ProcartaPlex®
Contents
1. H of Vials of Total Volume of Mixed Volume of Detection Antibody Detection Antibody Diluent to Add 1 60 uL 2940 uL 2 120 uL 2880 uL 3 180 uL 2820 uL 4 240 uL 2760 uL Example for using 48 wells of Vials of Total Volume of Mixed Volume of Detection Antibody Detection Antibody Diluent to Add 1 30 uL 1470 uL 2 60 uL 1440 uL 3 90 uL 1410 uL 4 120 uL 1380 uL Step 8 Add Detection A Antibody Mixture and B incubate Step 9 Wash the 96 Well Plate Step 10 Add SAPE and Wash plate twice using Step 4 Wash Antibody Magnetic Beads Add 25 uL of Detection Antibody Mixture 1X to each well Seal the plate with a new Plate Seal cover plate with Black Microplate Lid and incubate 30 min on a plate shaker at RT at 500 rpm Add 50 uL of SAPE solution to each well incubate B Sealthe plate with a new Plate Seal cover plate with Black Microplate Lid and incubate 30 min on a plate shaker at RT at 500 rpm Step 11 Wash the Wash plate twice using Step 4 Wash Antibody Magnetic Beads 96 Well Plate Step 12 Prepare the Add 120 uL of Reading Buffer into each well 96 Well Plate for B Seal the plate with a new Plate Seal cover plate with Black Microplate Analysis on a Luminex Lid and incubate 5 min on a plate shaker at RT at 500 rpm Instrument c Remove Plate Seal and run the plate on a Luminex Instrument Setup of the Luminex Instruments Instrument Sample Size DD Gate Timeout B2. O affymetrix eBioscience Multiplex Immunoassay Using Magnetic Beads For Serum Plasma and Cell Culture Supernatant Samples Instructions for Human Assays 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 43 1796 40 40 305 Clinical Products europe eBioscience com 877 126 8559 Fax 43 1 796 40 40 400 858 642 2058 tech eBioscience com aj Bender MedSystems GmbH Customer Service Campus Vienna Biocenter 2 898 9099 1371 1030 Vienna Austria 958 642 2058 www eBioscience com nfo e Bi oscience com Customers i counties waere alee oes are not eval my contact their eBioscience distributor listed at www eBioscinece Fax 858 642 2046 com distributors 19 10 2015 Rev 21 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 eBioscience is conveyed or implied by this e
3. Samples and antigen standards were not stored on ice 14 Recommend Solution Remove the 96 Well Plate and perform a wash and rinse cycle Prepare the samples and standards on ice before setting up the assay Use a new Plate Seal for each incubation step After adding the standards and samples it is very important that any excess standards are removed during the wash step Avoid splashing the Wash Buffer during wash steps into adjacent wells Use a multichannel pipettor and careful pipette techniques Avoid touching pipette tips to sides of the wells when adding Wash Buffer Calibrate the instrument using the CAL2 Low RP1 target value Add 120 uL Reading Buffer into each well and shake at 500 rom for 5 min at room temperature to resuspend beads prior to reading on the Luminex Instrument Vortex the bead suspension well before using in the assay and ensure that the beads are properly mixed during the incubation steps Store bead solution and the 96 well plate in the dark Remove the 96 Well Flat Bottom Plate and perform a wash and rinse to the instrument Rerun the assay with further dilution of samples Refer to the Luminex Manual for proper adjustment of the needle height Replace or clean needle according to the manufacturer s recommendations Contirm that the plate shaker is set to 500 rom and shaking for at least 5 min before reading Refer to the Luminex manual for proper removal of the air bubble
