Magnetic Luminex Performance Assay Human MMP
Contents
1. Magnetic Luminex Performance Assay multiplex kits are designed for use with the Luminex MAGPIX CCD Imager Alternatively kits can be used with the Luminex 100 200 or Bio Rad Bio Plex dual laser flow based sorting and detection platforms Analyte specific antibodies are pre coated onto color coded magnetic microparticles Microparticles standards and samples are pipetted into wells and the immobilized antibodies bind the analytes of interest After washing away any unbound substances a biotinylated antibody cocktail specific to the analytes of interest is added to each well Following a wash to remove any unbound biotinylated antibody streptavidin phycoerythrin conjugate Streptavidin PE which binds to the biotinylated antibody is added to each well A final wash removes unbound Streptavidin PE the microparticles are resuspended in buffer and read using the Luminex MAGPIX Analyzer A magnet in the analyzer captures and holds the superparamagnetic microparticles in a monolayer Two spectrally distinct Light Emitting Diodes LEDs illuminate the beads One LED identifies the analyte that is being detected and the second LED determines the magnitude of the PE derived signal which is in direct proportion to the amount of analyte bound Each well is imaged with a CCD camera Kits can also be used with Luminex 100 200 or Bio Rad Bio Plex dual laser flow based systems www RnDSystems com 1 LIMITATIONS OF THE PROCEDURE FOR RESEARCH2. and LMPM51 1 respectively Cell culture supernate serum plasma and platelet poor plasma samples require a 5 fold dilution A suggested 5 fold dilution is 40 uL of sample 160 uL of Calibrator Diluent RD5 37 Mix thoroughly MMP 2 MMP 8 MMP 9 and MMP 12 serum and plasma samples must be further diluted 10 fold to a final 50 fold dilution A suggested 50 fold dilution is 20 uL of the 5 fold diluted sample 180 uL of Calibrator Diluent RD5 37 Mix thoroughly Saliva samples require a 40 fold dilution A suggested 40 fold dilution is 10 uL of sample 390 uL of Calibrator Diluent RD5 37 Mix thoroughly Urine samples require a 10 fold dilution A suggested 10 fold dilution is 15 uL of sample 4 135 uL of Calibrator Diluent RD5 37 Mix thoroughly REAGENT PREPARATION Bring all reagents to room temperature before use Wash Buffer If crystals have formed in the concentrate warm to room temperature and mix gently until the crystals have completely dissolved Add 20 mL of Wash Buffer Concentrate to deionized or distilled water to prepare 500 mL of Wash Buffer Standard Reconstitute the Standard with Calibrator Diluent RD5 37 Refer to the Standard Value Card for the reconstitution volume and assigned values Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making dilutions Use polypropylene tubes Pipette 500 uL of the reconstituted Standard into the Standard 1 tube Pipette 200 uL of Calibrator
3. R amp D Systems All trademarks and registered trademarks are the property of their respective owners www RnDSystems com REFERENCES 1 2 3 4 Shapiro S D 1998 Curr Opin Cell Biol 10 602 Overall C M and C Lopez Otin 2002 Nat Rev Cancer 2 657 Sternlicht M D and Z Werb 2001 Annu Rev Cell Dev Biol 17 463 Stamenkovic I 2003 J Pathol 200 448 5 Visse R and H Nagase 2003 Circ Res 92 827 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 01 13 McCawley L J and L M Matrisian 2001 Curr Opin Cell Biol 13 534 Kayagaki N et al 1995 J Exp Med 182 1777 Gearing A J et al 1994 Nature 370 555 Powell W C et al 1999 Curr Biol 9 1441 Sch nbeck U et al 1998 J Immunol 161 3340 Overall C M et al 2002 Biol Chem 383 1059 Fowlkes J L et al 1995 Prog Growth Factor Res 6 255 Suzuki M et al 1997 J Biol Chem 272 31730 Hashimoto G et al 2002 J Biol Chem 277 36288 Miyamoto S et al 2004 Cancer Res 64 665 No V et al 2001 J Cell Sci 114 111 Sato H etal 1994 Nature 370 61 Mochizuki S et al 2004 Biochem Biophys Res Commun 315 79 Lijnen H R 2001 Thromb Haemost 86 324 Nguyen M et al 1999 Lab Invest 79 467 Moilanen M et al 2003 Biochemist
