Rat Premixed Multi-Analyte Kit
Contents
1.2. Protect the 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 Streptavidin PE Concentrate to a 1X concentration by adding 220 uL of Streptavidin PE to 5 35 mL of Wash Buffer This provides enough Streptavidin PE to assay one 96 well microplate If assaying less than 96 wells adjust these volumes accordingly 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 Certificate of Analysis for details 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 microparticle region for each analyte being measured see Certificate of Analysis for details b 50 events bead c Minimum events 0 d Flow rate 60 uL minute fast e Sample size 50 uL f Doublet Discriminator gates at approximately 8000 and 16 500 g Collect MFI 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 tempe
3. e Deionized or distilled water e Multi channel pipette manifold dispenser or automated dispensing unit e 500 mL graduated cylinder e Polypropylene test tubes for dilution of standards and samples e Horizontal orbital microplate shaker 0 12 orbit capable of maintaining a speed of 800 50 rpm e Microcentrifuge www RnDSystems com 3 SAMPLE COLLECTION amp 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 Serum Allow blood samples to clot for 2 hours at room temperature before centrifuging for 20 minutes at 2000 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 EDTA or heparin as an anticoagulant Centrifuge for 20 minutes at 2000 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 SAMPLE PREPARATION To determine the appropriate dilution for each analyte refer to the table located in the following link http www rndsystems com Products LXSARM Serum and plasma samples require at least a 2 fold dilution A suggeste
4. Magnetic Luminex Screening Assay Rat Premixed Multi Analyte Kit Catalog Number LXSARM For the simultaneous detection of multiple rat biomarkers in cell culture supernates serum and plasma 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 ATRODU CTION se sans ese GEE RE oe ee GEE ee ek eo ER ee ee EG PRINGIELE OF EIE AS SA dees meisie sees ego sd gee ee see ee E ee ee Ve een ees ee ee LIMITATIONS OF THE PROCE DU cere enn ees ee ordes ee ee ese ee ee es ee ae ie Ml ER EE EE OE EA PRECAUTION es is E ee ee OE E MATERIALS PROVIDED amp STORAGE CONDITIONS esse se se seen se se se se een Ge Ge ee ee Ge ee OTHER SUPPLIES REQUIRED sr eende Ge ee re err er ee ie ee ie de oe ee ee SAMPLE COLLECTION amp STORAGE sees esse se se see eene se eene Gee se ee gee ee ee ee Se Se eg ee ee eg SAMPLE PREPARATION ie messe eie eine ene oe oe e eien eek ese sees ese ee ee ede KEAGENTPREPARATION es iedere etes de ese een eed er ee ee Pe ie ee oe seed ie DILUTED MICROPARTICLE COCKTAIL PREPARATION sesse se sesse se se see es se se se bee gee Gee ee DILUTED BIOTIN ANTIBODY COCKTAIL PREPARATION sesse sesse se se see ee se se se seges Ge se ee SIREPTAVIDIN PE PREPARAIIO N sesse eie segesse ee esse ee ende seksie see gewees be ees ee ees ed INSTRUMENT SETTINGS si esse ie E N ee ie E De rrr AS PROCEDURE is oie ee GE GR ed ee Ede E
5. 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 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 www RnDSystems com 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 e Do not mix or substitute reagents with those from other lots or sources e If samples generat
6. ator Diluent RD6 52 serves as the blank Refer to the Certificate of Analysis for values of Standard 1 100uL 100 uL 100uL 100 pL 100 uL RA OR A OR RA 500 uL Std D STANDARD Standard Cocktail Standard 1 Standard2 Standard3 Standard4 Standard5 Standard 6 www RnDSystems com 5 DILUTED MICROPARTICLE COCKTAIL PREPARATION 1 Centrifuge the Microparticle Cocktail vial for 30 seconds at 1000 x g prior to removing the cap 2 Gently vortex the vial to resuspend the microparticles taking precautions not to invert the vial 3 Dilute the Microparticle Cocktail using Assay Diluent RD1W in the mixing bottle provided Number of Wells Used Microparticle Cocktail Assay Diluent RD1W 96 500 uL 5 00 mL 48 250 uL 2 50 mL 24 125 uL 1 25 mL Note Protect microparticles from light during handling Prepare microparticles within 30 minutes of use Diluted microparticles cannot be stored DILUTED BIOTIN ANTIBODY COCKTAIL PREPARATION 1 Centrifuge the Biotin Antibody Cocktail 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 Biotin Antibody Cocktail in Assay Diluent RD1W Mix gently Number of Wells Used Biotin Antibody Cocktail Assay Diluent RD1W 96 500 uL 5 00 mL 48 250 uL 2 50 mL 24 125 uL 1 25 mL STREPTAVIDIN PE PREPARATION Use a polypropylene amber bottle or a polypropylene test tube wrapped with aluminum foil
