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Preface

This is the report of the thirty-fifth of a seriesof workshops organised by the European Cen-tre for the Validation of Alternative Methods(ECVAM). ECVAM�s main goal, as defined in1993 by its Scientific Advisory Committee, isto promote the scientific and regulatoryacceptance of alternative methods which areof importance to the biosciences and whichreduce, refine or replace the use of laboratoryanimals. One of the first priorities set byECVAM was the implementation of proce-dures which would enable it to become well-

informed about the state-of-the-art of non-animal test development and validation, andthe potential for the possible incorporation ofalternative tests into regulatory procedures.It was decided that this would be bestachieved by the organisation of ECVAMworkshops on specific topics, at which smallgroups of invited experts would review thecurrent status of various types of in vitro testsand their potential uses, and make recom-mendations about the best ways forward (1).

This joint ECVAM/FELASA (Federation ofEuropean Laboratory Animal Science Asso-ciations) workshop on The Immunisation of

The Production of Polyclonal Antibodies inLaboratory Animals

The Report and Recommendations of ECVAM Workshop 351,2

P.P.A. Marlies Leenaars,3 Coenraad F.M. Hendriksen,3 Wim A. de Leeuw,4Florina 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 BABilthoven, The Netherlands; 4Inspectorate for Health Protection, Commodities andVeterinary Public Health, De Stoven 22, 7206 AX Zutphen, The Netherlands; 5StallergenesSA, 6 Rue Alexis de Tocqueville, 92183 Antony Cedex Paris, France; 6Centre D�EconomieRurale, Rue du Point du Jour 8, 6900 Marloie, Belgium; 7Department of Biochemistry,Swiss Federal Institute of Technology, 8092 Zurich, Switzerland; 8ECVAM, JRCEnvironment Institute, 21020 Ispra (VA), Italy; 9Department of Microbiology andImmunology (M/C 790), University of Illinois, 835 S. Wolcott, Chicago, IL 60612, USA;10Paul-Ehrlich-Institut, Bundesamt für Sera und Impfstoffe, Paul-Ehrlich-Strasse 51�59,63225 Langen, Germany; 11Department of Comparative Medicine, Biomedical Centre, Box570, 75123 Uppsala, Sweden; 12Superfos Biosector a/s, Elsenbakken 23, 3600 Frederikssund,Denmark; 13Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120Heidelberg, Germany; 14Instituto de Salud Carlos III, Unidad Respuesta Immune, CentroNacional de Biol. Fundamental, 28220 Madrid, Spain; 15Division of Infection andImmunity, Institute of Biomedical and Life Sciences, Joseph Black Building, Glasgow G128QQ, UK

ATLA 27, 79�102, 1999 79

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 Institute for Health & Consumer Protection, 21020 Ispra (VA), Italy.1ECVAM � The European Centre for the Validation of Alternative Methods. 2This document represents theagreed report of the participants as individual scientists.

Laboratory Animals for the Production ofPolyclonal Antibodies was held in Utrecht(The Netherlands), on 20�22 March 1998,under the co-chairmanship of Coenraad Hen-driksen (RIVM, Bilthoven, The Netherlands)and Wim de Leeuw (Inspectorate for HealthProtection, The Netherlands). The partic-ipants, all experts in the fields of immunol-ogy, laboratory animal science, or regulation,came from universities, industry and regula-tory bodies. The aims of the workshop were:a) to discuss and evaluate current immunisa-tion procedures for the production of poly-clonal antibodies (including route ofinjection, animal species and adjuvant); andb) to draft recommendations and guidelinesto improve the immunisation procedures,with regard both to animal welfare and tothe optimisation of immunisation protocols.This report summarises the outcome of thediscussions and includes a number of recom-mendations and a set of draft guidelines(included in Appendix 1).

Introduction

The immunisation of laboratory animals toinduce a humoral and/or cellular immuneresponse, is a routine procedure performedworldwide. Consequently, the use of animalsis substantial, although no precise figuresare available. Researchers from many disci-plines need to produce antibodies. Often,they do not have specific knowledge ofimmunology, and are not sufficiently experi-enced in procedures for immunisation of lab-oratory animals. Therefore, specificguidelines on immunisation protocols arerequired.

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

Certain immunisation procedures are cur-rently under discussion for animal welfarereasons. For example, the adjuvant productsused to enhance the immune response areknown to cause local inflammation, andsome immunisation protocols are associatedwith pain and distress.

Some European countries, individualorganisations and institutes have issuedguidelines setting out criteria, for example,

on the maximum volume to be injected, theroute of inoculation and the number of injec-tions. The aim of these guidelines is toensure proper immunisation procedures,which combine acceptable immunologicalresults with minimal discomfort for the ani-mals. The immunisation protocol (includingprimary and booster injections and blood col-lection) has a great impact on both aspects.It is therefore crucial to carefully design thisprotocol.

The workshop focused on the productionof pAbs and did not address the cellularimmune response. Various aspects were dis-cussed which could influence the inductionof pAbs and affect the welfare of the animals.

Polyclonal Antibodies Versus Monoclonal Antibodies

The immune systems of most mammals arebelieved to be comprised of approximately1000 clonal populations of lymphocytes, ascharacterised by their antigen-receptorspecificity. This diversity permits immuneresponses to a broad range of immunogens,for example, foreign proteins, carbohydrates,peptides and bacterial and viral components.The lymphoid organs (spleen, lymph nodes,and gut-associated lymphoid tissue, includ-ing tonsils) are the production sites of a vastrange of antibodies by stimulated B lympho-cytes (plasma cells). Each antibody moleculerecognises a specific antigenic epitope, possi-bly as small as 5�6 amino acids or betweenone and two glucose or other monosaccha-ride units, and is able to bind to the immuno-gen. A polyclonal humoral response, makinguse of the entire range of antibodies (pAbs),results in high avidity (defined as the prod-uct of the affinity constants of all bindingantibodies) and gives the organism the abil-ity to defend itself successfully againstpathogens.

Köhler & Milstein (2) were the first tomake B-cell hybridomas by using a fusiontechnique with Sendai virus. Subsequentworkers have carried out successful fusionsby using other agents, for example, polyeth-ylene glycol. The fusion technique permitsthe immortalisation of single lymphocytes.The mAbs produced by such hybridoma cellclones originating from a single B lympho-cyte are identical, and specific for a singleepitope. There are some cases in which the

80 P.P.A.M. Leenaars et al.

extreme monospecificity of mAbs can be adisadvantage. If for any reason the antigenicsite is altered, which could be the case inmany experiments, the mAb might not con-tinue to bind. Nor are individual mAbs of usein precipitation assays such as immunoelec-trophoresis. In contrast, the specificity ofpAbs depends on a combination of hundredsor even thousands of clonal products, whichbind to a number of antigenic determinants.As a result, small changes in the structure ofthe antigen due to genetic polymorphism,heterogeneity of glycosylation or slightdenaturation, will usually have little notice-able effect on pAb binding. Whether thesecharacteristics are seen as a problem or anadvantage will depend on individual circum-stances.

The avidity of a mAb is generally equal toits affinity for a protein antigen, a fact whichis sometimes considered to be a disadvan-tage. MAbs are sometimes used in combina-tions, to increase the heterogeneity.

In making a choice between the generationof mAbs or pAbs, the desired application ofthe antibody and the time and money avail-able for its production should be considered.The production of mAbs is tedious and takes3�6 months. Cell cultures are required inaddition to the animal immunisation. Exam-ples of the use of mAbs include the immuno-staining of western blots, ELISA, the affinitypurification of proteins, and the immuno-staining of thin tissue sections visualised bylight microscopy or electron microscopy. Theimmortalised hybridoma serves as an inex-haustible antibody source of standardisedquality. The induction of pAbs usually takes4�8 weeks. The serum is suitable for manyapplications, for example, the immunostain-ing of western blots, ELISA and immunopre-cipitation complement fixation. In mostcases, polyclonal sera are of high titre, andpermit substantial dilution; however, theremay be batch-to-batch variability. The factthat a polyclonal antiserum can be obtainedwithin a short time with little financialinvestment favours its use. In research,many questions can be answered with theassistance of a polyclonal antiserum.

General Considerations

Of primary importance for pAb productionare factors such as the antigen used, route of

immunisation, animal species, type and qual-ity of the adjuvant, and method of blood col-lection. However, a number of other aspectscan also have an impact on the successfuloutcome of an immunisation procedureand/or on the welfare of the animals, includ-ing the health and genetic status of the ani-mals, the expertise and competence of thestaff, and the hygiene, diet and housing ofthe animals.

Immunosuppressive effects have beenreported for various agents which infectrodents (for example, the Sendai virus,mouse hepatitis virus, minute virus of mice;3) or rabbits (Encephalitozoon; 4). Infectionsin the donor animals might also reduce thespecificity of the antiserum, and, in the caseof zoonotic agents, pose a human health risk.In general, the workshop participants recom-mended the use of specific pathogen-free ani-mals in the case of small laboratory animals,and the implementation of a microbiologicalmonitoring system according to the recom-mendations of FELASA (5, 6). However, itwas also felt that, in some cases, more-con-ventional housing might be acceptable. Inaddition, it was recommended that the end-user of the animals should ask the supplierfor an animal health and diet record, sincespecific tolerance to an antigen of interestmay be generated by an animal�s earlydietary exposure to the antigen of an ana-logue. Immunosuppressive effects of dietshave also been reported (7, 8).

