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ProcartaPlex™

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2. NOTE For the most current version of user documentation go to our website at www ebioscience com Summary Antibodies are soluble immunoglobulins Ig produced by plasma cells in response to an immunogen and are critically involved in the immune response Each immunoglobulin actually binds to a specific antigenic determinant epitope All immunoglobulins have a four chain structure as their basic unit They are composed of two identical light chains lambda or kappa 23kD and two identical heavy chains alpha delta gamma epsilon or mu 50 70kD connected by disulfide bonds Based on the differences in the amino acid sequences in the constant region Fc part of the heavy chains the immunoglobulins can be divided into five different classes IgG IgM IgA IgD and IgE IgGs can additionally be divided into 4 subclasses based on the number of disulfide bonds and length of the hinge region If different immunoglobulin classes or isotypes recognize an antigen at the same epitope they still differ in the way the immune system reacts to the antigen The specific Fc part will promote a specific effector function of the immune system to the antigen The isotype of an antibody determines its distribution to the different compartments of the body in which their particular effector functions are appropriate How it Works ProcartaPlex Immunoassays use the xMAP technology multi analyte profiling beads to enable the detection a
3. affymetrix eBioscience ProcartaPlex Multiplex IMmunoassay Using Magnetic Beads For Serum Plasma and Cell Culture Supernatant Samples Instructions for Human Isotyping Panel North America Europe International Technical Support Technical Support Research Products 43 1796 40 40 120 888 810 6168 tech eBioscience com 858 642 2058 Customer Service tech eBioscience com 143 1796 40 40 305 Clinical Products europe eBioscience com 877 726 8559 Fax 43 1 796 40 40 400 858 642 2058 tech eBioscience com ud Bender MedSystems GmbH Customer Service Campus Vienna Biocenter 2 888 999 1371 1030 Vienna Austria 858 642 2058 www eBioscience com info eB ioscience com Customers in countries where direct sales are not avaliable may contact their eBioscience distributor listed at www eBioscinece Fax 858 642 2046 com distributors 21 05 2014 Rev 3 For research use only Not for use in diagnostic 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 patent or other intellectual property owned or licensable by eBioscienc
4. 0 at ebioscience com ppx analyst Components 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 Expiration of the kit is stated on the kit label Expiration of the kit components can only be guaranteed if the components are stored properly between 2 8 C and if in case of repeated use of one component this reagent is not contaminated by the first handling Component Antigen Standards premixed lyophilized 2 vials each set Description Please note that more than 1 set of vials may be shipped with each kit for certain products Detection Antibody premixed 50X Concentrated biotinylated detection antibodies Antibody 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 Concentrate 10X For analytes with high serum and plasma concentration additional UAB 10X will be included Reading Buffer Aqueous buffered solution PCR 8 Tube Strip 2X 0 2 mL 8 Tube Strip for preparing standard curve Filter Plate 96 Well Filter Plate for use with a vacuum system Flat Bottom Plate 96 Well Flat Bottom Plate for
5. Buffer for cell culture supernatants use cell culture medium into the vial 3 Vortex gently for 30 sec 4 Incubate on ice for 5 10 min Preparing a 3 Fold Serial Dilution 1 Prepare a 3 fold serial dilution of the standard mix reconstituted according to the text above using the PCR 8 Tube Strip provided Add 200 uL of the reconstituted antigen standard mix into the first strip tube and label the tube 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 and label them accordingly Transfer 75 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 75 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 Add Universal Assay Buffer or cell culture medium into tube 8 that serves as blank Keep on ice until ready to use Transfer 200uL 75 ul 75 pL 75 pL 75 ul 75 yL 75 ul beat LJ LI Lu J m E i y wwe Antigen KJ NI J w NI LL Standard Vial Std 1 std 2 Std 3 Std 4 Std 5 Std 6 std7 Use Assay Buffer or Cell Culture Media for Blanks Det
6. Plate For frozen samples thaw completely on ice 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 are intended for research use only and are not for use in diagnostic or therapeutic procedures 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 conjugate
