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

Bio Plex Pro

Acute Phase

Instruction Manual

Bio-Plex Manager™ software

recommended

Assays

For technical support, contact your local Bio-Rad office or in the US, call 1-800-4BIORAD (1-800-424-6723).

For research use only. Not for diagnostic procedures.

Table of Contents

Section 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Section 2 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Section 3 Bio-Plex

Section 4 Required Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Section 5 Recommended or Optional Materials . . . . . . . . . . . . . 6

Section 6 Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Section 7 Standard Preparation . . . . . . . . . . . . . . . . . . . . . . . . 10

Section 8 Control Preparation . . . . . . . . . . . . . . . . . . . . . . . . . 13

Section 9 Assay Instructions . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Section 10 Data Acquisition . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Section 11 Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . 30

Section 12 Safety Considerations . . . . . . . . . . . . . . . . . . . . . . . .34

Acute Phase Reagent Kit Product Description .4

Section 1 Introduction

Acute phase proteins are a class of proteins that have altered levels in response to inflammation and are thus relevant biomarkers in many disease processes. Typically, initial release of cytokines at the site of damage, most notably IL-1, IL-6, IL-8 and TNF-움, triggers the liver to release a number of acute phase proteins into the bloodstream. In turn, the increased levels of these proteins stimulate the release of more cytokines, presumably to initiate processes to prevent further tissue damage. Positive acute phase proteins—those that increase in response to tissue damage—are associated with infection, physical trauma, cardiac injury, diabetes, cancer, and other diseases.

Bio-Plex Pro™ assays are bead-based multiplex assays that allow the measurement of nine positive acute phase biomarkers in diverse matrices, including serum, plasma, and tissue culture supernatant. The biomarkers selected are circulating proteins that are known to be present in elevated levels during the acute phase response. The multiplexing feature makes it possible to quantify the levels of multiple target proteins simultaneously in a single well of a 96-well microplate in just 3 hours with as little as 5 µl of sample.

As one of the most recent additions to the Bio-Plex family of products, Bio-Plex Pro assays incorporate magnetic beads into their design. The magnetic beads allow the use of an assay protocol similar to nonmagnetic Bio-Plex assays, with the option of using magnetic separation during wash steps instead of vacuum filtration. The magnetic washing option also enables automated assay preparation.

Bio-Plex Manager™ software is recommended for Bio-Plex Pro acute phase assays. Instructions for Luminex xPONENT software are also provided. For instructions using other xMAP system software packages, contact Bio-Rad Technical Support or your Bio-Rad Field Applications Scientist.

For a current listing of Bio-Plex Pro acute phase assays, visit us on the Web at www.bio-rad.com/bio-plex/

Section 2 Principle

Technology

The Bio-Plex technologies. The first is a novel technology that uses up to 100 unique fluorescently dyed beads (xMAP technology) that permit the simultaneous detection of up to 100 different types of molecules in a single well of a 96-well microplate. The second is a flow cytometer with two lasers and associated optics to measure the different molecules bound to the surface of the beads. The third is a high-speed digital signal processor that efficiently manages the fluorescent output.

Assay Format

The principle of these 96-well plate-formatted, bead-based assays is similar to a capture sandwich immunoassay. An antibody directed against the desired target protein is covalently coupled to internally dyed beads. The coupled beads are allowed to react with a sample containing the target protein. After a series of washes to remove unbound protein, a biotinylated detection antibody specific for a different epitope is added to the reaction. The result is the formation of a sandwich of antibodies around the target protein. Streptavidin-phycoerythrin (streptavidin-PE) is then added to bind to the biotinylated detection antibodies on the bead surface for flourescencebased detection.

Data Acquisition and Analysis

Data from the reaction are then acquired using the Bio-Plex suspension array system, dual-laser, flow-based microplate reader system. The contents of the well are drawn up into the reader. The lasers and associated optics detect the internal fluorescence of the individual dyed beads as well as the fluorescent signal on the bead surface that corresponds to the amount of detected target protein. Intensity of fluorescence detected on the beads indicates the relative quantity of targeted molecules. A high-speed digital processor efficiently manages the data output, which is further analyzed and presented as fluorescence intensity on Bio-Plex Manager™ software.

suspension array system is built around three core

Assay Workflow

Prewet wells

Add beads

Wash

Add standards, controls,

and samples, 60 min

Wash

Add detection antibody, 30 min

Wash

Add streptavidin-PE, 10 min

Wash

Resuspend, acquire data

Section 3 Bio-Plex®Acute Phase Reagent Kit Product Description

Bio-Plex Pro™ acute phase assays require the use of the Bio-Plex acute phase reagent kit.

Component of the

Bio-Plex Acute Phase

Reagent Kit

Bio-Plex assay buffer Store at 4ºC. Do not freeze.

Bio-Plex wash buffer Store at 4ºC. Do not freeze.

Bio-Plex detection antibody diluent Store at 4ºC. Do not freeze.

Streptavidin-PE (100x) Store at 4ºC. Do not freeze.

