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User Manual: AimPlex Multiplex Immunoassay

1. Standard 7 1 729 80 160 Blank 0 160 Note If very low levels less than 10 pg mL of cytokines are expected in the samples we recommend adding 1 or even 2 more standard points Standard 8 1 2187 and Standard 9 1 6561 in the assay for better quantitation at low concentrations Standard 1 may be omitted when assaying those samples Note Serial dilution factor may be different for custom assay panels Refer to the Standard Info Sheet enclosed in the kit for details 9 Performing the assay 9 1 Prepare the plate template Mark the standard sample and blank wells Standards and samples should be run in duplicates or triplicates If the whole plate will not be used seal the unused well with a plate seal Important Place the filter plate on top of the filter plate lid during the entire assay process to prevent touching the plate bottom on any surface 9 2 Vortex working bead suspension for 15 second 9 3 Add 45 uL of capture bead working suspension to each well Note Save the remaining capture bead working suspension and store at 2 8 C with light protection It can be used for setting up acquisition parameters on the flow cytometer 9 4 Remove buffer in the wells by using the EZPrep Filter Plate Washer 14 Rev 1 3 11 7 L Bioprocess e AimPlex E Development Co bier bueren tor Fo 9 5 9 6 9 7 9 8 9 9 9 10 9 11 9 12 9 13 9 14 9 15 9 16 9 17 9 18 9 19 9 20 9 21 9 22 9 23 9
2. 417 658 uL of ddH o 7 1 7 Add the appropriate volume determined in Step 7 1 6 of ddH O to a test tube labeled with Working dAb Solution 7 1 8 Add the appropriate volume determined in Step 7 1 5 of 3x NR or Mouse Rat dAb Diluent 7 1 9 Add the appropriate volume determined in Step 7 1 4 of each dAb stock into the Working dAb Solution tube 7 1 10 Mix by gentle vortexing The working dAb solution can be stored at 2 8 C for up to 24 hrs 12 Rev 1 3 11 Y Bioprocess Ai e SL 4 Development Co O AimPlex W Multiplex Immunoas 7 2 Premixed Multiplex Kits Transfer the entire content of 2x NR or Mouse Rat dAb diluent to the 2x Premixed Biotin dAb vial Mix by gentle vortexing The working solution can be stored at 2 8 C for up to 24 hrs 8 Preparing Antigen Standards 8 1 Reconstitution of the lyophilized standards 8 1 1 Ifthereis only one standard vial in the kit 8 1 1 1 Centrifuge the antigen standard vial at 2 000x g for 10 sec 8 1 1 2 Add 250 uL of CCS cell culture supernatant SPB serum plasma bodily fluid or TL Tissue cell lysate standard diluent into the vial Note Reconstituted volume may be different for custom assay panels Refer to the Standard Info Sheet enclosed in the kit for details 8 1 1 3 Vortex gently for 15 sec 8 1 1 4 Incubate on ice for 5 10 min 8 1 1 5 Vortex gently for 15 sec before Serial Dilution Preparation Step 8 2 8 1 2 Ifthere are multiple standard vials in th
3. L a Bioprocess e AimPlex O Development Co Aani immunoassays for Flow User Manual ye AimPlex d Multiplex Immunoassays for Flow Non Magnetic Beads PN UM P20121001 For research use only Not for use in diagnostic procedures Rev 1 3 11 Multiplex Immun L a Bioprocess e AimPlex y e Development Co 2 passays for Flow AimPlex assay kits and any related reagents and documentation are provided as is without warranty of any kind Although we have tried our best to ensure accuracy of this user manual and any related documentation YSL Bioprocess Development Co does not warrant guarantee or make any representations regarding the use or the results of the use of AimPlex assay kits and related documentation in terms of correctness accuracy reliability or omissions The entire risk as to the use results and performance of AimPlex assay kits and related documentation is assumed by the user YSL Bioprocess Development Co assumes no liability of any kind whether oral or written expressed or implied for any direct indirect special incidental or consequential damages resulting from any defects in its documentation This user manual and related documentation are subject to change without notice AimPlex is a trademark licensed to YSL Bioprocess Development Co All other trademarks appearing in this user manual are owned by their respective owners Please contact Customer Service at contact yslbio com with any q
