ProcartaPlex Multiplex Immunoassays
Contents
1. Low bead counts during data acquisition Probe height is incorrect Refer to the Luminex manual for proper adjustment of the needle height Reading buffer volume added in the last step to resuspend the beads is too low Add 120 uL Reading Buffer into each well and shake at 500 rpm for 5 min at room temperature to resuspend the beads prior to reading on the Luminex instrument Make sure sample size is set at 100 uL in the acquisition protocol High bead aggregation Vortex the bead suspension well before using in the assay and ensure that the beads are properly mixed during the incubation steps 32 Probable Cause Solution Low bead counts during data acquisition High bead aggregation Verify that beads were added at the correct concentration and that correct bead regions and wells were selected during acquisition setup Dyes contained in the beads are photo bleached from overexposure to light Beads are falling outside the bead region gates due to photo bleaching Store bead solution in the dark and protect the 96 Well Plate from light by wrapping the 96 Well Plate with aluminum foil Do not use expired beads Do not expose the beads to ambient light for prolonged time Avoid intense light Beads settle on the bottom of the well Confirm that the plate shaker is set to 500 rpm and shaking for at least 5 min before reading Partial blockage of the fl2. EPX010 26038 901 EPX010 26008 901 EPX010 26034 901 EPX010 20612 901 EPX010 26031 901 EPX010 20606 901 EPX010 26002 901 EPX010 20601 901 EPX010 26035 901 EPX010 20613 901 EPX010 20610 901 EPX010 20603 901 Adiponectin Eotaxin CCL11 G CSF CSF 3 GM CSF Gro alpha IFN gamma IL 1beta IL 2 IL 3 IL 4 IL 5 IL 6 14 62 12 42 43 38 19 20 14 26 27 28 EPXO10 26041 901 EPXO10 20614 901 EPX010 26033 901 EPX010 26004 901 EPX010 26015 901 EPX010 26001 901 EPX010 20618 901 EPX010 26022 901 EPX010 26017 901 EPX010 26024 901 EPX010 26040 901 EPX010 26036 901 IL 9 IL 10 IL 12p40 IL 12p70 IL 13 IL 17A CTLA 8 IL 18 IL 22 IL 23 IL 27 LIF Leptin 34 13 39 39 35 52 66 33 37 36 18 65 Available Simplex Kits and respective Bead Regions EPX010 26018 901 IP 10 22 EPX010 26005 901 MCP 1 51 EPX010 26006 901 MCP 3 48 EPX010 26039 901 M CSF 21 EPX010 26013 901 MIP 1alpha 47 EPX010 26014 901 MIP 1beta 72 EPX010 26032 901 MIP 2 55 EPX010 26037 901 RANKL 46 EPX010 26009 901 RANTES 44 EPX010 20607 901 TNF alpha 45 EPX010 20619 901 VEGF A 25 New Analytes are released regularly all products listed on our website www eBioscience com 10 6 PROCARTAPLEX Kit CONTENTS ProcartaPlex Immunoassay Kits contain the components listed below Refer to the Certificate of Analysis included in the kit for quantities and details of components supplied Expiry of
3. 10 2 1 Assay panels or combinations of Simplexes requiring only one premixed standard in the kit 1 Centrifuge the antigen standard vial at 2000 x g for 10 sec Add 250 uL of sample type specific standard buffer for serum or plasma use Universal Assay Buffer for cell culture supernatants use cell culture medium into the vial 3 Vortex gentiv for 30 sec 4 Incubate on ice for 5 10 min 17 10 2 2 Panels or combinations of Simplexes with 2 or more different premixed standards in the kit 1 Centrifuge all the different antigen standard vials at 2000 g for 10 sec Add 250 uL of sample type specific standard buffer for serum or plasma use Universal Assay Buffer for cell culture use cell culture medium into one of the vials 3 Incubate the vial on ice for 5 10 min 4 Vortex gently for 30 sec 5 Transfer the entire content into the second vial with a different standard 6 Incubate on ice for 5 10 min 7 Vortex gently for 30 sec 8 If more than 2 of antigen standards are in the kit repeat steps 5 7 until all the vials with different lot numbers are reconstituted In cases where Simplex Kits are combined with other Simplex or Multiplex Kits check the contents of the Standard Mix in order to avoid that the same standard protein is added twice to the standard mix If the combination of analytes you want to analyse in your multiplex assay includes two analytes that require the same premixed standard use only one of these s
4. KITS ProcartaPlex Immunoassay Kits are available as Pre mixed panels contain all the reagents required to run the respective multiplex assav Simplex Kits designed for the measurement of one analyte in an immunoassay using xMAP technology Together with a ProcartaPlex Basic Kit Simplex kits can be used to detect one analyte alone or can be multiplexed with other Simplex Kits to measure a variety of analytes Simplex Kits can also be combined with premixed panels as long as no Bead Region occurs twice in one experimental set up New custom assay development for analytes not listed on our website Please contact your local eBioscience sales representative for new custom assay development for analytes not listed on our website www eBioscience com 3 How IT WORKS ProcartaPlex Mouse Immunoassays use the Luminex technology multi analyte profiling beads to enable the detection and quantitation of multiple protein targets simultaneously in diverse matrices All ProcartaPlex assays use magnetic beads The xMAP system combines a flow cytometer fluorescent dye microspheres beads dual laser design and digital signal processing to effectively allow multiplexing of up to 100 50 for MAGPIX unique assays within a single sample The ProcartaPlex Immunoassay kits are compatible with all Luminex 100 200 MAGPIX and FLEXMAP 3D currently available 4 PROCARTAPLEX M IMMUNOASSAY WORKFLOW ddd ee T
