Measurement in Science Bioassays and Immunoassays

Measurement in Science

Bioassays and Immunoassays

Karim Meeran

23 October 2009

Assay

• Method of measuring concentration of a hormone in body fluid (usually serum).

• Previously used “bioassay”

Bioassay

• Administer extract of pituitary to animal and observe

• (eg: LH would very slowly cause testicular enlargement).

Bioassay

• Cells in vitro

• Grow

Immunoassay

• Because antibody could bind to a particular protein, this could be used to measure the amount of a substance.

• Make antibody by giving protein to animal of different species

• Assay for human adrenomedullin

• Give adrenomedullin to rabbits until they make antibodies (few months)

• Collect blood from the rabbits

• Use that blood to mop up antigen

Principles of RIA

• Have known amount of radioactive adrenomedullin.

• Add unknown amount of unlabelled adrenomedullin

• Add measured amount of antibody

• Add a second antibody against the first

• Mix and spin

Principles of RIA

• The second antibody may be a mouse anti rabbit antibody.

• This will be bound to a solid particle that will sink when centrifuged.

Example

• Adrenomedullin paper

Immunoassays

• Method of measuring concentration of a hormone in a body

• Two methods: - Competitive Assays

- Sandwich Assays

• Both work on the principle of enzyme specificity.

Competitive assays

• For a given peptide (P) we are able to produce antibodies by administering the peptide to a different species.

E.g.: human peptide to rabbit.

• Let the rabbit make antibodies (Abr), after a couple months remove the blood and it will have Ab for this P

Competitive assays

• Have Ab to P and we need to be able to accurately measure P. How do we do this:

• Make a Radioactive version of the peptide (Phot) and an Abm to the rabbit produce

• Abr into a mouse. But this Abm we also covalently attach to a particle of charcoal so it is heavier.

Competitive Assays

A mixture of P and Phot

Competitive Assays

A mixture of P and Phot

Addition of Abr which bind specifically to the peptides but cannot discriminate which ones are hot

Competitive Assays

A mixture of P and Phot

Addition of Abr which bind specifically to the peptides but cannot discriminate which ones are hot

Competitive Assays

A mixture of P and Phot

Addition of Abr which bind specifically to the peptides but cannot discriminate which ones are hot

Addition of excess Abm which binds to the Abr

Competitive Assays

A mixture of P and Phot

Addition of Abr which bind specifically to the peptides but cannot discriminate which ones are hot

Addition of excess Abm which binds to the Abr

Competitive Assays

A mixture of P and Phot

Addition of Abr which bind specifically to the peptides but cannot discriminate which ones are hot

Addition of excess Abm which binds to the Abr

Spin

Competitive Assays

Following centrifugation, the supernatant and pellet need to be separated.

Competitive Assays

Following centrifugation, the supernatant and pellet need to be separated.

Competitive Assays

Following centrifugation, the supernatant and pellet need to be separated.

Competitive Assays

Following centrifugation, the supernatant and pellet need to be separated.

Count BOUND radiation.

Count FREE radiation

Competitive Assays

Following centrifugation, the supernatant and pellet need to be separated.

Count BOUND radiation.

50%

Count FREE radiation

50%

Competitive Assays

Following centrifugation, the supernatant and pellet need to be separated.

Count BOUND radiation.

50%

Count FREE radiation

50%

Repeat for different concentrations of P

Separating bound from free

Bound to antibody + charcoal

Competitive Assays

A mixture of P and Phot

Addition of Abr which bind specifically to the peptides but cannot discriminate which ones are hot

Competitive Assays

A mixture of P and Phot

Addition of Abr which bind specifically to the peptides but cannot discriminate which ones are hot

Competitive Assays

A mixture of P and Phot

Addition of Abr which bind specifically to the peptides but cannot discriminate which ones are hot

Addition of excess Abm which binds to the Abr

Spin

Competitive Assays

Following centrifugation, the supernatant and pellet need to be separated.

Competitive Assays

Following centrifugation, the supernatant and pellet need to be separated.

Competitive Assays

Following centrifugation, the supernatant and pellet need to be separated.

Competitive Assays

Following centrifugation, the supernatant and pellet need to be separated.

Count BOUND radiation.

