Techniques for Measurement of Drug Concentration

Question 1

In terms of measures, what is the difference between the concentration and activity?

Answer 1

Concentration (preferred measure)

• SI unit is Molar
• Relies on knowing the molecular mass of a substance

Activity

• Molecular mass of a substance is not known
• There’s no SI unit for activity; measured in ‘units of acticvity’

Question 2

When is concentration used and when is activity used?

Answer 2

Activity is used when the molecular mass of the substance is not entirely known.

Usually, the concentration is the preferred measure.

Question 3

What is immunoassay?

Answer 3

Using antibodies in order to measure a substances functional activity

‘Immuno-‘ referring to the immune system
‘assay’ referring to the measurement of the functional activity of a substance

Question 4

What is the procedure of radioimmunoassay?

Answer 4

Treat specialised plastic dish containing antibodies with AN UNKNOWN AMOUNT sample of molecule that binds to the antibody and then wash off the excess
Treat the dish with A KNOWN AMOUNT of radiolabelled hormone and then wash off the excess.

The amount of radioactivity detect at the end is inversely proportional to the activity of the antibodies.
High radioactivity = low activity
Low radioactivity = high activity

Question 5

What is radioimmunoassay?

Answer 5

RIA is an immunoassay technique utilising radiolabelled molecules.

Question 6

How do you raise antibodies for RIA or ELISA?

Answer 6

Inject human protein into an animal (horse or sheep).
The animals immune system will recognise the protein as foreign and will develop antibodies in order to try to ‘fight off’ the foreign protein. The antibodies produced can be cultivated.

Question 7

What is a calibration curve?

Answer 7

A calibration curve (or standard curve) is calculated by carrying out assays using samples of known amounts of hormone.
This provides the relationship between radioactivity measured and amount of hormone present.

Question 8

What is an ELISA?

Answer 8

Enzyme-Linked Immunosorbent Assay

Utilises two sets of enzymes (primary and secondary) to carry out assays. Detection is through colour change.

Question 9

Why might you want to use ELISA over RIA?

Answer 9

ELISA is much safer than RIA, as RIA uses radioactivity to detect activity whereas ELISA uses colour change.

Question 10

What is the procedure of ELISA?

Answer 10

Incubate sample of molecules in a specialised dish containing antibodies and wash off excess.
Incubate the dish with secondary enzyme-linked antibodies and wash off excess.
Incubate the dish with a colourless enzyme substrate.

If the molecule was present, then the secondary antibody containing the enzyme will have adhered to the dish and so the substrate will change colour. If the molecule was not present, the secondary antibody will not adhere to the dish so the substrate will not change colour.

Question 11

In ELISA, what relationship must the molecule containing the antigens and the two types of antibodies have?

Answer 11

The primary and secondary antibodies must adhere to different regions of the molecules. For example, the primary antibodies must be able to adhere to the N-terminus regions of the molecule and the secondary antibodies must be able to adhere to the C-terminus region.

Question 12

What is non-competitive ELISA to detect the presence of an antibody?

Answer 12

Incubate a sample of antibodies (primary) in a specialised dish containing antigens for the antibody you’re trying to detect, then wash off the excess.
Incubate the dish with secondary enzyme-linked antibodies which have been raised against the primary antibody and then wash off the excess.
Incubate the dish with a colourless enzyme substrate.

If the primary antibody was present, then the secondary antibody containing the enzyme will have adhered to the dish and so the substrate will change colour. If the primary antibody was not present, the secondary antibody will not adhere to the dish so the substrate will not change colour.

Question 13

What is competitive ELISA to detect the presence of an antibody?

Answer 13

Competitive ELISA worked based on antibodies competing for the same antigen.

Incubate a sample of antibodies (primary) in a specialised dish containing antigens for the antibody you’re trying to detect, then wash off the excess.
Incubate the dish with secondary enzyme-linked antibodies which have been raised against the primary antibody and then wash off the excess.
Incubate the dish with a colourless enzyme substrate.

