Bead Coupling Technology and Applications

Bead Coupling Technology and Applications
Wenming Liu
Field Application Specialist
Content
the protein assay development
the nucleic acid assay development

Capture Sandwich Immunoassay
Most common
approach
SAPE
Used in most
biotin
commercial assays,
including Bio-Rad
Cytokine, Phospho-
protein and Diabetes
kits
Requires two
antibodies (capture
and detection)
Protein Assay Development Workflow
1. Obtain reagents for assay
Analytes
biotin
Detection Antibodies
Capture Antibodies
Beads
77
73
34
2. Couple the proteins (or, peptides) to bead

Attachment of protein molecules to the microsphere
surface (most often through formation of covalent bond)
3. Confirm the coupling with anti-species antibody.
SAPE
4.Test high standard or high positive control
5. Select the best performers
6. Moderate optimization of each simplex

7. Begin Multiplex. Use multiplexed bead sets but separate standards,

controls, and detection antibodies
Prepare standards and/or controls in sample matrix.

Optimize:
Sample and reaction volumes
Number of beads per reaction (2000-5000 per set)
Incubation times 77
Detection antibody and reporter concentration 73
34
Coupling amount for capture reagents
SAPE
Best assay format (washed vs. no wash)
8. Re-test reagents with new conditions.
9. Test optimized assay with true samples.

Selection Of Bead Regions
Avoid the low and

high bead numbers
(least and most
amount of dye,
respectively)
Avoid the bead

regions located at the
edge of the map
It is perfectly OK to
use neighbouring
bead regions e.g. 25,
26 and 27
Incompatible Buffers and Solvents
Certain solvents should not be used with microspheres. They may affect
the classification dyes in the microspheres.
High Salt As the salt concentration of the buffer increases, the

microspheres will tend to spread out on the bead map. High salt buffers
(6X SSC, > 0.2 M NaCl) should be diluted or exchanged prior to analysis
as they may interfere with microsphere classification.
Aromatic hydrocarbons
Benzene
Hydrocarbons
Toluene
Methylene chloride
Xylene
Chloroform
Ethylbenzene
Carbon tetrachloride
Chlorinated aliphatic
Incompatible Buffers and Solvents
Others
Pyridine Ethyl acetate
Dioxane Butyl acetate
Dimethylformamide 1-nitro-propane
Methyl ethyl ketone Carbon disulfide
Diisopropyl ketone Tributyl phosphate
Cyclohexanone Cyclohexane
Tetrahydrofuran Methylcyclohexane
N-butyl phthalate Ethylcyclohexane
Methyl phthalate Acetone
Ethyl phthalate DMSO
Tetrahydrofurfuryl alcohol
tested 10% DMSO at 37C for 1 month with no obvious change in microsphere
properties. 20% DMSO was shown to have no effect over a period of several hours,
but no long-term studies have been done at this concentration.
Protein Coupling
The Goal: Attachment of protein molecules to the

microsphere surface (most often through formation of
covalent bond between COOH group on bead and NH2
group on protein)
Selection Of Capture Antibodies For
Sandwich Assay
Monoclonal antibody is recommended for capture
Where possible, select antibodies that have been tested and found
suitable for capture in ELISA. Many companies include reference to
which clones are suitable as capture antibodies in ELISA.
Choose antibodies appropriate for protein configuration e.g. antibodies

approved for use in Westerns probably detect the denatured form of
the protein and are unlikely to be usable for detecting native proteins
Linscott Directory lists available commercial antibodies and sources, as

well as suitability for ELISA, Immunoprecipitation, Western blotting, etc
Protein Coupling
Part 1: Microsphere Activation

Microspheres are washed to remove antimicrobials and storage
solution. Activate microspheres via EDC and Sulfo-NHS to yield a
long lived intermediate Sulfo-NHS Ester.
EDC Sulfo-NHS
Carboxylate- Semi-stable amine-

