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Affinity Measurement with B iomolecular I nteraction A nalysis Bia core. Kinetics. Affinity. Thermodynamics. What SPR Biosensors Measures. Concentration. Specificity. How fast, strong & why… Is the binding of a lead compound. How specific & selective...

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what spr biosensors measures

Kinetics

Affinity

Thermodynamics

What SPR Biosensors Measures

Concentration

Specificity

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?

biacore history
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
total internal reflection spr
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

SPR detection
sensor chips
Sensor Chips

Sensor Chip specific matrix

Glass

Gold 50 nm

sensor chip cm5
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
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
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
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
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
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
The Steps in the Biacore Assay

Surface preparation

Analysis Cycle

surface preparation immobilization

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Surface Preparation: Immobilization

Direct

Capture

amine coupling sensorgram
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

analysis cycle
Analysis Cycle

Generates the desired data

Sample injection

Regeneration

Evaluation

analysis cycle1
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

Sample injection

Regeneration

Evaluation

experiments without kinetics

Experiments without Kinetics

Specificity

Multi layer structure

Concentration assays

Affinity constants

specificity
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
Specificity Analysis

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

multiple binding
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 binding1

Analyte

Ligand

2nd Binder

31000

30000

29000

28000

27000

26000

50

100

150

200

250

300

350

400

Multiple Binding

Response [RU]

Time [s]

epitope specificity of two mabs against hiv1 p24
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 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

Sample

Calibration Curves

Response

x

x

x

x

x

Concentration

=

Sample matrix for

calibration curve

Sample matrix for

unknown samples

affinity analysis
Affinity Analysis
  • How STRONG is the binding at equilibrium?
  • » Quantify KD
  • » Rank Antibodies
  • » Find best Ab pairs
affinity and equilibrium

Signal [RU]

20

15

10

5

0

Time [s]

60

120

0

Affinity and Equilibrium
  • Furosemide binding to carbonic anhydrase
  • Referenced data
  • Report Point towards end of injection
  • Do secondary plot
determining affinity constants
Determining Affinity Constants
  • Plot Req against C
  • Steady state model
  • Concentration at 50% saturation is KD
slide37

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 target sites occupied

1 µM

100 nM

30 min

60 min

30 min

60 min

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]

106

10-2

105

10-3

104

10-4

103

10-5

KD 10 nM

equilibrium constants

kd

(A).(B)

=

ka

(AB)

Equilibrium Constants

ka

(AB)

=

kd

(A).(B)

extracting rate constants from sensograms

dR

= ka. C . (Rmax-R) – kd . R

dt

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
biacore and other methods

Biacore and other Methods

Conventional

Biacore

Assays

Time

Method

Time

One Day

ELISA

Day 1

Isotyping

RIA

Weeks + labelling

Affinity

Day 1&2

Na

Na

Kinetics

Day 1&2

Weeks + labelling

ELISA

Overnight

Epitope Map

Biacore is much quicker than conventional methods

summary

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