4. 24 9 25 9 26 9 27 9 28 9 29 9 30 9 31 Rev 1 3 11 Gently tap the plate bottom onto several layers of paper towels to remove residual buffer after removal of the buffer Add 30 uL of CCS SPB or TL Assay Buffer to each sample well Note Cell culture supernatant samples can be run without diluting in Assay Buffer if very low levels less than 20 pg mL of cytokines are expected If it is the case skip this step and add 45 uL of cell culture supernatant samples to each sample well in Step 9 7 Add 15 uL of samples to each sample well Add 45 uL of standards to each standard well Cover the plate with a plate seal Incubate on the shaker set at 700 rpm for 60 min at room temperature Protect from light Remove the plate seal Remove solutions in the wells by using the EZPrep Filter Plate Washer Wash the wells three times with 100 uL 1x Wash Buffer using the EZPrep Filter Plate Washer Gently tap the plate bottom onto several layers of paper towels to remove residual buffer on the plate bottom after the last wash Add 25 uL of biotinylated antibody working solution to each well Cover the plate with a plate seal Incubate on the shaker set at 700 rpm for 30 min at room temperature Protect from light Remove the plate seal Remove solutions in the wells by using the EZPrep Filter Plate Washer Wash the wells three times with 100 uL 1x Wash Buffer using the EZPrep Filter Plate Washer Gently tap the plate bottom onto
5. 7 of the 44m beads 25x Analyte Specific biotinylated detection Ab e g 25x Biotin human IL 2 35 uL dAb Lyophilized Antigen Standard 1 vial Each vial may contain one or multiple antigens refer to lot specific Standard Info Sheet for details 3 1 2 Basic Kit There are 2 species specific non rodent NR and mouse rat Basic Kits 3 1 2 1 NR Non Rodent Basic Kits These Basic Kits are for human non human primate and other non rodent species assays Component Vol 32 Tests Vol 96 Tests 3x NR Detection Ab Diluent 0 3 mL 0 85 mL 1x Streptavidin PE SAPE 1 mt 3 mL Rev 1 3 11 e Y Bioprocess est AimPlex e Development Co L Multiplex immunoassays for Flow W 10x Reading Buffer 1 5 mL 5mL 10x Wash Buffer 5mL 15 mL PCR 8 tube Strip 2 each 2 each Filter Plate and Lid 1 each 1 each Plate Seals 3 sheets 6 sheets User Manual 1 each 1 each Packaging insert 1 each 1 each 3 1 2 2 Mouse Rat Basic Kits These Basic Kits is for mouse and rat assays Component Vol 32 Tests Vol 96 Tests 3x Mouse Rat Detection Ab Diluent 0 3 mL 0 85 mL 1x Streptavidin PE SAPE 1mL 3 mL 10x Reading Buffer 1 5 mL 5mL 10x Wash Buffer 5mL 15 mL PCR 8 tube Strip 2 each 2 each 3 2 Premixed Multiplex Kits Each Premixed Muliplex Kit consists of a Premixed Analyte Kit a Basic Kit for premixed panels and a sample type specific Diluent Kit order separately Refer to section 3 3 for availabl
6. Troubleshooting Issue Low event count Low assay signal or sensitivity High Background Low Precision Clogged filter plate Rev 1 3 11 Possible cause Bead aggregate Bead settle on the well bottom Vacuum too strong AimPlex Multiplex Immunoassays for Flow Recommended actions Vortex stock and working bead suspensions well before pipetting Shake plates at 700 rpm for 30 seconds prior to acquisition or resuspend the beads in a well by pipetting up and down 6 8 times with a P200 pipette prior to transferring to a sample tube for acquisition Adjust the vacuum pressure so that 100 uL of 1x Wash Buffer in the wells can be clear in 3 5 second Standard not reconstituted well Incubation time too short Excess exposure to light Standard s should be incubated on ice for 5min after the addition of standard diluent Follow recommended incubation time in each step During incubation cover the plate with aluminum foil to minimize exposure of the beads to light Well to well contamination Change pipette tips after every transfer Remove plate seal carefully and avoid contents from one well to mix with another Poor pipetting precision Contamination from adjacent wells Use calibrated pipettes Avoid well to well contamination during pipetting and removal of plate seal High lipid content in the serum plasma or bodily fluid samples Centrifuge the samples at 10 000
