The Production of Polyclonal Antibodies in Laboratory Animals

The Report and Recommendations of ECVAM Workshop 35^1,2

Reprinted with permission from ECVAM and ATLA

P.P.A. Marlies Leenaars,³ Coenraad F.M. Hendriksen,³ Wim A. de Leeuw,⁴ Florina Carat,⁵ Philippe Delahaut,⁶ René Fischer,⁷ Marlies Halder,⁸ W. Carey Hanly,⁹ Joachim Hartinger,¹⁰ Jann Hau,¹¹ Erik B. Lindblad,¹² Werner Nicklas,¹³ Ingrid M. Outschoorn¹⁴ and Duncan E.S. Stewart-Tull¹⁵
³National Institute of Public Health and the Environment (RIVM), P.O. Box 1, 3720 BA Bilthoven, The Netherlands; ⁴Inspectorate for Health Protection, Commodities and Veterinary Public Health, De Stouen 22, 7206 AX Zutphen, The Netherlands; ⁵Stallergenes SA, 6 Rue Alexis de Tocqueville, 92183 Antony Cedex Paris, France; ⁶Centre D'Economie Rurale, Rue du Point du Jour 8, 6900 Marloie, Belgium; ⁷Department of Biochemistry Swiss Federal Institute of Technology, 8092 Zurich, Switzerland; ⁸ECVAM, JRC Environment Institute, 21020 Ispra (VA), Italy; ⁹Department of Microbiology and Immunology (M/C 790), University of Illinois, 835 S. Wolcott, Chicago, IL 60612; ¹⁰Paul-Ehrlich-Institut, Bundesamt für Sera und Impfstoffe, Paul-Ehrlich-Strasse 51-59, 63225 Langen, Germany; ¹¹Department of Comparative Medicine, Biomedical Centre, Box 570, 75123 Uppsala, Sweden; ¹²Superfos Biosector a/s, Elsenbakken 23, 3600 Frederikssund, Denmark; ¹³Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany; ¹⁴Instituto de Salud Carlos III, Unidad Respuesta Immune, Centro Nacional de Biol. Fundamental, 28220 Madrid, Spain; ¹⁵Division of Infection and Immunity, Institute of Biomedical and Life Sciences, Joseph Black Building, Glasgow G12 8QQ UK

¹ECVAM - The European Centre for the Validation of Alternative Methods. ²This document represents the agreed report of the participants as individual scientists.

Address for correspondence: Dr. P.P.A.M. Leenaars, National Institute of Public Health and the Environment (RIVM), P.O. Box 1, 3720 BA Bilthoven, The Netherlands.

Address for reprints: ECVAM, TP 580, JRC Environment Institute, 21020 Ispra (VA), Italy

Preface:

This is the report of the thirty-fifth of a series of workshops organised by the European Centre for the Validation of Alternative Methods (ECVAM). ECVAM's main goal, as defined in 1993 by its Scientific Advisory Committee, is to promote the scientific and regulatory acceptance of alternative methods which are of importance to the biosciences and which reduce, refine or replace the use of laboratory animals One of the first priorities set by ECVAM was the implementation of procedures which would enable it to become well informed about the state-of-the-art of non-animal test development and validation, and the potential for the possible incorporation of alternative tests into regulatory procedures It was decided that this would be best achieved by the organisation of ECVAM workshops on specific topics, at which small groups of invited experts would review the current status of various types of in vitro tests and their potential uses, and make recommendations about the best ways forward (1).

This joint ECVAM/FELASA (Federation of European Laboratory Animal Science Associations) workshop on The Immunisation of Laboratory Animals for the Production of Polyclonal Antibodies was held in Utrecht (The Netherlands), on 2022 March 1998, under the co-chairmanship of Coenraad Hendriksen (RIVM, Bilthoven, The Netherlands) and Wim de Leeuw (Inspectorate for Health Protection, The Netherlands). The participants, all experts in the fields of immunology, laboratory animal science, or regulation, came from universities, industry and regulatory bodies. The aims of the workshop were: a) to discuss and evaluate current immunisation procedures for the production of polyclonal antibodies (including route of injection, animal species and adjuvant); and b) to draft recommendations and guidelines to improve the immunisation procedures, with regard both to animal welfare and to the optimization of immunisation protocols. This report summarises the outcome of the discussions and includes a number of recommendations and a set of draft guidelines (included in Appendix 1).

Introduction

The immunisation of laboratory animals to induce a humoral and/or cellular immune response, is a routine procedure performed worldwide. Consequently, the use of animals is substantial, although no precise figures are available. Researchers from many disciplines need to produce antibodies. Often, they do not have specific knowledge of immunology, and are not sufficiently experienced in procedures for immunisation of laboratory animals. Therefore, specific guidelines on immunisation protocols are required.

