Affinity Measurement with

Biomolecular Interaction

Analysis

Biacore

What SPR Biosensors Measures

How specific & selective...

Is this drug binding to its receptor?

How much...

Biologically active compound is in a production batch?

SpecificitySpecificitySpecificitySpecificity ConcentrationConcentrationConcentrationConcentration

How fast, strong & why…

Is the binding of a lead compound

KineticsKineticsKineticsKinetics AffinityAffinityAffinityAffinity

ThermodynamicsThermodynamicsThermodynamicsThermodynamics

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

Sensor Chips

SPR Detection

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

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

Sensor Chips

Sensor Chip specific matrixGlass 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.

Surface preparation

Analysis Cycle

The Steps in the Biacore Assay

Surface Preparation: Immobilization

Direct Capture

a n a ly t e

l ig a n d

a n a ly te

l ig a n d

c a p tu r in g

m o le c u le

Covalent coupling of

Regeneration down toligand capture molecule

Direct Immobilization

Various Coupling Chemistries

Activation

Ligand contact

Blocking

Amine Coupling - Sensorgram

• Activation = EDC/NHS injection surface esters• Ligand contact = reaction with amine groups on ligand• Blocking = deactivation of free esters with ethanolamine

High Affinity Capture

Capture Surfaces and Molecules

Type Product/Molecule Comment

Anti-AntibodyRaM Fcanti-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-bindingAvidin family

StrepTactin

Streptavidin / Neutravidin

See IBA

Oligos Sequence specific Home made

Sensor Chip SA, NTA, L1 Available from Biacore

Sample injection

Regeneration

Evaluation

Analysis Cycle

Generates the desired data

Sample injection

Regeneration

Evaluation

Analysis Cycle

• Done by• 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

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

26000

27000

28000

29000

30000

31000

50 100 150 200 250 300 350 400

Time [s]

Res

po

nse

[R

U]

Multiple Binding

Analyte

Ligand

2nd Binder

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

x

x

xxx

Response

Concentration

Sample

Sample matrix forcalibration curve

Sample matrix forunknown 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

0

5

10

15

20

0 60 120

Signal [RU]

Time [s]

Determining Affinity Constants

• Plot Req against C• Steady state model

• Concentration at 50% saturation is KD

How FAST is the binding ?

» ka kon (recognition)

» kd koff (stability)

» KD = kd/ka

» Ab selection; wash steps

Kinetic Analysis

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

All target sites

occupied

30 min 60 min

100 nM 1 µM

30 min 60 min

10-5103

10-4104

10-3105

10-2106

kd

[s-1]

ka

[M-1s-1]

KD 10 nM

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

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

ka(AB)(A).(B)

= kd

Equilibrium and Kinetic Constants are related

A + B ABka

dk

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.

• 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

dRdt

= ka. C . (Rmax-R) – kd . R

Biacore and other Methods

Biacore

Assays

Isotyping

Affinity

Kinetics

Epitope Map

Time

Conventional

Method Time

Day 1

Day 1&2

Day 1&2

Overnight

ELISA One Day

RIA Weeks + labelling

Na Na

ELISA Weeks + labelling

Biacore is much quicker than conventional methods

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

Summary