Antibodies are proteins that are produced by the immune systems of animals in
response to foreign substances. The immune system has the ability to recognize material as nonself or foreign and mount a response to them. Substances that elicit this are
known as immunogens or antigens, and one of the outcomes of the immune response is
the production of antibodies that will recognize and bind to the eliciting substance.
The immune response results in the degradation of the antigen by cells called macrophages and its presentation in fragments to B lymphocytes that are found in the lymphatic tissues of the animal. On presentation of the antigen fragments the B lymphocytes
mature and “learn” to produce antibody molecules that have a specific affinity to the
antigen fragment that they have been presented with. This process gives rise to populations of B lymphocyte clones each of which produces antibody molecules to different
locations known as epitopes on the target antigen. As individual clones produce the
antibody molecules and there are many of them, the resulting antibody mix in the blood
is known as polyclonal and the fluid derived from the clotted blood is known as
polyclonal antiserum.
Artificial exposure to antigens to produce antibodies in animals is known as immunization, and repeated exposure leads to a condition known as hyperimmunity, in which
a significantly large proportion of the circulating antibodies in the animals blood will
be directed toward the antigen of interest. Immunization gives rise to stable but quiescent populations of B lymphocytes known as memory cells that will respond to the
presence of the antigen by dividing to increase their number and the level of circulating
antibody in the blood.
The primary exposure to an antigen gives rise to a pentameric form of antibody
known as immunoglobulin M (IgM). Subsequent exposure to the antigen causes a shift
in the antibody type to the more stable immunoglobulin G (IgG). This shift in antibody
type happens as affinity maturation of the B lymphocyte clones occurs.
Some antigens, particularly highly glycosylated proteins, do not elicit the production of memory B lymphocytes and also may not give rise to IgG antibodies even after
repeated immunization. These are known as anamnestic antigens, and as no memory B lymphocytes are produced, the immune system always interprets exposure to them as a primary one and mounts a primary IgM response. It is possible to produce IgG to
anamnestic antigens by treating them to remove the carbohydrate moieties prior
to immunization, but this may lead to antibodies that do not recognize the native protein structure.
Antibodies have two qualities known as avidity and affinity. Affinity is a measure of
how well the binding site on the antibody molecule fits the epitope, and antibodies,
which have high affinity also, have high specificity to the target epitope. Avidity is a
measure of how strong the interaction between the epitope and the antibody molecule is.
For research purposes, antibodies to the antigen of interest should be expressed at
fairly high levels in the sera of donor animals, should be of high affinity, and should
predominantly be composed of IgG molecules, which are stable and easily purified.
This can be achieved for most antigens providing they are not too highly glycosylated,
have a molecular weight >5000, and are not toxic or induce immuneparesis in the
donor animal.
Monoclonal antibodies are used very successfully in many areas of research and can
either replace, complement, or be used in conjunction with immunoglobulins obtained
from donor serum. Monoclonal antibodies are produced by hybridoma cell lines and
can be grown in tissue culture in the laboratory. Hybridomas are recombinant cell lines
produced from the fusion of B cell clones derived from the lymphatic tissue of donor
animals and a myeloma cell line that imparts immortality to the cells. As each
hybridoma is descended from a single B cell clone the antibody expressed by it is of a
single specificity and immunoglobulin type, and is thus termed a monoclonal antibody.
Each monoclonal antibody is monospecific and will recognize only one epitope on
the antigen to which it has been raised. This may lead to practical problems if the
epitope is not highly conserved on the native protein or where conformational changes
may occur in the because of to shifts in pH or other environmental factors. Monoclonal
antibodies are highly specific and will rarely if ever produce cross-reactions with other
proteins. Polyclonal antibodies may cross-react with other closely related proteins
where there are shared epitopes.
Polyclonal antisera contain a heterogeneic mixture of antibody molecules, many of
which will recognize different epitopes on the protein, and their binding is much less
likely to be affected by poorly conserved epitopes or changes in the protein shape.
Antisera are derived from individual animal bleeds and because of this are subject to
batch variation. Individual animals can have very different immune responses to the
same antigen, and individual bleeds from the same animal may vary in antibody content quite markedly. Monoclonal antibodies are produced from highly cloned cell lines
that are stable and reliably produce a defined antibody product.
Polyclonal antibodies are generally less specific than monoclonals, which can be a
disadvantage, as cross-reactivity may occur with nontarget proteins. Monoclonal antibodies can be too specific, as they will recognize only a single epitope that may vary on
the protein of interest.
Both antibody types have their place in the research laboratory, and a careful evaluation of the required use should be undertaken before deciding which would be most
applicable.
Most polyclonal antibodies for research purposes are produced in domestic rabbits unless very large quantities are required, and then sheep, goats, donkeys, and even horses are used. Antibodies can also be produced in chickens; the antibodies are conveniently produced in the eggs. Rats and mice can also be used to produce antiserum but yield much smaller quantities of antibody owing to their relatively small size. Immunized mice can be used to produce 5–10 mL of polyclonal ascitic fluid by induction of ascites. Ascites is induced by the introduction of a nonsecretory myeloma cell line into the peritoneal cavity after priming with Pristane. Polyclonal antibodies are secreted into the peritoneum from the blood plasma of the animal and can achieve levels of 2–5 mg/mL. Ascitic fluid is aspirated from the peritoneum of the mouse when ascites has developed, indicated by bloating of the abdomen. The UK Home Office and other regulatory authorities now regard induction of ascites in mice as a moderate/severe procedure and this method is not normally used unless no alternative methods are available.