Modified Unstable reactive
o-Acylisourea reactive NHS Ester
Micropshere Intermediate
Intermediate
Protein Coupling
Part 2: Washing
All unreacted EDC and Sulfo-NHS is then removed by several washes

to prevent activation of carboxyl groups on the protein molecule which
would result in protein-protein coupling rather than protein-
microsphere coupling.
Protein Coupling
Part 3: Coupling
Sulfo-NHS esters on the surface are combined with a
protein solution and allowed to mix for a minimum of two
hours. Free amines on the protein side chains interact with
the intermediate to form a covalent bond with the
microsphere.
Semi-stable Coupled
amine-reactive Microsphere
NHS Ester Sulfo-NHS
Intermediate
Protein Coupling
Part 4: Blocking
Remove excess unbound protein and block microspheres with
detergent and BSA
Protein Stock
Protein stock should not contain foreign protein, azide, glycine,

Tris, Bovine Serum Albumin (BSA), or any primary amines.
If any of these agents exist in the protein preparation, remove
them through dialysis, or chromatography.
Microcentrifuge Tubes and
Transfer Pipettes
Uncoupled microspheres tend to stick to the walls of most tubes,
resulting in poor post-coupling microsphere recovery.
ALWAYS use USA Scientific microcentrifuge tubes (cat. #: 1415-

25000) for coupling. They yield the highest microsphere recoveries
post-coupling.
ALWAYS centrifuge the tubes with the hinge facing outwards and
remove the liquid from the opposite side of the tube using transfer
pipettes with a narrow bore/tip and large bulb.
The recommended transfer pipettes are made by Samco and can

be obtained from VWR (cat. #: 14670-333), or Fisher Scientific (cat.
#: 13-711-29).
How Much Protein Should Be Coupled To
The Beads?
Antibody Coupling to xMAP Microspheres

5-12 g per 1x Microsphere Stock (Amine Coupling Manual)
Needs to be tested empirically
Antigen Coupling to xMAP microspheres

Start with 5x less protein than antibody coupling
Needs to be tested empirically
Microsphere stock is 1.25 x 107 beads/ml

1x Stock is 100 ul i.e. 1.25 x 106 beads
Coupling Confirmation
Goal: To determine coupling efficiency via a biotin-labeled anti-species IgG
detection antibody, followed by streptavidin-PE (or a PE-labeled anti-species
IgG detection antibody)
Coupling Confirmation (Indirect)
Assay Setup: Streptavidin-PE Dilution
(50 ul)
Bi-labeled Anti-Species
SA-PE
IgG Dilution (50 ul)
Bi
SA_PE
30 minute incubation,
followed by three
washes with each
component. Analyze
on the Bio-Plex
Coupled Microspheres System
(50 ul)
Coupling Optimization
Choose the coupling concentration that gives the best sensitivity and dynamic range
for your needs.
Signals decrease as capture antibody concentrations increase

Microspheres may be over conjugated.
More is not always better.
Select the capture antibody concentration that provides the best result
Not necessarily the concentration with the most efficient coupling.
Potential Coupling Problems
Microsphere loss
Biggest contribution is the brand of microcentrifuge tube used
USA Scientific (catalog # 1415-2500)
Eppendorf Protein LoBind (catalog # 022431081)
Removing too much supernatant during wash steps

Pellet is very fluffy
Free amine groups

Any available amine will couple to the microspheres
BSA or other carrier proteins
Amine containing buffers, Glycine
TRIS and Azide should also be avoided
Amine containing detergents
Reagent Stability
EDC and Sulfo NHS are both water sensitive
Store desiccated at -20C
Sometimes proteins are poor quality

A good coupling is vital before moving forward with developing the rest of the assay
Coupling Confimation
30000
25000
Good Coupling
20000
MFI
15000
10000
5000 Bad Coupling

0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
PE-Anti-Species IgG (ug/mL)
Selection Of Detection Antibodies
A monoclonal to a different part of the protein is recommended for

detection. Polyclonal antibodies are also usable in many instances
Where possible, select antibodies that have been tested and found
suitable for detection in ELISA. Many companies include reference to
which clones are suitable as detection antibodies in ELISA. They often
sell these antibodies in a biotinylated form.
Choose antibodies appropriate for protein configuration e.g. antibodies

approved for use in Westerns probably detect the denatured form of
the protein and are unlikely to be usable for detecting native proteins
Linscott Directory lists available commercial antibodies and sources, as

well as suitability for ELISA, Immunoprecipitation, Western blotting, etc
Detection Tips
Capture Sandwich immunoassays

