Bio-Rad Human Metabolic and Hormone Assays User Manual

Bio-Plex Pro

™

RBM

Metabolic and Hormone
Assays

Instruction Manual

For technical support, call your local Bio-Rad office, or in the U.S., call 1-800-424-6723.
For research use only. Not for diagnostic procedures.

FPO

Table of Contents

Introduction 1

Principle 2

Kit Contents and Storage 4

Recommended Materials 5

Assay Workflow 6

lmportant Considerations 7

Detailed Instructions 7

1. Plan Plate Layout 8

2. Prepare Instrument 9

3. Prepare Wash Method 10

4. Prepare Reagents 11

5. Prepare Samples 13

6. Run the Assay 16

7. Read Plate 18

Troubleshooting Guide 24

Plate Layout Template 29

Safety Considerations 30

Legal Notices 30

Ordering Information 31

Introduction

Approximately 65% of individuals with diabetes die from heart disease
and stroke. Individuals with type 2 diabetes also have an increased rate of
high blood pressure, lipid problems, and obesity, which contribute to their
high rates of cardiovascular disease (CVD). Given the complexity of these
conditions, it is not surprising that assessment of a single biomarker alone has
not yielded more than an incremental advantage over traditional measures
of assessing risk. In diabetes, further complexity is added by the common
coincidence of metabolic syndrome, which includes insulin resistance,
dyslipidemia, hypertension, and systemic inflammation, all of which contribute
to the pathogenesis of CVD (Iqbal et al. 2012). As such, researchers studying
CVD and/or diabetes need to monitor multiple biomarkers for cardiovascular
risk factors along with biomarkers directly linked to the metabolic disorders
mentioned above. These markers include gut and pituitary hormones,
adipokines, growth factors, and cardiovascular and acute phase–related
proteins. Since these hormones and proteins frequently act in concert, it
is advantageous to have the capacity to measure a number of different
biomarkers simultaneously in a single blood sample.

Bio-Plex Pro™ RBM Metabolic and Hormone Assays

The Bio-Plex Pro RBM metabolic and hormone assays include a wide
range of proteins implicated in the pathophysiology of diabetes, obesity,
and metabolic syndrome, as well as biomarkers for associated conditions
such as cardiovascular disease and inflammation.

For researchers working with limited sample volume, the capacity to
multiplex provides an effective option over the traditional ELISA method.
The use of magnetic (MagPlex) beads allows researchers to automate
wash steps on a Bio-Plex Pro (or similar) wash station.

References

Iqbal N et al. (2012). Cardiac biomarkers: New tools for heart failure
management. Cardiovas Diagn Ther 2, 147–164.

1

Principle

Technology

The Bio-Plex® multiplex system is built upon the three core elements of
xMAP technology:

n

Fluorescently dyed magnetic microspheres (also called beads), each

with a distinct color code or spectral address to permit discrimination of
individual tests within a multiplex suspension. This allows simultaneous
detection of up to 500 different molecules in a single well of a 96-well
microplate on the Bio-Plex® 3D system, up to 100 different molecules
on the Bio-Plex® 200 system, and up to 50 different molecules on the
Bio-Plex® MAGPIX™ system

n

A dedicated plate reader. The Bio-Plex 200 and Bio-Plex 3D systems

are flow cytometry–based instruments with two lasers and associated
optics to measure the different molecules bound to the surface of the
beads. In the Bio-Plex MAGPIX system, the sample is injected into a
chamber where the beads are imaged using LED and CCD technology

n

A high-speed digital signal processor that efficiently manages the

fluorescence data

Assay Format

The Bio-Plex Pro™ RBM metabolic and hormone assays are essentially
immunoassays formatted on magnetic beads. The assay principle is similar
to that of a sandwich ELISA (Figure 1). Capture antibodies directed against
the desired biomarker are covalently coupled to the beads. Coupled beads
react with the sample containing the biomarker of interest. After a series
of washes to remove unbound protein, a biotinylated detection antibody
is added to create a sandwich complex. The final detection complex is
formed with the addition of streptavidin-phycoerythrin (SA-PE) conjugate.
Phycoerythrin serves as a fluorescent indicator, or reporter. The use of
magnetic (MagPlex®) beads allows researchers to automate wash steps on
a Bio-Plex Pro (or similar) wash station. Magnetic separation offers greater
convenience, productivity, and reproducibility compared to vacuum filtration.

Note: The cortisol assay in Metabolic Panel 1 is a competitive
immunoassay (schematic not shown).