If the primary antibody was present, then the secondary antibody containing the enzyme WILL NOT have adhered to the dish and so the substrate will change colour. If the primary antibody was not present, the secondary antibody WILL adhere to the dish so the substrate will not change colour.

Question 14

How is the calibration curve calculated for ELISAs?

Answer 14

The density of the colour is what is measured, however the density of the colour is affected by the time for incubation and the amount of secondary enzyme-linked antibody that was present during incubation.

‘96 well plates’ are used in order to construct the calibration curve. Some of the wells contain known amounts of the substance being detected and time isn’t as much of a factor if at all. This is because each well is loaded, washed incubated and measured at the same time.

Question 15

What is 1 standard unit of insulin?

Answer 15

41.67 mg of 52% ox insulin and 48% pig insulin

Question 16

What is 1 standard unit of oxytocin?

Answer 16

0.5 mg of acetone-dried oxytocin sample from posterior pituitary gland of cattle

Question 17

What is radioligand binding?

Answer 17

Labelling a drug (or any other ligand) with a radioactive isotope so that the amount of drug bound to sample can be detected/measured.

Question 18

How do you use radioligand binding to detect affinity?

procedure

Answer 18

Homogenise a tissue/organ and divide equally between two sets of test tubes.
Incubate the FIRST set of test tubes with varying concentrations of radiolabelled drug.
Incubate the SECOND set of test tubes with a large excess of a cold (no radioactive isotope) version of the drug in addiction to varying concentrations of radiolabelled drug.
Filter and wash samples to leave behind only radiolabelled drug which had been bound.
Measure the amount of radioactivity per sample.

Subtract the second set of test tubes’ ‘amount of radioactivity’ from the first set of test tubes’ ‘amount of radioactivity’. The plot will give you the affinity of the drug to the receptor.

Question 19

Why do we need two sets of test tubes when using radioligand binding to detect affinity?

Answer 19

Non-specific binding occurs, which is where the ligand may bind to proteins other than their intended receptors.
The graph for the first set of test tubes will show a plot for ‘total binding’ (specific and non-specific binding).
The excess cold version of the drug added in the second set of test tubes will occupy all of the receptors before the radiolabelled drugs, meaning that is a graph were made for the second set of test tubes, the plot would show ‘non-specific binding’ only.
So in order to get a graph showing ‘specific binding’ only, we just subtract the values for the second set of test tubes from the first set of test tubes.

Question 20

What is the dose ratio?

Answer 20

A way of measuring the shift in the curve for a competitive reversible agonist

Question 21

What is pA2?

Answer 21

Just a definition that needs to be known.

‘The negative logarithm of the molar concentration of antagonist that necessitates that you double the agonist concentration to produce the same response.’

pA2 = -log(molar concentration of antagonist that doubles agonist concentration needed to produce the same response)

Question 22

What is the Schild equation?

Answer 22

log (dose ratio -1) = log (antagonist concentration) – log KB

Question 23

What is KB? (B is subscript)

Answer 23

KB is the dissociate equilibrium constant for an antagonist.

Question 24

How do you produce a Schild plot?

Answer 24

Construct log concentration-response curves of different concentrations of antagonist.
Find the logEC50 and calculate the EC50 for each of the curves.
Calculate the dose ration of each of the concentrations of antagonist.
Plot the log(dose-1) against the log([antagonist])
What you should find is a linear graph.

Question 25

What is significant about the Schild plot?

How do you calculate KB from Schild plot?

Answer 25

It is a linear graph where the gradient is (almost) 1
y = mx + c
y = log(dose ratio - 1)
x = log([antagonist])
c = -logKB

As the ‘m’ is (almost) equal to 1, we know that the y intercept (which isn’t shown on the Schild plot) is -logKB and the x intercept (which is shown on the Schild plot) is logKB.

Question 26

How do you calculate pA2 from KB

Answer 26

pA2 = -logKB