Elisa

ELISA Enzyme Linked Immunosorbent Assay Dr Janet Paterson SIBE Edinburgh University

What is ELISA? • Technique used to detect (assay) specific molecules (e.g. proteins & carbohydrates) in samples. • Immunological technique: uses antibodies. • Quantitative. • Very sensitive. • Commonly used in medicine and scientific research.

Antibodies  Proteins secreted by B-lymphocytes (type of white blood cell), in vertebrates.  Recognise and bind to molecules (antigens) on foreign particles, marking them for destruction by T-lymphocytes.

Fab fragments

 Each antigen may generate several antibodies for different sites (epitopes) on antigen.

Fc fragments

IgG molecule

Basic steps of ELISA

Enzyme Linked Immunosorbent Assay 1. Antigen of interest is absorbed on to plastic surface (‘sorbent’). 2. Antigen is recognised by specific antibody (‘immuno’). 3. This antibody is recognised by second antibody (‘immuno’) which has enzyme attached (‘enzyme-linked’). 4. Substrate reacts with enzyme to produce product, usually coloured.

Coloured product = measure (assay) of antigen present

Secondary antibody

Substrate ym z n E

e

Coloured product

Primary antibody

Different antigens in sample

Botrytis cinerea conidiophore

Effect of relative humidity on development of Botrytis infection in raspberries Experiment compares the following: 1. Control = no fruit 2. Infected fruit kept in low humidity 3. Infected fruit kept in medium humidity 4. Infected fruit kept in high humidity

Today, each group will prepare one fruit sample, then share extracts with other groups.

Prepare fruit samples

Place half a raspberry in tube

Add 2ml PBS

Break up tissue with glass rod

Filter into new tube using muslin

Coating the wells 

Label microwells 1, 2, 3 and 4

Using pastettes, transfer 4 drops of the appropriate extract into microwells: 1. PBS only 2. low humidity 3. medium humidity 4. high humidity

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Leave on bench for ten minutes to allow samples to absorb on to plastic surface.

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Inoculation of raspberries & humidity chamber set-up

Wash wells of excess sample Using pipette, fill wells with PBS-Tween then empty out.

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Tap wells upside down on paper towel.

Repeat twice more, making sure no liquid remains after the last wash.

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Label the pipette PBST and keep for later PBST pipetting.

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Add primary antibody Using a clean pastette, add 4 drops of primary antibody (mouse monoclonal) to each well. 

Leave on bench for ten minutes. 

Monoclonal antibody production (hybridoma technology) Inject mouse with antigen Obtain Mouse spleen B-lymphocytes

Grow mouse myeloma (tumour) cells in culture Fuse B-lymphocytes with myeloma cells

Antibody-producing hybridoma cells

B-lymphocyte and myeloma mixture

Select fused and reproducing hybridoma cells via growth medium

Screen hybridomas for antibody production

Unlimited supply of antibody specific for single epitope Keep clone producing antibody which best detects antigen

Make clones from individual antibodyproducing cells

Secondary antibody production Mouse serum injected into a different species, e.g. rabbit, goat.

Polyclonal antibodies which can recognise any mouse antibody

Animal makes various antibodies against the different antigens in serum

Select anti-mouse antibodies from plasma Take blood from animal

Wash wells of excess primary antibody Empty out contents of wells into waste container.

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Using pipette, fill wells with PBST then empty out.

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Tap wells upside down on paper towel.

Repeat twice more, making sure no liquid remains after the last wash.

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Add secondary antibody  Conjugated

to the enzyme horseradish peroxidase. Using a clean pastette, add 4 drops of secondary antibody (anti-mouse polyclonal) to each well. 

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Leave on bench for twenty minutes.

8. Observe colour development 1. Add antigen

7. Add substrate for enzyme

2. Wash with PBST

6. Wash with PBST

4. Wash with PBST

3. Add primary antibody

5. Add secondary antibody

Uses of ELISA outside the classroom • Disease detection in people, animals and plants (e.g. HIV in humans). • Detection of allergens in food, e.g. peanuts. • Detection of illegal drugs in humans. • Detection of hormones, e.g. pregnancy testing kits.

ELISA in the curriculum  Higher

Biology, Biotechnology and Human Biology E.g. Biotechnology: production & use of monoclonal antibodies

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Advanced Higher Biology: Biotechnology Unit Environmental Biology Unit Investigations

Advanced Higher Biology Investigation ideas  Detection of Botrytis in fruit and vegetables from

market or garden.  Quantification of Botrytis as infection develops.  Detection of Botrytis in tissue before symptoms

are observed.  Investigation on effect of temperature on rate of

Botrytis development.

 Based in the Institute of Cell Biology at

the University of Edinburgh.  Role: to enhance engagement with

biotechnology through interactions with the scientific community, school students, teachers and the general public.

School workshops

Supporting biology teachers

Science communication training

Science exhibitions

[email protected]

Enzymes used in ELISA  React with a colourless substrate to produce a

coloured product.  Must work fast at room temperature so the colour

develops quickly.  Have minimal interference from factors in sample.

Peroxidase from horseradish Alkaline phosphatase from E. coli β -galactosidase from E. coli

Wash wells of excess secondary antibody Empty out contents of wells into waste container.

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Using pipette, fill wells with PBST then empty out.

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Tap wells upside down on paper towel.

Repeat twice more, making sure no liquid remains after the last wash.

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Add substrate for enzyme  Wearing gloves, and

using a clean pastette, add 4 drops of tetramethyl benzidine (TMB) to each well.  Observe colour development (blue product

formed) and compare with colour chart.  Intensity of colour indicates amount of Botrytis

present in sample. Conclusions?

Results Effect of relative humidity on Botrytis infection in raspberries Infection level (arbitrary units)

100 80 60 40 20 0 0

20

40

60

Relative humidity (%)

80

Science and Plants for Schools SAPS Head Office Homerton College, Cambridge CB2 2PH Paul Beaumont [email protected] SAPS Biotechnology Scotland Project 2 Pitreavie Court, Dunfermline KY11 8UB Kath Crawford [email protected]

http://www-saps.plantsci.cam.ac.uk

ELISA Kit Available (£40 + VAT) from: Scottish Agricultural Science Agency (SASA) Antibody Unit East Craigs Edinburgh EH12 9FJ Tel. 0131 244 8911 [email protected]