Stress in animals should be avoided, sincethis can result in immune suppression, aswell as discomfort for the animals. Immunesuppression can result from stress generatedbefore, as well as during, the actual period ofimmunisation (9). The quality of immunisa-tion procedures can be optimised by theestablishment of central facilities or unitswith responsible scientists (for example, aveterinarian) with experience in animal hus-bandry and immunisation processes.

The workshop participants emphasisedthe importance of training programmes forthe personnel engaged in immunisation pro-cedures and, in particular, for the animaltechnician. Reference was made to FELASArecommendations on education, which arenow available for animal caretakers (Cate-gory A) and researchers (Category C; 10),and are being drafted for animal technicians(Category B) and animal welfare officers/lab-oratory animal specialists (Category D). For

ECVAM Workshop 35: polyclonal antibodies 81

instance, a curriculum for animal techni-cians should include the basic principles ofimmune response, injection and bleedingtechniques, and the development of skills forobservation, anaesthesia and euthanasia ofthe animals.

Another important aspect is animal hous-ing. It is recommended that, whenever possi-ble, animals should be housed underconditions that encourage their naturalbehaviour. The requirements of the Councilof Europe (11) can be used as a point of ref-erence. However, it was also noted that, inmost laboratories, animals are housed indi-vidually without environmental enrichment,for example, rabbits. In general, the grouphousing of animals is recommended,although in specific situations, individualhousing might be justified. There might alsobe microbiological considerations in favourof individual housing, because the more-intensive direct contact between individualshoused in groups encourages the transmis-sion of pathogens (for example, coccidia).Some participants reported the successfulgroup housing of castrated bucks. However,the ethical implications should be balancedagainst the advantages of group housing, andin some countries castration is not permit-ted, because it is stressful to the animals andis considered to be an unethical interferencewith the integrity of the animal.

The principles of Good Animal Experimen-tation Practice, as laid down in EuropeanUnion legislation, Council Directive86/609/EEC (12), must be followed in allaspects of the immunisation process. A num-ber of crucial steps were identified: thepreparation of the antigen, from its initialpurification to its eventual mixing with adju-vant, the injection of antigen/adjuvant, thebleeding of the animal, and the processing ofantiserum. It is recommended that particu-lar attention should be paid to the quality ofthe antigen preparation (for example,removal of endotoxin, formaldehyde orsodium axide), the storage of the reagents,and the sterility of the instruments used forinjection and bleeding (13).

Choice of Laboratory Animal

The selection of the animal species for theproduction of pAbs depends, at least in part,on the amount of antiserum needed and the

ease of obtaining blood samples. The intendeduse of the pAbs can also play a role. In anELISA, the antibody which binds to the anti-gen (the primary antibody) should be from adifferent species to the conjugated (sec-ondary) antibody used in the next step of theassay. When there is no need for a specificspecies, the animals from which samples ofblood are relatively easy to obtain (rabbitsand mice) should be preferred over thosewhich are difficult to bleed, for example,guinea-pigs and hamsters. The selectionmight also be related to the purpose of theexperiment, as each species, strain, stock orbreed of animal might differ in its immuneresponse. An example is the differencebetween Balb/c mice and C57BL/6 mice, thefirst strain generally being a Th2-like respon-der, and the latter a Th1-like responder,although these may not always be hard andfast rules. The choice of a strain of mice orrats might also be influenced by the availabil-ity of inbred strains or outbred stocks. Whenan inbred strain is used, genetic variationbetween animals is restricted, such that hypo-responsiveness or hyper-responsiveness to aparticular antigen might occur quite uni-formly among members of the strain (uniformfailure or uniform success). When an outbredstock is used, genetic variation among the ani-mals may lead to a range of responses amongmembers of the group. Although many envi-ronmental factors and a number of geneticfactors can influence an animal�s immuneresponse, the major histocompatibility com-plex genotype plays a major role, in that itdefines an individual�s potential for antigenpresentation and thus the individual�s anti-body response potential to a particular anti-gen. Chickens could be used as an alternativeto mammals for the production of pAbs. Theproduction of avian antibodies (IgY) in chick-ens is considered to be a refinement, since thecollection of blood in the production of IgYhas been replaced by the extraction of anti-bodies from egg yolk. In addition, chickensmight be preferred for scientific reasons, forexample, their phylogenetic distance frommammals. Unfortunately, the production ofIgY in chickens is not widespread. This isprobably due to a number of factors, such astraditions among researchers, the infrequentuse of the chicken as a laboratory animal ingeneral, the limited availability of conjugatedantibodies, specific requirements for housing,and lack of experience with chickens and


chicken antibodies. Also, chicken antibodiesare more difficult to purify than are mam-malian antibodies. Further information onIgY production is given in ECVAM workshop21 (14).

Traditionally, female animals are most fre-quently used in pAb production. Female ani-mals are generally more docile for handlingpurposes, and are less aggressive in socialinteractions, and can therefore be group-housed more successfully. Although there issome evidence that androgens can slightlydampen the antibody response, there are nooverriding scientific reasons for not usingmale animals.

The immune status of the immunised indi-vidual may also determine the outcome of animmunisation procedure. Young adultsshould be used for pAb production, as theimmune response is immature at an earlyage and drops with age after the period ofyoung adulthood. When animals are re-usedafter other procedures (only one use for pAbproduction is appropriate), it is important tokeep the age of the animals in mind. Whenchickens are immunised, they should be ofegg-laying age by the time antibody is to beharvested. Recommendations as to the age atwhich animals should first be immunised aregiven in the Swiss guidelines (15) and byHanly et al. (13; Table I). Some guidelinesrecommend the use of animals at an earlierage (16). In addition, it is important to con-sider the weight of the animals.

Immunisation Protocol

The nature of the antigen, as well as theintended purpose of the antiserum produced,should be reflected in the choice of theimmunisation protocol. Before proceedingwith the immunisation, the investigatorshould consider the toxicity of the antigenpreparation due to, for example, contaminat-ing lipopolysaccharide (also called endo-toxin), or chemical residues used toinactivate micro-organisms (for example,sodium azide, formaldehyde, or β-propiolac-tone), or an extreme pH, and should makeadjustments, if appropriate. Essential factorsto be considered in the preparation of animmunisation, including the selection of anadjuvant, the route and volume of injection,and the immunisation schedule, aredescribed below.

Selection of immunological adjuvants

Adjuvants are used to enhance the immuneresponse. The ideal adjuvant can be charac-terised as a substance which stimulates highand sustainable antibody titres (even withsmall quantities of antigen), is efficient in avariety of species, applicable to a broad rangeof antigens, is easily and reproducibly pre-pared in an injection mixture, is easilyinjectable, is effective in a small number ofinjections, has low toxicity for the immu-nised subject, and is not harmful to theinvestigator. Unfortunately, the adjuvantthat meets all these criteria still does notseem to exist.

There are more than 100 known adju-vants, but many of them would not be rou-tinely used for the production of pAbs, due tocost or difficulty in the preparation of theinjection mixture. The different adjuvantcategories that can be used for pAb produc-tion are briefly described in Appendix 2.These categories include oil emulsions, min-eral salts, saponins, microbial products, syn-thetic products, and adjuvant formulationscontaining mixtures of products. Commer-cially available adjuvants that are used forroutine pAb production include Freund�scomplete adjuvant (FCA), Freund�s incom-plete adjuvant, adjuvants from the Mon-tanide® ISA series, GerbuTM, Quil A,aluminum salts, TiterMaxTM, and RIBITM.More-detailed information on adjuvants canbe obtained from several review articles andbooks (17�20).


Table I: Recommended age after whichanimals should be used for poly-clonal antibody production

Animal Age

Mice 6 weeksRats 6 weeksRabbits 3 monthsGuinea-pigs 3 monthsChickens 3�5 monthsGoats 6�7 monthsSheep 7�9 months

Data from references 13 and 15.

The choice of adjuvant is, in principle, leftto the investigator, but the workshop partic-ipants agreed that the overall welfare of thelaboratory animal to be immunised should beof primary importance when selecting anadjuvant. It is recommended that animalethics committees should be involved in theevaluation of immunisation protocols withregard to animal welfare aspects.

The (antigen/adjuvant) inoculation mix-ture should be prepared aseptically to min-imise the risk of possible contamination. It isalso essential to check whether the antigento be used requires the presence of an adju-vant or possesses innate adjuvanticity. Adju-vants are usually not necessary when wholebacteria, whole cells or other particulateantigens (for example, cell fractions and bac-terial cell walls) are used, but they are oftennecessary in the case of soluble antigens(proteins, peptides, polysaccharides). Anadjuvant might also be necessary when onlya very limited amount of antigen is available,when native antigens are used, or when aspecific type of response is required.

When an oil emulsion is used in an immu-nisation experiment, the stability and qual-ity of the emulsion should be checked. It isnot difficult for an investigator to testwhether a water-in-oil emulsion has been

formed, because a drop of the mixture placedon the surface of water in a dish will retainits shape and not disperse. On the otherhand, dispersion of the emulsion droplet overthe surface of the water is indicative of anoil-in-water emulsion.

Selection of route of injection

Suggested injection routes for antigens withor without adjuvants in experimental mix-tures are given in Table II.