4. disruption of the interaction between the catalytic site zinc and a cysteine thiol group in the pro peptide domain This is followed by cleavage ofthe pro peptide 5 Activation can be mediated by several serine proteases 19 21 MMPs 4 17 21 22 or potentially via NO mediated S nitrosylation of the pro peptide cysteine thiol group 23 In some cases activation can take place intracellularly via a furin like serine protease 24 25 MMPs are expressed by many cell types and can be upregulated in response to adhesion molecules growth factors cytokines and hormones 2 5 They have been implicated in several physiological processes including tissue morphogenesis 26 28 cell migration 29 31 wound healing 32 bone remodeling 33 34 and angiogenesis 35 37 MMP activities are modulated on several levels including transcription pro enzyme activation or by their endogenous inhibitors tissue inhibitors of metalloproteinases TIMPs 5 38 Imbalances in MMP regulation have been implicated in several pathological processes including cancer 39 40 cardiovascular disorders 41 42 and arthritis 43 45 MMPs included in this panel Analyte Catalog Number Microparticle Region Analyte Catalog Number Microparticle Region EMMPRIN CD147 LMPM972 30 MMP 8 LMPM908 25 MMP 1 LMPM901 20 MMP 9 LMPM911 26 MMP 2 LMPM902 19 MMP 10 LMPM910 27 MMP 3 LMPM513 21 MMP 12 LMPM919 28 MMP 7 LMPM907 22 MMP 13 LMPM511 29 PRINCIPLE OF THE ASSAY
5. 1 Centrifuge each Biotin Antibody Concentrate vial for 30 seconds at 1000 x g prior to removing the cap 2 Gently vortex the vials taking precautions not to invert the vials 3 Add 50 uL of each Biotin Antibody Concentrate to the vial of Biotin Antibody Diluent 2 Mix gently STREPTAVIDIN PE PREPARATION Use a polypropylene amber bottle or a polypropylene tube wrapped with aluminum foil Protect Streptavidin PE from light during handling and storage 1 Centrifuge the Streptavidin PE vial for 30 seconds at 1000 x g prior to removing the cap 2 Gently vortex the vial taking precautions not to invert the vial 3 Dilute the 100X Streptavidin PE to a 1X concentration by adding 55 uL of Streptavidin PE to 5 5 mL of Wash Buffer 6 For research use only Not for use in diagnostic procedures INSTRUMENT SETTINGS Luminex MAGPIX analyzer a Assign the microparticle region for each analyte being measured see Introduction on page 1 b 50 events bead C Sample size 50 uL d Collect Median Fluorescence Intensity MFI Luminex 100 200 and Bio Rad Bio Plex analyzers Note Calibrate the analyzer using the proper reagents for superparamagnetic microparticles refer to instrument manual a Assign the bead region for each analyte being measured see Introduction on page 1 b 50 events bead c Minimum events 0 d Flow rate 60 uL minute fast e f Doublet Discriminator gates at approximately 8000 and 16 500 9 Collect M
6. Diluent RD5 37 into the remaining tubes Use Standard 1 to produce a 3 fold dilution series below See analyte specific datasheets for details Mix each tube thoroughly before the next transfer Standard 1 serves as the high standard Calibrator Diluent RD5 37 serves as the blank 100 uL 100uL 100uL 100uL 100uL 100 uL an a a aan 500 uL Std mE STANDARD Standard Standard 1 Standard2 Standard3 Standard4 Standard 5 Standard6 Standard 7 www RnDSystems com 5 DILUTED MICROPARTICLE COCKTAIL PREPARATION 1 Centrifuge each Microparticle Concentrate vial for 30 seconds at 1000 x g prior to removing the cap 2 Gently vortex the vials to resuspend the microparticles taking precautions not to invert the vials 3 Dilute the Microparticle Concentrates in the mixing bottle provided The volume of the Microparticle Concentrate listed in the table below is for each analyte e g if measuring a full plate of MMP 1 and MMP 9 add 50 uL of MMP 1 Microparticle Concentrate and 50 uL of MMP 9 Microparticle Concentrate to 5 mL of Microparticle Diluent 3 Number of Wells Used Microparticle Concentrate Microparticle Diluent 3 96 50 0 uL 5 00 mL 72 37 5 uL 3 75 mL 48 25 0 uL 2 50 mL 24 12 5 pL 1 25 mL Note Protect microparticles from light during handling Diluted microparticles cannot be stored Prepare microparticles within 30 minutes of use DILUTED BIOTIN ANTIBODY COCKTAIL PREPARATION
