Canadian Council on Animal Careguidelines on:
antibody
production

This document, the CCAC guidelines on: antibody production, has been developed by thead hoc subcommittee on immunological procedures of the Canadian Council on Animal Care (CCAC)
Guidelines Committee:
Dr Albert Clark, Queen's University (Chair)
Dr Dean Befus, University of Alberta (Canadian Society for Immunology representative)
Dr Pamela O'Hashi, University of Toronto
Dr Fred Hart, Aventis Pasteur
Dr Michael Schunk, Aventis Pasteur
Dr Andrew Fletch, McMaster University
Dr Gilly Griffin, Canadian Council on Animal CareIn addition, the CCAC is grateful to the many individuals, organizations and associations that provided comments on earlier drafts of this guidelines' document. In particular thanks are extended to: theCanadian Society of Immunology; the Canadian Association for Laboratory Animal Science and theCanadian Association for Laboratory Animal Medicine; Dr Terry Pearson, University of Victoria; Dr
Mavanur Suresh, University of Alberta; Drs Ernest Olfert and Barry Ziola, University of Saskatchewan; andDr Patricia Shewen, University of Guelph.

© Canadian Council on Animal Care, 2002

ISBN: 0-919087-37-X

Canadian Council on Animal Care
315-350 Albert Street
Ottawa ON CANADA
K1R 1B1
http://www.ccac.ca

CCAC guidelines on: antibody production, 2002

TABLE OF CONTENTSA.

PREFACE . . . . . . . . . . . . . . . . . . . . . . . . . . .1

B.

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . .2

C.

POLYCLONAL ANTIBODY PRODUCTION 3
1.
2.
3.
4.
5.
6.
7.

Animal Selection and Care . . . . . . . . . . .3
Immunization Protocol . . . . . . . . . . . . . .6
Standard Operating Procedures . . . . . . .6
Immunogen Preparation . . . . . . . . . . . . .6
Choice of Adjuvant . . . . . . . . . . . . . . . . .7
Route of Injection . . . . . . . . . . . . . . . . . .9
Volume and Number of Injection
Sites . . . . . . . . . . . . . . . . . . . . . . . . . .10
8. Blood Collection . . . . . . . . . . . . . . . . . .12
9. Monitoring of Animals . . . . . . . . . . . . . .13
10. Disposition of the Animals . . . . . . . . . .13

D.

MONOCLONAL ANTIBODYPRODUCTION . . . . . . . . . . . . . . . . . . . .14
1.
2.
3.

Animal Selection and Care . . . . . . . . . .16
Production of Hybridoma Clones . . . . .17
Ascites Production . . . . . . . . . . . . . . . .17
3.1 Priming . . . . . . . . . . . . . . . . . . . . .17
3.2 Contamination . . . . . . . . . . . . . . .18
3.3 Hybridoma implantation . . . . . . . .18
3.4 Monitoring the animals andendpoints . . . . . . . . . . . . . . . . . .19
3.5 Ascites tumor growth . . . . . . . . . .19
3.6 Ascites fluid collection . . . . . . . . .20

E.

REFERENCES . . . . . . . . . . . . . . . . . . . . . . .20
1.
2.
3.

F.

General . . . . . . . . . . . . . . . . . . . . . . . . .20
Polyclonal Antibody Production . . . . . .21
Monoclonal Antibody Production . . . . .25
3.1 Other useful references . . . . . . . . .27
3.2 Additional useful information . . . . .27

GLOSSARY . . . . . . . . . . . . . . . . . . . . . . . . .27

APPENDIX ACOMMON ADJUVANTS . . . . . . . . . . . . . . .31
APPENDIX B
IMMUNIZATION – RECOMMENDED
ROUTES AND VOLUMES(adapted from Leenars, et al., 1999) . . . . . .33

