Ligand Binding Assay & Scratchard Plot

Question 1

What is ligand binding assay?

Answer 1

It is an analytical procedure that measures binding of ligands to receptors or other molecules.

Question 2

Mention 5 types of ligand binding assay with two examples each.

Answer 2

1. Labelled ligand binding assay
   - Radioligand LBA
   - Fluorescent LBA
2. Label-free ligand binding assay
   - Mass spectrometry
   - Surface plasmon resonance
3. Structure-based Ligand binding assay
   - NMR
   - X-ray crystallography
4. Thermodynamic ligand binding assay
   - Thermal denaturation
   - Isothermal titration calorimetry
5. Whole cell ligand binding assay
   - Surface acoustic wave biosensor

Question 3

What is a radioligand?

Answer 3

It is a radioactively labelled ligand that can bind to any target of interest.

Question 4

What is Scratchard plot?

Answer 4

It is a graphical method for binding assay analysis.

Question 4

What is the significance of radioligand binding assay?

Answer 4

i. To determine the rate and extent of ligand binding
ii. Provides info on number of binding sites as well as their affinity for various drugs.

Question 5

What is the significance of the scratchard plot?

Answer 5

i. Provides useful parameters on drug-receptor interactions
ii. Gives estimates of Kd and [R] in ligand binding assay
iii. Shows if ligand binds to just 1 type of receptor
iv. Shows changes in Bmax and Kd in case of receptor desensitization or down-regulation

Question 6

Derive the equation of the graph for scratchard plot. Identify the parameters on the graph.

Answer 6

[L]bound/[L] = -[L]bound/kd + RT/kd

Question 7

Define Ki.

Answer 7

The molar concentration of the unknown that binds to 50% of the receptors in the absence of a radioligand.

Question 7

How do you determine the receptor affinity for an unknown unlabeled drug?

Answer 7

A radioligand binding assay is carried out with the unlabeled unknown drug (displacer) in the presence of the radiolabeled ligand.

The unknown competes with the radioligand for binding.

Question 7

Define IC50.

Answer 7

The molar concentration of the unlabeled unknown drug which decreases radioligand binding by 50%

Question 7

Define EC50.

Answer 7

The molar conc of agonist that produces a response half way between baseline and maximum response.

Question 8

Define PD2

Answer 8

Also called pEC50, it is the negative log of EC50.
i.e. The negative log of the molar conc of agonist that produces a response half way between baseline and maximum response.

Question 9

What is the formula for Ki?

Answer 9

Ki = IC50/ (1 + [s]/km)