Biacore system application

Proteins interaction and Binding Affinity Analysis

Baijun Kou, M.D, Ph.DBaylor College of Medicine

July17-18,2012. San Diego

•Surface Plasmon resonance (SPR) detects refractive index changes close to the surface•The accumulation of 1 pg/mm2 gives a change of 1 RU

Analysis Cycle

Surface preparation

Analysis Cycle

The Steps in the Biacore Assay

Sample injection

Regeneration

Evaluation

Optimization of pH for immobilization

Immobilization of ligand to Sensor chip

Immobilization

• What to immobilize Ligand vs. analyte

• Available sensor chip surface Ligand /analyte properties to decide

• How to create a ligand surface Direct vs. capture

• Method to immobilize Amine coupling vs. others

• Control

How to create a ligand surface

Direct Capture

a n alyte

lig a n d

a n alyte

lig a n d

ca pturin g

m ole cule

The advantage of capture technique • Always a fresh surface

• Homogeneous presentation

• Regeneration conditions maybe easier to determine

• Complete purification is not necessary

Sensor surface

•Covalent chemistry

•Often heterogenous orientation

•Higher binding capacity

•Orientation-specific

•Selective ligand capture

•Lower binding capacity

Immobilization procedure

• pH scouting

Antibody in variable pH of Acetate contact with chip for 2min NaOH to remove binding

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

Immobilization

Activation

Blocking

Ligand contact

RL

Immobilization level

• The binding capacity of the surface(Rmax) depends on the immobilization level

• Different applications require different immobilization levels

Kinetic analysis Rmax is between 50-250RU

• Rmax describes the binding capacity of the surface

Rmax=analyte MW / ligand MW *RL*Sm RL=the immobilization level Sm= number of binding sites on ligand for analyte.

• The theoretical Rmax is often higher than the experimental Rmax

Analyte

• The relationship of Concentration, KD and Rmax

Mass transport limitation

• Mass-transfer limited binding can cause of deviation from 1:1 binding

Balance between analyte consuption and analyte supply

Overcome• Fast flow rates reduce the diffusion distance

•Low Rmax reduces the comsuption

•Mass transport correction included in all kinetic models

Regeneration

• Remove bound analyte completely from the surface• Maintain the activity of the surface• Efficient regeneration is crucial for high-quality data

• Start with mild regeneration conditions• Increase flow rate to 50-100ul/min Follow injection with Extraclean• Use short contact times with extreme

conditions• Be creative Regeneration cocltail

Strategy

Regeneration Scouting

•Efficient regeneration removes all bound analyte

•A second injection of analyte reveals whether the ligand is still fully active

•Repeated cycles of analyte and regeneration injections are required to fully assess the conditions selected

Monitor baseline!!!

Kinetics v.s Affinity

•Kinetics•How fast do things happen?-Time-dependent

•Association-how fast molecules bind

•Dissociation-how fast complexes fall apart

•Kinetics determine whether a complex forms or dissociates within a given time span

•Affinity•How strong is a complex?-Time-independent

•Affinity determines how much complex is formed at equilibrium (steady state where association balances dissociation)

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

Kinetic vs. affinity• Kinetic rate analysis

Concentration analysis- find out active concentration

Ka /kd

• Affinity analysis Ligand fishing, relative ranking KD /KA

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

Same affinity but different kinetic rate

Affinity analysis

• Steady state model over a range of analyte

concentration

• Analyte C=20-80% saturation

• Plot Req against C

• C at 50% saturation is KD

• Use referenc e surface

• Include zero concentration sample

• At least one concentration in duplicate

0

5

10

15

20

0 60 120

Signal [RU]

Time [s]