Sterile filter plate (96-well) with cover and tray

Sealing tape 1 pack of 4 10 packs of 4 (40)

Acute Phase instruction manual

Storage and Stability

Kit components should be stored at 4ºC and never frozen. Coupled magnetic beads and streptavidin-PE should be stored in the dark. All components are guaranteed for up to 6 months from the date of purchase when stored as specified in this manual.

Catalog #

171-304050

1 x 96-Well

Format

1 x 75 ml 1 x 750 ml

1 x 150 ml 2 x 750 ml

1 x 15 ml 1 x 150 ml

1 vial 1 vial

1 plate 10 plates

1 1

Catalog #

171-304051

10 x 96-Well

Format

Section 4 Required Materials

The Bio-Plex Pro™ acute phase assay panel is divided into two separate multiplex assay kits based on the sample dilution required for optimal performance.

• Each assay requires one Bio Plex

• The Bio-Plex acute phase diluent kit (see next section) is recommended for serum and plasma samples. One diluent kit contains sufficient volumes for performing a 5-plex assay and a 4-plex assay or two 5-plex assays or two 4-plex assays, as long as dilutions are performed as instructed.

The Bio Plex Pro acute phase assay kits contain the following components:

Multiplex assays

• Antibody-conjugated beads (25x concentration)

• Detection antibody (10x concentration)

• Acute phase standard (2 vials/lyophilized)

• Acute phase control (2 vials/lyophilized)

Please visit the Bio-Plex web site at www.bio-rad.com/bio-plex/ for our current list of assays and panels.

acute phase reagent kit.

Bio-PlexManager™ software version 3.0 and Luminex IS Software Users

Contact Bio-Rad Technical Support or your Bio-Rad Field Applications Scientist. Additional supplies are required to run the acute phase assays using these systems.

Section 5 Recommended or Optional Materials

Item Catalog #

Bio-Plex®Acute Phase Diluent Kit

Serum-based diluent Serum-free diluent

Bio-Plex Pro™ Human Acute Phase Standards

Optional additional standards sold separately

Item Ordering Information

Bio-Plex 200 System with HTF (or Luminex System)

Bio-Plex Validation Kit 4.0 Bio-Rad catalog #171-203001

Bio-Plex Calibration Kit Bio-Rad catalog #171-203060

Microtiter Plate Shaker

IKA-Schuttler MTS-4 shaker for 4 microplates or Lab-Line Model 4625 plate shaker (or equivalent, capable of 300-1,100 rpm)

Filter Plate Vacuum Apparatus

Aurum™ Vacuum Manifold IMPORTANT: The use of filter plate manifolds other than the one specified may result in diminished assay performance; see section 8 for instructions specific to this assay

171-305050 (1x96)

(10 ml)

(300 ml)

171-D40002

(2 vials, lyophilized)

171-D00006

(50 vials, lyophilized)

Bio-Rad catalog #171-000205

IKA catalog #3208000

VWR catalog #57019-600

Bio-Rad catalog #732-6470

Vortexer

VWR mini-vortexer Scientific Instruments Vortex-Genie 2 mixer

VWR catalog #58816-121 VWR catalog #58815-234

Item (continued) Ordering Information

Reagent Reservoir

Corning, Inc. Costar 50 ml reagent reservoir 4870

1.2 ml Tubes in 96 Rack

For performing sample dilutions in 96-well format

Other Materials

Pipets and pipet tips, sterile distilled water, aluminum foil, absorbent paper towels, 1.5 ml microcentrifuge tubes, 15 ml culture tubes

Bio-Rad catalog #224-4872

VWR catalog #82006-698

(tubes in rack)

VWR catalog #83009-678

(refill tubes)

Section 6 Sample Preparation

This section provides instructions for preparing samples derived from serum, plasma, and culture supernatant. For sample preparations not mentioned here, consult the publications listed in Bio-Rad bulletin 5297, available for download at www.discover.bio-rad.com

Serum and Plasma Samples

Collect and process the serum or plasma samples and assay immediately or freeze at –20ºC. Avoid repeated freeze-thaw cycles. EDTA is the recommended anticoagulant for preparing plasma samples. Extremely lipemic samples may be filtered with a 0.22 µm filter to prevent clogging. Hemolyzed samples are not suitable for Bio-Plex Pro™ acute phase assays.

1. After collecting blood samples, immediately add appropriate inhibitors according to manufacturer’s instructions if necessary, invert tube several times to mix.

2. Prior to assay setup, centrifuge the samples at 13,200 rpm for 10 min at 4ºC to clear the samples of precipitate. Alternatively, carefully filter the samples with a 0.22 µm filter to prevent instrument clogging. Keep samples on ice until ready for use.

3. Refer to the steps and the table below for sample dilution instructions for preparing the 5-plex assay, the 4-plex assay, or for preparing samples for the 4-plex and 5-plex assays at the same time.

5-plex assay: Dilute 5 µl of sample in 495 µl of serum free diluent. Mix by vortexing gently or pipetting. This results in a final 1:100 sample dilution.

4-plex assay: First dilute 5 µl of sample in 495 µl of serum-free diluent to create a 1:100 dilution. Mix gently. Next, dilute 5 µl of this mixture into an additional 495 µl of serum-free diluent. Mix gently. This results in a final dilution of 1:10,000.

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