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

T

Bio Plex Pro

-

Acute Phase

Instruction Manual

Bio-Plex Manager™ software

recommended

M

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

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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/

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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.

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suspension array system is built around three core

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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

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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

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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.

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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.

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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

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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)

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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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