Molecular Biology

Bio-321
 ELISA
Enzyme-Linked Immuno-Sorbent Assay
LAB 2
❖ What is ELISA?
It stands for Enzyme-linked immunosorbent assay (measurement), a rapid
immunochemical test that involves an enzyme used for measuring a wide variety of tests
of body fluids.

✓ ELISA is considered to be a quantitative analysis .

✓ It is a solid phase (plate based assay).

❖ Uses
ELISA tests detect substances that have antigenic properties:
• primarily large molecules as; Proteins , growth factors, inflammatory factors and
Hormones.
• Small molecules and ions, such as glucose and potassium.
• Some of other substances as; Bacterial Antigens, Antibodies and Drugs
❖ Principle
The test depends on the interaction between components of the human body
(Antigen) with an (Antibody)
An antigen binds to a solid surface, then recognized specifically by an antibody
conjugated with an enzyme.

After then it is incubated with the enzyme substrate to give a measurable product since
color intensity reflect amount of antigen measured

N.B:

1. Antigens are the substances to which antibodies are produced as they stimulate
an immune response.

2. Antibodies are proteins produced by the body to identify and neutralize any foreign
substances that may be encountered, such as viruses and bacteria.
❖ Advantages
1) Fast (many samples can be processed at once).

2) ELISA tests are generally highly sensitive and specific

3) Very little sample volume is needed( <10 μl in most cases)

4) Easy to learn (simple procedures)

❖ Basic immunological protocols :

Immobilization of antigen\ antibody to solid surface

96 well or 384 well polystyrene plates (microplates)
Addition of reagents
Specific binding mostly via antigen antibody reaction
Washing steps
Separation bound from nonbounded material elimination of non
specifically bound material as excess antibody or antigen added
 ELISA Types

D) Special:
A) Direct B) Indirect C) Sandwich
Competitive
A. DIRECT ELISA
It is a direct detection involve attachment of the antigen (sample) to the solid
phase followed by primary antibody is conjugated with the enzyme to give a
measurable product for antigen detection
B. INDIRECT ELISA
It is an indirect detection also involve attachment of antigen to the solid phase but the
primary antibody is not labelled as in the direct ELISA method then we add labeled a
secondary enzyme conjugated antibody to the primary antibody bound to antigen to
be detected.
C . sandwich ELISA
It is more efficient in signal
amplification, rapid, easy , highly specific
principle: we measure the antigen (sample)
between two layers of antibodies (capture and
detection antibody).
Capture antibody is attached to a solid phase
support then adding antigen samples with a
buffer to minimize attachment to solid phase
then an enzyme labelled antibody is added for
detection
The target antigen must contain at least two
antigenic sites capable of binding to antibodies.
D . Special ELISA
competitive ELISA
(inhibition ELISA)
principle: labelling a purified antigen instead of the
antibody since competition between unlabeled antigen
(sample) and the labelled antigen (reference) for binding to
the capture antibody common in small antigens of only one
binding site.
• competitive ELISA assays measure the concentration of an
antigen by detection of signal interference.
• The sample antigen competes with a reference antigen for
binding to a specific amount of labeled antibody.
• The reference antigen is pre-coated on a multi-well plate
and sample is pre-incubated with labeled antibody and
added to the wells.
• SO Depending on the amount of antigen in the sample, more or less free
antibodies will be available to bind the reference antigen. This means the more
antigen there is in the sample, the less reference antigen will be detected and the
weaker the signal.
N.B
Some competitive ELISA kits use labeled antigen instead of a labeled antibody. The
labeled antigen and the sample antigen (unlabeled) compete for binding to the
primary antibody. The lower the amount of antigen in the sample, the stronger the
signal due to more labeled antigen in the well.
Application of ELISA

❑Presence of antigen or the presence of antibody in

a sample can be evaluated.
❑Determination of serum antibody concentrations
in a virus test.
❑used in food industry when detecting potential
food allergens.
❑Applied in disease outbreaks- tracking the spread
of disease e.g. HIV, bird flu, common, colds,
cholera, STD etc.
SUM UP:
Thank You