5. Well Plate 1 Seal the 96 Well Plate using a Plate Seal provided 2 Remove the 96 Well Plate from the filtration manifold and cover the plate with the black microplate lid provided in the kit 3 Incubation A Shake the 96 Well Plate at 500 rom for 60 to 120 min at room temperature B Alternatively the 96 Well Plate can be incubated overnight Shake the 96 Well Plate at room temperature for 30 min Transfer the plate to 4 and store on a level surface u After incubation remove the 96 Well Plate from 4 and shake for 30 min at room temperature and proceed to next step NOTE We recommend 120 min at room temperature or overnight incubation at 4 C for assays that require higher sensitivities 11 2 6 Wash the 96 Well Plate 1 Carefully remove the Plate Seal to avoid splashing the plate contents 2 Remove the liquid in the wells by aspiration using the vacuum filtration manifold Blot the bottom of the plate after filtration 3 Add 150 uL of 1X Wash Buffer into each well 4 Remove the liquid in the wells by aspiration using the vacuum filtration manifold Blot the bottom of the plate after filtration 5 Repeat actions 3 4 two more times for a total of 3 washes NOTE When washing the 96 Well Plate we recommend using a multi channel pipette or a multi channel automatic liquid dispenser Avoid touching the pipette tips to the sides of the wells when adding wash buffer using a multi channel pipette 28 11
6. a sink The magnetic frame is white polycarbonate with a corrosion resistant plate seal below both set up to polypropylene base The clip system provided is capable of holding a variety of microplates It is recommended that the Hand Held Magnet is used with the black 96 well flat bottom plate from Greiner included with Multiplex and Basic Kits The ring on the base plate facilitates gripping for all sizes of hands Refer to section 11 1 for detailed information The Hand Held Magnetic Plate Washer can be ordered via eBioscience in combination with the ProcartaPlex Kit Cat No EPX 55555 000 B Inthe case of the test procedure using the filter plate being chosen a Filtration manifold and a vacuum pump is required Refer to section 11 2 for detailed information We recommend using the Multi Well Plate Vacuum Manifold PALL cat 5017 or directly from eBioscience cat BMS497FF for bead washing 13 8 PRECAUTIONS AND TECHNICAL HINTS Thoroughly read this user manual and certificate of analysis that is included with the assay kit Before starting the assay turn on the Luminex machine and initiate the startup protocol It takes 30 min for the lasers to warm up Make sure the Luminex machine is calibrated according to the manufacturer s instructions Some samples may contain high analyte concentrations and require sample dilution for accurate quantitation Please use Cell culture mediu
7. coe Capture Beads Capture Target Analytes E Add analyte specific capture beads coated with target specific capture antibodies to Analyte prepared sample Incubate 1 2 hours and wash See Detection Antibody Detect Captured Analyte E Incubate captured bead analytes with biotinylated analyte specific detection antibody Immuno sandwich Complex Incubate 30 min and wash Label Detection l bati v ncupation E For analyte quantitation 7 Wash l 4 ae na incubate with a fluorescent detection label SA PE E Wash sample E Quantitate sample Incubate 30 min and wash Read amp Analyze E Read with Luminex 100 200 Magpix or Bio Plex instrument E Analyze results THE rower XMAP INSIDE TECHNOLOGY in Becouse you want to k re Luminex Secreted proteins simultaneously measured using ProcartaPlex Cytokine Assay 5 ANALYTE OVERVIEW ProcartaPlex Immunoassays are available as predefined Multiplexing Panels or as Simplex assays Together with a ProcartaPlex Basic Kit Simplex kits can be used to detect one analyte alone or can be multiplexed with other Simplex Kits to measure a variety of analytes Simplex Kits can also be combined with premixed panels as long as no Bead Region occurs twice in one experimental set up ProcartaPlex Mouse Panels are designed to detect up to 26 Mouse cytokines found in Mouse serum plasma and cell culture supernatants sa