The intramuscular route of injection was amajor point of discussion. Some participantsargued that the intramuscular route is fre-quently used without problems, while othersconsidered this route neither acceptable nornecessary for injections with adjuvant, espe-cially for small rodents such as mice. In ani-mals with large muscles, large volumes ofmaterial can be accommodated. Antigen canbe absorbed by lymphatics in this region, butantigen and adjuvant can spread and residealong interfacial planes between muscle bun-dles (because of leakage from the musclebundle or because of misplacement of theinjection mixture) and establish contact withnerve bundles, where serious pathology con-sequent to inflammatory processes can occur(13, 21). This is especially true for small ani-


Table II: Suggested routes of injection with or without adjuvant

Primary injection Booster injection(s)Day 0 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 shouldbe allowed for certain purposes in the rabbit and in large animals, to stimulate the requiredimmune response.bWith i.v. or i.p. booster injections, there is a risk of inducing anaphylactic shock in the ani-mals.

mals such as mice, for which the intramus-cular route should only be used by experi-enced researchers and biotechnicians.Furthermore, local reactions after intramus-cular injections can easily be overlooked (22).

The intraperitoneal injection of adjuvantmixtures is not recommended, since it isknown to induce inflammation (macroscopi-cally evident), peritonitis (with the risk ofascites formation), and behavioural changes(for example, decreased activity and weightloss).

Herbert (23) considered intravenousadministration to be the route of choice forsmall particulate antigens such as viruses,bacteria or cells (where danger of anaphy-laxis is low), because the antigen distributionis broad and capture by lymphoid tissues ishigh. However, due to the risk of embolism,it is inappropriate for oil adjuvants (oil emul-sions), viscous gel adjuvants or large particu-late antigens (for example, bacterialaggregates, heterologous lymphocytes whenraising anti-lymphocyte sera, or other het-erologous whole mammalian cells [23]).

The workshop participants agreed thatalternative injection sites, such as the foot-pad, are not to be recommended for pAb pro-duction. With precious antigens in very smallquantities or with protein bands from elec-trophoretic separating gels (for example, pre-cipitating bands from immunoelectrophoresisexperiments), there could be scientific reasonsfor the use of the intra-lymph node injection

procedure with an ocular grade needle, asdescribed by Goudie et al. (24). Some authorshave recommended intra-splenic injection forsimilar reasons (25). The participants agreedthat investigators should provide scientificjustification to ethical committees for suchprotocols (for example, the need to useextremely valuable or unique and irreplace-able antigens, or extremely small quantities ofantigen).

Determination of volume of injection

To ensure that animals experience minimaldiscomfort at the antigen injection site, theinjection volume should be as small as possi-ble. Agreed maximum volumes per site ofinjection are shown in Table III. These vol-umes are based on the use of an injectionmixture that forms a depot at the site ofinjection, for example, an immunostimula-tory oil emulsion or a viscous gel. If theinoculum contains an immuno-potentiator,for example, mycobacteria or a muramyldipeptide derivative, the amount of antigenin the injection mixture should generally notexceed 25µg for a mouse or 200µg for guinea-pigs, rats or rabbits. The inoculum should bespread among multiple injection sites inlarger animals.

If the intraperitoneal route of injection isrequired for adjuvants such as oil emulsionsand viscous gels, the maximum volumeinjected should not exceed 0.2ml. If the intra-dermal route is required (use should be limited


Table III: Maximum volumes for injection of antigen/depot-forming adjuvantmixtures per site of injection for different animal species

Maximum volume SubsequentSpecies per site Primary injection injections

Mice, hamsters 100µl s.c. s.c.Mice, hamsters 50µl i.m.a i.m.Guinea-pigs, rats 200µl s.c., i.m. s.c., i.m.Rabbits 250µl s.c., i.m. s.c., i.m.Sheep, goats, 500µl (if in multiple s.c., i.m. s.c., i.m.

donkeys, pigs sites 250µl/site)Chickens 500µl s.c., i.m. s.c., i.m.

s.c. = subcutaneous, i.m. = intramuscular.aOne hind limb.

to rabbits and large animals), the maximumdose of adjuvant/antigen mixture would be25µl per site at no more than four sites.

Maximum volumes that are allowed forinjections of antigen solutions without adju-vant have been described by Iwarsson et al.(26) and van Zutphen et al. (27) and aregiven in Table IV.

Design of boosting protocol/immunisationschedule

The boosting protocol can have a decisiveinfluence on the result of the immunisation.The time between two immunisation stepscan influence both the induction of B mem-ory cells and the class switch of B cells (fromIgM to other antibody classes and sub-

classes). Specific recommendations for theinterval between primary and booster immu-nisations are usually not cited. In general, abooster can be considered after the antibodytitre has plateaued or begun to decline. If thefirst immunisation is performed without adepot-forming adjuvant, the antibody titrewill usually peak 2�3 weeks after immunisa-tion. When a depot-forming adjuvant is used,a booster injection can follow at least 4 weeksafter the first immunisation. For boosterimmunisations, an adjuvant is not alwaysnecessary.

The participants agreed that, in mostcases, the endpoint of pAb production shouldbe judged when the antibody titre hasreached an acceptable level. This should usu-ally occur after a maximum of two boosters.


Table IV: Maximum volume of injection and needle size used for injection ofantigen without adjuvant per route of injection for different animalspecies

s.c. i.m. i.d. i.p. i.v.

Species Vol.a Needleb Vol. Needle Vol. Needle Vol. Needle Vol. Needle

Mice 0.5 ≤ 25G 0.05 25�27G 0.05 26G 1 ≤ 23G 0.2 ≤ 25G

Hamsters 1 25G 0.1 ≤ 25G 26G 2�3 ≤ 23G 0.3 ≤ 25G

Guinea-pigs 1 ≤ 20G 0.1 25G 0.05 26G 10 23G 0.5 ≤ 27G

Rats 1 23�25G 0.1 25G 0.05 26G 5 23G 0.5 25�27G

Rabbits 1.5 20G 0.5 23G 0.05 26G 20 21G 1�5 20�21G

Sheep or goats 5 20G 0.05 26G 10 21G 30

Pigs (> 50kg) 3 19�21G 2 20G 0.1 26G 250 20G 20 21�23G

Chickens 4 23G 0.5 21�23G 26G 10 21G 0.5c 23G

s.c. = subcutaneous, i.m. = intramuscular, i.p. = intraperitoneal, i.d. = intradermal, i.v. =intravenous.aMaximum injection volume in ml.bNeedle size in G (gauge: 27G = 0.40mm, 26G = 0.45mm, 25G = 0.50mm, 24G = 0.55mm,23G = 0.60mm, 22G = 0.70, 21G = 0.80mm, 20G = 0.90mm, 19G = 1.00mm).cMI/kg body weight.

Data from references 26 and 27.

If the antibody response is still insufficientfor laboratory purposes at this time, theexperiment should, in general, be termi-nated. In the case of antigens of interest witha low molecular weight, such as peptides orsteroids, injections might have to be repeatedseveral times before the antiserum reachesthe titre and specificity required for theapplication. However, long immunisationschemes, with repeated boosting, not onlyresult in the production of antibodies withincreased affinity for the antigen of interest,but also in the production of more antibodiesspecific for contaminants in the antigenpreparation. Such multispecific antiserarequire absorption before they are monospe-cific, a process with some inherent difficul-ties.

Animals can be rested for long intervalsbetween boosting. Even when serum anti-body titres have dropped to relatively lowlevels, a booster injection into an animal thathas previously established a memoryresponse will usually re-establish a highserum antibody titre (13). Intermittentbleeding of a hyper-immunised animalappears to help maintain a high serum anti-body level. Thus, regular interval blood col-lections after a sufficient serum antibodytitre has been reached could facilitate thecollection of adequate amounts of pAbs to anantigen that is in very limited supply. How-ever, animals must not be kept in antibodyproduction programmes unnecessarily (asalready discussed).

Primary injections with very low amountsof antigen (picograms) are not recom-mended, since this does not stimulate theimmunologic memory sufficiently, and mightinduce tolerance to the antigen. Frequentimmunisations with relatively low amountsof antigen can be counterproductive. In addi-tion, animal welfare argues against suchschedules. However, low amounts of antigenfor booster immunisation may help raise theaverage affinity of the antibodies subse-quently produced (28).

In general, booster injection sites shouldbe distant from previous injection sites.Booster antigen mixtures should never beinoculated into granulomas or swellingsinduced by earlier immunisations. Boosterimmunisations do not need to be adminis-tered by the same route used for the primaryimmunisation (29). Adjuvants that containmycobacteria or their components (for exam-

ple, cell walls) should only be used once peranimal, because severe hypersensitivity reac-tions may result following re-exposure of thehost to mycobacteria (30, 31). Booster immu-nisations applied intravenously or intraperi-toneally with aqueous soluble antigensmight result in systemic anaphylaxis, causedby the rapid release of histamine and othermediators from basophils and perivascularmast cells.

Blood Collection

Blood samples should be taken with mini-mum stress for the animal. Animals to beimmunised should be conditioned to be con-fident with the animal care staff. This is notonly important for animal well-being, butalso ensures that the animals do not exhibitstress-mediated vasoconstriction, whichwould make blood sampling difficult.

Blood collection should be performedunder conditions where it is possible to keepthe animals warm, a prerequisite for ensur-ing good blood supply to the periphery. It isalso important that the animals are not sub-jected to sudden noises or other environmen-tal stress-inducers during blood collection,or, for that matter, during their routinehousing.

The application of organic solvents toinduce vasodilation is not recommended,because of the toxic and carcinogenic poten-tial of the solvents for laboratory animalsand humans.

