Bio-Plex Pro Human Cancer Biomarker Assays ™ Instruction Manual For technical support, call your local Bio-Rad office, or in the U.S., call 1-800-424-6723. For research use only. Not for diagnostic procedures.
Table of Contents Introduction 1 Principle 2 Kit Contents and Storage 4 Recommended Materials 5 Assay Workflow 6 lmportant Considerations 7 Detailed Instructions 7 1. Plan Plate Layout 8 2. Prepare Instrument 9 3. Prepare Wash Method 10 4. Prepare Standards and Controls 11 5. Prepare Samples 13 6. Prepare Coupled Beads 16 7. Run Assay 17 8.
Introduction Bio-Plex Pro™ Human Cancer Biomarker Assays Bio-Plex Pro human cancer biomarker assays are designed to meet the needs of the most demanding preclinical and clinical research environments. These magnetic bead–based multiplex immunoassays include a unique blend of soluble biomarkers involved in disease processes such as angiogenesis, metastasis, inflammation, cell proliferation, cell adhesion, and apoptosis.
Principle Technology The Bio-Plex® suspension array system is built upon the three core elements of xMAP technology: n n n Fluorescently dyed microspheres (also called beads), each with a distinct color code or spectral address to permit discrimination of individual tests within a multiplex suspension.
Biomarker of Interest Streptavidin Magnetic Bead Biotinylated Detection Antibody Capture Antibody Phycoerythrin Fluorescent Reporter Fig. 1. Bio-Plex sandwich immunoassay. Data Acquisition and Analysis Data from the reactions are acquired using a Bio-Plex system or similar Luminex-based reader.
Kit Contents and Storage Reagents Supplied Bio-Plex Pro™ human cancer biomarker assays are available in a convenient all-in-one kit format that includes assay, reagent, and diluent components in a single box. Table 1. Contents of 1 x 96-well premixed and custom x-Plex™ kits.
Table 2. Recommended materials. Item Ordering Information Bio-Plex Pro Assays Quick Guide 1 Bulletin #100-22581 (download at www.bio-rad.
Assay Workflow Prewet wells (for filter plate only) Add 50 μl 1x beads to wells Wash 2 x 100 μl Add 50 μl standards, samples, controls, incubate 1 hr at RT with shaking at 850 rpm Wash 3 x 100 μl Add 25 μl 1x detection antibody, incubate 30 min at RT with shaking at 850 rpm Wash 3 x 100 μl Add 50 μl 1x streptavidin-PE, incubate 10 min at RT with shaking at 850 rpm Wash 3 x 100 μl Resuspend in 125 μl assay buffer, shake at 850 rpm for 30 sec Acquire data on Bio-Plex system 6
lmportant Considerations Instruments and Software The Bio-Plex Pro™ assays described in this manual are compatible with all currently available Luminex-based life science research instruments. Assays can be read and analyzed with either Bio-Plex Manager™ software or Luminex xPonent software (section 8). Assay Procedures Please pay close attention to vortexing, shaking, and incubation times and to Bio-Plex® reader PMT (RP1) setting, as these have been optimized specifically for each assay panel.
1. Plan Plate Layout Prior to running the assay, determine the total number of wells in the experiment using the Plate Layout Template on page 34 or the Plate Formatting tab in Bio-Plex Manager™. A suggested plate layout is shown in Figure 2, with all conditions in duplicate. 1. Assign standards to columns 1 and 2, with the highest concentration in row A and the lowest concentration in row H. 2. Assign the blank to wells A3 and A4. The blank should consist of your chosen standard diluent.
2. Prepare Instrument Start up and calibrate the Bio-Plex® system with Bio-Plex Manager™ software prior to setting up the assay. The calibration kit should be run daily or before each use of the instrument to standardize the fluorescent signal. For instructions on using other xMAP system software packages, contact Bio-Rad Technical Support. The validation kit should be run monthly to ensure optimal performance of fluidics and optics systems.
3. Prepare Wash Method Bio-Plex Pro assays are compatible with both magnetic separation and vacuum filtration methods. However, for best results, we recommend performing the assays in a flat bottom plate with magnetic separation. Table 3. Summary of compatible wash stations and plate types.
4. Prepare Standards and Controls General Instructions n n It is essential to prepare standards and quality controls (if included) exactly as described in this section. Incorrect preparation may lead to low signal or variable measurements from plate to plate The product data sheet or peel-off sticker provided with the standards lists the most concentrated point on the standard curve (S1).
Reconstitute Standards and Quality Controls This procedure prepares enough standard to run each dilution in duplicate. 1. Gently tap the vial containing the lyophilized standards on a solid surface to ensure the pellet is at the bottom of the vial. 2. Reconstitute a single vial of standards with 781 µl of the appropriate diluent. Optional: at the same time, reconstitue the two control vials with 250 µl of the appropriate diluent as summarized in Table 4. Controls do not require further dilution. 3.
