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1. C W ed pp 65 103 Springer Verlag Berlin 2 Dunnington D J 1984 The Development and Study of Single Cell cloned Metastasizi Tumour Cell Systems in the Rat Ph D thesis University of London w Ebralidze A Tulchinsky E Grigorian M Afanayeva A Senin V Revazova E and L din E 1989 Genes Dev 3 1086 1093 4 Nikitenko L Lloyd B Rudland P Fear S and Barraclough R 2000 Int J Q in press Davies B Davies M Gibbs F Barraclough R and Rudland P 1993 Me 8 999 1008 6 Lloyd B Platt Higgins A Rudland P and Barraclough R 1998 O 465 473 7 Davies M Rudland P Robertson L Parry E Jolicoeur P an gh R 1996 Oncogene 13 1631 1637 Nn 8 Ambartsumian N Grigorian M Larsen F Karlstrom O eni Rygaard J Georgiev G and Lukanidin E 1996 Oncogene 13 1621 1630 9 Takenaga K Nakanishi H Wada K Suzuki M Matsuura A and Endo H 1997 Clin Cancer Res 3 2309 2316 Related Products CircuLex S100A13 ELISA Kit Cat CY CircuLex S100A12 ELISA Kit Cat CY CircuLex S100P ELISA Kit Cat CY CircuLex S100A8 MRP8 ELISA Kit t CY 8061 CircuLex S100A9 MRP14 ELISA at CY 8062 CircuLex S100A11 ELISA Kit 63 CircuLex S100A14 ELISA Kit 8064 CircuLex S100A7 Psoriasin EL Cat CY 8073 CircuLex S100A4 ELISA at CY 8086 Anti Human S100A3 Clon E3 Cat CY M1039 Anti Human S102. Invert the plate and blot it towels Note 2 Reliabl ard curves are obtained when either O D values do not exceed 0 2 units for the oncentration or 2 5 units for the highest standard concentration The plate nitored at 5 minute intervals for approximately 30 minutes plate reader is not capable of reading absorbance greater than the absorbance of the andard perform a second reading at 405 nm A new standard curve constructed e values measured at 405 nm is used to determine 100A4 concentration of off scale amples The readings at 405 nm should not replace the on scale readings at 450 nm Note 1 Complete remoya CY 8086 Version 120726 CircuLex 100A4 ELISA Kit Ver 2 n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Calculations Average the duplicate readings for each Standard Solution control and sample and subtrac average zero standard optical density Plot the optical density for the standards versus the conce of the standards and draw the best curve The data can be linearized by using log logespa regression analysis may be applied to the log transformation To determine the 100A4 co each sample first find the absorbance value on the y axis and extend a horizontal line t curve At the point of intersection extend a vertical line to the x axis and read the S100A4 concentration If the samples have been diluted the concentration read from the standard curve must b
3. calculating the O Sensitivity corresponding concentration 0 5 10 15 20 25 30 G 100A4 conc ng ml C CY 8086 11 Version 120726 CircuLex 100A4 ELISA Kit Ver 2 TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures 2 Specificity The antibodies in the Circulex S100A4 ELISA Kit are highly specific of S100A4 with no detectab cross reactivities to all S100 proteins except S100A4 100 ng ml of recombinant S100 proteins measured by the CircuLex S100A4 ELISA kit Specificity of S100A4 ELISA Kit Dee eee ae ee T E E E EE DG peas Se ie E E E E se AE ee Bid crete erat orer er nrorrememeererereroeneree A450 eee T ese ose Bese E E E E E E ies ses ip p aE 0 0 S100A1 S100A2 S100A3 100A4 S100A5 jf S100A6 J S100A7 jf S100A8 J S100A9 I S100A10 S100A11 S100A12 S100A13 S100A14 S100A16 3 Precision Intra assay Precision Precision within an assay Three samples of known concentration CO eight times on one plate to assess intra assay precision e Intra assay Within Run n 8 CV 5 8 Inter assay Precision Precision bet says Three samples of known con ation were tested in four separate assays to assess inter assay precision e Inter assay Run to Ru 7 2 4 Spiking Recover Serum samples wer ith different amounts of S100A4 and assayed The recovery of d to levels throughout