When blood is collected for antibody pro-duction, it can be advantageous to prepareplasma (use of an anticoagulant such asheparin, citrate, or EDTA) instead of serumsince the fluid yield can be increased signifi-cantly.

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

When possible, a collection method notrequiring anaesthesia should be preferred.This may favour the choice of a species inwhich blood sampling in conscious animals iseasy for the operator and unstressful for theanimal. Small ruminants and rabbits arethus advantageous compared to smallrodents.

If an animal is not stressed during bleed-ing, the use of a sedative to facilitate blood


sampling is usually unnecessary. However,in large-scale production systems, operatorsmay find it advantageous to use sedatives tomaintain low stress levels and to achieverapid blood collection.

The needle used should be matched to thevessel size and should preferably be of rela-tively large bore to facilitate rapid collection.Vacutainers may be used, provided that thevein does not collapse. The bleeding of rabbitsthrough an ear vein should be performed witha needle rather than with a scalpel, because ofthe difficulties inherent in stopping bleedingof a cut vessel. Butterfly needles are recom-mended, as they allow the animal to move itshead during the procedure.

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

International guidelines differ with respectto frequency of blood collection (1�4 weeks),irrespective of the bleeding interval. The max-imum volume allowed should not be removedmore frequently than once a fortnight.

Exsanguination should be performedunder general anaesthesia and is best carriedout by heart puncture. After exsanguinationis completed, small rodents should be sub-jected to cervical dislocation and larger ani-mals should be euthanised by an overdose ofan appropriate anaesthetic agent.

Assessment of Side-effects

In order for investigators to minimise painand distress in immunisation procedures,the side-effects induced by immunisationhave to be assessed. Some assessment proce-dures for quantifying discomfort in animalshave been developed; these include the pro-tocol proposed by Morton & Griffiths (34),and systems which measure changes in activ-ity and behavioural patterns for a givenperiod (35�37).

Animals should be checked daily and, inaddition to the routine check-up whichincludes observance of the animal�s generalappearance and food and water intake, the


Table V: Vessels appropriate for blood collection

Animal

Ham- Guinea- Dog, Goat,Vessel Mouse Rat sters pig Rabbit cat sheep Horse Chicken

Vena jugularis E E B B & E B BAorta E E E E EVena saphena B B B B BVena cephalica B

antebrachiiVena cava E E E E E

Tail vein/artery B BLateral ear vein BCentral ear artery BHeart puncture E E E B & E E B & EPeriorbital vein B & E B & E B & E

plexusa

B = blood collection, E = exsanguination.aSampling from the periorbital vein plexus is aesthetically questionable, requires both skill andgeneral anaesthesia, and is not permitted in some countries.

Data from reference 32.

site of injection should be inspected. How-ever, it should be noted that checking thefood and water intake of individual animalsmay not be possible when animals are group-housed. When comparative studies or newexperiments are performed (for example,with new combinations of antigen and adju-vant), the assessment of side-effects shouldalso include studies of pathological changesat the end of the experiment; for this pur-pose, animals should be necropsied and mul-tiple tissues should be examined.Pathological changes may not always be evi-dent during clinical observation, dependingon the route of injection (for example, theinjection site cannot be examined afterintraperitoneal injection). Pathologicalchanges might not be confined to the site ofinjection.

Blood collection can also cause side-effects,especially when the animals are anaes-thetised. Although blood samples shouldpreferably be taken from unanaesthetisedanimals, in some cases anaesthesia might beneeded (for example, for exsanguination,bleeding from the orbital sinus and heartpuncture). When HypnormTM is used as ananaesthetic in blood sampling, the animalsshould be checked afterwards, since bleedingmay continue. After a blood sampling proce-dure, the puncture site should be monitoredfor closing and healing of the wound. Specificattention is necessary when the ear artery ofa rabbit is used. The artery must be com-pressed for a sufficiently long period of time(sometimes up to 5 minutes) for reliable clo-sure and to avoid leakage.

Advanced Techniques and Alternatives

The traditional ways of adjuvanting proteinhave sometimes failed to result in polyclonalantiserum, for example, against small pep-tides and carbohydrate antigens. Therefore,it has been necessary to develop alternativeapproaches for antigen preparation. The con-jugation technology and the multiple antigenpeptides (MAP) procedure are describedbelow. By coupling synthetic peptides or car-bohydrates to protein carriers, it is possibleto promote the T-cell helper function thatsupports a vigorous antibody response. Ingeneral, proteins (for example, keyholelimpet haemocyanin and bovine serum albu-min) which are highly immunogenic them-

selves, are used as carriers. Therefore, anantibody response is also elicited against thecarrier molecule (38), and the antiserum willneed to be absorbed to render it monospe-cific. The MAP procedure overcomes some ofthe shortcomings of the classical conjugationapproach described above for eliciting anantibody response against peptides. In placeof a large protein carrier (which in itselfwould be immunogenic), a relatively non-immunogenic core matrix, consisting of tri-functional amino acids (such as lysine), isused as the �carrier�. Lysine, with its extraamino group, can be used to form a lysine�tree� (core matrix) to which a number ofpeptides can be attached (39). The entireconstruct, including the peptides, can be gen-erated with a peptide synthesiser. The num-ber of peptides to be incorporated will beproportional to the number of lysine residuesin the core matrix. The molecular weight ofthe resulting MAP will also reflect this.Accordingly, the density of B-cell epitopes issignificantly higher with the MAP approachthan with traditional protein carriers. TheMAP approach has been successfully usedwith aluminum adjuvant (Alhydrogel), aswell as with oil emulsion adjuvants (40) toraise antibodies to peptides. However, for theMAP construct to be immunogenic, it mustcontain both T-cell receptor epitopes and B-cell epitopes within the peptide of interest,because the lysine matrix itself does not pro-vide the T-cell receptor epitopes necessaryfor eliciting the all-important T-cell help. Avariant of the MAP procedure has beendesigned to allow synthesis of a multipleantigen construct which contains two differ-ent peptides, one of which is the antigen ofinterest for eliciting antibodies, while theother is a peptide with a strong T-cell recep-tor epitope (41).

Recently, Glenn et al. (42) demonstratedthat it is possible to induce a systemichumoral immune response by transcuta-neous immunisation. Cholera toxin was usedas adjuvant, and bovine serum albumin,tetanus toxoid and diphtheria toxoid wereused as antigens. No redness, swelling orother signs of inflammation were seen. Tak-ing into account the large area of the skin, itsaccessibility, and the presence therein ofpotent immune cells (Langerhans cells), onecould presume that this route could be apractical alternative to the most commoninjection routes, at least in some species. The


immunising solution could be applied for acertain period with plaster or with patchessimilar to those developed for transcuta-neous drug delivery. However, with regard tothe technique, further research should beundertaken involving other types of antigenand other adjuvants, and on the developmentof memory, species variations, and classes ofantibodies elicited.

Oral immunisation aimed at inducing asimultaneous peripheral and mucosalimmune response may be an attractive alter-native to subcutaneous or intramuscularimmunisation for animal welfare reasons.Oral immunisation has been conducted suc-cessfully in studies of new vaccine deliverysystems in which antigens have been admin-istered orally in biodegradable biospheres orassociated with carrier proteins derived fromcholera toxin or Escherichia coli heat-labileprotein.

The development of novel DNA-basedtechnologies, such as phage display libraries,direct cloning of antibody genes into plas-mids, and molecular engineering, has intro-duced some very interesting possibilities forthe production of antibodies which combinea high specificity with certain functionalcharacteristics that are important for theirfurther use (43�45). Although these possibil-ities are primarily seen as an alternative tothe in vivo and in vitro methods currentlyused for the production of mAbs, under cer-tain conditions these techniques could alsobe used as an alternative for the productionof pAbs. At present, only a small number oflaboratories have phage display libraries andthe expertise needed to make use of them.However, it is evident that the availabilityand accessibility of these phage displaylibraries are growing.

Existing Guidelines on the Productionof Polyclonal Antibodies

In the last decade, many sets of guidelines onthe production of pAbs have been establishedby various national control authorities,organisations, and institutions. The increas-ing number of guidelines demonstrates theincreased awareness of undesirable side-effects caused by some adjuvants and injec-tion routes, but also that efforts should bemade to harmonise these guidelines. Itshould be borne in mind that many of the

immunisation protocols used for pAb produc-tion were (or are still) based on habit andtradition, rather than on scientific princi-ples. The common aims of the establishedguidelines are to ensure appropriate proce-dures for the production of pAbs which givesatisfactory results, and minimise discom-fort, distress and pain in the animalsinvolved. The guidelines are considered to bea tool for scientists, animal technicians, ani-mal welfare officers and ethical review com-mittees.

In 1988, the US National Institutes ofHealth issued their intramural recommenda-tions for the research use of FCA (46). Thiswas followed by Canadian Council on AnimalCare guidelines on acceptable immunologicalprocedures (30). These two sets of guidelineshave had a major impact on the establish-ment of guidelines or codes of practice inother countries.

