Bio-rad Bio-Plex Pro Human Acute Phase Reagent and Diluent User Manual

Acute PhaseInstruction ManualFor technical support, contact your local Bio-Rad office orin the US, call 1-800-4BIORAD (1-800-424-6723).For research us

Section 5Recommended or Optional Materials6Item Catalog #Bio-Plex®Acute Phase Diluent KitSerum-based diluentSerum-free diluent171-305050 (1x96)(10 ml)

7Item (continued) Ordering InformationReagent ReservoirCorning, Inc. Costar 50 ml reagentreservoir 4870Bio-Rad catalog #224-48721.2 ml Tubes in 96 Rac

Section 6Sample PreparationThis section provides instructions for preparing samples derived fromserum, plasma, and culture supernatant. For sample pre

95-plex and 4-plex assays For the 5-plex assay, dilute 5 µl ofsample in 495 µl of serum-free diluent. Mix by vortexing gentlyor pipetting. This result

10Section 7Standard PreparationTwo vials of Iyophilized acute phase standards are provided in eachmultiplex Bio-Plex Pro™ acute phase assay kit and on

114. Gently vortex 1-3 sec and incubate on ice for 60 min. For optimalassay performance, be consistent with the incubation time.5. Save the standard/c

4-plex standard dilution series: Add 50 µl of reconstituted 4-plexstandard to the first 1.5 ml tube containing 350 µl 4-plex standarddiluent. Vortex g

Section 8Control PreparationTwo vials of lyophilized acute phase controls are provided in eachmultiplex Bio-Plex Pro™ acute phase assay kit. This sect

14Section 9Assay InstructionsThe following instructions apply to both the 5-plex and 4-plex assays. Allof the necessary components are provided premix

15Prepare Coupled Magnetic BeadsProtect the beads from light by covering the tubes with aluminum foil.Keep all tubes on ice until ready to use.1. Vort

16Assay ProcedureBring all buffers to room temperature. Avoid bubbles when pipetting.The following terms are repeated throughout the assay procedure.

176. While the samples are incubating, perform a 30 sec quick-spincentrifugation of the detection antibody (10x for multiplex assays,100x for singlepl

1811. While the detection antibodies are incubating, perform a 30 secquick-spin centrifugation of the streptavidin-PE (100x) prior topipetting to coll

19Section 10Data AcquisitionBio-Plex Manager™ software is recommended for Bio-Plex Pro™ acutephase assays. Refer to the details in the appropriate sec

201. Select Calibrate and confirm that the default values for CAL1and CAL2 are the same as the values on the Bio-Plex calibrationbead labels. Use the

NOTE: If the preset protocol was downloaded from the web site, thestandards will already be added to the plate format. Make anynecessary changes to th

227. Select Step 5 (Enter Controls Info) to enter controls information. Thisis where the concentration of the user-specified controls is enteredinto t

Acquire Data1. Shake the assay plate at 900 rpm for 30 sec immediately beforeacquiring data. Failure to do so will result in increased read time dueto

Bio-Plex Manager Software Version 4.1 and 4.1.1Prepare System1. Empty the waste bottle and make sure that there is sufficient sheathfluid in the bottl

3. Select Step 2 (Select Analytes) and choose the appropriate acutephase panel name, if available in the pull-down menu. If the panelname is not avail

Table of ContentsSection 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1Section 2 Principle . . . . . . . . . . . .

5. Select Step 4 (Enter Standards Info) to enter standards information.Note that this information will already be entered with the presetdownload prot

Acquire Data1. Shake the assay plate at 900 rpm for 30 sec immediately beforeacquiring data. Failure to do so will result in increased dataacquisition

Reaquire DataIt is possible to acquire data from a well or plate a second time using theRerun/Recovery mode located below Start in Step 7 (Run Protoco

5. If acquiring data from more than one plate, empty the waste bottleand refill the sheath bottle after each plate (if HTF not present). SelectWash Be

Section 11Troubleshooting GuideThis troubleshooting guide addresses problems that may be encountered withBio-Plex Pro™ acute phase assays. If you expe

31Possible Causes Possible SolutionsPipetting techniquePipet carefully and slowly whenadding standards, samples,detection antibodies, andstreptavidin-

32Possible Causes Possible SolutionsLow Bead CountMiscalculation of bead dilutionCheck your calculations and becareful to add the correct volumes.Bead

33Possible Causes Possible SolutionsHigh Background SignalIncorrect buffer was used(for example, assay bufferused to dilute standards)Use sample matri

34Section 12Safety ConsiderationsEye 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 ofEppendorf-Neth

Bio-Rad Laboratories, Inc.2000 Alfred Nobel Dr.Hercules, CA 94547 USA1-800-424-6723 (in the US)Life ScienceGroupSig 110610011597 US/EG Rev ABio-RadLab

Section 1IntroductionAcute phase proteins are a class of proteins that have altered levels inresponse to inflammation and are thus relevant biomarkers

Section 2PrincipleTechnologyThe Bio-Plex®suspension array system is built around three coretechnologies. The first is a novel technology that uses up

Assay Workflow3Add beadsWashAdd standards, controls,and samples, 60 minWashAdd detection antibody, 30 minWashAdd streptavidin-PE, 10 minWashResuspend,

Section 3Bio-Plex®Acute Phase Reagent KitProduct DescriptionBio-Plex Pro™ acute phase assays require the use of the Bio-Plex acute phasereagent kit.St

Section 4Required MaterialsThe Bio-Plex Pro™ acute phase assay panel is divided into two separatemultiplex assay kits based on the sample dilution req