7. d 2 fold dilution is 75 uL of sample 75 uL of Calibrator Diluent RD6 52 Mix thoroughly High abundance biomarkers may require additional dilution such as 50 or 200 fold A suggested 50 fold dilution is 10 uL of sample 490 uL of Calibrator Diluent RD6 52 Mix thoroughly A suggested 200 fold dilution can be achieved by adding 10 uL of sample to 90 uL of Calibrator Diluent RD6 52 Complete the 200 fold dilution by adding 10 uL of the diluted sample to 190 uL Calibrator Diluent RD6 52 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 Standard Reconstitute the Standard Cocktail with Calibrator Diluent RD6 52 Refer to the Certificate of Analysis for the reconstitution volume 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 Cocktail into a tube labeled Standard 1 Pipette 200 uL of Calibrator Diluent RD6 52 into the remaining tubes Use Standard 1 to produce a 3 fold dilution series below Mix each tube thoroughly before the next transfer Standard 1 serves as the high standard Calibr
8. e values higher than the highest standard 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 Discepancies may exist in values obtained for the same analyte utilizing different technologies Magnetic Luminex Screening Assays afford the user the benefit of multianalyte analysis of biomarkers in a single sample A multipurpose diluent is used to dilute samples if necessary and provide accurate estimates of natural analytes in cell culture supernates serum and plasma Only the analytes listed on the enclosed Certificate of Analysis can be measured with this kit TECHNICAL HINTS 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 Some components in this kit contain ProClin which may cause an allergic skin reaction Avoid breat
9. ere oes EALEULATIO N OF BESLIS ss ie Dee ei ee ee ee de ee sey Ge EALIBRATION osse ee ee ee Ge OEA Ge E NE VEE iS ER OE tre verre A re rrrrrer terre rrrtrer ir rerrrrerr frre trier ire errr erect 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 This kit contains the components required to screen multiple rat biomarkers in cell culture supernate serum and plasma samples in multiplexed sandwich ELISAs Magnetic Luminex Screening Assays can be used to assess the levels of biomarkers of your choosing in a single sample For ease of use the microparticles are premixed in one vial as are the biotinylated detection antibodies 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 or Bio Rad Bio Plex dual laser flow based sorting
10. hing 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 This kit contains sufficient materials to run multiplex assays on one 96 well plate STORAGE OF OPENED DILUTED PART PART DESCRIPTION OR RECONSTITUTED MATERIAL Rat Standard 893115 2 vials of recombinant rat biomarkers in a buffered Once reconstituted any Cocktail A protein base with preservatives lyophilized remaining standard must be discarded Use fresh a standard for each assay Microparticle Cocktail preservatives 1 month at 2 8 C Rat Premixed 894783 0 6 mL ofa concentrated biotin antibody cocktail with Streptavidin PE 893535 0 25 mL of a concentrated streptavidin phycoerythrin ae ES EN Calibrator Diluent 895438 2 vials 21 mL vial of a buffered protein base with oe eean ayes rt Concentrate surfactant with preservative May turn yellow over time volumes and concentrations for the provided Standard s Provided this is within the expiration date of the kit OTHER SUPPLIES REQUIRED e Luminex MAGPIX Luminex 100 200 or Bio Rad Bio Plex analyzer with X Y platform Hand held microplate magnet or platewasher with a magnetic platform e Pipettes and pipette tips
11. rature 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 a N 3 10 Prepare all reagents 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 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 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 flat bottom microplate Add 50 uL of diluted Biotin Antibody Cocktail to each well 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 each well Securely cover with a foil plate sealer and incubate for 30 minutes at
12. 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 a Luminex or Bio Rad analyzer Note Resuspend microparticles immediately prior to reading Samples may 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 Certificate of Analysis 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 If 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 rat biomarkers produced at R amp D Systems All trademarks and registered trademarks are the property of their respective owners www RnDSystems com 9 PLATE LAYOUT to record standards and samples assayed ECX XXXXXX eC OOOO O00 ECX XXXXXX ECX XXXXXX 6 OOOOOOOO 7 DOOOOOOO EESSESSSD OOOOOOOO 7 OOOO O00 6 OOOOOOOO OOOO OOOO 7
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