7. one extra well of Standard 1 for instrument setup purpose 16 L a Bioprocess e AimPlex O Development Co L Multiplex immunoassays for Flow 10 3 Setting up a display layout template 10 3 1 Perform start up and verification of fluidic stability and optical alignment by following cytometer manufacturer s recommendations 10 3 2 Open a new protocol 10 3 3 Create the following plots and histograms 1 A dot plot with FS X axis and SS Y axis in linear display mode 2 2 histograms of PE Cy5 in Log display mode 3 2 dot plots with PE X axis and PE Cy5 Y axis in Log display mode 4 If PE Cy7 or APC channel is available create 2 histograms of PE Cy5 in Log display mode and 2 dot plots with PE X axis and PE Cy7 or APC Y axis in Log display mode 10 3 4 Set all compensation to zero 10 3 5 Both Areas and Heights of the FL parameters should be collected 10 3 6 Save the protocol 10 4 Running the setup beads 10 4 1 Run the Blank beads prepared in Step 10 2 1 1 Adjust FS and SS gains so that the bead populations are on scale Figure 1 2 Create Gate 1 for the smaller 4 micron size S4 beads and Gate 2 for the larger 5 micron size S5 beads Figure 1 a Hames Figure 1 D 16384 32768 49152 65536 FS 3 Apply Gate 1 to one of the PE Cy5 histograms and one of the PE PE Cy5 dot plots 4 Apply Gate 2 to the other PE Cy5 histogram and the other PE PE Cy5 dot plot 5 Adjust P
8. x g for 10 min at 2 8 C Collect the serum plasma or bodily fluid fraction 21 Y L Bioprocess gt Development Co v 14 Blank Plate L ayout ZIILOL 6 8L 9SHrEZ
9. E Cy5 PMT voltage so that all bead populations are clearly separated on the histograms and dot plots Figure 2 17 Rev 1 3 11 SL a Bioprocess e AimPlex y e Development Co C W Multiplex Immunoassays for Flow In this example S4 has 4 bead populations S4P3 S4P7 S4P9 and S4P11 S4P3 Size 4 micron Peak 3 is the dimmest and S4P11 Size 4 micron Peak 11 is the brightest 6 Adjust PE PMT voltage so that the dimmest bead population is positioned within the first decade on the PE axis of the plot Figure 2 c w eg 10 teg PE Cy5 Figure 2 7 Save the protocol 10 4 2 Run the Standard 1 beads prepared in Step 10 2 2 1 Verify all the bead populations on the PE axis are on scale Figure 3 2 Adjust PE PMT voltage if needed If adjustment is needed make sure rerun the Standard 8 and make sure the dimmest bead population is still visible on the PE PE Cy5 dot plots PE Cy5 Figure 3 3 Apply proper PE Cy5 PE color compensation so that the bead populations are in a horizontal position see Figure 4 as an example of proper color compensation setting 18 Rev 1 3 11 L a Bioprocess e AimPlex y e Development Co L wie immunoassays for Flow Figure 4 Figure 5 4 Save the protocol Note If PE Cy7 or APC fluorescence channel is available carry out Steps 10 4 1 and 10 4 2 for the PE Cy7 or APC channel for the proper PMT voltage and
10. NR or Mouse Rat dAb Diluent is needed 7 1 1 Determine the number of analytes in the panel e g a 7 plex panel 7 1 2 Determine the number of wells in the experiment We recommend adding an additional 2 wells to account for pipetting recovery For example if a total of 48 wells is needed in the experiment prepare enough for 50 wells 7 1 3 Determine the total volume of working dAb solution needed for the experiment Each tube well needs 25 uL of the working detection antibody solution The total volume is calculated by multiplying the number of wells calculated in Step 7 1 2 by 25 uL For example 50 wells x 25 uL 1 250 uL total working detection antibody solution 7 1 4 Determine the volume needed for each 25x Biotinylated Detection Antibody stock i e 1 0 uL needed for each well Therefore for example a total of 50 wells needs 50 uL of each 25x detection antibody stock 7 1 5 Determine the volume of 3x NR or Mouse Rat dAb Diluent needed to prepare the working dAb solution ___ uL from Step 7 1 3 3 wLof 3x NR or Mouse Rat dAb Diluent For example a total of for 50 wells 1 250 3 417 uL of 3x NR or Mouse Rat dAb Diluent 7 1 6 Calculate the ddH O volume by subtracting the volume for each capture bead stock and 3x 3x NR or Mouse Rat dAb Diluent _____ ul from Step 7 13 uL from Step 7 1 4x number of Plex Step 7 1 1 ul from Step 7 1 5 uL of ddH O For example a 7 plex panel for 50 wells 1 250 50 x 7