Adjuvants are substances that increase the immune response to antigen by an
animal. They may be simple chemicals such as alum, which adsorbs and aggregates
proteins, increasing their effective molecular weight, or they may be specific
immunestimulators such as derivatives of bacterial cell walls. Saponins such as
Quil-A derived from the tree Quillaja saponaria may also be used to increase the
effective immunogenicity of the antigen. Ideally an adjuvant should not induce an
antibody response to itself to ensure that antibodies generated are specific to the
antigen of interest.
The most popular adjuvant, which has been used very successfully for many years,
is Freund’s adjuvant. It has two formulations, complete and incomplete, which are
used for the primary and subsequent immunizations, respectively. Freund’s incomplete
is a mixture of 85% paraffin oil and 15% mannide monooleate. The complete formulation additionally contains 1 mg/mL of heat-killed Mycobacterium tuberculosis. In
recent years the use of Freund’s adjuvant has declined owing to animal welfare concerns and also the risk that it poses to workers, as it can cause localized soft tissue
damage following accidental needlestick injuries. Injection preparations that contain
Freund’s adjuvant are also difficult to work with, as the resultant emulsion can be too
thick to administer easily and it interacts with plastic syringes, preventing easy depression of the plunger.
Several adjuvant formulations are available that contain cell wall derivatives of
bacteria without the intact organisms; they are much easier to administer and will achieve
a similar immunostimulatory effect to Freund’s without its attendant problems.
Most countries have legislation governing the use of animals for all experimental
work, and antibody production is no exception. Although the methods used are usually
mild in terms of severity they must be undertaken with appropriate documentation and
always by trained, authorized staff.
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.
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. 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.
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). 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.
Antibody Production
1.At least two rabbits should be used for each polyclonal antibody production project, as
they may have different immune responses to the antigen. They should be purchased from
a reputable source and be parasite and disease free. New Zealand whites are often used but
any of the domestic breeds will make acceptable donors.
2.The antigen should be in a buffer of pH 6.5–7.5 and be free of toxic additives (sodium
azide is often added as a preservative). A concentration of 1 mg/mL is desirable but anything above 100 µg/mL is acceptable.
3.Complete and incomplete Freund’s adjuvant or any of the propriety preparations containing purified bacterial cell wall components such as Titermax and RIBI.
1. Prior to a course of immunizations a test bleed should be taken from the rabbits to provide
a source of preimmune antiserum for each animal. It is usual to take
only 2–3 mL for the test bleeds, which will yield 1–1.5 mL of serum. The blood should
be allowed to clot at 4°C for 12 h and the serum gently aspirated from the tube.
2. The antigen should be mixed with the appropriate adjuvant according to the manufacturer’s instructions to achieve a final volume of 0.5 mL/injection containing 50–500 µg of
antigen. If Freund’s adjuvant is to be used, the first injection only should
contain the complete formulation and the incomplete one should be used for all subsequent immunizations.
3. The rabbit should be restrained and the antigen–adjuvant mixture injected into the thigh muscle. Alternate legs should be used for each injection.
4.The immunization should be repeated 14 d after the primary one and a test bleed taken 30 d after that.
5. If the antiserum shows that the desired immune status and antibody quality has been
achieved then donor bleeds can be taken. The volume of blood collected and frequency of
bleeding depends on animal welfare legislation and must be adhered to. Each bleed should
be assayed individually, and once the antibody titer has started to fall a further immunization can be given, followed by donor bleeds, or the animal can be terminally anesthetized
and exsanguinated by cardiac puncture or by severing the carotid artery.
6. Antiserum collected from rabbits can be stored for extended periods of time at 4°C but the
addition of 0.02% sodium azide is recommended to prevent adventitious bacterial growth.
Antiserum quite commonly has functional antibodies even after years of refrigerated storage, but storage at –20°C is recommended for long-term preservation.
7. Antiserum will often yield in excess of 5 mg/mL of antibody and can be purified to give
the immunoglobulin fraction. This can be achieved with ammonium sulfate precipitation
or by affinity chromatography using either the immobilized antigen or protein A. The
antibody fraction can then be adjusted to 1 mg/mL, which is an ideal concentration both
for its stability and for many practical applications. To purified antibody 0.02% sodium
azide or some other preservative should be added to prevent the growth of adventitious
organisms. Sodium azide can interfere with enzyme reactions and with photometric measurement, and this should be taken into account with regard to the final assay. Purified
antibodies diluted to 1 mg/mL can be stored for long periods at 4°C with no loss of activity but for extended storage –20°C is recommended.
1. Some antigens will consistently fail to induce an antibody response in certain animals, and
other species should be investigated as potential donors. In very rare occasions antigens will not elicit an immune response in a range of species and the nature of the antigen will
then have to be investigated with a view to modifying it to increase its immunogenicity.
2. Female rabbits are less aggressive, and although smaller and yielding smaller quantities of
antiserum are preferable to male rabbits for antiserum production. Many biomedical
facilities use communal floor pens for donor animals and female rabbits adapt better to
this form of housing.
3. The use of excessive amounts of antigen in immunizations should be avoided, as this can
lead to a poor immunological response. Swamping of the system can lead to selective
deletion of the B cell clones of interest and a reduction in the specific antibody titer. High
doses of antigen in the secondary and subsequent immunizations can cause anaphylactic
shock and death of the donor animal.
4. It has been reported that increased stress levels in animals can depress the immune
response, and appropriate measures should be taken to ensure that immunizations and
bleeds are performed with the minimum of stress to the animals. General husbandry in
terms of housing, noise levels, and other environmental factors should also be examined
to ensure that animals for polyclonal production are maintained under suitable conditions.
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