Immunoassays: PD Dr. J. Graßmann PD Dr. T. Letzel

Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt

Technische Universität München
Immunoassays
PD Dr. J. Graßmann; PD Dr. T. Letzel

Immunoassays
Immunoassays or „ligand binding assays“ employ antibodies to

specifically bind an antigen, which is the analyte of interest (or use an
antigen to quantify an specific antibody). Immunoassays are highly
sensitive and specific. The extent of antigen-antibody-complexes
formation is used as a means for quantification.

Immunoassays
ELISA
ELISA (enzyme-linked immunosorbent assay) is a plate-based method, which utilizes

the specifity of antibodies to detect and quantify substances such as proteins, peptides,
nucleic acids or hormones.
The presence of a substance of interest is detected by its specific binding to the
respective antibody, which ultimately causes a colour change (e.g. generated by the
action of an enzyme) detectable with appropriate instrumentation.
One can distinguish between „direct/indirect ELISA“, „sandwich ELISA“ and „competitive
ELISA“.
The detection can be either achieved via visible color changes, generation of a
fluorescent, chemiluminescent or electrochemical signal.

Immunoassays
Direct ELISA
Addition of the enzymatic
substrate and the generation of
Substrate Product product results in the generation
of a detectable signal.
Antibody specific to the

antigen is added with an
enzyme attached
Antigens, i.e. substance of

interest attached to the
surface

Immunoassays
Indirect ELISA
Substrate Product
Secondary
antibody
Detection
antibody

Immunoassays
Sandwich ELISA
Substrate is converted to
product by the enzyme, which
Substrate Product can be detected.
Secondary antibody with

attached enzyme is added and
Secondary binds to the detection antibody
antibody
Antibody specific to the antigen
Detection binds the antigen
antibody
Antigens, i.e. substance of
interest binds to the
Capture antibody attached to the
antibody surface

Immunoassays
Quantification
To be able to quantity the substance of interest, a calibration is performed beforehand.

For this purpose increasing concentrations of the analyte are added to the antibodies.
The intensity of the detected signal corresponds to the known analyte concentration.
The signal obtained with an unknown analyte concentration can now be referred to the
calibration curve to conclude on the analyte quantity within the unknown sample.

Immunoassays
Calibration curve

Immunoassays
Competitive ELISA
Known concentration of
Sample containing protein of interest which
various proteins is conjugated with a
Plate coated with antibodies including an unknown reporter enzyme
specific to the protein of interest quantity of the protein (=enzyme conjugate)
of interest
+ +

Immunoassays
Competitive ELISA
Sample with the unknown amount of protein
of interest is mixed with the enzyme
conjugated protein
Mixed sample is added to the plate coated
with specific antibodies
 The non-conjugated and the conjugated
protein of interest will compete for the
binding to the limited number of antibodies
Unbound protein and enzyme-

PD Dr. J. Graßmann; PD Dr. T. Letzel conjugate will be removed by washing
Immunoassays
Competitive ELISA
Substrate Product
The substrate added will then be converted to detectable product

by the enzyme conjugated proteins, but not from the proteins
originating from the unknown sample

Immunoassays
High concentration of protein of interest in the unknown sample results in less

conjugated enzyme able to bind to an antibody, which results in a lower product
generation and a lower detectable signal.
Substrate Product

Immunoassays
Low concentrations of sample protein allow a higher quantity of enzyme conjugate

to bind to the antibodies, which results in a high detectable signal.
So the amount of colour produced in inversely related to the amount of protein of

interest (in contrast to conventional direct, indirect of sandwich ELISA techniques).
Substrate Product

Immunoassays
Production of specific monoclonal antibodies
Monoclonal antibodies are produced by identical immune cells, resulting in them being
monospecific, i.e. they bind one specific antigen. They can be used to detect (e.g.
ELISA) or purify antigens of interest.
One way to obtain monoclonal antibodies specific to the desired antigen is to fuse
mouse spleen cells, which has been immunized with the antigen of interest, with
myeloma cells (= cancer of antibody producing plasma cells) to produce hybridoma
cells. Those are able to secret an antibody, which is able to bind the desired antigen.
Further steps to purify the antibody include centrifugation, filtration, chromatographic
separation and/or electrophoretic methods.
An especially high purity can be achieved by employing affinity chromatography. The
immobilized antigen is hereby used to specifically bind and retain the desired antibody,
whereas impurities will be washed away. The antibody can then be recovered by means
of e.g. a high salt buffer, which would break the bond between antigen and antibody.

