Bio-Plex Pro Human Isotyping 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 1.1. Plan Plate Layout 2.2. Prepare Instrument 3.3. Prepare Wash Method 4.4. Prepare Wash Buffer 5.5. Prepare Standards and Controls 6. Prepare Samples 8 9 10 11 11 14 7.7. Prepare Coupled Beads 8.8. Run Assay 9.9.
Introduction Bio-Plex Pro™ Human Isotyping Assays Bio-Plex Pro isotyping assays enable you to measure multiple immunoglobulin isotypes in only 5 μl of sample. A detailed profile of the immune response to infection, disease, vaccination, or drug therapy can now be obtained in a single three hour experiment. Bio-Plex Pro isotyping assays are multiplex bead-based assays designed to quantitate multiple immunoglobulin isotypes in diverse matrices, including serum, plasma, and tissue culture supernatants.
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™ isotyping 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 kits.
Table 2. Recommended materials.
Assay Workflow Prewet wells (for filter plate only) Add 50 µl 1x beads to wells Wash 2 x 100 μl Add 50 μl diluted 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 9). Assay Procedures 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 33 or the Plate Formatting tab in Bio-Plex Manager™ software. 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.
2. Prepare Instrument These directions are specific for the Bio-Plex® 100/200 reader. To prepare either a Bio-Plex 3D or Bio-Plex® MAGPIX™ reader, consult their respective user manuals. Note: While the instrument is warming up, bring the 10x wash buffer, assay buffer, and isotyping diluent to room temperature. Keep other items on ice until needed. Also, begin to thaw frozen samples. Start up and calibrate the Bio-Plex system with Bio-Plex Manager™ software prior to setting up the assay.
2. Select OK and follow the software prompts for step-by-step instructions for CAL1 and CAL2 calibration. Note: In Bio-Plex Manager version 6.1 and higher, startup, warm up, and calibration can be performed together by selecting the “Start up and calibrate” icon. 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.
Setting up a Vacuum Manifold Calibrate the vacuum manifold by placing a standard 96-well flat bottom plate on the unit and adjusting the pressure to –1 to –3" Hg. In general, 100 µl liquid should take 3–4 sec to clear the well. For more detailed instructions, refer to bulletin #10005042. 4. Prepare Wash Buffer 1. Bring the 10x stock solution to room temperature. 2. If crystals exist, ensure that they are completely dissolved. Mix the 10x stock solution by inversion before preparing the 1x wash buffer.
Selecting a Diluent for Standards and Controls Refer to Table 4 for recommended diluents based on different sample types. In order to meet the lot-specific control ranges provided on the product data sheet, both the standards and controls should be reconstituted in Bio-Plex® isotyping diluent. If reconstituting in a different diluent, users will need to establish/validate their own control ranges or acceptance criteria. Table 4. Summary of recommended diluents for samples.
Prepare the Standard Dilution Series The following procedure produces an eight-point standard curve with a fourfold dilution between each point. Pipet carefully using calibrated pipets and use a new pipet tip for every volume transfer. 1. Label eight 1.5 ml microcentrifuge tubes S2 through S8 and Blank. Alternatively, using Titertube® micro test tubes may prove to be more convenient if a multichannel pipet will be used to load the plate. 2. Add 150 µl of the appropriate diluent to tubes S2–S8 and Blank.
6. Prepare Samples These assays are designed to quantitate classes and subclasses of immunoglobulins in serum, plasma, and cell culture media. For optimal recovery and sensitivity, it is important to properly prepare samples. n Once thawed, keep samples on ice. Prepare dilutions just prior to the start of the assay and equilibrate to room temperature before use Prepare sample dilutions in 1.5 or 2 ml microcentrifuge tubes.
4. Extremely lipemic samples may be filtered through a 0.2 µm filter to prevent clogging. 5. Dilute samples with isotyping diluent following a 2-step dilution scheme as noted below, vortexing for 5 sec between each dilution step. 1:40,000 dilution for IgG1, IgG2, IgG3, IgG4, IgA, IgM, and IgG total Step 1. 5 µl of sample + 995 µl of diluent Step 2. 5 µl of solution from first step + 995 µl of diluent 1:500 dilution for IgE Step 1. 5 µl of sample + 120 µl of diluent Step 2.
