Serology: Fluorescent antibody tests and other tests employing conjugated antibodies
Fluorescent antibody tests and other tests employing
conjugated antibodies
Authors: Adapted by Prof M van Vuuren. Originally compiled by Dr RW Worthington. (Retired)
Licensed under a Creative Commons Attribution license.
Introduction ................................................................................................................................................. 2
Immunofluorescence assays ..................................................................................................................... 2
Fluorescent antibody and immunohistochemical tests for the demonstration of antigen ........................ 2
Anti-IgM/FITC conjugates ........................................................................................................................ 5
Monoclonal antibodies for us in FA tests ................................................................................................. 5
Procedure for fluorescent antibody staining of virus-infected cell cultures in plastic plates .................... 6
Trouble shooting ...................................................................................................................................... 6
Immunoblotting and immunohistochemistry ........................................................................................... 7
Immunoblots for antibody detection ......................................................................................................... 7
Immunoblots for antigen detection ........................................................................................................... 9
Serology: Fluorescent antibody tests and other tests employing conjugated antibodies
Fluorescent antibody (FA) tests, immunoenzyme assays and radioimmunoassays are primary binding
assays. This implies that antigen-antibody complexes are formed during the test procedures, followed
by detection or measurement of the amount of immune complexes formed. A period of time is allowed
to enable the reaction of antigens and antibodies to reach an equilibrium phase after which the
unbound antibody is separated.
Antibodies linked to fluorescent markers or enzymes can be used to detect antigen in tissues or tissue
cultures or for the detection of antibody in indirect tests. Fluorescein isothiocyanate is readily coupled
to proteins and is the marker most commonly used for attachment to antibodies. When exposed to
ultraviolet light, fluorescein radiates longer wavelength apple green coloured light. A variety of dyes
e.g. rhodamine are used today but will not be discussed here. Sections or smears of tissues may be
stained with fluorescein labelled antibodies and examined under an ultraviolet light microscope to
locate antigenic material to which the antibody has attached. When enzyme linked antibodies are
used to locate antigen, the section or smear is flooded with antibody coupled to an enzyme.
Alternatively an uncoupled positive serum can be used and a second antibody coupled to an enzyme
can be used to identify the attached antibody in a sandwich test similar to those used in ELISA tests.
Sections are then washed and flooded with a substrate, which undergoes a chemical reaction
catalysed by the enzyme. The substrate changes colour and is deposited in the tissues. Stained
areas can then be visualised by conventional light microscopy. The enzyme usually used for this
technique is horseradish peroxidase.
Direct and indirect immunofluorescence procedures provide means whereby the site of an antigenantibody reaction can be observed by microscopic examination. Specific antibody molecules are
chemically linked to a fluorescent dye, the most common of which is fluorescein isothiocyanate
(FITC). These labelled antibodies retain their specificity for their respective antigens, and after a short
period, the reaction site can be visually detected with the aid of a fluorescence microscope.
Fluorescent antibody and immunohistochemical tests for the demonstration of
Direct fluorescent antibody tests
The most elementary form of the FA test is the direct test where specific antibodies (polyclonal
or monoclonal) are marked with (conjugated to) a fluorescent dye and then placed directly onto
the target or capture antigens. After an appropriate incubation period of 30-45 minutes, the
excess unbound antibodies are washed away with phosphate buffered saline (PBS). The PBS
is then washed off with distilled water and the preparation covered with a suitable mounting
fluid such as buffered glycerol and examined with a fluorescence microscope. The direct FA
Serology: Fluorescent antibody tests and other tests employing conjugated antibodies
test can only be used for the detection of specific antigens and not for the detection of
Fluorescent antibody tests (FAT) are frequently used for the demonstration of antigens in tissue
and tissue culture preparations. An example is the demonstration of classical swine fever virus
in tissues of infected pigs. In a typical case tonsil from suspected infected pigs would be used
as follows:
Sections are cut from frozen tissue and acetone fixed to microscope slides using
standard histological techniques.
The sections are washed in PBS, flooded with a classical swine fever antibody
conjugated to fluorescein and incubated in a humid chamber for 30-minutes at 370C.
The conjugate is used at a dilution previously determined by titration.
