Serology: Fluorescent antibody tests and other tests employing conjugated antibodies
Serology:
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.
IMMUNOBLOTTING AND IMMUNOHISTOCHEMISTRY
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
antiserum.
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
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Serology: Fluorescent antibody tests and other tests employing conjugated antibodies
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
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
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Serology: Fluorescent antibody tests and other tests employing conjugated antibodies
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
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
antibody.
Immunoblots for antigen detection
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Serology: Fluorescent antibody tests and other tests employing conjugated antibodies
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).
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
tissues.
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Serology: Fluorescent antibody tests and other tests employing conjugated antibodies
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
(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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