8. 2 7 Add Detection Antibodies Mix 1x 1 Add 25 uL of Detection Antibodies Mix 1x into each well 2 Seal the 96 Well Plate with a new Plate Seal 3 Cover the plate with the black microplate lid provided in the kit 4 Shake at 500 rpm for 30 min at room temperature 11 2 8 Wash the 96 Well Plate Repeat step 11 2 6 11 2 9 Add SA PE Add 50 uL of SA PE solution into each well 2 Seal the 96 Well Plate with a new Plate Seal and cover the plate with the black microplate lid provided in the kit 3 Shake at 500 rpm for 30 min at room temperature 11 2 10 Wash the 96 Well Plate Repeat step 11 2 6 11 2 11 Prepare the Plate for Analysis on a Luminex Instrument Add 120 uL of Reading Buffer into each well Seal the 96 Well Plate with a new Plate Seal Cover the plate with the black microplate lid provided in the kit Shake at 500 rpm for 5 min at room temperature Remove the Plate Seal prior to reading on the Luminex instrument KON gt 29 12 SETUP OF THE LUMINEX INSTRUMENT Sample Size DD Gate Timeout Bead Event Region 100 5 000 25 000 45sec 50 100 If you are running assays on your Luminex instrument that uses both the 96 Well Flat Bottom Plates for Magnetic Beads and Filter Plates for Polystyrene Beads verify the probe height for each plate type before reading Failure to adjust the probe height can cause damage to the instrument The Luminex system allows for calibration of Low and High RP1 t
9. B affymetrix eBioscience ProcartaPlex Multiplex Immunoassavs For research use oniv Not for diagnostic or therapeutic procedures Using Magnetic Beads For serum plasma and cell culture supernatants North America Technical Support Research Products 888 810 6168 858 642 2058 tech eBioscience com Clinical Products 877 726 8559 858 642 2058 tech eBioscience com Customer Service 888 999 1371 858 642 2058 info eBioscience com Fax 858 642 204 Instructions for Mouse Assays Europe International Technical Support 43 1 796 40 40 120 tech eBioscience com Customer Service 43 1 796 40 40 304 europe eBioscience com Fax 43 1 796 40 40 400 Bender MedSystems GmbH ul Campus Vienna Biocenter 2 1030 Vienna Austria www eBioscience com Customers outside North America and Europe may contact their eBioscience distributor listed on our website at www eBioscience com distributors For Research Use Only Not for use in diagnostic or therapeutic procedures Limited License Subject to the eBioscience an Affymetrix Company terms and conditions that govern your use of eBioscience products eBioscience grants you a non exclusive non transferable non sub licensable license to use this eBioscience product only in accordance with the manual and written instructions provided by eBioscience You understand and agree that except as expressly set forth in the eBioscience terms and conditions no right or license to any
10. a Filter Plate In this case a filtration manifold is required 11 2 1 Prepare the 96 Well Filter Plate Mark the standard sample and blank wells For your convenience a blank layout is provided in the Layout section 11 2 2 Add the Antibody Magnetic Beads 1 Add 50 ul Reading Buffer 1x to the filter plate to pre wet the wells Aspirate using the vacuum filtration manifold Blot the bottom of the plate after filtration 2 Vortex the Antibody Magnetic Beads for 30 sec Add 50 uL of the Antibody Magnetic Beads to each well 4 Remove the liquid in the wells by aspiration using the vacuum filtration manifold Blot the bottom of the plate after filtration 5 Repeat actions 3 4 using the same plate with all Antibody coated beads you want to include in your Multiplexing assay w 11 2 3 Wash Antibody Magnetic Beads 1 Add 150 uL of 1X Wash Buffer into each well 2 Remove the liquid in the wells by aspiration using the vacuum filtration manifold Blot the bottom of the plate after filtration 11 2 4 Add Antigen Standards and Samples 11 2 4 1 Serum and Plasma Samples 1 Add 25 uL of Universal Assay Buffer into each well 2 Add 25 uL of standards or samples into dedicated wells 3 Add 25 uL of Universal Assay Buffer to the blank wells 21 11 2 4 2 Cell Culture Supernatant Samples 1 Add 50 uL of samples or standards into dedicated wells 2 Add 50 uL of cell culture medium to the blank wells 11 2 5 Incubate the 96
11. andard 2 10 10 18 18 26 26 34 34 Standard 3 Standard 3 11 11 19 19 27 27 35 35 Standard 4 Standard 4 12 12 20 20 28 28 36 36 Standard 5 Standard 5 13 13 21 21 29 29 37 37 Standard 6 Standard 6 14 14 22 22 30 30 38 38 Standard 7 Standard 7 15 15 23 23 31 31 39 39 Blank Blank 16 16 24 24 32 32 40 40 35 16 BLANK PLATE LAYOUT 36
12. ant in the wells by quickly inverting the 96 Well Plate over a sink or waste container Repeat actions 4 7 two more times for a total of 3 washes After the last wash blot the assembly onto several layers of paper towels to remove any residual solution 11 1 7 Add Detection Antibodies Mix 1x Add 25 uL of Detection Antibodies Mix 1x into each well Seal the 96 Well Plate with a new Plate Seal Remove the 96 Well Plate from the Hand Held Magnetic Plate Washer and cover the plate with the black microplate lid provided in the kit Shake at 500 rpm for 30 min at room temperature 25 11 1 8 Wash the 96 Well Plate Repeat step 11 1 6 11 1 9 Add SA PE Add 50 uL of SA PE solution into each well 2 Seal the 96 Well Plate with a new Plate Seal 3 Remove the 96 Well Plate from the Hand Held Magnetic Plate Washer and cover the plate with the black microplate lid provided in the kit 4 Shake at 500 rpm for 30 min at room temperature 11 1 10 Wash the 96 Well Plate Repeat step 11 1 6 11 1 11 Prepare the Plate for Analysis on a Luminex Instrument Add 120 uL of Reading Buffer into each well 2 Seal the 96 Well Plate with a new Plate Seal 3 Remove the 96 Well Plate from the Hand Held Magnetic Plate Washer and cover the plate with the black microplate lid provided in the kit Shake at 500 rpm for 5 min at room temperature 5 Remove the Plate Seal prior to reading on the Luminex instrument 26 11 2 PROTOCOL using