Known positive and negative control sections are similarly tested
The slides are washed in PBS to remove all unbound conjugate, blotted dry and
covered with mounting buffer and a coverslip and examined under an UV
The presence of brilliant green fluorescing cells indicates the presence of virus in the tissues. In
the case of classical swine fever, cross-reactions can occur with other pestiviruses when
polyclonal sera are used as the conjugate. For this reason highly specific monoclonal
antibodies that are specific for swine fever and for other pestiviruses can be used on additional
sections to differentiate the viruses. In principle the specificity and sensitivity of tests depends
on the antibody used. Where high specificity is a prime requirement of the test, high quality
MAbs are generally preferred if they are available. Polyclonal antibodies may give results of
similar or greater sensitivity but are less specific than MAbs.
Similar tests can be done using antibody conjugated to horseradish peroxidase instead of
fluorescein. In this case after washing to remove unattached conjugate the slide must be
incubated with substrate and then examined by conventional light microscopy to identify
stained antigen. Substrates that deposit as a coloured product in the tissues when oxidised by
peroxidase must be used. Some of the substrates suitable for use with horseradish peroxidase
3-amino-9 ethyl carbazole; N,N- dimethyl-formamide
Diaminobenzidine tetrahydrochloride
FAT and immunohistochemical methods using enzyme conjugated antibodies are used to
identify virus in tissues and in tissue culture including tissue culture used in virus neutralisation
tests. Use of these methods in tissue culture is useful for identifying virus, when it grows
Serology: Fluorescent antibody tests and other tests employing conjugated antibodies
without an easily identifiable cytopathic effect on the tissue cells. The methods are also used to
identify bacterial infections such as venereal campylobacteriosis and clostridial infections. A
major advantage of these methods over tissue culture methods is that the results are obtained
more rapidly.
Indirect fluorescent antibody tests
Indirect fluorescent antibody tests (IFA) are used for detecting antibody in serum. An antigen
preparation such as a smear that contains identifiable antigen particles must be available for
the test. IFA is widely used for protozoal diseases where bloodsmears or parasite smears can
be used as antigen. The test is also commonly used in a wide variety of viral diseases where
tissue culture preparations are used as capture antigens.
The indirect test is more versatile in that it can be employed for the detection of both antibodies
and antigens. It can for certain applications be more sensitive than the direct test in that its
fluorescent signal is amplified as a result of stacking of antibodies on top of each other as a
result of more binding sites being available, thus leading to more FITC molecules being
detected with an UV microscope.
To be able to use the indirect FA test for the detection of antigens, one needs good quality
known antiserum against the specific antigen in question and an anti-species IgG-FITC
conjugate. In other words, if we want to detect the presence of antigens in smears or tissues,
we have to react our specific antibodies with the target or capture antigens in the same way as
described for the direct test. However, after this step, the antigen-antibody reaction will not be
visible microscopically as a result of the absence of fluorescing molecules. To achieve that
objective, one must add an anti-species antibody/FITC conjugate prepared in a different
species as the one under investigation. This secondary antibody will attach to the primary
antibody and give rise to the fluorescent signal. To enhance the contrast between negative and
positive organisms or infected and non-infected cells, a counter-stain such as Evan’s blue is
added in conjunction with the FITC conjugate.
The indirect FA test can similarly be employed to detect antibodies. In this approach however,
the unknown element is the serum specimen and the known entity is the target cell or
organism. The target or capture antigens are prepared in the laboratory in the form of tissue
culture-infected cells (in the case of intracellular microorganisms), or smears of larger
microorganisms such as protozoa. These targets are fixed onto microscope slides, and
combined/reacted with the known primary antibodies. The antigen-antibody complexes are then
again made visible by adding an anti-species IgG/FITC conjugate diluted in Evan’s blue
The example given below describes an IFA test for detection of anti-viral antibodies.
Protocol for the indirect fluorescent antibody test
1. Remove slides to be used from the -20 ˚C freezer (chest freezer) or -80 ˚C freezer to thaw
Serology: Fluorescent antibody tests and other tests employing conjugated antibodies
2. Make two-fold serial dilutions of the animal serum sample starting at 1:10 and ending at 1:5
3. Place 10µl of each serum dilution on wells on the slide. To be able to use only one pipette
tip, start with the highest dilution (1:5 120) and work backwards to fill the spots on the slide.
