assay technques

examples of biochemical assay

• saturation

• competition

• scintillation proximity

• GPCR

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

radioligand binding assays

• 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

competition curve

• IC50

• non specific binding is seen more clearly

• only one radioligand is needed, availability is a less of a restriction

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

applications of bioassay

• organ bath

• 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

• 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

biochemical assays

• binding assays and reporter gene assays - are more widely used than bioassays

  • bioassays will only be used once the number of potential drug target compounds have been narrowed down

  • indicate the non selectivity