Several European countries have issuednational guidelines on the production ofpAbs, i.e. Switzerland (15), Denmark (16),The Netherlands (31), the UK (47) and Swe-den (48). Variations in the legal status of theguidelines are evident between these coun-tries. The Dutch and Swedish guidelines arenot mandatory in the strict sense of theword; however, they are followed by the eth-ical review committees, and, in the case ofThe Netherlands, by the Veterinary Inspec-torate. Thus, Dutch scientists have to justifyexplicitly when their immunisation protocoldeviates from the Code of Practice for theImmunisation of Laboratory Animals (31).In the UK, immunisation protocols arereviewed and authorised by the Home OfficeInspectorate, and experts in the productionof pAbs are consulted when necessary. Thesituation in Denmark is comparable. InSwitzerland, scientists are obliged to set uptheir immunisation protocols in accordancewith the guidelines. Modification of the pro-tocols can be required by the Swiss Compe-tent Authority, the Bundesamt fürVeterinärwesen.

Guidelines on the production of pAbs havebeen published by various organisations, forexample the Scientists Centre for AnimalWelfare (49), the Australian and NewZealand Council for the Care of Animals inResearch and Teaching (50), theTierärztliche Vereinigung für Tierschutz(22) and the Arbeitsgemeinschaft der Tier-schutzbeauftragten in Baden-Württemberg


(51) and are included in several handbookson immunological procedures (23, 38, 52,53). In the USA in particular, but also inEurope, Australia and New Zealand,research institutes, and especially universi-ties, have established their own guidelines,many of which are available on the Internet.Most of these institutional guidelines are notmandatory. In the USA, the InstitutionalAnimal Care and Use Committees (IACUCs)provide guidelines that are not intended todictate procedures, but are intended toassure proper treatment of animals that areused for pAb production. The IACUCs canrequest that investigators modify immunisa-tion protocols and allow deviations fromtheir guidelines, provided that these are forscientific reasons. This has clearly broughtabout some changes, which have improvedattention to the welfare of the animals con-cerned. However, there is still some dis-agreement about the best immunisationprotocols for the production of pAbs.

The recommendations given in some of theguidelines mentioned above were comparedfor the purposes of this report. Several guide-lines include a lot of background informationand a list of references. All of them cover, todifferent extents, the following importantaspects of pAb production: choice of species,antigen preparation, injection route, injec-tion volume, choice and use of adjuvants,injection technique, immunisation sched-ules, blood sampling and post-injectionobservation (Table VI). All put specialemphasis on the limitations involved in theuse of FCA, and similar recommendationsare evident with respect to the permittedmaximum volume of an FCA antigen prepa-ration (Table VII). Some of the guidelinesstate explicitly which injection routes shouldbe preferred, discouraged or not allowed(Tables VI and VII). There is a general con-sensus that footpad injection is not necessaryfor pAb production.

There is no doubt that these guidelineshave had a positive impact on pAb produc-tion and have increased the attention givento animal welfare. In The Netherlands, theeffect of the guidelines was evaluated in 1996(54). It became evident that the guidelineshad initiated discussions within institutes,which led in turn to the modification ofimmunisation protocols. However, this eval-uation (and also a symposium) showed thatthe guidelines have to be amended in some

specific areas. In Sweden, the set of guide-lines have proved to be a valuable and prac-ticable tool for both ethical committees andscientists.

Concluding Remarks

Current immunisation procedures for theproduction of pAbs are often based on habitand tradition. To ensure that appropriateprocedures are used, many sets of guidelineshave been established. The harmonisation ofthese guidelines is needed. In the workshop,various aspects of immunisation protocolsand existing guidelines were discussed. InAppendix 1, draft guidelines for the produc-tion of pAbs are given, based on all availableinformation. These guidelines should beregarded as a tool for use by scientists andethical committees to improve immunisationprocedures in order to minimise pain anddistress to the animals involved. The guide-lines should initiate discussions, whichshould lead to the further modification ofimmunisation protocols.

References

1. ECVAM (1994). ECVAM News & Views. ATLA22, 7�11.

2. Köhler, G. & Milstein, C. (1975). Continuous cul-tures of fused cells secreting antibody of prede-fined specificity. Nature, London 256, 495�497.

3. National Research Council. (1991). Committee onInfectious Diseases of Mice and Rats. 397pp.Washington, DC, USA: National Academy Press.

4. Cox, J.C. (1977). Altered immune responsivenessassociated with Encephalitozoon cuniculi infec-tion in rabbits. Infection and Immunity 15,392�395.

5. Kraft, V., Deeny, A.A., Blanchet, H.M., Boot, R.,Hansen, A.K., Hem, A., van Herck, H., Kunstýr,I., Milite, G., Needham, J.R., Nicklas, W., Perrot,A., Rehbinder, C., Richard, Y. & de Vroey, G.(1994). Recommendations for the health moni-toring of mouse, rat, hamster, guinea-pig andrabbit breeding colonies. Report of the Federa-tion of European Laboratory Animal ScienceAccociations (FELASA) Working Group on Ani-mal Health. Laboratory Animals 28, 1�12.

6. Rehbinder, C., Baneux, P., Forbes, D., vanHerck, H., Nicklas, W., Rugaya, Z. & Winkler, G.(1996). FELASA recommendations for the healthmonitoring of mouse, rat, hamster, gerbil,guinea-pig and rabbit experimental units. Reportof the Federation of European Laboratory Ani-mal Science Associations (FELASA) WorkingGroup on Animal Health. Laboratory Animals30, 193�208.

7. Knight, P.A. & Lucken, R.N. (1979). The effectsof laboratory animal diets on the potency tests of


Tab

le V

I: A

spec

ts t

hat

are

in

clu

ded

in

a n

um

ber

of

exis

tin

g gu

idel

ines

Pre

ferr

edO

bse

rvat

ion

Bac

kgr

oun

dU

se o

f ot

her

Dif

fere

nt

Pre

ferr

edIm

mu

nis

atio

nin

terv

alaf

ter

info

rmat

ion

an

dG

uid

elin

eU

se o

f F

CA

adju

van

tsro

ute

sro

ute

sp

roto

cols

pri

me-

boo

stin

ject

ion

refe

ren

ces

AN

ZCC

AR

T+

++

++

+A

TB

W+

++

s.c.

≥4

wee

ksB

VE

T+

++

s.c.

+≥

4 w

eeks

++

CC

AC

++

+H

O+

+s.

c.+

NIH

++

TV

T+

++

+?

+≥

4 w

eeks

++

VP

HI

++

+s.

c.+

≥ 4

wee

ks+

+D

anis

h+

++

s.c.

or

++

+gu

idel

ines

ade

ep i.

m.

s.c.

= s

ubc

uta

neo

us,

i.m

. = i

ntr

amu

scu

lar.

AN

ZC

CA

RT

= A

ust

rali

an a

nd

New

Zea

lan

d C

oun

cil

for

the

Car

e of

An

imal

s in

Res

earc

h a

nd

Tea

chin

g (A

ust

rali

a/N

ew Z

eala

nd;

50)

,A

TB

W =

Arb

eits

gem

ein

sch

aft

der

Tie

rsch

utz

beau

ftra

gten

in

Bad

en-W

ürt

tem

berg

(G

erm

any;

51)

, BV

ET

= B

un

desa

mt

für

Vet

erin

ärw

esen

(Sw

itze

rlan

d; 1

5),

CC

AC

= C

anad

ian

Cou

nci

l on

An

imal

Car

e (C

anad

a; 3

0),

HO

= H

ome

Off

ice

(UK

; 47

), N

IH =

Nat

ion

al I

nst

itu

tes

ofH

ealt

h (

US

A;

46),

TV

T =

Tie

rärz

tlic

he

Ver

ein

igu

ng

für

Tie

rsch

utz

(G

erm

any;

22)

, V

PH

I =

Vet

erin

ary

Pu

blic

Hea

lth

In

spec

tora

te (

Th

eN

eth

erla

nds

; 31)

.a D

ata

from

ref

eren

ce 1

6.

Tab

le V

II:

Max

imal

vol

um

e (m

l) o

f F

reu

nd

�s c

omp

lete

ad

juva

nt

con

tain

ing

inoc

ulu

m p

er i

nje

ctio

n s

itea

s.c.

i.m

.i.

p.

i.d

.f.

p.

Gu

idel

ine

Rab

bit

Rat

Mou

seR

abb

itR

atM

ouse

Rab

bit

Rat

Mou

seR

abb

itR

atM

ouse

Rab

bit

Rat

Mou

se

AN

ZCC

AR

T0.

250.

250.

20.

10.

50.

10.

10.

05nr

nrnp

0.05

0.05

AT

BW

0.1

0.1

0.1

0.02

0.05

npnp

npu

npu

BV

ET

0.1

0.1

0.1

0.2

0.2

npnp

unp

uC

CA

C0.

250.

10.

10.

5nr

nrnp

0.1

0.1

0.05

nrnp

npnp

unp

uH

O0.

250.

20.

1N

IH0.

10.

10.

10.

50.

05nr

npu

npu

TV

T0.

10.

10.

10.

5nr

nr0.

20.

20.

05np

unp

unp

uV

PH

I0.

10.

10.

10.

50.

20.

20.