Use 2-4 g/mL detection antibody.
Optimize detection antibody concentration. Start with 4 g/mL and titrate

down to 1 g/mL by two-fold dilutions.
Optimal detection antibody concentration depends on reagents and level

of multiplexing.
Concentrations often need to be increased when increasing the number

of multiplexed assays and when converting to a no-wash assay format.
Relative Reporter Intensities
Streptavidin-Phycoerythrin, Alexa 532, and Cyanine 3 as the most
suitable reporter fluorochromes.
General Assay Tips
Run background samples, at least in duplicate.
Run all samples at least in duplicate, whenever sample allows.
Streptavidin-Phycoerythrin (SA-PE) gives the highest signal of the

dyes have compared.
Dilute concentrated biological samples at least 1:5.

This will overcome matrix effects which can interfere with analysis of the
microspheres.
If samples cannot be diluted, try using a small initial reaction volume and
diluting the final reaction prior to analysis on the Bio-Plex instrument.
Minimize the presence of detergents in samples.

Washed Assay Tips
Use wash steps to increase sensitivity.
Wash after each incubation, or at the end of the assay.
Immediately before adding samples, wet the filter plate with 100 L
Assay Buffer and vacuum.
Avoid vacuuming to dryness.

The filter membrane should appear glossy once all of the solution has been
vacuumed through.
To resuspend the beads, add wash buffer and mix with a multi-channel
pipette.
Blot the bottom of the plate after washing and before resuspending the
beads.
If buffer is beneath the membrane, it will act like a wick and pull the solution
through.
x-Reactivity Test
Biotin SAPE
Biotin SAPE
x-Reactivity Test
x-Reactivity Studies
Multiplex beads/capture Ab, multiplex detection Ab and multiplex
antigen
Multiplexing beads/capture Ab, multiplex detection Ab and single

antigen
Multiplexing beads/capture Ab, single detection Ab and multiplex

antigen
Multiplex beads/capture Ab, multiplex detection Ab and multiplex

antigen minus one target
Single Antigen x-Reactivity Test
Multiplexing beads/capture Ab, multiplex detection Ab and

single antigen
Each point in the standard curve must be at least in duplicate.
The standard curve will have all components in multiplex (beads,
antigen and detection).
Any single Ag to be tested must be at least in duplicate at any
given concentration.
The zero antigen point (background) is not part of the standard
curve.
The test antigen is usually tested at the concentration equivalent
to the second highest concentration of the standard curve (S2)
Cross-reactivity analysis based on MFI, or concentration values
is possible.
Ag layout for Single Ag Component Cross-Reactivity
SAPE 2 g/mL
2
Ab Multiplexed 2o Ab
Ag Multiplex Ag Individual Ag as designated
1
Ab Multiplexed 1 Ab beads
S1 S1 0
S2 S2 0
S3 S3 1
S4 S4 1
S5 S5 2
S6 S6 2
S7 S7 3
S8 S8 3
(Positive Signal Background) single antigen solution

% Cross Reactivity MFI = X 100%
(Positive Signal Background)standard curve
Calculated Conc in single Ag Solution

% Cross-Reactivity conc = X 100%
Observed Conc. in standard curve
Single Detection Ab x-Reactivity Test
Multiplexing beads/capture Ab, single detection Ab and multiplex

antigen
All data points on plate (single detection Ab and standard curve) must
be at least in duplicate.
For single detection Ab cross-reactivity, a single concentration point
of antigens (the equivalent of the second highest point of the
standard curve) is sufficient.
Background can not be defined as blank because there is no single
background that can apply to all conditions. Each condition with a
different detection Ab must have its own background control, as
different bead and detection Ab combination results in different
background signal. Background must be later subtracted during data
analysis.
The standard curve will be an 8-point standard curve with all

components in multiplex (beads, antigen and detection).
Cross-reactivity analysis based on MFI values is adequate for
detection antibody cross-reactivity evaluation.
Secondary layout for Single Detection Ab Component Cross-Reactivity for Detection Ab
SAPE 2 g/mL
Multiplexed 2o
2
Ab Ab Individual 2 Ab as designated
Ag Multiplex Ag/background
1
Ab Multiplexed 1 Ab beads
S1 S1 0 0
A-no antigen
S2 S2 0 0
B-S2 antigen
S3 S3 1A 1A
S4 S4 1B 1B
S5 S5 2A 2A
S6 S6 2B 2B
S7 S7 3A 3A
S8 S8 3B 3B
(Positive Signal Background)target with single detection Ab