4. the range of the assay was evaluated Sample Average Range Cell culture medi 103 93 84 113 C CY 8086 12 Version 120726 a CircuLex S100A4 ELISA Kit Ver 2 Circulex User s Manual For Research Use Only Not for use in diagnostic procedures 5 Linearity To assess the linearity of the assay samples containing and or spiked with high concentrations S100A4 were serially diluted with the Dilution Buffer to produce samples with values withi dynamic range of the assay Linearity 25 N S100A4 conc ng ml 0 0 2 0 4 0 6 0 8 1 1 2 Sample Dilution Ratio Example of Test Results Fig l S100A4 level in culture supernatant of several c Measured S100A4 level in Culture supernatant of dilution buffer ines confluent cultured cell diluted 2 fold with 29o a aa aa aa aaa E OO IEEE E eee ee ee eee nena menial eae ell aie eleanor 1 5 fB 10 lt x OS p i am aaa 0 0 m m E e e E ee a a mm Te o 5 N N N 2 2 g e z F 5 T a 0 o D l d x o 3 lt lt He x 2 T 5 4 N a I a r E gt C CY 8086 13 Version 120726 CircuLex 100A4 ELISA Kit Ver 2 n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures References Hilt D and Kligman D 1991 in Novel Calcium binding Proteins Fundamentals and Cli Implications Heizmann
5. 0A4 Cat CY P1026 Anti Human S100P P1028 Anti Human t CY P1033 Anti Human A t CY P1034 Anti Human S Cat CY P1039 Anti Huma Cat CY P1040 t CY R2250 1 Cat CY R2251 2 Cat CY R2252 0043 Cat CY R2253 CY 8086 Version 120726 CircuLex 100A4 ELISA Kit Ver 2 n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Human S100A4 Cat CY R2254 Human S100A5 Cat CY R2255 Human S100A6 Cat CY R2256 Human S100A7 Cat CY R2257 Human S100A8 Cat CY R2258 Human S100A9 Cat CY R2259 G Human S100A9 Cat CY R2259 H Human S100A10 Cat CY R2260 Human S100A12 Cat CY R2262 G Human 8100A12 Cat CY R2262 H Human S100A13 Cat CY R2263 Human 8100A14 Cat CY R2264 Human S100A16 Cat CY R2266 Human S100P Cat CY R2267 Human 8100A11 Cat CY R2269 Human S100A1 Low Endotoxin Cat CY R2451 Human S100A3 Low Endotoxin Cat CY R2453 Human S100A4 Low Endotoxin Cat CY R2454 Human S100A7 Low Endotoxin Cat CY R2457 Human S100A8 Low Endotoxin Cat CY R2458 Human S100A9 Low Endotoxin Cat CY R2459 G Human S100A11 Low Endotoxin Cat CY R2461 Human S100412 Low Endotoxin Cat CY R2462 G Human S100A14 Low Endotoxin Cat CY R2 Human S100P Low Endotoxin Cat CY R24 PRODUCED BY CycLex Co Ltd 1063 103 Terasawaoka Ina Nagano 396 0002 Japan Fax 81 265 76 7618 e mail info cyclex co jp U
6. 26 CircuLex 100A4 ELISA Kit Ver 2 n TM Circulex User s Manual For Research Use Only Not for use in diagnostic procedures Introduction Members of the S100 protein family are low molecular mass acidic proteins characterize cell type specific expression and the presence of 2 EF hand calcium binding domains with di affinities for calcium 1 Elevated levels of one S100 protein S100A4 are closely associate process of metastasis in breast and other cancer cells in rodent animal models and in specimens S100A4 or its mRNA is found at an elevated level in metastatic relative to no 2 and mouse 3 tumor cell lines and malignant relative to benign human breast tumors of the level of rat 5 or human 6 S100A4 in benign rat mammary tumor cells results in the aequisition of metastatic capability by some of the cells In transgenic mouse models of breastscancer elevated levels of S100A4 in neu oncogene induced 7 or in murine mammary tumor virus 8 benign mammary tumors yield lung metastases In colorectal adenocarcinoma specimens levated levels of immunocytochemically detected S100A4 are associated with the more malign atous regions of the primary tumors and with liver metastases 9 e immunoassay technique A monoclonal antibody specific for S100A4 has been pre coate microplate Standards and samples are pipetted into the wells and the immobilized anti ds any S100A4 present After washing away any unbound substan