APPENDIX CSTAGES OF MONOCLONALANTIBODY PRODUCTION . . . . . . . . . . . . .35

APPENDIX D
INFORMATION ON IN VITROTECHNIQUES FOR MONOCLONALANTIBODY PRODUCTION . . . . . . . . . . . . .37

antibody
production
A. PREFACEThe Canadian Council on Animal Care
(CCAC) is responsible for overseeing
animal use in research, teaching and testing. In addition to the Guide to the Care andUse of Experimental Animals,Vol. 1, 2ndEdn., 1993 and Vol. 2, 1984, which lay
down general principles for the care anduse of animals, the CCAC also publishes
guidelines on issues of current and emerging concerns. The CCAC guidelines on:
antibody production is the fifth of this series,
and has been developed by the CCAC ad
hoc subcommittee on immunological procedures.
The purpose of this document is to present
guidelines for production of both polyclonal(pAb) and monoclonal antibodies (mAb) thatassist investigators and research support
personnel to achieve an acceptable
immunological result with minimal discomfort for the animals involved. These guidelines are also provided to assist animalcare committee (ACC) members to evaluate
protocols involving the production of
antibodies, to ensure that the highest
standards of animal care and use are met.
These guidelines supercede the guidanceon antibody production given in the 1991
CCAC policy statement on Acceptable
Immunological Procedures.
Many institutions already have excellent
standard operating procedures (SOPs) inplace for the production of both pAbs andmAbs. These CCAC guidelines borrow
much from the experience of these institutions, and from the recent international
initiatives to refine protocols for antibody
production.
The refinement of animal use in research,teaching and testing is an ongoing process
which is never complete. The CCAC recognizes that in moving towards implementation of these guidelines, considerable
expenditure may be involved; for example,
to establish central services for productionof mAbs in vitro. As with the implementationof other CCAC guidelines, institutions must
recognize such expenditures as responsibilities if sound humane research is to be
carried out within their facilities. The CCAC
is committed to assisting institutions byproviding information on production of mAbs
in vitro as well as on best practices foranimal-based production for pAbs andmAbs where necessary.

B. INTRODUCTION

ccac guidelines

Antibodies are produced by the immune
system of an animal in a specific response
to a challenge by an immunogen. The
immune system acts through two principal
mechanisms: humoral type responses(production of antibodies) and cell-mediatedresponses. Immunogens (antigens) aremolecules which can induce a specificimmune response and are usually foreign
proteins or carbohydrates, or sometimes
lipids and nucleic acids. The immune
systems of mammals are comprisedof large numbers of lymphocytes, eachcharacterized by its unique antigen-receptor
specificity. This receptor diversity permits
immune responses to a broad range of
immunogens. The B lymphocytes, characterized by the presence of specific immunoglobulin receptors on their surface, areresponsible for production of the humoral(antibody) response. Antibodies are secreted from plasma cells which have differentiated from B lymphocytes after appropriate
stimulation by the foreign immunogen.
Each antibody molecule is capable ofrecognizing a specific epitope (antigenicsite), usually 5-6 amino acids or monosaccharide units which are either linear or
topographically assembled, and is capableof binding to this epitope.
A polyclonal humoral response is comprisedof antibodies, derived from various clonal
populations having varying specificities (fordifferent epitopes, even on the same molecule), affinities and classes, and hence
provides an effective defense against
pathogens. Polyclonal antisera are difficult
to reproduce because of the variety ofantibodies made in the polyclonal response.
The level and quality of the antibodies
produced will vary from animal to animal,2

and from a single animal over time. Therefore, pAbs have a finite availability and are
subject to possible character change during
the period of production. On the other hand,monoclonal antibodies (mAbs) are derived
from a single clone and hence are specificfor a single epitope and have a defined
affinity for that epitope. Thus if the rightmAb is obtained, it can be extremely specific for the relevant immunogen, and underappropriate conditions, an almost limitless
production of a constant product is possible.
Polyclonal antisera can be obtained in a
relatively short time frame (1-2 months), in
contrast to standard mAb production
procedures that can be tedious and require
3-6 months. (Newer procedures, however,can shorten the time for mAb derivation toas little as one month.) Polyclonal antibodies show different affinities for different
epitopes and thus may demonstrate overall
excellent binding achieved by adherence toa number of different sites on a complex
immunogen or antigen. In contrast thesingle epitope specificity of mAbs may
mean that a slight change in the structure of
the epitope, for example by antigen denaturation (such as occurs in immunoblotting
experiments or by sterilization of theimmunogen prior to injection into theanimal), can result in loss of antibody
binding. For this reason two or three mAbs
are sometimes combined.
Consideration should be given at the outset
to procuring commercially produced antibodies from sources that comply with CCAC
guidelines, or if international, are accreditedby the Association for Assessment andAccreditation of Laboratory Animal Care,
International (AAALAC). The Antibody
Resource Site http://www.antibodyresource.
com may assist in this regard as well asimmunogens or antigens (Hanly, et al.,1995). A number of references to suitable
immunology texts are given in the reference
list: Abbas, et al., 2001; Anderson, 1999;
Kuby, 2000; Roitt, et al., 1998; Sharon,
1998; Sompayrac, 1999.
antibody production, 2002

provide background information on antibody
production. A list of suppliers of mAbs
produced in vitro has been compiled by the
Focus on Alternatives Group, UK, and asecond list of US suppliers has been compiled by the Alternatives Research andDevelopment Foundation, US. Both lists
can be found at http://www.frame.org.uk/
Monoclonal_Suppliers.htm. Additional
material, for example SOPs, are available
on the CCAC website http://www.ccac.ca.

General guideline: In the production ofpolyclonal antibodies, the overriding
consideration must be to minimize painand distress for the animals used.

