Bio-Plex Manager™ software recommended TM Bio-Plex Pro Assays Acute Phase Instruction Manual For technical support, contact your local Bio-Rad office or in the US, call 1-800-4BIORAD (1-800-424-6723). For research use only. Not for diagnostic procedures.
Table of Contents Section 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Section 2 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Section 3 Bio-Plex® Acute Phase Reagent Kit Product Description .4 Section 4 Required Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Section 5 Recommended or Optional Materials . . . . . . . . . . . . . 6 Section 6 Sample Preparation . . . . . . . . . . . . . . . . . . . . . . .
Section 1 Introduction Acute phase proteins are a class of proteins that have altered levels in response to inflammation and are thus relevant biomarkers in many disease processes. Typically, initial release of cytokines at the site of damage, most notably IL-1, IL-6, IL-8 and TNF-움, triggers the liver to release a number of acute phase proteins into the bloodstream.
Section 2 Principle Technology The Bio-Plex® suspension array system is built around three core technologies. The first is a novel technology that uses up to 100 unique fluorescently dyed beads (xMAP technology) that permit the simultaneous detection of up to 100 different types of molecules in a single well of a 96-well microplate. The second is a flow cytometer with two lasers and associated optics to measure the different molecules bound to the surface of the beads.
Assay Workflow Prewet wells Add beads Wash Add standards, controls, and samples, 60 min Wash Add detection antibody, 30 min Wash Add streptavidin-PE, 10 min Wash Resuspend, acquire data 3
Section 3 Bio-Plex® Acute Phase Reagent Kit Product Description Bio-Plex Pro™ acute phase assays require the use of the Bio-Plex acute phase reagent kit. Catalog # 171-304050 Catalog # 171-304051 1 x 96-Well Format 10 x 96-Well Format Bio-Plex assay buffer Store at 4ºC. Do not freeze. 1 x 75 ml 1 x 750 ml Bio-Plex wash buffer Store at 4ºC. Do not freeze. 1 x 150 ml 2 x 750 ml Bio-Plex detection antibody diluent Store at 4ºC. Do not freeze.
Section 4 Required Materials The Bio-Plex Pro™ acute phase assay panel is divided into two separate multiplex assay kits based on the sample dilution required for optimal performance. • Each assay requires one Bio Plex® acute phase reagent kit. • The Bio-Plex acute phase diluent kit (see next section) is recommended for serum and plasma samples.
Section 5 Recommended or Optional Materials Item Catalog # ® Bio-Plex Acute Phase Diluent Kit Serum-based diluent Serum-free diluent Bio-Plex Pro™ Human Acute Phase Standards Optional additional standards sold separately Item (300 ml) 171-D40002 (2 vials, lyophilized) 171-D00006 (50 vials, lyophilized) Ordering Information Bio-Plex 200 System with HTF (or Luminex System) Bio-Rad catalog #171-000205 Bio-Plex Validation Kit 4.
Item (continued) Reagent Reservoir Corning, Inc. Costar 50 ml reagent reservoir 4870 1.2 ml Tubes in 96 Rack For performing sample dilutions in 96-well format Ordering Information Bio-Rad catalog #224-4872 VWR catalog #82006-698 (tubes in rack) VWR catalog #83009-678 (refill tubes) Other Materials Pipets and pipet tips, sterile distilled water, aluminum foil, absorbent paper towels, 1.
Section 6 Sample Preparation This section provides instructions for preparing samples derived from serum, plasma, and culture supernatant. For sample preparations not mentioned here, consult the publications listed in Bio-Rad bulletin 5297, available for download at www.discover.bio-rad.com Serum and Plasma Samples Collect and process the serum or plasma samples and assay immediately or freeze at –20ºC. Avoid repeated freeze-thaw cycles. EDTA is the recommended anticoagulant for preparing plasma samples.
5-plex and 4-plex assays For the 5-plex assay, dilute 5 µl of sample in 495 µl of serum-free diluent. Mix by vortexing gently or pipetting. This results in a final 1:100 sample dilution. For the 4-plex assay, dilute 5 µl of the 1:100 mixture into an additional 495 µl of serum-free diluent. Mix gently. This results in a final sample dilution of 1:10,000.
Section 7 Standard Preparation Two vials of Iyophilized acute phase standards are provided in each multiplex Bio-Plex Pro™ acute phase assay kit and one vial is provided in each singleplex assay kit. Only one vial is required per 96-well plate. The product insert provided with the assay lists the concentration of the most concentrated dilution point of the standard curve. This procedure will prepare enough standard to run each dilution in duplicate. Reconstitute Standards 1.
4. Gently vortex 1-3 sec and incubate on ice for 60 min. For optimal assay performance, be consistent with the incubation time. 5. Save the standard/control diluents for preparation of the controls (Section 8). Prepare Standard Dilution Series The standard concentrations specified for the 8-point standard dilution set have been selected for optimized curve fitting using the 5-parameter logistic (5PL) or 4-parameter logistic (4PL) regression in Bio-Plex Manager™ software.