% Cross Reactivity MFI = X 100%
(Positive Signal Background)target in standard curve with multiplex detection Ab
B
B
protein nucleic acid

Nucleic Acids-DNA Overview
DNA Analysis
Direct Hybridization to Hybridization of

Sequences of Interest Enzymatically Generated
Products to xTAG
(FlexMAP) Microspheres
Direct Hybridization
Direct Hybridization to
Sequences of Interest
5
3
PCR: Biotin
5 3
Precursor Step - Perform PCR amplification of Genomic DNA. One

primer should be labeled with biotin.
Direct Hybridization to
Sequences of Interest
5
3
PCR: Biotin
5 3
Microsphere:
Oligo Probe
Step 1- Denature at 95C, TMAC buffer
5
3 Biotin
5 3
Oligo Probe
Direct Hybridization Experiment
Step 2 - Anneal Probe to Biotinylated Complementary DNA Sequence at

appropriate melting temperature e.g. 52C
5
3 Biotin
Step 3 - Label complex with Streptavidin-Phycoerythrin (SA-PE) at

appropriate Hybridization Temperature and detect on Bio-Plex System
5
3 Biotin
SAPE
Eventually you can multiplex up

to100 different PCR targets,100
microspheres and 100 probes
Nucleic Acids-DNA Overview
DNA Analysis
Direct Hybridization to Hybridization of

Sequences of Interest Enzymatically Generated
Products to xTAG
(FlexMAP) Microspheres
xTAG Allele-Specific Primer Extension
(ASPE)
1. Generate PCR product containing sequence of interest

(ASPE)
2. Enzymatic reaction with allele specific (SNP Specific) primer to

create product with xTAG complement
SNP
Allele Specific Primer
xTAG Anti-TAG
(ASPE)
2 (cont). Enzymatic reaction extension with allele specific (SNP

Specific) primers to create product with xTAG complement.
Biotin is added as biotin-dCTP (the C is omitted from the
dNTP mix).
SNP biotin biotin
xTAG Anti-TAG
(ASPE)
3. Hybridize the enzymatic product to the xTAG microspheres

at 37C and then incubate with SA-PE at the same
temperature. Analyze on Bio-Plex System
xTAG TAG
SNP
biotin biotin SAPE

SAPE
xTAG Anti-TAG
(ASPE)
Eventually you can multiplex up
to100 different PCR targets,100
microspheres and 100 probes
xTAG Microspheres-Overview
Why Use xTAG Microspheres Instead of Direct Hybridization?
Past ten to twelve targets, optimizing a direct hybridization assay

becomes more difficult
-All probes must be designed to hybridize at the same temperature

-Cross hybridization must be minimized
-Size restraints on PCR products (<300 bp)
-Secondary structure in PCR products
-Competition from other PCR strand
Intended Use
Direct Hybridization xTAG
Single nucleotide discrimination Single nucleotide discrimination
Two SNPs within 8 nucleotides Two SNPs

of each other or more than 20 (ASPE primers on opposite
nucleotides apart strands)
Multiple polymorphisms spaced Multiple polymorphisms spaced

less than 15 20 nucleotides more than 15 20 nucleotides
apart apart
(far enough apart for annealing
Unrelated sequences of TAG-ASPE primers)
Coupling
Couple sequence specific No Coupling

probes to microspheres
Microspheres are pre-coupled to
Hybridizing to a PCR-amplified the capture sequences (anti-
target TAGs)
One primer is biotinylated to

label the target strand
PCR
Works best with a small PCR- PCR with unlabeled primers to

amplified target up to 300 make a genotyping template
base pairs
Size is unrestricted
Larger targets can be used, but
how well they work often Clean up PCR with an Exo-SAP
depends on the sequence reaction (for ASPE)
Put into an enzymatic

genotyping reaction (ASPE)
where the corresponding
address sequence (TAG) is
incorporated into the genotyping
product
Temperature
Single nucleotide discrimination Hybridization temperature is

