Enzyme-Linked Immunosorbent Assay

Reported by:
RIVERA, Rein Casey C.
SERRANO, Mary Blesilda D.
TUA, Marphil Joyce A.
VILLALUNA, Donna Lalaine P.
BSP 4PHI-O

Enzyme-Linked Immunosorbent Assay:

Introduction
A biochemical assay that
uses
antibodies
and
an
enzyme-mediated color change
to detect the presence of
either antigen or antibody in
a given sample

Enzyme-Linked Immunosorbent Assay:

Basic Principle
Uses an enzyme to detect the
binding of antigen (Ag) antibody
(Ab)
The enzyme coverts a colorless
substrate (chromogen) to a
colored product, indicating the
presence of Ag:Ab binding
An ELISA can be used to detect
either the presence of Antigen or
Antibodies in a sample depending
how the test is designed.

Enzyme-Linked Immunosorbent Assay:

BASIC PRINCIPLE

Enzyme-Linked Immunosorbent Assay:

Parts
1. Enzymes
Horseradish Peroxidase
- found in the roots of horseradish,
is used extensively in biochemistry
applications primarily for its ability to
amplify a weak signal and increase
detectability of a target molecule
Alkaline Phosphatase
- is a protein found in
all the tissues of the liver,
bile ducts, and bone.

Enzyme-Linked Immunosorbent Assay:

Parts
2. Microtiter Plate
Microplate
Microwell Plate
Multiwell
Is a flat plate made of
polystyrene with multiple
"wells" used as small test
tubes.
Has become a standard
tool in analytical research
and clinical diagnostic
testing laboratories.

Enzyme-Linked Immunosorbent Assay:

Parts
3. ELISA Reader
Installed in a personal

computer
Detect and process biological
and chemical data using
absorbance luminescence,
and fluorescence detection
modes, including intensity,
TRF, and polarization.
It is where the plate is
positioned in the carrier
between the light source board
and scanning assembly

Enzyme-Linked Immunosorbent Assay:

Applications
Detecting infections
Detecting allergens
Measuring rheumatoid
factors
Detecting autoimmune
diseases
Measuring toxins in
contaminated food
Screening donated blood
Measuring hormone levels
Detect many bacterial and
viral antigens

Enzyme-Linked Immunosorbent Assay:

Applications

Drugs that undergo ELISA testing:

Amphetamine
Barbiturates
Benzodiazepines
Dextrometorphan
Opiates
Zolpidem

Enzyme-linked Immunosorbent Assay:

Importance Of Incubation
For the proper binding between antigen and
antibody and also binding with conjugate and color
development of substrate
Incubation temp: not above 25C
Importance of WASHING: for the removal of any
Antibody/Antigen proper washing and taping is
required otherwise we get the incorrect result
Incubation and Washing is much important for
good results

Enzyme-linked Immunosorbent Assay:

Principle Of Instrument

Enzyme-Linked Immunosorbent Assay:

Advantages
Tests are generally highly SENSITIVE and
SPECIFIC
Doesnt need isotopes
Reagents are likely to have a long shelf life
No radiation hazards occur during labelling
and disposal of waste

Enzyme-Linked Immunosorbent Assay:

Advantages

A versatile tool for physicians, laboratories, and

medical professionals all over the world

Easy to perform and quick procedures
Large no. of samples can be processed at a time
Equipment can be inexpensive and widely
available
Can be used to variety of infections

Enzyme-linked Immunosorbent Assay:

Disadvantages
Requires skilled laboratory
technicians laboratory
equipments
Measurement of enzyme
activity can be more complex
Enzyme activity may be
affected by plasma
constituents
False positives/negatives
possible, especially with

Enzyme-linked Immunosorbent Assay:

Limitations
Results may not be
absolute
Antibody must be
available
Concentration may be
unclear
False positive possible
False negative possible

Enzyme-Linked Immunosorbent Assay:

Sample Specimen

Serum
CSF
Sputum
Urine
Semen

Enzyme-linked Immunosorbent Assay:

Materials Needed
Testing sample
Antibody (1st, 2nd) /
Antigen
Polystyrene microtiter
plate
Blocking buffer
Washing buffer
Substrate
Enzyme
Pipette

Enzyme-Linked Immunosorbent Assay:

Results

Enzyme-Linked Immunosorbent Assay:

Types

1.Direct ELISA
An antigen coated to a multiwell
plate detected by an antibody that
has been directly conjugated to
an enzyme.
Reversible, with an antibody
coated to the plate and labelled
antigen used for detection

Enzyme-Linked Immunosorbent Assay:

Types

Direct ELISAs involve

attachment of the
antigen to the
polystyrene plate
followed by an
enzyme-labeled
antibody

Enzyme-Linked Immunosorbent Assay:

Types
ADVANTAGES
Quick methodology since only
ONE antibody is used.
Cross reactivity of secondary
antibody is eliminated.