4. Magnetic Beads Securely insert the 96 Well Flat Bottom Plate into the Hand Held Magnetic Plate Washer and ensure that the plate is held in place by the tabs and 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 Flat Bottom Plate assembly over a sink or waste container Do not remove the 96 Well Flat Bottom Plate from the Hand Held Magnetic Plate Washer Blot the inverted assembly onto several layers of paper towels or absorbent surface to remove any residual solution Add 150 uL of Wash Buffer 1X into each well and wait 30 seconds to allow the beads to accumulate on the bottom of each well Remove the Wash Buffer in the wells by quickly inverting the plate and then blotting onto an absorbent towel to remove any residual solution Remove the 96 Well Flat Bottom Plate from the Hand Held Magnetic Plate Washer and proceed to the next step Step 5 Add sample type specific buffer samples standards and blanks and incubate Universal Assay Buffer for most of the kits is provided at 1X concentration If the kit is supplied with 10X Universal Assay Buffer prepare a 1X working concentration of Universal Assay Buffer by mixing 10 mL of the 10X Universal Assay Buffer with 90 mL ddH O Add 25 uL of Universal Assay Butter 1X to each well for serum and plasma followed by 25 uL of standards or sample
5. Sample Preparation 0 0 0 0 eee 5 Assay Protocol Overview 0 0 0 0 ce eee 7 Preparation of Reagents 0 2 00 00 ee 8 Assay Protocol ccsnongansmikasas CARAS PEGA niBsa add na 10 Setup of the Luminex Instruments 0 00 00 ee eee 13 Analyzing Results ona eaee eave de GRAVAR FLS ANDA LAR ASAE 13 WHOUDICSNOOUNG 264 c54 wues gees ten4245 0080 Ge ub Erri gaii 14 Recommended and Blank Plate Layout 0 15 Intended Use This user manual is for a ProcartaPlex Immunoassay Kit from the eBioscience division of Affymetrix to perform quantitative multiplexed protein measurements from serum plasma and cell culture supernatant samples using magnetic beads technology from Luminex Other biological samples might be suitable for use in the assay NOTE For the most current version of user documentation go to our website at www ebioscience com How it Works ProcartaPlex Immunoassays incorporate magnetic microsphere technology licensed from the Luminex Corporation to enable the simultaneous detection and quantitation of multiple protein targets in diverse matrices The platform allows the simultaneous detection from a single sample of up to 100 protein targets on the Luminex 200 100 and FLEXMAP 3D platforms and 50 protein targets on the MAGPIX platform Materials Provided and Storage Conditions ProcartaPlex Immunoassay Kits contain the components listed below Refer to the Certificate of Analysis for quant
6. label as Standard 1 Std1 D Add 150 uL of sample type specific standard buffer into Std tubes 2 7 Use Universal Assay Buffer for serum or plasma samples and cell culture media for culture supernatant samples E 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 G Change the pipette tip and transfer 50 uL of the mixed standards from Tube 2 into Tube 3 H Mix by pipette up and down 10 times Repeat steps E H for Std tubes 4 7 J Add 200 uL of Universal Assay Buffer or cell culture media into tube 8 which serves as a blank Keep on ice until ready to use Transfer 200uL 50uL 50uL 50uL 50uL 50uL 50uL Antigen wy 7 em P S es 5 Standard Vial RE as Std 1 Std 2 Std 3 Std 4 Std 5 Std 6 Std 7 Use Assay Buffer or Cell Culture Media for Blanks Assay Protocol Step 1 Prepare 1X Wash Buffer Step 2 Define the plate map Bring the Wash Buffer Concentrate 10X to room temperature and vortex for 15 seconds Mix 20 mL of the Wash Buffer Concentrate 10X with 180 mL ddH O Wash Buffer 1X can be stored at 2 8 C for up to 6 months Mark the standard sample and blank wells using the plate map at the end of this manual Step 3a Add the Antibody Magnetic Beads only 1 bead set A Vortex the Antibody Magnetic Beads for 30 sec B Add 50 uL of the Antibody Magnetic Beads to each well Use a multi chann
7. Bioscience 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 Luminex 100 200 and FLEXMAP 3D are registered trademarks of the Luminex Corporation All other trademarks are the property of their respective owners Contents Intended Use cccccc es 4 How it VVOFKS weak paes EREa a RAd DE DOIS AS DD ene 4 Materials Provided and Storage Conditions 4 Precautions and Technical Hints 0 0 0000 eee 5 Required Equipment and Materials Not Supplied 5
8. Prepare fresh antigen standards following the instructions in the Preparing Antigen Standards section Use the same cell culture media that is used to culture the cells Prepare the samples and standards on ice before setting up the assay Recommended and Blank Plate Layout Standards aja 4 a o 2 0 2 2 2 is f ema o u u 2 2 2 5 jax foe e e ufefapapapa o a 15
