Bio-Plex Cytokine Assay

1 Bio-Plex Cytokine AssayInstruction ManualFor technical service, call your local Bio-Rad office, or in the US, call 1-800-4 BIORAD (1-800-424-6723).For research use only. Not for diagnostic of ContentsSection 1 Introduction1 Section 2 Principle3 Section 3 Product Description4 Section 4 Materials Required or Recommended but Not Supplied5 Section 5 Sample Preparation and Premixed Standard Dilution8 Section 6 Assay Procedure for Premixed MultiplexPanels and Singleplex Assays14 Section 7 Mixing Multiplex Assays: Bead, Standard,and Detection Antibody Preparation21 Section 8 Bio-Plex Suspension Array System Operation27 Section 9 Data Analysis30 Section 10 Troubleshooting Guide31 Section 11 Safety Considerations36 Section 12 Publications Citing the Bio-Plex Cytokine Assay37 Section 13 Bio-Plex Multiplex Cytokine Assay Templateand Dilution Worksheet39 Section 14 Legal Notices42 Bio-Plex Cytokine Assay WorkflowAdd beadsAdd standardsAdd samplesIncubate/shake(30 min)*Add detectionantibodiesIncubate/shake(30 min)Add streptavidin-PEIncubate/shake(10 min)Resuspend beadsRead platePrewet filter platePrepare standards(30 min)

2 Prepare samplesPrepare beadsPrepare detectionantibodyPrepare streptavidin-PEFilterFilter-wash 2xFilter-wash 3xFilter-wash 3xFilter-wash 3x* For the human Th1/Th2 magnetic panel, the incubation time is 60 1 IntroductionCytokines are important cell signaling proteins, mediating a wide range of physiological responses, including immunity, inflammation, andhematopoiesis. They are also associated with a spectrum of diseasesranging from tumor growth to infections to Parkinson s disease. Cytokines are typically measured either by bioassay or immunoassay. Both techniques are time consuming and can facilitate the analysis of only a single Cytokine at a time. The Bio-Plex suspension array system,which incorporates novel technology using color-coded beads, permitsthe simultaneous detection of up to 100 cytokines in a single well of a 96-well Cytokine assays are multiplex bead-based assays designed toquantitate multiple cytokines in diverse matrices, including serum samples,plasma samples, and tissue culture supernatants.

3 For a brief overview of the protocol, see the Bio-Plex Cytokine Assay Workflow. The 96-wellmicroplate-format Bio-Plex assays are optimized for the Bio-Plexsuspension array system, which utilizes xMAP detection technology. By multiplexing, it is possible to quantitate the level of multiple cytokines in a single well in just 3 hr, using as little as l of serum or 50 l oftissue culture sample. The advantages over traditional immunoassays that analyze only a single Cytokine at a time include the ability to create a complete Cytokine profile from limited sample, reduce samplepreparation time, and increase throughput. For a current listing of Bio-Plex Cytokine assays, panels, and reagents, visit us on the Web As Premixed or Unmixed Multiplex AssaysBio-Rad offers both mixed-to-order panels for multiplex Cytokine assays(panels include premixed beads, detection antibody, and standard) andsingleplex configurations. Premixed multiplex panels test for the presenceof a predetermined set of cytokines in a single sample.

4 All the necessarypanel components are provided premixed for ease of use. Procedures forthis configuration are provided in Section 6, Assay Procedure for PremixedMultiplex Panels and Singleplex technology allows end users to combine multiplex or singleplexreagents. By choosing among a series of available singleplex cytokineassays, components can be combined to create a tailored multiplex singleplex configuration provides maximum flexibility, enabling endusers to choose the cytokines that they wish to combine to meet theirspecific analysis needs. Procedures for mixing assays are provided inSection 7, Mixing Multiplex Assays: Bead, Standard, and DetectionAntibody research use only. Not for diagnostic 2 PrincipleThe Bio-Plex suspension array system is built around three coretechnologies. The first is the family of fluorescently dyed microspheres(beads) to which biomolecules are bound. The second is a flow cytometerwith two lasers and associated optics to measure biochemical reactionsthat occur on the surface of the microspheres.

5 The third is a high-speeddigital signal processor that efficiently manages the fluorescent Bio-Plex suspension array system employs patented multiplexingtechnology that uses up to 100 color-coded bead sets, each of which canbe conjugated with a specific reactant. Each reactant is specific for adifferent target molecule. Bio-Plex Cytokine assays are designed in acapture sandwich immunoassay format. Antibody specifically directedagainst the Cytokine of interest is covalently coupled to color-coded m polystyrene beads. The antibody-coupled beads are allowed to react with a sample containing an unknown amount of Cytokine ,or with a standard solution containing a known amount of Cytokine . After performing a series of washes to remove unbound protein, abiotinylated detection antibody specific for a different epitope on thecytokine is added to the beads. The result is the formation of a sandwichof antibodies around the Cytokine .

6 The reaction mixture is detected by theaddition of streptavidin-phycoerythrin (streptavidin-PE), which binds to the biotinylated detection antibodies. The constituents of each well aredrawn up into the flow-based Bio-Plex suspension array system, whichidentifies and quantitates each specific reaction based on bead color and fluorescence. The magnitude of the reaction is measured usingfluorescently labeled reporter molecules associated with each targetprotein. Unknown Cytokine concentrations are automatically calculated by Bio-Plex Manager software using a standard curve derived from arecombinant Cytokine standard. By using colored beads as the solidphase instead of a coated well, up to 100 differently colored beads can be mixed and used for quantitating up to 100 different analytes 3 Product DescriptionCytokine testing requires the Bio-Plex Cytokine reagent kit to run anysingleplex Assay , any multiplex panel, or any x-Plex custom panel.