7. bead regions and wells were selected during acquisition setup Store bead solution in the dark and protect the 96 Well Plate from light by wrapping the 96 Well Plate with aluminum foil Remove the 96 Well Plate and perform a wash and rinse to the instrument Replace or clean needle according to the manufacturer s recommendations Confirm that the plate shaker is set to 500 rpm and shaking for at least 5 min before reading Observation Sample measurements not falling on the standard curve Low signal or sensitivity Poor accuracy Probable Cause Recommend Solution Partial blockage of the flow cell Remove the 96 Well Plate and perform a wash and Beads were exposed to organic solvents Did not use supplied 96 well microtiter plate Air bubble in the sample loop Dilution of sample is too low too high or Target concentration is below detection Standards not reconstituted and diluted correctly Expired reagents were used Suboptimal assay conditions Did not use the appropriate assay diluents 28 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 shaking the plate for 3 5 minutes at 500 rom immediately before analysis Microbial growth in buffers can cause beads to stick to the filter
8. sec and add 5 mL of each Bead Mixture to an appropriate vial 3 Vortex the Antibody Magnetic Beads for 30 sec 4 Add the appropriate volume of Antibody Magnetic Beads to each well a 100 uL to each well if 2 different Antibody Magnetic Beads have been mixed a 150 uL to each well if 3 different Antibody Magnetic Beads have been mixed a 200 uL to each well if 4 different Antibody Magnetic Beads have been mixed a 250 uL to each well if 5 different Antibody Magnetic Beads have been mixed m 300 uL to each well if 6 different Antibody Magnetic Beads have been mixed 5 Wait 2 min to allow the Antibody Magnetic Beads to accumulate on the bottom of each well 6 Remove the liquid in the wells by quickly inverting the Hand Held Magnetic Plate Washer and 96 Well Flat Bottom Plate assembly over a sink or waste container Blot the inverted assembly onto several layers of paper towels to remove any residual solution 7 Leave 96 Well Flat Bottom Plate on Hand Held Magnetic Plate Washer and proceed to the step Wash Antibody Magnetic Beads using a Hand Held Magnetic Plate Washer on page 18 If a suitable tube Magnetic Bead Separator is available premix the beads using the following protocol 1 Vortex the Antibody Magnetic Beads for 30 sec 2 Add 5 mL for 96 wells of each Bead Mixture to an appropriate vial 3 Let the beads accumulate according to the instructions of the separator 4 Remove the liquid If necessary repeat 1 4 until al
9. ash Buffer using a multi channel pipette Add Detection Antibodies Mixture 1X 1 Add 25 uL of Detection Antibodies Mixture 1X into each well 2 Seal the 96 Well Filter Plate with a new Plate Seal and cover the plate with the Black Microplate Lid provided in the kit 23 3 Shake at 500 rpm for 30 min at room temperature Wash the 96 Well Filter Plate Repeat steps indicated under Wash the 96 Well Filter Plate Add Streptavidin PE SA PE 1 Add 50 uL of SA PE solution into each well 2 Seal the 96 Well Filter 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 Wash the 96 Well Filter Plate Repeat steps indicated under Wash the 96 Well Filter Plate Prepare the Plate for Analysis on a Luminex Instrument 1 Add 120 uL of Reading Buffer into each well Seal the 96 Well Filter 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 U DS WN 24 Setup of the Luminex Instrument Sample Size DD Gate Timeout Bead Event Bead Region 50 uL 5 000 25 000 45 sec 50 100 If you are running assays on your Luminex Instrument that uses both the 96 Well Flat Bottom Plate and 96 Well Filter Plate for Antibody Magnetic Beads verify the probe height fo
10. before starting the test procedure If crystals have formed in the Wash Buffer Concentrates 10X warm them gently until they have completely dissolved 1 Dilute the Wash Buffer Concentrate 10X 1 10 with deionized water E g mix 20mL of the Wash Buffer Concentrate 10X with 180 mL deionized water 2 Store the Wash Buffer 1X at 2 8 C The Wash Buffer is stable for 6 months when stored properly Universal Assay Buffer 10X Dilute the Universal Assay Buffer Concentrate 10X with deionized water E g mix 10 mL of Universal Assay Buffer Concentrate 10X with 90 mL deionized water Store the Universal Assay Buffer Concentrate 10X at 2 to 8 C When stored properly the Universal Assay Buffer 1X is stable for 30 days Standard This section provides instructions on how to make a 7 point standard curve for the assay panel Each kit is shipped with two vials of identical premixed antigen 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 x NOTE Standard Range vary from lot to lot The Certificate of Analysis CoA includes the S1 data of your kit For antigen standard preparation follow the steps indicated below 1 Centrifuge the antigen standard vial at 2000 x g for 10 sec 2 Add 250 uL of sample type specific standard buffer for serum or plasma use Universal Assay