Although several animal species are used for the production of antibodies, rabbits and mice are the species most frequently used for the production of polyclonal antibodies (pAbs) and monoclonal antibodies (mAbs), respectively.

Certain immunisation procedures are currently under discussion for animal welfare reasons. For example, the adjuvant products used to enhance the immune response are known to cause local inflammation, and some immunisation protocols are associated with pain and distress.

Some European countries, individual organizations and institutes have issued guidelines setting out criteria, for example, on the maximum volume to be injected, the route of inoculation and the number of injections. The aim of these guidelines is to ensure proper immunisation procedures, which combine acceptable immunological results with minimal discomfort for the animals. The immunisation protocol (including primary and booster injections and blood collection) has a great impact on both aspects. It is therefore crucial to carefully design this protocol.

The workshop focused on the production of pAbs and did not address the cellular immune response. Various aspects were discussed which could influence the induction of pAbs and affect the welfare of the animals.

Polyclonal Antibodies Versus Monoclonal Antibodies

The immune systems of most mammals are believed to be comprised of approximately 1000 clonal populations of Iymphocytes, as characterized by their antigen-receptor specificity. This diversity permits immune responses to a broad range of immunogens, for example, foreign proteins, carbohydrates, peptides and bacterial and viral components. The Iymphoid organs (spleen, lymph nodes, and gut-associated Iymphoid tissue, including tonsils) are the production sites of a vast range of antibodies by stimulated B Iymphocytes (plasma cells). Each antibody molecule recognises a specific antigenic epitope, possibly as small as 5-6 amino acids or between one and two glucose or other monosaccharide units, and is able to bind to the immunogen. A polyclonal humoral response, making use of the entire range of antibodies (pAbs), results in high avidity (defined as the product of the affinity constants of all binding antibodies) and gives the organism the ability to defend itself successfully against pathogens.

Köhler & Milstein (2) were the first to make B-cell hybridomas by using a fusion technique with Sendai virus. Subsequent workers have carried out successful fusions by using other agents, for example, polyethylene glycol. The fusion technique permits the immortalization of single Iymphocytes. The mAbs produced by such hybridoma cell clones originating from a single B Iymphocyte are identical, and specific for a single epitope. There are some cases in which the extreme monospecificity of mAbscan be a disadvantage. If for any reason the antigenic site is altered, which could be the case in many experiments, the mAb might not continue to bind. Nor are individual mAbs of use in precipitation assays such as immunoelectrophoresis. In contrast, the specificity of pAbs depends on a combination of hundreds or even thousands of clonal products, which bind to a number of antigenic determinants. As a result, small changes in the structure of the antigen due to genetic polymorphism, heterogeneity of glycosylation or slight denaturation, will usually have little noticeable effect on pAb binding. Whether these characteristics are seen as a problem or an advantage will depend on individual circumstances.

The avidity of a mAb is generally equal to its affinity for a protein antigen, a fact which is sometimes considered to be a disadvantage. MAbs are sometimes used in combinations, to increase the heterogeneity.

In making a choice between the generation of mAbs or pAbs, the desired application of the antibody and the time and money available for its production should be considered. The production of mAbs is tedious and takes 3-6 months. Cell cultures are required in addition to the animal immunisation. Examples of the use of mAbs include the immunostaining of western blots, ELISA, the affinity purification of proteins, and the immunostaining of thin tissue sections visualised by light microscopy or electron microscopy. The immortalized hybridoma serves as an inexhaustible antibody source of standardized quality. The induction of pAbs usually takes 4-8 weeks. The serum is suitable for many applications, for example, the immunostaining of western blots, ELISA and immunoprecipitation complement fixation. In most cases, polyclonal sera are of high titre, and permit substantial dilution; however, there may be batch-to-batch variability. The fact that a polyclonal antiserum can be obtained within a short time with little financial investment favours its use. In research many questions can be answered with the assistance of a polyclonal antiserum.

General Considerations

Of primary importance for pAb production are factors such as the antigen used, route of immunisation, animal species, type and quality of the adjuvant, and method of blood collection. However, a number of other aspects can also have an impact on the successful outcome of an immunisation procedure and/or on the welfare of the animals, including the health and genetic status of the animals, the expertise and competence of the staff, and the hygiene, diet and housing of the animals.

