Transcript ELISA - Nobel

ELISA
Enzyme Linked Immunosorbent Assay
Fariba mazrouei
Definitions

Antibodies (also known as immunoglobulins
abbreviated Ig) are gamma globulin proteins that
are found in blood and are used by the immune
system to identify and neutralize foreign objects,
such as bacteria and viruses.
Definitions- cont
 Antigens
A substance that when introduced into the body
stimulates the production of an antibody
Immunoassay
A laboratory technique that makes use of the
binding between an antigen and its
homologous antibody in order to identify and
quantify the specific antigen or antibody in a
sample

Definitions- cont
Analyte
The
sample
being
analyzed
and
in
immunoasssays the analyte is either Antibody
or Antigen

Antigen

Is present naturally in the body like hormones

Is manufactured in special disease status for
example human chorionic gonadotrophin
hormone (HCG) which is normally produced
by cells of the placenta in pregnancy is found in
the body in some types of cancer

Is not present in the body in normal condition
like drugs
Introduction

The Antibody: An immunoglobulin, a
specialized immune protein, produced because
of the introduction of an antigen into the body,
and which possesses the remarkable ability to
combine with the very antigen that triggered
its production (specific affinity)

The antibody recognises and bind to the
antigenic determinant region of the antigen
Labeled Immunoassays

Some antigen/antibody reactions not detected
by precipitation or agglutination.

Looking for very small amounts.

Measured indirectly using a labeled reactant.

Referred to as receptor-ligand assays
Labels

Used to detect reaction which has occurred.

Most common are:
 Radioactive
 Enzymes
 Fluorescent
 Chemiluminescent
Enzyme Immunoassay
 Enzymes occur naturally and catalyze
biochemical reactions.

Enzymes are
 Cheap
 Readily available
 Have a long shelf life
 Easily adaptable to automation.
 Automation relatively inexpensive.
Enzyme Immunoassay

Enzymes used include:
 Horseradish peroxidase
 Glucose-6-phosphate dehydrogenase
 Alkaline phosphatase
 Β-D-galactosidase

Horseradish peroxidase and alkaline
phosphatase are the most popular.
ELISA technique
Is a biochemical technique used mainly in
immunology to detect the presence of an
antibody or an antigen in a sample.

The technique is divided into:
1- Competitive ELISA
2- Sandwich ELISA
3- Indirect ELISA
Competitive ELISA

The labelled antigen competes for primary
antibody binding sites with the sample antigen
(unlabeled). The more antigen in the sample,
the less labelled antigen is retained in the well
and the weaker the signal.
Sandwich ELISA

The ELISA plate is coated with Antibody to
detect specific antigen
Sandwich ELISA-Cont
Antibody bound to solid phase.
 If looking for antigen must have multiple
epitopes, bound antibody specific for one
epitope, second labeled antibody added
specific for a different epitope.

Sandwich ELISA-Cont
Antigens detected can be
 Antibodies
 Hormones
 Proteins
 Tumor markers
 Microorganisms especially viruses
 Enzyme label used to detect reaction

Sandwich ELISA-Cont
1.
2.
3.
4.
5.
Add patient sample with antigen.
Antigen will bind to antibody bound to solid
phase.
Add enzyme labeled antibody directed against
a different epitope on the antigen.
Wash the plate, so that the unbound
antibody-enzyme conjugates are removed.
Add substrate, measure intensity of color.
Sandwich ELISA
Indirect ELISA

The protein antigen to be tested for is added
to each well of ELISA plate, where it is given
time to adhere to the plastic through charge
interactions

A solution of non-reacting protein is added to
block any plastic surface in the well that
remains uncoated by the protein antigen
Indirect ELISA-Cont
 Then the serum is added, which contains a
mixture of the serum antibodies, of unknown
concentration, some of which may bind
specifically to the test antigen that is coating
the well.

