Prior to the development of the EIA/ELISA, the only
option for conducting an immunoassay was
radioimmunoassay, a technique using radioactively-
labeled antigens or antibodies. Avrameas (1966,
1969) and Pierce (1967) developed methods to
chemically link antibodies to biological enzymes
whose activities produce a measurable signal with
solutions containing appropriate substrates.This
signal has to be associated with the presence of
antibody or antigen
4. INTRODUCTION TO ELISA
The term ELISA was first used by Engvall & Perlma
ELISA, or enzyme-linked immunosorbent assay,
are quantitative immunological procedures in which
the Ag-Ab reaction is monitored by enzyme
The ELISA test, or the enzyme immunoassay (EIA),
was the first screening test commonly employed for
HIV. It has a high sensitivity.
A test that uses antibodies and color change to
identify a substance.
ELISA involves at least one antibody with specificity
for a particular antigen.
5. COMPONENTS OF ELISA
Enzyme: Horse Radish Peroxidase (HRP) MW
44,000,glycoprotein with 4 lysine residues.
Substrate: TMB (3,3',5,5', tetramethylbenzidine)
The enzyme acts as a catalyst to oxidize substrate
in the presence of Hydrogen peroxide to produce a
The basic principle of an ELISA is to use an
enzyme to detect the Ag-Ab binding (antigen-
antibody binding). The enzyme converts a colorless
substrate (chromogen) to a colored product,
indicating the presence of Ag:Ab binding.
7. WHY KNOWN AS ......?
Enzyme Linked Immunosorbent Assay
1. Antigen of interest is absorbed on to plastic
2. Antigen is recognized by specific antibody
3. This antibody is recognized by second antibody
("immuno') which has enzyme attached (enzyme-
4. Substrate reacts with enzyme to produce
product, usually colored.
9. TYPES OF ELISA
10. INDIRECT ELISA
Antigen is added to plate.
Suitable primary antibody is added.
Secondary antibody- HRPO is then added which
recognizes and binds to primary antibody.TMB
substrate is added, is converted to detectable form.
11. DIRECT ELISA
Apply a sample of known antigen to a surface.
Enzyme linked primary antibody is applied to the
Washed, After this wash, only the antibody-antigen
complexes remain attached.
Apply a substrate which is converted by the
enzyme to elicit a chromogenic signal.
13. SANDWICH ASSAY
Antigens such as tumor markers, hormones and
serum proteins may be determined
Antigen in the sample binds with the capture
antibody on the microwell and becomes
The antibody of the enzyme conjugate binds with
the immobilized antigen to form a sandwich of
antibody-antigen-antibody/enzyme bound to the
Enzyme reaction product is directly proportional to
concentration of standard or analytical antigen
14. COMPETITIVE ELISA
Used to determine small molecule antigens. (T3,
T4,progesterone etc.) antibody coated microwell
Serum antigen and labelled antigen added
Antibody-antigen-enzyme complex bound is
inversely related to the concentration of antigen
present in the sample.
The bound enzyme conjugate reacts with the
chromogenic substrate added produce a color
reaction (blue to yellow color)..
16. TEST PERFORMANCE
Using a clean Pipette, add 100 μL of diluted serum
sample (Dilute the sera to be tested 1:100 in the
sample diluents) to each well.
Incubate for 1 hour at 37°C.
17. •After incubation empty out contents
of wells into waste container..
•Using pipette, fill wells with
washing buffer then empty out.
•Tap wells upside down on paper
towel.. Wash the wells 5 times.
•At the end of the washing process,
the wells must be entirely dry after
the last wash.
•Distribute 100μL of anti-human
immunoglobulin-POD conjugate in
each well. Incubate 30 minutes at
19. ELISA PLATE READY FOR READING
Measures the absorbance at 450nm with the help
of ELISA READER.
Calculate the absorbance for each sample and
We used Ascent Software for Calculation of the
20. ADVANTAGES OF ELISA
Reagents are relatively cheap & have a long shelf
ELISA is highly specific and sensitive.
No radiation hazards occur during labelling or
disposal of waste.
Easy to perform and quick procedures.
Equipment can be inexpensive and widely
ELISA can be used to a variety of infections.
21. DISADVANTAGES OF ELISA
Measurement of enzyme activity can be more
complex than measurement of activity of some type
Enzyme activity may be affected by plasma
Kits are commercially available, but not cheap.
Very specific to a particular antigen. Won't
recognize any other antigen.
False positives/negatives possible, especially with
Results may not be absolute.
Antibody must be available.
Concentration may be unclear.
False positive possible.
False negative possible
Serum Antibody Concentrations
Detecting potential food allergens (milk, peanuts,
walnuts, almonds and eggs)
Disease outbreaks- tracking the spread of
diseasee.g. HIV, bird flu, common, colds, cholera,
Detections of antigens e.g. pregnancy hormones,
drug allergen, GMO, mad cowdisease
Detection of antibodies in blood sample for past
exposure to disease e.g. Lyme Disease, trichinosis,
HIV, bird flu.