11. e 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 Add Detection Antibodies Mixture 1X 1 Add 25 uL of Detection Antibodies Mixture 1X 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 Wash the 96 Well Flat Bottom Plate Repeat steps indicated under Wash the 96 Well Plate using a Hand Held Magnetic Plate Washer Add Streptavidin PE SA PE 1 2 di 4 Add 50 uL of SA PE solution 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 Wash the 96 Well Flat Bottom Plate Repeat steps indicated under Wash the 96 Well Plate using a Hand Held Magnetic Plate Washer Prepare the Plate for Analysis on a Luminex Instrument 1 2 3 Add 120 uL of Reading Buffer 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 prov
12. e 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 Immunoassay in Publications When describing a procedure for publication using this product please refer to it as the ProcartaPlex Multiplex Immunoassay from eBioscience Disclaimer 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 Trademarks 2014 Affymetrix Inc All rights reserved Affymetrix eBioscience and ProcartaPlex are trademarks or registered trademarks of Affymetrix Inc Luminex xMAP MagPix FlexMap 3D and xPonent are registered trademarks of the Luminex Corporation All other trademarks are the property of their respective owners Contents Intended Use aiar fi A EE E oo oa 4 SUMMAN ili RE 5 AI APRE RARO PETITE TICO 5 Analyte Overview LL 5 ProcartaPlex Workflow 6 LGOMPONCHE siglati Lo 7 Required Equipment and Materials Not Supplied 8 Precautions and Techn
13. ection Antibody Mixture 1X Detection Antibodies in Simplex Kits and Panels are provided as 50X concentrate 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 Detection Antibody Concentrate provided in the Simplex Kits must also be included in the Detection Antibody Mixture 1X If your kit includes Detection Antibody Concentrate 50X prepare the Detection Antibody Mixture 1X according to the following calculation 1 Each well uses 25 uL of the Working Detection Antibody Mixture 1X Prepare an appropriate volume of Working Detection Antibody Mixture 1X by combining the concentrated Detection Antibody 50X with the Detection Antibody Diluent 2 Scale according to the number of assays to be run and include sufficient overage Use the table below as a guide When calculating the number of wells make sure to consider the total number of wells needed that include the standard curves blanks and samples Calculate the final volume of the Detection Antibody Mixture 1X needed and estimate for 25 overage to enable use of a reagent reservoir and multi channel pipette Example for 1 vial of Detection Antibody 24 Well 48 Wells 96 Wells Detection Antibody 15 uL 30 uL 60 uL Detection Antibody 735 uL 1470 uL 2940 uL Diluent Total 750 uL 1500 uL 3000 pL Example for 2 vials of Detection Antib
14. ghly read this user manual and product insert that is included with the assay kit The product insert may contain specific instructions for proper use of your 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 When working with samples and standards change the pipette tips after every transfer and avoid creating bubbles when pipetting Some samples may contain high analyte concentrations and require sample dilution for accurate quantitation Please use cell culture medium for cell culture supernatants or Universal Assay Buffer for serum and plasma samples During the incubation steps cover the 96 Well Plate with the Black Microplate Lid provided in the kit to minimize exposure of the beads to light Be careful not to invert the ProcartaPlex 96 Well Flat Bottom 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 Store the Detection Antibody Antibody Magnetic Beads Streptavidin PE samples and reconstituted standards including standard diluent sets on ice before adding to the 96 Well
15. ical Hits aoaaa aaa aaa aana aaraa 8 Sample Preparation sails been idro et 10 Assay Protocol Overview 12 Preparation of Reagents x sissi parlare NG 13 Protocol using a Hand Held Magnetic Plate Washer 16 Protocol using a Filter Plate 21 Setup of the Luminex Instrument 000 25 Analyzing Results aaa KAANAK AB AA NAKAKA AA bka kA 25 Specii oud pea cit een eeepc SRST terri 26 Troubleshooting naam ewe doe oe LANG KOK KA HERA dee Ae ws 27 Recommended Plate Layout siii a 29 Blank Plate Layout ipod AIA 30 Intended Use This user manual is for a ProcartaPlex Human Isotyping 7plex Kit Magnetic Beads from eBioscience to perform quantitative detection of Human IgG1 IgG2 IgG3 IgG4 IgA IgM and IgE by using 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 ProcartaPlex Human Antibody Isotyping Panel cannot be combined with other ProcartaPlex Simplex or Multiplex Kits NOTE Simplex Kits can only be combined with analytes included in ProcartaPlex Human Isotyping Panel