1. Animal Selection and Care

C. POLYCLONAL ANTIBODYPRODUCTION
Polyclonal antibodies (pAbs; antisera) havea number of uses in research, for example:
for detection of molecules in ELISA-type
assays; in Western blots; in immunohistochemical and immunoprecipitation
procedures; and in immunofluorescenceand immunoelectron microscopy.
Antisera are commonly produced by injection of the immunogen (antigen) of interest
into an animal, often in combination with an
adjuvant to increase the immune response.
The antibody response can be enhanced bysubsequent booster injections of the antigen
with or without adjuvant. Blood samples are
obtained from the animal to assess thelevel of antibodies produced, and once a
sufficiently high titre has been reached, theantiserum is prepared by blood collection
followed by serum preparation, with subsequent purification of antibodies from theserum if required.
Background knowledge of the mechanisms
involved in generating a humoral immune
response is essential in the development of
the most appropriate SOPs for particular

Guideline: Careful consideration should
be given to the selection of the species
to be used for polyclonal antibody
production.
Careful consideration should be given to theappropriateness of the species and strain
chosen. The investigator should consider
the following factors: 1) the quantity of Ab orantiserum required (larger animals should
be considered when larger quantities of Ab
are required); 2) the phylogenetic relationship between the species from which theprotein antigen originated and the species
used to raise the Ab; 3) the effector functionof the pAbs made by the species raising the
Ab (such as complement fixing ability – see
Hanly, et al., 1995); and 4) the intended useof the antibodies (e.g., in an ELISA, theantibody which binds to the antigen may
need to be derived from a different species
than the secondary antibody used in thenext step of the assay). Different strains of a
species may also react differently, due togenetic variation in presentation of theantigen in major histocompatibility complex
molecules and in immune response regulatory mechanisms (Hanly, et al., 1995;
Coligan, et al., 1997).

3

ccac guidelines

The rabbit is the most commonly usedanimal for the production of pAbs, as it iseasy to handle and to bleed, and for mostapplications will produce an adequate
volume of high-titre, high affinity, antiserum(Stills, 1994). A typical bleed from a rabbit
should yield approximately 250mg of pAb; aterminal bleed using saline displacement
can yield approximately 1g of pAb. It is
important to use disease-free rabbits for
all immunological procedures to reduce
the likelihood of Pasteurella multocida
abscesses at injection sites, and to minimize the likelihood of cross-reactivity toother antigens the rabbits’ immune systems
may previously have encountered.
For protocols requiring the production oflarge volumes of pAbs, or where theanimals are to be maintained as antibody
producers for a long period of time, theuse of whiffle balls or serum pockets for
antibody collection may be considered(Whary, et al., 1998). Investigators and
ACCs should evaluate whether the subsequent ease of handling for serum sampling
will outweigh the initial requirement for
surgical intervention to implant the whiffleball chamber.
The chicken is phylogenetically distant frommammals and, therefore, can be useful for
raising pAbs to mammalian proteins, andin particular, to intracellular mammalianproteins, as the amino acid sequence ofmany intracellular proteins tends to beconserved between mammalian species.
The product, IgY, is for almost all purposes
equivalent to the mammalian IgG class ofAbs. The chicken may be considered as arefinement of technique as pAbs can beextracted from the egg yolk, removing thenecessity for blood collection. The use of
the chicken can also represent a reduction

4in animal use as chickens produce larger
quantities of antibodies than laboratoryrodents (Schade, et al., 1996; Bollen, et al.,1995). Typically, a single egg will contain up
to 250mg pAb in the egg yolk (Erhard, et al.,2000). In general, chickens are potent
antibody producers and their immunologicalresponsiveness is similar to that of
mammals. However, it is important toemphasize that the chicken is not suitable
for all applications, and appropriate facilities
for housing chickens must be available ifthey are to be used.
In general, rodents are used less frequently
than rabbits for pAb production, but may
be suitable when small volumes of pAbs
are required. Blood volumes that can becollected from these animals are considerably smaller, and thus collection of areasonable volume may require cardiac
puncture under terminal anesthesia. The
rat can be used when Abs of restricted
specificity to mouse proteins are required,or for IgE production (Garvey, et al., 1977).
The hamster is used to produce anti-mouseprotein Abs when such pAbs cannot bereadily produced in the rat or when broader
specificity of pAbs is required.
Historically, the guinea pig was commonlyused for antibody production, in particularfor use in insulin assays, but otherwise it
does not appear to have any significant
advantage over the use of rodents.
Larger species are used when large
volumes of antisera are required, in particular for commercial production. Horses,
sheep and goats, for example, have a longlife span, are relatively easy to handle,
and can be bled from the jugular vein.
Requirement for large animal facilities andthe expense of maintaining larger animals