11. ad stock 6 1 5 Determine the volume of 1x Reading Buffer needed to prepare the working bead suspension Calculate the 1x Reading Buffer volume by subtracting the volume for each capture bead stock uL from Step 6 1 3 ___ uL from Step 6 1 4x number of Plex Step 6 1 1 uL of 1x Reading Buffer For example a 7 plex panel for 50 wells 2 250 50 x 7 1900 uL of 1x Reading Buffer 6 1 6 Add the appropriate volume determined in Step 6 1 5 of 1x Reading Buffer to a test tube labeled with Working Bead Suspension 6 1 7 Centrifuge each capture bead vial at 2 000x g for 10 sec 6 1 8 Vortex each capture bead vial for 15 second 6 1 9 Add the appropriate volume determined in Step 6 1 4 of each capture bead stock into the Working Bead Suspension tube 6 1 10 Vortex gently to mix If not used immediately store the working bead suspension at 2 8 C with light protection It can be stored at 2 8 C for up to 1 week 6 2 Premixed Multiplex Kits The antibody coupled beads are provided as premixed ready to use 1x suspension No preparation is needed 11 Rev 1 3 11 7 L a Bioprocess AimPlex O Development Co CH wes immunoassays for Flow 7 Preparing Biotinylated Detection dAb Antibody Working Solution 7 1 Single Plex Kits The Biotinylated Detection dAb Antibody stock provided with each kit is a 25x concentrated stock 1 uL per test Dilution of the stock biotinylated dAb stocks to working solution with
12. color compensation usually zero settings 11 Analyzing the samples If the flow cytometer does not equipped with a 96 well plate loader resuspend the beads in a well by pipetting up and down 6 8 times then transfer to a test tube for acquisition Acquire 100 events each bead population of the larger beads Gate 2 For example if there are 3 bead populations in Gate 2 larger beads acquire 3 x 100 300 events per sample We have found that as few as 50 events for a bead population is sufficient Save the FCS files for data analysis FCAP Array 3 0 is recommended for data analysis 19 Rev 1 3 11 Multiplex Immun SL a Bioprocess AimPlex y Development Co 2 passays for Flow 12 Technical hints 12 3 1 Set up the EZPrep Filter Plate Washer according to the instruction in the Product Insert Adjust the vacuum pressure so that 100 uL of 1x Wash Buffer in the wells can be clear in 3 5 second 12 3 2 When working with samples and standards change the pipette tips after every transfer and avoid creating bubbles when pipetting 12 3 3 We recommend whenever possible using a multi channel pipette for reagent additions to achieve optimal assay precision 12 3 4 When applying plate seal to the filter plate do not use a rubber roller Use finger to gently press over the plate seal to seal the plate 12 3 5 Sample handling a Cell Culture Supernatant Remove particles by centrifugation and assay immediat
13. e Diluent kits for 32 or 96 tests 3 2 1 Premixed Analyte Kit Component Vol 32 Tests Vol 96 Tests 1x Premixed Ab conjugated beads 1 5 mL 5 mL 2x Premixed Biotin dAbs 0 45 mL 1 5 mL 2x NR or Mouse Rat dAb Diluent depending on the assay panel 0 45 mL 1 5 mL Lyophilized Antigen Standards with Standard Info Sheet s 1 or multiple vials 1 or multiple vials Antigen standards are supplied as premixed but can be in a single vial or multiple vials depending on the panels Refer to Standard Info Sheet for details Rev 1 3 11 e e Y S e Bioprocess e Ke AimPlex O eve opment oO 6 Multiplex immunoassays for Flow 3 2 2 Basic Kit for Premixed Panels Component Vol 32 Tests Vol 96 Tests 1x Streptavidin PE SAPE 1 mt 3 mL 10x Reading Buffer 1 5 mL 5 mi 10x Wash Buffer 5 mL 15 mL PCR 8 tube Strip 2 each 2 each Filter Plate and Lid 1 each 1 each Plate Seals 3 sheets 6 sheets User Manual 1 each 1 each Packaging insert 1 each 1 each 3 3 Diluent Kits Diluent Kits are sample type specific Please indicate the sample type of your assay panel when placing an order 3 3 1 CCS Cell culture supernatant Diluent kit This kit is for cell culture supernatant samples It is universal for all species Component Vol 96 Tests CCS Standard Diluent 2 5 mL CCS Assay Buffer 8 mL 3 3 2 NR Non Rodent SPB Serum Plasma Bodily Fluid Diluent kit This kit is for serum plasma and bodily flu
