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.
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 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.
Related reading： Introduction To Antibody Production Summary Of ELISA Protocols