Where species-specific considerations are
not an issue, the species should be chosen
to minimize pain and distress, bearing in
mind ease of handling for injection andblood sampling. The expected requirementof the pAb will influence the volume ofserum required and this will impact on the
animal species and number of animals
used. In general, young adult animals are
better pAb producers than older animals, as
immune function peaks at puberty and thenslowly declines. In addition, females are
preferred as they often produce a stronger
immune response and tend to be moredocile, easier to handle, and easier to pairor group house. For larger farm animal
species, castrated males may also be suitable. Grossman (1989) has documented
the enhanced production of pAbs by adultfemales.
Other factors which influence pAb production include: the nature of the immunogen to
be used; the route and timing of administration; the type and quality of adjuvant; thenature of the immunogen/adjuvant formulation (i.e., emulsion, liposome formation,
immunostimulatory complex [iscom] formation or adsorption); the method of bloodcollection; the strain, health and genetic

status of the animals; the training, expertiseand competence of the animal care staff;
and the diet and housing. In addition, it
should be noted that the presence of infectious agents and/or stress can suppress
the immune response, and thus reduce thequantities of pAbs produced, or even resultin no significant response.

antibody production, 2002

has limited their use principally to commercial production of antisera. It should be
noted that horses, which have been used
to produce antisera for toxins and venoms
for clinical applications, are particularly
intolerant of oil-based adjuvants, such as
Freund’s Complete Adjuvant (FCA) andsaponin (Quil A). Antibodies can also be
harvested from the milk of cattle, sheep andgoats and represent a non-invasive means
to repeatedly acquire large volumes of pAbs
(Brussow, et al., 1987; Tacket, et al., 1988;
and Sarker, et al., 1998).

The pre-immunization status of the animals,with respect to the immunogen, or othercross-reacting antigens to be administered,should be determined. Immunogen-specificpre-existing Abs can influence both thequality and the quantity of the Abs, especially where a monospecific pAb is required.
Usually a pre-immunization (pre-bleed)sample of serum is prepared in sufficientquantity to use as a control throughout
testing and subsequent use of the immune
serum.
General guidance on appropriate care foranimals to be used in Ab production isprovided in other CCAC guidelines. Animals
should be housed under conditions thatbest meet their social and behavioral needs
to permit natural behavior, with group
housing being preferred (CCAC, 1990).
Where that is not possible, the decision
should be justified to an ACC.
Guideline: Investigators, researchsupport and animal care staff involved inpolyclonal antibody production musthave the appropriate training andcompetence.
The training and competence of the animal
users and animal care staff are of great
importance in minimizing pain and/or
distress for the animals. In addition tohaving knowledge of animal care, thestaff should understand basic immuno-

5

ccac guidelines

logical principles and be trained andcompetent in immunization procedures
and in blood collection. For some species,
this will require knowledge of anesthetic
procedures. The staff must also be capableof recognizing pain and/or distress in thespecies used (CCAC, 1993, 1999).

2. Immunization Protocol
Guideline: Animal Care Committees are
expected to evaluate immunization
protocols with respect to animal welfare
aspects; particular attention should begiven to atypical immunogens and/or
adjuvants likely to cause pain and/or
distress.
The proposal for antibody production foreach new immunogen or immunogen/adjuvant combination must be reviewed by
the local ACC, and should include reasonable efforts to determine whether the
product required is available commercially,or from another research group. The review
can be brief if the type of immunogenhas been used previously and SOPs are
already in place. Protocols involving novel
immunogens should be given a more
careful review, and the advice of an immunologist should be sought in these
instances. SOPs should be in place toaddress the routine procedures, but may
need slight modification depending on theparticular proposal. The use of FCA cancause considerable pain and distress forthe animals concerned. Therefore, protocols
which involve the use of FCA, or where
the investigator anticipates that the animals
may experience an adverse reaction, mustreceive particular attention and must
be assigned to Category of Invasivenesslevel D (CCAC, 1991). This category may

6

be changed to Category of Invasivenesslevel C following a favorable report on thecondition of the animals by those responsible for their day to day care.

3. Standard Operating Procedures
Guideline: Each facility should establish
Standard Operating Procedures for pAb
production in each species.
The immunization protocol raises several
significant issues that must be considered:
1) method of antigen preparation; 2) use of
adjuvant – whether to use one and thenwhich one; 3) route of injection, and numberof sites to be used; 4) volume to be injected;
and 5) schedule of injections. SOPs should
also be implemented to limit the frequency
of use of individual animals and to limit thelength of time that an individual animal
can be held for the purpose of antibody
production.