13. arget values We recommend RPI Low target value settings for ProcartaPlex Immunoassavs Please refer to the Certificate of Analvsis provided with the kit for bead region and analvte associations when entering the information into the Luminex acquisition software Please also refer to the Certificate of Analysis when assigning the Standard 1 Std 1 concentration into the analysis software Each analyte may have a different Std 1 concentration A 4 fold dilution should be applied to each subsequent standard Standard 2 7 For example if the starting concentration was 20 000 then a 4 fold dilution for Std 1 7 would be 20 000 5000 1250 312 78 19 5 and 4 8 pg ml NOTE If there is a malfunction of the Luminex instrument or software during the run the 96 Well Plate can be re read Remove the 96 Well Plate from the instrument insert the 96 Well Plate into the Hand Held Magnetic Plate Washer wait 2 min then remove the buffer in the wells by quickly inverting the 96 Well Plate over a sink or waste container Blot the assembly onto several layers of paper towels to remove any residual solution Resuspend the beads in 120 uL of Reading Buffer remove from the Hand Held Magnetic Plate Washer seal the 96 Well Plate with a new Plate Seal and shake at 500 rpm for 5 min at room temperature The assayed samples may take longer to read since there will be less beads in the wells 30 13 ANALYZING RESULTS The concentration of the sam
14. at 2 8 C and collect the plasma fraction 5 Use immediately or aliquot and store below 20 C 9 2 Preparing Serum Samples Allow blood to clot for 20 30 min at 20 25 C Centrifuge at 1 000 x g for 10 min at 20 25 C Collect the serum fraction Alternatively use any standard serum separator tube following the manufacturer s instructions 4 Optional If there is a high lipid content in the sample centrifuge at 10 000 x g for 10 min at 2 8 C Collect the serum fraction 5 Use immediately or aliquot and store below 20 16 ey 10 PREPARATION OF REAGENTS 10 1 Wash Buffer Dilute the wash buffer concentrate 10x 1 10 with deionized water E g mix 20ml of the wash buffer concentrate 10x with 180ml deionized water Store the wash buffer 1x at 2 8 C Please note that the wash buffer is stable for up to 6 months 10 2 Standard This section provides instructions on how to make a 4 fold 7 point standard curve for the assay panel Each kit is shipped with two vials of identical premixed standards Some higher plexed kits contain two or more different premixed standards Please refer to the Certificate of Analysis provided in the kit for the content of analytes in each premixed Standard and when assigning Standard antigen concentrations for each analyte It is recommended spinning down vials for a few seconds in a microcentrifuge before opening to collect lyophilized standard at the bottom
15. d mix In case of mixing two or more Simplex Kits or Simplex in combination with Multiplex Kits please check the bead regions assigned to the analytes you intend to combine Some analytes use the same bead region and thus cannot be combined together in one multiplex assay 21 11 TEST PROTOCOL 4 Qwest the beads Seal Plate Shake and incubate 60 120 min at room temperature in the dark Wash plate 3x Seal Plate Shake and incubate 30 min at room temperature in the dark Wash plate 3x Seal Plate Shake and incubate 30 min at room temperature in the dark Wash plate 3x 4 Shake 5 minutes at room temperature 1 For assays that require higher sensitivity 120 min or overnight incubation is recommended 22 11 1 PROTOCOL using Hand Held Magnetic Plate Washer 11 1 1 Prepare the 96 Well Flat Bottom Plate Mark the standard sample and blank wells For your convenience a blank layout is provided in the Layout section 11 1 2 Add the Antibody Magnetic Beads 1 2 3 Vortex the Antibody Magnetic Beads for 30 sec Add 50 uL of the Antibody Magnetic Beads to each well Insert the Procarta 96 Well Flat Bottom Plate into the Hand Held Magnetic Plate Washer so that the A1 location of the 96 Well Plate matches up with the A1 on the washer Lock the 96 Well Plate in place by pushing the 2 securing tabs located on each end of the washer towards the 96 Well Plate until they overlap