14. e kit 8 1 2 1 Centrifuge the antigen standard vials at 2 000x g for 10 sec 8 1 2 2 Add 250 uL of CCS cell culture supernatant SPB serum plasma bodily fluid or TL Tissue cell lysate standard diluent into the first vial Note Reconstituted volume may be different for custom assay panels Refer to the Standard Info Sheet enclosed in the kit for details 8 1 2 3 Vortex gently for 15 sec 8 1 2 4 Incubate on ice for 5 min 8 1 2 5 Vortex gently for 15 sec 8 1 2 6 Transfer the entire content to the next vial 8 1 2 7 Vortex gently for 15 sec 8 1 2 8 Incubate on ice for 5 min 8 1 2 9 Vortex gently for 15 sec 8 1 2 10 If more than 2 standard vials in the kit repeat Steps 8 1 2 6 to 8 1 2 9 for the rest of the vials 13 Rev 1 3 11 Y L Bioprocess se AimPlex O Development Co L Multiplex immunoassays for Flow 8 2 Serial dilution preparation Prepare 3x serial dilutions 160 ul in total enough for duplicated wells according to Table 1 Mix each addition by pipetting up and down 6 8 times Change pipette tips after each addition to avoid contamination from one concentration to the other Keep the standards on ice until use Table 1 Preparation of antigen Standard Curve Standard Amount from Previous Standard Diluent uL Standard pL Standard 1 Undiluted Prepared in Section 8 1 Standard 2 1 3 80 160 Standard 3 1 9 80 160 Standard 4 1 27 80 160 Standard 5 1 81 80 160 Standard 6 1 243 80 160
15. ed at 2 8 C for up to 3 months 5 2 1x Wash Buffer Bring the 10x Wash Buffer to room temperature and vortex for 15 seconds Mix 15 mL or 5 mL of the 10x Wash Buffer with 135 mL or 45 mL ddH 0 respectively The 1x Wash Buffer can be stored at room temperature for up to 3 months 10 Rev 1 3 11 Y SL Bioprocess e AimPlex O Development Co CH wes immunoassays for Fiow 6 Preparing Antibody Coupled Bead Working Suspension 6 1 Single Plex Kits The capture bead stock provided with each kit is a 45x concentrated stock 1 uL per test Dilution of the stock beads to working suspension with 1x Reading Buffer is needed 6 1 1 Determine the number of analytes in the panel e g a 7 plex panel 6 1 2 Determine the number of wells in the experiment We recommend adding at least 2 additional wells to account for pipetting recovery For example if a total of 48 wells is needed in the experiment prepare enough for 50 wells 6 1 3 Determine the total volume of working bead suspension needed for the experiment Each tube well needs 45 uL of the working bead suspension The total volume is calculated by multiplying the number of wells calculated in Step 6 1 2 by 45 uL For example 50 wells x 45 uL 2 250 uL total working bead suspension 6 1 4 Determine the volume needed for each 45x Analyte Specific Ab Conjugated Beads i e 1 0 uL needed for each well Therefore for example a total of 50 wells needs 50 uL of each 45x capture be
16. ely or aliquot and store samples at lt 80 C Avoid repeated freeze thaw cycles Serum If using serum separator tubes allow samples to clot for 30 minutes at room temperature Centrifuge for 10 minutes at 1 000x g Collect the serum fraction and assay promptly or aliquot and store the samples at 80 C Avoid multiple freeze thaw cycles If serum separator tubes are not being used allow samples to clot overnight at 2 8 C Centrifuge for 10 minutes at 1 000x g Remove serum and assay promptly or aliquot and store the samples at 80 C Avoid multiple freeze thaw cycles Plasma Centrifuge samples at 1 000x g at 4 C for 10 min within 30 min of blood collection Collect the plasma fraction and assay promptly or aliquot and store the samples at 80 C Avoid multiple freeze thaw cycles Thaw frozen samples on ice and mix well prior to adding to the assay wells If there is a high lipid content in serum plasma or bodily fluid samples centrifuge at 10 000 x g for 10 min at 2 8 C Collect the serum plasma or bodily fluid fraction for the assays If samples contain high analyte concentrations and need dilution for the assays use Sample Dilution Buffer Part P830100 for sample dilution The exact dilution must be determined empirically 12 3 6 If 30 uL of Assay Buffer and 15 uL of sample are added to a well dilution factor is 3 when calculating the final concentration Rev 1 3 11 20 Y SL Bioprocess e Development Co W 13