7. FI Sample size 50 uL Note The CAL2 setting for the Bio Rad Bio Plex analyzer should be set at the low RP1 target value www RnDSystems com 7 ASSAY PROCEDURE Bring all reagents and samples to room temperature before use It is recommended that all samples and standards be assayed in duplicate Note Protect microparticles and Streptavidin PE from light at all times N UJ Ui M p O MMPs are detectable in saliva Take precautionary measures to prevent contamination of kit reagents while running this assay Prepare all reagents working standards and samples as directed in the previous sections Resuspend the diluted microparticle cocktail by inversion or vortexing Add 50 uL of the microparticle cocktail to each well of the microplate Add 50 uL of Standard or sample per well Pipette assay within 15 minutes Securely cover with a foil plate sealer Incubate for 2 hours at room temperature on a horizontal orbital microplate shaker 0 12 orbit set at 800 50 rpm Using a magnetic device designed to accommodate a microplate wash by applying the magnet to the bottom of the microplate removing the liquid filling each well with Wash Buffer 100 uL and removing the liquid again Complete removal of liquid is essential for good performance Perform the wash procedure three times Note Refer to the magnetic device user manual for proper wash technique using a round bottom micropla
8. Magnetic Luminex Performance Assay Human MMP Base Kit Catalog Number LMPM000 For the simultaneous quantitative determination of multiple human matrix metalloproteinase MMP concentrations in cell culture supernates serum plasma platelet poor plasma saliva and urine This package insert must be read in its entirety before using this product For research use only Not for use in diagnostic procedures TABLE OF CONTENTS SECTION INTRODUCTION o Se Te PRINCIPLE OF TEIE ASSAY isset ie qui itte tata RUE M trc io tr S aa une da ar dM Ud LIMITATIONS DE THE PROCEDURE tent taret bt Re teend ba ttpu ita tk tsi tu Re pa Rt toco tnane Lis ML PRECAUTIONS t iSe MATERIALS PROVIDED amp STORAGE CONDITIONS uu ssssssssssessessssssssssesecssssessseessnssssensees OTHER SUPPLIES BEQUIBED eret eei inni traten etit sand ete ananebbaniced as snas etta cea SAMPLE COLLECTION AND STORAGE eiiteerisir ttt bant t etota etas netndisa intrent SAMPLE PREPARATION m REAGENT PREPARATION m DILUTED MICROPARTICLE COCKTAIL PREPARATION eere tente tenenntnntnnnn DILUTED BIOTIN ANTIBODY COCKTAIL PREPARATION eerte tenentes STREPTAVIDIN PE PREPARATION uiii diiit ticae pp ie eaten occi o btts INSTRUMENT SETTINGS isaciicesctietcotinstdsittuts Dette etienne tait bate niian on serbe htnc ASSAY PROCEDURE m CADCUEATIO
9. N OF RESULTS sissit CALIBRATION c M I HP REFERENCES acutum Uu ido QU ten ui eni deuda teu ae en EIN DNE MANUFACTURED AND DISTRIBUTED BY USA amp Canada R amp D Systems Inc 614 McKinley Place NE Minneapolis MN 55413 USA TEL 800 343 7475 612 379 2956 FAX 612 656 4400 E MAIL info RnDSystems com DISTRIBUTED BY UK amp Europe R amp D Systems Europe Ltd 19 Barton Lane Abingdon Science Park Abingdon OX14 3NB UK TEL 44 0 1235 529449 FAX 44 0 1235 533420 E MAIL info RnDSystems co uk China R amp D Systems China Co Ltd 24A1 Hua Min Empire Plaza 726 West Yan An Road Shanghai PRC 200050 TEL 86 21 52380373 FAX 86 21 52371001 E MAIL info RnDSystemsChina com cn INTRODUCTION The matrix metalloproteinases MMPs consist of 24 known human zinc proteases with essential roles in breaking down components of the extracellular matrix ECM 1 5 In addition to ECM proteins other potential MMP substrates include cytokines 6 10 chemokines 11 growth factors and binding proteins 12 15 cell cell adhesion molecules 16 and other proteinases 17 18 With afew exceptions MMPs share common structural motifs including a pro peptide domain a catalytic domain a hinge region and a hemopexin like domain 2 4 5 Synthesized as pro enzymes most are secreted before conversion to their active forms In general the activation mechanism is thought to occur in a stepwise fashion involving
10. USE ONLY NOT FOR USE IN DIAGNOSTIC PROCEDURES e The kit should not be used beyond the expiration date on the kit label e Do not mix or substitute reagents with those from other lots or sources e If samples fall outside the dynamic range of the assay further dilute the samples with Calibrator Diluent and repeat the assay Any variation in standard diluent operator pipetting technique washing technique incubation time or temperature and kit age can cause variation in binding e Variations in sample collection processing and storage may cause sample value differences e This assay is designed to eliminate interference by other factors present in biological samples Until these factors have been tested in the Luminex Performance Assay the possibility of interference cannot be excluded e Magnetic Luminex Performance Assays afford the user the benefit of multianalyte analysis of biomarkers in a single complex sample A single multipurpose diluent is used to optimize recovery linearity and reproducibility Such a multipurpose diluent may not optimize any single analyte to the same degree that a unique diluent selected for analysis of that analyte can optimize conditions Therefore some performance characteristics may be more variable than those for assays designed specifically for single analyte analysis Only the analytes listed on the Standard Value Card can be measured with this base kit TECHNICAL HINTS e When mixing or rec