hybridization temperature is always the same - 37C
usually 45 55C
Works well for setting up many
Multiple polymorphisms, or samples because the
unrelated sequences hybridization is always at 37C
hybridization temperature is
usually 37 45C Plates can be held without
worrying about evaporation or
decreasing reporter signal
Time
PCR = 2 hrs PCR = 2 hrs
Assay = 35 mins excluding wash Exo-SAP = 30 mins

steps which may not be needed
ASPE = 2 3 hrs
Reading Time = about 20 40
minutes per plate Assay = 47 mins excluding wash
steps which are needed
TOTAL = 3 hrs 15 mins
Reading time = 20 40 mins
TOTAL = 6 hrs 40 mins

Cost
About $0.10 per sequence About $0.25 per sequence
Costs more because of the

additional enzymes
The microspheres cost more as

well because they are pre-
coupled
Probe/Primer Design Strategy
Recommendations for Probe/Primer Design for Direct Hyb Assay
C-12 Linker
Probe sequence
3
Probe Design for SNP Analysis

18 to 24 nucleotides (start at 20)
For each target, probes are the same length
For each target, all probes from the same strand
SNP centered, multiple SNPs spread evenly
Carbon spacer (C12 or UniLink) on Oligos
5 Amino (NH2) modifier on Oligo
Probes for multiple targets have similar melting temps
DO NOT RESUSPEND IN TE!
Probe Design for non-SNP applications e.g.Gene expression,

bacterial detection
50 to 70 nucleotides
HPLC purification
Probes for multiple targets have similar melting temps
DO NOT RESUSPEND IN TE!
Direct Hybridization-Coupling Oligos
Use standard conjugation techniques (for detailed info check out

Pierce website, or Bioconjugation Techniques book)
Microspheres contain carboxyl (COOH) groups. Couple to amine
(NH2) group on biomolecule
5 Amino modifiers added during oligonucleotide synthesis
AmMC12: 5 amino C12 modifier
Image from www.idtdna.com

PCR Primer Design Strategy
Primer Design
Amplicons should be in the 100 to 300 base pair range.
Longer targets may be used successfully.

Hybridization efficiency is dependent on sequence and secondary structure in
addition to length.
One PCR primer must be labeled with a 5-biotin (to bind streptavidin-
phycoerythrin reporter)
The labeled strand of the amplicon must be complementary to the

capture probes on the microspheres.
The Importance Of Primer Design
Like any PCR-based assay, the key to creating a successful

multiplex assay is choosing the right oligonucleotide primers.
Coupling Tips
Amine-substituted oligonucleotide probes should ALWAYS be

resuspended and diluted in dH20.
No Tris, sodium azide or other amine-containing buffers should be

used.
The amine groups on these molecules will compete with the amine groups
on the oligonucleotide for coupling to the carboxyl group on the beads.
For oligonucleotide coupling, TE buffer is the most common problem.

Coupling Tips
Uncoupled microspheres tend to stick to the walls of most tubes,
resulting in poor post-coupling microsphere recovery.
ALWAYS use USA Scientific microcentrifuge tubes (cat. #: 1415-

25000) for coupling. They yield the highest microsphere recoveries
post-coupling.
ALWAYS centrifuge the tubes with the hinge facing outwards and
remove the liquid from the opposite side of the tube using transfer
pipettes with a narrow bore/tip and large bulb.
The recommended transfer pipettes are made by Samco and can

be obtained from VWR (cat. #: 14670-333), or Fisher Scientific (cat.
#: 13-711-29).
Coupling Tips
100 mM MES, pH 4.5 (Coupling buffer)

It should be filter-sterilized and either prepared fresh, or stored at 4C
between uses.
Do not store at room temperature. If left at room temperature, the pH
of the MES buffer will increase and result in less efficient coupling
The pH must remain in the 4.5-4.7 range for optimal coupling efficiency
Warm to room temperature just prior to use.
Development Reagents
Procedure: Protein purification (10 min)
1.4x106 beads
Activated by EDC and S-NHS

(20 min incubation)
Add protein (2 hrs incubation)
Blocking (30 mins incubation)
Resuspending the beads in storage buffer

(success of coupling reaction can then be assessed)
Nucleic Acid Application
Protocol for of probe oligonucleotides to microspheres.