7. CircuLex 100A4 ELISA Kit Ver 2 TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures ELISA Kit for Measuring Human S100A4 CircuLex S100A4 ELISA Kit Ver 2 cA Cat CY 8086 O Intended Use cisssiesadstatsntcssaasaaaiaaabalenatonatsons 1 AOI aT EEEE 1 ntroduction s ssseeeeeeeeeeeeseesssssrsssresesessrssee 2 Principle of the Assay 2 3 Materials Provided c cccesssceeessteeeeeeeees 4 Materials Required but not Provided 5 Precautions and Recommendations 6 O Sample Collection and Preparation 7 Detailed Protocol eececcesesteeeeeeteeeeees 8 9 CALCU LAGIOIIS cecancasaacentesassasnassaexconeanwndeetteausde 10 Measurement Rative ccssesccccersnsvssaacenss 10 Troubleshooting vis ccsacsesseicecaccsnansasasanaysevenen 10 Reagent SCADA vesccdseases ccstavrvecaccaronsasaaedoays 11 Assay Characteristics eee 11 Example of Test Results ceeeeeeeeeeeeee 1 RGLELCHCES sasctssscnisvsinccedaqaniebosaainanadatailues 1 Related PROGUCIS antenccsattgscsstasteasasaspustareansds 14 1 Intended Use The CycLex Research Product Circulex LISA Kit is used for the quantitative measurement of human S100A4 in cell extract tissue remedium and other biological media This assay kit is for research use olfa t for use in diagnostic or therapeutic procedures Storage e Upon receipt store all co e Don t expose reagents t e C CY 8086 1 Version 1207
8. Cl 0 2 NP 40 5 mM EDTA 2Na pH 7 5 10 Glycerol 0 5 mM DTT protease inhibitor cocktail 1 pg ml pepstatin 1 ug ml in 0 3 uM aprotinin 15 uM E 64 C CY 8086 T Version 120726 CircuLex 100A4 ELISA Kit Ver 2 n TM Circulex User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol The CycLex Research Product Circulex S100A4 ELISA Kit is provided with removable stri wells so the assay can be carried out on separate occasions using only the number of strips requi the particular determination Since experimental conditions may vary an aliquot of the S100A within the kit should be included in each assay as a calibrator Disposable pipette tip troughs should be used for all liquid transfers to avoid cross contamination of reagents or s Preparation of Working Solutions All reagents need to be brought to room temperature prior to the assay Assay r ready to use with the exception of 10X Wash Buffer and S100A4 Standard 1 Prepare a working solution of Wash Buffer by adding 100 mL of the 10X W deionized distilled water ddH2O Mix well Store at 4 C for two wee storage er to 900 mL of C for long term 2 Reconstitute 100A4 Standard with 0 9 mL of ddH O The concent the human S100A4 in vial should be 960 ng mL which is referred as a Master Standar S100A4 Prepare Standard Solutions as follows Use the Master Standard to produce a dilution series b ach tube thoroughl
9. RL http www cyclex co jp CycLex CircuLex products reg ied for research use only CycLex CircuLex products and components thereof may e 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 4 ontact us via email C CY 8086 15 Version 120726
10. ces an HRP conjugated anti ic for S100A4 is added to the wells Following a wash to remove any unbound antibody jugate the remaining conjugate is allowed to react with the substrate H O tetramethylbenzidi i acidic solution and absorbance of the resulting yello 9 neasured at 450 nm The absorbance is proportional to the concentration of S100A4 A is constructed by plotting absorbance values versus S100A4 concentrations of calibrat and concentrations of unknown samples are determined using this standard curve The Circulex S100A4 ELISA Kit is designed to meastire the concentration of S100A4 from human cell extract cultured human macrophages or itioned medium Q N C CY 8086 2 Version 120726 CircuLex 100A4 ELISA Kit Ver 2 TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Summary of Procedure Add 100 uL of diluted sample to the wells Y Incubate for 1 hour at room temp cA Wash the wells O v Add 100 uL of HRP conjugated anti S100A4 antibody v Incubate for 1hour at room temp Wash the wells Add 100 uL of Substrate Reagent y Add 100 uL of Stop Solution v Measure absorbance at 450 nm C CY 8086 3 Version 120726 CircuLex 100A4 ELISA Kit Ver 2 TM CircuLex User s Manual 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