17. id SPB samples from human non human primate and other non rodent NR species Component Vol 96 Tests NR SPB Standard Diluent 2 5 mL NR SPB Assay Buffer 8 mL 3 3 3 Mouse Rat SPB Serum Plasma Bodily Fluid Diluent kit This kit is for mouse and rat serum plasma and bodily fluid samples Component Vol 96 Tests Mouse Rat SPB Standard Diluent 2 5 mL Mouse Rat SPB Assay Buffer 8 mL Rev 1 3 11 YSJ Bioprocess o AimPlex e Development Co d itncten keemonoasiaya tor Hie W 3 3 4 TL Tissue cell lysate Diluent kit This kit is for tissue and cell lysate samples It is universal for all species Component Vol 96 Tests TL Standard Diluent 2 5 mL TL Assay Buffer 8 mL 4 Required Equipment Not Supplied e Barnstead Lab Line Titer Plate Shaker Thermo Scientific Waltham MA or equivalent The shaker should have a 3mm orbit with ability to maintain 600 800 rpm e AimPlex EZPrep Filter Plate Washer PN VM1001 e Vacuum source for the filter plate washer An economy vacuum pump e g Barnant Model 400 1901 or equivalent is recommended e Flow cytometer capable of detecting forward scatter side scatter PE and APC or PE Cy5 5 Preparing 1x Reading Buffer and 1x Wash Buffer 5 1 1x Reading Buffer Bring the 10x Reading Buffer to room temperature and vortex for 15 seconds Mix 5 mLor 1 5 mL of the 10x Reading Buffer with 45 mL or 13 5 mL ddH 0 respectively The 1x Reading Buffer can be stor
18. nan an theeeasttsteeen Oi la ankete tees 10 5 2 DKW a SAB EE 10 6 Preparing Antibody Coupled Bead Working Suspenslon 11 6 1 KT WT EE 11 6 2 Premixed Multiplex Kits Yow pete eta ak yabe ai aneyee so due van eee sos aa s oton Eza da Eed depan ses bien dp Ee 11 7 Preparing Biotinylated Detection dAb Antibody Working Solution 12 7 1 Sinple EE 12 7 2 Premixed Multiplex Kits wiii keaktksaat ae ae sara sike es ae ake dak Ge raai da ako spes a d EES ad asse n site t s sis EE 13 8 Preparing Antigen Standarde eee aa aaaaat aaa ae a aaa aaatestese se toaooooeossesesetooooooeoeoeseeseuoooooooosenenon 13 8 1 Reconstitution of the lyophilized standards nnsneseeeensnssnsesenresnssssrsennrssssesrrennessssesrreesnssssenns 13 Rev 1 3 11 7 L a Bioprocess e AimPlex O Development Co CH wes immunoassays for Fiow 8 1 1 If there is only one standard Vial in the Kit ccccccessececssssececsesaeceesesaeeeeseseeeeeseaeeeeseaaes 13 8 1 2 If there are multiple standard vials in the Kit cccccsesssecccecessessaeceeeeseessesseaeeeeeesseeeees 13 8 2 Serial dilution preparation ccccccccccecssssssssececececsesecesecececeseesesaeeeeeessesseseeaeeeseessesseseaeeeseeseeeeees 14 9 PErFOFMING THE ASSAY Zeene REESEN EENS a n ae dan es manke 14 10 Setting Up FlOW CYt M ETENS getesselt ageet is e pate ki ii n ae e eet a pale ea aa 16 10 1 Fluorescence CHAN NE IS vie kanon bes kke dok man kana a EAR ESA EEN 16 10 2 Preparing in
19. on of the bead classification dye is at 700 nm It can be detected on PE Cy5 channels of most of the flow cytometers with blue 488 nm excitation It can also be detected on PE Cy7 channels with blue 488 nm excitation or APC channel with red 635 or 640nm excitation if such a florescence channel is available The reporter dye of the AimPlex assays is PE and can be detected on the PE channel with blue 488 nm excitation 10 2 Preparing instrument setup beads 10 2 1 10 2 2 Rev 1 3 11 Blank beads Aliquot half e g 75 uL if resuspension volume in Step 9 29 is 150 uL of bead suspension from one of Blank wells from Step 9 32 into a sample tube or a well of a 96 well plate depending on the sample loading mechanism of a flow cytomter Add 100 to 300 uL of 1x Reading Buffer to the tube well Remaining capture bead working suspension from Step 9 3 can also be used for this purpose Standard 1 beads Aliquot half e g 75 uL if resuspension volume in Step 9 29 is 150 uL bead suspension from one of the Standard 1 wells from Step 9 32 into a sample tube ora well of a 96 well plate depending on the sample loading mechanism of a flow cytomter Add 100 to 300 uL of 1x Reading Buffer to the tube well Note Add 75 uL 150 uL of 1x Reading Buffer to the Blank and Standard 1 wells Acquisition for both wells will be slower less bead concentrations during the sample acquisition step When running a panel the first time we recommend running