9. al and Certificate of Analysis that is included with the assay kit The product insert may contain specific instructions for proper use of your kit For Luminex 100 200 and FLEXMAP 3D instruments initiate the startup protocol to warm up the lasers for at least 30 minutes Ensure that the Luminex machine is calibrated according to the manufacturer s instructions MAGPIX instrument doesn t require additional warm up When working with samples and standards change the pipette tips after every transfer and avoid creating bubbles when pipetting During the incubation steps cover the 96 Well Flat Bottom Plate with the Black Microplate Lid provided in the kit to minimize exposure of the beads to light Be careful not to invert the 96 Well Flat Bottom Plate during the assay or allow contents from one well to mix with another well Use a multi channel pipette and reagent reservoirs whenever possible to achieve optimal assay precision Store the reconstituted standards including standard diluent sets on ice before adding to the 96 Well Flat Bottom Plate Required Equipment and Materials Not Supplied MAGPIX Luminex 100 200 FLEXMAP 3D or Luminex based Instrument Glass distilled or deionized water Adjustable single and multi channel pipettes with disposable tips s Multichannel pipette reservoir Beakers flasks cylinders necessary for preparation of reagents Hand Held Magnetic Plate Washer Vortex mixer and Microtiter p
10. ead Event Bead Region Luminex 100 200 50 uL 5 000 25 000 60 sec 50 100 FLEXMAP 3D MAGPIX 50 uL N A N A 50 100 Prior to running the assay ensure that the probe height has been calibrated with 96 Well Flat Bottom Plate supplied with the kit Failure to adjust the probe height can cause damage to the instrument or low bead count 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 Flat Bottom Plate can be re read Remove the 96 Well Flat Bottom Plate from the instrument insert the 96 Well Flat Bottom Plate into the Hand Held Magnetic Plate Washer wait 2 min then remove the buffer in the wells by quickly inverting the 96 Well Flat Bottom 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 Flat Bottom Plate with a new Plate Seal and Lid 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 well Analyzin
11. el pipette for this step as well as the steps below Step 3b Add the Antibody Magnetic Beads 2 or more beads sets A Vortex each of the Antibody Magnetic Bead sets for 30 sec and add 5 IMPORTANT ProcartaPlex pre mixed panel and simplex kits can be mixed together for enhanced flexibility Ensure that the bead regions from your ProcartaPlex panels and simplex kits do not overlap Some analytes use the same bead region and cannot be combined together in one multiplex assay Please check the compatibility of our analytes using our online panel configurator or contact our technical service at tech ebioscience com Both simplex and pre mixed kits are supplied with Antibody Magnetic Beads at working concentration that require 50 uL beads per well or 5 mLs per 96 wells For running a partial plate adjust the volume accordingly mL of each bead set for 96 wells to an appropriate sized tube After all of the bead sets are added vortex the tube for another 30 sec B Add the bead mix to a disposable reservoir and add the appropriate volume of Antibody Magnetic Beads to each well of the 96 Well Flat Bottom Plate using the table below of different Magnetic Bead Amount added to each sets to be mixed well in uL 2 100 uL 150 uL 200 uL 250 uL 300 uL If more than 6 bead sets are to be mixed proceed to Step 4 and repeat Step 3b until all Antibody Magnetic Beads have been added and washed Step 4 Wash Antibody