05np

npnp

unp

u

AN

ZC

CA

RT

= A

ust

rali

an a

nd

New

Zea

lan

d C

oun

cil

for

the

Car

e of

An

imal

s in

Res

earc

h a

nd

Tea

chin

g (A

ust

rali

a/N

ew Z

eala

nd;

50)

,A

TB

W =

Arb

eits

gem

ein

sch

aft

der

Tie

rsch

utz

beau

ftra

gten

in

Bad

en-W

ürt

tem

berg

(G

erm

any;

51)

, BV

ET

= B

un

desa

mt

für

Vet

erin

ärw

esen

(Sw

itze

rlan

d; 1

5),

CC

AC

= C

anad

ian

Cou

nci

l on

An

imal

Car

e (C

anad

a; 3

0),

HO

= H

ome

Off

ice

(UK

; 47

), N

IH =

Nat

ion

al I

nst

itu

tes

ofH

ealt

h (

US

A;

46),

TV

T =

Tie

rärz

tlic

he

Ver

ein

igu

ng

für

Tie

rsch

utz

(G

erm

any;

22)

, V

PH

I =

Vet

erin

ary

Pu

blic

Hea

lth

In

spec

tora

te (

Th

eN

eth

erla

nds

; 31)

.

s.c.

= s

ubc

uta

neo

us,

i.m

. = i

ntr

amu

scu

lar,

i.p

. = i

ntr

aper

iton

eal,

i.d.

= i

ntr

ader

mal

, f.p

. = f

ootp

ad.

nr

= n

ot r

ecom

men

ded,

np

= n

ot p

erm

itte

d, n

pu =

not

per

mit

ted

un

less

.a M

axim

um

vol

um

es o

f in

ocu

lum

wit

hou

t an

adj

uva

nt

can

be

fou

nd

in s

ever

al h

andb

ooks

(23

, 38,

52,

53)

.

bacterial vaccines. Developments in BiologicalStandardization 45, 143�149.

8. Strobel, S. & Mowat, A.M. (1998). Immuneresponses to dietary antigens: oral tolerance.Immunology Today 19, 173�181.

9. Zalcman, S., Minkiewicz-Janda, A., Richter, M.& Anisman, H. (1988). Critical periods associatedwith stressor effects on antibody titers and onthe plaque-forming cell response to sheep redblood cells. Brain Behaviour in Immunology 2,254�266.

10. Wilson, M.S., Berge, W., Maess, J., Mahouy, G.,Natoff, I., Nevalainen, T., van Zutphen, L.F.M. &Zaninelli, P. (1995). FELASA recommendationson the education and training of persons workingwith laboratory animals: categories A and C.Report of the Federation of European Labora-tory Animal Science Associations (FELASA)Working Group on Education. Laboratory Ani-mals 29, 121�131.

11. Council of Europe (1986). European Conventionfor the Protection of Vertebrate Animals Used forthe Experimental and Other Scientific Purposes,51pp. Strasbourg, France: Council of Europe.

12. EEC (1986). Council Directive 86/609/EEC of 24November 1986 on the approximation of laws,regulations and administrative provisions of theMember States regarding the protection of ani-mals used for experimental and other scientificpurposes. Official Journal of the European Com-munities L358, 1�29.

13. Hanly, W.C., Artwohl, J.E. & Bennett, B.T.(1995). Review of polyclonal antibody productionprocedures in mammals and poultry. ILAR Jour-nal 37, 93�118.

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35. Barclay, R.J., Herbert, W.J. & Poole, T.B. (1988).The Disturbance Index: A Behavioural Method ofAssessing the Severity of Common LaboratoryProcedures on Rodents. Animal Welfare ResearchReport No 2, 36pp. Potters Bar, Herts., UK:UFAW.


36. Jansen van �t Land, C. & Hendriksen, C.F.M.(1995). Change in locomotor activity pattern inmice: a model for recognition of distress? Labo-ratory Animals 29, 286�293.

37. Bulthuis, R.J.A., Bergman, A.F., Nijessen, S.,Schlingmann, F., Tolboom, J., Remie, R., Van deWeerd, H.A., Van Loo, P.L.P., Baumans, V. &van Zutphen, L.F.M. (1997). Automated behav-iour classification: the LABORAS project. InHarmonization of Laboratory Animal Husbandry(ed. P.N. O�Donoghue), pp. 17�18. London, UK:Royal Society of Medicine Press.

38. Harlow, E. & Lane, D. (1988). Adjuvants. InAntibodies: A Laboratory Manual, pp. 96�124.New York, USA: Cold Spring Harbor Laboratory.

39. Tam, J.P. (1988). Synthetic peptide vaccinedesign: synthesis and properties of a high-den-sity multiple antigenic peptide system. Proceed-ings of the National Academy of Sciences USA85, 5409�5413.

40. Chai, S.K., Clavijo, P., Tam, J.P. & Zavala, F.(1992). Immunogenic properties of multiple anti-gen peptide systems containing defined T and Bepitopes. Journal of Immunology 149,2385�2390.

41. Reed, R.C., Louis-Wileman, V., Cosmai, E.V.,Fang, S., Jue, D.L., Wohlhueter, R.M., Hunter,R.L. & Lal, A.A. (1997). Multiple antigen con-structs (MACs): induction of sterile immunityagainst sporozoite stage of rodent malaria para-sites, Plasmodium berghei and Plasmodiumyoelii. Vaccine 15, 482�488.

42. Glenn, G.M., Rao, M., Matyas, G.R. & Ray, C.R.(1998). Skin immunisation made possible bycholera toxin. Nature, London 391, 851.

43. Winter, G., Griffiths, A.D., Hawkins, R.E. &Hoogenboom, H.R. (1994). Making antibodies byphage display technology. Annual Review ofImmunology 12, 433�455.

44. de Kruif, J., van der Vuurst de Vries, A.R.,Cilenti, L., Boel, E., van Ewijk, W. & Logtenberg,T. (1996). New perspectives on recombinanthuman antibodies. Immunology Today 17,453�455.

45. Vaughan, T.J., Williams, A.J., Pritchard, K.,Osbourn, J.K., Pope, A.R., Earnshaw, J.C.,McCafferty, J., Hodits, R.A., Wilton, J. & John-son, K.S. (1996). Human antibodies with sub-nanomolar affinities isolated from a large

non-immunised phage display library. NatureBiotechnology 14, 309�314.

46. Grumstrupp-Scott, J. & Greenhouse, D.D.(1988). NIH intramural recommendations forthe research use of complete Freund�s adjuvant.ILAR News 30, 9.

47. Home Office Inspectorate. (1991). Antibody pro-duction: advice on protocols for minimal sever-ity. In Report of the Animal ProceduresCommittee for 1991, p. 26. London, UK: HMSO.

48. Swedish National Board for Laboratory Animals.(1994). General recommendations of theNational Board for Laboratory Animals on thetreatment of certain matters relating to ethicalreviews of the use of animals for scientific pur-poses (LSFS/Statute book of the National Boardof Agriculture/1990: 22 & 23, Subject No. L29).In Provisions and General RecommendationsRelating to the Use of Animals for Scientific Pur-poses (ed. O. Lundgren), pp. 46�49. Stockholm,Sweden: Karl Olov Öster

49. SCAW (1989). Scientists Center for Animal Wel-fare: guidelines on immunological procedures.SCAW Newsletter 11, 2�6.

50. ANZCCART (1995). The Use of Immuno-adju-vants in Animals in Australia and New Zealand,27pp. Glen Osmond, Australia: ANZCCART.

51. ATBW. (1998). Empfehlungen über das Immu-nisieren von Versuchstieren, 1p. Heidelberg,Germany: Arbeitsgemeinschaft der Tier-schutzbeauftragten in Baden-Württemberg.

52. Herbert, W.J. & Kristensen, F. (1986). Labora-tory animal techniques for immunology. InHandbook of Experimental Immunology, Vol. 1,Immunology � Laboratory Manuals (ed. D.M.Weir), pp. 133.1�133.13. Oxford, UK: Blackwell.

53. Baumans, V., ten Berg, R.G.M., Bertens,A.P.M.G., Hackbarth, H.J. & Timmerman, A.(1993). Experimental techniques. In Principlesof Laboratory Animal Science (ed. L.F.M. vanZutphen, V. Baumans & A.C. Beynen), pp.299�318. Amsterdam, The Netherlands: Else-vier.

54. de Leeuw, W.A. & De Greeve, P. (1997). Produc-tion of polyclonal and monoclonal antibodies inThe Netherlands. In Animal Alternatives, Wel-fare and Ethics (ed. L.F.M. van Zutphen & M.Balls), pp. 1053�1059. Amsterdam, The Nether-lands: Elsevier.


General Considerations

Training

Qualified technical staff (Category B of theFELASA recommendations) should per-form immunisation procedures in labora-tory animals. The technical staff shouldhave a basic understanding of immunologi-cal principles and be experienced in thehandling, injecting, anaesthetising andbleeding of the animal species used for anti-body production. In particular, aspects ofasepsis and anaesthesia are very important.Furthermore, the technical staff should becapable of recognising signs of pain and dis-tress in the animals, and must be responsi-ble for taking action when necessary. Theinvestigator should have biomedical train-ing and should be qualified in laboratoryanimal science according to the FELASArecommendations (Category C).

Hygiene

The general principles of asepsis should beapplied to immunisation protocols at allstages with regard to: the preparation of theantigen/adjuvant mixture; the administra-tion of the mixture to laboratory animals;and the bleeding of the animals.

Housing of animals

Animals should be kept under conditionsthat allow important natural behaviour.Immunisation protocols are generally formedium-term to long-term experiments.Therefore, depending on the animal speciesused, group housing of animals is prefer-able.

Laboratory animals

The number of animals used, and thus thevolume of antiserum produced, should be inaccordance with the expected need for thepolyclonal antibody (pAb). Priority should begiven to a reduction of distress in the ani-mals, rather than to a limitation of the num-ber of animals.