Immunoassays
Radioimmunoassay
RIA allows the quantification of small quantities of molecules like e.g. hormons,
enzymes, pharmaceuticals or DNA. The detection is specific since antibodies are used,
which bind to the substance (=antigen) of interest. The experimental setup and
prodecure is comprable to „competitive ELISA“. Radioactive-labelled and non-labelled
antigens are mixed with specific antibodies. The more non-labelled antigens present in
the sample, the less radioactive-labelled antigen can bind to the antibodies. Unbound
antigens are washed away and the radioactivity of the sample is detected.
Another possibility to detect specific antigens is to radioactive-label the antibodies which

bind the antigen (comparable to direct or indirect ELISA).
However radioactive methods require specially equipped laboratories, which is often

cost-intensive. For this reason ELISA is often preferred over RIA.

Immunoassays
Radiobinding assay
Radiobinding assay is used for the detection and quantification of antibodies.
A sample, which might contain the antibody of interest is mixed with a radioactive-
labelled antigen. The antigen specifically binds the desired antibody, thus allowing the
assessment of antibody presence and quantity in the sample.
+ Radiolabelled Removal of
antigen Centrifugation+ unprecipitated
precipitation antibodies
Detection of
PD Dr. J. Graßmann; PD Dr. T. Letzel radioactivity
Immunoassays
Immunoprecipitation
Immunoprecipitation is used for the detection and quanitification of specific antigens,

e.g. protein from a cell lysate. An antibody, which binds the desired antigen, is added.
After binding of the antibody to the antigen, the sample is centrifuged, which results in
the precipitation of the antigen-antibody-complex. Other substances are thereupon
removed. The purified sample can then be analysed using e.g. Western Blot.
Specific antibody Removal of

Centrifugation+ unprecipitated
precipitation antibodies

Immunoassays
Immunoprecipitation
The precipitation step either involves the immobilization of antigen-antibody complexes
by e.g. magnetic beads and/or the binding of special proteins to the antibody. The latter
results in the insolubility and thus precipitation of the antigen-antibody-protein
complexes.
Magnetic beads are coupled to the specific antibody and allow to capture and
immobilize the antigen-antibody complex until the sample is washed and purified.
Separation/
Immobilization Washing Purification

Immunoassays
Magnetic immunoassay
Magnetism can also be used to quantify an antigen of interest instead of using

conventional radioactive labelling or fluorescent detection.
The quanity of magnetic beads, which are bound to the antigen is detected by
measuring the magnetic field using a magnetometer.
The detected signal is proportional to the analyte/antigen within the sample.

Immunoassays
Dot Blot
Dot Blot is an easy and fast method to detect whether or not a substance of interest is
contained in a sample.
A protein sample is applied directly onto a membrane, whereupon e.g. vaccuum is
applied, which results in other impurities to pass whereas the proteins bind the
membrane. It can be considered similar to Western Blot, however no electrophoretic
separation of the proteins is included.
A primary antibody specific to the protein of interest is added and binds the respective
protein. The secondary antibody, which is conjugated with a reporter enzyme, then
binds the primary antibody. A signal is generated by the addition of the enzymatic
substrate and the generation of the detectable product (comparable to ELISA or
Western Blot detection).
Dot Blot is either used for qualitative analysis or employed as a fast screening method
for a large quanity of samples. Protein concentrations can however be estimated semi-
quantitatively by direct comparing the strength of the detected signal of different
samples or samples with known protein concentrations.
Immunoassays
Dot Blot
1 2 3
Samples are applied to the membrane,
addition of primary, secondary antibody and
enzymatic substrate
Samples containing a mixture of

proteins and varying quantity of
the protein of interest

Immunoassays
Dot Blot

Immunoassays
FACS (= fluorescence activated cell sorting)
FACS provides the possibility to sort and quantify cells according to their size,
inner cellular structure or specific surface molecules (e.g. receptors) using light
scattering and fluorescence. Molecules of interest, either on the cell surface or
within the cell are marked with specific antibodies, which are conjugated with
fluorophores. By means of these markers, FACS helps to to characterize a
large population of different cells or identify particular cells with special
properties.

Immunoassays
FACS

Immunoassays
FACS Side scatter
Side scatter provides insight into the

cells structural complexity.
Laser beam

Immunoassays
FACS

Immunoassays
FACS
Cells can be sorted according
to their surface molecules
(„specific antigen“),
e.g. receptors, by marking
those structures with antibodies
conjudated with fluorophores.

