ELISA

What is ELISA?
ELISA is the basic assay technique, known as enzyme-linked immunosorbent assay, that is
carried out to detect and measure antibodies, hormones, peptides and proteins in the blood.
Antibodies are blood proteins produced in response to a specific antigen. It helps to examine
the presence of antibodies in the body, in case of certain infectious diseases.

There are three types of ELISA.

1. Indirect ELISA
2. Direct ELISA
3. Sandwich ELISA

Principle of ELISA

ELISA works on the principle that specific antibodies bind the target antigen and detect
the presence and quantity of antigens binding. In order to increase the sensitivity and
precision of the assay, the plate must be coated with antibodies with high affinity. ELISA
can provide a useful measurement of antigen-antibody concentration.

Material

There many substrates available for use ELISA test detection. However, the commonly
used horseradish peroxidase (HRP) and alkaline phosphatase (ALP).

ELISA Procedure

ELISA is one of the easiest blood tests that can be carried out. It is rapid, quick and requires a
blood sample of the patient. The entire procedure of ELISA is mentioned below.
 An antibody is attached to a polystyrene plate which is a solid surface and is attracted
or has an affinity towards bacteria, other antibodies and hormones.
 A microtiter coated with antigen is filled with this antigen-antibody mixture after
which free antibodies are removed by washing.
 A second antibody specific to primary antibody is added which is usually conjugated
with an enzyme.
 Free enzyme-linked secondary antibodies are removed by washing the plate.
 Finally, the substrate is added. The substrate is converted by the enzyme to form a
coloured product, which can be measured by spectrophotometry.
Indirect ELISA

Indirect ELISA (Enzyme-linked immunosorbent assay) is a type of immunoassay used to

detect the presence of antigens or antibodies in a sample. It is called “indirect” because
the detection of the target molecule is achieved through the detection of a secondary
antibody or protein, rather than the target molecule itself.

In an indirect ELISA, a sample is added to plate that has been coated with an antigen. If
the sample contains the corresponding antibody, it will bind to the antigen on the plate. A
secondary antibody, conjugated to an enzyme, is then added to plate and will bind to the
primary antibody. The final step involves the addition of a substrate for the enzyme,
resulting in the production of a detectable signal, such as change in color.

Indirect ELISA is widely used in research, diagnostics, and quality control for the
detection of a variety of antigens and antibodies, including those related to infection
disease, autoimmune disorder, and allergies.

Direct ELISA

Direct ELISA (Enzyme-linked immunosorbent assay) is a type of immunoassay used to

detect the presence of antigens or antibodies in a sample. It is called “direct” because the
detection of the target molecule is achieved directly, without the need for a secondary
antibody or protein.

In a direct ELISA, a sample is added to the plate that has been coated with an antibody. If
the sample contains the corresponding antigen, it will bind to the antibody on the plate. A
secondary antibody, conjugated to an enzyme, is the added to the plate and will bind to
antigen-antibody complex. The final step involves the addition of a substrate for the
enzyme, resulting in the production of a detectable signal, such as a change in color.

Direct ELISA are commonly used for the detection of specific antigens in a sample,
such as for the diagnosis of infection diseases or for the detection of food allergens. They
are also useful for the detection of antibodies in a sample, such as in the diagnosis of
autoimmune disorders.

Sandwich ELISA

Sandwich ELISA (Enzyme-linked immunosorbent assay) is a type of immunoassay used

to detect the presence of target molecule, such as an antigen or antibody, in a sample. The
name “sandwich” comes from the way the target molecule is captured between two
specific binding partners, an antibody or protein, in the sample and on the surface of the
plate.

In a sandwich ELISA, a sample is added to the plate that has been coated with a capture
antibody. If the sample contains the target molecule, it will bind to the capture antibody
on the plate. A secondary antibody, conjugated to an enzyme, is then added to the plate
and will bind to the target molecule, forming a “sandwich” of the target molecule
between the capture antibody and the secondary antibody. The final step involves the
addition of a substrate for the enzyme, resulting in the production of detectable signal,
such as a change in color.

Sandwich ELISA are widely used in research, diagnostics, and quality control for the
detection of a variety of target molecule, including proteins, hormones, and infectious
agents. They are particularly useful for the detection of low-abundance target molecules
in complex samples.