2. 1.DEFENTION OF ELISA
2.PRINCIPLES OF ELISA
3.EQUIPMENT
4.REAGENTS
5.GENERAL ELISA PROCEDURE
6.ADVANTAGES AND DISADVANTEGES
7.APPLICATION OF ELISA TEST
8.TYPES OF ELISA
A.DIRECT ELISA
B.INDIRECT ELISA
C.SANDWICH ELISA
D.COMPETETITVE ELISA
9.TROUBLESHOOTING IN ELISA
3. DEFENITION
ELISA is the abbreviation of Enzyme-Linked Immunosorbent Assay:
Antigen of interest is adsorbed onto a solid phase (sorbent )
Antigen is recognized by specific antibody (Immuno)
The antibody is conjugated with an enzyme which acts on certain
substrate to produce product, usually colored .
4. DEFENITION
ELISA is considered the gold standard of immunoassays.
This immunological test is very sensitive
Used to detect and quantify substances, including antibodies, antigens,
proteins, glycoproteins, and hormones.
The detection of these products is done by complexing antibodies and
antigens to produce a measurable result.
5.
6. ELISA Principle
1.Antigens from
the sample are
captured by
corresponding
antibodies
coated on to the
solid surface.
2.After incubation
period the plate is
washed to remove
sample and
unbound antigens
or antibodies
with wash buffer
3.To detect the
antibody antigen
complex another
antibody that is
linked to an enzyme
as peroxidase is
added to each well
7. ELISA Principle
4.After an
incubation
period, the
unbound
secondary
antibodies are
washed of.
5.When a
suitable
substrate is
added, the
enzyme acts on
it to produce a
colored product
6.The intensity
of the color
gives an
indication of
the amount of
antigen
8. There are many substrates available for use in ELISA detection.
However, the most commonly used
• horseradish peroxidase (HRP).The substrate for HRP is hydrogen
peroxide and results in a blue color change.
• alkaline phosphatase (ALP) which give the yellow color.
17. REQUIRED REAGENTS
1. Enzyme
Enzymes used in ELISA should
meet requirements such as
• high purity,
• favorable specificity,
• stable properties,
• rich resources,
• cheap price
horseradish peroxidase (HRP) and
alkaline phosphatase (AP) are
usually used.
2. Antigen and antibody
High purified IgG is
usually used in conjugate
in order to avoid the
interference of other
proteins when it is linked
with enzyme. These
enzyme conjugates are
all specific in
immunocompetence and
can react in low
concentration.
3.Substrates:
There are many
substrates available for
use in ELISA detection.
However, the most
commonly used
horseradish peroxidase
(HRP) and alkaline
phosphatase (ALP)
18. REQUIRED REAGENTS
4.Controls
Most kits are formulated with
prediluted controls. However,
some require that you dilute
them in the same manner as
your sample. Controls should be
added to the plate in the same
method and at the same time as
the samples.
5. Wash Solution
Make sure wash solution have
come to room temperature
(18–25°C) before use. It is
usually the largest bottle in a
kit and require the most time
to equilibrate. If the wash
solution still shows crystal
formation after reaching room
temperature, mix it by inverting
it several times.
5. Stop solution
The stop solution should be
at room temperature before
use.. The stop solution may
crystalize at lower
temperatures. Before use,
make sure that it is
completely dissolved and
appears clear. H2SO4 is
widely used as stop solution
for HRP reaction.
21. Advantages
• High sensitivity and specificity
• Easy to perform: protocols are easy to follow and involve
little hands-on time.
• Quantitative: it can determine the concentration of antigen
in a sample.
• Possibility to test various sample types: serum, plasma,
cellular and tissue extracts, urine, and saliva among others.
22. Disadvantages
• Temporary readouts: detection is based on
enzyme/substrate reactions and therefore readout must be
obtained in a short time span.
• Limited antigen information: information limited to the
amount or presence of the antigen in the sample.
23. APPLICATION OF ELISA TEST
• Detect and Estimate the Levels of
Tumor Markers
Prostate-specific antigen (PSA)
Carcinoembryonic Antigen (CEA)
• Detect and Estimate Hormone
Levels
Prolactin
Testosterone
• Tracking Disease Outbreaks
Cholera
Influenza
• Detecting Past Exposures
HIV
Hepatitis
• Screening Donated Blood for Possible Viral
Contaminants
• Detecting Drug Abuse
24.
25. 1. Direct ELISA:
• An antigen coated to a multi-well plate is detected by an antibody
that has been directly conjugated to an enzyme. This detection
method is a good option if there are no commercially available ELISA
kits for your target protein.
• Direct ELISAs are suitable for Immunohistochemical staining of cells &
tissue .
26. 2.Indirect ELISA
• It uses a two-step process for detection: a primary antibody specific
for the antigen binds to the target, and a labeled secondary antibody
against the primary antibody
• Used to detect specific antibodies in a serum sample by substituting
the serum for the primary antibody.
27.
28. 3.SANDWICH ELISA
• It quantify antigens between two layers of antibodies. The sandwich assay uses
two different antibodies that are reactive with different epitopes on the antigen
• The antigen to be measured must contain at least two antigenic epitope
capable of binding to antibody
• Used for detection of antigen like tumor markers, hormones, serum protein
29. Result
The yellow color indicate that the target protein is present
The higher degree of the color, the higher conc. Of the target protein .
30.
31. 4.Competitive ELISA
Competitive ELISAs are great at detecting small analytes such as lipids
and can detect these analytes in complex mixtures like plasma, serum,
or cell extracts.
Principle of the test : two specific antibodies, one conjugated with
enzyme and the other present in test serum (if serum is positive for
antibodies), are used.
- Competition occurs between the two antibodies for the same antigen.
- Appearance of color indicates a negative test (absence of antibodies),
while the absence of color indicates a positive test (presence of
antibodies).
32. In this test, microtiter wells are coated with antigen.
The sera to be tested are added to these wells and incubated at 37°C and
then washed.
If antibodies are present in the test serum, antigen–antibody reaction occurs.
The antigen– antibody reaction is detected by adding enzyme-labeled-specific
antibodies:
Procedures:
The procedures of competitive ELISA are different in some respects
compared with other forms of ELISA.
33.
34. 1. In a positive test:
no antigen is left for these
antibodies to act.
Hence, the antibodies remain free
and are washed away during the
process of washing.
When substrate is added, no
enzyme is available to act on it.
Therefore, positive result is
indicated by absence of color
reaction.
• . In a negative test: in which
no antibodies are present in the
serum,
antigen in the coated wells is
available to combine with
enzyme-conjugated antibodies
and the enzyme acts on the
substrate to produce color.
38. Troubleshooting in ELISA
Contamination of the substrate solution , enzyme labelled antibody or the
control themselves
Insufficient washing
Inadequate blocking of the plate
If negative controls are giving positive result
If no color has developed for positive control or the sample
Check all reagent for storage and dating condition
Microwell plates not coated properly
Reagent applied in wrong order
Contamination of enzyme conjugate
39. If little color has developed for positive control and sample
1.Check the dilution of the enzyme labelled antibody
2.The concentration of substrate
3.Inadequate incubation time
4.Improper washing
5.Poorly coated Microwell plates
6.Contamintion of the reagent
If color has developed for the test sample but not for
positive control
check the source of positive control their expiry date and storage
If the color can be seen but the absorbance is not high as expected
Check the wave length
