THE INSTITUTE OF MEDICAL & VETERINARY SCIENCE
ANIMAL ETHICS COMMITTEE
GUIDELINES FOR POLYCLONAL ANTIBODY PRODUCTION
- RABBIT, SHEEP, CHICKEN, GOAT
1. Use non-inflammatory adjuvants or those adjuvants known to produce less intensive
inflammatory responses whenever possible and appropriate to experimental goals.
2. Preparations for injection should be sterile.
3. If using Freund’s Complete Adjuvant (FCA), injection sites lumps or lesions may
appear 2 or 3 weeks post injection. Animals should be checked daily for general health
by a trained animal technician and observations recorded. Injection sites in rabbits or
sheep are examined 2 or 3 weeks post injection and receive appropriate Veterinary
treatment if lesions are found. This response is less likely in chickens. In some cases
more frequent examination may be required – eg where gel segments or other
irritating antigen are administered.
4. The use of FCA is permitted for the initial inoculation only.
5. The maximum total injection volume using FCA or Incomplete Freunds adjuvant (FIA)
is One ml in rabbits and hens and volume per injection site should not exceed 150;l.
The maximum total injection volume using FCA or FIA is Four ml in sheep and goats
and volume per injection site should not exceed 800;l
1. Young adult animals are usually used. Paddock or floor pen accommodation is
preferred, but if it is not available or suitable, cages must be of sufficient size to allow
free movement of the animal - depending upon its age and weight.
2. If no measurable antibody is detected by test bleed after 3-4 immunisations the antigen
preparation and immunisation protocol should be reviewed.
3. Injection sites in the rabbit should be the neck (forward of the shoulder blades) and
along the back on either side of the spine. In the sheep/goat injections should be given
at the inside of the top of all four legs, and/or the base of both sides of the neck. In
chickens, injection sites can be into or above the pectoral muscles and the leg muscles
– care must be taken to ensure injection is not too deep, or in a location that may harm
4. Injections should be subcutaneous and not intradermal.
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Animal Ethics Committee
5. Footpad injection of rabbits is inappropriate.
6. Intravenous inoculation with soluble antigen is associated with a risk of anaphylaxis.
Animals so inoculated should be carefully observed post injection, and appropriate
therapeutics should be available to treat such emergencies.
7. Occasionally small subcutaneous lumps are seen at the site of previous injections.
These do not appear to cause pain or distress but further injections into these areas
should be avoided, such lesions should be recorded and reported and receive
appropriate Veterinary treatment if necessary.
8. Animals should need only gentle restraint (by a second person) while the injections
are being given and do not require anaesthesia.
RECOMMENDATIONS FOR COLLECTION OF ANTISERA
1. After the desired titre is reached, the Committee recommends complete
exsanguination under general anaesthesia of rabbits and sheep/goats. If the
experimental goals do not allow this, the following schedule should be followed:
FREQUENCY VOLUME PERMISSIBLE BLOOD BLOOD VOLUME
AS % OF VOLUME FOR
TOTAL BLOOD FOR 55 KG SHEEP OR
VOLUME 3.5 KG RABBIT GOAT
Weekly 5% 10mls 200mls
3 weekly 10% 20mls 400mls
maximum for 30mls 800mls ；15%
(Blood volume of a rabbit/chicken is ； 55ml/kg ; Blood volume of a sheep/goat is ； 75ml/kg)
The preferred method of antibody collection in chickens is via the extraction of IgY from egg yolk. If repeated blood collection is required, up to of 15ml can be taken from a 3Kg
chicken every 3 or 4 weeks.
2. Blood samples from rabbits are collected via the marginal ear vein (warming under a
light source for 5 or 10 minutes beforehand will assist blood flow). Rabbits should be
restrained for blood collection by tight wrapping in a towel or similar restraint (hard
plastic or wooden restraints are not recommended as these may cause injury to the
animal). Light tranquillisation with a phenothiazime tranquilliser or other analeptic
will reduce distress and may facilitate blood collection.
3. Blood collection from the sheep is via the jugular vein. Small volumes may be
collected by restraining the sheep in a standing position, but for larger volumes a
cradle restraint is best used.
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4. Blood samples from chicken are taken from the brachial (wing) vein but the animal
must be immobilised and great care taken as slight movement may cause haematoma
formation. If this occurs, blood collection should cease, and pressure applied until the
animal stops bleeding.
5. Exsanguination of any animal must be conducted under anaesthesia and once
complete, euthanased immediately. Cardiac puncture is performed in rabbits (and
chickens where required).
6. Special consideration by the Committee is required for any antibody production
rabbits to be held longer than six months. The committee should be advised if
sheep or chickens are to be held for an extended period.
7. Rabbits must be weighed monthly and weights recorded. Any reduction in body
weight should be investigated by a Veterinary Surgeon.
8. Rabbits must be euthanased if they become cachectic, develop and unkempt coat and
are reluctant to move, or when they reach twelve months of experimental age.
ILAR News:Biological Effects of Blood Loss, Implications for Sampling Volumes
and Techniques, Vol. 31, Number 4 1989.
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