12. g 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 Instrument e g MAGPIX Luminex 100 200 FLEXMAP 3D We offer a free and robust analysis software package for data analysis ProcartaPlex Analyst 1 0 can be download at www ebioscience com resources procartaplex analyst 1 0 software htm Troubleshooting Observation Low Flow Rate High CVs Limited dynamic range for BioPlex software users Low bead count Low signal or sensitivity Poor recovery Probable Cause Samples beads are stuck in flow cell Samples and antigen standards not stored on ice Contamination from re using the Plate Seal Incomplete washing Contamination from contents from adjacent wells Poor pipetting techniques Instrument calibrated at high PMT settings Volume of bead solution is too low High bead aggregation Dyes contained in the beads are photo bleached trom overexposure to light Samples causing the instrument to clog Probe height is incorrect Instrument needle is partially clogged Beads stuck to the bottom of the plate Air bubble in the sample loop Standards not reconstituted and diluted correctly Did not use appropriate cell culture media to prepare the standards
13. ities and details of components supplied Expiration date is stated on the kit when stored between 2 8 C Do not use past kit expiration date Components Supplied Pre mixed Simplex Basic Kit Custom Panels Kit Panels Antigen Standards premixed y y V Detection Antibody premixed 50X J y Detection Antibody premixed 1X V Antibody Magnetic Beads premixed y y y Streptavidin PE SA PE 1X y V V Wash Buffer Concentrate 10X y y V Detection Antibody Diluent y V Universal Assay Buffer 1X y y V Universal Assay Buffer Concentrate 10X y V v optional Reading Buffer y V V PCR 8 Tube Strip V y V 96 Well Flat Bottom Plate y V V Black Microplate Lid y V V Plate Seals y V V 1 Contains sodium azide See WARNING 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 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 Precautions and Technical Hints Thoroughly read this user manu
14. late shaker Sample Preparation For frozen samples thaw samples on ice and mix well by vortexing followed by centrifugation to remove particulates Avoid multiple freeze thaw cycles It there is a high lipid content in the sample centrifuge at 10 000 x g for 10 min at 2 8 C and transfer contents to a new tube For preparation instruction of tissue homogenates and lysates from cultured cells refer to www ebioscience com resources best protocols multiplexing htm Plasma Sample Preparation A 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 B Centrifuge samples at 1 000 x g at 4 C for 10 min within 30 min of blood collection Collect the plasma Traction D Use immediately or aliquot and store at 80 C Serum Sample Preparation We recommend to spin down serum samples at 1000 x g for 10 min at 20 25 C before running the assay A Allow blood to clot for 20 30 min at 20 25 C B Centrifuge at 1 000 x g for 10 min at 20 25 C cC Collect the serum fraction Alternatively use any standard serum separator tube following the manufacturer s instructions D Use immediately or aliquot and store at 80 C Diluting Samples with High Concentration of Target Analytes You may need to further dilute your sa
15. mples if the analyte concentration Is above the assay upper limit of quantitation ULOQ When preparing dilution of serum and plasma samples we recommend using Universal Assay Buffer For cell culture supernatant samples we recommend using the medium that was used to culture the cells You can find recommended dilution factors for analytes with high normal serum or plasma concentration in the table below NOTE For analytes with high serum and plasma concentration additional Universal Assay Buffer 10X will be included in the kit Species Analytes Recommended Sample Dilution Factor Human Adiponectin 500 Human CRP 500 Human CD44var var6 20 Human Fibrinogen 200000 Human Endoglin 20 Human ICAM 1 200 Human MMP 2 50 Human MMP 3 50 Human MMP 9 50 Human Osteopontin 20 Human RANTES 50 Human SAA 200 Human SAP 4000 Human SCGF beta zo Human L Selectin 200 Human TIMP 1 20 Human VCAM 1 200 Dilution required only for plasma samples For the most current version of user documentation go to our website at www ebioscience com resources best protocols multiplexing htm Assay Protocol Overview 4 Fas the plate Seal Plate Incubate with shaking for 60 120 min at room temperature Wash beads 2X Seal Plate Incubate with shaking for 30 min at room temperature Wash beads 2X Seal Plate Incubate with shaking for 30 min at room temperature Wash beads 2X Seal plate a