Stock microspheres (1 ml; 1.25107 microspheres)
Vortex for 30 s, sonicate for 60 s, and centrifuging at 8,000g for 5 min, then removing supernatant
Resuspending microspheres in MES (50ul, 0.1M, pH 4.5), vortex, and sonicate
Adding probe (10ul; 50uM in MES) and vortex
Adding aqueous solution of EDC(5 l, 10 mg/ml) ,vortex, and gently agitating for 30 min in the dark.
Second adding aqueous solution of EDC(5 l, 10 mg/ml) ,vortex, and gently agitating for 30 min in the dark.
Adding Tween 20 (1 ml, 0.02% [vol/vol]) vortex, and centrifuge, removing the supernatant
washing repeatedly microspheres by SDS (1 ml, 0.1% [mass/vol]) and then TE buffer.
Resuspending the probe-conjugated microspheres in TEbuffer(250 ul), vortex, then storing at 4C in the dark.

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Bead Coupling Technology and Applications

the protein assay development

the nucleic acid assay development

1. Obtain reagents for assay

2. Couple the proteins (or, peptides) to bead

3. Confirm the coupling with anti-species antibody.

4.Test high standard or high positive control

5. Select the best performers

6. Moderate optimization of each simplex

7. Begin Multiplex. Use multiplexed bead sets but separate standards,

Prepare standards and/or controls in sample matrix.

8. Re-test reagents with new conditions.

9. Test optimized assay with true samples.

Avoid the low and

Avoid the bead

High Salt As the salt concentration of the buffer increases, the

The Goal: Attachment of protein molecules to the

Choose antibodies appropriate for protein configuration e.g. antibodies

Linscott Directory lists available commercial antibodies and sources, as

Part 1: Microsphere Activation

Carboxylate- Semi-stable amine-

All unreacted EDC and Sulfo-NHS is then removed by several washes

Protein stock should not contain foreign protein, azide, glycine,

ALWAYS use USA Scientific microcentrifuge tubes (cat. #: 1415-

The recommended transfer pipettes are made by Samco and can

Antibody Coupling to xMAP Microspheres

Antigen Coupling to xMAP microspheres

Microsphere stock is 1.25 x 107 beads/ml

Signals decrease as capture antibody concentrations increase

More is not always better.

Removing too much supernatant during wash steps

Free amine groups

Sometimes proteins are poor quality

5000 Bad Coupling

A monoclonal to a different part of the protein is recommended for

Choose antibodies appropriate for protein configuration e.g. antibodies

Linscott Directory lists available commercial antibodies and sources, as

Capture Sandwich immunoassays

Optimize detection antibody concentration. Start with 4 g/mL and titrate

Optimal detection antibody concentration depends on reagents and level

Concentrations often need to be increased when increasing the number

Run all samples at least in duplicate, whenever sample allows.

Streptavidin-Phycoerythrin (SA-PE) gives the highest signal of the

Dilute concentrated biological samples at least 1:5.

Minimize the presence of detergents in samples.

Avoid vacuuming to dryness.

Multiplexing beads/capture Ab, multiplex detection Ab and single

Multiplexing beads/capture Ab, single detection Ab and multiplex

Multiplex beads/capture Ab, multiplex detection Ab and multiplex

Multiplexing beads/capture Ab, multiplex detection Ab and

Ag layout for Single Ag Component Cross-Reactivity

Ag Multiplex Ag Individual Ag as designated

(Positive Signal Background) single antigen solution

Calculated Conc in single Ag Solution

Multiplexing beads/capture Ab, single detection Ab and multiplex

The standard curve will be an 8-point standard curve with all

Secondary layout for Single Detection Ab Component Cross-Reactivity for Detection Ab

(Positive Signal Background)target with single detection Ab

protein nucleic acid

Direct Hybridization to Hybridization of

Precursor Step - Perform PCR amplification of Genomic DNA. One

Step 1- Denature at 95C, TMAC buffer

Step 2 - Anneal Probe to Biotinylated Complementary DNA Sequence at

Step 3 - Label complex with Streptavidin-Phycoerythrin (SA-PE) at