7. Prepare Coupled Beads 1. Use Table 6 or the Calculation Worksheet on page 34 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 medium 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.
8. Run Assay Considerations n n n n Bring all buffers, diluents, diluted standards, diluted coupled beads, and samples to room temperature before use Use calibrated pipets and pipet carefully, avoiding bubbles. Use new pipet tips for every volume transfer 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 at 850 ± 50 rpm.
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. a) Prewet the wells with 100 µl assay buffer and remove the liquid by vacuum filtration. Dry the bottom of the filter plate thoroughly by blotting on a clean paper towel. 3. Vortex the diluted (1x) beads for 30 sec at medium speed.
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. Table 8. Preparing 1x detection antibodies from 20x stock (includes 25% excess volume). # of Wells 20x Detection Antibodies, µl Detection Antibody Diluent, µl 96 150 2,850 3,000 48 75 1,425 1,500 Total Volume, µl 5.
4. Dilute SA-PE to 1x by pipetting the required volume into the 15 ml tube. Vortex and protect from light until ready to use. Each well of the assay requires 0.5 μl of the 100x stock adjusted to a final volume of 50 μl in assay buffer. Table 9. Preparing 1x SA-PE from 100x stock (includes 25% excess volume). # of Wells 100x SA-PE, µl Assay Buffer, µl Total Volume, µl 96 60 5,940 6,000 48 30 2,970 3,000 5. After detection antibody incubation, slowly remove and discard the sealing tape. 6.
9. Read Plate Bio-Plex Manager™ software is recommended for all Bio-Plex Pro™ assay data acquisition and analysis. Instructions for Luminex xPONENT software are also included. For instructions using other xMAP system software packages, contact Bio-Rad Technical Support or your regional Bio-Rad field applications specialist. Prepare Protocol in Bio-Plex Manager Software Version 6.0 and Higher The protocol should be prepared in advance so that the plate is read as soon as the experiment is complete.
b. Click the Add button. Enter the bead region number and name for the first analyte. Click Add Continue to repeat for each analyte in the assay. For reference, bead regions are shown in Table 10. Table 10. Bead regions for Bio-Plex Pro isotyping assays. Analyte Bead Region Analyte Bead Region lgG1 lgG2 lgG3 lgG4 54 21 36 72 lgA lgM lgE lgG Total 12 56 38 66 c. Click the Add button when the last analyte has been added and click OK to save the new panel. d.
Fig. 4. Plate formatting. 4. Click Enter Standards Info. a. Enter the highest concentration and units of each analyte in the top row (labeled S1) of the table. S1 concentration information is listed in the product data sheet. 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.
b. For the vial of quality controls supplied, format the appropriate wells as controls and enter descriptions, but leave the concentrations blank. Alternatively, the quality controls can be formatted as samples with clear descriptions such as “quality control.” In any case, the expected control ranges provided on the product data sheet are not entered into Bio-Plex Manager software version 6.1 and earlier. 6. Click Enter Sample Info and enter sample information and the appropriate dilution factor. 7.
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. If acquiring data from more than one plate, empty the waste bottle and refill the sheath bottle after each plate (if HTF are not present). Select Wash Between Plates and follow the instructions. Then repeat the Prepare Protocol and Acquire Data instructions. 5.
Note: Expected control ranges are provided for reference and should be used as general guidelines. Actual results may vary for some operators. If the controls do not fall within the expected ranges, please refer to the troubleshooting section for possible causes and solutions. Removing Outliers Outliers are identified as standard data points that do not meet accuracy or precision requirements and should be considered invalid when performing curve fitting.
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 or Bio-Plex 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 isotyping diluent to dilute standards. High Background Signal Accidentally spiked blank wells 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 33
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.
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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