Bio-Rad Bio-Plex Precision Pro Human Cytokine Assays User Manual

Requires Bio-Plex

Manager™ 4.1 software

(or later versions)

Bio Plex®Precision Pro

-

Cytokine Assay

Instruction Manual

TM

For technical support, call 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 Required Materials . . . . . . . . . . . . . . . . . . . . .4

Section 4 Recommended Materials . . . . . . . . . . . . . . . . 5

Section 5 Sample Preparation . . . . . . . . . . . . . . . . . . . . 6

Section 6 Standard Preparation . . . . . . . . . . . . . . . . . . . 7

Section 7 Control Preparation (Optional) . . . . . . . . . . . . 9

Section 8 Assay Instructions . . . . . . . . . . . . . . . . . . . . 10

Plan Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Prepare Coupled Magnetic Beads . . . . . . . . . . . . . 11

Calibrate Vacuum

Assay Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Section 9 Data Acquisition . . . . . . . . . . . . . . . . . . . . . . 15

Apparatus . . . . . . . . . . . . . . . . . 11

Section 10 Troubleshooting

Section 11 Safety Considerations . . . . . . . . . . . . . . . . . .24

. . . . . . . . . . . . . . . . . . . . . . 20

Section 1
Introduction

Bio-Plex®Precision Pro™ cytokine assays are highly sensitive magnetic
bead-based multiplex assays that allow the accurate measurement of low
levels of cytokines in diverse matrices including serum, plasma, and culture
supernatant. The multiplexing feature makes it possible to quantitate the
level of multiple cytokines in a single well of a 96-well microplate in just
3 hr, using as little as 12.5 µl of serum or plasma, or 50 µl of culture
supernatant.

As one of the most recent additions to the Bio-Plex suspension array
system, these assays incorporate magnetic beads into their design. The
magnetic beads allow the use of an assay protocol similar to non­magnetic Bio-Plex cytokine assays, with the option of using magnetic
separation of wash steps instead of vacuum filtration (and allows
automation of many of the steps). The 25-bead map in Bio-Plex
Manager™ 4.1 software (or later versions) is required for data acquisition.

These assays are offered in a convenient kit format that includes assay,
reagent, and diluent components in a single box. Standard diluents for
serum and plasma are included, as are additional Iylophilized cytokines
which can be used to prepare user-specified quality controls.

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

1

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 captur
desired target cytokine is covalently coupled to internally dyed beads. The
coupled beads are allowed to react with a sample containing the target
cytokine. 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 cytokine. Streptavidin-phycoerythrin (streptavidin-PE) is then
added to bind to the biotinylated detection antibodies on the bead surface.

Data Acquisition and Analysis

Data from the reaction are then acquired using the Bio-Plex suspension
array system (or Luminex system), a dual-laser
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. This identifies each assay and reports the level of target protein
in the well. 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, the
accompanying software package.

2

®

suspension array system is built around three core

e sandwich immunoassay. An antibody directed against the

, flow-based microplate

Assay Workflow

Prewet wells

Add beads

Wash

Add standards, controls,

and samples, 1hr

Wash

Add detection antibody, 30 min

Wash

Add streptavidin-PE, 10 min

Wash

Resuspend, acquire data

3

Section 3

Coupled magnetic beads (25x) 1 vial

Detection antibodies (10x) 1 vial

Standard 2 vials

Control 1 vial

Standard diluent (serum) 10 ml

Standard diluent (plasma) 10 ml

Sample diluent 15 ml

Assay buffer 75 ml

Wash buffer 150 ml

Detection antibody diluent 15 ml

Streptavidin-PE (100x) 1 vial

Sterile filter plate (96-well) 1 plate

Sealing tape 1 pack of 4

Component Units

Required Materials

Bio-Plex®Precision Pro™ assays are offered in a convenient kit format
that includes assay, reagent, and diluent components all in a single box
(does not require separate reagent and diluent kits). These assays require
the use of Bio-Plex Manager™ software version 4.1 or higher.

Storage and Stability

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

4

eptavidin-PE should be stored in the

Section 4

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

Recommended Materials

For optimal results, the use of the items below is recommended.

5

Section 5
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 discover.bio-rad.com

Serum and Plasma Samples

Note that for plasma samples, EDTA tubes are recommended; however,
sodium citrate tubes ar
filtered with a 0.22 µm filter to prevent clogging. Hemolyzed samples are
not suitable for Bio-Plex

1. Collect and process the serum or plasma samples and assay
immediately or freeze at –20ºC. Avoid repeat freezing and thawing.

2. 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.

3. Immediately dilute 1 volume of sample with 3 volumes of sample
diluent. Keep the samples on ice until ready for use.

Culture Supernatant Samples

1. Collect and process the culture supernatant samples and assay

immediately or fr

e acceptable. Extremely lipemic samples may be

®

Precision Pro™ cytokine assays.

eeze at –20ºC. Avoid repeat freezing and thawing.

2. If required, dilute the culture supernatant with culture medium.

Serum-free culture medium should contain carrier protein (such as
BSA) at a concentration of at least 0.5%. Keep the samples on ice
until ready for use.

6

Section 6

Serum Serum standard diluent

Plasma Plasma standard diluent

Culture supernatant Same culture medium used

to prepare samples

Sample Standard Diluent

Standard Preparation

Two tubes of Iyophilized cytokine standard are provided in each Bio-Plex
Precision Pro™ cytokine assay. However, only one of the tubes is required per
96-well plate. The product insert provided with the assay lists the
concentration of the reconstituted standard. This procedure will prepare
enough standard to run each dilution in duplicate.

Reconstitute Standards

1. Gently tap the glass vial containing the lyophilized cytokine standard
on a solid surface to ensur

2. Reconstitute 1 vial of lyophilized standard with 500 µl of the
appropriate standard diluent. Do not use assay buffer to dilute
standards.

3. Gently vortex 1–3 sec and incubate on ice for 30 min. Be
consistent with the incubation time for optimal assay performance.

Prepare Standard Dilution Series

The cytokine concentrations specified for the 8-point standard dilution set
have been selected for optimized curve fitting using the 5-parameter
logistic (5PL) or 4-parameter logistic (4PL) r
Manager™ software. Results generated using dilution points other than
those listed in this manual have not been optimized.

1. Label a set of 1.5 ml Eppendorf tubes as shown in the diagram on
the next page.

e the pellet is at the bottom.

egression in Bio-Plex

®

7