Antigen Antibody Reactions
The
interaction between antigen and antibody is called antigen-antibody reactions.
It is abbreviated as Ag- Ab reaction. This reaction is the basis of humoral
immunity. The antigen and the antibody react to form immune complex.
Ag + Ab
------------ Ag − Ab complex
The reaction between antigen and antibody is highly specific. It is compared to the lock and key system. The part of the antigen that combines with the antibody is called epitope or antigenic determinant. The part of antibody which combines with the antigen is called paratope or antigen determining site. Most of the antibodies have two binding sites and IgM has 5–10 binding sites.
When
antibodies are mixed with the fluorescent dyes such as fluorescein or
rhodamine, they emit radiation. This phenomenon of emitting radiation by
antibodies labelled with fluorescent dye is called immuno fluorescence. This
reaction is well observed under fluorescent microscope. It is used to locate
and identify antigens in tissues.
• Direct
method
• Indirect
method
In this
method, the antibody labelled with fluorescent dye is directly applied on the
tissue section. The labelled antibody binds with specific antigen. This can be
observed under the fluorescent microscope.
In this
method, unlabelled antibodies are directly applied on the tissue sections which
bind with the specific antigens. Then the antibody labelled with the
fluorescent dye is added to the tissue. Anti -antibody specifically binds with
already added or linked unlabelled antibody (Figure 11.1)
ELISA (Enzyme-Linked Immuno Sorbent Assay) is a plate-based assay technique designed for detecting and
quantifying substances such as peptides, proteins, antibodies and hormones. It
is also known as Enzyme Immuno Assay (EIA)
In 1971,
after the descriptions of Peter Perlmann and Eva Engvall at Stockholm
University in Sweden, ELISA has become the system of choice when assaying
soluble antigens and antibodies. All assays for antibody production depend upon
the measurement of interaction of elicited antibody with antigen.
The principle of ELISA is very simple. The
test is generally conducted in micro titre plates. (Figure 11.2 Micro titre
plate).
If the
antigen is to be detected the antibody is fixed in the micro titre plate and
vice versa. Test sample is added in the microtitre plate, if there is presence
of Ag or Ab in the test sample, there will be Ag-Ab reactions (with immobilized
Ab or Ag) . Later enzyme labelled antibody is added in the reaction mixture,
which will combine with either test antigen or Fc portion of test antibody.
The
enzyme system consists of:
1. An
enzyme: Horse Radish Peroxidase(HRP),alkalinephosphatase
which is labelled or linked, to a specific antibody.
2. A
specific substrate:
• O-Phenyl-diamine -dihydrochloride for peroxidase
• P
nitrophenyl Phosphate- for Alkaline Phosphatase
Substrate
is added after the antigen-antibody reaction. The enzyme hydrolyses the
substrate to give a yellow colour compound in case of alkaline phosphatase
(Figure 11.3) . The intensity of the colour is proportional to the amount of
antibody or antigen present in the test sample, which can be quantified using
ELISA reader (Figure 11.4 ELISA reader)
There are
four kinds of ELISA assay tests.
They are: Direct ELISA, Indirect ELISA, Sandwich ELISA and Competitive ELISA
(Figure 11.5).
An
antigen is immobilized in the well of an ELISA plate. The antigen is then
detected by an antibody directly conjugated to an enzyme such as HRP. Direct
ELISA detection is much faster than other ELISA techniques as fewer steps are
required. The assay is also less prone to error since fewer reagents and steps
are needed, i.e. no potentially cross-reacting secondary antibody needed.
Finally, the direct ELISA technique is typically used when the immune response
to an antigen needs to be analyzed.
Indirect
ELISA is used to detect antibody. A known antigen is coated on the micro titre
plate. If the patient’s serum contains antibody specific to the antigen, the
antibody will bind to the antigen. After incubation the wells are washed and
the enzyme labelled anti Human Gamma Globulin (HGG) is added to the well.
Anti-HGG can react with antigen antibody complex. The substrate for the enzyme
is added finally which is hydrolysed by the enzyme which develops a colour.
Sandwich
ELISA is used to detect antigen. A known antibody is coated on the micro titre
plate. A test antigen is added to each well and allowed to react with the bound
antibody.
If the
patient’s serum contains antigen specific to the antibody, the antigen will
bind to the antibody. Specifically bound antigen and antibody will remain in
the wells even after washing. The second antibody is added and allowed to react
with bound antigen. Substrate is added to measure colour reaction
It is used for the detection of antigens. Antibody is first incubated with a sample-containing antigen. The antigen and antibody complex is added to the antigen coated microtitre well. If more antigen present in the sample, the less free antibody will be available to bind to the antigen coated well. Addition of an enzyme conjugated secondary antibody specific to the primary antibody can be used to determine the amount of primary antibody bound to the well. It is a quantitative test for the antigen detection.
An ELISA test may be used to diagnose: HIV, Lyme disease, pernicious anaemia, Rocky Mountain spotted fever, rotavirus, squamous cell carcinoma, syphilis, toxoplasmosis, varicella-zoster virus, which causes chickenpox and Zika virus.
Related Topics
Privacy Policy, Terms and Conditions, DMCA Policy and Compliant
Copyright © 2018-2023 BrainKart.com; All Rights Reserved. Developed by Therithal info, Chennai.