Count FREE radiation

Competitive Assays

Following centrifugation, the supernatant and pellet need to be separated.

Count BOUND radiation.

100%

Count FREE radiation

0%

Competitive Assays

Following centrifugation, the supernatant and pellet need to be separated.

Count BOUND radiation.

100%

Count FREE radiation

0%

Repeat for different concentrations of P

Competitive Assays

A mixture of P and Phot

Addition of Abr which bind specifically to the peptides but cannot discriminate which ones are hot

Competitive Assays

A mixture of P and Phot

Addition of Abr which bind specifically to the peptides but cannot discriminate which ones are hot

Competitive Assays

A mixture of P and Phot

Addition of Abr which bind specifically to the peptides but cannot discriminate which ones are hot

Addition of excess Abm which binds to the Abr

Spin

Competitive Assays

Following centrifugation, the supernatant and pellet need to be separated.

Count BOUND radiation.

66%

Count FREE radiation

33%

Repeat for different concentrations of P

Summary

Zero blue peptide gives 100% bound radiation.

Three blue peptide gives 66% bound radiation.

Twelve blue peptides gives 33% bound radiation.

This is the basis of how it works but for each assay we do using the antibodies we have we must run a series of these to obtain a reference curve.

Six blue peptide gives 50% bound radiation.

What is serial dilution?

Competitive Assays

Set up a series of tubes with known varying concentrations of P (achieved by serial dilution).

1 2 3 4

Competitive Assays

Set up a series of tubes with known varying concentrations of P (achieved by serial dilution).

To all of these we add a set amount of Phot.1 2 3 4

Competitive Assays• To each of these we add a set amount of Abr.

• This will bind randomly to the hot and cold peptides.

• Then add second antibody

1 2 3 4

Competitive Assays• If the amount of Phot causes 1000 counts

• When we add Abm and centrifuge the mixtures we will be able to detect different amounts of radiation from the light and heavy regions of the mixtures.

1 23 4

667 counts

333 counts 500 counts 667 counts

500 counts 333 counts 0 counts

1000 counts

Competitive Assays• If the amount of Phot causes 1000 counts

• When we add Abm and centrifuge the mixtures we will be able to detect different amounts of radiation from the light and heavy regions of the mixtures.

12 3

12 added

667 counts

None added

0 counts

1000 counts

B/F = infinite

6 added

500 counts

500 counts

B/F=1.0

3 added

333 counts

Heavy

Light

667 counts

B/F =2.0

333 counts

B/F=0.5

Competitive assays

• This has allowed us to set up a graph which shows the counts against concentration of P.Counts

B/F ratio

2

1

0.5

0 3 6 12 Conc. Of P

Competitive assays• We perform the same thing to a P of

unknown concentration, adding same amount of Phot and Abr and Abm as before.

• The count we get will allow us to extrapolate the value of P off the graph.

Counts

B/F ratio

2

1

0.5

0 3 6 12 Conc. Of P

Immunoradiometric assay

• Two antibodies (monoclonal) to different epitopes of the protein.

• The first is bound to the tube.

• The second is radioactive.

Sandwich Assays

• This is a different assay type but using the same principle of antibody specificity.

• Except we use MONOCLONAL antibodies.

• Therefore more expensive and takes longer.

• But more precise.

Sandwich Assays• Here we are attaching one antibody (Abc)

to a surface.

• This Abc is specific to one part of the peptide (P) ie: the N-terminus

• We then expose the surface to P

• We wash way any remaining solution

Sandwich Assays

• We then create an antibody (Abh) which is Radioactive

• This Abh is specific to the C-terminus of P.

• This is added to the surface.

• The excess solution is then washed away.

Sandwich Assays

• As we did in the competetive assays we need to create a refrence chart for this assay. This is done by using different concentrations of P but set amount of Abc and Abh.

• We can then measure the concentration of P in our unknown solution.

Sandwich Assays

• But in this type of assay we get a different sort of graph to extrapolate from.

Radioactive Count

Conc. Of P

Practical planning

• Aim: to enable you to write up a practical that you have carried out

• The write up needs to be in the format of a paper for a journal.

• Abstract, aim, introduction, methods, results, discussion, references.

• You need to use refworks to reference it.