Selection of animal species

The species of animals to be selected shouldbe related to the amount of serum needed,the application of the antibodies to be pro-duced, and the characteristics of the antigenconcerned.

Microbiological status

In the case of small laboratory animals, themicrobiological status should preferably bespecific pathogen-free.

Immunisation protocol

Only the least harmful materials and theleast severe protocols necessary should beused. To minimise the potential for induc-tion of side-effects and to maximise thepotential for successful pAb production, theinvestigator should check the quality of theantigen and of the antigen/adjuvant mixtureprior to its injection.

Use of adjuvant

The first question is whether an adjuvant isneeded at all. Selection of adjuvant should begiven very high priority, since it affects thewelfare of the animals to be immunised, as wellas the type and level of immune response. Theselection of, and need for, an adjuvant shouldbe determined on the basis of the nature of theantigen. Mineral oil adjuvants combined withbacterial components may induce considerableside-effects. Adjuvants that contain mycobac-teria or their cell wall components should beused only once per animal.

Route of injection

Suggested routes of injection are given inTable I and comments regarding injectionroutes are given below.

Subcutaneous injectionThe subcutaneous route is preferred for theinjection of oil adjuvants or viscous gel adju-vants.


Appendix 1

Draft Guidelines for the Production of Polyclonal Antibodies

Intramuscular injectionThe intramuscular route should not be thefirst choice for injection of oil adjuvants orviscous gel adjuvants in small animals suchas mice and rats.

Intradermal injectionThe intradermal route should only be used inrabbits or large animals. If intradermal injec-tion is carried out at multiple sites, the max-imum volume administered per site shouldbe 25µl and the maximum number of sitesshould be four.

Intraperitoneal injectionThe intraperitoneal route is not recom-mended when oil adjuvants or viscous geladjuvants are used. Its use should be scien-tifically justified to the ethical committee ona case-by-case basis.

Intravenous injectionThe intravenous route is not recommendedwhen oil adjuvants or viscous gel adjuvantsare used, because there is a high risk oflethal complications due to embolism.

Footpad, intra-lymph node or intra-splenal injectionFootpad, intra-lymph node and intra-splenal

injection are not necessary for routine pAbproduction. The use of these routes must bescientifically justified on a case-by-case basis.If a footpad injection is given, only one hindfoot should be used, with a maximum volumeof 50�100µl, and the animals should behoused on soft bedding.

Volume of injection

The volume of injection should be as small aspossible. Maximum dosages of depot-formingadjuvants (for example oil adjuvants, viscousgel adjuvants) per site of injection are shownin Table II.

Immunisation schedule

Number of sitesA single site of injection is preferred and amaximum of four injection sites per immu-nisation per animal should be allowed. Ifmore than one injection site is used, thedistance between each site should be max-imised.

Booster scheduleWhen an adjuvant is used, the time betweenprimary and secondary injection should be atleast 4 weeks. A maximum of two boosterinjections is recommended.


Table I: Suggested routes of injection with or without adjuvant

Primary injection Booster injection(s)Day 0 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 shouldbe allowed for certain purposes in the rabbit and in large animals, to stimulate the requiredimmune response.bWith i.v. or i.p. booster injections, there is a risk of inducing anaphylactic shock in the ani-mals.

Blood collection

Blood should be collected according to therecommendations given by the BVA/FRAME/RSPCA/UFAW Joint WorkingGroup on Refinement (1) or by McGuill &Rowan (2).

When frequent blood sampling is per-formed, the health status of the animalsshould be carefully observed. The collectionof blood should not exceed more than 15% ofthe total blood volume, which in practice,represents an amount up to 1% of total bodyweight. Animals should not be bled more fre-quently than once every fortnight when max-imum blood volumes are collected.

Assessment of side-effects

During the entire experiment immunisedanimals should be checked daily for gen-

eral appearance, as well as for food andwater intake. In addition to this routinecheck-up, the injection site should be mon-itored. When comparative studies are per-formed, the assessment of side-effectsshould include pathological studies at theend of the experiment. Pathological studiesshould include dissection for gross exami-nation of organs and tissues, and histologi-cal examination of specific tissues. Theresults should be used to optimise thedesign of future experiments.

References

1. BVA/FRAME/RSPCA/UFAW (1993). Joint Work-ing Group on Refinement. Removal of blood fromlaboratory mammals and birds. Laboratory Ani-mals 27, 1�22.

2. McGuill, M.W. & Rowan, A.N. (1989). Biologicaleffects of blood loss: implication for sampling vol-umes and techniques. ILAR News 31, 5�20.

Table II: Maximum volumes for injection of antigen/depot-forming adjuvantmixtures per site of injection for different animal species

Maximum volume SubsequentSpecies per site Primary injection injections

Mice, hamsters 100µl s.c. s.c.Mice, hamsters 50µl i.m.a i.m.Guinea-pigs, rats 200µl s.c., i.m. s.c., i.m.Rabbits 250µl s.c., i.m. s.c., i.m.Sheep, goats, 500µl (if in multiple s.c., i.m. s.c., i.m.

donkeys, pigs sites 250µl/site)Chickens 500µl s.c., i.m. s.c., i.m.

s.c. = subcutaneous, i.m. = intramuscular.aOne hind limb.


An overview of adjuvant categories used forroutine polyclonal antibody (pAb) productionis given in Table I.

Immunostimulatory Oil Emulsions

Water-in-oil emulsions, which include Fre-und-type adjuvants, are the adjuvants mostcommonly used to produce pAbs in labora-tory animals. Although most investigatorsand commercial vendors still refer to theFreund-type adjuvants in use today as Fre-und�s complete adjuvant (FCA, i.e. it con-tains mycobacteria) or Freund�s incompleteadjuvant (FIA, i.e. it does not containmycobacteria), it would be desirable to

replace these terms to reflect the differ-ences between the components in the origi-nal formulation and those in the modernformulation, as well as the differences inreactogenicity of the different formulations.Few laboratories would be in a position tomake the original FCA, because it was for-mulated with heat-killed Mycobacteriumtuberculosis of high virulence, a mineral oilof low quality manufactured before 1969,and a surfactant, predominantly mannidemono-oleate, of variable purity and quality.Due to a change in the oil refining proce-dure in the early 1970s, the mineral oil com-ponent of the original FCA is no longeravailable (1). It has been replaced by ahigher quality oil with less-irritant proper-

Table I: Overview of categories of adjuvants that may be used for routinepolyclonal antibody production

Category Examples (references)

Immunostimulatory oil emulsions Freund�s incomplete adjuvant, (for example, water-in-oil, oil-in-water, Montanide®, Specol (18)water-in-oil-in-water)

Mineral salts Al(OH)3, AlPO4

Microbial (like) products LPS, MDP, MPL, TDM (10)

Saponins Quil A (19)

Synthetic products DDA (13)ISCOMsNBP (12)

Adjuvant formulations Oil emulsion + NBP (TiterMaxTM)Oil emulsion + bacterial products

(Freund�s complete adjuvant; RIBITM)Gerbu (11)

LPS = lipopolysaccharide; MDP = muramyl dipeptide; MPL = monophosphoryl lipid A; TDM= trehalose dimycolate; DDA = dimethyldioctadecylammonium bromide; NBP = non-ionicblock polymer; ISCOMs = immune stimulating complexes.

Appendix 2

Overview of Adjuvants


ties. The mannide mono-oleate currently inuse is also of higher quality. Today, only afew adjuvant immunologists retain the useof the old FCA, which is manufactured bythe Statens Serum Institute (Copenhagen,Denmark) as a �gold standard� for compar-ison against a new adjuvant. The workshopparticipants agreed that this formulation isunsuitable as an adjuvant for use in routinepAb production, due to severe side-effects.The product that is quoted as FCA in themore recent literature can be obtainedfrom, for example, Difco Laboratories,Sigma, ICN Biomedicals, and Pierce, andconsists of a refined oil and a high qualitymannide mono-oleate preparation, withheat-killed M. butyricum or M. tuberculosisH37Ra, an avirulent human strain. ThisFCA has less-irritant and less-inflammatoryproperties than the original FCA, but stillinduces considerable side-effects in animals(2, 3).

Currently, there are immunostimulatoryoil emulsions that are acceptable or evensuperior to the original FIA with regard toenhancing antibody responses. Moreover,the purified components in these formula-tions produce fewer and less-severe adversereactions after injection, for example, theMontanide® ISA (Incomplete Seppic Adju-vant) series (Seppic, Paris, France) andNUFCA Guildhay oil (Guildhay, Guildford,Surrey, UK).

Montanide ISA 740 adjuvant is composedof highly purified mannitol octadecenoicesters (Montanide ISA 80) as surfactant, ina mixture of a metabolisable oil and arefined non-metabolisable light mineral oilclassified pharmacologically as an excipient.This mixture can form a stable emulsion(especially under nitrogen storage), in theweight ratio 70:30 Montanide ISA 740:aque-ous phase antigen. When properly formu-lated, the emulsion will remain in a singlephase for at least 2 years. This adjuvantemulsion is easy to inject and is well-toler-ated by the recipient animal. When injectedsubcutaneously into mice or guinea-pigs inaccordance with the European Pharma-copoeia, there are no serious adverseeffects. The Montanide ISA series has beenaccepted for use in all food-producingspecies (4), as a pharmacologically activesubstance generally regarded as safe.