Immunosuppressive effects have been reported for various agents which infect rodents (for example, the Sendai virus mouse hepatitis virus, minute virus of mice, 3) or rabbits (Encephalitozoon; 4). Infections in the donor animals might also reduce the specificity of the antiserum, and, in the case of zoonotic agents, pose a human health risk. In general, the workshop participants recommended the use of specific pathogen-free animals in the case of small laboratory animals, and the implementation of a microbiological monitoring system according to the recommendations of FELASA (5, 6). However, it was also felt that, in some cases, more-conventional housing might be acceptable. In addition, it was recommended that the enduser of the animals should ask the supplier for an animal health and diet record, since specific tolerance to an antigen of interest may be generated by an animal's early dietary exposure to the antigen of an analogue. Immunosuppressive effects of diets have also been reported (7, 8).

Stress in animals should be avoided, since this can result in immune suppression, as well as discomfort for the animals. Immune suppression can result from stress generated before, as well as during, the actual period of immunisation (9). The quality of immunisation procedures can be optimised by the establishment of central facilities or units with responsible scientists (for example, a veterinarian) with experience in animal hus~andry and immunisation processes.

The workshop participants emphasised the importance of training programmes for The personnel engaged in immunisation pro:edures and, in particular, for the animal technician. Reference was made to FELASA recommendations on education, which are now available for animal caretakers (Category A) and researchers (Category C; 10), and are being drafted for animal technicians (Category B) and animal welfare officers/laboratory animal specialists (Category D). For instance, a curriculum for animal technicians should include the basic principles of immune response, injection and bleeding techniques, and the development of skills for observation, anaesthesiaandeuthanasia of the animals.

Another important aspect is animal housing. It is recommended that, whenever possible, animals should be housed under conditions that encourage their natural behaviour. The requirements of the Council of Europe (11) can be used as a point of reference. However, it was also noted that, in most laboratories, animals are housed individually without environmental enrichment, for example, rabbits. In general, the group housing of animals is recommended although in specific situations, individual housing might be justified. There might also be microbiological considerations in favour of individual housing, because the moreintensive direct contact between individuals housed in groups encourages the transmission of pathogens (for example, coccidia). Some participants reported the successful group housing of castrated bucks. However the ethical implications should be balanced against the advantages of group housing, and in some countries castration is not permitted, because it is stressful to the animals and is considered to be an unethical interference with the integrity of the animal.

The principles of Good Animal Experimentation Practice, as laid down in European Union legislation, Council Directive 86/609/EEC (12), must be followed in all aspects of the immunization process. A number of crucial steps were identified: the preparation of the antigen, from its initial purlfication to its eventual mixing with adjuvant, the injection of antigen/adjuvant, the bleeding of the animal, and the processing of antiserum. It is recommended that particular attention should be paid to the quality of the antigen preparation (for example removal of endotoxin, formaldehyde or sodium axide), the storage of the reagents, and the sterility of the instruments used for injection and bleeding (13).

Choice of Laboratory Animal

The selection of the animal species for the production of pAbs depends, at least in part, on the amount of antiserum needed and the ease of obtaining blood samples. The intended use of the pAbs can also play a role. In an ELISA, the antibody which binds to the antigen (the primary antibody) should be from a different species to the conjugated (secondary) antibody used in the next step of the assay. When there is no need for a specific species, the animals from which samples of blood are relatively easy to obtain (rabbits and mice) should be preferred over those which are difficult to bleed, for example, guinea-pigs and hamsters. The selection might also be related to the purpose of the experiment, as each species, strain, stock or breed of animal might differ in its immune response. An example is the difference between Balb/c mice and C57BL/6 mice, the first strain generally being a Th2-like responder, and the latter a Th1-like responder, although these may not always be hard and fast rules. The choice of a strain of mice or rats might also be influenced by the availability of inbred strains or outbred stocks. When an inbred strain is used, genetic variation between animals is restricted, such that hyporesponsiveness or hyper-responsiveness to a particular antigen might occur quite uniformly among members of the strain (uniform failure or uniform success). When an outbred stock is used, genetic variation among the animals may lead to a range of responses among members of the group. Although many environmental factors and a number of genetic factors can influence an animal's immune response, the major histocompatibility complex genotype plays a major role, in that it defines an individual's potential for antigen presentation and thus the individual's antibody response potential to a particular antigen. Chickens could be used as an alternative to mammals for the production of pAbs. The production of avian antibodies (IgY) in chickens is considered to be a refinement, since the collection of blood in the production of IgY has been replaced by the extraction of antibodies from egg yolk. In addition, chickens might be preferred for scientific reasons, for example, their phylogenetic distance from mammals. Unfortunately, the production of IgY in chickens is not widespread. This is probably due to a number of factors, such as traditions among researchers, the infrequent use of the chicken as a laboratory animal in general, the limited availability of conjugated antibodies, specific requirements for housing, and lack of experience with chickens and chicken antibodies. Also, chicken antibodies are more difficult than are mammalian antibodies. Further information on IgY production is given in ECVAM workshop 21 (14).