Immunoassays
FACS
FACS also allows the detection of two different fluorophores at a time.

Immunoassays
FRET (= Förster/Fluorescence resonance energy transfer)
Is a mechanism by which energy is transferred from one chromophore to another. When two
chromophores get within a certain distance to each other, the excited donor chromophore is able to
transfer its energy to a so-called acceptor chromophore. The change of emission wavelength (here
from orange to blue) reveals the closed distance between both chromophores.
Chromophore 1 Chromophore 2
Light absorption
Emission
Wavelength at x nm
wavelength at y nm
No FRET
Energy transfer
Light absorption Emission
Wavelength at x nm wavelength at z nm FRET
Chromophores in close proximity

Immunoassays
FRET
An important regulatory mechanism in cells is the phosphorylation of proteins. With FRET the
identification of particular protein phosphorylation becomes possible. By using two antibodies,
which for one thing bind to the phosphorlyation side and for another thing bind to a specific
structure of the protein of interest, the investigation of cellular regulation becomes possible
Detection wavelength
P P
Both antibodies bind to the Antibody binds to the

One antibody binds to the
phosphorylated protein of interest phosphorylated side of
non-phosphorylated protein
another protein, which is
of interest.
 FRET however not recognized by
the second antibody
 No FRET
PD Dr. J. Graßmann; PD Dr. T. Letzel  No FRET
Immunoassays
FRET

Immunoassays: PD Dr. J. Graßmann PD Dr. T. Letzel

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Lehrstuhl für Siedlungswasserwirtschaft

Ingenieurfakultät Bau Geo Umwelt

PD Dr. J. Graßmann; PD Dr. T. Letzel

Immunoassays or „ligand binding assays“ employ antibodies to

PD Dr. J. Graßmann; PD Dr. T. Letzel

ELISA (enzyme-linked immunosorbent assay) is a plate-based method, which utilizes

PD Dr. J. Graßmann; PD Dr. T. Letzel

Antibody specific to the

Antigens, i.e. substance of

PD Dr. J. Graßmann; PD Dr. T. Letzel

PD Dr. J. Graßmann; PD Dr. T. Letzel

Secondary antibody with

PD Dr. J. Graßmann; PD Dr. T. Letzel

To be able to quantity the substance of interest, a calibration is performed beforehand.

PD Dr. J. Graßmann; PD Dr. T. Letzel

PD Dr. J. Graßmann; PD Dr. T. Letzel

PD Dr. J. Graßmann; PD Dr. T. Letzel

Unbound protein and enzyme-

The substrate added will then be converted to detectable product

PD Dr. J. Graßmann; PD Dr. T. Letzel

High concentration of protein of interest in the unknown sample results in less

PD Dr. J. Graßmann; PD Dr. T. Letzel

Low concentrations of sample protein allow a higher quantity of enzyme conjugate

So the amount of colour produced in inversely related to the amount of protein of

PD Dr. J. Graßmann; PD Dr. T. Letzel

PD Dr. J. Graßmann; PD Dr. T. Letzel

Another possibility to detect specific antigens is to radioactive-label the antibodies which

However radioactive methods require specially equipped laboratories, which is often

PD Dr. J. Graßmann; PD Dr. T. Letzel

Immunoprecipitation is used for the detection and quanitification of specific antigens,

Specific antibody Removal of

PD Dr. J. Graßmann; PD Dr. T. Letzel

PD Dr. J. Graßmann; PD Dr. T. Letzel

Magnetism can also be used to quantify an antigen of interest instead of using

The detected signal is proportional to the analyte/antigen within the sample.

PD Dr. J. Graßmann; PD Dr. T. Letzel

Samples containing a mixture of

PD Dr. J. Graßmann; PD Dr. T. Letzel

PD Dr. J. Graßmann; PD Dr. T. Letzel

PD Dr. J. Graßmann; PD Dr. T. Letzel

PD Dr. J. Graßmann; PD Dr. T. Letzel

Side scatter provides insight into the

PD Dr. J. Graßmann; PD Dr. T. Letzel

PD Dr. J. Graßmann; PD Dr. T. Letzel

PD Dr. J. Graßmann; PD Dr. T. Letzel

PD Dr. J. Graßmann; PD Dr. T. Letzel

Chromophores in close proximity

Both antibodies bind to the Antibody binds to the

PD Dr. J. Graßmann; PD Dr. T. Letzel

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