Magnetic Luminex Performance Assay Human
Contents
1. PLATE LAYOU MANUFACTURED AND DISTRIBUTED 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 kidneys play important roles in organismal homeostasis by regulating osmolality and blood pressure aiding in the reabsorption of water and nutrients excreting wastes and secreting hormones Renal function is also important in the metabolism and excretion of drugs 1 Therefore analyzing nephrotoxicity using renal markers is an important experimental step during drug development Historically renal function has been evaluated by measuring serum creatinine and blood urea nitrogen levels 2 Recently more sensitive kidney biomarkers have been identified and renal function can be assessed contextually by analyzing multiple proteins simultaneously In addition renal markers can be used to assess kidney development during embryogenesis as well as patholog2. serum and assay immediately or aliquot and store samples at lt 20 C Avoid repeated freeze thaw cycles Plasma Collect plasma using EDTA or 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 Note Citrate plasma has not been validated for use in this assay Urine Aseptically collect the first urine of the day mid stream voided directly into a sterile container Centrifuge to remove particulate matter and assay immediately or aliquot and store at lt 20 C Avoid repeated freeze thaw cycles SAMPLE PREPARATION Use polypropylene tubes Urine samples require a 10 fold dilution A suggested 10 fold dilution is 20 uL of sample 180 uL of Calibrator Diluent RD6 62 diluted 1 5 Mix thoroughly When assaying IP 10 Lipocalin 2 OPN TIM 1 and TFF3 serum and plasma samples require 10 fold dilution A suggested 10 fold dilution is 20 uL of sample 180 uL of Calibrator Diluent RD6 62 diluted 1 5 Mix thoroughly When assaying Clusterin Cystatin C Fetuin A and RBP4 serum and plasma samples must be diluted to a final 4000 fold dilution A suggested 4000 fold dilution is 10 uL of sample 990 uL of Calibrator Diluent RD6 62 diluted 1 5 Add 25 uL of the diluted sample to 975 uL of Calibrator Diluent RD6 62 diluted 1 5 to complete the 4000 fold dilution Mix thoroughly Se
3. to each well Incubate for 2 minutes on the shaker set at 800 50 rpm Read within 90 minutes using a Luminex or Bio Rad analyzer Note Resuspend microparticles immediately prior to reading Samples require dilution See Sample Preparation section 8 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 kidney biomarkers produced at R amp D Systems REFERENCES 1 Mehta R L et al 2004 Kidney Int 66 1613 2 Ozer J S et al 2010 Nat Biotech 28 486 3 Bonventre J V et al 2010 Nat Biotech 28 436 4 Goodsaid F M et al 2009 Clin Pharmacol Ther 86 490 5 Vaidya V S et al 2010 Nat Biotech 28 478 6 Dieterle et al 2010 Nat Biotech 28 455 All trademarks and registered trademarks are the property of their respective owners www RnDSystems com PLAT
4. 3 g Prepare all reagents working standards and samples as directed in the previous sections Resuspend the diluted microparticle cocktail by inversion or vortexing Add 100 uL of the microparticle cocktail to each well of the microplate Add 50 of Standard or sample per well Securely cover with a foil plate sealer Incubate for 3 hours at room temperature on a horizontal orbital microplate shaker 0 12 orbit set at 800 50 rpm A plate layout is provided to record standards and samples assayed 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 microplate Add 50 uL of diluted Biotin Antibody Cocktail to each well Securely cover with 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 each well 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
5. DB O 2 Standard Standard1 Standard2 Standard3 Standard4 StandardS 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 OPN and TFF3 50 uL of OPN Microparticle Concentrate and 50 uL of TFF3 Microparticle Concentrate to 10 mL of Microparticle Diluent Number of Wells Used Microparticle Concentrate Microparticle Diluent 96 50 0 uL 10 0 mL 72 37 5 uL 7 50 mL 48 25 0 uL 5 0 mL 24 12 5 pL 2 50 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 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 5 5 mL of Diluent RD2 1 Mix gently STREPTAVIDIN PE PREPARATION Use a polypropylene amber bottle or a polypropylene tube wrapped with aluminum foil Protect Strep
6. E LAYOUT Use this plate layout to record st 1000000002 EOOOOOOOO 07 409000000002 409000000002 007 4000000002 4000000002 2013 R amp D Systems 3 752829 3 10 13
7. Magnetic Luminex Performance Assay Human Kidney Biomarker Base Kit Catalog Number For the simultaneous quantitative determination of multiple human kidney biomarker concentrations in serum plasma 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 PRINCIPLE OF THEASS AY LIMITATIONS OF THE PROCEDURE TECHNICAE HINTS PRECAUTIONS wee mentee MATERIALS PROVIDED amp STORAGE CONDITIONS OTHER SUPPLIES REQUIRED SAMPLE COLLECTION AND STORAGE REAGENT PREPARATION DILUTED MICROPARTICLE COCKTAIL DILUTED BIOTIN ANTIBODY COCKTAIL STREP TAVIDIBI PE PREPARATION INSTRUMENT ASSAY PROCEDURE CALCULATION COP cesthas aiii CALIBRATION REFERENCES