A non-ulcerative oil (NUFCA Guildhay oil)that can be administered by the intramuscu-

lar, subcutaneous or intradermal routes atmultiple sites has been introduced by Guild-hay. The intramuscular site creates a focusof stimulus with fewer adverse reactionsthan the classical FIA on the market today.However, as NUFCA Guildhay oil has onlyrecently been introduced, limited informa-tion on its use is currently available. Mineral Salts

Aluminum adjuvants in the form of alu-minum hydroxide or aluminum phosphatehydrated gels can be injected subcutaneouslyor intramuscularly for priming an immuneresponse in the recipient. These adjuvantsare generally regarded as safe and they havebeen used for human vaccination for morethan 50 years (5). Priming immunisationswith aluminum adjuvants can be followed byboosters with or without adjuvant (6, 7). Thebiological function of these adjuvants isrelated to their ability to adsorb proteinantigens, thereby ensuring that soluble pro-teins will be taken up as particulate antigensby antigen-presenting cells (8). Due to thisadsorption/function relationship, it isstrongly recommended that investigatorsascertain that adsorption of the antigen tothe gel has been successfully accomplishedprior to its injection (9).

Microbial (like) Products

Micro-organisms such as M. butyricum andmicrobial products can exhibit strong adju-vant activity. The innate vertebrate immunesystem has evolved mechanisms for therecognition of, and response to, certainmicrobial products. Although the innateimmune system itself is not highly efficient,some of its response components, once stim-ulated, help energise the specific antibodyresponse. The microbial products involved(primarily cell wall components) usuallyinduce considerable undesirable inflamma-tory side-effects, as well as an adjuvanteffect. Investigators have identified activefractions or subunits of bacterial products,for example, trehalose dimycolate, and havein some cases modified the bacterial prod-ucts, for example, threonyl-muramyl dipep-tide, or monophosphoryl lipid A, to achieve abalance of immunostimulatory propertiesand diminished inflammatory properties (10,11).


Saponins

Saponins are triperpene glycosides which arederived from the bark of the Quillajasaponaria tree and which have detergent andadjuvant properties. Saponin preparationsintended for use as immunological adjuvants(for example, Quil A or QS-21) are purified toreduce the presence of components whichcause adverse local reactions. Food-gradesaponin preparations should not be used forimmunisation schemes. In general, saponinsshould not be injected intraperitoneally orintravenously, but only subcutaneously orintramuscularly, due to their haemolyticactivity.

Synthetic Products

Synthetic adjuvants are a rather heteroge-neous group of products, because their clas-sification has no single chemical, physical, orfunctional basis. This group includes non-ionic block polymers (NBP), dimethyldioc-tadecylammonium bromide (DDA), immunestimulating complexes (ISCOMs), and lipo-somes. NBPs can contain differenthydrophobic and hydrophilic regions, whichinfluence their surfactant and immunopo-tentiating properties (12). The adjuvanteffect of a given NBP also depends on theantigen used in combination with it, and, assuch, different NBPs may be needed for dif-ferent antigens for optimal effects. DDA isnot an optimal adjuvant for antibodyresponses (13), but is rather better for T-cell-mediated cytotoxic responses. DDA has alipophilic character, which might be respon-sible for its capacity to enhance T-cellresponses. It is a representative of the qua-ternary amines (also classified as a cationicdetergent). ISCOMs are small (40nm diame-ter) cage-like structures prepared from QuilA, cholesterol, and phospholipids. The anti-gen to be inserted into ISCOMs must beamphipathic (14). ISCOMs can be recom-mended as an excellent first choice for viralvaccines, based on past successes. In part,this is because ISCOMs can deliver the anti-gen to the cytosolic compartment of antigen-processing/antigen-presenting cells, and thusdirect the immune response to a cytotoxic T-cell response, which is effective against manyviruses. However, ISCOMs may also facili-tate antibody responses. There is generally

some resistance to the use of ISCOMs,because of the perceived, but misconceived,difficulty in their preparation; as an alterna-tive, there are commercially available �hon-eycomb structures�, to which the antigencan be added (15). Liposomes are unilamellaror multilamellar vesicles artificially con-structed from natural products. The bilayermembranes mimic those of cells. The adju-vanticity of liposomes is influenced bycharge, composition, and method of prepara-tion. The antigen can be encapsulated in thewater phase or the lipid phase, or it can becoupled to the surface (see reviews by Buit-ing et al. [16] and Alving [17]).

Adjuvant Formulations

Combining different immunostimulatoryagents can increase the potency of an adju-vant. Oil emulsions are frequently combinedwith other agents (for example NBP in Titer-MaxTM, or bacterial products in FCA andRIBITM adjuvants). Immunostimulatoryagents such as muramyl dipeptide can beincorporated along with antigen into lipo-somes. In fact, many adjuvants have morethan one immunostimulatory substance andmore than one mechanism of action.

References

1. Stewart-Tull, D.E.S. (1995). The Theory andPractical Application of Adjuvants, 380pp. Chich-ester, UK: John Wiley & Sons.

2. Smith, D.E., O�Brien, M.E., Palmer, V.J. & Sad-owski, J.A. (1992). The selection of an adjuvantemulsion for polyclonal antibody productionusing a low molecular weight antigen in rabbits.Laboratory Animal Science 42, 599�601.

3. Leenaars, P.P.A.M., Koedam, M.A., Wester, P.W.,Baumans, V., Claassen, E. & Hendriksen, C.F.M.(1998). Assessment of side-effects induced byinjection of different adjuvant/antigen combina-tions in rabbits and mice. Laboratory Animals 32,387�406.

4. EC (1995). Commission Regulation (EC) No.2796/95 of 4 December 1995 amending Annex IIof Council Regulation (EEC) No. 2377/90 layingdown a Community procedure for the establish-ment of maximum residue limits of veterinarymedicinal products in foodstuffs of animal origin(Text with EEA relevance). Official Journal of theEuropean Communities L290, 1�4.

5. Gupta, R.K., Relyveld, E.H., Lindblad, E.B.,Bizzini, B., Ben-Efraim, S. & Gupta, C.K. (1993).Adjuvants: a balance between toxicity and adju-vanticity. Vaccine 11, 293�306.

6. Collier, L.H., Polakoff, S. & Mortimer, J. (1979).Reactions and antibody responses to reinforcing


doses of adsorbed and plain tetanus vaccines.Lancet 8131, 1364�1367.

7. Bomford, R. (1980). The comparative selectivityof adjuvants for humoral and cell-mediatedimmunity. I. Effect on the antibody response tobovine serum albumin and sheep red blood cells ofFreund�s incomplete and complete adjuvants,alhydrogel, Corynebacterium parvum, Bordetellapertussis, muramyl dipeptide and saponin. Clini-cal and Experimental Immunology 39, 426�434.

8. Mannhalter, J.W., Neychev, H.O., Zlabinger, G.J.,Ahmad, R. & Eibl, M.N. (1985). Modulation of thehuman immune response by the non-toxic andnon-pyrogenic adjuvant aluminium hydroxide:effect on antigen uptake and antigen presenta-tion. Clinical and Experimental Immunology 61,143�151.

9. Lindblad, E.B. (1995). Aluminium adjuvants. InThe Theory and Practical Application of Adju-vants (ed. D.E.S. Stewart-Tull), pp. 21�35. Chich-ester, UK: John Wiley & Sons.

10. Warren, H.S., Vogel, F.R. & Chedid, L.A. (1986).Current status of immunological adjuvants.Annual Review of Immunology 4, 369�418.

11. Grubhofer, N. (1995). An adjuvant formulationbased on N-acetylglucosaminyl-N-acetylmu-ramyl-L-alanyl-D-isoglutamine with dimethyl-dioctadecylammonium chloride and zinc-L-proline complex as synergists. Immunology Let-ters 44, 19�24.

12. Verheul, A.F.M. & Snippe, H. (1992). Non-ionic

block polymer surfactants as immunological adju-vant. Research in Immunology 143, 512�519.

13. Hilgers, L.A.T. & Snippe, H. (1992). DDA as animmunological adjuvant. Research in Immunol-ogy 143, 494�503.

14. Dalsgaard, K., Lovgren, K. & Stewart-Tull, D.E.S.(1995). Immune stimulating complexes with QuilA. In The Theory and Practical Application ofAdjuvants (ed. D.E.S. Stewart-Tull), pp. 129�144.Chichester, UK: John Wiley & Sons.

15. Morein, B., Sundquist, B., Höglund, S., Dals-gaard, K. & Osterhaus, A.D.M.E. (1984). ISCOM,a novel structure for antigenic presentation ofmembrane proteins from enveloped viruses.Nature, London 308, 457�459.

16. Buiting, A.M.J., Van Rooijen, N. & Claassen, E.(1992). Liposomes as antigen carriers and adju-vants in vivo. Research in Immunology 143,541�548.

17. Alving, C.R. (1997). Liposomes as adjuvants forvaccines. In New Generation Vaccines, 2nd edn(ed. M.M. Levine, G.C. Woodrow, J.B. Kaper &G.S. Cobon), pp. 207�213. New York, USA: Mar-cel Dekker.

18. Boersma, W.J.A., Bogaerts, W.J.C., Bianchi,A.T.J. & Claassen, E. (1992). Adjuvant propertiesof stable water-in-oil emulsions: evaluation of theexperience with Specol. Research in Immunology143, 503�512.

19. Campbell, J.B. & Peerbaye, Y.A. (1992). Saponin.Research in Immunology 143, 526�530.


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