42715699

8/12/2019 42715699

1/24

Characterisation of Molecular Interactions

Using Surface Plasmon Resonance: BIAcore

Nathan Scott

Wednesday 21stMay

Technique Workshop: Protein-Ligand Interactions

Centre for Structural Biology

Department of Life Sciences

8/12/2019 42715699

2/24

Overview

Scope of the technique

Principle of SPR

Application

Advantages and Limitations

8/12/2019 42715699

3/24

Molecular Interactions

Interactomics

Signal

Transduction

Cell Adhesion

Antibody Binding

Endogenous

Receptor-Ligand

Interactions

DNA BindingProteins

DrugTarget

Interactions

8/12/2019 42715699

4/24

What can be studied?

Scope of

Technique

Proteins

Nucleic AcidsLipids &

membrane

molecules

Carbohydrates

Small-Molecule

Drugs

Whole Cells

Viruses Bacteria

8/12/2019 42715699

5/24

Biomolecular Interaction Analysis: BIAcore

Detection principle: Surface Plasmon Resonance: SPR

One binding partner (LIGAND) immobilised on chip

Other (ANALYTE) injected: microfluidics

PC collects binding data in real time

Chip is regenerated to remove analyte

Cycle is repeated

8/12/2019 42715699

6/24

Comprehensive information

Molecular interactions in real time:

Detect

Yes/No

Identify

Specificity

Binding partners

Characterize

Affinity

Kinetics

Epitope mapping

Concentration

Thermodynamics

Mass Spec Link-Up

8/12/2019 42715699

7/24

Versatility

Range of chip surfaces

Range of coupling chemistries for different needs

8/12/2019 42715699

8/24

Surface Plasmon Resonance

Critical angle of polarised light total internal reflection

Results in excitation of surface plasmons in the gold

Creates evanescent wave fielddissipates into sample matrix

Intensity of reflected light depletes

Critical angle changes with changes in sample matrix (binding)

Change in angle is converted to resonance signal directly proportional to mass bound at surface

8/12/2019 42715699

9/24

Case Study

Antibody Kinetics

8/12/2019 42715699

10/24

Kinetics provides unique information

Two molecules with

identical affinity

Kinetic profiling differssignificantly

Cannot be seen with

end-point assays

Physiological relevance

8/12/2019 42715699

11/24

Antibody Engineering

Molecular Engineering

Whole IgGWhole IgG

Single-Chain Fv

VHVL

Retains monovalent binding affinity

Improved biodistribution/tissue penetration

Can be manipulated significantly

Can be selected from combinatorial libraries

8/12/2019 42715699

12/24

AssayFLOW

100mM Sodium Phosphate + 300mM NaCl + Surfactant p20 (0.005%) + Azide (0.005%) pH 7.4

Streptavidin

Chip

Platform (SA)

Gold surface

Carboxymethylated

dextran

Streptavidin

Optical Interface

10mM Glycine-HCl (pH 2.5)

8/12/2019 42715699

13/24

Toxin Immbolisation and

Stability Assessment

-100

0

100

200

300

400

500

600

700

800

900

1000

0 500 1000 1500 2000

sTime

RU

Response

8/12/2019 42715699

14/24

700

800

900

1000

1100

1200

0 100 200 300 400 500 600 700 800

Time s

Resp.Diff.

RU

Testing scFv Binding & Regeneration

8/12/2019 42715699

15/24

Testing scFv Binding & Regeneration

8/12/2019 42715699

16/24

When there are problems....

600

700

800

900

1000

1100

1200

1300

1400

1500

1600

0 1000 2000 3000 4000 5000 6000

Time s

Resp.Diff.

RU

8/12/2019 42715699

17/24

8/12/2019 42715699

18/24

BIA-Evaluation Software

8/12/2019 42715699

19/24

Results

Parameter Value

KD 136nM

koff 1.45 x 10-03 S-1

kon 1.07 x 104 M-1S-1

T-value (koff) 120

T-value (kon) 3.6 x 103

Chi-Squared 1.07

Model: Langmuir 1:1

8/12/2019 42715699

20/24

So you want to play on the BIAcore

Express & Purify

Your Ligand/AnalyteLabel if necessary

Dialyse/exchange

into

appropriate buffer

Immobilise ligand

on chip

- Be careful!!

Determine optimal

regeneration buffer

Dissociate your

ligand/analyte

without damaging

or removing ligand

Kinetics

Studies

Concentration Determination

Specificity

Equilibrium Studies

Check binding

by ELISA*

What buffers are

both your analyte

& ligand stable in?

Immobilisation buffer

may be different

From running buffer

Running buffer

should

contain a surfactant

Optimise buffer and

amount of ligand

Kinetics Experiment!!

LOW

HIGH

8/12/2019 42715699

21/24

8/12/2019 42715699

22/24

8/12/2019 42715699

23/24

Contacts and Booking

Dr Lisa Jennings: Application Specialist GE Healthcare

E-mail: [email protected]

Book the BIAcore at: www3.imperial.ac.uk/cmmi/core

Training strongly advised before using machine

Onsite (Discounted):

Contact Prof. Kurt Drickamer; E-mail: [email protected]

Will be a waiting time until enough people for course to take place

Externally (Cambridge):

Book at www.biacore.com
mailto:[email protected]:[email protected]://www.biacore.com/http://www.biacore.com/mailto:[email protected]:[email protected]

8/12/2019 42715699

24/24

Acknowledgements

Wellcome Trust

Dr Mahendra Deonarain

All the R.A.T lab members!

Dr Lisa Jennings

42715699

Documents

Transcript of 42715699