4-plex standard dilution series: Add 50 µl of reconstituted 4-plex standard to the first 1.5 ml tube containing 350 µl 4-plex standard diluent. Vortex gently. This mixture is identified as S1 in the diagram below and in the standard and control values product insert provided with the assay. Continue making serial dilutions of the standards as shown in the diagram. Before making each dilution, vortex gently and change the pipet tip.
Section 8 Control Preparation Two vials of lyophilized acute phase controls are provided in each multiplex Bio-Plex Pro™ acute phase assay kit. This section provides instructions on how to reconstitute the Iyophilized control. The product insert provided with the assay lists the concentration ranges of the prepared controls. Prepare Acute Phase Controls To ensure optimal assay performance, the acute phase controls should be prepared in a manner consistent with that used to prepare the acute phase standards.
Section 9 Assay Instructions The following instructions apply to both the 5-plex and 4-plex assays. All of the necessary components are provided premixed for ease of use. Plan Experiment 1. Assign which wells of a 96-well plate will be used for each standard, control, and sample (see the example plate layout below). 2. Determine the total number of wells that will be used in the assay.
Prepare Coupled Magnetic Beads Protect the beads from light by covering the tubes with aluminum foil. Keep all tubes on ice until ready to use. 1. Vortex the coupled beads (25x) at medium speed for 30 sec. 2. Prepare a sufficient volume of coupled beads (1x) using assay buffer. Each well requires 2 µl of coupled beads (25x) adjusted to a final volume of 50 µl with assay buffer (refer to the example below).
Assay Procedure Bring all buffers to room temperature. Avoid bubbles when pipetting. The following terms are repeated throughout the assay procedure. Refer to these detailed instructions when wash, incubate, and vacuum-filter are shown in bold. Detailed Directions Add 100µL of wash buffer to each well. Place the filter plate on a calibrated vacuum apparatus and remove the buffer by vacuum filtration. Blot the bottom of the filter plate with a clean paper towel. Repeat as specified.
6. While the samples are incubating, perform a 30 sec quick-spin centrifugation of the detection antibody (10x for multiplex assays, 100x for singleplex assays) prior to pipetting to collect the entire required volume at the bottom of the vial. 7. Prepare a sufficient volume of detection antibodies (1x) using detection antibody diluent. Refer to the example tables below. NOTE: The detection antibody stock concentration is 10x in the multiplex assays and 100x in the singleplex assays.
11. While the detection antibodies are incubating, perform a 30 sec quick-spin centrifugation of the streptavidin-PE (100x) prior to pipetting to collect the entire volume at the bottom of the vial. 12. Prepare a sufficient volume of streptavidin-PE (1x) using assay buffer. Each well requires 0.5 µl of streptavidin-PE (100x) adjusted to a final volume of 50 µl with assay buffer (refer to the example below).
Section 10 Data Acquisition Bio-Plex Manager™ software is recommended for Bio-Plex Pro™ acute phase assays. Refer to the details in the appropriate section below for your xMAP system software package. Instructions for Luminex xPONENT software are also provided. For instructions using other xMAP system software packages, contact Bio-Rad Technical Support or your Bio-Rad Field Applications Scientist. NOTE: Bio-Plex Pro acute phase assays are analyzed using the low RP1 target value (low PMT).
1. Select Calibrate and confirm that the default values for CAL1 and CAL2 are the same as the values on the Bio-Plex calibration bead labels. Use the Bio-Plex low RP1 target value. 2. Select OK and follow the instructions for CAL1 and CAL 2 calibration. Prepare Protocol Lot-specific assay panel protocols are available for download at www.bio-rad.com/bio-plex/standards If the desired protocol is not available for download, create a new protocol: 1.
NOTE: If the preset protocol was downloaded from the web site, the standards will already be added to the plate format. Make any necessary changes to the plate layout to match your plate requirements. Plate Formatting Example 6. Select Step 4 (Enter Standards Info) to enter standards information. Note that this information will already be entered when using a download protocol. Otherwise: a) Deselect the box labeled “Same concentration values for all analytes”.
7. Select Step 5 (Enter Controls Info) to enter controls information. This is where the concentration of the user-specified controls is entered into the protocol. a) If necessary, deselect the box for same concentration values for all analytes. b) Select an analyte from the pull-down cell. c) Enter the description and dilution information for each userspecified control. d) Repeat steps 7b and 7c for each additional analyte in the assay. 8.
