Scintillation proximity assay (SPA)
Radioligand is bound in
close proximity stimulating
the bead to emit light
principl.ppt
Unbound radioligand does
not stimulate the bead
Applications of the SPA technology
!Radio-immunoassays
!Receptor
!Enzyme
binding assays
assays
!Molecular
!Cell
applic.ppt
interaction assays
based assays (Cytostar-T)
Review information in literature/current
practices in the laboratory
!
Most if not all of your present assay
conditions can be transferred to SPA
!
SPA assay development
Optimization
Reference
Development
Application area
of
reference assay
Receptors - 3H, 125I
DNA - 3H, 33P
RIA - 125I
Enzymes - 3H, 33P, 35S
Kinases - 33P
Comp A/B - 3H, 125I, 35S
Capture method
Bead selection
Binding capacity
Label/NSB ratio
Bead addition
format
Delivery
Validation
Plate format:
96 or 384
Matrix experiments:
Bead amount
versus
assay components
Optimal reagent concentration
Reduce NSB/NPE
Stable counting window
Adequate signal to noise
Assay validation
Colour quench
curves
Automation
and
High
Throughput
Screening
SPA assay development
Reference
Application area
of reference
assay
Receptors - 3H, 125I
DNA - 3H, 33P
RIA - 125I
Enzymes - 3H, 33P, 35S
Kinases - 33P
Comp A/B - 3H, 125I, 35S
Development of
SPA assays
should be
referenced to an
existing assay
e.g. filter or gel
based assay
Common radioisotopes used in SPA
Mean Path Length
[3H]
[125I]
[14C]
[35S]
[33P]
[32P]
istop2.ppt
1.5µm
2e- 1.0µm ; 17µm
50µm
65µm
125µm
2100µm
Choice of isotope: general considerations
!
Specific activity
– 1-100 mCi/mmol - 14C
– 1-85 Ci/mmol - 3H
– 600-2000 Ci/mmol - 125I
– 1000-3000 Ci/mmol - 33P
– ~1000 Ci/mmol - 35S
!
High energy isotopes, 125I, 33P, 35S, and 14C
– Low expression levels, low purity and
concentration of reagents
! Low energy isotopes, 3H
– Low affinities, high expression levels, high
purity and concentration of reagents
SPA Assays: points of consideration
!
Receptor binding assays - 3H and 125I
–Expression level of receptor influences selection of
radioligand
–Typical levels of expression in tissue or cell-line
are:• 200fmol/mg membrane protein for 125I ligands
• 2pmol/mg membrane protein for 3H ligands
–Affinity for receptor, Kd’s
!
Enzyme assays - 3H, 33P, 35S, 125I
–Activation / co-factors
–Purity of substrate and enzyme
–Specific or generic substrate
–Km of enzyme for chosen substrate
SPA Assays: points of consideration
!
Molecular binding assays - 3H, 125I, 35S, 33P
– Expression levels of individual components
– Concentration and purity of binding components
– Kd of assay components
– Capture method
!
Nucleic acid assays - 3H, 33P
– Experimental design and labeling options
– Concentration and purity of DNA, RNA and
oligonucleotide
!