Traditionally, female animals are most frequently used in pAb production. Female animals are generally more docile for handling purposes, and are less aggressive in social interactions, and can therefore be grouphoused more successfully. Although there is some evidence that androgens can slightly dampen the antibody response, there are no overriding scientific reasons for not using male animals.

The immune status of the immunised individual may also determine the outcome of an immunization procedure. Young adults should be used for pAb production, as the immune response is immature at an early age and drops with age after the period of young adulthood. When animals are re-used after other procedures (only one use for pAb production is appropriate), it is important to keep the age of the animals in mind. When chickens are immunised, they should be of egglaying age by the time antibody is to be harvested. Recommendations as to the age at which animals should first be immunised are given in the Swiss guidelines (15) and by Hanly et al (13; Table I). Some guidelines recommend the use of animals at an earlier age (16). In addition, it is important to consider the weight of the animals.

Table I: Recommended Age After which Animals Should Be Used for Polyclonal Antibody Production

Animal	Age
Mice	6 weeks
Rats	6 weeks
Rabbits	3 months
Guinea-pigs	3 months
Chickens	3-5 months
Goats	6-7 months
Sheep	7-9 months

Data from references 13 and 15.

Immunisation Protocol

The nature of the antigen, as well as the intended purpose of the antiserum produced, should be reflected in the choice of the immunization protocol. Before proceeding with the immunization, the investigator should consider the toxicity of the antigen preparation due to, for example, contaminating lipopolysaccharide (also called endotoxin), or chemical residues used to inactivate micro-organisms (for example, sodium azide, formaldehyde, or 5-propiolactone), or an extreme pH, and should make adjustments, if appropriate. Essential factors to be considered in the preparation of an immunization, including the selection of an adjuvant, the route and volume of injection, and the immunization schedule, are described below.

Selection of immunological adjuvants

Adjuvants are used to enhance the immune response. The ideal adjuvant can be characterised as a substance which stimulates high and sustainable antibody titres (even with small quantities of antigen), is efficient in a variety of species, applicable to a broad range of antigens, is easily and reproducibly prepared in an injection mixture, is easily injectable, is effective in a small number of injections, has low toxicity for the immunised subject, and is not harmful to the investigator. Unfortunately, the adjuvant that meets all these criteria still does not seem to exist.

There are more than 100 known adjuvants, but many of them would not be routinely used for the production of pAbs, due to cost or difficulty in the preparation of the injection mixture. The different adjuvant categories that can be used for pAb production are briefly described in Appendix 2. These categories include oil emulsions, mineral salts, saponins, microbial products, synthetic products, and adjuvant formulations containing mixtures of products. Commercially available adjuvants that are used for routine pAb production include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant, adjuvants from the Montanide® ISA series, Gerbu^TM, Quil A, aluminum salts, TiterMax^TM, and RIBI^TM. More-detailed information on adjuvants can be obtained from several review articles and books (17-20).

The choice of adjuvantis, in principle, left to the investigator, but the workshop participants agreed that the overall welfare of the laboratory animal to be immunised should be of primary importance when selecting an adjuvant. It is recommended that animal ethics committees should be involved in the evaluation of immunization protocols with regard to animal welfare aspects.

The (antigen/adjuvant) inoculation mixture should be prepared aseptically to minimise the risk of possible contamination. It is also essential to check whether the antigen to be used requires the presence of an adjuvant or possesses innate adjuvanticity. Adjuvants are usually not necessary when whole bacteria, whole cells or other particulate antigens (for example, cell fractions and bacterial cell walls) are used, but they are often necessary in the case of soluble antigens (proteins, peptides, polysaccharides). An adjuvant might also be necessary when only a very limited amount of antigen is available, when native antigens are used, or when a specific type of response is required.

When an oil emulsion is used in an immunisation experiment, the stability and quality of the emulsion should be checked. It is not difficult for an investigator to test whether a water-in-oil emulsion has been formed, because a drop of the mixture placed on the surface of water in a dish will retain its shape and not disperse. On the other hand, dispersion of the emulsion droplet over the surface of the water is indicative of an oil-in-water emulsion.

Selection of route of injection

Suggested injection routes for antigens with or without adjuvants in experimental mixtures are given in Table II.

Table II: 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 thge 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.