16. nd shake for 5 minutes at room temperature For assays that require higher sensitivity 120 min or overnight incubation is recommended Preparation of Reagents Antigen Standard Carefully review the Certificate of Analysis for kit specific Antigen Standard preparation instructions The majority of kits is supplied with lyophilized multi standards containing a mix of multiple standard proteins Some kits contain multiple sets of standards each with a unigue lot number that require pooling prior to use Each kit is shipped with two identical vials of each premixed antigen standard set from the same lot to permit the user to run the assay twice if running a partial plate When preparing Antigen Standards the final volume after reconstitution and pooling should be 250 uL When combining multiple kits ensure that the Antigen Standards of your analytes of interest are only present in one of the used standard vials Alternative preparation instructions for combination of pre mixed panels with simplex kits or different simplex kits with more then 5 antigen standard sets are available upon request or can be downloaded under www ebioscience com resources best protocols multiplexing htm In Custom Panels with more then 5 antigen standard sets alternative preparation instruction will be included as an extra data sheet Step Action Step 1 A Centrifuge each different antigen standard set vial s at 2000 x g for 10 Reconstitution SEC and pooli
17. ng of B Add 50 uL of sample type specific buffer into each standard set vial For Standards serum or plasma samples use Universal Assay Buffer and for cell culture supernatant samples use the cell culture media that was used to culture the cells C Gently vortex the vial s for 30 seconds and centrifuge at 2000 x g for 10 seconds to collect contents at the bottom of the vial s D Incubate on ice for 10 min to ensure complete reconstitution E Pool entire contents of each vial into one of the vials and add sample type specific buffer to quantity sufficient q s to 250 uL See table below for example F Gently vortex the vial for 30 seconds and centrifuge at 2000 x g for 10 seconds to collect contents at the bottom of the vial of Standard Reconstitution Pooled Buffer to q s Total Volume Sets Volume per vial Volume 1 50 uL 50 uL 200 uL 250 uL 2 50 uL 100 uL 150 uL 250 uL 3 50 uL 150 uL 100 uL 250 uL 4 50 uL 200 uL 50 uL 250 uL 5 50 uL 250 uL O uL 250 uL Step Action Step 2 Prepare A Refer to Certificate of Analysis for the value of each premixed standard 4 Fold Serial with assigning S1 values for each analyte for the current lot Dilution B Prepare a 4 fold serial dilution of the reconstituted standard s using the PCR 8 tube strip provided Label tubes Std1 Std2 Std3 Std4 Std5 Std6 and Std7 c Add 200 uL of the reconstituted antigen standards into the first tube of the strip tube and
18. s into dedicated wells For cell culture supernatant samples add 50 uL standards or samples into dedicated wells For wells designated as blanks add an additional 25 uL of Universal Assay Buffer for serum or plasma samples For cell culture supernatant samples add 50 uL of cell culture medium Seal the plate with the provided Plate Seal Cover the plate with the Black Microplate Lid and shake at 500 rpm for 60 to 120 min at room temperature RT Alternatively the 96 well plate can be incubated overnight Shake the 96 well plate for 30 min at RT at 500 rom then transfer the plate to 4 C and store on a level surface After overnight incubation shake the plate for an additional 30 min at RT at 500 rom Step 6 Wash the 96 Well Plate Wash the plate twice using Step 4 Wash Antibody Magnetic Beads Step 7 Prepare 1X Detection Antibody Mixture A Prepare fresh prior to use detection antibodies If intending overnight incubation do not prepare detection antibodies at this point Detection antibodies for Custom Panels are provided at a 1X concentration and do not require dilution For simplex and pre mixed panels detection antibody is provided at 50X concentration Add 60 uL of each detection antibody concentrate to the mixing bottle and bring volume of the mixing bottle to a total of 3 mL using detection antibody diluent Tables below are an example for 48 and 96 wells Example for using 96 wells
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