Affinity and kinetics Flashcards
Affinity
the binding strength of a ligand to one binding site on
the antibody
Kinetics
the speed whereby antigen-antibody complex forms
and dissociates
Avidity
the combined strength of interaction of an antigen with
an antibody’s binding sites
AgAb interaction: common values
ka normally 10^3 - 10^7 M-1s-1
kd normally 10^-5 – 10^-2 s-1
KA : association constant (commonly used as the aff. constant)
Ka = ka / kd = [AB] / [A] [B]
dissociation constant
KD = 1 / KA
ka
association rate constant
kd
dissociation rate constant
At equilibrium: d[AB] / dt =
0
–>
ka [A] [B] - kd [AB] = 0
–>
Ka = ka / kd = [AB] / [A] [B]
Why analyse affinity and kinetics?
- 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
The term half maximal effective concentration (EC50)
the concentration of a substance which induces a response halfway between
the baseline and maximum.
Slow dissociation ->
less antibody required for neutralization
The same affinity can be reached by antibodies with very different kinetic properties
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
Often high affinity is advantageous, but that is not always the case, give an example
High affinity results in
poor tumour penetration away from blood vessel
Affinity relates to function in vivo!
High affinity allows an antibody to…
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
How do you determine the affinity ?
- Equilibrium dialysis
- QCM-d
- Surface plasmon resonance (SPR) (Biacore)
- Isothermal titration calorimetry
- etc.
Equilibrium dialysis
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.
how do you calculate the association constant in Equilibrium dialysis?
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.
Scatchard Plot Basics
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.
Isothermal titration calorimetry (ITC)
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)
Quartz Crystal Microbalance with Dissipation monotoring
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)
Surface Plasmon Resonance (SPR)
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)
name the several methods available to immobilize ligands
on the carboxymethylated dextran surface in SPR
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
what are Binding curves used for?
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)
Examples of potential problems when determining binding constants that must be addressed
- 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
LigandTracer technology
determining affinity of
molecule (e.g. an antibody) for its cell-bound target