The intramuscular route of injection was a major point of discussion. Some participants argued that the intramuscular route is frequently used without problems, while others considered this route neither acceptable nor necessary for injections with adjuvant, especially for small rodents such as mice. In animals with large muscles, large volumes of material can be accommodated. Antigen can be absorbed by Iymphatics in this region, but antigen and adjuvant can spread and reside along interracial planes between muscle bundles (because of leakage from the muscle bundle or because of misplacement of the injection mixture) and establish contact with nerve bundles, where serious pathology consequent to inflammatory processes can occur (13, 21). This is especially true for small animals such as mice, for which the intramuscular route should only be used by experienced researchers and biotechnicians. Furthermore, local reactions after intramuscular injections can easily be overlooked (22).

The intraperitoneal injection of adjuvant mixtures is not recommended, since it is known to induce inflammation (macroscopically evident), peritonitis (with the risk of ascites formation), and behavioural changes (for example, decreased activity and weight loss).

Herbert (23) considered intravenous administration to be the route of choice for small particulate antigens such as viruses, bacteria or cells (where danger of anaphylaxis is low), because the antigen distribution is broad and capture by Iymphoid tissues is high. However, due to the risk of embolism, it is inappropriate for oil adjuvants (oil emulsions), viscous gel adjuvants or large particulate antigens (for example, bacterial aggregates, heterologous lymphocytes when raising anti-lymphocyte sera, or other heterologous whole mammalian cells [23]).

The workshop participants agreed that alternative injection sites, such as the footpad, are not to be recommended for pAb production. With precious antigens in very small quantities or with protein bands from electrophoretic separating gels (for example, precipitating bands from immunoelectrophoresis experiments), there could be scientific reasons for the use of the intra-lymph node injection procedure with an ocular grade needle, as described by Goudie et al (24). Some authors have recommended intra-splenic injection for similar reasons (25). The participants agreed that investigators should provide scientific justification to ethical committees for such protocols (for example, the need to use extremely valuable or unique and irreplaceable antigens, or extremely small quantities of antigen).

Determination of volume of injection

To ensure that animals experience minimal discomfort at the antigen injection site, the injection volume should be as small as possible. Agreed maximum volumes per site of injection are shown in Table III. These volumes are based on the use of an injection mixture that forms a depot at the site of injection, for example, an immunostimulatory oil emulsion or a viscous gel. If the inoculum contains an immuno-potentiator, for example, mycobacteria or a muramyl dipeptide derivative, the amount of antigen in the injection mixture should generally not exceed 25 µg for a mouse or 200 µg for guinea pigs, rats or rabbits. The inoculum should be spread among multiple injection sites in larger animals.

Table III: 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

If the intraperitoneal route of injection is required for adjuvants such as oil emulsions and viscous gels, the maximum volume injected should not exceed 0.2 ml. If the intradermal route is required (use should be limited to rabbits and large animals), the maximum dose of adjuvant/antigen mixture would be 25 µl per site at no more than four sites.

Maximum volumes that are allowed for injections of antigen solutions without adjuvant have been described by Iwarsson et al (26) and van Zutphenet et al (27) and are given in Table IV.

Design of boosting protocol/immunisation schedule

The boosting protocol can have a decisive influence on the result of the immunization. The time between two immunization steps can influence both the induction of B memory cells and the class switch of B cells (from IgM to other antibody classes and subclasses). Specific recommendations for the interval between primary and booster immunisations are usually not cited. In general, a booster can be considered after the antibody titre has plateaued or begun to decline. If the first immunization is performed without a depot-forming adjuvant, the antibody titre will usually peak 2-3 weeks after immunisation. When a depot-forming adjuvant is used, a booster injection can follow at least 4 weeks after the first immunization. For booster immunizations, an adjuvant is not always necessary.

The participants agreed that, in most cases, the endpoint of pAb production should be judged when the antibody titre has reached an acceptable level. This should usually occur after a maximum of two boosters. If the antibody response is still insufficient for laboratory purposes at this time, the experiment should, in general, be terminated. In the case of antigens of interest with a low molecular weight, such as peptidesor steroids, injections might have to be repeated several times before the antiserum reaches the titre and specificity required for the application. However, long immunization schemes, with repeated boosting, not only result in the production of antibodies with increased affinity for the antigen of interest, but also in the production of more antibodies specific for contaminants in the antigen preparation. Such multispecific antisera require absorption before they are monospecific, a process with some inherent difficulties.