4. Immunogen Preparation
Guideline: The immunogen should
be prepared in such a manner to elicit
an acceptable response without adversely affecting the well-being of the animals.
The quality of the immunogen preparation must be a prime consideration. The
immunogen must be non-toxic and it must
be prepared aseptically, or otherwise
rendered sterile and free of toxins andpyrogens. In particular, any chemical
residues, contaminating endotoxins or othertoxic contaminants must be minimized (e.g.,<1ng/ml for immunogens derived fromgram -ve bacteria) and the pH must be
adjusted within physiological limits. Most

The quantity of immunogen to be injected toelicit a good antibody response varies
greatly depending on the species and strainof the animal to be immunized, the adjuvant
used, the route and frequency of injection
and the immunogenicity of the immunogenitself.
The quality and quantity of the pAb produced is dependent on the size and thestate of the immunogen. Small polypeptides and non-protein molecules may
require conjugation with a larger immunogenic carrier protein in order to provoke an
immune response. Appropriate carrier molecules must be selected, so as not to induce
suppression of the immune response to theantigen (carrier-induced epitopic suppression) due to pre-existing antibodies to thecarrier (Schutz, et al., 1985).
It should also be noted that the conformation of the immunogen may be important, as
antibodies raised against native proteins
may react best with native proteins, andthose raised against denatured proteins
may react best with denatured proteins. For
example, if the pAbs are to be used to blockan active site on a protein, the immunogenshould not be denatured prior to injection. In
this regard it should be noted that apreformed oil-in-water emulsion adjuvantwith adsorbed immunogen will maintain agreater amount of native conformation than

the immunogen prepared in a water-in-oil
emulsion requiring vigorous shaking (Allison& Byars, 1991). Antigen adsorbed to alum
can also give a good Ab response to native
configuration (Chen, et al., 1964; Dardiri &Delay, 1964; Butler, et al., 1969; Cohen, etal., 1970; Weeke, et al., 1975; and Ziola, B.,
pers. comm.).

antibody production, 2002

protein immunogens can be filter sterilized
through a microporous cellulose acetate
filter (0.22µm pore size). In a recent surveyof Canadian institutions that routinely
prepare pAbs (Craig & Bayans, 1999),
respondents stated that immunization withproteins in polyacrylamide gel was associated with adverse reactions at the site of
injection.

5. Choice of Adjuvant
Guideline: The decision as to whether an
adjuvant is required should be carefully
considered and justified. Investigators
should seek the most appropriate adjuvant for the antigen of interest, bearing
in mind the current state of knowledge
on immunogen/adjuvant preparation.
The advice of a knowledgeable immunologist should be obtained for each
protocol involving a new adjuvant ornovel immunogen. The use of Freund’sComplete Adjuvant should be avoided
where possible, and when necessary
should only be used for the primary
immunization, and should contain lessthan 0.5mg/ml mycobacteria.
Investigators and ACC members should not
overlook the considerable pain and/or
distress that adjuvants can cause forlaboratory animals. Most of the undesirable
side-effects of pAb production that range inseverity and duration are caused by the
adjuvant (Jennings, 1995). Therefore, it
should be determined first whether an
adjuvant is required. Adjuvants are used toenhance the immune response and whenused should result in enhanced andsustained Ab levels.
Adjuvants work in several ways. They may
form a depot of immunogen at the injection

7

ccac guidelines

site leading to a slow release over a periodof time giving sustained stimulation to theimmune system. The adjuvant can also help
deliver the immunogen to the spleen and/or
lymph nodes where many of the cell-cellinteractions in the immune response occur.
They may directly or indirectly activate
the various cells, such as macrophages orT-helper lymphocytes, involved in the
immune response. Adjuvants may also
influence the duration, subclass and avidityof the antibodies produced and may affectcell-mediated immunity (Hunter, et al.,1995).
The use of an adjuvant is usually necessary
for soluble, relatively pure or pure immunogen preparations. If the immunogen is asmall molecule, it usually does not
induce an immune response by itself.
The use of an adjuvant can also meanthat less immunogen is required, which
may be a consideration for molecules which
are scarce or are particularly valuable.
Highly aggregated antigens may not
require the use of an adjuvant andmay in fact induce a more authentic
immune response in the absence ofadjuvant.
The use of adjuvant with whiffle balls is
not necessary as the whiffle ball itself
serves as an immunogen depot, leading toprolonged release of the antigen (Clemons,et al., 1992).
The immunogen/adjuvant mixture should be
easily injectable in small volumes andshould have low toxicity. The commonlyused adjuvants include both Freund’sComplete and Incomplete Adjuvants (FCAand FIA), Quil A, RibiTM and TiterMaxTM, andmineral-based adjuvants – aluminiumhydroxide, aluminium phosphate and8calcium phosphate. However, all desirable
characteristics of an adjuvant are not foundtogether in any of the 100 or so known
adjuvants. In addition to the brief descriptions of the commonly used alternatives
to FCA/FIA used in Canada which areprovided in Appendix A, see Leenars, et al.
(1999); Jennings (1995); CEDARLANE
Laboratories Limited "The Adjuvant Guide"(Hornby, Ontario), or the European Adjuvant Database (Stewart-Tull, 1995) for moredetailed information.
The CCAC recognizes that many investigators have been reluctant to discontinue
the use of FCA because it is used as a"gold standard" due to its well known effectiveness with a wide variety of antigens(Craig & Bayans, 1999). While FCA is oneof the most effective adjuvants, it can cause
a greater chronic inflammatory response,
and therefore, should be used only when
there is evidence that other adjuvants
will not work (for example, when only
small amounts of soluble immunogens areavailable, or when the antibody responseis weak, e.g., Jennings, 1995). Broderson(1989) has shown that if the concentrationof mycobacteria in the FCA preparationis less than 0.1mg/ml, less severe inflammatory reactions result. If large amounts
of a particulate immunogen are available,
or if the antigen is highly immunogenic,
immunization without adjuvant or withother adjuvants should be used. If FCA
must be used, it should be used only forthe initial subcutaneous immunization,
following the procedures detailed in Section
C.7 – Volume and Number of Injection
Sites. FIA should be used for subsequent
booster immunizations, if an adjuvant isrequired. Secondary (booster) immunizations can sometimes be carried out using asaline buffer solution.

