1. PRESENTER: LAIBA KHAN (18)
ALINA FATIMA (22)
PRESENTED TO: DR. SIBTAIN AHMAD
Degree: MS Biochemistry
Semester: 2nd
Subject: Advance Techniques In biochemistry

2. ENZYME LINKED IMMUNOSORBANT ASSAY

3. HISTORY
 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
in 1971.
 ELISA, or enzyme-linked immunosorbent assay,
are quantitative immunological procedures in which
the Ag-Ab reaction is monitored by enzyme
measurements.
 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
 Antibody
 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
blue color.

6. PRINCIPLE
 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
surface ('sorbent').
 2. Antigen is recognized by specific antibody
('immuno').
 3. This antibody is recognized by second antibody
("immuno') which has enzyme attached (enzyme-
linked').
 4. Substrate reacts with enzyme to produce
product, usually colored.

9. TYPES OF ELISA
 INDIRECT ELISA
 DIRECT ELISA
 SANDWICH ELISA
 COMPETETIVE ELISA

10. INDIRECT ELISA
 Antigen is added to plate.
 Added buffer.
 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
plate.
 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
immobilized.
 The antibody of the enzyme conjugate binds with
the immobilized antigen to form a sandwich of
antibody-antigen-antibody/enzyme bound to the
microwell.
 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
together--competition.
 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
37°C.

18. ASSAY PROCEDURE

19. ELISA PLATE READY FOR READING
 Measures the absorbance at 450nm with the help
of ELISA READER.
 Calculate the absorbance for each sample and
reference.
 We used Ascent Software for Calculation of the
result

20. ADVANTAGES OF ELISA
 Reagents are relatively cheap & have a long shelf
life.
 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
available.
 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
of radioisotopes.
 Enzyme activity may be affected by plasma
constituents.
 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
mutated/altered antigen.

22. LIMITATIONS
 Results may not be absolute.
 Antibody must be available.
 Concentration may be unclear.
 False positive possible.
 False negative possible

23. APPLICATIONS
 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,
STD etc
 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.