Bio-rad Bio-Plex Pro™ Human Cancer Biomarker Assays User Manual

Bio-Plex Pro™ Human Cancer Biomarker AssaysInstruction ManualFor technical support, call your local Bio-Rad office, or in the U.S., call 1-800-424-6

8 1. Plan Plate LayoutPrior to running the assay, determine the total number of wells in the experiment using the Plate Layout Template on page 34

9 2. Prepare InstrumentStart up and calibrate the Bio-Plex® system with Bio-Plex Manager™ software prior to setting up the assay. The calibration k

10 3. Prepare Wash MethodBio-Plex Pro assays are compatible with both magnetic separation and vacuum filtration methods. However, for best results,

11 4. Prepare Standards and ControlsGeneral Instructions n It is essential to prepare standards and quality controls (if included) exactly as des

12 Reconstitute Standards and Quality ControlsThis procedure prepares enough standard to run each dilution in duplicate. 1. Gently tap the

13 6. Use reconstituted and diluted standards and controls immediately. Do not freeze for future use.Fig. 3. Preparing a fourfold dilutio

14 Sample PreparationSerum and Plasma EDTA or citrate is preferred as an anticoagulant. Heparin-treated plasma, while compatible with Bio-Plex Pro

Lavage, Sputum, and Other Biological Fluid Samples Keep all samples on ice until ready for use. The appropriate sample dilution factor should be optim

16 6. Prepare Coupled Beads1. Use Tables 6–7 or the Calculation Worksheet on page 35 to calculate the volume of coupled beads and assay buffer n

7. Run AssayThe following instructions apply to premixed multiplex, singleplex, x-Plex™, and Express assay formats.Considerationsn Bring all assay c

Table of ContentsIntroduction 1Principle 2Kit Contents and Storage 4Recommended Materials 5Assay Workflow 6lmportant Considerations 7Detailed Instr

18 n Place the assay plate on the plastic plate holder/tray as neededn Before each incubation, gently cover the plate with a new sheet of seali

19 2. Add the required volume of Bio-Plex detection antibody diluent to a 15 ml polypropylene tube.3. Vortex the 20x stock of detection anti

20 Prepare and Add Streptavidin-PE (SA-PE)1. While detection antibodies are incubating, use Table 11 or the Calculation Worksheet on page 35 to

21 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,

22 Bio-Plex Manager software versions 6.0 and higher contain protocols for most Bio-Plex® assays. Choose from available protocols or create a new p

23 3. Format the plate in Bio-Plex Manager according to the plate layout created in section 1 (Plan Plate Layout). To modify the plate layout, f

24 4. Enter Standards Info into Bio-Plex Manager. a. Enter the highest concentration of each analyte in the top row (labeled S1) of the

25 5. Enter Controls Info into Bio-Plex Manager. a. For user-specified controls, select an analyte from the dropdown menu, then enter a des

26 Acquire Data1. Shake the assay plate at 850 ± 50 rpm for 30 sec, and visually inspect the plate to ensure that the assay wells are filled w

27 Data AnalysisQuality ControlsIf the quality controls were run in the assay plate, open the results (.rbx) file, click on Report Table, and locate

IntroductionBio-Plex Pro™ Human Cancer Biomarker Assays Bio-Plex Pro human cancer biomarker assays are designed to meet the needs of the most demandin

28 Luminex xPONENT SoftwareLuminex xPONENT software may be used to analyze Bio-Plex assays. Although guidelines are provided here, consult the xPON

29 Possible CausesHigh Inter-Assay CV Standards and controls were not reconstituted consistently between assaysReconstituted standards, controls,

30 Possible CausesHigh Intra-Assay CV Improper pipetting technique Reagents and assay componentsnot equilibrated to room temperature prio

31 Possible CausesLow Bead Count Miscalculation of bead dilutionBeads clumped in multiplexbead stock tubeVacuum on for too long whenaspirating b

32 Possible CausesHigh Background Signal Incorrect buffer was used (for example, assay buffer used to dilute standards)Accidentally spiked bla

33 Possible SolutionsPipet carefully when adding standards, samples, detection antibodies, and streptavidin-PE, especially when using a multichanne

34 Plate Layout Template

35 Calculation WorksheetIf using either a premixed panel or one singleplex assay, follow these directions.Plan the plate layout and enter the numbe

36 If mixing singleplex assays, follow these directions.Enter the number of wells to be used in the assay:_______ 11. Determine the volume of 1x

37 Safety ConsiderationsEye protection and gloves are recommended when using these products.Consult the MSDS for additional information. The Bio-Pl

PrincipleTechnologyThe Bio-Plex® suspension array system is built upon the three core elements of xMAP technology:n Fluorescently dyed microspheres

38 Ordering InformationDetailed ordering information can be found at www.bio-rad.com/bio-plex.Bio-Plex® x-Plex™ Assays (We Mix)Premium custom assay

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Life ScienceGroup Sig 121110022186 Rev B Bio-Rad Laboratories, Inc.Web site www.bio-rad.com USA 800 424 6723 Australia 61 2 9914 2800 Austri

3 Fig. 1. Bio-Plex sandwich immunoassay. Data Acquisition and AnalysisData from the reactions are acquired using a Bio-Plex system or similar Lumin

4 Kit Contents and StorageReagents SuppliedBio-Plex Pro™ human cancer biomarker assays are available in a convenient all-in-one kit format that inc

5 ItemBio-Plex Pro Assays Quick Guide 1Bio-Plex® 200 system or Luminex system with HTFBio-Plex validation kit Note: Run the validation kit monthl

6 Prewet wells (for lter plate only)Add 50 μl 1x beads to wellsWash 2 x 100 μlAdd 50 μl standards, samples, controls, incubate 1 hr at RT with sh

7 lmportant ConsiderationsInstruments and Software The Bio-Plex Pro™ assays described in this manual are compatible with all currently available Lu