PATIENT SAFETY & EARLY INTERVENTION AND MANAGEMENT

1. ENZYME LINKED IMMUNOSORBENT
ASSAY (ELISA)

2. INTRODUCTION
Enzyme Linked Immunosorbent Assay
• Antigen of interest is absorbed on to plastic surface (‘sorbent’)
• Antigen is recognized by specific antibody (‘immuno’)
• This antibody is recognized by second antibody (‘immuno’) which has
enzyme attached (‘enzyme-linked’)
• Substrate reacts with enzyme to produce product, usually coloured.

3. PRINCIPLE OF ELISA
• A sensitive immunoassay that uses an enzyme linked to an antibody or
antigen as a marker for the detection of a specific protein, especially
an antigen or antibody.
• ELISA involves detection of “analyte” in a liquid sample using liquid
reagent (wet lab) or dry strips (dry lab).
• In dry analysis, strip can be read in reflectometry.The quantitative
reading usually based on detection of intensity of transmitted light by
spectrophotometry at specific wavelength.

4. HISTORY OF ELISA
Radioimmunoassay was first described in
a scientific paper by Rosalyn Sussman
Yalow and Solomon Berson published in
1960.
In 1971, Peter Perlmann and Eva Engvall
at Stockholm University in Sweden, and
Anton Schuurs and Bauke vanWeemen in
the Netherlands independently published
papers that synthesized this knowledge
into methods to perform EIA/ELISA.

5. EQUIPMENT REQUIRED
• In order to perform the ELISA
technique, the following equipment is
required :
1. Micro plate reader
2. Micro plate washer
3. Liquid dispensing system ( multi-
channel pipettes may be used )
4. Incubator to incubate the plates

6. TYPES OF ELISA
i) Direct ELISA :-
In a direct ELISA, an antigen or sample is immobilized directly on the plate and a conjugated
detection antibody binds to the target protein. Substrate is then added, producing a signal that
is proportional to the amount of analyte in the sample. Since only one antibody is used in a
direct ELISA, they are less specific than a sandwich ELISA.

7. ii) Indirect ELISA :-
An indirect ELISA is similar to a direct ELISA in that an antigen is immobilized on a plate, but it
includes an additional amplification detection step. First, an unconjugated primary detection
antibody is added and binds to the specific antigen.A conjugated secondary antibody directed
against the host species of the primary antibody is then added. Substrate then produces a signal
proportional to the amount of antigen bound in the well.

8. iii) Sandwich ELISA :-
Sandwich ELISAs are the most common type of ELISA.Two specific antibodies are
used to sandwich the antigen, commonly referred to as matched antibody pairs.
Capture antibody is coated on a microplate, sample is added, and the protein of
interest binds and is immobilized on the plate. A conjugated-detection antibody is
then added and binds to an additional epitope on the target protein. Substrate is
added and produces a signal that is proportional to the amount of analyte present in
the sample. Sandwich ELISAs are highly specific, since two antibodies are required to
bind to the protein of interest.

9. iv) Competitive ELISA :-
Competitive ELISAs are commonly used for small molecules, when the protein of
interest is too small to efficiently sandwich with two antibodies. Similar to a sandwich
ELISA, a capture antibody is coated on a microplate. Instead of using a conjugated
detection antibody, a conjugated antigen is used to complete for binding with the
antigen present in the sample.The more antigen present in the sample, the less
conjugated antigen will bind to the capture antibody. Substrate is added and the
signal produced is inversely proportional to the amount of protein present in the
sample.

10. APPLICATIONS OF ELISA
• Serum Antibody Concentrations.
• Detecting potential food allergens -
Milk, Peanuts, Walnuts, Almonds, Eggs.
• Disease outbreaks- tracking the spread of disease -
HIV, Bird Flu, Common,Colds, Cholera, STD etc.
• Detection of antigens -
Pregnancy hormones, Drug allergen, GMO, Mad cow disease.
• Detection of antibodies in blood sample for past exposure to
disease -
Lyme disease,Trichinosis, HIV, Bird flu.

11. ADVANTAGES AND DISADVANTAGES
TYPES ADVANTAGES DISADVATAGES
Direct ELISA ● A straightforward and fast method
● No risk of cross-reactivity
● Not very specific results due to using only
one antibody
● Risk of high background reading
Indirect ELISA ● Higher sensitivity due to amplifying the
signal with a second antibody
● Higher specificity
● High flexibility
●The secondary antibody increases the risk
of cross-reactivity.
● Potential cross-reactivity could cause high
background noise.
Sandwich ELISA ● Highest level of sensitivity
● Highest specificity due to using two
antibodies
● High flexibility
●Takes more time than any other type of
ELISA
● Can be more expensive
Competitive ELISA ● High sensitivity and flexibility
● Ability to measure tiny molecules
● Great for measuring immune responses
● High sensitivity and flexibility
● Ability to measure tiny molecules
● Great for measuring immune responses

12. THANK YOU