Immunological, plate-based assay used for quantifying and detecting soluble substances in biological samples.

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ELISA
INTRODUCTION
 Enzyme-linked immunosorbent assay depends on enzyme.
 An enzyme conjugated to an antibody reacts with a colorless
substrate to generate a colored reaction product.
 A number of enzymes have been employed for ELISA, including
alkaline phosphatase, horseradish peroxidase, and p-nitrophenyl
phosphatase.
 Enzyme + Substrate = Product (Colored)
 ELISA can detect antigen or antibody, quantitatively as well as
qualitatively.
 Standard curve based on known concentrations of antibody or
antigen is prepared from which the unknown concentration of
sample can be determined.

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TYPES
1. Indirect ELISA
 Detect or quantitate antibody
 Sample containing primary antibody is added to an antigen-
coated microtiter well and allowed to react with the bound
antigen.
 After washing, free primary antibody washed away.
 The presence of antibody bound to the antigen is detected by
adding an enzyme-conjugated secondary anti-isotype antibody,
which binds to the primary antibody.
 Any free enzyme-conjugated secondary anti-isotype antibody
then is washed away, and a substrate for the enzyme is added.
 The colored reaction product that forms is measured by
spectrophotometric plate reader which can measure the
absorbance of a 96-well plate.
Antigen-coated well -> wash -> add specific antibody -> wash ->
add enzyme-conjugated secondary antibody -> wash -> add
substrate and measure color

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2. Sandwich ELISA
 Detect or quantitate antigen
 Antibody is immobilized on a microtiter well.
 Sample is added and allowed to react with the bound antibody.
 After the well is washed, a second enzyme-linked antibody
specific for a different epitope on the antigen is added and
allowed to react with the bound antigen.
 After any free second antibody is removed by washing, substrate
is added and the colored reaction product is measured.
Antibody-coated well -> wash -> add antigen -> wash -> add
enzyme-conjugated secondary antibody -> wash -> add substrate
and measure color

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3. Competitive ELISA
 Detect or quantitate antigen
 Inhibition-type assay
 Antibody is first incubated in solution with a sample containing
antigen
 The Ag-Ab mixture is then added to an Ag-coated microtiter well
 The more Ag present in the sample, the less free Ab will be
available to bind to the Ag-coated well
 Addition of an enzyme-conjugated secondary antibody specific for
the isotype of the primary Ab can be used to quantitate the
amount of primary Ab bound to the well as in an indirect ELISA.
 Antigen concentration is inversely proportional to the colored
produced
 The higher the antigen concentration in the sample, the lower the
absorbance
Incubate antibody with antigen -> add antigen-antibody to the
antigen-coated well -> wash -> add enzyme-conjugated secondary
antibody -> wash -> add substrate no color