Immunization schedules

11.2 Immunization schedules

Common sense and experience provide the best guide to effective immunization. For example, if immunization is being used to obtain antibody-secreting plasma cells or an antiserum, the inter- val between immunization and use will be much shorter than if the intention is to generate mem- ory B lymphocytes.

Serum should be obtained from the animal(s) prior to immunization. If the animal is large (rabbit, goat, etc.) a blood sample of 50 ml or more will yield a useful quantity of normal or pre- immunization serum. If the animal is small (mouse, guinea pig, etc.) it is not usually feasible to prebleed the individual intended for immunization. In this case a pool of serum is a practical alternative, derived from non-immunized animals of the same strain and the same colony (i.e. same environmental antigens) and of approximately the same age.

Important: do NOT take more than 15% of the total blood volume from normal laboratory species over any 4-week period. Blood volume averages about 65 ml/kg. Therefore one can take

11.2IMMUNIZATION SCHEDULES

11.2.1 Rabbits

1 Use between 50 and 500 µg foreign protein antigen, in Freund’s complete adjuvant, injected subcutaneously into areas of loose skin, up to 0.1 ml at each of the two sites.

2 Repeat the inoculation after 2 weeks using Freund’s incomplete adjuvant, being careful to inject into different sites from those previously used.

3 If a good precipitating antiserum is not obtained 2 weeks after the second inoculation of antigen, continue boosting with 300 µg of alum-absorbed antigen given subcutaneously.

Sample bleed It is relatively easy to obtain a 10–30 ml blood sample (under aseptic conditions) by bleeding

from the central ear artery using a hypodermic syringe and needle. When required, exsanguina- tion can be accomplished by cardiac puncture under phenobarbital anaesthesia. A 2.5-kg rabbit should yield 100–120 ml blood, equivalent to 70–80 ml serum, when killed.

11.2.2 Rats and mice

Good B-cell responses in mice can be obtained by intraperitoneal immunization with 0.1 ml alum-precipitated protein (400 µg) mixed with standard ‘whooping cough’ vaccine (equivalent

to 2 × 10 9 killed Bordetella pertussis organisms). IgG and IgM plaque-forming cells can be detected in the spleen after 8 days and the serum antibody levels are maximal by 10–14 days (with slight variations, depending on the antigen used). If memory B cells are required then at least 2–3 months should be allowed to elapse after the last immunization. If T-cell priming is required, immunization in Freund’s complete adjuvant is advised. Use 10–100 µg of antigen in 100 µl Freund’s complete adjuvant and inject the emul- sion subcutaneously near the nape of the neck.

Immunization of mice against membrane antigens is particularly effective when saponin (Quill A purified extract) is used as an adjuvant. Mix 15–20 µg of saponin with 10–100 µg of anti- gen in phosphate-buffered saline and inject the mixture subcutaneously near the nape of the neck. The adjuvant effects of saponin are highly dose dependent a20 µg is about the maximum subtoxic dose that can be given to a mouse.

Mice can be selectively immunized for an IgE response by immunization with the antigen of choice and infection with the nematode Nippostrongylus brasiliensis. Animals are primed with alum-precipitated antigen, followed 2–3 weeks later with infection with 300–500 N. brasiliensis larvae. On further boosting with antigen, pronounced IgE responses are induced.

Exsanguinate mice either by cardiac puncture or by severing the underarm vessels (see Fig.

6.2) under ether anaesthesia. Rats may be immunized by subcutaneous or intramuscular injection of 10–100 µg antigen in 500 µl Freund’s complete adjuvant, distributed at three sites. Exsanguinate by cardiac puncture under general anaesthesia.

C H A P T E R 1 1: Immunological manipulations in vivo

11.2.3 Goats, sheep and pigs

These large animals can be immunized easily and provide relatively large volumes of serum (up to

1.5 l) even when done commercially as a customer request. They are immunized by subcutaneous injection with up to 1 mg of foreign protein antigen in Freund’s complete adjuvant to a maximum of 0.25 ml at each of the four sites, and boosted for repeated bleeding. Test bleeds can

be taken from the jugular vein and exsanguination accomplished by insertion of an arterial shunt.

The size of these animals puts the inexperienced immunizer at a considerable disadvantage. It is wise to seek expert help or a commercial supplier.