Animals can be rested for long intervals between boosting. Even when serum antibody titres have dropped to relatively low levels, a booster injection into an animal that has previously established a memory response will usually re-establish a high serum antibody titre (13). Intermittent bleeding of a hyper-immunised animal appears to help maintain a high serum antibody level. Thus, regular interval blood collections after a sufficient serum antibody titre has been reached could facilitate the collection of adequate amounts of pAbs to an antigen that is in very limited supply. However, animals must not be kept in antibody production programmes unnecessarily (as already discussed).

Primary injections with very low amounts of antigen (picograms) are not recommended, since this does not stimulate the immunologic memory aufficiently, and might induce tolerance to the antigen. Frequent immunizations with relatively low amounts of antigen can be counterproductive. In addition, animal welfare argues against such schedules. However, low amounts of antigen for booster immunization may help raise the average affinity of the antibodies subsequently produced (28).

In general, booster injection sites should be distant from previous injection sites. Booster antigen mixtures should never be inoculated into granulomas or swellings induced by earlier immunizations. Booster immunizations do not need to be administered by the same route used for the primary immunization (29). Adjuvants that contain mycobacteria or their components (for example, cell walls) should only be used once per animal, because severe hypersensitivity reactions may result following reexposure of the host to mycobacteria (30, 31). Booster immunisations applied intravenously or intraperitoneally with aqueous soluble antigens might result in systemic anaphylaxis, caused by the rapid release of histamine and other mediators from basophils and perivascular mast cells.

Blood Collection

Blood samples should be taken with minimum stress for the animal. Animals to be immunised should be conditioned to be confident with the animal care staff. This is not only important for animal well-being, but also ensures that the animals do not exhibit stress-mediated vasoconstriction, which would make blood sampling difficult.

Blood collection should be performed under conditions where it is possible to keep the animals warm, a prerequisite for ensuring good blood supply to the periphery. It is also important that the animals are not subjected to sudden noises or other environmental stress-inducers during blood collection, or, for that matter, during their routine housing.

The application of organic solvents to induce vasodilation is not recommended because of the toxic and carcinogenic potential of the solvents for laboratory animals and humans.

When blood is collected for antibody production, it can be advantageous to prepare plasma (use of an anticoagulant such as heparin, citrate, or EDTA) instead of serum since the fluid yield can be increased significantly.

Blood should be collected only from the sites recommended by the BVA/FRAME/ RSPCA/UFAW Joint Working Group on Refinement (32; see Table V).

When possible, a collection method not requiring anaesthesia should be preferred. This may favour the choice of a species in which blood sampling in conscious animals is easy for the operator and unstressful for the animal. Small ruminants and rabbits are thus advantageous compared to small rodents.

If an animal is not stressed during bleeding, the use of a sedative to facilitate blood sampling is usually unnecessary. However, in large-scale production systems, operators may find it advantageous to use sedatives to maintain low stress levels and to achieve rapid blood collection.

The needle used should be matched to the vessel size and should preferably be of relatively large bore to facilitate rapid collection. Vacutainers may be used, provided that the vein does not collapse. The bleeding of rabbits through an ear vein should be performed with a needle rather than with a scalpel, because of the difficulties inherent in stopping bleeding of a cut vessel. Butterfly needles are recommended, as they allow the animal to move its head during the procedure.

The volume to be removed per bleeding should not exceed 15% of the total blood volume; in practice, an amount up to 1% of the total body weight can safely be removed (33).

International guidelines differ with respect to frequency of blood collection (1 4 weeks), irrespective of the bleeding interval. The maximum volume allowed should not be removed more frequently than once a fortnight.

Exsanguination should be performed under general anaesthesia and is best carried out by heart puncture. After exsanguination is completed, small rodents should be subjected to cervical dislocation and larger animals should be euthanised by an overdose of an appropriate anaesthetic agent.

Assessment of Side-effects

In order for investigators to minimise pain and distress in immunization procedures, the side-effects induced by immunization have to be assessed. Some assessment procedures for quantifying discomfort in animals have been developed; these include the protocol proposed by Morton & Griffiths (34), and systems which measure changes in activity and behavioural patterns for a given period (35-37).

Animals should be checked daily and, in addition to the routine check-up which includes observance of the animal's general appearance and food and.water intake, the site of injection should be inspected. However, it should be noted that checking the food and water intake of individual animals may not be possible when animals are group-housed. When comparative studies or new experiments are performed (for example, with new combinations of antigen and adjuvant), the assessment of side-effects should also include studies of pathological changes at the end of the experiment; for this purpose, animals should be necropsied and multiple tissues should be examined. Pathological changes may not always be evident during clinical observation, depending on the route of injection (for example, the injection site cannot be examined after intraperitoneal injection). Pathological changes might not be confined to the site of injection.