16. ided in the kit Shake at 500 rpm for 5 min at room temperature Remove the Plate Seal prior to reading on the Luminex Instrument 20 Protocol using a Filter Plate The Antibody Magnetic Beads can be used with a Vacuum Filtration Manifold and the 96 Well Filter Plate The Vacuum Manifold can be properly calibrated by placing a standard 96 Well Filter Plate on top of the manifold and then turning on the vacuum Press down on all 4 corners of the standard 96 Well Filter Plate to form a tight seal and adjust the pressure so that it takes 4 6 seconds to evacuate 150 uL of Wash Buffer from the wells If the vacuum is too high beads can get trapped or pulled through the filter Turn off vacuum as soon as the solution filters through the wells and remove the plate from the manifold Prepare the 96 Well Filter Plate 1 For your convenience please use the blank layout provided in the Layout section to define your plate scheme blanks standards and samples 2 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 Add Antibody Magnetic Beads If only one Antibody Magnetic Bead Mixture is included in the kit please follow the steps indicated below 1 Vortex the Antibody Magnetic Beads for 30 sec 2 Add 50 uL of the Antibody Magnetic Beads to each well 3 Remove the liquid in the wells by aspiration using the Vacuum Filtrat
17. ion Manifold Blot the bottom of the plate after filtration 4 Proceed to the step Wash Antibody Magnetic Beads using a 96 Well Filter Plate Combining Antibody Magnetic Beads from multiple kits Multiple Simplex Kits can be combined with one another as well as with Premixed Panels When mixing two or more Simplex Kits or Premixed Panels ensure that the bead regions assigned to the analytes do not overlap Some analytes use the same bead region and thus cannot be combined together in one multiplex assay Please check the compatibility of analytes online Panel Configurator or contact your local sale representative Both Simplex and Premixed Kits are supplied with beads at working concentration Our test protocol requires of 50 uL beads per well The 96 Well Filter Plate used can hold up to 300 ul per well so 6 different Antibody 21 Magnetic Beads can be premixed at one time using the procedure below for use with the Vacuum Filtration Manifold If more than 6 bead mixtures are to be mixed then this procedure can be repeated If two or more Antibody Magnetic Bead Mixtures are included in the kit please follow the steps indicated below 1 Vortex the Antibody Magnetic Beads for 30 sec and add 5 mL for 96 wells of each Bead Mixture to an appropriate vial 2 Vortex the Antibody Magnetic Beads for 30 sec 3 Add the appropriate volume of Antibody Magnetic Beads to each well a 100 uL to each well if 2 different Antibody Magne
18. iquid in the wells by aspiration using the Vacuum Filtration Manifold Blot the bottom of the plate after filtration 22 Add Standards and Samples 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 Cell Culture Supernatant Samples 1 Add 50 uL of standards or samples into dedicated wells 2 Add 50 uL of cell culture medium to the blank wells Incubate the 96 Well Filter Plate 1 Seal the 96 Well Filter Plate using a Plate Seal provided 2 Cover the plate with the Black Microplate Lid provided in the kit 3 During incubation shake the 96 Well Filter Plate at 500 rom for 60 to 120 min at room temperature Wash the 96 Well Filter 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 one more time for a total of 2 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 W
19. l beads are in the tube 5 Place the 96 Well Flat Bottom Plate onto the Hand Held Magnetic Washer and securely lock into place 6 If using the whole plate 96 wells re suspend the beads in 16 mL Wash Buffer Vortex the Antibody Magnetic Beads for 30 sec and add 150 uL of the Antibody Magnetic Beads to each well of the plate 7 Wait 2 min to allow the Antibody Magnetic Beads to accumulate on the bottom of each well 8 Remove the liquid in the wells by quickly inverting the Hand Held Magnetic Plate Washer and 96 Well Flat Bottom Plate assembly over a sink or waste container Blot the inverted assembly onto several layers of paper towels to remove any residual solution 9 Proceed to the step Add Standards and Samples Wash Antibody Magnetic Beads using a Hand Held Magnetic Plate Washer 1 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 Flat Bottom Plate assembly over a sink or waste container 4 Blot the inverted assembly onto several layers of paper towels to remove any residual solution Add Standards and Samples 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 Cell Culture S