2

Biomarker
of Interest

Streptavidin

Magnetic Bead

Capture

Antibody

Fig. 1. Bio-Plex sandwich immunoassay.

Biotinylated

Detection

Antibody

Phycoerythrin

Fluorescent

Reporter

Data Acquisition and Analysis

Data from the reactions are acquired using a Bio-Plex system or similar
Luminex-based reader. When a multiplex assay suspension is drawn into
the Bio-Plex 200 reader, for example, a red (635 nm) laser illuminates the
fluorescent dyes within each bead to provide bead classification and thus
assay identification. At the same time, a green (532 nm) laser excites PE
to generate a reporter signal, which is detected by a photomultiplier tube
(PMT). A high-speed digital processor manages data output and
Bio-Plex Manager™ software presents data as median fluorescence
intensity (MFI) as well as concentration. The concentration of analyte
bound to each bead is proportional to the MFI of reporter signal.

3

Kit Contents and Storage

The Bio-Plex Pro™ RBM metabolic and hormone assays are available
in a convenient kit format that includes assay, reagent, and diluent
components in a single box (Table 1). All other recommended materials
are listed in Table 2.

Table 1. Contents of 1 x 96-well kits.

Volume after
Component Quantity Volume Reconstitution or Dilution

Capture beads (1x) 1 tube 1.4 ml

Detection antibodies 1 vial Lyophilized 4.8 ml

Standards mix 1 vial Lyophilized 150 µl

Control 1 (high) 1 vial Lyophilized 100 μl

Control 2 (low) 1 vial Lyophilized 100 μl

Blocking buffer 1 vial Lyophilized 1.5 ml

Standard diluent 1 vial Lyophilized 1.0 ml

Sample dilution buffer 1 bottle 100 ml

Assay buffer (10x) 1 bottle 60 ml 600 ml

Streptavidin-PE (10x) 1 tube 250 μl 2.5 ml

Assay plate (96-well flat bottom) 1 plate

Plate seals 1 pack of 3

Assay quick guide 1 sheet

Product data sheet 1 sheet

Storage and Stability

Kit contents should be stored at 2–8°C and never frozen. Coupled
magnetic capture beads and streptavidin-PE should be stored in the
dark. All components are guaranteed for a minimum of six months from
the date of purchase when stored as specified.

4

Tab l e 2. Recommended materials.

Item

Bio-Plex® 200 system or Luminex system with HTF

Bio-Plex validation kit

Note: Run the validation kit monthly to ensure optimal
performance of fluidics and optics systems

Bio-Plex calibration kit

Note: Run the calibration kit daily to standardize
fluorescence signal

Bio-Plex Pro wash station

For use with magnetic bead–based assays only

Bio-Plex Pro II wash station

For use with both polystyrene (nonmagnetic) and magnetic
bead–based assays

Bio-Plex handheld magnetic washer

For use with magnetic bead–based assays only

Bio-Plex Pro flat bottom plates (40 x 96-well)

For magnetic separation on the Bio-Plex Pro wash station

Titertube® micro test tubes

For preparing replicate standards, samples, and controls
prior to loading the plate

Microtiter plate shaker

IKA MTS 2/4 shaker for 2 or 4 microplates
or
Barnstead/Lab-Line Model 4625 plate
shaker (or equivalent capable of 300–1,100 rpm)

Aurum™ vacuum manifold

For vacuum filtration

BR-2000 vortexer

Reagent reservoirs, 25 ml

For capture beads and detection antibodies

Reagent reservoir, 50 ml (for reagents and buffers)

Pall Life Science Acrodisc: 25 mm PF syringe filter

(0.8/0.2 µm Supor membrane)

Filter plate, 1 x 96 with clear plastic lid and tray

Other: 15 ml polypropylene tubes for reagent dilutions, calibrated pipets, pipet tips, sterile

distilled water, aluminum foil, absorbent paper towels, 1.5 or 2 ml microcentrifuge tubes, and
standard flat bottom microplate (for calibrating vacuum manifold).

Ordering Information

Bio-Rad catalog #171-000205

Bio-Rad catalog #171-203001

Bio-Rad catalog #171-203060

Bio-Rad catalog #300-34376

Bio-Rad catalog #300-34377

Bio-Rad catalog #170-20100

Bio-Rad catalog #171-025001

Bio-Rad catalog #223-9390

IKA catalog #320-8000

VWR catalog #57019-600

Bio-Rad catalog #732-6470

Bio-Rad catalog #166-0610

VistaLab catalog #3054-1002
or

VistaLab catalog #3054-1004

VistaLab catalog #3054-1006

Pall Life Sciences
catalog #4187

Bio-Rad catalog #171-304502

5

Assay Workflow

Prepare samples, reconstitute lyophilized

reagents, dilute assay buffer to 1x,

prepare standards

Add 10 µl blocking buffer to all wells

Add 30 μl standards, blank, samples,

and controls to appropriate wells

Add 10 μl 1x capture beads per well.