20. ope of the protein followed by a streptavidin R phycoerythrin treatment The fluorescent intensity of R phycoerythrin on the beads is quantified on a flow cytometer Concentrations of a protein of interest in the samples can be obtained by comparing the fluorescent signals to those of a standard curve generated from a serial dilution of a known concentration of the analyte The assay procedure consists of a 60 min antigen and capture antibody conjugated bead incubation step a 30 min biotinylated detection incubation step and a 20 min streptavidin PE incubation step Assay Protocol Overview Add Ab beads to wells Total Assay time 2 hours 1 Remove S Add standards and samples to wells 1 1hour RT make N Add biotinylated 2 Wash 3X antibodies to wells 1 30 min RT dile Se 2 Wash 3X 1 20 min RT shaking 2 Wash 2x 3 Add Reading Buffer Add streptavidin PE Read ona flow cytometer Data analyzed by Soft Flow s FCAP Array v3 RT room temperature Rev 1 3 11 L a Bioprocess e AimPlex O Development Co CH wes immunoassays for Fiow 2 Available AimPlex Immunoassay Kit format 2 1 Single Plex Kits All available AimPlex immunoassays are grouped as 12 24 analytes per group Each analyte in a Group has a unique bead region and can be multiplexed together in any combination Analytes in different groups may be multiplexed together but may have some cross reactivity because cros
21. opment for analytes not currently available We will work with you to design the panels and assays Please contact us for pricing lead time and details of those offerings We also offer custom bulk packaging usually in 5 plate size Please contact us for details Rev 1 3 11 Bioprocess Ai Y SL g Ben yen AimP lex 3 Supplied reagents and materials 3 1 Single Plex Kits Each Single Plex Kit consists of 1 or more user selectable Analyte Kits a Basic Kit and a sample matrix specific Diluent Kit refer to section 3 3 for available Diluent kits for 96 tests Some of the single plex kits are available as 32 tests 3 1 1 Analyte Kit Each 96 test Analyte Kit consists of the following components If multiple analyte kits are ordered all the components are shipped in a single Analyte Kit box Component 45x Analyte Specific Ab conjugated beads e g 45x S4P7 human IL 2 S4 Size 4um beads P7 Peak 7 of the 44m beads 25x Analyte Specific biotinylated detection Ab e g 25x Biotin human IL 2 0 1 mL dAb Lyophilized Antigen Standard 1 vial Each vial may contain one or multiple antigens refer to lot specific Standard Info Sheet for details Each 32 test Analyte Kit if available consists of the following components If multiple analyte kits are ordered all the components are shipped in a single Analyte Kit box Component 45x Analyte Specific Ab conjugated beads e g 45x S4P7 human IL 2 S4 Size 4um beads P7 Peak
22. s reactivity of all the assays across different groups was not validated We offer custom multiplex assay panel design and development Please contact us for details including pricing and lead time Note Bead set assignment may vary in a panel Refer to packaging product inserts and kit labels for details Each Single Plex Kit consists of 1 or more user selectable Analyte Kits a Basic Kit and a sample matrix specific Diluent Kit Each Analyte Kit contains analyte specific Ab conjugated beads detection antibody ies and corresponding antigen standard s The Basic Kit has species specific detection antibody diluent reading buffer wash buffer Streptavidin PE SAPE filter plate and plate seals The Diluent Kit contains sample type specific Standard Diluent and sample Assay Buffer Some of the single plex kits are available in both 96 and 32 test sizes 2 2 Premixed Multiplex Kits Each Premixed Multiplex Kit has a pre defined multiplex panel with premixed antibody conjugated beads antigens and detection antibodies Except the Reading Buffer 10x and Wash Buffer 10x all the reagents are supplied as ready to use Most of the premixed multiplex kits are available in both 96 and 32 test sizes Note Bead set assignment may vary in a panel Refer to packaging product inserts and kit labels for details 2 3 Custom panel and assay development and custom bulk packaging We offer custom premixed multiplex panels as well as custom assay devel