16. e intended for research use only and are not for use in diagnostic or therapeutic procedures 14 Do not mix or substitute reagents with those from other lots or other sources Do not use kit reagents beyond expiration date on label Do not pipette by mouth Do not eat or smoke in areas where kit reagents or samples are handled Avoid contact of skin or mucous membranes with kit reagents or specimens Rubber or disposable latex gloves should be worn while handling kit reagents or specimens Avoid splashing or generation of aerosols In order to avoid microbial contamination or cross contamination of reagents or specimens which may invalidate the test use disposable pipette tips and or pipettes Use clean dedicated reagent trays for dispensing the conjugates Glass distilled water or deionized water must be used for reagent preparation Decontaminate and dispose of specimens and all potentially contaminated materials as if they could contain infectious agents The preferred method of decontamination is autoclaving for a minimum of 1 hour at 121 5 C Liquid wastes not containing acid and neutralized waste may be mixed with sodium hypochlorite in volumes such that the final mixture contains 1 0 sodium hypochlorite Allow 30 minutes for effective decontamination Liquid waste containing acid must be neutralized prior to the addition of sodium hypochlorite 15 9 SAMPLE PREPARATION Cell culture supernatant
17. eded PBS can be used for the final bead resuspension step Sample Dilution of sample is too low or If values are higher than the measurements not falling on the standard curve too high standard curve dilute samples further in appropriate Sample Diluent Target concentration is below detection Verify that curve fitting at the lower end of the standard curve is accurate Not all serum plasma samples contain detectable levels of all analytes Low signal or sensitivity Standards not reconstituted and diluted correctly Prepare fresh antigen standards following the instructions in the Preparing Antigen Standards Section Expired reagents were used Expiry of the kit and reagents is stated on labels Do not use expired reagents Suboptimal assay conditions Follow the recommended incubation times and temperature Shake the 96 Well Plate during all incubations except during optional overnight incubation step Poor accuracy Did not use the appropriate assay diluents Use the same sample type specific standard and assay buffers for standard and sample preparations Universal Assay Buffer for serum and plasma Samples or Cell culture medium for cell culture supernatant samples 34 Example Plate Layout 1 2 5 6 7 8 9 10 11 12 Standards Samples Standard 1 Standard 1 9 9 17 17 25 25 33 33 Standard 2 St
18. eeenenznsnzznn 6 b Analyte OVGIVIOW six dies re ae i e d Ka 7 6 ProcartaPlex Kit Contents ssseeemenennzzznnnnzzznnezznznzzzni 11 7 Required Equipment and Materials Not Supplied 13 8 Precautions and Technical Hints s seen 14 9 Sample Preparation aga seecerescesdveeviuvinuedtentedeteaeaosaesaeceN 16 10 Preparation of Reagents nn rneeeeeee 17 DL MOGI ErOOCO uaia a a a i a 22 12 Setup of the Luminex Instrument sse 30 TS ANALYZING RESUS tie desi a i a 31 14 SPECIMEN iri i a e a ak 31 t9 MHOUDIGSNOOUNGs ciao sia taa a Gee tee aes 32 16 Bank Plate Lay OUl aceite biased Mot nai 36 3 12 08 2013 05 1 INTENDED USE This user manual is for a ProcartaPlex Immunoassay Kit Magnetic Beads from eBioscience to perform quantitative multiplexed immunoassays based on the Luminex technology The procedure is for simultaneous measurements of multiple protein biomarkers in serum plasma and cell culture supernatant samples Other biological samples might be suitable for use in the assay The assay protocol and reagents supplied are not compatible with other manufacturer s reagents Each 96 well plate kit is configured to allow for the following usage 14 wells for a 7 point standard curve in duplicate 2 wells for blanks and up to 80 wells for samples NOTE For the most current version of user documentation go to our website at www eBioscience com 2 ABOUT PROCARTAPLEX M MousE IMMUNOASSAY
19. m for cell culture supernatants or Universal Assay Buffer for serum and plasma samples When working with samples and standards change the pipette tips after every transfer and avoid creating bubbles when pipetting Do not expose kit reagents to light during storage or incubation the beads and Streptavidin Phycoerythrin are photosensitive During the incubation steps cover the 96 well plate with the black lid Be careful not to invert the 96 well Plate during the assay or allow contents from one well to mix with another well Ensure that the Hand Held Magnetic Plate Washer is securely locked into place prior to inverting when performing the wash steps Use a multi channel pipette and reagent reservoirs whenever possible to achieve optimal assay precision For frozen samples thaw completely and mix well prior to running the assay All chemicals should be considered as potentially hazardous We therefore recommend that this product is handled only by those persons who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice Wear suitable protective clothing such as laboratory overalls safety glasses and gloves Care should be taken to avoid contact with skin or eyes In the case of contact with skin or eyes wash immediately with water See material safety data sheet s and or safety statement s for specific advice www eBioscience com Reagents ar