Acquire Data 1. Shake the assay plate at 900 rpm for 30 sec immediately before acquiring data. Failure to do so will result in increased read time due to bead settling. 2. Visually inspect the plate and ensure that the assay wells are filled with buffer prior to placing the plate in the Bio-Plex microplate platform. 3. Slowly remove the sealing tape and any plate cover before placing the plate in the reader. 4. Select the appropriate protocol (.pbx) file. 5. Select Start and save the report (.
Bio-Plex Manager Software Version 4.1 and 4.1.1 Prepare System 1. Empty the waste bottle and make sure that there is sufficient sheath fluid in the bottle or cube before proceeding. 2. Turn on the reader, XY platform and HTF (if included). Allow the system to warm up for 30 min. 3. Select Start up and follow the instructions to prepare the reader to acquire data. If the system is idle for 4 hr without aquiring data, the lasers will automatically turn off.
3. Select Step 2 (Select Analytes) and choose the appropriate acute phase panel name, if available in the pull-down menu. If the panel name is not available, create a new panel. See the Bio-Plex Manager software instruction manual for details. Choose the analytes to be selected from the available list of targets. Note that the targets will already be entered in the selected view when using a downloaded protocol.
5. 6. 7. 26 Select Step 4 (Enter Standards Info) to enter standards information. Note that this information will already be entered with the preset download protocol. a) Select each analyte individually from the pull-down cell. Deselect the box labeled “Same concentration values for all analytes”. If analyzing a 5-plex assay, also deselect the box labeled “Same units for all analytes”. b) Select the Enter Automatically option and then select the most concentrated value as S1.
Acquire Data 1. Shake the assay plate at 900 rpm for 30 sec immediately before acquiring data. Failure to do so will result in increased data acquisition time due to bead settling. 2. Visually inspect the plate and ensure that the assay wells are filled with buffer prior to placing the plate in the Bio-Plex microplate platform. 3. Slowly remove the sealing tape and any plate cover before placing the plate in the reader. 4. Select Step 7 (Run Protocol): a) Choose 100 beads per region.
Reaquire Data It is possible to acquire data from a well or plate a second time using the Rerun/Recovery mode located below Start in Step 7 (Run Protocol). For details, refer to the Bio-Plex Manager instruction manual. Bio-Plex Manager Software Version 4.0 Bio-Plex Manager version 4.0 does not include the 25-bead map. Hence, the 100-bead map is used. Follow the instructions above for Bio-Plex Manager version 4.1 and 4.1.1 for preparing and calibrating the system.
5. If acquiring data from more than one plate, empty the waste bottle and refill the sheath bottle after each plate (if HTF not present). Select Wash Between Plates and follow the instructions for fluidic maintenance. Then repeat the Prepare Protocol and Acquire Data steps. NOTE: Use the Wash Between Plates command after every plate run to reduce the possibility of clogging the instrument. 6. When data acquisition is complete, select Shut Down the instructions.
Section 11 Troubleshooting Guide This troubleshooting guide addresses problems that may be encountered with Bio-Plex Pro™ acute phase assays. If you experience any of the problems listed below, review the possible causes and solutions provided. This will assist you in resolving problems directly related to how the assay steps should be performed. Poor assay performance may also be due to the Bio-Plex array reader. To eliminate this possibility, we highly recommend use of the Bio-Plex validation kit.
Possible Causes Possible Solutions Pipetting technique Pipet carefully and slowly when adding standards, samples, detection antibodies, and streptavidin-PE, especially when using a multichannel pipet. Use a calibrated pipet. Change pipet tip after every volume transfer. Reagents and assay components were not equilibrated to room temperature prior to plating All reagents and assay components should be equilibrated to room temperature prior to plating.
Possible Causes Possible Solutions Low Bead Count Miscalculation of bead dilution Check your calculations and be careful to add the correct volumes. Beads clumped in multiplex bead stock tube Vortex for 15–20 sec at medium speed before aliquoting beads. Vacuum on for too long when aspirating buffer from wells Do not apply vacuum to the filter plate for longer than 10 sec after the buffer is completely drained from each well.
Possible Causes Possible Solutions High Background Signal Incorrect buffer was used (for example, assay buffer used to dilute standards) Use sample matrix or serum-based diluent to dilute standards. Spiked 0 pg/ml wells by mistake Be careful when spiking standards. Do not add any antigens in the 0 pg/ml (blank) point. Streptavidin-PE incubated too long Follow the procedure incubation time. Poor Recovery Expired Bio-Plex reagents were used Check that reagents have not expired.
Section 12 Safety Considerations Eye protection and gloves are recommended while using this product. Consult the MSDS for additional information.
xMAP, xPONENT and MagPlex are trademarks of Luminex Corp. Costar is a trademark of Coming Costar Corporation. Eppendorf is a trademark of Eppendorf-Netheler-Hinz GmbH. Luminex 100 and xMAP are trademarks of Luminex Corporation. Multiscreen is a trademark of Millipore Corporation. Vortex-Genie is a trademark of Scientific Industries, Inc.
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