Shake at 850 ± 50 rpm for 1 hr at RT

Wash 3x with 100 µl assay buffer (1x)

Add 40 μl reconstituted detection antibodies.

Shake at 850 ± 50 rpm for 1 hr at RT

Do not aspirate after incubation

Add 20 μl 1x streptavidin-PE.

Shake at 850 ± 50 rpm for 30 min at RT

Wash 3x with 100 µl assay buffer (1x)

Resuspend beads in 100 μl assay buffer (1x).

Shake at 850 ± 50 rpm for 30 sec

Read plate on Bio-Plex system

6

lmportant Considerations

Instruments and Software

The assays described in this manual are compatible with all currently
available Luminex-based life science research instruments. Assays can
be read and analyzed with either Bio-Plex Manager™ software or Luminex
xPONENT software.

Assay Procedures

Pay close attention to vortexing, shaking, and incubation times and to
Bio-Plex® reader PMT (RP1) setting, as these have been optimized specifically
for each assay panel.

Assay Quick Guide

Each assay kit comes complete with a printed Bio-Plex Pro RBM
Metabolic and Hormone Assays Quick Guide (bulletin #10041817), which
can be used to set up and prepare a full 1 x 96-well assay plate. Users
can also download a copy at www.bio-rad.com/bio-plex.

Bead Regions and Multiplexing Compatibility

n

Bead regions for all analytes are listed in the Read Plate section

n

Do not mix analytes between different panels, or with other Bio-Plex

assay panels or reagents

Detailed Instructions

The following pages provide detailed instructions for each step of the
assay procedure, including preparation, running the assay, and reading the
plate with Bio-Plex Manager™ and Luminex xPONENT software.

7

1. Plan Plate Layout

Determine the total number of wells in the experiment using the Plate
Layout Template on page 29 or the Plate Formatting tab in Bio-Plex
Manager™ software. A suggested plate layout is shown in Figure 2, with
all conditions in duplicate.

1. Assign standards to columns 1 and 2, with the highest
concentration in row A and the lowest concentration in row H.

2. Assign the blank to wells A3 and A4. The blank should consist of
standard diluent alone and be processed in the same manner as
sample and standard wells. Bio-Plex Manager software automatically
subtracts the assay blank (B) MFI value from all other assay wells.

3. User-specific controls, as well as the quality controls supplied in the
kits, are assigned to wells in columns 3 and 4.

4. The remainder of the plate is available for samples.

Legend

S Standards

B Blank

X Samples

C Controls

Fig. 2. Suggested plate layout. For detailed instructions on
plate formatting in Bio-Plex Manager, see section Read Plate.

8

2. Prepare Instrument

These directions are specific for the Bio-Plex® 100/200 reader. To prepare
either a Bio-Plex 3D or Bio-Plex® MAGPIX™ reader, consult their respective
user manuals.

Start up and calibrate the Bio-Plex system with Bio-Plex Manager™
software prior to setting up the assay. The calibration kit should be run
daily or before each use of the instrument to standardize the fluorescent
signal. For instructions on using other xMAP system software packages,
contact Bio-Rad Technical Support.

Note: While the instrument is warming up, bring the 10x assay buffer and
sample dilution buffer to room temperature. Keep other items on ice until
needed. Also, begin to thaw frozen samples.

The validation kit should be run monthly to ensure performance of fluidics
and optics systems. Refer to either the software manual or online Help for
directions on how to conduct validation.

Start Up System (Bio-Plex 100, 200, or Similar)

1. Empty the waste bottle and fill the sheath fluid bottle before starting
if high throughput fluidics (HTF) are not present. This will prevent fluidic
system backup and potential data loss.

2. Turn on the reader, XY platform, and HTF (if included). Allow the
system to warm up for 30 min (if not already done).

3. Select Start up and follow the instructions. If the system is idle
for 4 hr without acquiring data, the lasers will automatically turn off.
To reset the 4-hr countdown, select Warm up and wait for the
lasers/optics to reach operational temperature.

Calibrate System

1. Select Calibrate and confirm that the default values for CAL1
and CAL2 are the same as the values printed on the bottle of Bio-Plex
calibration beads. Use the Bio-Plex system low RP1 target value.

9