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The Production of Polyclonal Antibodies in Laboratory Animals
The Report and Recommendations of ECVAM Workshop 351,2
Reprinted with minor amendments from ATLA 27, 79-102.
Appendix 1
Draft Guidelines for the Production of Polyclonal Antibodies
General Considerations
Training
Qualified technical staff (Category B of the FELASA recommendations) should perform immunization procedures in laboratory animals. The technical staff should have a basic understanding of immunological principles and be experienced in the handling, injecting, anaesthetising and bleeding of the animal species used for antibody production. In particular, aspects of asepsis and anaesthesia are very important. Furthermore, the technical staff should be capable of recognising signs of pain and distress in the animals, and must be responsible for taking action when necessary. The investigator should have biomedical training and should be qualified in laboratory animal science according to the FELASA recommendations (Category C).
Hygiene
The general principles of asepsis should be applied to immunization protocols at all stages with regard to: the preparation of the antigen/adjuvant mixture; the administration of the mixture to laboratory animals; and the bleeding of the animals.
Housing of animals
Animals should be kept under conditions that allow important natural behaviour. Immunisation protocols are generally for medium-term to long-term experiments. Therefore, depending on the animal species used, group housing of animals is preferable.
Laboratory animals
The number of animals used, and thus the volume of antiserum produced, should be in accordance with the expected need for the polyclonal antibody (pAb). Priority should be given to a reduction of distress in the animals, rather than to a limitation of the number of animals.
Selection of animal species
The species of animals to be selected should be related to the amount of serum needed, the application of the antibodies to be produced, and the characteristics of the antigen concerned.
Microbiological status
In the case of small laboratory animals, the microbiological status should preferably be specific pathogen-free.
Immunisation protocol
Only the least harmful materials and the least severe protocols necessary should be used. To minimise the potential for induction of side-effects and to maximise the potential for successful pAb production, the investigator should check the quality of the antigen and of the antigen/adjuvant mixture prior to its injection.
Use of adjucant
The first question is whether an adjuvant is needed at all. Selection of adjuvant should be given very high priority, since it affects the welfare of the animals to be immunised, as well as the type and level of immune response. The selection of, and need for, an adjuvant should be determined on the basis of the nature of the antigen. Mineral oil adjuvants combined with bacterial components may induce considerable side-effects. Adjuvants that contain mycobacteria or their cell wall components should be used only once per animal.
Route of injection
Suggested routes of injection are given in Table I and comments regarding injection routes are given below.
Table I: Suggested Routes of Injection with or without Adjuvant
| Primary Injection Day 0 |
Booster Injection(s) Day 28 and/or later |
||
| With Adjuvant | Without Adjuvant | With Adjuvant | Without Adjuvant |
| s.c. | i.v. | s.c. | s.c. |
| i.m. | s.c. | i.m. | i.m. |
| i.d.a | i.m. | i.d.a | i.v.b |
| i.p. | i.p.b | ||
| i.d.a | i.d.a | ||
s.c. = subcutaneous, i.m. = intramuscular, i.p. = intraperitoneal, i.d. = intradermal, i.v. = intravenous.
afor i.d. injection at multiple sites, it was the opinion of the participants that this route should be allowed for certain purposes in the rabbit and in large animals to stimulate the required immune response.
bWith i.v. and i.p. booster injections, there is a risk of inducing anaphylactice shock in the animals.
Subcutaneous injection
The subcutaneous route is preferred for the injection of oil adjuvants or viscous gel adjuvants.
Intramuscular injection
The intramuscular route should not be the first choice for injection of oil adjuvants or viscous gel adjuvants in small animals such as mice and rats.
Intradermal injection
The intradermal route should only be used in rabbits or large animals. If intradermal injection is carried out at multiple sites, the maximum volume administered per site should be 25 µl and the maximum number of sites should be four.
Intraperitoneal injection
The intraperitoneal route is not recommended when oil adjuvants or viscous gel adjuvants are used. Its use should be scientifically justified to the ethical committee on a case-by-case basis.
Intravenous
The intravenous route is not recommended when oil adjuvants or viscous gel adjuvants are used, because there is a high risk of lethal complications due to embolism.
Footpad, intra-lymph node or intrasplenal injection
Footpad, intra-lymph node and intra-splenal injection are not necessary for routine pAb production. The use of these routes must be scientifically just)fied on a case-bycase basis. If a footpad injection is given, only one hind foot should be used, with a maximum volume of 50-100 µl, and the animals should be housed on soft bedding.
Volume of injection
The volume of injection should be as small as possible. Maximum dosages of depot-forming adjuvants (for example oil adjuvants, viscous gel adjuvants) per site of injection are shown in Table II.
Table II: Maximum Volumes for Injection of Antigen/Depot-Forming Adjuvant Mixtures per Site of Injection for Different Animal Species
| Species | Maximum Volume per Site | Primary Injection | Subsequent Injections |
| Mice, hamsters | 100 µl | s.c. | s.c. |
| Mice, hamsters | 50 µl | i.m.a | i.m. |
| Guinea-pigs, rats | 200 µl | s.c., i.m. | s.c., i.m. |
| Rabbits | 250 µl | s.c., i.m. | s.c., i.m. |
| Sheep, goats, donkeys, pigs | 500 µl (if in multiple sites 250 µl/site | s.c., i.m. | s.c., i.m. |
| Chickens | 500 µl | s.c., i.m. | s.c., i.m. |
s.c. = subcutaneous, i.m. = intramuscular.
aOne hind limb
Immunisation schedule
Number of sites
A single site of injection is preferred and a maximum of four injection sites per immunisation per animal should be allowed. If more than one injection site is used, the distance between each site should be maximised.
Booster schedule
When an adjuvant is used, the time between primary and secondary injection should be at least 4 weeks. A maximum of two booster injections is recommended.
Blood Collection
Blood should be collected according to the recommendations given by the BVA/ FRAME/RSPCA/UFAW Joint Working Group on Refinement (1) or by McGuill & Rowan (2).
When frequent blood sampling is performed, the health status of the animals should be carefully observed. The collection of blood should not exceed more than 15% of the total blood volume, which in practice, represents an amount up to 1% of total body weight. Animals should not be bled more frequently than once every fortnight when maximum blood volumes are collected.
Assessment of Side-Effects
During the entire experiment immunised animals should be checked daily for general appearance, as well as for food and water intake. In addition to this routine check-up, the injection site should be monitored. When comparative studies are performed, the assessment of side-effects should include pathological studies at the end of the experiment. Pathological studies should include dissection for gross examination of organs and tissues, and histological examination of specific tissues. The results should be used to optimise the design of future experiments.
References
- BVA/FRAME/RSPCA/UFAW (1993). Joint Working Group on Refinement. Removal of blood from laboratory mammals and birds. Laboratory Animals 27: 1-22.
- McGuill, M.W. & Rowan, A.N. (1989). Biological effects of blood loss: implication for sampling volumes and techniques ILAR News 31: 520.