Afterwards, a secondary antibody is added,
which will bind to the antibody bound to the
test antigen in the well. This secondary
antibody often has an enzyme attached to it
Indirect ELISA-Cont

A substrate for this enzyme is then added.
Often, this substrate changes colour upon
reaction with the enzyme. The colour change
shows that secondary antibody has bound to
primary antibody, which strongly implies that
the donor has had an immune reaction to the
test antigen.
Indirect ELISA-Cont

The higher the concentration of the primary
antibody that was present in the serum, the
stronger the colour change. Often a
spectrometer is used to give quantitative
values for colour strength
Indirect ELISA
An example of an ELISA experiment

Before starting the work read kit instruction
carefully

1- The 96 well plate is labeled carefully and the
first wells are used to draw the standard curve
An example of an ELISA
experiment-Cont

The sample is added to plate in duplicate or
triplicate and then the mean result is calculated

The quality control sample which is provided
with the kit is treated as the test samples
Standards or Calibrators

Substance of exact known concentration.

Usually run for each new lot number

Based on results create standard curve.

Standard curve used to “read” results or built
into machine to provide results.
Results

After reading the results the standard curve is
drawn were the concentration is blotted on
the X-axis and the absorbance on the Y-axis
Absorption
nm
Concentration ng/ml
Results-cont

The standards concentrations is specified on
the x-axis and the reading of each standard is
specified on the y-axis and the standard curve
is drawn
Results-cont

This standard curve is used to determine the
unknown concentration of each sample by
finding the opposite concentration to the
absorbance
Absorption
nm
Concentration ng/ml
Results-cont

The quality control sample concentration is
determined from the standard curve and if the
result is in the range given by the kit
manufacturer the results could be accepted
Common Problems
The negative controls can give positive results
when the blocking solution isn’t effective,
therefore the secondary antibody or antigen of
interest can bind to the open sites in the well.
 If the positive controls or standards give no
signal, check your chemicals and be aware that
the enzyme reaction is short-term, so the plate
should be read as quickly as possible.

© 2007 NOVUS BIOLOGICALS
Common Problems
 Running your samples in duplicate and
triplicate will allow for a more accurate
determination of concentration.
 Applying your primary antibody in a dilution
range increases the likelihood that you will get
a signal that is neither too weak nor too
strong.
 Past a certain limit, the strength of a signal
gives useless information. Dilute your sample
or primary antibody if this occurs.
© 2007 NOVUS BIOLOGICALS
Useful sites



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http://www.edumedia-sciences.com/en/a543-directenzyme-linked-immunosorbent-assay-elisa
ELISA
http://www.sumanasinc.com/webcontent/animations/cont
ent/ELISA.html
http://www.biology.ualberta.ca/facilities/multimedia/upload
s/procedures/elisa-sound.swf
Competitive and Noncompetitive
Immunoassays



The measurement of analyte in an
immunoassay is achieved by using either
a competitive
or
a noncompetitive format.
Competitive format

unlabelled analyte (usually antigen) in the test
sample is measured by its ability to compete
with labeled antigen in the immunoassay.

The unlabeled antigen blocks the ability of the
labeled antigen to bind because that binding
site on the antibody is already occupied.
Con’ted

Thus, in a competitive immunoassay, less label
measured in the assay means more of the
unlabeled (test sample) antigen is present. The
amount of antigen in the test sample is inversely
related to the amount of label measured in the
competitive format (Figure 1-7).
one step competitive format

In the one step competitive format (see Figure 1-8),
both the labeled antigen reagent (Ag*) and the
unlabeled specimen (or test sample analyte) compete
for a limited amount of antibody.
two step competitive format

In the two step competitive format, the antibody
concentration of the reaction solution is present in
excess in comparison to the concentration of
antigen. Antibody reagent is first incubated with
specimen containing antigens of interest; then in
the second step, labeled antigen is added.
Remember that in the competitive format, less
bound labeled antigen indicates more antigen
present in the test sample. Two step competitive
assay formats provide several fold improved assay
sensitivity compared to one step assay formats.
Noncompetitive (Sandwich)
Method
 Noncompetitive assay formats generally
provide the highest level of assay sensitivity
and specificity and are applied to the
measurement of critical analytes such as
cardiac and hepatitis markers. This format is
referred to as a “sandwich” assay because
analyte is bound (sandwiched) between two
highly specific antibody reagents (Figure 1-10).
Noncompetitive assay

Noncompetitive assay formats can also utilize
either one step or two step methods, as with the
competitive assay.
 The two step assay format employs wash steps
in which the sandwich binding complex is
isolated and washed to remove excess unbound
labeled reagent and any other interfering
substances.
 The two step noncompetitive format usually
offers the highest specificity and sensitivity of
all the assay formats discussed here.