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 (Ab r), 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 (P hot ) and an Ab m to the rabbit produce Ab r into a mouse. But this Ab m we also covalently attach to a particle of charcoal so it is heavier.

Competitive Assays A mixture of P and P hot

Competitive Assays A mixture of P and P hot Addition of Ab r which bind specifically to the peptides but cannot discriminate which ones are hot

Competitive Assays A mixture of P and P hot Addition of Ab r which bind specifically to the peptides but cannot discriminate which ones are hot

Competitive Assays A mixture of P and P hot Addition of Ab r which bind specifically to the peptides but cannot discriminate which ones are hot Addition of excess Ab m which binds to the Ab r

Competitive Assays A mixture of P and P hot Addition of Ab r which bind specifically to the peptides but cannot discriminate which ones are hot Addition of excess Ab m which binds to the Ab r

Competitive Assays A mixture of P and P hot Addition of Ab r which bind specifically to the peptides but cannot discriminate which ones are hot Addition of excess Ab m which binds to the Ab r 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 P hot Addition of Ab r which bind specifically to the peptides but cannot discriminate which ones are hot

Competitive Assays A mixture of P and P hot Addition of Ab r which bind specifically to the peptides but cannot discriminate which ones are hot

Competitive Assays A mixture of P and P hot Addition of Ab r which bind specifically to the peptides but cannot discriminate which ones are hot Addition of excess Ab m which binds to the Ab r 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 P hot Addition of Ab r which bind specifically to the peptides but cannot discriminate which ones are hot

Competitive Assays A mixture of P and P hot Addition of Ab r which bind specifically to the peptides but cannot discriminate which ones are hot

Competitive Assays A mixture of P and P hot Addition of Ab r which bind specifically to the peptides but cannot discriminate which ones are hot Addition of excess Ab m which binds to the Ab r 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)

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 P hot

Competitive Assays To each of these we add a set amount of Ab r. This will bind randomly to the hot and cold peptides. Then add second antibody

Competitive Assays If the amount of P hot causes 1000 counts When we add Ab m and centrifuge the mixtures we will be able to detect different amounts of radiation from the light and heavy regions of the mixtures counts 333 counts 500 counts667 counts 500 counts 333 counts0 counts 1000 counts

Competitive Assays If the amount of P hot causes 1000 counts When we add Ab m and centrifuge the mixtures we will be able to detect different amounts of radiation from the light and heavy regions of the mixtures added 667 counts None added 0 counts 1000 counts B/F = infinite 6 added 500 counts B/F=1.0 3 added 333 counts Heavy Light 667 counts B/F = 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 Conc. Of P

Competitive assays We perform the same thing to a P of unknown concentration, adding same amount of P hot and Ab r and Ab m as before. The count we get will allow us to extrapolate the value of P off the graph. Counts B/F ratio 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 (Ab h ) which is Radioactive This Ab h 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 Ab c and Ab h. 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.