DISADVANTAGES
Immunoreactivity of the primary
antibody maybe reduced as a
result of labelling
Labelling every primary antibody
is TIME-CONSUMING and
EXPENSIVE
No flexibility in choice of primary
antibody label from one
experiment to another
Little signal amplification

Direct ELISA

Enzyme-Linked Immunosorbent Assay:

Types

2. Indirect ELISA
An antigen coated to a polystyrene
multiwell plate is detected in two
stages:
Unlabelled primary antibody
(antigen specific)
Enzyme-labelled secondary antibody
(anti-species and often
polyclonal)

Enzyme-Linked Immunosorbent Assay:

Types
Involves attachment of
the antigen to the
polystyrene plate, but in
this case, the primary
antibody is not labeled.
An enzyme-conjugated
secondary antibody,
directed at the first
antibody, is then added.
This format it used most
often to detect specific
antibodies in sera.

Enzyme-Linked Immunosorbent Assay:

Types
ADVANTAGES
Wide variety of secondary
antibody are commercially
available
Immuno-reactivity of the
primary antibody is not
affected by labelling
Different visualization
markers can be used with
the same primary antibody

DISADVANTAGES
Cross reactivity may occur
with the secondary antibody
resulting in non-specific
signal
An extra incubation step is
required in the procedure

Indirect ELISA

Enzyme-Linked Immunosorbent Assay:

Types
3. DOT ELISA
uses NITROCELLULOSE MEMBRANE instead of
polystyrene plate and the substrate is
Bromocreso indoylpyrophosphate
this gives blue color
useful in immunoblot technique

Enzyme-Linked Immunosorbent Assay:

Types
ADVANTAGES
could detect as little as
0.055 ng/mL microbial
antigen
does not require
radioactivity or
sophisticated equipment

DISADVANTAGES
quantification is not
possible

DOT ELISA

Enzyme-Linked Immunosorbent Assay:

Types
4. Sandwhich ELISA
Require the use of matched antibody
pairs (each antibody is specific for an
epitope)
The first antibody (capture
antibody), is coated to the
polystyrene plate. Next, the analyte or
sample solution is added to the well.
A second antibody (detection
antibody), measures the
concentration of the analyte
Direct sandwhich - detection
antibody is conjugated to an enzyme

Advantages:

High specificity
Suitable for
complex
samples
Flexibility
Sensitivity

Enzyme-Linked Immunosorbent Assay:

Types
Involve attachment of a
capture antibody to the
polystyrene plate.
Samples containing
known or unknown
antigen are then added
in a matrix or buffer that
will minimize
attachment of the solid
phase. An enzymelabeled antibody is then
added for detection

Enzyme-Linked Immunosorbent Assay:

Types

Enzyme-Linked Immunosorbent Assay:

Types
Sensitivity depends on
4 factors:
1) Number of mol. of the first
antibody that are bound to
the solid phase
2) Avidity of the first
antibody for the antigen
3) Avidity of the second
antibody for the antigen
4) Specific activity of the
second antibody.

Enzyme-Linked Immunosorbent Assay:

Types
APPLICATIONS:
to detect and quantitate large molecules with
multiple antigenic sites, usually a protein in a
sample
to test recognition or binding between Ag and Ab
where polyclonals are usually used
to make an ELISA when a pair of monoclonal
antibody is not immediately available

Sandwhich ELISA

Enzyme-Linked Immunosorbent Assay:

Types
5. Competitive ELISA
Most complex ELISA
Measures the concentration of
an antigen (or antibody) in a
sample by observing
interference in an expected
signal output
Aka Inhibition ELISA
Most often used when only one
antibody is available to the
antigen of interest or when the
analyte is small

Advantage:

Maximum
flexibility in setup

Enzyme-Linked Immunosorbent Assay:

Types

Competitive ELISA

Enzyme-Linked Immunosorbent Assay:

Types
6. Reverse ELISA
Uses a strong stage
created up of an
immunosorbent
polystyrene rod with 412 sticking out ogives
The whole system is
engrossed in a test
tube containing the
gathered example

Advantages:

gives can every

be sharpened to
an alternate
reagent
Specimen
volume can be
expanded

Enzyme-Linked Immunosorbent Assay:

Types

Reverse ELISA

Enzyme-Linked Immunosorbent Assay:

