Protein Protocols Protein Protocols

938 Burns
The legislation in terms of animal housing, immunization procedures, bleeding
regimens, and choice of adjuvant vary widely according to local legislation, and it is
imperative that advice be taken from the appropriate authorities prior to undertaking
any antibody production work.
1.4. Antigens
The antigen chosen for an immunization program should be as close in structure and
chemical identity to the target protein as possible. An exception to this is when synthetic
peptides are produced to mimic parts of the native protein, an approach that is
invaluable when the native antigen may be toxic to animals or nonimmunogenic.
The antigen should be soluble, stable at dilutions of approx 1 mg/mL, and capable of
being administered in a liquid of close to physiological pH (6.5–7.5). The antigen
should also be in as pure a form as practically possible to avoid the generation of antibodies
to contaminating materials.
Many proteins are highly immunogenic in donor animals, particularly when the
antigen is derived from a different species (see Note 1). Raising antibodies in the same
species from which the antigen is derived from can be extremely difficult but can
be overcome by conjugating the antigen to a carrier protein from another species prior
to immunization. Carrier proteins such as hemoglobin, thyroglobulin, and keyole
limpet hemocyanin are commonly used. Animals immunized with the conjugated form
of the antigen will produce antibodies to both the protein of interest and the carrier
protein. Apart from a lower specific antibody titer in the serum there should be no
interference from the carrier protein antibodies.
1.5. Test Protocol
Ideally antiserum should be tested using the procedure in which it is to be used, as
antibodies may perform well using one assay but not with another. This, however, is
not always practical and so a number of tests can be carried out on antiserum to test
its suitability for final use. The affinity of the antiserum to the antigen can be assayed
by plate-trapped double-antibody sandwich enzyme-linked immunosorbent assay (DAS
ELISA) (5). The antigen is bound to a microtiter plate and then challenged with dilutions
of the test antiserum. A secondary antispecies antibody enzyme conjugate is then
added to the plate and will bind to any antibody molecules present. A chromogenic
enzyme substrate is then added and the degree of color development indicates the quantity
of antibody bound by the antigen.
Ouchterlony double-immunodiffusion can be used to observe the ability of the antiserum
to produce immune complexes with the antigen in a semisolid matrix. This
method can also be used to test cross-reactivity of the antiserum to other proteins closely
related to the antigen. Radioimmunoassay and other related techniques can also be
used to test the avidity of the antiserum to the antigen and will also give a measure of
antibody titer.
In all the above tests preimmune antisera should be included to ensure that results
obtained are a true reflection of antibodies produced by immunization and not due to
nonspecific interactions.