7 If serum or plasma samples are to be tested, Bio-Rad recommendsspecies-specific diluent kits for optimum recovery (refer to Section 4).Storage and StabilityKit components should be stored at 4 C. Keep the streptavidin-PE in thedark. Do not freeze. All components are guaranteed for 6 months from thedate of purchase when stored as specified in this x 96-Well Format171-30400110 x 96-Well Format1 x 75 ml1 x 150 ml1 x 15 ml1 vial1 plate1 pack of 411 x 750 ml2 x 750 ml1 x 150 ml1 vial10 plates10 packs of 4 (40)1 The Bio-Plex Cytokine reagent kitcontains the following components: Bio-Plex Assay bufferStore at 4 C. Do not wash bufferStore at 4 C. Do not detection antibody at 4 C. Do not (100x)Store at 4 C. Do not filter plate (96-well) with cover and traySealing tapeCytokine instruction manualSection 4 Materials Required or Recommended but Not SuppliedRequired Materials: Cytokine Singleplex Assay or Multiplex PanelCytokine testing requires the Bio-Plex Cytokine reagent kit and either asingleplex Assay , a multiplex panel, or an x-Plex multiplex panel.

8 If serumor plasma samples are to be tested, Bio-Rad recommends species-specific diluent kits for optimum visit the Bio-Plex web site at our list of assays and Bio-Plex Cytokine assays and panels contain thefollowing components:Anti- Cytokine conjugated beads (25x concentration) Cytokine detection antibody (check vial for concentration prior to dilution and/or mixing) Cytokine standard (2 vials, lyophilized)Required Materials: Instrument and AccessoriesIn addition to the reagents and kits listed above, the following materialsare required to run Bio-Plex assays or panels. For optimal results, we recommend the use of these specific items:* See p. 13 for directions for using Bio-Plex Cytokine assays on the Luminex 100 or 200 Materials: Serum DiluentIf serum or plasma samples are to be tested, Bio-Rad recommends thesespecies-specific diluent kits for optimum recovery: Catalog # Bio-Plex Human Serum Diluent Kit171-305000 (1 x 96)171-305001 (10 x 96) Bio-Plex human serum sample diluent15 ml/150 mlBio-Plex human serum standard diluent10 ml/100 mlBio-Plex Mouse Serum Diluent Kit171-305004 (1 x 96)171-305005 (10 x 96) Bio-Plex mouse serum sample diluent15 ml/150 mlBio-Plex mouse serum standard diluent10 ml/100 mlBio-Plex Rat Serum Diluent Kit171-305008 (1 x 96) Bio-Plex rat serum sample diluent15 mlBio-Plex rat serum standard diluent10 mlCatalog # Bio-Plex 200 Suspension Array 171-000201 System or Luminex System* Bio-Plex 200 Suspension Array 171-000205 System With High-Throughput FluidicsBio-Plex Validation KitIncludes optics validation, classify validation,171-203001reporter validation, and fluidics validation(for Bio-Plex Manager )

9 Bead set for approximately 50 validationroutines using Bio-Plex Manager andMCV plateBio-Plex Calibration Kit171-203060 Microplate ShakersIKA MTS 2/4 shaker for 2 or 4 microplates IKA MTS 2/4 digital microtiteror Barnstead/Lab-Line Model 4625 plate(IKA catalog #3208000)shaker (or equivalent, capable ofModel 4625300 1,100 rpm)(VWR catalog #57019-600)7 MultiScreen Resist Vacuum Manifold,(Millipore catalog #MAVM0960R)available through Millipore, or Aurum Vacuum Manifold,732-6470available through Bio-RadWarning: The use of filter plate manifoldsother than the ones specified may result infilter plate leakage. See VacuumCalibration Procedure in Section 7 for instructions specific to this brand vortex mixer(VWR catalog #58816-121)Scientific Instruments Vortex-Genie 2 mixer(VWR catalog #58815-234)Sterilized Reagent Reservoirs224-4872 Costar 50 ml reagent reservoir, availablethrough Bio-RadOtherPipets and pipet tips, sterile distilled water,aluminum foil, absorbent paper towels,and ml or 2 ml microcentrifuge tubesNote Regarding Magnetic Bead Cytokine Assay PanelsFormagnetic bead panels, filter plates can be used as with polystyrenebeads.

10 For advice on atuomating magnetic bead panels, please contactTechnical Support. Bio-Plex magnetic bead panels will only work onBio-Plex or Luminex instruments using Bio-Plex Manager software or 5 Sample Preparation and PremixedStandard DilutionBio-Plex Cytokine assays are designed to quantitate multiple cytokines indiverse matrices including serum samples, plasma samples, and tissueculture supernatants. For optimal recovery and sensitivity, it is importantto properly prepare samples and standard curve dilutions. This sectionprovides instructions for preparing sample and standard curve sample preparations not mentioned here (including tissue,branchoaleovar lavage, cerebrospinal fluid, and others), consult thepublications listed in Bio-Rad bulletin 5297, available for download PreparationCell Culture SamplesKeep all samples on ice until ready for use. Culture medium isrecommended if dilution is required. Serum-free culture medium shouldcontain carrier protein (such as BSA) at a concentration of at least and store the samples at 70 C and avoid repeated freezing andthawing.