Blood collection can also cause side-effects, especially when the animals are anaesthetised. Although blood samples should preferably be taken from unanaesthetised animals, in some cases anaesthesia might be needed (for example, for exsanguination, bleeding from the orbital sinus and heart puncture). When Hypnorm^TM is used as an anaesthetic in blood sampling, the animals should be checked afterwards, since bleeding may continue. After a blood sampling procedure, the puncture site should be monitored for closing and healing of the wound. Specific attention is necessary when the ear artery of a rabbit is used. The artery must be compressed for a aufficiently long period of time (sometimes up to 5 minutes) for reliable closure and to avoid leakage.

Advanced Techniques and Alternatives

The traditional ways of adjuvanting protein have sometimes failed to result in polyclonal antiserum, for example, against small peptides and carbohydrate antigens. Therefore, it has been necessary to develop alternative approaches for antigen preparation. The conjugation technology and the multiple antigen peptides (MAP) procedure are described below. By coupling synthetic peptides or carbohydrates to protein carriers, it is possible to promote the T-cell helper function that supports a vigorous antibody response. In general, proteins (for example, keyhole limpet haemocyanin and bovine serum albumin) which are highly immunogenic themselves, are used as carriers. Therefore, an antibody response is also elicited against the carrier molecule (38), and the antiserum will need to be absorbed to render it monospecific. The MAP procedure overcomes some of the shortcomings of the classical conjugation approach described above for eliciting an antibody response against peptides. In place of a large protein carrier (which in itself would be immunogenic), a relatively nonimmunogenic core matrix, consisting of trifunctional amino acids (such as Iysine), is used as the "carrier". Lysine, with its extra amino group, can be used to form a Iysine "tree" (core matrix) to which a number of peptides can be attached (39). The entire construct, including the peptides, can be generated with a peptide synthesizer. The number of peptides to be incorporated will be proportional to the number of Iysine residues in the core matrix. The molecular weight of the resulting MAP will also reflect this. Accordingly, the density of B-cell epitopes is sign)ficantly higher with the MAP approach than with traditional protein carriers. The MAP approach has been successfully used with aluminum adjuvant (Alhydrogel), as well as with oil emulsion adjuvants (40) to raise antibodies to peptides. However, for the MAP construct to be immunogenic, it must contain both T-cell receptor epitopes and B-cell epitopes within the peptide of interest, because the Iysine matrix itself does not provide the T-cell receptor epitopes necessary for eliciting the all-important T-cell help. A variant of the MAP procedure has been designed to allow synthesis of a multiple antigen construct which contains two different peptides, one of which is the antigen of interest for eliciting antibodies, while the other is a peptide with a strong T-cell receptor epitope (41).

Recently, Glenn et al (42) demonstrated that it is possible to induce a systemic humoral immune response by transcutaneous immunization. Cholera toxin was used as adjuvant, and bovine serum albumin tetanus toxoid and diphtheria toxoid were used as antigens. No redness, swelling or other signs of inflammation were seen. Taking into account the large area of the skin, its accessibility, and the presence therein of potent immune cells (Langerhans cells), one could presume that this route could be a practical alternative to the most common injection routes, at least in some species. The immunising solution could be applied for a certain period with plaster or with patches similar to those developed for transcutaneous drug delivery. However, with regard to the technique, further research should be undertaken involving other types of antigen and other adjuvants, and on the development of memory, species variations, and classes of antibodies elicited.

Oral immunisation aimed at inducing a simultaneous peripheral and mucosal immune response may be an attractive alternative to subcutaneous or intramuscular immunisation for animal welfare reasons. Oral immunisation has been conducted successfully in studies of new vaccine delivery systems in which antigens have been administered orally in biodegradable biospheres or associated with carrier proteins derived from cholera toxin or Escherichia coli heat-labile protein.

The development of novel DNA-based technologies, such as phage display libraries direct cloning of antibody genes into plasmids, and molecular engineering, has introduced some very interesting possibilities for the production of antibodies which combine a high specificity with certain functional characteristics that are important for their further use (43-45). Although these possibilitles are primarily seen as an alternative to the in vivo and in vitro methods currently used for the production of mAbs, under certain conditions these techniques could also be used as an alternative for the production of pAbs. At present, only a small number of laboratories have phage display libraries and the expertise needed to make use of them. However, it is evident that the availability and accessibility of these phage display libraries are growing.