RIA assays - 125I, 3H
• availability of antibody
• epitope recognised by the antibody
• affinity of antibody for target molecule
SPA assay development
Reference
Development
Application area
Capture method
of
Bead selection
reference assay
Binding capacity
Label/NSB ratio
Receptors - 3H, 125I
Bead addition
DNA - 3H, 33P
format
RIA - 125I
Enzymes - 3H, 33P, 35S
Kinases - 33P
Comp A/B - 3H, 125I, 35S
Bead coupling/capture strategies
germ agglutinin - N acetyl β
D- glucosamine (NAG) moieties
–Receptor ligand binding
!Streptavidin - biotin
!Antibody capture
!Fusion protein specific beads
–GST and His tagged proteins
!Physico-chemical interactions
–charged based coupling
!Wheat
SPA Bead Type
Applications
Binding capacity
(based on model assays)
Donkey-anti rabbit, PVT and YSi
Sheep-anti mouse, PVT and YSi
Donkey-anti sheep, PVT and YSi
Protein A, PVT and YSi
Radioimmunoassays,
molecular interactions
~0.4µg antibody/mg bead
Wheat germ agglutinin, PVT and YSi Cell membrane
PEI-WGA Type A, PVT
bound receptors
PEI-WGA Type B, PVT
Polylysine, YSi
10-30µg protein/mg bead
10-30µg protein/mg bead
10-30µg protein/mg bead
10µg protein/mg bead
Streptavidin, PVT
Streptavidin, YSi
Enzymes assays,
molecular interactions,
receptor binding,
100pmol/mg bead
200pmol/mg bead
Glutathione, PVT
Glutathione, YSi
Enzymes assays,
molecular interactions,
Application dependent
Copper His-tag, PVT
Copper His-tag, YSi
Enzymes assays,
molecular interactions
Application dependent
RNA binding beads
Charged interactions
Application dependent
beadtype.ppt
Coupling strategies for SPA beads
Wheatgerm Agglutinin beads
(WGA)
Wheatgerm agglutinin binds
to n-acetyl glucosamine residues
on glycoproteins and glycolipids
Streptavidin beads
Streptavidin binds to biotinylated
proteins, peptides and oligonucleotides
Coupling Strategies for SPA Beads
GST fusion
protein
6x-His fusion protein
Copper
Direct
radiolabel
Cu2+
*
Glutathione
*
Indirect radiolabel
binding partner
His
His
His
His
His
His
Direct
radiolabel
*
*
Indirect
radiolabel
binding partner
The Select-a-bead kit for receptor binding
assays
! A pack of 5 different SPA bead types
(RPNQ0250)
! A convenient method for assessing which bead
types are appropriate for a new receptor binding
assay
! Each kit contains 100mg each of:
–PVT WGA
–PVT WGA (PEI Type A)
–PVT WGA (PEI Type B)
–YSi WGA
–YSi Polylysine
Determine non specific binding (NSB) from
label / bead interactions
! Determine counts bound to bead as a
percentage of total counts added to well
(label/bead NSB)
– combine bead, buffer and label
– consult NSB table
! Examine non proximity effect (NPE)
– signal generated from non bound label or
ligand
Reagents that can reduce NSB
!NSB reduction is assay dependent
!Various additives include:
– Proteins such as BSA, peptone, casein, (0-1.0%)
– Salts such as NaCl, MgCl2, MnCl2, KCl, ZnSo4
(0-500mM)
– Detergents such as Tween-20, CHAPS, SDS, Nadeoxycholate, PEI, octylglucoside, Triton X-100
(0-1.0%)
– Nucleic acids such as tRNA, salmon sperm DNA,
oligonucleotides (0-2.5mg/ml)
– Ligand or assay mimics, ‘cold’ ligand
– Plate type, Corning NBS plates found to reduce
NSB for various ligands
Non proximity effect (NPE)
(NPE
Unbound isotope is close
enough to stimulate bead
radiolabelled ligand
npefig.ppt
High energy isotopes and SPA
!In
suspension, two
effects are apparent:
!High energy particles
from a specific binding
interaction passing
through the bead
!Non proximity effect
(NPE) of high energy
particle
!Example would be 33P,
35S, 14C, 125I
33psusp.ppt
The effect of bead packing on specific
signal
!Concentrating
the beads
by either centrifugation,
floating or settling:
!Reduces
the non-proximity
effect
!Harvests the energy of the
specific binding
interactions more
efficiently
33psettl.ppt
Specific SPA (cpm x 10-3)
Comparison of [33P] SPA signal
generation techniques
80
Centrifuged
Settled (1 hour)
CsCl (1 hour)
Settled (o/n)
CsCl (o/n)
60
40
20
0
0.00
0.25
0.50
Erk 1 (µ
µg)
sigcomp.ppt
0.75
1.00
Effect of SPA PVT bead settling on cpm
using a Packard TopCount
10000
cpm
8000
6000
4000
[3H] PVT beads
2000
[125I] PVT beads
0
0
2
4
6
8
Time (hours)
10
12
14
Example of a partial matrix experiment to
reduce NSB
Major variables
Minor variables
Various salt
concentrations
A
B
C
e.g. NaCl2 MgCl2 MnCl2
D
Cold ligand
Ligand mimics
E
F
G
H
topcount.ppt
0.5% BSA
1.0% BSA
0.5% Triton
0.5% BSA
1
4
7
2
3
5
6
8
9
1.0% Triton
1.0% BSA
10 11 12
Various capture formats for SPA assays
Pre-coupled bead
Delayed addition
recformat.ppt
T=0 addition
Pre-coupled format
!Potential
!Assay
improvement of signal to noise ratio
kinetics can be followed
!Proteases/inhibitors/competitors
may be
removed by washing after coupling
!If
aggregation is seen,
!Sonicate, vortex, add small amount of
detergents
precoup.ppt
T=0 format
All components are added to the assay at
the same time
!
