ELISA and its type

1. Enzyme linked immuno sorbent
assay (ELISA)

2. Enzyme-linked immunosorbent assay (ELISA), also known as
an enzyme immunoassay (EIA), is a biochemical technique
used mainly in immunology to detect the presence of an
antibody or an antigen in a sample.
What is ELISA:-
Some examples include:
diagnosis of HIV infection,
pregnancy tests, and
measurement of cytokines or
soluble receptors in cell
supernatant or serum.
,

3. In ELISA, an unknown amount of antigen is affixed to
a surface, and then a specific antibody is applied over
the surface so that it can bind to the antigen. This
antibody is linked to an enzyme, and in the final step a
substance is added so that the enzyme can convert to
some detectable signal , most commonly a colour
change in a chemical substrate.
Contd…

4. A . Anti-cytokine abs are coated on
microtitre plate.
B . PBMNC suspension is added to
wells,
after incubation the secretor cells
produce cytokines , which attached to
the abs.
C . The wells are washed.
D . Mouse Mabs directed to cytokines
are added which are probed with
enzyme linked anti-mouse Ig abs ,
intract with the secreted cytokines.
E . Substrate is added and color spots
will be developed at place where
each secretor cell was present.

5. Types of ELISA:-
Direct ELISA
 Indirect ELISA
 Sandwich ELISA

6. DIRECT ELISA
In direct ELISA the antigen is adsorbed to a plastic
plate, then an excess of another protein (normally bovine
serum albumin) is added to block all the other binding
sites. While an enzyme is linked to an antibody in a
separate reaction, the enzyme-antibody complex is
applied to adsorb to the antigen. After excess enzyme-
antibody complex is washed off, enzyme-antibody bound
to antigen is left. By adding in the enzyme's substrate,
the enzyme is detected illustrating the signal of the
antigen.
Direct ELISA, when compared to other
forms of ELISA testing, is performed faster
because only one antibody is being used
and fewer steps are required.

7. Advantages of Direct ELISA:-
The direct ELISA uses the method of direct
labeling of antibody itself.
 Microwell plates are coated with a sample
containing the target antigen.
the direct ELISA is relatively quick , as no
secondary antibody is used.
 Also avoids potential problems of cross-reactivity
of the secondary antibody with components in the
antigen sample.

8. Disadvantages of Direct ELISA:-
 The direct ELISA requires the labeling of every
antibody to be used, which can be a time-consuming
and expensive proposition.
 In addition, certain antibodies may be unsuitable for
direct labeling.
 Direct methods also lack the additional signal
amplification that can be achieved with the use of a
secondary antibody.

9. Indirect elisa
Indirect ELISA is a two-step ELISA which involves two
binding process of primary antibody and labeled
secondary antibody. The primary antibody is incubated
with the antigen followed by the incubation with the
secondary antibody.
Indirect ELISA capture antigen should be of high
affinity for the target antibodies and should be
screened for unwanted cross-reactions with antibodies
from individuals .
Use of more than one appropriate antigen will
capture more antibodies from the sample.

11. INDIRECT ELISA
http://microvet.arizona.edu/Courses/MIC419/ToolBox/elisa.

12. SANDWICH ELISA
The sandwich ELISA quantify antigens between two
layers of antibodies (i.e. capture and detection
antibody). The antigen to be measured must contain at
least two antigenic epitope capable of binding to
antibody, since at least two antibodies act in the
sandwich. Either monoclonal or polyclonal antibodies
can be used as the capture and detection antibodies in
Sandwich ELISA systems. Monoclonal antibodies
recognize a single epitope that allows fine detection
and quantification of small differences in antigen. A
polyclonal is often used as the capture antibody to pull
down as much of the antigen as possible.

14. ELISA Advantages
Compared to other immunoassay methods, there are
many advantages of ELISA. ELISA tests are more
accurate. They are
considered highly sensitive, specific and compare
favorably with other methods used to Detect
substances in the body, such as radio-immuno assay
(RIA) tests. ELISA possesses the
added advantages of not needing radioisotopes
(radioactive substances) or a costly radiation counter (a
radiation-counting apparatus).
 High sensitivity.
Strong specificity.

15. ELISA Applications :-
ELISA test is a useful tool since either the presence of
antigen or thepresence of antibody in a sample can be
evaluated by the application of ELISA.
ELISA can be applied to determination ofserum antibody
concentrations in a virus test (such as HIV testor ).
Applications of ELISA have also been found in the food
industry The ELISA was widely used in various of areas
sach as Immunology, Biological Pharmacy, Diagnostic
industry, and so on.

16. Principle of Radioimmunoassay
Principle: Uses an immune reaction [Antigen – Antibody
reaction] to estimate a ligand
Unbound Ag* and Ag washed out
Radioactivity of bound residue measured
Ligand conc is inversely related to radioactivity
[Ag : ligand to be measured ; Ag* radiolabelled
ligand
Advantages & Disadvantages of RIA
Advantages
Highly specific: Immune reactions are specific
High sensitivity : Immune reactions are sensitive

17. Disadvantages
Radiation hazards: Uses radiolabelled reagents
Requires specially trained persons
Labs require special license to handle radioactive
material

18. By –yashi jain