8. ative 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 6 adhesive foil strips Standard Value Card 749829 1 card listing the Standard Cocktail reconstitution volume and working standard concentrations for this lot of base kit Provided this is within the expiration date of the kit OTHER SUPPLIES REQUIRED e Luminex Performance Assay analyte specific kit s see Introduction on page 1 e Luminex 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 100 mL and 500 mL graduated cylinders e Horizontal orbital microplate shaker 0 12 orbit capable of maintaining a speed of 800 50 rpm e Microcentrifuge 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 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
9. d other factors present in biological samples Until these proteins 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 e Only the analytes listed on the Standard Value Card can be measured with this base kit TECHNICAL HINTS e When mixing or reconstituting protein solutions always avoid foaming e 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 Calibrator Diluent RD6 62 contains sodium azide which may react with lead and copper plumbing to form explosive metallic azides Flush with large volumes of water during di
10. e Reagent Preparation section 4 For research use only Not for use in diagnostic procedures 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 Calibrator Diluent RD6 62 diluted 1 5 Add 20 mL of Calibrator Diluent RD6 62 concentrate to 80 mL of deionized or distilled water to prepare 100 mL of Calibrator Diluent RD6 62 diluted 1 5 Standard Reconstitute the Kidney Biomarker Standard Cocktail with Calibrator Diluent RD6 62 diluted 1 5 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 300 uL of the reconstituted Standard into a tube labeled standard 1 Pipette 200 uL of Calibrator Diluent RD6 62 diluted 1 5 into the remaining tubes Use standard 1 to produce a 3 fold dilution series below Refer to analyte specific datasheets for details Mix each tube thoroughly before the next transfer Standard 1 serves as the high standard Calibrator Diluent RD6 62 1X serves as the blank 100 uL 100 uL 100 uL 100 uL 100 uL 100 uL gt gt _ gt 300 uL Std
11. ical conditions such as renal failure and renal cell carcinoma 2 6 This kit is an excellent tool for drug toxicology studies because it can simultaneously assess the levels of 9 Kidney Biomarkers in a single serum plasma or urine sample Any combination of the following bead sets are suitable for use with this base kit Analyte Catalog Number Microparticle Region Clusterin LHK2937 20 ystatin LHK1196 19 CXCL10 IP 10 LHK266 25 Fetuin A AHSG LHK1184 21 Lipocalin 2 NGAL LHK1757 27 Osteopontin OPN LHK1433 28 RBP4 LHK3378 29 TFF3 LHK4407 30 TIM 1 KIM 1 HAVCR LHK1750 26 PRINCIPLE OF THE ASSAY 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 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 resuspe
12. nded 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 microparticles 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 a Bio Rad Bio Plex dual laser flow based systems This product is covered by one or more of the following US Patents 7 300 652 7 041 290 6 664 385 and other US and foreign patents pending or issued www RnDSystems com 1 LIMITATIONS OF THE PROCEDURE FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC PROCEDURES e The kit should not be used beyond the expiration date on the kit label Do not mix or substitute reagents with those from other lots or sources If samples fall outside the dynamic range of the assay further dilute the samples with Calibrator Diluent and repeat the assay e 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 soluble receptors binding proteins an
13. sposal 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 kit expiration date STORAGE OF OPENED DILUTED PART PART DESCRIPTION OR RECONSTITUTED MATERIAL Kidney Biomarker 894311 2 vials of recombinant human kidney Standard Cocktail biomarkers in a buffered protein base with preservatives lyophilized Microparticle Diluent 895529 2 vials 6 mL vial of a buffered protein base be stored for up to 1 month at 2 8 C Discard after use Use a fresh standard for each assay with blue dye and preservative Once diluted any unused microparticle cocktail must be discarded Diluent RD2 1 895970 11 ml buffered protein base with preservatives Calibrator Diluent 895986 21 ofa concentrated buffered animal RD6 62 serum with preservatives Used diluted 1 5 in this assay Streptavidin PE 892525 0 07 ml ofa 100 fold concentrated May be stored for up to 1 month at 2 8 C streptavidin phycoerythrin conjugate with preservatives Wash Buffer Concentrate 895003 21 mLofa25 fold concentrated solution of buffered surfactant with preserv
14. tavidin 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 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 Flow rate 60 uL minute fast Doublet Discriminator gates at approximately 8000 and 16 500 g Collect MFI Sample size 50 uL Note The CAL2 setting for the Bio Rad Bio Plex analyzer should be set at the low 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
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