!Excess
of bead required to capture all
reactants
!Common
!Easy
toform.ppt
format for HTS
to configure and automate
Delayed addition format
!Used
in most enzyme and kinase assays,
combined with stop reagents (EDTA, pH
change)
!Excess
of bead required for capture
!Volume
changes in delayed addition format
may affect kinetics
toform.ppt
!Allows
sampling from assay
!Similar
to traditional fluid phase assay
Common assay formats for enzyme
scintillation proximity assay
!
!
!
!
enzymcon.ppt
Signal increase
Transferase and
polymerase
activities
Signal decrease
Hydrolase activities
e.g. proteases,
nucleases
SPA assay development
Optimization
Reference
Development
Application area
of
reference assay
Receptors - 3H, 125I
DNA - 3H, 33P
RIA - 125I
Enzymes - 3H, 33P, 35S
Kinases - 33P
Comp A/B - 3H, 125I, 35S
Capture method
Bead selection
Binding capacity
Label/NSB ratio
Bead addition
format
Plate format:
96 or 384
Matrix experiments:
Bead amount
versus
assay components
Optimal reagent concentration
Reduce NSB/NPE
Stable counting window
Adequate signal to noise
Plate format
!Choice of plate format
– 96 well plate
– 384 well plate
!Consider potential signal, signal
window, reagent usage, cost and
counting time
topcount.ppt
Matrix experiments
!Bead optimization
!Capture molecule optimization
– Membrane/receptor
– Substrate and enzyme
– Binding component A
!Isotope optimization
– Ligand
– Label
– Binding component B or more
Matrix experiments
! Approximate amount of bead used per
application
– Receptor binding assays: 500µ
µg - 2.0mg
– Enzyme assays: 250µ
µg - 1mg
– Radioimmunoassays: 250µ
µg - 1.5mg
– Molecular interaction assays: 250µ
µg - 2.0mg
– Ionic interaction assays: 250µ
µg - 1.5mg
! Matrix experiments will define exact amount
Example of a partial matrix experiment
for a receptor binding assay
Membrane protein
Constant amount of
labeled ligand
g
0µ
1
Vary amount of
reagents to
reduce NSB,
inhibitors, etc.
in rows B, D, F
and H
A
B
C
D
E
F
G
H
topcount.ppt
2
3
10
4
5
µg
6
20
7
8
µg
9
40
µg
10 11 12
0.25 mg
bead
0.5 mg
bead
1.0 mg
bead
2.0 mg
bead
Optimal assay conditions
Chose appropriate plate format
! Identify from matrix experiments
– concentration of bead
– concentration of capture molecule
– concentration of label or ligand
! Maximize assay performance
– incubation, temperature, shaking, reagent
stability
– reduce NSB and/or NPE, if needed
! Achieve acceptable signal to noise
!
assay1.ppt
Determination of stable counting
window
Must be established experimentally for
each application and assay (cycle count
plate)
! Binding of membrane to bead requires 30
minutes
! Streptavidin/biotin, ionic interaction, GST
and 6x His tag-based binding usually
occur within 30 minutes
! Antibody based assays require longer
equilibration which should be established
experimentally
!
recctwin.ppt
Determination of stable counting window
!Kinetic
assays
– receptor binding assays
– enzyme assays
!Stabilize
assay components
– protease inhibitors (cocktail)
– phosphatase inhibitors
– other factors (BSA, RNA, DNA, assay
mimics)
recctwin.ppt
Trouble shooting - low counts
If low counts are encountered, should examine
!Ability of bead to bind capture molecule
–Streptavidin, glutathione, copper, antibody,
ionic
!Ability of captured molecule to interact with label
–receptor level, affinity of assay components,
interfering substance, stability of complex
!Non-isotopic labeling of the substrate
(biotinylation)
–biological activity
–specificity for substrate
Trouble shooting - low counts
If low counts are encountered, should
examine
!Specific activity
–1-100 mCi/mmol - 14C
–1-85 Ci/mmol - 3H
–600-2000 Ci/mmol - 125I
!Counter settings
–Optimize window settings
–Nuclide library
Trouble shooting - high background
!Ability of bead to bind label directly
–examine bead/label NSB, less than 10%?