11. e multiplied by the dilution factor 1 The dose response curve of this assay fits best to a sigmoidal four parameter results of unknown samples can be calculated with any computer program logistic function It is important to make an appropriate mathematical adjus the dilution factor our parameter 2 Most microtiter plate readers perform automatic calculations of analyte entration The calibration curve is constructed by plotting the absorbance Y of calib sus log of the known concentration X of calibrators using the four parameter fi tion Alternatively the logit log function can be used to linearize the calibration curve i e logit efyabsorbance Y is plotted versus log of the known concentration X of calibrators Measurement Range The measurement range is 0 375 ng mL to 24 standard should be diluted with Dilution Buffer in to be taken into consideration in calculating the hum ample reading higher than the highest dilution and re assayed Dilution factors need 00A4 concentration Troubleshooting 1 The Standard Solutions should be mn cate using the protocol described in the Detailed Protocol Incubation times or tempe fatureg significantly different from those specified may give erroneous results 2 Poor duplicates accompanied by elev values for wells containing no sample indicate insufficient washing If all instructions i iled Protocol were followed accurately such results indicate a ne
12. ed for washer maintenan 3 Overall low signal may ndic at desiccation of the plate has occurred between the final wash and addition of Substrate Re t Do not allow the plate to dry out Add Substrate Reagent immediately after wash O C CY 8086 10 Version 120726 CircuLex 100A4 ELISA Kit Ver 2 TM Circubex 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 100A4 ELISA Kit have tested for stability Reagents should not be used beyond the stated expiration date Upon recei reagents should be stored at 4 C except the reconstituted S100A4 Standard must be storedya below 70 C Coated assay plates should be stored in the original foil bag sealed by the containing a desiccant pack For research use only not for use in diagnostic or therapeutic procedures Assay Characteristics i 2 1 Sensitivity The limit of detection defined as such a concentration of S100A4 hon higher than mean absorbance of blank plus three standard deviations of the absorb A blank 3 SD blank is better than 0 28 ng ml of sample O Dilution Buffer is pipetted into blank wells Eighty assays were evaluated and the minimum detectable dos f S100A4 ranged from 0 196 0 352 ng mL The mean MDD was 0 282 ng mL The MDD determined by adding three standard deviations to the mean optical density value of twenty zefOgst d replicates and
13. o be read at a single wavelength of 450 nm which will give a somewhat higher readi Software package facilitating data generation and analysis opti 9 500 or 1000 mL graduated cylinder e Reagent reservoirs e Deionized water of the highest quality e Disposable paper towels C CY 8086 5 Version 120726 CircuLex 100A4 ELISA Kit Ver 2 n TM CircuLex User s Manual A For Research Use Only Not for use in diagnostic procedures Precautions and Recommendations 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 h 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 Qy e Use deionized water of the highest quality e Do not mix reagents from different kits The buffers and reagents used in this kit contain NaN as preservati hould 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 N where samples or reagents are handled e Dispose of tetra methylbenzidine TMB containi ions in compliance with local regulations e Avoid contact with the acidic Stop Solution and Subs Solution which contains hydrogen peroxide e Wear glove
14. oom temperature ca 25 C for 1 hour shaking at ca 3 on an orbital microplate shaker 5 Wash 4 times by filling each well with Wash Buffer 350 uL using a s le multi channel pipette manifold dispenser or microplate washer 6 Add 100 uL of HRP conjugated Detection Antibody into each well N Incubate the wells at room temperature ca 25 C for 1 hour at ca 300 rpm on an orbital microplate shaker 8 Wash 4 times by filling each well with Wash Buffer 350 u pipette manifold dispenser or microplate washer sin squirt bottle multi channel 9 Add 100 uL of Substrate Reagent Avoid expos the plate with e g aluminum foil is recommende etutn Substrate Reagent to 4 C immediately after the necessary volume is removed 10 Incubate the wells at room temperature ca orbital microplate shaker The incubation temperature is below than 20 C ay be extended up to 30 minutes if the reaction 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 well usi pectrophotometric microplate reader at dual wavelengths of 450 540 nm Dual wavelen 0 550 or 450 595 nm can also be used Read the microplate at 450 nm if only a single wa an be used Wells must be read within 30 minutes of adding the Stop Solution liquid at each step is essential to good performance After the last wash g Wash Buffer by aspirating or decanting
