepare appropriate volume for your assay Disfi any unused HRP conjugated Detection Antibody after diluted 3 Reconstitute Human Calprotectin Standard 1 mL of Dilution Buffer After dissolved by gently mixing immediately dispense in small aliquots e g 200 uL to micro centrifuge tubes and store at below 70 C to avoid repeated figo ng and thawing The concentration of the human calprotectin in a vial should be 640 ng L which is referred as a Master Standard of human calprotectin v Prepare Standard Solutions as follows Use the Master Standard to p duce a dilution series below Mix each tube thoroughly before the next transfer The 32 joe standard Std 1 serves as the highest standard The Dilution Buffer serves as the zero st d Blank V of Standard Dilution Buffer Concentration Std 1 100 uL of M ster Standard 640 ng mL 900 uL 64 ng mL Std 2 300 uL 1 64 ng mL 300 uL 32 ng mL Std 3 300 Std 2 32 ng mL 300 uL 16 ng mL Std 4 300 uL of Std 3 16 ng mL 300 uL 8 ng mL Std 5 3 L of Std 4 8 ng mL 300 uL 4 ng mL Std 6 0 uL of Std 5 4 ng mL 300 uL 2 ng mL Std 7 Boo uL of Std 6 2 ng
10. c ug mL x le 1 le 2 le 3 le 4 F 1 1 45 7 89 10 35 15 09 2 1 71 7 58 9 57 16 04 3 1 75 7 74 10 59 16 32 4 1 63 8 93 11 80 16 85 MAX 1 75 8 93 11 80 16 85 MIN 1 45 7 58 9 57 15 09 MEAN 1 63 8 04 10 57 16 08 S D 0 14 0 61 0 92 0 74 CY 8 3 7 6 8 7 4 6 a 3 Spiking Recovery 4 Recombinant human calprotectin was added to el ap ad at different concentrations SS Sample diluted human serum a Spiked Ovserved Expected oe Concentration aad ee ng ml ng ml ng ml 0 Ta Low 4 9 5 Hi 116 5 6 11 5 13 1 114 3 i E SENEE 2 N 1073 0 11 1 Medium 1 4 15 5 15 1 96 9 8 18 6 17 1 91 7 eS 10 IN lt 0 17 9 Medium 2 4 23 1 21 9 95 0 6 26 6 23 9 89 8 8 315 25 9 81 3 Ni Human Calprotectin ELISA Kit CircuLex Users Manial g For Research Use Only Not for use in diagnostic procedures 4 4 Linearity Three samples were diluted with Dilution Buffer and assayed after dilution The neat sample WA set to 1 The results are summarized in the figure below RA Sample hum Linearity Sample 1l Sample 2 Sample3 Calprotectin cone ug mL 0 4 0 6 1 Dilution factor A CircuLex Users Manual For Research Use Only Not for use in diagnostic procedures 4 Example of Test Results 3 Fig l Human calprotectin concentrations in sera from Crohn s disease patients CRP positi tients and healthy volunteers X S Human Calprotectin ELISA Kit i 5 Z c 0 volunteers
11. es at below 70 C Avoid repeated freeze thaw cy Ches Other biological samples Remove any particulates by cent jugation and assay immediately or aliquot and store samples at below 70 C Avoid repeated freeze cycles NOTE Although we suggest to conduct experigets as outlined above the optimal experimental conditions will vary depending on the parafheters being investigated and must be determined by the individual user NO WARRAXTY OR GUARANTEE OF PERFORMANCE USING THESE PROCEDURES R DE OR IMPLIED c Human Calprotectin 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 CircuLex Human Calprotectin ELISA Kit is pro with removable strips of wells so the assay can be carried out on separate occasions using only thesaumber of strips required for the particular determination Since experimental conditions may vary hice of the Standard within the kit should be included in each assay as a calibrator Disposable pette tips and reagent troughs should be used for all liquid transfers to avoid cross contaminati f reagents or samples Y Preparation of Working Solutions All reagents need to be brought to room temperature prior to the assay AS reagents are supplied ready to use with the exception of 10X Wash Buffer 100x HRP conjugated etection Antibody and Human Calprotectin Standard 1 Prepare a working solution of
12. ical line to the x axis and read the c ponding human calprotectin concentration If the samples have been diluted the concentration r rom the standard curve must be multiplied by the dilution factor R Y 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 lyte concentration The calibration curve is constructed by plotting the absorbance Y of Morais 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 ofghRorbance Y is plotted versus log of the known concentration X of calibrators RS Measurement Range O The measurement range is 1 ng mL to 64 ng m Sy sample reading higher than the highest standard should be diluted with Dilution Buffer in hig ilution and re assayed Dilution factors need to be taken into consideration in calculating the hungan calprotectin concentration Troubleshooting Q 1 All samples and standards should b assayed in duplicate using the protocol described in the Detailed Protocol Incubation times or peratures significantly different from those specified ma