20. mples See the following tables for a list of analytes detectable by these panels and a list of available Simplex assays and the respective bead regions ProcartaPlex Mouse Pre mixed panel overview Cytokine amp Chemokine Panel 1 26Plex EPX260 26088 901 Th9 Th17 Th22 Treg amp Chemokine Panel 15plex EPX150 26089 90 1 Th1 Th2 amp Chemokine Panel 1 20plex EPX200 26090 901 Th1 Th2 Th9 Th17 Th22 Treg 17plex EPX170 26087 901 Essential Th1 Th2 6plex EPXO60 20831 901 Th1 Th2 11plex EPX110 20820 901 IFN gamma 38 IL 12p70 39 IL 4 26 IL 5 27 IL 6 28 TNF alpha 45 IFN gamma 38 IL 12p70 39 IL 13 35 IL 18 19 IL 2 20 IL 4 26 IL 5 27 IL 6 28 TNF alpha 45 GM CSF 42 IL 18 66 IL 10 13 IL 17A 52 IL 22 33 IL 23 37 IL 27 36 IL 9 34 GRO alpha 43 IP 10 22 MCP 1 51 MCP 3 48 MIP 1alpha 47 MIP 1beta 72 MIP 2 55 RANTES 44 Eotaxin 62 IFN gamma 38 Eotaxin 62 GM CSF 42 GRO alpha 43 IL 18 19 IL 2 20 IL 4 26 IL 5 27 IL 6 28 IL 12p70 39 IL 13 35 IL 18 66 IP 10 22 MCP 1 51 MCP 3 48 MIP 1alpha 47 MIP 1beta 72 MIP 2 55 RANTES 44 TNF alpha 45 Available Simplex Kits and respective Bead Regions ProcartaPlex Mouse Simplex kit overview Available Simplex Kits and respective Bead Regions
21. on antibody concentrate provided in the simplex kits must also be included in the Detection Antibody Mixture 1x Prepare the Detection Antibody Mixture 1x according to the following calculation a Per test well 25 ul of the Detection Antibody Mixture 1x is required Consider tests for standard curves blanks and samples Calculate the final volume V fin of the Detection Antibody Mixture 1x needed V fin number of wells x 25 ul Round up for pipetting reservoir e g 96 tests x 25 ul 2400 ul round up V fin 3000 ul b Pipette 1 50 of final volume V fin of each premixed or simplex Detection Antibody Concentrate to a vial labelled Detection Antibody Mixture 1x e g V fin 3000 ul 1 50 of final volume 60 ul c Fill up to the final volume V fin with Detection Antibody Diluent 20 10 4 Bead Mixture Please note that the Certificate of Analysis provided with the assay lists the premixed antibody coated beads provided in the Kit 10 4 1 Kits Including one Capture Bead Mix Follow the steps indicated under Assay Protocol 11 1 2 or 11 2 2 10 4 2 Kits Including more than one Capture Bead Mix in one single Kit or combination of Kits For assays that are shipped with two or more bead mixtures included in the kit or if you want to add additional simplexes to your multiplex kit refer to 11 1 2 or 11 2 2 for the first bead mix and repeat the same procedure under 11 1 2 or 11 2 2 for each additional bea
22. ow cell Remove the 96 Well Plate and perform a wash and rinse to the instrument Clear system of clogs or air using maintenance steps described in the instrument user manual sanitize alcohol flush probe sonication etc Make sure that the probe height is set correctly Make sure that beads are in suspension by incubating plate for 3 5 minutes on plate shaker 750 rpm immediately before analysis Microbial growth in buffers can cause beads to stick to the filter plate membrane Do not use contaminated reagents Instrument needle is partially clogged Replace or clean needle according to the manufacturer s recommendations Air bubble in the sample loop Refer to the Luminex manual for proper removal of the air bubble Did not use supplied 96 well microtiter plate Only use the ProcartaPlex 96 Well Flat Bottom Plate supplied with the kit Beads were exposed to organic solvents Do not use organic solvents in the immunoassay as they will damage beads irreversibly Timeout limit is set too low 50 100 events per bead region should be acquired within the 60 second timeout limit If necessary the timeout limit can be set higher e g 75 seconds 33 Problem Probable Cause Solution Insufficient Solutions were not prepared Confirm correct buffer dilutions volume of an or used as described in and use immunoassay protocol If additional Universal Assay reagent Buffer is ne
23. patent or other intellectual property owned or licensable by eBioscience is conveyed or implied by this eBioscience product In particular no right or license is conveyed or implied to use this eBioscience product in combination with a product not provided licensed or specifically recommended by eBioscience for such use Citing ProcartaPlex Multiplex Immunoassays in Publications When describing a procedure for publication using this product please refer to it as the ProcartaPlex Multiplex Immunoassay from eBioscience eBioscience Inc reserves the right to change its products and services at any time to incorporate technological developments This manual is subject to change without notice Although this manual has been prepared with every precaution to ensure accuracy eBioscience Inc assumes no liability for any errors or omissions not for any damages resulting from the application or use of this information 2013 Affymetrix Inc All rights reserved Affymetrix eBioscience and ProcartaPlex are trademarks or registered trademarks of Affymetrix Inc Luminex and xMAP are registered trademarks of the Luminex Corporation All other trademarks are the property of their respective owners Table of Contents A tended Se iii pi ra a va A 4 2 About ProcartaPlex Mouse Immunoassay Kits 0 00 ssmeen 4 FAOW EVV OI Si i ijiet ra tana ktibt E ui den aka a 5 4 ProcartaPlex Immunoassay Workflow ss se