The first well on top left is reserved for the positive control serum, and the first well bottom
left for the negative control serum
4. Incubate at 37˚ C for 30 minutes in a moist chamber
5. Wash slide for 5 minutes in PBS (preferably sterile but not compulsory) with a magnetic
stirrer. Rinse slide in distilled water and blow dry
6. Add 10µl of an anti-species IgG or IgM conjugate (e.g. anti-cat IgG conjugated with
fluorescein isothiocyanate (FITC) diluted in 0.05% Evans Blue solution
 The freeze-dried commercial conjugate is reconstituted in distilled water as
recommended by the manufacturer (usually 1 ml) and aliquoted into 0.25µl volumes in
Nunc tubes and stored at -20 ˚C
Evans Blue is prepared from 0.5% stock that is stored in a dark bottle at room
temperature. Dilute 1:10 in PBS to obtain a 0.05% solution
To use in the test, take a vial with 25µl conjugate and add 2 ml of the 0.05% Evans
Blue to obtain a 1:80 dilution. If some remains after the test, it can be left in the fridge
at 4˚C for up to 10 days maximum
7. Incubate at 37˚ C for 30 minutes in a moist chamber
8. Wash slide for 5 minutes with a magnetic stirrer using PBS (preferably sterile but not
compulsory). Rinse slide in distilled water and blow dry
9. Place 2-3 drops of glycerol-containing mounting fluid on slide and cover with a coverslip to
allow mounting fluid to fill all wells
10. Examine slide with an UV microscope
Anti-IgM/FITC conjugates
Detection of pathogen-specific IgM is used to diagnose recent infections with a single serum sample.
Different antibody classes behave differently during the course of an infectious disease. Antibodies of
the primary response belong to the IgM class, and fall below detectable levels after 2-3 months.
Already in the first two weeks following infection, they are starting to be replaced with antibodies of
the IgG class, the latter which may persist for years, but often drop to undetectable levels after 14-18
months. Detection of pathogen-specific IgM antibodies therefore usually indicates a recent infection.
Monoclonal antibodies for us in FA tests
Monoclonal antibodies result from the fusion of normal antibody-producing plasma cells and nonantibody-producing myeloma cells. The hybrid cells are permanently adapted to grow in cell cultures
and are capable of producing antibodies of desired specificity. In contrast, polyclonal antibodies have
traditionally been limited by difficulty in generating reagents of high quality and specificity. Purified
antigens used to induce polyclonal antibody formation often are contaminated with small amounts of
macromolecules that induce large amounts of unwanted antibodies. Even high titre specific antiserum
is composed of antibodies heterogenous in their affinity, biological activity and cross-reactivity.
Serology: Fluorescent antibody tests and other tests employing conjugated antibodies
Procedure for fluorescent antibody staining of virus-infected cell cultures in
plastic plates
Plastic multi-well plates are useful when testing a large number of samples. The effect of acetone on
plastic plates however, must be considered when using acetone to fix cells in the plates. Acetone
alone or as an 80% solution in PBS will etch the plate, making it impossible to read the results. To
overcome this problem, the PBS must be added to the wells before adding the acetone. PBS is added
in 0.2 ml volumes to each well of a 24-well plated to be stained. Acetone is then gently added to the
PBS using a Pasteur pipette to fill the wells about three-quarters full.
Trouble shooting
Non-specific staining
The IFA is a subjective test method which does not always allow an easy interpretation. Ideally
one should include three positive sera (high, medium and low) and a negative serum to serve
as controls. Serum dilutions less than 1:20 (or 1:40 for certain preparations) may lead to nonspecific staining.
Look critically at positive controls to determine:
The ratio of negative to positive cells/organisms
The nature of the fluorescence
Trapping of conjugate among the cells or organisms
The presence of anti-nuclear or anti-cytoplasmic fluorescence
Control wells may give an incorrect result
Check capture/target antigens by reacting it with antigen-specific conjugates with the aid of a
direct FA test
Check anti-species conjugates with known antigens and known primary antibody. Once
reconstituted, it can only be kept for up to 10 days at 4 ˚C.
Check positive control sera with known antigens and known secondary antibody. Serum can
deteriorate following several freeze and thaw cycles. Use at a dilution of four-fold less than the
antibody titre.
Check pH of wash buffer
Check for presence of mounting fluid under coverslip
Check that the fluorescence objective of the microscope is working
Check the life-expectancy (in working hours) of the fluorescent light source (bulb)
Serology: Fluorescent antibody tests and other tests employing conjugated antibodies
Immunoblots for antibody detection
With most serological tests when using impure antigens the exact antigen or epitope with which an
antibody is reacting may be in doubt. Immunoblotting tests utilise the resolving power of
polyacrylamide gel electrophoresis to separate antigen components before testing them with the
Western blotting
Proteins are separated on sodium dodecyl sulphate polyacrylamide gels according to their
molecular mass. The separated proteins are transferred electrophoretically onto a nitrocellulose
membrane. Membranes are incubated together with a blocking solution such as Nonidet P-40,
Tween-20, non-fat dried milk or bovine serum albumin to block free sites on the membrane.