11. asma samples are not suitable for use in the MMP 13 assay EDTA and Citrate are not recommended for use in this assay due to their chelating properties Hemolyzed and icteric samples are not suitable for use in this assay Some MMPs may be released upon platelet activation For example to measure circulating levels of MMP 9 platelet poor plasma should be used It should be noted that many protocols for plasma preparation including procedures recommended by the Clinical and Laboratory Standards Institute CLSI result in incomplete removal of platelets or platelet activation This may cause variable and irreproducible results for assays of factors contained in platelets and released by platelet activation Saliva Collect saliva in a tube and centrifuge for 5 minutes at 10 000 x g Collect the aqueous layer and assay immediately or aliquot and store samples at lt 20 C Avoid repeated freeze thaw cycles Urine Aseptically collect the first urine of the day mid stream voided directly into a sterile container Centrifuge to remove particulate matter assay immediately or aliquot and store at lt 20 C Avoid repeated freeze thaw cycles 4 For research use only Not for use in diagnostic procedures SAMPLE PREPARATION Use polypropylene tubes Note When assaying serum and plasma samples EMMPRIN cannot be multiplexed with MMP 7 MMP 8 MMP 10 MMP 12 or MMP 13 R amp D Systems Catalog s LMPM907 LMPM908 LMPM910 LMPM919
12. body Diluent2 895832 5 5 mL ofa buffered protein base with preservatives Calibrator Diluent RD5 37 895853 21mLofa buffered protein base with preservatives Streptavidin PE 892525 0 07 mL of a 100 fold concentrated streptavidin phycoerythrin conjugate with preservatives Wash Buffer Concentrate 895003 21 mL of a25 fold concentrated solution of buffered surfactant with preservative May turn yellow over time 641385 1 flat bottomed 96 well microplate used as a vessel for the assay Mixing Bottles 895505 2 empty 8 mL bottles used for mixing microparticles with Microparticle Diluent Plate Sealers 640445 4 adhesive foil strips Standard Value Card 749407 1 card listing the Standard reconstitution volume and working standard concentrations for this lot of base kit Provided this is within the expiration date of the kit Discard after use Use a fresh standard for each assay May be stored for up to 1 month at 2 8 C OTHER SUPPLIES REQUIRED e Luminex Performance Assay analyte specific kit s see Introduction on page 1 e Luminex MAGPIX Luminex 100 200 or Bio Rad Bio Plex analyzer with X Y platform e Hand held microplate magnet or platewasher with a magnetic platform e Pipettes and pipette tips e Deionized or distilled water e Multi channel pipette manifold dispenser or automated dispensing unit 50 mL and 500 mL graduated cylinders e Horizontal orbital microplate shaker 0 12 orbit capabl
13. e of maintaining a speed of 800 50 rpm e Microcentrifuge e Polypropylene test tubes for dilution of standards and samples www RnDSystems com 3 SAMPLE COLLECTION AND STORAGE The sample collection and storage conditions listed below are intended as general guidelines Sample stability has not been evaluated Cell Culture Supernates Remove particulates by centrifugation and assay immediately or aliquot and store samples at lt 20 C Avoid repeated freeze thaw cycles Note Cell culture supernate samples are not suitable for use in the MMP 2 assay Serum Use a serum separator tube SST and allow samples to clot for 30 minutes at room temperature before centrifuging for 15 minutes at 1000 x g Remove serum and assay immediately or aliquot and store samples at lt 20 C Avoid repeated freeze thaw cycles Plasma Collect plasma using heparin as an anticoagulant Centrifuge for 15 minutes at 1000 x g within 30 minutes of collection Assay immediately or aliquot and store samples at lt 20 C Avoid repeated freeze thaw cycles Platelet poor Plasma Collect plasma on ice using heparin as an anticoagulant Centrifuge at 2 8 C for 15 minutes at 1000 x g within 30 minutes of collection For complete platelet removal centrifuge the separated plasma at 10 000 x g for 10 minutes at 2 8 C Assay immediately or aliquot and store samples at lt 20 C Avoid repeated freeze thaw cycles Note Plasma and platelet poor pl