23. several layers of paper towels to remove residual buffer on the plate bottom after the last wash Add 25 uL of streptavidin PE working solution to each well Cover the plate with a plate seal Incubate on the shaker set at 700 rpm for 20 min at room temperature Protect from light Remove the plate seal Remove solutions in the wells by using the EZPrep Filter Plate Washer Wash the wells twice with 100 uL 1x Wash Buffer Gently tap the plate bottom onto several layers of paper towels to remove residual solution Add 150 uL to 300 uL of 1x Reading Buffer to each well depending on the sample loading mechanism of a flow cytomter to resuspend the beads Cover the plate with a plate seal Place the plate on the microtiter shaker and shake for 30 seconds at 700 rpm Note If the flow cytometer has no 96 well plate loader and more than 200 uL of 1x Reading Buffer is needed to resuspend the beads do not shake the plate Resuspend the beads in each well by pipetting up and down 6 8 times with a P200 pipette then transfer to a test tube for acquisition 15 7 L Bioprocess AimPlex E Development Co chter kiarigis or Fie 9 32 9 33 Remove the plate seal Read on a flow cytometer Note If the assayed plate is not read immediately it can be stored at 2 8 C for up to 16 hr The plate should be sealed with a plate seal and protected from light 10 Setting Up Flow cytometers 10 1 Fluorescence channels The maximum emissi
24. strument setup DEAS te aaaaaooseesesesooooooooseesesseoeoooooosoesenaaoonoon 16 10 3 Setting up a display layout template cccccccssscesssecsseceessececsseceseecsseecesaecesseeesseeceaeeeeaaeeesees 17 10 4 Running the setup Dead sivsesdscccicssscesetecaceg vanse ge onina dok elan pan Ea iaaea E EE 17 11 Analyzing the samples ue 19 12 UE nl Ru ne 20 13 leif aile el NO kone e tike ket leaves ki e kn va n ou Nen n to ki e te n kaa a sane e an En n ki a ee ea aaa ete e a 21 14 Blank Plate Lay OU wk s v ai says kai aaa dw ea ia ti di n aa Fo kan plim sann a ame od a D pa inn A den Yn 22 4 Rev 1 3 11 Y Bioprocess Al SL e Development Co 2 AimPlex kK4 1 How It Works AimPlex multiplex assay technology utilizes multiple bead populations differentiated by size and different levels of fluorescence intensity With multiple sizes of beads and multiple levels of fluorescence intensity in each bead size the AimPlex technology can measure up to 24 analytes simultaneously in a single reaction The bead populations in the reaction are determined by a flow cytometer equipped with a 488nm laser The maximum emission of the bead classification dye is at 700 nm Similar to the principle of a sandwich ELISA each bead population conjugated with a specific capture antibody traps the protein of interest such as a cytokine in the sample The amount of the analyte captured is detected via a biotinylated antibody against a secondary epit
25. uestions or comments Copyright 2012 YSL Bioprocess Development Co All rights reserved Rev 1 3 11 7 L a Bioprocess AimPlex E Development Co attirer tor F k Contents 1 HOW e 5 2 Available AimPlex Immunoassay Kit format rete aetaatattsseaeaoaoooooossesesetooooooooeseseesenosooooonn 6 2 1 KINN WC 6 2 2 Premixed Multiplex Kifer ebe soon end ks eege ee ere ege vi aaa e 6 2 3 Custom panel and assay development and custom bulk packaging sssesssessnssesesssenrnssesssereene 6 3 Supplied reagents and materials 7 3 1 Sinell TE WEE H 3 1 1 Analyte EE 7 3 1 2 Baie KI E E disi E ke ke ai oke PO ke DA kan ees eee ees 7 3 2 Premixed Multiplex Kits ccccccccccsssssssecececeesesecseseeeeecessesesaeeeeeeeceeseseeaeseeecsceeesaeaeeeeeeesseseaeaeeeesens 8 3 2 1 Premixed Analyt it ccs ieren eng Ee Een Ee no ai de a ees 8 3 2 2 Basic Kit for Premixed Panels sis cc s cuiscecsscceesedeace akse ake vote caus REENEN skin da EES NEO 9 3 3 DIEM dE 9 3 3 1 CCS Cell culture supernatant Diluent Kit 9 3 3 2 NR Non Rodent SPB Serum Plasma Bodily Fluid Diluent kt 9 3 3 3 Mouse Rat SPB Serum Plasma Bodily Fluid Diluent kp 9 3 3 4 TL Tissue cell lysate Diluent kt 10 4 Required Equipment Not Supplied eegene deed ee ceeesdssanteeieesceesesasaveel seneedveesandiei e geen 10 5 Preparing 1x Reading Buffer and 1x Wash Butter 10 5 1 Tx Reading BUR tccsscets castasecesisn cs e swa odyo sisan an fri fose taka save pa age ed a

User Manual: AimPlex Multiplex Immunoassay

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