15. s and eye protection when han unodiagnostic materials and samples of human origin and these reagents In case of conta th the Stop Solution and the Substrate Solution wash skin thoroughly with water and seek m l attention when necessary Biological samples may be cont ith infectious agents Do not ingest expose to open wounds or breathe aerosols W ective gloves and dispose of biological samples properly e CAUTION Sulfuric Acid Sutin g acid Wear disposable gloves and eye protection when amp Y C CY 8086 6 Version 120726 CircuLex 100A4 ELISA Kit Ver 2 n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Sample Collection and Preparation Cell culture medium Collect Cell culture medium and Remove any particulates by centrifugatio assay immediately or aliquot and store samples at below 70 C Avoid repeated freeze thaw cycle Other biological samples Remove any particulates by centrifugation and assay immedia iq and store samples at below 70 C Avoid repeated freeze thaw cycles Cell extract Prepare cell extract according to following protocol Preparation of cell extract from cells yy 1 Suspend 1 x 10 cells 100mm dish sub confluent into 0 5 1 ml of oF er 2 Vortex the cell suspension several times 3 Keep on ice for 30 min QO 4 c f g 15 000 rpm for 10 min 5 Keep the supernatant Store at below 70 C E1A Buffer 25 mM Hepes pH 7 2 7 6 250 mM Na
16. 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 S100A4 monoclonal antibody capture antibody 10X Wash Buffer One 100 mL bottle of 10X buffer containing 2 Tween 20 Dilution Buffer One bottle containing 50 mL of 1X buffer use for sample dilution o use S 100A4 Standard One vials containing 864 ng each of lyophilized win S100A4 HRP conjugated Detection Antibody One vial containing 12 mL of orseradish peroxidase conjugated anti S100A4 antibody Ready to use Substrate Reagent One bottle containing 20 mL of the chromo trate tetra methylbenzidine TMB Ready to use Stop Solution One bottle containing 20 mL of 1 N H2SO4 se C CY 8086 4 Version 120726 CircuLex 100A4 ELISA Kit Ver 2 n TM CircuLex User s Manual 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 Qy e Microplate washer optional Manual washing is possible but not preferable e Plate reader capable of measuring absorbance in 96 well plates at dual wave s of 450 nm 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be used can als
17. y before a the next transfer The 24 ng mL standard Std 1 ser highest standard The Dilution Buffer serves as the zero standard Blank Volume of Standard Dilution Buffer Concentration Std 1 15 uL of Master Standard 585 uL 24 ng mL Std 2 300 uL of Std 1 24 ng mL 300 pL 12 ng mL Std 3 300 uL of Std 2 12 ng 300 uL 6 ng mL Std 4 300 uL of Std 3 6 ng 300 uL 3 ng mL Std 5 300 uL of Std 4 3 300 uL 1 5 ng mL Std 6 300 uL of Std 5 300 uL 0 75 ng mL Std 7 300 uL of Std 300 uL 0 375 ng mL Blank 300 uL 0 ng mL Note Do not use a Repeat ette Change tips for every dilution Wet tip with Dilution Buffer before dispensing portions of Standards should be aliquoted and stored at below 70 C immediately Avo tiple freeze and thaw cycles Sample Diluti e Samples req two fold serial dilution C CY 8086 8 Version 120726 CircuLex 100A4 ELISA Kit Ver 2 n TM CircubLex User s Manual For Research Use Only Not for use in diagnostic procedures Assay Procedure Remove the appropriate number of microtiter wells from the foil pouch and place them into the 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 dup the appropriate wells 4 Incubate the wells at r
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