1. Affinity Measurement with Biomolecular Interaction Analysis Biacore

2. What SPR Biosensors Measures
Kinetics
Affinity
Thermodynamics
Specificity
Concentration
How fast, strong & why…
Is the binding of a lead compound
How specific & selective...
Is this drug binding to its receptor?
How much...
Biologically active compound is in a production batch?

3. 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

4. Probing Biological Affinities

5. The Corner-stones of the Technology
Sensor Chips
SPR Detection
IFC Microfluidic

6. The SPR Detector

7. 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

8. SPR detection Principle Result
SPR detects refractive index changes close to the surface
E.g. accumulation of 1 pg/mm2 gives a change of 1 µRIU or 1 RU
All biomolecules have refractive properties, so no labeling required
Result
No need to separate bound from free
This facilitates real-time measurements as a basis for taking kinetic data
Work with un-altered analytes possible

9. Sensor Chips
Sensor Chip specific matrix
Glass
Gold 50 nm

10. 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.

11. 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.

12. 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.

13. 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

14. 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

15. 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.

16. The Steps in the Biacore Assay
Surface preparation
Analysis Cycle

17. Surface Preparation: Immobilizationy t e i g d c p u r m o a n l y t e i g d
Direct
Capture
Covalent coupling of
Regeneration down to
ligand
capture molecule

18. Direct Immobilization

19. Various Coupling Chemistries

20. 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
Ligand contact

21. High Affinity Capture

22. 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
E.g. Penta-His
See IBA
Anti-Fc
Protein A / G / L -
Biotin-binding
Avidin family
StrepTactin
Streptavidin / Neutravidin
Oligos
Sequence specific
Home made
Sensor Chip
SA, NTA, L1

23. Analysis Cycle Generates the desired data Sample injection
Regeneration
Evaluation

24. Analysis Cycle Done by Sample injection Results Regeneration
Buffer flow, pH shift, salt & chaotrophic ions, detergents
Similar concept as in affinity chromatography
Results
Re-use of biospecific surface
Low amount of ligand needed
Sample injection
Regeneration
Evaluation

25. The Result: the Sensorgram

26. Experiments without Kinetics
Specificity
Multi layer structure
Concentration assays
Affinity constants

27. 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.

28. Specificity Analysis
Overplay plot of sensorgrams showing interaction between different lectins and immobilized thyroglobulin.

29. Multiple Binding Enhancement Sandwich assays Epitope mapping
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

30. Multiple Binding Analyte Ligand 2nd Binder 26000 27000 28000 29000
30000
31000
Response [RU] 50
100
150
200
250
300
350
400
Time [s]

31. 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

32. 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

33. = Calibration Curves Sample matrix for calibration curve
Response x
Sample x x x x
Concentration =
Sample matrix for
calibration curve
Sample matrix for
unknown samples

34. Affinity Analysis How STRONG is the binding at equilibrium?
» Quantify KD
» Rank Antibodies
» Find best Ab pairs

35. Affinity and Equilibrium
Furosemide binding to carbonic anhydrase
Referenced data
Report Point towards end of injection
Do secondary plot 5 10 15 20 60
120
Signal [RU]
Time [s]

36. Determining Affinity Constants
Plot Req against C
Steady state model
Concentration at 50% saturation is KD

37. Kinetic Analysis
How FAST is the binding ? » ka kon (recognition) » kd koff (stability) » KD = kd/ka » Ab selection; wash steps

38. 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
[M-1s-1] kd
[s-1]
All target sites occupied
30 min
60 min
100 nM
1 µM
30 min
60 min
106
10-2
105
10-3
104
10-4
103
10-5
KD 10 nM

39. Rate Constants Association rate constant ka
Dissociation rate constant kd
Definition ka
A + B AB kd
AB A + B
Unit
[M-1s-1]
[s-1]
Describes
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.
Typical range
1x10-3 – 1x107
1x10-1 – 5x10-6

40. Equilibrium Constants
Equilibrium dissociation constant KD
Equilibrium association constant KA
Definition
Unit
[M]
[M-1]
Describes
Dissociation tendency
High KD = low affinity
Association tendency
High KA = high affinity
Typical range
1x10-5 – 1x10-12
1x105 – 1x1012 kd
(A).(B)
(AB) = ka
(AB) ka = kd
(A).(B)

41. Equilibrium and Kinetic Constants are related

42. Equilibrium and Kinetics in Biacore

43. Information in a Sensorgram

44. 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.
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 dR dt
= ka. C . (Rmax-R) – kd . R

45. Biacore and other Methods
Conventional
Assays
Time
Method
Time
Isotyping
Day 1
ELISA
One Day
RIA
Weeks + labelling
Affinity
Day 1&2
Kinetics
Day 1&2 Na Na
ELISA
Weeks + labelling
Epitope Map
Overnight
Biacore is much quicker than conventional methods

46. 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