20. nd quantitation of multiple protein targets simultaneously in diverse matrices The xMAP system combines a flow cytometer fluorescent dyed 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 and Luminex based instruments currently available Analyte Overview All products listed on our website www eBioscience com ProcartaPlex Workflow Capture Target Analytes 0 p i Q E Add analyte specific AYE b gt capture beads coated Q Q s R with target specific Capture Beads Analyte capture antibodies to prepared sample Incubate 1 2 hours and wash Detect Captured Analyte M Incubate captured bead analytes with biotinylated analyte specific detection dee Detection Antibody Immuno sandwich Complex Incubate 30 min and wash o Label Detection 5s 3 7 7 Incubation a E For analyte quantitation pe Wash incubate with a yey mp fluorescent detection N2 HE label SA PE SA PE LHE E Wash sample E Quantitate sample Incubate 30 min and wash Read amp Analyze E Read with Luminex Instrument E Analyze results with ProcartaPlex Analyst Software 1 0 XMAP TECHNOLOGY Because you want to know mos POWER INSIDE UZZZA Free download of ProcartaPlex Analyst Software 1
21. nd the beads in 120 uL of Reading Buffer 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 Analyzing Results The concentration of the samples 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 For reliable and quick analysis of the data ProcartaPlex Analyst 1 0 can be used NOTE Dilutions factors of samples have to be entered manually as described in the ProcartaPlex Analyst 1 0 Software Manual Free download at ebioscience com ppx analyst 25 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 Panel Configurator on www eBioscience com 26 Troubleshooting Observation Low Flow Rate High CVs Low bead count Probable Cause Recommend Solution Partial Blockage of the flow cell Remove the 96 Well Plate and perform a wash and Instrument needle is partially clogged Contamination from re using the Plate Seal Incomplete washing Contamination from contents from adjacent wells Poor pi
22. odies 24 Well 48 Wells 96 Wells Detection Antibody 1 15 uL 30 uL 60 uL Detection Antibody 2 15 uL 30 uL 60 uL Detection Antibody 720yL 1440 uL 2880 uL Diluent Total 750 uL 1500 uL 3000 uL Protocol using a Hand Held Magnetic Plate Washer Prepare the 96 Well Flat Bottom Plate For your convenience please use the blank layout provided in the Layout section to define your plate scheme blanks standards and samples Add Antibody Magnetic Beads If only one Antibody Magnetic Bead Mixture is included in the kit please follow the steps indicated below 1 Vortex the Antibody Magnetic Beads for 30 sec 2 Add 50 uL of the Antibody Magnetic Beads to each well 3 Insert the ProcartaPlex 96 Well Flat Bottom Plate into the Hand Held Magnetic Plate Washer DI a II CUCL COTY COU 0600000 4 Place the 96 Well Plate by pushing the 2 securing tabs located on each end of the washer Rubber gasket surrounds the system and provides optimal mount of the 96 Well Flat Bottom Plate 5 Verify that the 96 Well Flat Bottom Plate is securely locked by holding the assembly in the palm of your hand and gently pulling up on the 96 Well Flat Bottom Plate 6 Wait 2 min to allow the Antibody Magnetic Beads to accumulate on the bottom of each well 7 Remove the liquid in the wells by quickly inverting the Hand Held Magnetic Plate Washer and 96 Well Flat Bottom Plate assembly over a sink or waste container Do n
23. oncentrations 1 20 000 fold dilution in Universal Assay Buffer 1x is appropriate for most serum and plasma samples For samples that yield results outside the range of the standard curve a lower or higher dilution might be required For cell culture supernatant a 1 200 dilution is suggested Each serum or plasma sample has to be diluted 1 20 000 according to following scheme Dilution 1 1 200 20 ul sample 1980 ul Universal Assay Buffer 1x Dilution 2 1 100 10 ul prediluted sample 1 200 990 ul Universal Assay Buffer 1x If the samples are taken from cell culture supernatant prepare a 1 200 dilution Dilution 1 1 10 10 ul sample 90 ul cell culture medium 1x Dilution 1 1 20 50u prediluted sample 1 10 950 ul cell culture medium 1x NOTE Cell culture supernatant concentrations are cell line dependent and range from 5 50 g mL Bioreactor supernatants may be as concentrated as 1 mg mL and thus need higher dilutions NOTE For analytes with high serum and plasma concentration additional Universal Assay Buffer 10X will be included in the kit Preparing Plasma Samples 1 Di 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 Centrifuge samples at 1 000 x g at 4 C for 10 min within 30 min of bl
