Magnetic Luminex Screening Assay Human
Contents
1. Standard Standard Standard Standard Cocktail 1 Cocktail 2 Cocktail 3 Cocktail 4 Cocktail 5 STANDARD STANDARD STANDARD STANDARD gem Standard Standard Standard Standard Standard Cocktail 6 Cocktail 7 Cocktail 8 Cocktail 9 Cocktail 10 www RnDSystems com REAGENT PREPARATION CONTINUED Pipette 200 uL of Calibrator Diluent RD6 52 into each of 5 test tubes labeled 2 6 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 Calibrator Diluent RD6 52 serves as the blank 100 iN 100 uL 100 uL 100 uL 100 uL Standard 1 Standard 2 Standard3 Standard4 Standard5 Standard 6 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 Diluent RD2 1 in the mixing bottle provided Number of Wells Used Microparticle Cocktail Diluent RD2 1 96 500 pL 5 00 mL 72 375 uL 3 75 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 4 To prepare the CCL5 RANTES microparticles add the CCL5 RANTES microparticle concentrate into previously diluted microparticles from Step 3 following the t2. face protection Wash hands thoroughly after handling Refer to the SDS 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 PART PART DESCRIPTION OR RECONSTITUTED MATERIAL Human Standard 893899 Human Standard 898127 Cocktail A Cocktail Rt Human Standard 893901 Human Standard 898128 Cocktail Bt Cocktail St Human Standard 894368 Human Standard 898129 Cocktail CO Cocktail Tt Human Standard 893985 Human Standard 898130 Cocktail D Cocktail Ur Human Standard 893986 Human Standard 898131 Cocktail E Cocktail Vt Human Standard 894162 Human Standard 898132 Cocktail F Cocktail W Human Standard 894625 Human Standard 898133 Cocktail Gt Cocktail X Human Standard 896017 Human Standard 898134 Cocktail Ht Cocktail Yt 2 vials of recombinant Human Standard 896018 Human Standard 898135 numan eeler d e Discard after use Use fresh er e buffered protein base Cocktail Cocktail Z standard s for each assay with preservatives Human Standard 896019 Human Standard 993545 lyophilized Cocktail Jt Cocktail 1 Human Standard 894824 Human Standard 998546 Cocktail K Cocktail 2t Human Standard 894863 Human Standard 998547 Cockt
3. Magnetic Luminex Assay Human Premixed Multi Analyte Kit Catalog Number LXSAHM For the simultaneous detection of multiple human 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 PAGE HN Wa TON KE 1 FPR Ee OF THEAS SA E 1 EATER HONS ele Ree RU Eorp en aia S EAEE EEEE EE E E A 2 TEC RNGAC UE 2 PRECAUTION Doriti E E E A E E N E E pees E A E anit aaneescet 2 MATERIALS PROVIDED amp STORAGE COIN DOIN Secrets es circruerscenetesoe aie nterre recente eres VANES N 3 TE EN BE e TR e E A TE T 4 AMPEE COLLE CHON e E 5 SAMPLE PREPARA HO DEE 6 REA CENTEPRERPRARATIO N ME 7 DILUTED MICROPARTICLE COCKTAIL ES LA Hl D 8 DILUTED BIOTIN ANTIBODY COCKTAIL PREPARATION E 9 STREP TAVIDIN PE PREPARA TION EE 9 Ja BT SC HERR EE 10 PY EN Le RR EE 11 ASSAY PROCEDURE aU IIPS N seipinn Eins en aana ENERE EEE AEAEE i TENENSA Spaa ERATES EE EE casey 12 Ee EIER HUET EE EE EE 13 CALIBRATION rrna en E E E E EEEE E AE 13 PEATE CAYO RE 14 Manufactured and Distributed by USA R amp D Systems Inc 614 McKinley Place NE Minneapolis MN 55413 TEL 800 343 7475 612 379 2956 FAX 612 656 4400 E MAIL info bio techne com Distributed by Europe Middle East Africa Bio Techne Ltd China Bio Techne China Co Ltd 19 Barton Lane Abingdon Science Park Unit 1901 Tower 3 Raffles City Cha
4. RnDSystems com Products LXSAHM Note On the day of the assay ALL fresh and previously frozen serum and plasma samples require centrifugation at 16 000 x g for 4 minutes immediately prior to use or dilution Cell culture supernate serum and plasma samples require at least a 2 fold dilution A suggested 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 50 200 4000 or 40 000 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 A suggested 4000 fold dilution can be achieved by adding 10 uL of 200 fold diluted sample to 190 uL of Calibrator Diluent RD6 52 A suggested 40 000 fold dilution can be achieved by adding 20 uL of 4000 fold diluted sample to 180 uL Calibrator Diluent RD6 52 6 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 480 mL of deionized or distilled water to prepare 500 mL of Wash B
