3. EQIPMENTS OF ELISA :
A. MICROWELL PLATE
B. MULTI CHANNEL PIPETTE
C. WASHING DEVICE
D. MICROPLATE WASHER
E. ELISA READER (PHOTOMETER)
A. Horseradish peroxidase (HRP)
B. Alkaline phosphatase (AP)
Enzyme Linked Immunosorbent Assays (ELISA) are those that are
based on the measurement of an enzymatic reaction associated
with immune complexes. In any particular assay, the enzyme may
be linked to either the antigen or the antibody.
The antigens and antibodies combine specifically with each
other. This interaction between them is called Antigen-Antibody
Three stages are occurred in the reactions between Ag and Ab :-
In first stage, the reaction involves formation of Ag-Ab
The second stage leads to visible events like precipitation,
The third stage includes destruction of Ag or its
EACH ANTIBODY BINDS TO A SPECIFIC ANTIGEN; AN INTERACTION
SIMILAR TO A LOCK AND KEY.
6. DIRECT ELISA
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. In
this way, the antigen of interest is detected.
PROCEDURE OF DIRECT ELISA
Apply a sample of known antigen to a surface .
Enzyme linked primary antibody is applied to
the plate. Then washed.
After washing only the antibody-antigen
complexes remain attached.
Apply a substance which is converted by the
enzyme to elicit a chromogenic signal.
7. PROCEDURE OF INDIRECT
1. Antigen is added to plate.
2. Added blocking buffer.
3. Suitable primary antibody is added.
4. Secondary antibody – HRPO is then
added which recognizes and binds to
5. TMB substrate is added, is
converted to detectable form.
8. The sandwich ELISA quantify antigens between two layers of antibodies (i.e. capture and
detection antibody). Sandwich ELISA is named so as antigen is sandwiched between two
antibodies. The sandwich assay uses two different antibodies that are reactive with
different epitopes on the antigen with a concentration that needs to be determined.
Prepare a surface to which a known quantity of antibody is bound.
Apply the antigen-containing sample to the plate.
Wash the plate, so that unbound antigen is removed.
Apply the enzyme-linked antibodies which are also specific to the antigen.
Wash the plate, so that unbound enzyme –linked antibodies are removed.
Apply a chemical which is converted by the enzyme into a fluorescent signal.
View the result : if it fluoresces, then the sample contained antigen.
9. The central event of competitive ELISA is a competitive binding process executed by
original antigen (sample antigen) and add-in antigen. The procedures of competitive
ELISA are different in some respects compared with Indirect ELISA, Sandwich ELISA and
1.Primary antibody (unlabeled) is incubated with sample antigen.
2.Antibody-antigen complexes are then added to 96-well plates which are pre-coated
with the same antigen.
3.Unbound antibody is removed by washing the plate. (The more antigen in the sample,
the less antibody will be able to bind to the antigen in the well, hence "competition.")
4.The secondary antibody that is specific to the primary antibody and conjugated with an
enzyme is added.
5.A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal.
6.For competitive ELISA, the higher the sample antigen concentration, the weaker
10. ELISA can detect both antigen and antibody. It is a useful tool for determining serum
It is also found applications in the food industry in detecting potential food allergens ,
such as milk, peanuts, walnuts, almonds and eggs.
Elisa can detect both antigen and antibody. It is a useful tool for determining serum
In food industry for detecting potential food allergens E.g., Milk, Peanuts, Almonds,
Eggs and Walnuts.
Measuring hormone levels : HCG ( as a test for Pregnancy); LH ( determining the time
of Ovulation); TSH, T3 and T4 (for Thyroid function).
To detect HIV, Syphilis, Chlamydial infections, Hepatitis-B and C, Toxoplasmosis, UTI,
To detect serum antibodies produced against a variety of parasites causing
Amoebiasis, Malaria disease.
To detect Monoclonal Antibody Screening.
To study drug toxicity.
Disease diagnosis E.g., HIV, Bird flu, Common colds, Cholera, STD, etc.
For Rapid Test.
To measure Rheumatoid factors and other Auto-Antibodies in autoimmune disease
such as Lupus Erythematosus (LE), Thyroiditis etc.
To detect Snake Poisoning, Fungal diseases.
To develop Vaccine.