24. ood collection Collect the plasma fraction Optional To minimize lipid and or platelets in the sample centrifuge the sample at 10 000 x g for 10 min at 2 8 C and collect the plasma fraction Use immediately or aliquot and store below 20 C Preparing Serum Samples 1 Allow blood to clot for 20 30 min at 20 25 C 2 a 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 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 Use immediately or aliquot and store below 20 C Assay Protocol Overview Wash the beads 1x 4 4 Wash the beads 1x Seal plate Shake and incubate 60 120 min at room temperature in the dark Wash plate 2x Seal plate Shake and incubate 30 min at room temperature in the dark Wash plate 2x Seal plate Shake and incubate 30 min at room temperature in the dark Wash plate 2x Shake 5 minutes at room temperature For assays that require higher sensitivity 120 min or overnight incubation at 4 C is recommended Concentration of Detection Antibody in Customized Kits is 1x therefore no further dilution is needed 12 Preparation of Reagents Wash Buffer Wash Buffer Concentrate 10X should be brought to room temperature and should be diluted
25. ot remove the 96 Well Flat Bottom Plate from the Hand Held Magnetic Plate Washer Blot the inverted assembly onto several layers of paper towels to remove any residual solution 8 Proceed to the step Wash Antibody Magnetic Beads using a Hand Held Magnetic Plate Washer Combining Antibody Magnetic Beads from multiple kits Multiple Simplex Kits can be combined with one another as well as with Premixed Panels When mixing two or more Simplex Kits or Premixed Panels ensure that the bead regions assigned to the analytes do not overlap Some analytes use the same bead region and thus cannot be combined together in one multiplex assay Please check the compatibility of analytes online Panel Configurator or contact your local sale representative Both Simplex and Premixed Kits are supplied with beads at working concentration Our test protocol requires 50 uL beads per well The 96 Well Flat Bottom Plate used can hold up to 300 uL per well so 6 different Antibody Magnetic Beads can be premixed at one time using the procedure below for use with the Hand Held Magnetic Washer If more than 6 bead mixtures are to be mixed then this procedure can be repeated If two or more Antibody Magnetic Bead Mixtures are included in the kit please follow the steps indicated below for 96 wells 1 Place the 96 Well Flat Bottom Plate onto the Hand Held Magnetic Washer and securely lock into place 2 Vortex each of the Antibody Magnetic Beads for 30
26. petting techniques Probe height is incorrect Reading buffer volume added in the last step to resuspend the beads is too low High bead aggregation Dyes contained in the beads are photo bleached from overexposure to light Beads are falling outside the bead region gates due to photo bleaching Partial blockage of the flow cell Instrument needle is partially clogged Beads settle on the bottom of the well 27 rinse cycle Replace or clean needle according to the manufacturer s recommendations Use a new Plate Seal for each incubation step Blot the 96 Well Plate onto several layers of paper towels to remove any residual solution after each wash step Avoid splashing the Wash Buffer during wash steps into adjacent wells 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 Refer to the Luminex Manual for proper adjustment of the needle height 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 Vortex the bead suspension well before using in the assay and ensure that the beads are properly mixed during the incubation steps Verify that beads were added at the correct concentration and that correct
27. plate membrane Do not use contaminated reagents Do not use organic solvents in the immunoassay as they will damage beads irreversibly Only use the ProcartaPlex 96 Well Flat Bottom Plate supplied with the kit Refer to the Luminex Manual for proper removal of the air bubble If values are higher than the standard curve dilute samples further in appropriate Sample Diluent 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 Prepare fresh antigen standards following the instructions in the Preparing Antigen Standards section Expiry of the kit and reagents is stated on labels Do not use expired reagents Follow the recommended incubation times and temperature Shake the 96 Well Plate during all incubations except during optional overnight incubation step 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 Recommended Plate Layout CTTCECECIEFEIPE Ec EEE paaa EEE EEN maamo Gea I I RAI RA NE NE e a EE O O CO Standard 7 Standard 7 29 Blank Plate Layout 1 2 3 10 11 12 10 11 12 30
