Background information
ELISA stands for
E
nzyme-
L
inked
I
mmuno
S
orbent
A
ssay. It is a very sensitive method for detecting small numbers of specific
molecules in a sample. There are different kinds of ELISAs. The main method described here is usually called an
‘indirect ELISA’ in which a primary antibody is attached to the antigen and then a secondary antibody attaches an enzyme to the
antigen-antibody complex. The kit can be adapted to simulate a ‘direct ELISA’ or a ‘sandwich ELISA’.
Applications:
- Testing for antigens. The target antigen could be a virus, a food allergen or a drug for example.
-
Testing for antibodies in blood serum, e.g. HIV test detects the presence of HIV antibodies in the blood of people
infected with the HIV virus.
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How the indirect ELISA works:
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The sample to be tested is added to a coated plastic well which binds the molecules of interest (antigen/antibody)
to its surface.
The well is washed with a weak detergent solution to remove unbound antigen. The detergent will also prevent new molecules
binding to the surface in subsequent stages of the procedure.
Specific antibodies are added to the well and bind to any of the molecules of interest. (In a direct ELISA these primary
antibodies have an enzyme attached in which case the addition of a secondary antibody is not necessary.)
The well is washed to remove unbound antibody.
A secondary antibody is added. This binds to the primary antibody and also carries an enzyme.
The well is washed to remove unbound antibody/enzyme.
The enzyme substrate is added. The enzyme reaction yields a coloured product. Only wells which contained the molecule of
interest bound to the well at step 1 will contain the enzyme so only they will show the positive result (colour change).
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The enzyme reaction
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The ELISA described here uses the enzyme HRP (horse-radish peroxidase) with the substrate TMB
(tetramethylbenzidine) which is oxidised to blue tetramethylbenzidine diimine.
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