ELISA

ELISA (Enzyme Linked Immuno Sorbent

Assay)
• ELISA is an important immunological assay method used to detect and
measure the concentration of antibody, antigen, glycoproteins, etc. in
biological samples.

• An immunoassay is a biochemical test that detects and measures the

concentration of analyte in a solution through the use of an antibody
(usually) or an antigen (sometimes).

• The principle behind the immunoassay test is the use of an antibody that
will specifically bind to the antigen of interest. The antibody used in the
immunoassay must have a high affinity for the antigen.
 Cont.
• ELISA works on main four principles

1) Antibodies and antigens interactions.

2) Antibodies and antigens can immobilize or attach to polystyrene plastic

surface or other solid support and maintain immunological activity.

3) Antibodies and antigens can be bonded to enzymes and the resulting

complexes are fully functional both immunologically as well as
enzymatically.

4) The bonded enzyme is detected by its ability to convert a colorless substance

into colored products.
 Requirements
• Antibodies and antigens can be produced by recombinant DNA
technology.
• ELISA assays use antibodies or antigens covalently bound to enzymes.
• The conjugated enzymes are selected on the basis of their ability to
catalyze the conversion of a substrate into a colored, fluorescent, or
chemiluminescent product.
• A number of variations on the basis ELISA assay have been developed
.
• Each type of ELISA can be prepared and used to determine the
concentration of a sample.
• Alternatively, a standard curve based on known concentrations of
antibody or antigen can be prepared and used to determine the
concentration of a sample
Steps to run an ELISA assay
 Enzymes and substrates used for ELISA

Both antibody and antigen can bind to enzyme.

Enzyme Chromogenic substrate Colour

developed
1 Alkaline phosphatase p-niitrophenyl phosphate yellow

2 Horseradish peroxidase o-phenylenediamine Orange-red color

dihydrochloride
Types of ELISA
• Direct
• Indirect
• Sandwitch
• Competetive
Micro titre plate containing 96 micro wells
 Direct ELISA
• The antigen of interest is detected.
• In direct ELISA, only an enzyme-labeled primary antibody is used, meaning that
secondary antibodies are not needed.

• The enzyme –labeled primary antibody directly binds to the target (antigen) that
is immobilized to the plate (solid surface).

• Next, the enzyme linked to the primary antibody reacts with its substrate to
produce a visible signal that can be measured .
 Advantages and disadvantages
Advantages
• Fast because only one antibody and fewer steps are involved

Disadvantages
 Indirect ELISA
• Antibody can be quantitatively determined by indirect ELISA.
• In this technique, antigen is coated on the microtitre well.
• Serum (target antibody) or some other sample containing primary antibody (Ab1) is added
to the micro titre well and allowed to react with the coated antigen.
• Any free primary antibody is washed away and the bound antibody to antigen is detected by
adding an enzyme conjugated secondary antibody (Ab2) that binds to the primary antibody
(ab1).
 Cont.
• Unbound secondary antibody (Ab2) is then washed away and a specific substrate for the
enzyme is added.
• Enzyme hydrolyses the substrate to form color products.
• The amount of colored end product is measured .
Indirect ELISA
 Indirect ELISA
• Advantages
• Increased sensitivity, since more than one labeled antibody is bound per
primary antibody
• A wide variety of secondary antibodies are available commercially.
• Maximum immunoreactivity of the primary antibody is retained because it is
not labeled.
• Versatile because many primary antibodies can be made in one species and the
same labeled secondary antibody can be used for detection.
• Flexibility, since different primary detection antibodies can be used with a
single labeled secondary antibody.
• Less costly, since fewer labeled antibodies are required.
• Disadvantages
• Cross reactive might occur with the secondary antibody, resulting in
nonspecific signal.
• An extra incubation step is required in this procedure.
 Sandwich ELISA
• Antigen can be detected by sandwich ELISA.
• In this technique antibody is coated on the micro titre well.
• A sample containing antigen is added to the well and allowed forming antibody to
react with the antibody attached to the well, forming antigen-antibodty complex.
 Cont.
• After the well is washed , a second enzyme –linked antibody specific for a different
epitopes on the antigen is added and allowed to react with the bound antigen.
• Then unbound secondary antibody is removed by washing.
• Finally substrate is added to the plate which is converted to color products by the
help of enzyme.
 Sandwitch ELISA
Advantages:
• High specificity , since two antibodies are used the antigen (analyte) is
specifically captured and detected.

• Suitable for complex samples, since the antigen does not require
purification prior to measurement.

• Flexibility and sensitivity, since both direct and indirect detection

methods can be used.
 Competitive ELISA
• Excess antibody is first incubated in solution with a sample containing antigen.
• The antigen-antibody mixture is then added to the micro titre well which is coated
with antigen.
• The more the antigen present in the sample the less free antibody will be available
to bind to the antigen coated well
• After the well is washed, enzyme conjugated secondary antibody specific for the
primary antibody is added to determine the amount of primary antibody bound to
the antigen.
• The higher the concentration of antigen in the sample, the lower the absorbance.
Competitive ELISA
 Advantages of ELISA
• ELISA gives an accurate diagnosis.
• Can be carried out for complex samples as the antigen is not required to
get purified to detect.
• It is a rapid test
• Easy to perform
• Less costly
 Applications of ELISA
• Disease detection:
Ebola, pernicious anaemia, AIDS ritrovirus, Syphilis. Carcinoma of the
epithelial cells, Covid 19 etc.
• Pregnancy test:
Human chorionic gonadotropin(hCG) hormone is produced during pregnancy.
• Cancer detection
• Vaccine development
• Drug monitoring
• transplantation