28. r 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 target values We recommend RP1 Low target value settings for ProcartaPlex Immunoassays Please refer to the Certificate of Analysis provided with the kit for bead region and analyte associations when entering the information into the Luminex Acquisition Software 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 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 and remove the liquid in the wells by aspiration using the Vacuum Filtration Manifold Blot the bottom of the plate after filtration Resuspe
29. s 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 Sample Preparation Cell culture supernatant serum and plasma samples have been tested and validated with the ProcartaPlex assay Although not validated other biological samples such as BAL saliva CSF tissue lysates and urine might be suitable for use in the assay Samples containing a visible precipitate must be clarified prior to use in the assay Do not use grossly lipemic specimens Fat causes agglutination of the beads Centrifugation of samples about 16 000 x g for 5 min before analysis is recommended For serum or plasma samples remove 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 The analytes included in the kit have high serum or plasma c
30. tic Beads have been mixed a 150 uL to each well if 3 different Antibody Magnetic Beads have been mixed a 200 uL to each well if 4 different Antibody Magnetic Beads have been mixed a 250 uL to each well if 5 different Antibody Magnetic Beads have been mixed a 300 uL to each well if 6 different Antibody Magnetic Beads have been mixed 4 Remove the liquid in the wells by aspiration using the Vacuum Filtration Manifold Blot the bottom of the plate after filtration 5 Proceedto the step Wash Antibody Magnetic Beads using a 96 Well Filter Plate below If a suitable tube Magnetic Bead Separator is available premix the beads using the following protocol 1 Vortex the Antibody Magnetic Beads for 30 sec 2 Add 5 mL for 96 wells of each Bead Mixture to an appropriate vial 3 Let the beads accumulate according to the instructions of the separator 4 Remove the liquid If necessary repeat 1 4 until all beads are in the tube 5 If using the whole plate 96 Wells re suspend the beads in 16 mL Wash Buffer Vortex the Antibody Magnetic Beads for 30 sec and add 150 uL of the Antibody Magnetic Beads to each well of the plate 6 Remove the liquid in the wells by aspiration using the Vacuum Filtration Manifold Blot the bottom of the plate after filtration 7 Proceed to the step Add Standards and Samples Wash Antibody Magnetic Beads using a 96 Well Filter Plate 1 Add 150 uL of 1X Wash Buffer into each well 2 Remove the l
31. upernatant Samples 1 Add 50 uL of standards or samples into dedicated wells 2 Add 50 uL of cell culture medium to the blank wells Incubate the 96 Well Flat Bottom Plate 1 Seal the 96 Well Flat Bottom Plate using a Plate Seal provided 2 Remove the 96 Well Flat Bottom 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 During incubation shake the 96 Well Flat Bottom Plate at 500 rpm for 60 min at room temperature Wash the 96 Well Plate using a Hand Held Magnetic Plate Washer 1 Insert the 96 Well Flat Bottom 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 2 Carefully remove the Plate Seal to avoid splashing the plate contents 3 Remove the solution in the wells by quickly inverting the Hand Held Magnetic Plate Washer and 96 Well Flat Bottom Plate assembly over a sink or waste container 4 Add 150 uL of 1X Wash Buffer into each well 5 Wait 30 sec to allow the Antibody Magnetic Beads to accumulate on the bottom of each well 6 Remove the supernatant in the wells by quickly inverting the 96 Well Plate over a sink or waste container Repeat actions 4 6 one more time for a total of 2 washes 7 After the last wash blot the assembly onto several layers of paper towels to remove any residual solution NOTE When washing th
32. use with hand held magnet Black Microplate Lid Protecting the assay from light during incubation Plate Seals Adhesive backed foil plate sealer 1 Contains Sodium Azide See WARNING on next page WARNING All chemicals should be considered potentially hazardous 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 Required Equipment and Materials Not Supplied Luminex 100 200 MAGPIX FlexMap 3D 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 Hand Held Magnetic Plate Washer Vacuum Filtration Manifold Precautions and Technical Hints Thorou

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