13. ing 200 uL of HRP horseradish peroxidase cogfygated anti human calprotectin monoclonal antibody Ready to use Conjugate tion Buffer One bottle containing 12 mL of Conjugate Dilution Buffer Substr te Reagent One bottle containing 20 mL of the chromogenic substrate tetra methylbenzidine TM Ready to use Solution One bottle containing 20 mL of 1 N H2SOx Ready to use CY 8107 3 Version 151027 A CircuLex Users Moniki S Human Calprotectin 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 200 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 oO e Optional Microplate washer Manual washing is possible but not predate 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 THE Plate can also be read at a single wavelength of 450 nm which will give a somewhat higher Ey Optional Software package facilitating data generation q analysis 500 or 1000 mL graduated cylinder KG e Reagent reservoirs amp Y Deionized water of the highest quality wW O e Disposable paper towels S Human Calprotectin ELISA Kit 9 CircuLex Users Manual For Research Use
14. mL 300 uL 1 ng mL gs a 300 uL 0 ng mL Nof Do not use a repeating pipette Change tips for every dilution Unused portions of Master Standard should be aliquoted and stored at below 70 C immediately Avoid multiple freeze and amp thaw cycles CY 8107 7 Version 151027 L ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures S Human Calprotectin ELISA Kit gt Sample Preparation l Dilute samples with Dilution Buffer Serum and plasma samples may require 500 to 1 000 fold dilution lt e Cell culture supernatant samples may require neat to 10 fold dilution Ww e Biological samples may require 50 to 5 000 fold dilution amp Saliva 5 000 fold Sweat 500 fold Urine 100 fold Tear 100 fold Milk 50 f g Standard Assay Procedure for Human Calprotectin K EN Remove the appropriate number of microtiter wells from the foil pouch and lace them into the well holder Return any unused wells to the foil pouch refold seal with tape and e at 4 C w 2 Dilute samples with Dilution Buffer See Sample Preparation above w Pipette 100 uL of Standard Solutions Std1 Std7 Blank and dilated samples in duplicates into the appropriate wells O 4 Incubate the plate at room temperature ca 25 C for 1 hou shaking at ca 300 rpm on an orbital microplate shaker n Wash 4 times by filling each well with Wash Bune
15. man calprotectin concentrati calibrators and concentrations of unknown samples are determined using this standard curve amp CircuLex Users Manial For Research Use Only Not for use in diagnostic procedures S Human Calprotectin ELISA Kit gt Summary of Procedure l 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 calprotectin seifbouy 4 Incubate for 1 hour at room temp y Wash the wells RA wW y gt Add 100 uL of Substrate Reagent wy 4 Incubate for 10 20 mj s at room temp Add 100 uL of Stop Solution N Measure absorbance at 450 nm 4 gt amp Materials Provided Ss 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 cated with anti human calprotectin monoclonal antibody as a capture antibody gt 10X Wash Buffer One bottle gine 100 mL of 10X buffer containing Tween 20 Dilution Buffer One aN onii 50 mL of 1X buffer use for reconstitution of Human Calprotectin Standard and ple dilution Ready to use Human Calprotecti Standard One vial containing 640 ng of lyophilized recombinant human calprotectin O 100x HRP conp Ated Detection Antibody One bottle contain
16. r for inflammatory gastrointestnal as well as neoplastic diseases It is often difficult to distinguish between IBD and irrita Lowel syndrome IBS This leads in many cases to extensive and unnecessary colonoscopic yaminations Fecal calprotectin may be a useful marker for discriminating between patients th IBD and IBS 8 In addition fecal calprotectin levels correlate significantly with histologic and endoscopic assessment of disease activity in Crohn s disease and ulcerative colitis ny b gt disorders including cell arteritis 4 cystic fibrosis G some an dermatoses chronic Principle of the Assay wS The CycLex Research Product CircuL fuman Calprotectin ELISA Kit employs the quantitative sandwich enzyme immunoassay techni An antibody specific for human calprotectin is pre coated onto a microplate Standards and sampl are pipetted into the wells and the immobilized antibody binds any human calprotectin present Af washing away any unbound substances an HRP conjugated antibody specific for human calprot amp fin is added to the wells Following a wash to remove any unbound antibody HRP conjugate th maining conjugate is allowed to react with the substrate Orteramethylbenzidine Bact is stopped by addition of acidic solution and absorbance of the resulting yellow product is sured at 450 nm The absorbance is proportional to the concentration of human calprotectin A st gard curve is constructed by plotting absorbance values versus hu
