Affinity and kinetics

Question 1

Affinity

Answer 1

the binding strength of a ligand to one binding site on
the antibody

Question 2

Kinetics

Answer 2

the speed whereby antigen-antibody complex forms
and dissociates

Question 3

Avidity

Answer 3

the combined strength of interaction of an antigen with
an antibody’s binding sites

Question 4

AgAb interaction: common values

Answer 4

ka normally 10^3 - 10^7 M-1s-1
kd normally 10^-5 – 10^-2 s-1

Question 5

KA : association constant (commonly used as the aff. constant)

Answer 5

Ka = ka / kd = [AB] / [A] [B]

Question 6

dissociation constant

Answer 6

KD = 1 / KA

Question 7

ka

Answer 7

association rate constant

Question 8

kd

Answer 8

dissociation rate constant

Question 9

At equilibrium: d[AB] / dt =

Answer 9

0
–>
ka [A] [B] - kd [AB] = 0
–>
Ka = ka / kd = [AB] / [A] [B]

Question 10

Why analyse affinity and kinetics?

Answer 10

• To further understand the function of an antibody in a
  biological context or in a particular assay.
• To further optimize the design and choice of an
  antibody for a particular application/assay

Question 11

The term half maximal effective concentration (EC50)

Answer 11

the concentration of a substance which induces a response halfway between
the baseline and maximum.

Question 12

Slow dissociation ->

Answer 12

less antibody required for neutralization

Question 13

The same affinity can be reached by antibodies with very different kinetic properties

Answer 13

ka high e.g. 10^6 and kd is 10^-2 gives Ka 10^8–> half life is in minutes
ka

ka low e.g 10^2 and kd 10^-6 also gives Ka 10^8 –> but half life is in days

Biosensor: short half life favoured to obtain fast response
Therapeutic activity: long half life favoured for sustained biological activity

Question 14

Often high affinity is advantageous, but that is not always the case, give an example

Answer 14

High affinity results in
poor tumour penetration away from blood vessel

Question 15

Affinity relates to function in vivo!
High affinity allows an antibody to…

Answer 15

remain on its target! –
beneficial for instance when a radiolabelled antibody is used to treat a cancer in which case its retention on the tumour allows for more tumor irradiation

High affinity/slow
dissociation results in better
retention of antibody on
target cell and thus more
irradiation of the tumour

Question 16

How do you determine the affinity ?

Answer 16

• Equilibrium dialysis
• QCM-d
• Surface plasmon resonance (SPR) (Biacore)
• Isothermal titration calorimetry
• etc.

Question 17

Equilibrium dialysis

Answer 17

Simple. Suitable for small antigens (e.g. haptens).
Known amount of ab is mixed with different known amounts of analyte.
Ab and AbAg cannot diffuse → equilibrium of free ag across the membrane.
Measure and determine the amount of free ag!
Affinity and number of binding site determined by Scatchard plot

Knows
- amount of ab.
- amount of total ag.
Measures
- amount of free ag.
Can calculate
- amount of bound ag.

Question 18

how do you calculate the association constant in Equilibrium dialysis?

Answer 18

r = mole ag bound per mole ab.
c = conc. of free ag.
Slope = association constant = r/c
X intercept =No. of binding sites per ab.

Question 19

Scatchard Plot Basics

Answer 19

Binding Analysis:

The plot is used to understand the binding interactions between a ligand (e.g., a hormone, drug, or antibody) and a receptor or binding protein. It helps determine the equilibrium dissociation constant (Kd) and the number of binding sites (Bmax).
Plot Construction:

The Scatchard plot is constructed by plotting the ratio of bound ligand to free ligand (B/F) on the y-axis versus the bound ligand (B) on the x-axis.
The data are typically derived from experiments where the ligand concentration is varied, and the amount of ligand bound to the receptor is measured.
Linear Relationship:

For a simple binding interaction, the Scatchard plot will yield a straight line.
The slope of the line is equal to -1/Kd, where Kd is the equilibrium dissociation constant.
The x-intercept of the plot represents Bmax, the total number of binding sites available on the receptor.

Question 20

Isothermal titration calorimetry (ITC)

Answer 20

Isothermal Titration Calorimetry (ITC) is a label-free quantification technique used in studies of a wide variety of biomolecular interactions. It works by directly measuring the heat that is either released or absorbed during a biomolecular binding event.

ITC can be utilized to study
biomolecular interactions,
since heat is absorbed or
generated in essentially all
binding events

Can be used to determine entropy and enthalpy of interaction!
(∆G = ∆H - T∆S)

Question 21

Quartz Crystal Microbalance with Dissipation monotoring

Answer 21

Surface sensitive technique enabling real-time measurements (label-free).
Alternating voltage is applied across a quartz crystal.
The crystal oscillates at a certain frequency in a mass dependent manner.
When something, e.g. a protein, binds to the crystal, the mass is changed, and
thereby the oscillating frequency.
QCM-d measures changes in frequency
energy dissipation (viscoelasticity)

Question 22

Surface Plasmon Resonance (SPR)

Answer 22

Gold surface (chip). The unit is sensitive for changes in refractive index.
When a protein is bound, the refractive index changes. This can be measured
and transformed into adsorbed mass. The measures are made in real-time.

Technology that makes it possible to study molecular interactions without
having to label any of the participating molecules.
The measurements are made in real-time.
Determines the affinity and rate constants.
The most commonly used method in antibody technology!
Can be used to determine entropy and enthalpy of interaction!
(∆G = ∆H - T∆S)

Question 23

name the several methods available to immobilize ligands
on the carboxymethylated dextran surface in SPR

Answer 23

direct covalent coupling via amine groups direct covalent coupling via sulfhydryl groups
* antibody capture
biotin attachment to avidin coated surfaces
aldehyde coupling to carbohydrate groups attachment through 6xhis tags with NTA chips

Question 24

what are Binding curves used for?

Answer 24

Binding curves at different free ligand concentrations are used to calculate
reaction rate constants and to deduce affinity
Equilibrium at different free ligand concentration can be used to calculate
affinity constant (e.g. if reaction is too fast to monitor on/off rates independently)

Question 25

Examples of potential problems when determining binding constants that must be addressed

Answer 25

• Avidity effects (presence of binders with > 1 binding site per molecule)
• Diffusion limitations (concentration at surface different from that in bulk solution)
• Re-binding of dissociated binder
• Steric hindrance
• Heterogeneous antigen

Question 26

LigandTracer technology

Answer 26

determining affinity of
molecule (e.g. an antibody) for its cell-bound target