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ELISA

Also known as an enzyme assay (EIA)


to detect the presence of an antibody / antigen in a
sample.
enzyms as indicator molecules can be used with
different indikator label
Common enzym : horseradish peroxxidase &
alkaline phosphatase
biotin / avidin enhanced antigen detection using
extremely small disease
ELISA
enzym + substrates a colored product
spectrophotometer
can be classed : non competitive or non
competitive
Types ELISA non competititve
Indirect ELISA
Direct ELISA
Sandwich ELISA
Indirect Types

to detect antibodies in biological sample


Antigen is affixed to the solid phase
The antigent will bind Fab regions of the
patients antibody
The Fc regions will for binding indicator labels
anti- Fc antibodies (secondary antibody)
Comparison of Direct and Indirect ELISA Detection Methods.

Quick because only one antibody and fewer steps are used.
Advantages
Cross-reactivity of secondary antibody is eliminated.

Direct Immunoreactivity of the primary antibody might be adversely affected by labeling with
Detection enzymes or tags.
Labeling primary antibodies for each specific ELISA system is time-consuming and
Disadvantages
expensive.
No flexibility in choice of primary antibody label from one experiment to another.
Minimal signal amplification.

A wide variety of labeled secondary antibodies are available commercially.


Versatile because many primary antibodies can be made in one species and the same
labeled secondary antibody can be used for detection.
Advantages Maximum immunoreactivity of the primary antibody is retained because it is not labeled.
Sensitivity is increased because each primary antibody contains several epitopes that can
Indirect
be bound by the labeled secondary antibody, allowing for signal amplification.
Detection
Different visualization markers can be used with the same primary antibody.

Cross-reactivity might occur with the secondary antibody, resulting in nonspecific signal.
Disadvantages
An extra incubation step is required in the procedure.
Indirect and direct ELISA

The method of antigen immobilization is


non-specific; when serum is used as the
source of test antigen, all proteins in the
sample may stick to the microtiter plate
well, so small concentrations of analyte in
serum must compete with other serum
proteins when binding to the well surface.
Sandwich ELISA
to detect antigen in biological sample
Antibody ass cappture antibody is affixed to
the solid phase
The antibody will bind antigent of the
patients
three layers of reagenst antibodi , antigen,
labeled antibody antigen is sandwich
Sandwich ellisa
Competitive ellisa
Unlabeled antibody is incubated in the presence
of its antigen (Sample).
These bound antibody/antigen complexes are
then added to an antigen-coated well.
The plate is washed, so that unbound antibody is
removed. (The more antigen in the sample, the
less antibody will be able to bind to the antigen in
the well, hence "competition.")
The secondary antibody, specific to the primary
antibody is added. This second antibody is
coupled to the enzyme.
A substrate is added, and remaining enzymes elicit
a chromogenic or fluorescent signal.