17. rcuLex High Sensitivity CRP ELISA Kit Cat CY 8071 CircuLex Human Chitotriosidase ELISA Kit Cat CY 80 CircuLex Mouse AIM CD5L Spa ELISA Kit Cat CY 8075 CircuLex Human AIM CD5L Spa ELISA Kit Cat CY 080 CircuLex Human soluble LOX 1 OLR1 ELISA Kit CY 8081 CircuLex Human AIF1 ELISA Kit Cat CY 8084 CircuLex Human YKL 39 ELISA Kit Cat CY 8087 CircuLex Human YKL 40 ELISA Kit Cat C 88 CircuLex Human Lactoferrin ELISA Kit Ca Y 8089 CircuLex Bovine Lactoferrin ELISA Kit G cY CircuLex Human TXNIP ELISA Kit Ca 8090 CircuLex Mouse CIRP ELISA Kit C CircuLex Human CIRP ELISA Kit CircuLex Human soluble VAP 1 E Kit Cat CY 8104 CircuLex Human Calprotectin Eg Kit Cat CY 8107 X Q WwW CycLex Co Ltd 1063 103 Terasawaghie Ina Nagano 396 0 Japan Fax eee E mail info clex co j URL http w cyclex co j PRODUCED BY CyL ReuLes products are supplied for research use only CycLex CircuLex products and com nts thereof may not be resold modified for resale or used to manufacture commercial pr s without prior written approval from CycLex Co Ltd To inquire about licensing for s commercial use please contact us via email CY 8107 20 Version 151027
18. tein family as prominent players in innate immunity 2003 a Res Tech 60 569 580 6 Foell D Frosch M Sorg C Roth J Phagocyte specific galcfum binding S100 proteins as clinical laboratory markers of inflammation 2004 Clin Chim Acta 37 51 7 Rammes A Roth J Goebeler M Klempt M Harsghann M amp Sorg C Myeloid related protein MRP 8 and MRP14 calcium binding proteins of the 00 family are secreted by activated monocytes via a novel tubulin dependent pathway 1997 J BS hem 272 9496 9502 8 Tibble J Teahon K Thjodleifsson B Rose Sigthorsson G Bridger S Foster R Sherwood R Fagerhol M and Bjarnason I A simple cai r assessing intestinal inflammation in Crohn s disease 2000 Gut 47 4 506 513 amp 4 o c Human Calprotectin ELISA Kit L ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures amp Related Products 3 100A8 S100A9 Related Kit aS CircuLex S100A8 MRP8 ELISA Kit Cat CY 8061 RY CircuLex S100A9 MRP14 ELISA Kit Cat CY 8062 CircuLex Human Calprotectin ELISA Kit Cat CY 8107 Y S100A8 S100A9 Related Recombinant Protein A Human S100A8 Cat CY R2258 Q Human S100A9 Cat CY R2259 G wU Human S100A9 Cat CY R2259 H Yon Human S100A8 Low Endotoxin Cat CY R2458 aS Human S100A9 Low Endotoxin Cat CY R2459 G gt N amp Inflammation Related Kit CircuLex Human NGAL Lipocalin 2 ELISA Kit Cat CY 807 Ci
19. uL using a squirt bottle multi channel pipette manifold dispenser or microplate washer G 6 Add 100 uL of HRP conjugated Detection Anty into each well Incubate the plate at room temperature calo for 1 hour shaking at ca 300 rpm on an orbital microplate shaker lt N oo Wash 4 times by filling each well wi ash Buffer 350 uL using a squirt bottle multi channel pipette manifold dispenser or micro washer 9 Add 100 uL of Substrate ReagenfAvoid exposing the microtiter plate to direct sunlight Covering the plate with e g aluminum fo msrecommended Return Substrate Reagent to 4 C immediately after the necessary volume is rem 10 Incubate the plate at ro mperature ca 25 C for 10 20 minutes shaking at ca 300 rpm on an orbital microplate ot incubation time may be extended up to 30 minutes if the reaction temperature is below2Zor 11 Add 100 uL of AN Solution to each well in the same order as the previously added Substrate Reagent amp 12 Measure ab ance in each well using a spectrophotometric microplate reader at dual wavelengths of 450 540 sig Dual wavelengths of 450 550 or 450 595 nm can also be used Read the microplate at N Complete removal of liquid at each step is essential to good performance After the last wash A remove any remaining Wash Buffer by aspirating or decanting Invert the plate and blot it e2 against clean paper towels CY 8107 8 Version 151027 A CircuLex
20. y give erroneous results CA 2 Poor duplicates accompafeed by elevated values for wells containing no sample indicate insufficient washing If all instructi s in the Detailed Protocol were followed accurately such results indicate a need for washer mai ce 3 Overall low signd aay 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 Reage Stability All Oe cece included in the CycLex Research Product CircuLex human Calprotectin ELISA Kit have been tested for stability Reagents should not be used beyond the stated expiration date Upon rece pt kit reagents should be stored at 4 C except the reconstituted Human Calprotectin Standard must b red at below 70 C Coated assay plates should be stored in the original foil bag sealed by the zip and containing a desiccant pack CY 8107 10 Version 151027 A CircuLex Users Manial For Research Use Only Not for use in diagnostic procedures y Human Calprotectin ELISA Kit 8 ee Assay Characteristics gt SSS oO 1 Sensitivity The limit of detection defined as such a concentration of human calprotectin cea cians higher than mean absorbance of blank plus three standard deviations of the absorbas of blank A blank 3SD blank is better than 0 145 ng mL of sample 4 Dilution Buffer was pipetted into blank
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