Download Kinetic and Affinity Analysis using Biacore

Affinity Measurement with
Biomolecular Interaction
Analysis
Biacore
What SPR Biosensors Measures
Kinetics
Affinity
Specificity
Concentration
Thermodynamics
How fast, strong
& why…
How specific &
selective...
Is the binding of a lead
compound
Is this drug binding
to its receptor?
How much...
Biologically active
compound is in a
production batch?
Biacore History
• Founded 1984 as Pharmacia Biosensor AB
• Biacore System launched October 1990
• Biacore Symposium 1991
• Inline referencing started 1994
• Became Biacore AB in 1996
• Support of regulated environments from 2002
• Entering the drug discovery market with S51 in 2002
• Going into protein arrays with Biacore A100 and
Flexchip in 2005
Probing Biological Affinities
The Corner-stones of the Technology
SPR Detection
Sensor Chips
IFC Microfluidic
The SPR Detector
Total Internal Reflection & SPR
• Gold layer
• Evanescent field
• Total Internal Reflected light
(TIR)
• TIR angle
• Incident Light
• High refractive index medium:
Prism
• Low refractive index medium:
Buffer
SPR detection
Principle
Result
SPR detects refractive index
changes close to the surface
No need to separate bound
from free
E.g. accumulation of 1
pg/mm2 gives a change of 1
µRIU or 1 RU
This facilitates real-time
measurements as a basis
for taking kinetic data
All biomolecules have
refractive properties, so no
labeling required
Work with un-altered analytes
possible
Sensor Chips
Glass
Sensor Chip specific
matrix
Gold 50 nm
Sensor Chip CM5
• Dextran matrix covered with carboxyl groupes (red circles)
• Captures ligands such as proteins, lipids, carbohydrates and nucleic
acids (irreversible)
• Study of analytes ranging in size from small organic molecules, e.g. drug
candidates, to large molecular assemblies or whole viruses.
Sensor Chip CM4
• Similar to CM5 but with a lower degree of carboxymethylation resulting in
low immobilization capacity and lower surface charge density.
• Allows to reduce non specific binding in case of complex mixture such as
cell extract or culture medium.
• Advantageous for kinetic experiments where low immobilization levels
are recommended.
Sensor Chip CM3
• Similar to CM5 but with shorter dextran chains, giving a lower
immobilization capacity of the surface.
• Allows the interaction to take place closer to the cell surface which can
improve sensitivity when working with large molecules, molecular
complexes, viruses or whole cells.
Sensor Chip SA
• CM dextran matrix pre-immobilized with streptavidin
• Captures biotinylated ligands such as carbohydrates, peptides, proteins
and DNA (irreversible)
• Ideal for capture of large biotinylated DNA fragments and study of nucleic
acid interactions
Sensor Chip NTA
• CM dextran matrix pre-immobilized with nitrilotriacetic acid (NTA)
• Capture of His-tagged ligands via metal chelation
• Controled steric orientation of ligand for optimal site exposure
• Regeneration by injection of EDTA to remove metal ions
Sensor Chip L1
• CM dextran matrix modified with lipophilic anchor molecules
• For rapid and reproducible capture of lipid membrane vesicles such as
liposomes, with retention of lipid bilayer structure
• Allows studies of transmembrane receptors in a membrane-like
environment , for example.
The Steps in the Biacore Assay
Surface preparation
Analysis Cycle
Surface Preparation: Immobilization
a n a l y te
a n a ly te
li g a n d
lig a n d
c a p t u r in g
m o le c u l e
Direct
Covalent coupling of
Regeneration down to
ligand
Capture
capture molecule
Direct Immobilization
Various Coupling Chemistries
Amine Coupling - Sensorgram
• Activation = EDC/NHS injection  surface esters
• Ligand contact = reaction with amine groups on ligand
• Blocking = deactivation of free esters with ethanolamine
Blocking
Activation
High Affinity Capture
Capture Surfaces and Molecules
Type
Product/Molecule
Comment
Anti-Antibody
RaM Fc
anti-human Fc
Available from Biacore
Use affinity-pure products
Anti-tag
anti-GST
anti-His
Strep-MAB
anti-Biotin
Available from Biacore
E.g. Penta-His
See IBA
Use affinity-pure products
Anti-Fc
Protein A / G / L
-
Biotin-binding
Avidin family
StrepTactin
Streptavidin / Neutravidin
See IBA
Oligos
Sequence specific
Home made
Sensor Chip
SA, NTA, L1
Available from Biacore
Analysis Cycle
Sample injection
Regeneration
Evaluation
Generates the
desired data
Analysis Cycle
• Done by
Sample injection
Regeneration
• Buffer flow, pH shift, salt
& chaotrophic ions,
detergents
• Similar concept as in
affinity chromatography
• Results
Evaluation
• Re-use of biospecific
surface
• Low amount of ligand
needed
The Result: the Sensorgram
Experiments without Kinetics
Specificity
Multi layer structure
Concentration assays
Affinity constants
Specificity
• Do two molecules interact with each other?