Types

7. Multiplex ELISA
A logical progression
of the microtiter plate
A protein array
format that allows
detection of multiple
analytes at multiple
array addresses within
a single well

Advantage:

Simultaneous
detection of
multiple
analytes

Enzyme-Linked Immunosorbent Assay:

Types

Enzyme-Linked Immunosorbent Assay:

Types

Multiplex ELISA

1.Apositiveresultonthe ELISA screening test

doesnotmeanthatthepersonhasHIVinfection.Certain
conditionsmayleadtoafalsepositive result,suchasLyme
disease,syphilis,andlupus.Apositive ELISA test isalways
followedbyaWesternblottest.ApositiveWesternblot
confirmsanHIVinfection.
2.The Enzyme-Linked Immunosorbent Assay (ELISA) is
a technique used to detectantibodiesor infectious
agents in a sample.Antibodiesare made in response
to infection and so an antibody ELISA can indicate
whether or not an animal has been in contact with a
certain virus.

1.

Enzyme-Linked Immunosorbent Assay

Results
Quantitative
3. Semi-quantitative

be interpreted in
comparison to a standard
curve (a serial dilution of a
known, purified antigen) in
order to precisely calculate
the concentrations of
antigen in various samples.
2. Qualitative
o
a yes or no answer
indicating whether a
particular antigen is present
in a sample, as compared to
a blank well containing no
antigen or an unrelated

o to compare the relative levels of

antigen in assay samples, since the
intensity of signal will vary directly
with antigen concentration.
o ELISA data is typically graphed with
optical density vs log concentration
to produce a sigmoidal curve as
shown in Figure

What is?
Antibody
An immunoglobulin
molecule having a specific
amino acid sequence that
gives each antibody the
ability to adhere to and
interact only with the
antigen

Antigen
Any substance capable,
under appropriate
conditions, of inducing a
specific immune
response and reacting
with the products of that
response (antibody)
May be soluble substance
(toxins, foreign proteins),
particulates (bacteria and

Applications of ELISA
Flow cytometry- used to detect and distinguish between
benign and malignant hematopoietic disorders such as
various leukemia and lymphoma types.

Functional assay
Immunofluorescence -experiment a primary antibody
binds specifically to a protein of interest present in a
sample (e.g. fixed cells, tissue sections).
Immunohistochemistry -to determine morphological
abnormalities and the presence of biomarkers indicative of
certain diseases such as cancer.

Immunoprecipitation

ELISA Kits
ELISA Kitscontain pre-coated
antibody-plates, detection antibodies,
buffers, diluents, standards, and
substrates.
Antibody Pair Kitscontain only
matched antibodies and standard (no
plates or detection reagents).
Human ELISA Kits
Cynomolgous ELISA Kits
Mouse ELISA Kits
Rat ELISA Kits
Influenza ELISA Kits
HIV ELISA Kits
RSV ELISA Kits

ELISA Kits Components

Store in a lowtemperature (2~8)
and drying condition
for six months
Store in a roomtemperature
(18~25) before
use
*0.05% of tween
20 of PBST is

ELISA Kits Components

Controls should be
added to the plate in
the same method
and at the same
time as the samples
Conjugate consists:
1. Enzymes
2. Antigen and
antibody
*do not save leftover
solution for future
use

ELISA Kits Components

Enzymes
Horseradish Peroxidase
- found in the roots of horseradish,
is used extensively in biochemistry
applications primarily for its ability to
amplify a weak signal and increase
detectability of a target molecule
Alkaline Phosphatase
- is a protein found in
all the tissues of the liver,
bile ducts, and bone.

ELISA Kits Components

Protect this solution
by storing it in a dark
container until ready
for use
Stop solution
Store in a roomtemperature (18~25)
before use
*H2SO4 is widely used
as stop solution
*its conc is based on

ELISA Equipments

ELISA Equipments

Maintenance and Calibration

Schedule of ELISA Equipments

Maintenance and Calibration

Schedule of ELISA Equipments

Maintenance and Calibration

Schedule of ELISA Equipments

Definition of terms

Definition of terms

Definition of terms

 https://
www.idexx.com/pdf/en_us/livestock-poultry/eli
sa-technical-guide.pdf
http://
www.elisa-antibody.com/ELISA-Introduction/eli
sa-reagents
http://www.raybiotech.com/elisa-kits.html
https://
www.abdserotec.com/immunohistochemistry-an
tibodies-immunohistology-kits-reagents.htm
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http://www.elisa-antibody.com/elisa-antibody
http://
www.elisa-antibody.com/ELISA-Introduction/eli
sa-terms
https://