24. ples can be calculated by plotting the expected concentration of the standards against the MFI generated by each standard A 4PL or 5PL algorithm is recommended for the best curve fit Analyze the assayed samples according to the operation manual for the Luminex MAGPIX or Luminex based instrument 14 SPECIFICITY Cross reactivity was tested with combinable analytes of Simplex and Multiplex ProcartaPlex Assays There was no relevant cross reactivity observed For detailed information refer to Combination Table on www eBioscience com 31 15 TROUBLESHOOTING Problem Probable Cause Solution Low Flow Rate Partial blockage of flow cell Remove the 96 well plate and perform a wash and rinse cycle Instrument needle is partially clogged Replace or clean needle according to the manufacturer s recommendations Contamination from re using the Plate Seal Use a new Plate Seal for each incubation step Incomplete washing Blot the 96 Well Plate onto several layers of paper towels to remove any residual solution after each wash step Contamination from contents from adjacent wells Avoid splashing the Wash Buffer during wash steps into adjacent wells Poor pipetting techniques Use appropriate pipetting techniques Use new pipette tips for each well during sample and standard addition Avoid touching pipette tips to sides of the wells when adding wash buffer
25. rdous We recommend that this product and its components be handled by those trained in laboratory techniques and be used according to the principles of good laboratory practice WARNING This kit contains small quantities of sodium azide Sodium azide is highly toxic and reactive in the pure form At this product s concentration though not classified as hazardous build up of sodium azide may react with lead and copper plumbing to form highly reactive explosive metal azide Dispose of the product in accordance with all State and local regulations 12 7 REQUIRED EQUIPMENT AND MATERIALS NOT SUPPLIED Luminex 100 200 MAGPIX or other Luminex based Instrument Glass distilled or deionized water 5 ml and 10 ml graduated pipettes 10 1000 ul adjustable single channel micropipettes with disposable tips 20 ul to 300 ul adjustable multichannel micropipettes with disposable tips Multichannel micropipette reservoir Beakers flasks cylinders necessary for preparation of reagents Vortex mixer and Microplate shaker Note Manual Wash Solutions for Magnetic Beads A The Hand Held Magnetic Plate Washer provides you a low cost opinion for magnetic separation and automated washing of magnetic bead based assays without loss of assay performance The Magnet employs a magnet layer located around the bottom of the well capable to pull the beads to the side thus retaining them in the well while decanting the plate contents into
26. serum and plasma and were tested with this assay Other biological samples might be suitable for use in the assay Remove serum or plasma from the clot or cells as soon as possible after clotting and separation A total volume of 25 uL per well of serum and plasma or 50 uL per well of cell culture supernatant samples is needed and a minimum of 2 replicates is recommended Some samples may contain high concentrations of the analytes Dilution of the samples may be needed if the analyte concentration is above the assay upper limit of quantitation Serial dilution of the samples may need to be prepared to determine the appropriate dilution factor for accurately measuring the analytes of interest Use Universal Assay Buffer to prepare dilutions of serum and plasma samples Cell culture supernatants samples will be prepared by using appropriate Cell culture medium Refer to the table of Recommended Sample Dilution for Analytes at the end of this manual 9 1 Preparing Plasma Samples 1 Collect samples in sodium citrate or EDTA tubes When using heparin as an anticoagulant no more than 10 IU of heparin per mL of blood collected should be used since an excess of heparin may give falsely high values of some of the analytes 2 Centrifuge samples at 1 000 x g at 4 C for 10 min within 30 min of blood collection 3 Collect the plasma fraction 4 Optional To minimize lipid and or platelets in the sample centrifuge the sample at 10 000 x g for 10 min