6. Use reconstituted and diluted standards and controls immediately. Do not freeze for future use. Fig. 3. Preparing a fourfold dilution series with a single reconstituted standard. 50 50 50 50 50 50 50 Transfer Volume, µl Reconstituted Standard 150 150 150 150 150 150 150 150 S1 S2 S3 S4 S5 S6 S7 S8 Blank Diluent, µl 5. Prepare Samples General guidelines for preparing different sample types are provided here.
Sample Preparation Serum and Plasma EDTA or citrate is preferred as an anticoagulant. Heparin-treated plasma, while compatible with Bio-Plex Pro™ assays, may absorb certain soluble proteins of interest. Avoid using hemolyzed samples as this may lead to false positive results. 1. Draw whole blood into collection tubes containing anticoagulant. Invert tubes several times to mix. 2. For serum, allow blood to clot at room temperature for 30 to 45 min.
Lavage, Sputum, and Other Biological Fluid Samples Keep all samples on ice until ready for use. The appropriate sample dilution factor should be optimized by the user. 1. If required, dilute the sample in Bio-Plex sample diluent with BSA added to a final concentration of 0.5%. 2. Centrifugation at 10,000 x g for 10 min at 4°C may be required to clarify the sample. Lysates The Bio-Plex cell lysis kit is required for lysate preparation (available separately, catalog #171-304011 and #171-304012).
6. Prepare Coupled Beads 1. Use Tables 6–7 or the Calculation Worksheet on page 35 to calculate the volume of coupled beads and assay buffer needed. 2. Add the required volume of Bio-Plex® assay buffer to a 15 ml polypropylene tube. 3. Vortex the 20x stock of coupled beads at mid speed for 30 sec. Carefully open the cap and pipet any liquid trapped in the cap back into the tube. This is important to ensure maximum bead recovery. Do not centrifuge the vial; doing so will cause the beads to pellet. 4.
7. Run Assay The following instructions apply to premixed multiplex, singleplex, x-Plex™, and Express assay formats. Considerations n Bring all assay components and samples to room temperature before use n Use calibrated pipets and pipet carefully, avoiding bubbles n n Pay close attention to vortexing, shaking, and incubation instructions. Deviation from the protocol may result in low assay signal and assay variability Assay incubations are carried out in the dark.
n n Place the assay plate on the plastic plate holder/tray as needed Before each incubation, gently cover the plate with a new sheet of sealing tape. Avoid pressing down over the wells to prevent leaking from the bottom Add Coupled Beads, Samples, Standards, Blank, and Controls 1. Cover unused wells of the assay plate with sealing tape. 2. Prewet the filter plate. Skip this step if using a flat bottom plate.
2. Add the required volume of Bio-Plex detection antibody diluent to a 15 ml polypropylene tube. 3. Vortex the 20x stock of detection antibodies for 15–20 sec at medium speed, then perform a 30 sec spin to collect the entire volume at the bottom of the tube. 4. Dilute detection antibodies to 1x by pipetting the required volume into the 15 ml tube. Vortex. Each well of the assay requires 1.25 μl of the 20x stock adjusted to a final volume of 25 μl in detection antibody diluent.
Prepare and Add Streptavidin-PE (SA-PE) 1. While detection antibodies are incubating, use Table 11 or the Calculation Worksheet on page 35 to calculate the volume of SA-PE and assay buffer needed. SA-PE should be prepared 10 min before use. 2. Add the required volume of assay buffer to a 15 ml polypropylene tube. 3. Vortex the 100x stock of SA-PE for 5 sec at medium speed. Perform a 30 sec spin to collect the entire volume at the bottom of the vial. 4.
10. Wash the plate three times with 100 µl of wash buffer according to the wash method of choice. 11. To resuspend beads for plate reading, add 125 µl assay buffer to each well. Cover the plate with a new sheet of sealing tape. Shake at room temperature at 850 ± 50 rpm for 30 sec and slowly remove the sealing tape. Ensure that the plate cover has been removed before placing the plate on the reader. Table 12. Read the plate using the appropriate instrument settings.
Bio-Plex Manager software versions 6.0 and higher contain protocols for most Bio-Plex® assays. Choose from available protocols or create a new protocol. To create a new protocol, select File, then New from the main menu. Locate and follow the steps under Protocol Settings. 1. Describe Protocol and enter information about the assay (optional). 2. Select Analytes and create a new panel. Visually confirm the selected analytes and proceed to step 3. a.
Table 13. Bead regions for human cancer biomarker panels. Panel 1 Analyte Bead Region Analyte Bead Region sEGFR FGF-basic Follistatin G-CSF sHER2/neu HGF 15 44 26 57 12 62 sIL-6Ra Leptin Osteopontin PDGF-AB/BB PECAM-1 Angiopoietin-2 sCD40L EGF Endoglin sFASL HB-EGF 47 29 14 26 35 57 IGFBP-1 IL-6 IL-8 IL-18 PAI-1 PLGF Analyte Bead Region 19 78 77 47 46 Prolactin SCF TIE-2 sVEGFR-1 sVEGFR-2 52 65 64 76 45 62 19 54 42 61 52 TGF-a TNF-a uPA VEGF-A VEGF-C VEGF-D 76 36 78 45 64 43 Panel 2 3.