6. Route of Injection
Guideline: The route of injection must be
selected with the objective of causing
the least possible distress for the
animal.
The route of injection may be subcutaneous(SC), intramuscular (IM), intradermal (ID),intraperitoneal (IP), or intravenous (IV), as
detailed in Appendix B. Footpad, intralymph node or intrasplenic routes arestrongly discouraged and if required
must be justified on a case by casebasis. Injection into any closed spaceis painful, so even IM and ID can bequestioned as a route of choice. No
other routes are permitted. Footpad
injections have been used in the past tofollow the immune response to a specificantigen by studying the cells of the immune
system responsible for processing theantigen. Immunogens injected into thefootpad are processed by the popliteal
node, making it possible to routinely andaccurately acquire the desired cells.
However, this can be achieved with muchless distress to the animal by injecting at thebase of the tail, or in the popliteal area.
Intravenous administration can be the routeof choice for small particulate immunogens
because the immunogen is distributed
throughout the body and hence, capture bylymphoid cells is high. The IV route should
not be used for oil-based or viscous gel
adjuvants or for large particulate immunogens due to the risk of pulmonary embolism(Herbert, 1978).

antibody production, 2002

In general, an immunogen should be mixed
with an equal volume of adjuvant and emulsified. Emulsification can best be achieved
using two Luer-Lok syringes and a locking
connector, passing the adjuvant andimmunogen back and forth until it becomes
paste-like. Sonication using a narrow probe
is an alternative method of emulsification;
however, care must be taken to avoid overheating by keeping the sonication mixture
on ice. Proper preparation of the immunogen/adjuvant mixture is important as amajor cause of immunization failure is due
to inappropriate emulsification. Dispersal of
the emulsion with an equal volume of
2% Tween 80 reduces the viscosity of thesolution for easier injection (Herbert, 1965).

Subcutaneous injections should be used foroil or viscous gel adjuvants, in particularfor FCA, in order to minimize the formationof sterile abscesses. The IM route should
not be used for the injection of oil-based or
viscous gel adjuvants in small animals
such as mice or rats. Immunogen/adjuvant
injected IM can spread along interfascialplanes between muscle bundles and irritate
the nerve bundles leading to serious pathology due to the inflammatory process.
The ID route should not be used in small
animals, and should be restricted to cases
where it is absolutely necessary. For rabbits
or larger animals where the ID route isused, the volumes administered should besmall (i.e., 25µl in rabbits) (Halliday, et al.,2000).
Intraperitoneal injection of adjuvant mixtures
is not recommended for pAb production,
since it is known to induce inflammation,
peritonitis, and behavioral changes(Leenars, et al., 1999; Toth, et al., 1989).
See Section C.7 – Volume and Number of
Injection Sites, and Appendix B for further
recommendations, should the IP route berequired.

9Sites of injection should not interfere withsubsequent handling of the animals forblood sampling, etc.

