Elisa
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.
IgG molecule
Basic steps of ELISA
Enzyme Linked Immunosorbent Assay
Coloured
product
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
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
Conjugatedto the
enzyme horseradish
peroxidase.
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).
Higher
Biology, Biotechnology and
Human Biology
E.g. Biotechnology: production & use of monoclonal
antibodies
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.
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
http://www-saps.plantsci.cam.ac.uk
ELISA Kit
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