assay techniques

examples of biochemical assay

• saturation

• competition

• scintillation proximity

• GPCR

What piece of equipment is commonly used in assays to optimise efficiency?

• microplates with isolated enzymes and membrane fragments

• more assays can be done simultaneously with multiwell pipettes or robot automation

• improved efficiency and reduced costs

Outline the principles of radioligand binding assay used to screen compounds and explain how IC50 and Ki for drug binding can be determined from the results. (10 marks)

• tissue sample with target protein is homogenised

• incubation 1 - sample is incubated with single concentration of radioactive labelled ligand and is washed to filter

• radioactivity is measured by counter

• incubation 2 - excess cold unlabelled ligand (drug) at increasing conc is added to the sample and radioligand and washed to be measured again

• specific binding = incubation 1 - incubation 2

• radioactviity is plotted against ligand concentration concentration for each drug

• IC50 is conc of drug which inhibits 50% of radioligand binding

• Ki derived from cheng presoff equation

ligand binding assay

• ability of a ligand to bind to a target

• first type of assay used in search of compounds that interact with a target

Advantages of radioligand binding assays, and what radiolabels are used?

• simple, easy automated for high input

• tritium, iodine-125 are most common, sulfur has more specific usage

requirements for radioligand binding assay

• tissue containing target protein

• radiolabelled ligand

• instrument to detect and measure radioactivity

• method to separate bound ligand from free ligand

separation of specific binding from non specific

• incubation 1 - radioligand and tissues

• incubation 2 - radioligand, tissue and excess unlabelled (cold) ligand

  • bound radioactivity = non specific binding

• radioligand at low conc cannot compete with excess cold ligand for receptor

  • non specific binding not displaced as not saturable

  • specific binding = incubation 1 - incubation 2

displacement assays

• critical part of drug screening

• a single concentration of radioligand used, usually below Kd

• specific binding determined by adding competing ligands at a range of concentrations

  • evaluate potency of displacing radioligand

What does competition curve measure and advantage?

• IC50

• non specific binding is seen more clearly - plateau not at zero

key advantage of scintillation proximity assay

• developed to avoid the need to separate bound ligand from the free ligand

scintillation proximity assay characteristics

• does not use homogenous sample of the tissue

• membrane containing receptor is attached to microbead containing scintillant

• radioligand in proximity of bead evokes light emission

• beta particle is released

What do binding assays not usually measure? What is the exception?

• bind assays do not measure efficacy

  • except GTPyS assay

    • gives functional information, measures activity of ligand to bind to its specific receptor

GPCR activation and GTPyS assay

• GTP gamma s binds to GPCR instead of GTP

• no hydrolysis, permanent activation

• sulfur is radioactive and acts as a marker for agonist binding to receptor, and receptor activation

how do antagonists differentiate from agonists in GTPyS assays

• prevent incorporation of GTP gamma s into g protein

• no activation, no radioligand activity will be measured

miniaturisation of assays

• multiwell plates

• more wells, less reaction volume

• allows optomisation but compromises reproducibility and accuracy

What are the applications of a bioassay (4 marks)

• identification of unknown substance

• quantification of concentration of a substance

• determine potency

• study physiological and pharmacological function

identification of unknown substance with bioassays

• known agents must have unique effects which are well defined

  • eg insulin to promote glucose absorption, adrenaline to increase heart rate

  • bioassays need to be specific and sensitive - respond to low conc of unknown substance

NO identification with bioassay

• bioassay identifies NO as the EDRF

  • assay tissue relaxed when NO applied anywhere

  • ACh applied to donor tissue or endothelial cells

  • did not relax when ACh was applied directly to assay tissue

Which active substances cannot be measured by chemical means?

• clotting factors, immunoglobins, peptides, cytokines, growth factors, vaccines and monoclonal antibodies

• quantified by reproducible biological effect

quantification of concentration with bioassay example

doses of insulin are defines as international units , measured against a standard sample

why can same weight of insulin in different batches have different potencies?

• phosphorylation, glycosylation, post translational modifications etc

• lead to changes in molecular weights

determination of potency

• potency and efficacy can only be tested using bioassays

  • response at each conc plotted as function of log[X]

• EC50 - conc to give 50% of max response

• potency - pD2 - log 10 of molar EC50 conc

How are bioassay measurements conducted? For each state whether it a quantal or graded response. (4 marks)

• groups of animals - quantal behavioural

• individuals or humans - graded response

• organ or tissues - graded response

• cell cultures - graded response

graded response examples

• contraction of smooth muscle to ACh

• fall in blood glucose in response to insulin

• pain relief

quantal response examples

• % of animals given a drug that develops seizures

• % of people showing adverse reaction to a new drug

• % of population with COVID-19 symptoms

How drug potency is measured for quantal vs graded?

• graded - EC50

• quantal - ED50

relative potency

• complete dose response curve may not be necessary to compare potency of two drugs acting at the same receptor

• concentration below and above EC50 are applied, EC50 can be esitmated for each drug and relative potency can be determined

What 5 properties determine a good bioassay?

• sensitivity

• specificity

• precision

• accuracy

• efficiency

how is precision seen in bioassay

• concentration response curve will have a steep slope

  • response is apparent over a narrow range of conc, small increment in conc produces a large increase in response

Advantage of bioassays

measures biological activity

Disadvantages of bioassays

• less sensitive

• more variable

• slower

• more limited in the number of samples that can be tested

use of assays during drug development and discovery

• target validation - confirmation of biological activity of the target

  • requires in vitro and in vivo bioassays

  • genetically modified tissues and animals

Define biochemical assays and give 2 examples (2 marks)

• analytical methods used to measure the concentration, activity, or interactions of biological molecules (e.g., enzymes, proteins, DNA, and metabolites)

• binding assays and reporter gene assays