5. able below Number of Wells Used Diluted Microparticle Cocktail RANTES Microparticles 96 5 5 mL 55 ul 72 4 125 mL 41 25 uL 48 2 75 mL 27 5 uL 24 1 375 mL 13 75 uL 8 For research use only Not for use in diagnostic procedures 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 Diluent RD2 1 Mix gently Number of Wells Used Biotin Antibody Cocktail Diluent RD2 1 96 500 uL 5 00 mL 12 375 uL 3 75 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 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 in Wash Buffer Number of Wells Used Streptavidin PE Concentrate Wash Buffer 96 220 uL 5 35 mL 72 165 uL 4 00 mL 48 110 uL 2 65 mL 24 55 uL 1 35 mL www RnDSystems com INSTRUMENT SETTINGS Note Adjust the probe height setting on the analyzer to avoid puncturing the plate Calibrate the analyzer using the proper reagents for superparamagnetic microparticles refer to instru
6. ail L Cocktail 3t Human Standard 894909 Human Standard 993548 Cocktail Mr Cocktail A Human Standard 894908 Human Standard 998549 Cocktail Nt Cocktail 5t Human Standard 898124 Human Standard 998550 Cocktail 0t Cocktail 6t Human Standard 898125 Human Standard 998551 Cocktail Pt Cocktail 7 Human Standard 898126 Cocktail Qt Provided this is within the expiration date of the kit t Each premixed kit may contain 1 or more of the unique Standard Cocktails A Z 1 7 depending upon the analytes selected Note The reconstitution method outlined on the Certificate of Analysis enclosed in the kit results ina 10X standard cocktail If you received 11 14 standards refer to the revised reconstitution instructions supplied in the kit Part 753068 In the rare instance of 15 or more standards supplied in this assay two standard curves must be assayed One with up to 14 standards and the second with the remaining standards www RnDSystems com 3 MATERIALS PROVIDED amp STORAGE CONDITIONS CONTINUED STORAGE OF OPENED DILUTED PART PART DESCRIPTION OR RECONSTITUTED MATERIAL Human Magnetic 894723 0 6mL ofa concentrated microparticle cocktail with Premixed Microparticle preservatives May be stored for up to Cocktail 1 month at 2 8 C Biotin Ab Cocktail with preservatives be discarded Use fresh diluents Concentrate conjugate with preservatives Calibrator Diluent 895438 3 vials 21 ml Jual of a buffered protein base w
7. icroparticles 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 by shaking the plate for 2 minutes on the plate shaker at 800 50 rpm Samples may require dilution See Sample Preparation section www RnDSystems com 11 ASSAY PROCEDURE SUMMARY Note Protect microparticles and Streptavidin PE from light at all times Prepare all reagents as instructed Add 50 uL of standard or sample to each well 4 Add 50 uL of diluted Microparticle Cocktail to each well Incubate for 2 hours at RT on a shaker at 800 rpm Wash by removing the liquid from each well filling with 100 uL Wash Buffer and removing the liquid again Perform the wash 3 times Add 50 uL of diluted Biotin Antibody Cocktail to each well Cover and incubate for 1 hour at RT on the shaker at 800 rom Repeat the wash as in step 4 Add 50 uL of diluted Streptavidin PE to each well Incubate for 30 minutes at RT on the shaker at 800 rpm Repeat the wash as in step 4 Add 100 uL of Wash Buffer to each well Incubate for 2 minutes at RT on the shaker at 800 rpm 4 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 12 For
8. ith RD6 52 reservatives pepe eee P 1 month at 2 8 C Wash Buffer 895003 21 mL ofa 25 fold concentrated solution of buffered Concentrate surfactant with preservative May turn yellow over time 641385 1 flat bottomed 96 well microplate used as a vessel for the assay Certificate of Analysis 753436 1 sheet listing the selected analytes with the microparticle regions standard reconstitution volumes and concentrations for the provided Standard s Mixing Bottles 895505 2empty 8 mL bottles used for mixing microparticles with Assay Diluent RD2 1 Plate Sealers 640445 4adhesive foil strips Provided this is within the expiration date of the kit