Primary antibody is then applied to the membrane. Secondary antibody labeled with an enzyme
is added. The reaction is developed by incubation of the membrane in an appropriate substrate.
The catalytic product gives rise to an insoluble colour.
A typical example would be a test for Brucella ovis antibody in which there was some doubt
about whether the reaction was specific or non-specific. In this case an immunoblotting test
could be done to attempt to resolve the situation. A crude Brucella ovis antigen is subjected to
electrophoresis using a high-resolution polyacrylamide gel technique in a buffer containing
sodium dodecyl sulphate (SDS). The crude antigen will be resolved into many bands,
representing substances with varying electrophoretic mobility. In this system their mobility will
be mainly a function of their molecular weight with smaller molecules moving faster than larger
molecules. A set of proteins of known molecular weight are electrophoresed on the same gel
slab. Once the electrophoresis is complete the resolved components are transferred onto a
nitrocellulose membrane. The membrane is placed in contact with the polyacrylamide gel slab
and an electric current is passed through the gel and membrane at right angles to the direction
of the current in the original electrophoresis. The components are driven out of the gel and
immobilised onto the membrane. The strip containing the molecular weight markers is stained
with a protein stain. The rest of the membrane is washed, blocked with horse serum to prevent
non-specific binding of sheep serum components to the membrane, and strips of antigen are
flooded with the serum to be tested. Antibodies in the serum will react with and attach to the
molecules containing complementary epitopes. The membrane is then washed in blocking
buffer and flooded with antibody to sheep IgG, conjugated to horseradish peroxidase or alkaline
phosphatase. After further washing the membrane is immersed in substrate and the reaction
catalysed by the enzyme causes a coloured reaction product to be deposited on the
membrane. If the enzyme is alkaline phosphatase, the substrate is usually 5-bromo-4 chloro-3
indolyl phosphate and nitro blue tetrazolium and in the case of horseradish peroxidase it is
diaminobenzidine tetrahydrochloride and hydrogen peroxide. Antigen bands that have reacted
with antibody in the test serum are revealed as stained bands. The approximate molecular
Serology: Fluorescent antibody tests and other tests employing conjugated antibodies
weight of stained bands can be estimated by comparing the distance the particular antigen
component has migrated to the migration of the molecular weight control bands. A typical
example of an immunoblot for Brucella ovis is shown below.
An immunoblot done with a crude Brucella ovis antigen and sera from infected and non-infected
sheep. Those sera with clearly stained bands of 29 kDa (sera 5, 7, 11, and 13) are positive for
Brucella ovis antibody.
Immunoblotting is a powerful research and diagnostic tool but it is expensive and complicated
to perform and relatively small numbers can be tested at one time.
The results may be
complicated and difficult to interpret and it is necessary to know which antigen bands are
normally shown up by typical positive sera. There are usually several bands that are indicative
of the immunodominant antigens. For example for Brucella ovis bands with molecular weights
of 63, 29, 19, 17 and 12 kiloDaltons are commonly found in sera from infected animals. For
Brucella ovis the 29kDa band is considered the most important and whenever this band is
shown up, the serum is considered positive.
There may be several complicating factors and considerable experience and practice is needed
before interpretation of various immunoblot patterns can be made with confidence. Generally
several antigenic components are present in the crude antigen preparation used. A bacterium
may contain antigenic LPS on its surface and several outer membrane proteins or internal
proteins that are antigenic. Several or all of these may be found in the crude antigen
preparation. Some may be stronger antigens or present in larger amounts than others. The
antigens that evoke the best antibody response are known as immunodominant antigens and
these are the most commonly appearing and darkest bands identified by positive sera.
Individual animals may respond differently from others, and some animals appear to respond
more strongly to some antigens than others. Antigenic substances may be present in different
forms. An antigen may be present as an intact antigen visible as a single band or it may be
partially degraded so that some smaller molecular weight antigenic bands derived from the
Serology: Fluorescent antibody tests and other tests employing conjugated antibodies
same molecule may be present. Depending how an antigen has been fragmented when it is
degraded, different parts of the antigen containing different or similar epitopes may appear at
different positions on the electrophoretogram. Alternatively complexes of antigenic proteins or
peptides may occur as antigenic bands made up of several identical units or complexed to
other peptides or proteins thus giving rise to components of different molecular weight but
containing identical epitopes.