27. tandards If the kits you want to combine include two different batch numbers of the same premixed standard you may choose any of the two for your multiplex assay 18 10 2 3 Preparing a 4 Fold Serial Dilution 1 2 Prepare a 4 fold serial dilution of the standard mix reconstituted according to 10 2 1 or 10 2 2 using the PCR 8 tube strip provided Add 200 uL of the reconstituted antigen standard mix into the first tube of the strip tube and label as Standard 1 Std 1 Add 150 uL sample type specific standard buffer for serum or plasma use Universal Assay Buffer for cell culture use cell culture medium into Tubes 2 7 Transfer 50 uL of the reconstituted antigen standards from Tube 1 into Tube 2 Mix by pipetting up and down for a total of 10 times After changing the pipette tip transfer 50 uL of the mixed standards from Tube 2 into Tube 3 Mix by pipetting up and down 10 times Repeat Actions 4 to 7 for the rest of the tubes to prepare Std 4 7 Keep on ice until ready to use Transfer 200 uL 50 yL 50 ul 50 ul 50 yL 50 ul 50 ul An Standard Mix Vial ah NA A A Std 1 Std 2 Std 3 Std 4 Std 5 Std 6 Std 7 19 10 3 Detection Antibody Mixture 1x All detection antibodies are provided as 50x concentrate in the kits Some larger plexed panels include more than one premixed detection antibody concentrate in the kit If you want to include additional simplex assays in your multiplex assay the detecti
28. tandards into dedicated wells 2 Add 50 uL of cell culture medium to the blank wells 11 1 5 Incubate the 96 Well Plate Seal the 96 Well Plate using a Plate Seal provided 2 Remove the 96 Well Plate from the Hand Held Magnetic Plate Washer and cover the plate with the black microplate lid provided in the kit in order to protect it from light 3 Incubation A Shake the 96 Well Plate at 500 rom for 60 to 120 min at room temperature 24 B Alternatively the 96 Well Plate can be incubated overnight Shake the 96 Well Plate at room temperature for 30 min Transfer the plate to 4 C and store on a level surface After incubation remove the 96 Well Plate from 4 C and shake for 30 min at room temperature and proceed to next step NOTE We recommend 120 min at room temperature or overnight incubation at 4 C for assays that require higher sensitivities 11 1 6 Wash the 96 Well Plate 1 Insert the 96 Well Plate into the Hand Held Magnetic Plate Washer and wait 2 min to allow the Antibody Magnetic Beads to accumulate on the bottom of each well Carefully remove the Plate Seal to avoid splashing the plate contents Remove the solution in the wells by quickly inverting the Hand Held Magnetic Plate Washer and 96 Well Plate assembly over a sink or waste container Add 150 uL of 1X Wash Buffer into each well Wait 30 sec to allow the Antibody Magnetic Beads to accumulate on the bottom of each well Remove the supernat
29. the kit is stated on the kit label Expiry of the kit components can only be guaranteed if the components are stored properly and if in case of repeated use of one component this reagent is not contaminated by the first handling Component Description Included in Multiplex Simplex Basic Kits Kits Kits Antigen Standards premixed lyophilized 2 vials each set Please note that more than 1 set of vials may be shipped with each kit for certain products X X Detection Antibody premixed 50x Concentrated biotinylated detection antibodies Magnetic Beads premixed Magnetic Beads coated with specific antibodies Streptavidin PE SA PE Streptavidin conjugated R phycoerythrin Wash Buffer Concentrate 10x Concentrated aqueous buffered solution Detection Antibody Diluent For dilution of detection antibodies Universal Assay Buffer For dilution of standard and samples when analysing serum or plasma samples Reading Buffer Aqueous buffered solution PCR 8 Tube Strip 2x 0 2mL 8 tube strip for preparing standard curve Flat bottom and Filter Plate 96 well plates Plate seals Adhesive backed foil plate sealer Black Microplate Lid Protecting the assay from light during incubation Contains Sodium Azide See WARNING on next page 11 WARNING All chemicals should be considered potentially haza
30. the skirt of the 96 Well Plate Verify that the 96 Well Plate is securely locked by holding the assembly in the palm of your hand and gently pulling up on the 96 Well Plate Wait 2 min to allow the Antibody Magnetic Beads to accumulate on the bottom of each well Remove the liquid in the wells by quickly inverting the Hand Held Magnetic Plate Washer and 96 Well Plate assembly over a sink or waste container Do not remove the 96 Well Plate from the Hand Held Magnetic Plate Washer Blot the inverted assembly onto several layers of paper towels to re move any residual solution Repeat actions 2 7 using the same plate with all Antibody coated beads you want to include in your Multiplexing assay 23 11 1 3 Wash Antibody Magnetic Beads Add 150 uL of 1X Wash Buffer into each well 2 Wait 30 sec to allow the Antibody Magnetic Beads to accumulate on the bottom of each well 3 Remove the wash buffer in the wells by quickly inverting the Hand Held Magnetic Washer and 96 Well Plate assembly over a sink or waste container 4 Blot the inverted assembly onto several layers of paper towels to remove any residual solution 11 1 4 Add Antigen Standards and Samples 11 1 4 1 Serum and Plasma Samples 1 Add 25 uL of Universal Assay Buffer into each well 2 Add 25 uL of standards or samples into dedicated wells 3 Add 25 uL of Universal Assay Buffer to the blank wells 11 1 4 2 Cell Culture Supernatant Samples 1 Add 50 uL of samples or s
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