14. onstituting protein solutions always avoid foaming To avoid cross contamination change pipette tips between additions of each standard level between sample additions and between reagent additions Also use separate reservoirs for each reagent e To ensure accurate results proper adhesion of plate sealers during incubation steps is necessary e Protect microparticles and Streptavidin PE from light at all times to prevent photobleaching PRECAUTIONS MMPs are detectable in saliva Take precautionary measures to prevent contamination of kit reagents while running this assay Some components in this kit contain ProClin which may cause an allergic skin reaction Avoid breathing mist Wear protective gloves clothing eye and face protection Wash hands thoroughly after handling Please refer to the MSDS on our website prior to use 2 For research use only Not for use in diagnostic procedures MATERIALS PROVIDED amp STORAGE CONDITIONS Store the unopened kit at 2 8 C Do not use past the kit expiration date STORAGE OF OPENED DILUTED PART PART DESCRIPTION OR RECONSTITUTED MATERIAL MMP Panel 894339 2 vials of recombinant human MMPs in a Standard Cocktail buffered protein base with preservatives lyophilized Microparticle Diluent 3 8905857 6mLofa buffered protein base with May be stored for up to 1 month at 2 8 C preservatives Once diluted any unused microparticle cocktail must be discarded Biotin Anti
15. ry 42 5414 Suzuki K et al 1990 Biochemistry 29 10261 Gu Z et al 2002 Science 297 1186 Kang T et al 2002 Cancer Res 62 675 Pei D and S J Weis 1995 Nature 375 244 Simian M et al 2001 Development 128 3117 Lelongt B et al 1997 J Biol Chem 136 1363 Curry T E Jr and K G Osteen 2003 Endocr Rev 24 428 Faveeuw C et al 2001 Blood 98 688 Ratzinger G et al 2002 J Immunol 168 4361 Seiki M et al 2003 Biochem Soc Symp 70 253 Armstrong D G and E B Jude 2002 J Am Podiatr Med Assoc 92 12 Holmbeck K et al 2003 J Cell Biol 163 661 Delaisse J M et al 2003 Microsc Res Tech 61 504 Vu T H et al 1998 Cell 93 411 Nguyen M et al 2001 Int J Biochem Cell Biol 33 960 Seiki M and I Yana 2003 Cancer Sci 94 569 Baker A H et al 2002 J Cell Sci 115 3719 Hojilla C et al 2003 Br J Cancer 89 1817 Freije J M et al 2003 Adv Exp Med Biol 532 91 Sierevogel M J et al 2003 Curr Pharm Des 9 1033 Ikeda U and K Shimada 2003 Clin Cardiol 26 55 Mohammed F F et al 2003 Ann Rheum Dis 62 Suppl 2 ii43 Clark I M and A E Parker 2003 Expert Opin Ther Targets 7 19 Murphy G et al 2002 Arthritis Res 4 Suppl 3 539 2013 R amp D Systems Inc 752811 1 10 13 10 For research use only Not for use in diagnostic procedures
16. te Add 50 uL of diluted Biotin Antibody Cocktail to all wells Securely cover with a foil plate sealer and incubate for 1 hour at room temperature on the shaker set at 800 50 rpm Repeat the wash as in step 4 Add 50 uL of diluted Streptavidin PE to all wells Securely cover with a foil plate sealer and incubate for 30 minutes at room temperature on the shaker set at 800 50 rpm Repeat the wash as in step 4 Resuspend the microparticles by adding 100 uL of Wash Buffer to each well Incubate for 2 minutes on the shaker set at 800 50 rpm Read within 90 minutes using the Luminex or Bio Rad analyzer Note Resuspend microparticles immediately prior to reading Samples require dilution See Sample Preparation section For research use only Not for use in diagnostic procedures CALCULATION OF RESULTS Use the Standard concentrations on the Standard Value Card and calculate 3 fold dilutions for the remaining levels Average the duplicate readings for each standard and sample and subtract the average blank Median Fluorescence Intensity MFI Create a standard curve for each analyte by reducing the data using computer software capable of generating a five parameter logistic 5 PL curve fit Since samples have been diluted the concentration read from the standard curve must be multiplied by the dilution factor CALIBRATION This assay is calibrated against highly purified recombinant human MMPs produced at
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