ccac guidelines

If large volumes of pAb are required, theuse of whiffle balls should be considered.
Whiffle balls used in rabbits are perforated
plastic golf balls, available from a varietyof commercial suppliers. The balls should
be sterilized with ethylene oxide prior
to implantation. Whiffle balls must be
implanted using aseptic surgical techniques
described by Hillam, et al. (1974) and Reid,et al. (1992). Strict asepsis must be maintained during surgery to avoid secondary
infection of the implant. A minimum of fourweeks post-surgery is required for healingprior to experimental use, during which time
animals should be housed individually to
avoid injury. Once prepared, the whiffleball can be used both for primary andbooster injections as well as for serum
collection.
Immunization of chickens for pAb production is comparable to that of rabbits withrespect to route of injection, the amount of
antigen used and the kinetics of specificantibody generation (Haak-Frendscho,
1994; Song, et al., 1985). Recommendations for chicken immunization (Schade, etal., 1996), include the use of FCA, Specol,or preferably lipopeptide PCSL (Pam3-Cysser-[Lys] 4 ; 250µg). Erhard, et al. (1997)
found few side effects from the use of PCSLin comparison with multiple granulomas
after FCA or FCA/FIA treatment. Immunogen should be administered in the 10ng1mg range (preferably 10-100µg). Injection
should be IM for young laboratory chickens(neck or breast muscle), and intramuscular
injection in the leg should be avoided since
it can lead to lameness. Chickens should
be at least seven weeks of age. For older

10

laboratory chickens, the SC route should be
used with an injection volume <1ml; at least
two immunizations are generally given. A
primary vaccination and a booster should
be given before the laying period with aninterval of six weeks for emulsion-type
adjuvants and four weeks for lipopeptide
adjuvants. The stress induced by handlingcan have a negative effect on egg production; as can the nature of the antigen
or antibody used. Yolk Ab titres should bechecked 14 days after the last immunization, if Ab titres begin to decrease, boosters
can be given during the laying period at
4-8 week intervals. Antibody contained ineggs can be collected throughout the laying
period (about one year).
Other routes of administration of adjuvant
include aerosol, oral and intranasal administration. These routes have advantages forstimulation of an IgA response, and also
may be less stressful for the animals. For
examples involving mice, see Shen, et al.
(2000); Shen, et al. (2001); McCluskie andDavis (2001); McCluskie, et al. (2000);
Coste, et al. (2000); Falero-Diaz, et al.
(2000); and Holan, et al. (2000). Examples
involving rats are found in Papp, et al.
(1999); and Baxi, et al. (2000). For horses,
see Nally, et al. (2001); for pigs, see
Katinger, et al. (1999); and Tuboly & Nagy
(2001); and for poultry, see Sharma (1999).

7. Volume and Number of
Injection Sites
Guideline: The injection volume should
be kept as small as possible and should
be kept within the recommended limits.
Recommendations are given in the literature to limit the number of injection sites.

Some species may require sedation prior
to injection. This may be particularly
important if multiple injection sites are to be
used.
In all cases, the injection site should
be aseptically prepared 1 and allowed
to dry immediately prior to injection of
the immunogen or immunogen/adjuvant
mixture.
To minimize the chance of abscess formation when using FCA, it is important toensure that all material is injected aseptically into the subcutaneous space, not ID or
IM. In particular, care should be taken not to
contaminate the needle track. Once thesolution has been injected, and before it is1

Aseptic skin preparation means clipping the fur
and using a three-stage surgical preparation:
surgical soap, alcohol rinse and surgicalpreparation solution.

withdrawn from the subcutaneous space,
the plunger should be pulled back a few mm
and the needle quickly removed from thesubcutaneous space. In this manner,spillage into the dermal tissue will beminimized.
antibody production, 2002

However, in terms of minimizing adverse
reactions, it appears to be less stressful tothe animals in the post-immunization phase,to minimize the injection volume and touse multiple sites. Recommended injection volumes are given in Appendix B,
both for immunogen plus adjuvant or
immunogen alone. It should be noted thatin order to minimize immunization reactions,the volumes recommended per site forinjection of immunogen with adjuvant aresmaller than the generally recommended
volumes for injection. It is necessary to
use large gauge needles (20-21 gauge
for the rabbit), as immunogen/adjuvant
mixtures are quite viscous. Use of a double
emulsification as described by Herbert(1965), reduces the viscosity of thesolution enabling a smaller size needle(26-27 gauge) to be used.

Where there is no indication of reaction to
the initial injection, booster injections givenin the vicinity of the initial site take advantage of the memory cells established in thedraining lymph nodes. This may minimize
the number of injections needed per animal
and/or the number of animals required for
an effective immune response. Injection
sites should be sufficiently separated toprohibit the coalescing of the inflammatory
lesions, which may result in disruption ofblood supply to the area, with subsequent
formation of draining abscesses, or occasionally tissue sloughing. However, if there
are indications of local reactions, booster
injections should be distant from previous
injection sites and must never be given at
the site of a granuloma or swelling induced
by previous injections.
Animals must be closely monitored immediately following injection for any anaphylactic reactions, both after the primary
injection and after the subsequent booster
injections (see Section C.9 – Monitoringof Animals). Animals experiencing unrelievable pain and/or distress must be
euthanized.
For whiffle balls, injection of immunogenshould be directly into the whiffle ballchamber (Clemons, et al., 1992).
The boosting protocol can have a significant
effect on the result of the immunization.
The time between two immunization steps
can influence both the induction of B