Fig. 4. Plate formatting. 4. Enter Standards Info into Bio-Plex Manager. a. Enter the highest concentration of each analyte in the top row (labeled S1) of the table. S1 concentration information is included with each vial of standards. b. Enter a dilution factor of 4 and click Calculate. The concentrations for each standard point will be populated for all analytes in the table. c. Optional: enter the lot number of the vial of standards into the Standard Lot box and click Save.
5. Enter Controls Info into Bio-Plex Manager. a. For user-specified controls, select an analyte from the dropdown menu, then enter a description and concentration. Repeat for each additional analyte in the assay. b. For the quality controls supplied in premixed cancer biomarker kits, format the appropriate wells as controls, enter descriptions, but leave the concentrations blank.
Acquire Data 1. Shake the assay plate at 850 ± 50 rpm for 30 sec, and visually inspect the plate to ensure that the assay wells are filled with buffer. Slowly remove the sealing tape and any plate cover before placing the plate on the plate carrier. 2. Run Protocol – on the pop-up screen, select Load Plate and click OK to start acquiring data. 3. Use the Wash Between Plates command after every plate run to reduce the possibility of clogging the instrument. 4.
Data Analysis Quality Controls If the quality controls were run in the assay plate, open the results (.rbx) file, click on Report Table, and locate the control wells. Visually compare the observed concentrations of the high and low controls in the Report Table against the lot-specific control ranges shown in the product data sheet. Note! Expected control ranges are provided for reference and should be used as general guidelines. Actual results may vary for some operators.
Luminex xPONENT Software Luminex xPONENT software may be used to analyze Bio-Plex assays. Although guidelines are provided here, consult the xPONENT software manual for more details. Perform a system initialization with Luminex’s calibration and performance verification kit, as directed by Luminex. Select Batches to set up the protocol and follow the information under Settings. Note! The instrument settings described below apply to Luminex 100/200 and FLEXMAP 3D instruments.
Troubleshooting Guide This troubleshooting guide addresses problems that may be encountered with Bio-Plex Pro™ assays. If you experience any of the problems listed below, review the possible causes and solutions provided. Poor assay performance may also be due to the Bio-Plex® suspension array reader. To eliminate this possibility, use the validation kit to assist in determining if the array reader is functioning properly.
Possible Causes Possible Solutions High Intra-Assay CV Improper pipetting technique Reagents and assay components not equilibrated to room temperature prior to pipetting Contamination with wash buffer during wash steps Pipet carefully when adding standards, controls, samples, detection antibodies, and streptavidin-PE, especially when using a multichannel pipet. Use a calibrated pipet. Change pipet tip after every volume transfer.
Possible Causes Low Bead Count Miscalculation of bead dilution Possible Solutions Beads clumped in multiplex bead stock tube Vortex for 30 sec at medium speed before aliquoting beads. Vacuum on for too long when aspirating buffer from wells Assay plate not shaken enough during incubation steps and prior to reading 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 Incorrect buffer was used (for example, assay buffer used to dilute standards) Use sample matrix standard diluent to dilute standards. Accidentally spiked blank wells Be careful when spiking standards. Do not add any antigens to the blank wells. Detection antibodies or streptavidin-PE incubated too long Follow the procedure incubation time precisely.
Possible Causes Possible Solutions Poor Recovery Improper pipetting technique Pipet carefully 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. Impact of Sample Matrix Negative MFI values in samples or standards If samples contain little or no analyte, negative values observed may be due to statistical variation.
Plate Layout Template 34
Calculation Worksheet If using either a premixed panel or one singleplex assay, follow these directions. Plan the plate layout and enter the number of wells to be used in the assay:_______ 1 1. Determine the volume of 1x coupled beads needed. a. Each well requires 50 µl of coupled beads (1x): _______ x 50 µl = _______ µl 1 2 b. Include 20% excess to ensure enough volume: _______ µl x 0.20 = _______ µl 2 3 c. Total volume of 1x coupled beads: _______ µl + _______ µl = _______ µl 2 3 4 d.
If mixing singleplex assays, follow these directions. Enter the number of wells to be used in the assay:_______ 1 1. Determine the volume of 1x coupled beads needed. a. Each well requires 50 µl coupled beads (1x): _______ x 50 µl = _______ µl 1 2 b. Include 20% excess to ensure enough volume: _______ µl x 0.20 = _______ µl 2 3 c. Total volume of 1x coupled beads: _______ µl + _______ µl = _______ µl 2 3 4 d.
Safety Considerations Eye protection and gloves are recommended when using these products. Consult the MSDS for additional information. The Bio-Plex Pro™ assays contain components of animal origin. This material should be handled as if capable of transmitting infectious agents. Use universal precautions. These components should be handled at Biosafety Level 2 containment as defined by U.S. government publication, Biosafety in Microbiological and Biomedical Laboratories (Centers for Disease Control, 1999).
Ordering Information Detailed ordering information can be found at www.bio-rad.com/bio-plex.
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