Additional reagents supplied if CCL5 RANTES are ordered See page 5 for details OTHER SUPPLIES REQUIRED e Luminex MAGPIX Luminex 100 200 Luminex FLEXMAP 3D or Bio Rad Bio Plex analyzer with X Y platform e Hand held microplate magnet or plate washer with a magnetic platform e Pipettes and pipette tips e Deionized or distilled water e Multi channel pipette manifold dispenser or automated dispensing unit e 500 mL graduated cylinder e Horizontal orbital microplate shaker 0 12 orbit capable of maintaining a speed of 800 50 rpm e Microcentrifuge e Polypropylene test tubes for dilution of standards and samples 4 For research use only Not for use in diagnostic procedures MATERIALS PROVIDED amp STORAGE CONDITIONS CONTINUED Note Additional
9. ment manual Luminex MAGPIX analyzer a Sample volume i 1 25 analytes 50uL li gt 25 analytes 35 uL b Assign the microparticle region for each analyte being measured see Certificate of Analysis c 50 count region d Collect Median Fluorescence Intensity MFI Luminex 100 200 Luminex FLEXMAP 3D and Bio Rad Bio Plex analyzers Note Ensure that the instrument flow rate is set to the default of 60 uL minute fast for all flow based analyzers a Sample volume 50 uL b Bead Type i Luminex 100 200 and FLEXMAP 3D select MagPlex ii Bio Rad Bio Plex Manager use Bio Plex MagPlex Beads Magnetic c Doublet Discriminator gates i Luminex 100 200 and FLEXMAP 3D set at 8000 and 16 500 ii Bio Rad Bio Plex Manager set at 8000 and 23 000 d Reporter Gain Setting i Luminex 100 200 use Default setting ii Luminex FLEXMAP 3D use Enhanced PMT high setting iii Bio Rad Bio Plex Manager use the low RP1 target value for the CAL2 setting e Assign the microparticle region for each analyte being measured see Certificate of Analysis f 50 count region g Collect MFI 10 For research use only Not for use in diagnostic procedures ASSAY PROCEDURE Bring all reagents and samples to room temperature before use It is recommended that all standards and samples be assayed in duplicate Note Protect microparticles and Streptavidin PE from light at all times 10 Prepare all reagents
10. ngning Office Abingdon OX14 3NB UK 1193 Changning Road Shanghai PRC 200051 TEL 44 0 1235 529449 TEL 86 21 52380373 400 821 3475 FAX 44 0 1235 533420 FAX 86 21 52371001 E MAIL info emea bio techne com E MAIL info cn bio techne com INTRODUCTION This kit contains the components required to screen up to 50 human biomarkers in cell culture supernate serum and plasma samples in multiplexed sandwich ELISAs Magnetic Luminex Assays can be used to assess the levels of biomarkers of your choosing ina 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 Assay multiplex kits are designed for use with the Luminex MAGPIX CCD Imager Alternatively kits can be used with the Luminex 100 200 Luminex FLEXMAP 3D or Bio Rad Bio Plex dual laser flow based sorting and detection platforms Analyte specific antibodies are pre coated onto magnetic microparticles embedded with fluorophores at set ratios for each unique microparticle region 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
11. reagents supplied if CCL5 RANTES are ordered STORAGE OF OPENED DILUTED PART PART DESCRIPTION OR RECONSTITUTED MATERIAL Human CCL5 RANTES 899028 0 075 mLa concentrated microparticle stock May be stored for up to 1 month at 2 8 C Magnetic Microparticle with preservatives Once diluted 1X solutions must be discarded Use fresh diluents for each assay 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 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 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 have not been validated for use in this assay www RnDSystems com 5 SAMPLE PREPARATION Use polypropylene tubes To determine the appropriate dilution for each analyte refer to the table located in the following link http www
12. 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 a 5 PL curve fit is not available use a four parameter logistic 4 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 human biomarkers produced at R amp D Systems www RnDSystems com 13 PLATE LAYOUT yout to record standards and samples assayed CL NOOOOOOO et LC CL KCL CL LC CL LC et LC CL LC et LECH CL KCL CLC SL LIT