–label binding to bead via assay components,
breakdown products
–if a high energy isotope used, NPE?
–amount of label used, volume of assay, method
of bead concentration
!Plate type, NBS plates
!Cross-talk
SPA assay development
Optimization
Reference
Development
Application area
of
reference assay
Receptors - 3H, 125I
DNA - 3H, 33P
RIA - 125I
Enzymes - 3H, 33P, 35S
Kinases - 33P
Comp A/B - 3H, 125I, 35S
Capture method
Bead selection
Binding capacity
Label/NSB ratio
Bead addition
format
Validation
Plate format:
96 or 384
Matrix experiments:
Bead amount
versus
assay components
Optimal reagent concentration
Reduce NSB/NPE
Stable counting window
Adequate signal to noise
Assay validation
Colour quench
curves
Assay validation
!Comparison
with traditional assays
!Determination
of KD, Km values
!Inhibitor
profiles, IC50 values, competitive
binding curves with known drugs
!Establish
specificity of enzyme or
receptor-ligand interaction e.g. use of nonspecific substrate or cell line
valid.ppt
Colour quench correction
General guidelines for quench curves
!Use
labelled beads from colour quench kits or assay
“totals”
– PVT-[3H] (TRKQ 7080), PVT-[125I] (RPAQ 4030)
– YSi-[3H] (TRKQ 7150), YSi-[125I] (RPAQ 4040)
!Use
tartrazine solutions to construct the quench curve
(range 0-95% quench)
!Use
your assay for guidance
– Same weight of beads
– Same assay buffer
– Same assay volume
genguide1.ppt
Color quench correction
General guidelines for quench curves
Ensure identical bead packing conditions for your
assay and the quench curve
– Suspension (glycerol required?)
– Settled
– Floated
! Use a suitable figure for corrected counts (QCCPM)
- ‘Standard Set DPM’ or ‘Isotope Activity’ based on
the unquenched standard of the quench curve
(Refer to counter operator manual for guidance
! Install the quench curve on each counter using the
same plate
!
genguide2.ppt
SPA assay development
Optimization
Reference
Development
Application area
of
reference assay
Receptors - 3H, 125I
DNA - 3H, 33P
RIA - 125I
Enzymes - 3H, 33P, 35S
Kinases - 33P
Comp A/B - 3H, 125I, 35S
Capture method
Bead selection
Binding capacity
Label/NSB ratio
Bead addition
format
Delivery
Validation
Plate format:
96 or 384
Matrix experiments:
Bead amount
versus
assay components
Optimal reagent concentration
Reduce NSB/NPE
Stable counting window
Adequate signal to noise
Assay validation
Colour quench
curves
Automation
and
High
Throughput
Screening
Automation
♦ Liquid handing instrumentation
– Maintaining SPA bead suspension
♦ Bead mixing for SPA beads, PVT and YSi
– New bead mixing device in development
– Stirring mechanisms, troughs with baffles
to ensure uniform bead mixing
– Tip mixing
♦ Assay format
– Volumes
– Combinations
Amersham Biosciences
bead mixing device
Beckman Multimek
15.0
PVT
YSi
PS
YOx
% CV
12.5
10.0
7.5
5.0
2.5
0.0
15.0
10.0
µl
5.0
Summary of SPA assay development
♦ SPA
assay development follows a logical
flow
♦ Bubble
map can be configured to suit
individual style
♦A
defined method of assay development
can shortcut the design of new assays
and help trouble shoot existing assays
advanspa.ppt
SPA assay development
Optimization
Reference
Development
Application area
of
reference assay
Receptors - 3H, 125I
DNA - 3H, 33P
RIA - 125I
Enzymes - 3H, 33P, 35S
Kinases - 33P
Comp A/B - 3H, 125I, 35S
Capture method
Bead selection
Binding capacity
Label/NSB ratio
Bead addition
format
Delivery
Validation
Plate format:
96 or 384
Matrix experiments:
Bead amount
versus
assay components
Optimal reagent concentration
Reduce NSB/NPE
Stable counting window
Adequate signal to noise
Assay validation
Colour quench
curves
Automation
and
High
Throughput
Screening