Functional Assays Flashcards
What happens with a cell-based biochemical assay and when is this type of assay often used?
- In these, cells are grown in the laboratory in sterile culture plates.
- This type of assay is often used for G protein coupled receptors – receptor activation can be measured by measuring the concentration of intracellular messages such as cAMP, IP3 and calcium
What happens with an Organ bath assay and when is it usually used?
- In this type of assay an organ is removed from an animal and placed in a tissue bath.
- It is supplied with nutrients and oxygen to enable its function to be maintained during the experiment
- This type of assay measures how a normal physiological function of the organ changes in response to drugs
- E.g. an isolated heart will continue to beat in an organ bath, and the changes in the rate and force of contraction of the heart can be measured
What is a Cell-based electrophysiological assay and when do we use it?
- In electrophysiology we measure how the membrane potential of a cell changes in response to drugs.
- This requires placing electrodes on, or in the cell
- An important use of electrophysiology is to measure the activity of ion channels and ligand-gated channels
What is a whole animal assay, why is it powerful, what does it allow us to look at and what are the cons?
- In this type of assay we measure the changes a drug produces in the physiology, behaviour or disease state of living organisms
- This type of assay is powerful because it allows us to see a drug ‘in action’
- It allows us to look at factors such as drug absorption and metabolism
- If the experiment is done in humans it would be called a ‘clinical trial’
- There are many more ethical considerations for whole organism studies and they tend to be much more expensive than the other types of assay
How do we quantify how much drug has been given in a way that accounts for the size of our animal or volunteer?
By measuring the Dose of the drug
What are doses normalised to and why?
The subject’s body weight so that comparisons can be made between subjects of different size
What units will often be used for dose?
mg (of drug)/ kg subject body weight
Would you use dose or concentration for cell/ organ based assays?
Concentration
What is function?
changes in behaviour of cell, tissue, organism when drug administered
Are binding and function the same?
No
How do agonists vs antagonists show that binding doesn’t equate to function?
They both bind but agonists activate a receptor whereas antagonists inhibit a receptor
Why is the binding effect hard to measure?
Because the function we measure can often be quite distant from the binding effect (e.g. in nuclear hormone receptors we would measure the change in transcription which is quite a few steps away from the ligand binding to the NHCR
How do we deal with functional data?
Plotting an effect (logarithmic scale – sigmoid relationship)
Calculate maximum effect by estimating where the plato of the curve is
Work out the concentration that gives us 50% of maximum effect – EC50 (or ED50 in terms of dose)
What equation underlies functional data?
The functional Hill Langmuir equation
What is the functional hill Langmuir equation?
E = Emax. [D]/ EC50 + [D]
E = effect
E max = max effect
[D] = concentration of drug
EC50 = concentration giving 50% max effect
EC50 and Kd will often be different – not equivalent parameters