13. standards and samples as directed in the previous sections Add 50 uL of standard or sample per well A plate layout is provided to record standards and samples assayed Resuspend the diluted Microparticle Cocktail by inversion or vortexing Add 50 uL of the microparticle cocktail to each well of the microplate 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 allow 1 minute before removing the liquid filling each well with Wash Buffer 100 uL and allow 1 minute before removing the liquid again Complete removal of liquid is essential for good performance Note Do NOT blot this may cause a loss of microparticles 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 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 room temperature on the shaker set at 800 50 rpm Repeat the wash as in step 4 Resuspend the m
14. ther dilute the samples with calibrator diluent and repeat the assay e Any variation in 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 Discrepancies may exist in values obtained for the same analyte utilizing different technologies e Magnetic Luminex Assays afford the user the benefit of multi analyte 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 e Only the analytes listed on the enclosed Certificate of Analysis can be measured with this 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 photo bleaching PRECAUTIONS Some components in this kit contain a preservative which may cause an allergic skin reaction Avoid breathing mist Wear protective gloves clothing eye and
15. uffer Standards Refer to the Certificate of Analysis for reconstitution volumes and assigned values The standards provided in the kit will differ depending on the analytes selected but may include up to 20 unique Standard Cocktails A Z 1 7 Reconstitute 1 each of the unique Standard Cocktails provided in the kit with Calibrator Diluent RD6 52 Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making dilutions Upon reconstitution each Standard Cocktail is a 10X concentrate Use polypropylene tubes Create Standard 1 by combining 100 uL of each standard cocktail with Calibrator Diluent RD6 52 into your standard 1 tube The final volume in the standard 1 tube will be 1000 uL Use the table below to determine how much calibrator diluent to use based on the number of standard cocktails you are combining Number of Unique Standard Volume to Combine into Volume of Calibrator Total Volume of Cocktails Provided a Single Tube Diluent Required Standard 1 1 100 uL 900 uL 1000 uL 2 100 uL of each 800 ul 1000 uL 3 100 uL of each 700 uL 1000 uL 4 100 uL of each 600 uL 1000 uL 5 100 uL of each 500 uL 1000 uL 6 100 uL of each 400 uL 1000 uL 7 100 uL of each 300 ul 1000 uL 8 100 uL of each 200 uL 1000 uL 9 100 uL of each 100 uL 1000 uL 10 100 uL of each 0 uL 1000 uL Note f more than ten standard cocktails are supplied refer to the note on page 3 STANDARD STANDARD STANDARD STANDARD STANDARD Standard
16. which binds to the biotinylated antibody is added to each well Final washes remove 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 excites the dyes inside each microparticle to identify the region and the second LED excites the PE to measure the amount of analyte bound to the microparticle A sample from each well is imaged with a CCD camera with a set of filters to differentiate excitation levels Analysis with the Luminex 100 200 Luminex FLEXMAP 3D or Bio Rad Bio Plex uses one laser to excite the dyes inside each microparticle to identify the microparticle region and the second laser to excite the PE to measure the amount of analyte bound to the microparticle All fluorescence emissions from each microparticle as it passes through the flow cell is then analyzed to differentiate emission levels using a Photomultiplier Tube PMT and an Avalanche Photodiode www RnDSystems com LIMITATIONS OF THE PROCEDURE e 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 generate values higher than the highest standard fur
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