Yes/No Answers.
• Different analytes are tested with the same
ligand e.g. different lectins with immobilized
thyroglobulin.
• Quantitative measurements, test a range of
analyte concentration to determine the
concentration dependency of the response.
Specificity Analysis
Overplay plot of sensorgrams showing interaction between different lectins
and immobilized thyroglobulin.
Multiple Binding
• Enhancement
• Enhancing lower detection limit of assays
• Sandwich assays
• Enhancing selectivity of test
• Epitope mapping
• Charting the surface of antigens with antibodies
• Multimolecular complexes
• Identify the logical sequence of binding events
Multiple Binding
Analyte
Ligand
2nd Binder
Response [RU]
31000
30000
29000
28000
27000
26000
50
100
150
200
Time [s]
250
300
350
400
Epitope Specificity of two mAbs against HIV1-p24
Immobilization of rabbit rabbit anti-mouse IgG1
 A: baseline
 A-B: 1st mAb against HIV1-p24
 B-C: blocking antibody
 C-D: HIV1-p24
 D-E: 2nd mAb against HIV1-p24
Concentration Assays
• Concentration based on biological activity
• All concentration assays require a calibration curve
• Concentrations of unknowns samples are calculated from this
• 4 - 7 concentrations in duplicate
• Calibrants and unknowns in same matrix
• Moderate to high densities on sensor chip
• Direct binding formats
• Inhibition formats
Calibration Curves
Response
x
x
xx
x
Sample
Concentration
Sample matrix for
calibration curve
=
Sample matrix for
unknown samples
Affinity Analysis
• How STRONG is the binding at equilibrium?
• » Quantify KD
• » Rank Antibodies
• » Find best Ab pairs
Affinity and Equilibrium
• Furosemide binding to
carbonic anhydrase
• Referenced data
• Report Point towards end of
injection
• Do secondary plot
Signal [RU]
20
15
10
5
0
Time [s]
0
60
120
Determining Affinity Constants
• Plot Req against C
• Steady state model
• Concentration at 50% saturation is KD
Kinetic Analysis
How FAST is the binding ?
» ka kon (recognition)
» kd koff (stability)
» KD = kd/ka
» Ab selection; wash steps
Same Affinity but different Kinetics
• All four compounds have the same affinity KD = 10 nM = 10-8M
• The same affinity can be the result from different kinetics
ka
kd
[M-1s-1] [s-1]
All target
sites
occupied
100 nM
30 min
60 min
106
10-2
105
10-3
104
10-4
103
10-5
KD 10 nM
1 µM
30 min
60 min
Rate Constants
Association rate
constant ka
Definition
Unit
Describes
Typical range
A+B
ka
AB
Dissociation rate
constant kd
AB
kd
A+B
[M-1s-1]
[s-1]
Rate of complex
formation, i.e. the
number of AB formed
per second in a 1 molar
solution of A and B
Stability of the complex
i.e. the fraction of
complexes that decays
per second.
1x10-3 – 1x107
1x10-1 – 5x10-6
Equilibrium Constants
Equilibrium dissociation Equilibrium association
constant KD
constant KA
Definition
Unit
Describes
Typical range
(A).(B) kd
=k
a
(AB)
[M]
Dissociation tendency
High KD = low affinity
1x10-5 – 1x10-12
k
(AB)
= ka
(A).(B) d
[M-1]
Association tendency
High KA = high affinity
1x105 – 1x1012
Equilibrium and Kinetic Constants are related
A+B
ka
kd
AB
Equilibrium and Kinetics in Biacore
Information in a Sensorgram
Extracting Rate Constants from Sensograms
• Measure binding curves
• Decide on a model to describe the interaction
• Fit the curve to a mathematical rate equation describing the
model
 e.g.
dR = k . C . (R -R) – k . R
a
max
d
dt
• Obtain values for the constants ka, kd, Rmax
• Assess the fit
 overlay pots, residual plots
 acceptable statistics e.g. chi2 – curve fidelity
Biological and experimental relevance of the calculated parameters
Biacore and other Methods
Conventional
Biacore
Assays
Time
Method
Time
Isotyping
Day 1
ELISA
One Day
Affinity
Day 1&2
RIA
Weeks + labelling
Kinetics
Day 1&2
Na
Na
Epitope Map
Overnight
ELISA
Weeks + labelling
Biacore is much quicker than conventional methods
Summary
 Surface plasmon resonance detects binding
events as changes in mass at the chip surface
 Real-time kinetic measurements
 Qualitative rankings
 Measurement of concentrations
 Information about structure-activity relationships
 No labeling and low volumes samples needed