The above are theoretical possibilities and it may not be known what the structure of a
particular antigenic component of a crude antigen preparation is. The use of MAb helps to
resolve some difficulties but does not completely resolve all issues because a MAb may react
with more than one band, when components of differing electrophoretic mobility contain
identical epitopes. Animals that are to be tested may have had contact with infectious agents
that have epitopes in common with some of the antigenic components of the crude extract. If
they react with bands not usually identified by known positive sera the interpretation is easy but
if they cross react with some of the antigens usually regarded as markers for Brucella ovis the
findings may be confusing. Yet other animals may have had contact with both Brucella ovis and
with other cross-reacting organisms and may generate even more complicated patterns.
Clearly it is necessary to study the usual patterns generated by large numbers of known
positive, negative and non-specific reacting sera, and to work out rules for interpretation of
results using a particular test.
The above discussion of the difficulties of interpretation of immunoblots may appear daunting
but non-specific reactions only occur exceptionally and immunoblots provide a powerful tool for
resolving problems of this nature.
Dot blot
Antigen is applied as a dot on a nitrocellulose membrane and air-dried. Free sites are blocked
with blocking solutions. The specimens to be tested for antibodies are then applied. The
amount of antibody captured is detected by the addition of an enzyme-labelled anti-species
Immunoblots for antigen detection
Immunoblotting can also be used to identify antigens in tissues. The example given in Figure 2 shows
a test to identify rabbit calicivirus in the livers of suspected infected rabbits. Extracts of liver samples
are made and subjected to electrophoresis. When stained with Coomassie blue the complex nature of
these extracts is revealed. It would be impossible to identify virus antigen in these extracts. However,
a monoclonal antibody against the viral antigen band will attach to it and can be identified by an antimouse antibody coupled to an enzyme (See below).
Serology: Fluorescent antibody tests and other tests employing conjugated antibodies
Immunoblotting of rabbit liver extracts
Above are stained preparations of the liver extracts of five samples and negative and positive
controls. Below are the same extracts that have been blotted with a monoclonal antibody against
rabbit calicivirus VP60 antigen. Sample 5 is a positive sample showing the same antigen line as
the positive control. This figure has been taken from the following article: Motha MXJ., Kittelberger
R. 1998. Evaluation of three tests for the detection of rabbit haemorrhagic disease virus in wild
rabbits. Veterinary Record 143, 627-9. It has been used with the permission of the senior author
and the editor of the Veterinary Record.
Dot blot
This assay can also be used for antigen detection. Specimens to be tested for antigen are
directly dotted or applied to nitrocellulose membranes, and the presence of the antigen in the
samples is detected by addition of an enzyme-labelled antibody.
Enzyme immunohistochemical procedures
Immunohistochemical staining is the detection of antigens in tissue sections through the use of
specific antibodies that are labelled so that the sites of antibody attachment become
microscopically visible. Immunohistochemical stains used include FITC-labelled antibodies on
frozen sections of fresh tissue, or more commonly enzyme-labelled antibodies on formalin-fixed
With direct immunostains, the antiserum is incubated on the tissue sections followed by
addition of an insoluble, coloured reaction product at the sites of antibody binding in the tissues.
Indirect immunostaining methods utilize an enzyme-conjugated anti-immunoglobulin second
antibody to detect binding of the primary antibody to the tissue sections. Indirect stains
enhance the sensitivity of antigen detection because several secondary antibodies will bind to
each primary antibody, intensifying the visible signal produced by the binding of each primary
antibody. This phenomenon is especially exploited with the use of the avidin-biotin complex
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Serology: Fluorescent antibody tests and other tests employing conjugated antibodies
(ABC) method. The ABC immunostain relies upon the high affinity of the B-vitamin biotin for
the egg-white glycoprotein avidin. Antigens in tissue sections are detected by an unlabeled
primary antibody followed by a second antibody to immunoglobulin labelled with biotin.
Following application of the second antibody, the tissue is exposed to preformed complexes of
avidin and biotin. Each avidin has binding sites for 4 biotin molecules. The avidin-biotin
complexes are produced to ensure free biotin binding sites on the avidin molecules, which
promotes binding of the complexes to the biotinylated second antibody. The biotin molecules
are labelled with an enzyme that reacts with the enzyme substrate which is applied to the tissue
sections. A coloured reaction product forms on the slide at sites of antibody-enzyme complex
binding. The most commonly used chromagen is the peroxidase substrate diaminobenzadine
(DAB) which generates a dark brown precipitate.
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