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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
Fab
particles, marking them for fragments
destruction by T-lymphocytes.

 Each antigen may generate


Fc
several antibodies for different fragments
sites (epitopes) on antigen.

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 e
Enz

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 Add 2ml Filter into


raspberry in Break up new tube
PBS tissue with
tube using
glass rod 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

 Leave on bench for ten minutes to allow


samples to absorb on to plastic surface.
Inoculation of raspberries &
humidity chamber set-up
Wash wells of excess sample
 Using pipette, fill wells with PBS-Tween
then empty out.

 Tap wells upside down on paper towel.

 Repeat twice more, making sure no


liquid remains after the last wash.

 Label the pipette PBST and keep for


later PBST pipetting.
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 Grow mouse


Mouse spleen myeloma (tumour)
B-lymphocytes cells in culture

Fuse
B-lymphocytes with
myeloma cells

Antibody-producing
hybridoma cells
B-lymphocyte and Unlimited supply
myeloma mixture of antibody specific
for single epitope

Keep clone
Select fused producing antibody
and reproducing which best detects
hybridoma cells antigen
via growth medium

Make
Screen clones from
hybridomas individual
for antibody antibody-
production producing
cells
Secondary antibody production
Mouse serum Polyclonal
injected into a antibodies which
different species, can recognise any
e.g. rabbit, goat. mouse antibody

Animal makes
various antibodies
against the Select anti-mouse
different antigens antibodies from
in serum plasma

Take blood
from animal
Wash wells of excess primary antibody

 Empty out contents of wells into waste


container.

 Using pipette, fill wells with PBST then


empty out.

 Tap wells upside down on paper towel.

 Repeat twice more, making sure no


liquid remains after the last wash.
Add secondary antibody

 Conjugatedto the
enzyme horseradish
peroxidase.

 Using a clean pastette,


add 4 drops of secondary
antibody (anti-mouse
polyclonal) to each well.

 Leave on bench for twenty minutes.


8. Observe
colour
development
7. Add substrate
1. Add antigen for enzyme

2. Wash with 6. Wash with


PBST PBST

4. Wash with
PBST
3. Add primary 5. Add secondary
antibody 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

 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 Science communication
training

Supporting biology Science


teachers exhibitions

janet.paterson@ed.ac.uk
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.

 Using pipette, fill wells with PBST then


empty out.

 Tap wells upside down on paper towel.

 Repeat twice more, making sure no


liquid remains after the last wash.
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

100
(arbitrary units)

80
Infection level

60
40
20
0
0 20 40 60 80
Relative humidity (%)
Science and Plants
for Schools
SAPS Head Office
Homerton College, Cambridge CB2 2PH
Paul Beaumont pb288@cam.ac.uk

SAPS Biotechnology Scotland Project


2 Pitreavie Court, Dunfermline KY11 8UB
Kath Crawford kath.crawford@sserc.org.uk

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


Robert.Burns@sasa.gsi.gov.uk