Existing Guidelines on the Production of Polyclonal Antibodies

In the last decade, many sets of guidelines on the production of pAbs have been established by various national control authorities organizations, and institutions. The increasing number of guidelines demonstrates the increased awareness of undesirable sideeffects caused by some adjuvants and injection routes, but also that efforts should be made to harmonise these guidelines. It should be borne in mind that many of the immunisation protocols used for pAb production were (or are still) based on habit and tradition, rather than on scientific principles. The common aims of the established guidelines are to ensure appropriate procedures for the production of pAbs which give satisfactory results, and minimise discomfort, distress and pain in the animals involved. The guidelines are considered to be a tool for scientists, animal technicians, animal welfare officers and ethical review committees.

In 1988, the US National Institutes of Health issued their intramural recommendations for the research use of FCA (46). This was followed by Canadian Council on Animal Care guidelines on acceptable immunological procedures (30). These two sets of guidelines have had a major impact on the establishment of guidelines or codes of practice in other countries.

Several European countries have issued national guidelines on the production of pAbs, i.e. Switzerland (15), Denmark (16) The Netherlands (31), the UK (47) and Sweden (48). Variations in the legal status of the guidelines are evident between these countries. The Dutch and Swedish guidelines are not mandatory in the strict sense of the word; however, they are followed by the ethical review committees, and, in the case of The Netherlands, by the Veterinary Inspectorate. Thus, Dutch scientists have to justify explicitly when their immunisation protocol deviates from the Code of Practice for the Immunisation of Laboratory Animals (31). In the UK, immunisation protocols are reviewed and authorised by the Home Office Inspectorate, and experts in the production of pAbs are consulted when necessary. The situation in Denmark is comparable. In Switzerland, scientists are obliged to set up their immunisation protocols in accordance with the guidelines. Modification of the protocols can be required by the Swiss Competent Authority, the Bundesamt für Veterinärwesen.

Guidelines on the production of pAbs have been published by various organizations, for example the Scientists Centre for Animal Welfare (49), the Australian and New Zealand Council for the Care of Animals in Research and Teaching (50), the Tierärztliche Vereinigung für Tierschutz (22) and the Arbeitsgemeinschaft der Tierschutzbeauftragten in Baden-Württemberg (51) and are included in several handbooks on immunological procedures (23, 38, 52, 53). In the USA in particular, but also in Europe, Australia and New Zealand research institutes, and especially universities, have established their own guidelines, many of which are available on the Internet. Most of these institutional guidelines are not mandatory. In the USA, the Institutional Animal Care and Use Committees (IACUCs) provide guidelines that are not intended to dictate procedures, but are intended to assure proper treatment of animals that are used for pAb production. The IACUCs can request that investigators modify immunisation protocols and allow deviations from their guidelines, provided that these are for scientific reasons. This has clearly brought about some changes, which have improved attention to the welfare of the animals concerned. However, there is still some disagreement about the best immunisation protocols for the production of pAbs.

The recommendations given in some of the guidelines mentioned above were compared for the purposes of this report. Several guidelines include a lot of background information and a list of references. All of them cover, to different extents, the following important aspects of pAb production: choice of species, antigen preparation, injection route, injection volume, choice and use of adjuvants injection technique, immunisation schedules, blood sampling and post-injection observation (Table VI). All put special emphasis on the limitations involved in the use of FCA, and similar recommendations are evident with respect to the permitted maximum volume of an FCA antigen preparation Table VII. Some of the guidelines state explicitly which injection routes should be preferred, discouraged or not allowed (Tables VI and VII). There is a general consensus that footpad injection is not necessary for pAb production.

There is no doubt that these guidelines have had a positive impact on pAb production and have increased the attention given to animal welfare. In The Netherlands, the effect of the guidelines was evaluated in 1996 (54). It became evident that the guidelines had initiated discussions within institutes, which led in turn to the modification of immunisation protocols. However, this evaluation (and also a symposium) showed that the guidelines have to be amended in some specific areas. In Sweden, the set of guidelines have proved to be a valuable and practicable tool for both ethical committees and scientists.

Concluding Remarks

Current immunisation procedures for the production of pAbs are often based on habit and tradition. To ensure that appropriate procedures are used, many sets of guidelines have been established. The harmonization of these guidelines is needed. In the workshop, various aspects of immunisation protocols and existing guidelines were discussed. In Appendix 1, draft guidelines for the production of pAbs are given, based on all available information. These guidelines should be regarded as a tool for use by scientists and ethical committees to improve immunisation procedures in order to minimise pain and distress to the animals involved. The guidelines should initiate discussions, which should lead to the further mod)fication of immunisation protocols.

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The Production of Polyclonal Antibodies in Laboratory Animals

The Report and Recommendations of ECVAM Workshop 351,2

Appendix 1

Appendix 2

The Report and Recommendations of ECVAM Workshop 35^1,2