11

ccac guidelines

memory cells and the class switch of B cells(from IgM to other antibody classes andsubclasses). In general, a booster can beconsidered after the antibody titre haspeaked or begun to decline, and whenmemory cells can be expected to be
increasing. If the first immunization is
performed without a depot-forming adjuvant, the antibody titre will usually peak
2-3 weeks after immunization. When adepot-forming adjuvant is used, a booster
injection can follow at least four weeks
after the first immunization. An adjuvant
is not always necessary for booster
immunizations.
In most cases, the endpoint of Ab production should be considered as the point whenthe Ab titre has reached an acceptable level(generally a maximum of two boosters). A
long immunization schedule with repeatedboosting is not generally useful as it may
result not only in the production of Abs withincreased affinity for the immunogen ofinterest, but also in production of more Abs
specific for contaminants in the immunogenpreparation (if the immunogen is not a
purified protein or peptide) (Leenars, etal., 1999). Multiple boosters should not
require the use of adjuvant. In all cases thewelfare of the animals must be taken into
account.
Animals can be rested for long intervals
between boosting. In addition, regularintervals of blood collection, once a
sufficient serum titre has been reached,
could facilitate the collection of adequate
amounts of Abs for immunogens in limited
supply. However, animals must not be
kept in an Ab production program unnecessarily. Any animals kept long-term in an
animal facility should be under long-term
surveillance by a veterinarian.

12

8. Blood Collection
Guideline: The selection of bloodcollection procedure should aim tominimize stress for the animal andshould follow current CCAC guidelines.
The SOP for each pAb preparation should
include a section on blood collection. In
this respect, the training and expertise of
the staff carrying out the procedure isa significant factor (CCAC, 1999). The
animals should be kept warm and awayfrom environmentally-induced stresses
such as noise. The use of organic solvents
to induce vasodilation is not recommended.
The use of general anesthesia is notnecessary for blood collection in rabbits
and larger animals. It is also not necessary
for blood collection in rodents using
the tail vein or saphenous vein. However,terminal anesthesia is required for cardiac
puncture, which may be required to obtainsufficient volume. It may be useful to use
local anesthetics, for example EMLA TM
cream on rabbit ears, and/or parenteralanalgesics. The needle used should bematched to blood vessel size. Rabbits
should be bled by the ear vein using aneedle rather than a scalpel blade as
cutting may cause damage to the bloodvessels and surrounding tissues, which may
ultimately result in regional necrosis andsloughing of ear tissue. The maximum
blood volume removed should not exceed
10% of the total blood volume of the animal(approximately 1% body weight), if collected
every two weeks, and not more than 15%of the total blood volume if collected every
four weeks (Morton, et al., 1993; McGuill &Rowan, 1989; Diehl, et al., 2001).
Serum collection from implanted whiffle

For pAb production in chickens, eggs
are collected daily (usually one egg islaid per day) and marked for identification.
They can be stored for up to one year at
4°C prior to antibody (IgY) purification(Haak-Frendscho, 1994; Svendsen, et al.,1995).
Exsanguination must be performed under
non-recovery general anesthesia, using
a technique that results in a maximum
collection of blood. After exsanguination iscomplete, the death of the animal must be
assured following current CCAC guidelines
for euthanasia.

9. Monitoring of Animals
Guideline: Animals must be monitored
daily, and the Standard Operating
Procedures should include a checklist
for endpoints.
The SOP should include a requirement forrecord keeping. The immunization record
should include the agent, route, site or sites,
volume, date of injection and body weight of
the animal.
Animals may have inflammatory responses
to the injections of immunogen or immunogen/adjuvant mixtures and, therefore,
must be monitored daily for responses

at the injection sites in particular andfor overall health or distress in general.
Food and water intake, activity andgeneral appearance should be monitored.
The staff taking care of the animals shouldhave in their SOP a daily checklist formonitoring animals producing pAb withdefinitive endpoints for stopping theprocedure (euthanasia) if the animal
develops unrelievable distress due to the
inflammatory response. SOPs should
include a requirement for investigators tocontact the veterinary staff (and for theveterinary staff to contact the principal
investigator) if injection site lesions, orevidence of pain and/or distress, are identified in any animals. The veterinary staff
must be responsible for the implementationof timely and appropriate assessment of theanimals and for institution of therapy whenrequired. For immunization induced lesions,
supportive therapy may include topicalcleansing, antibiotic administration, and/or
analgesic administration. Fluid replacement
or nutritional supplements may be required
if animals have sustained anorexia ordecreased fluid intake. Animals mustalso be carefully monitored following bloodcollection, in particular if the procedure
is carried out under anesthetic (CCAC,
1998).

antibody production, 2002

balls is relatively easy and does not require
use of a local anesthetic. The skin over thewhiffle ball chamber must be aseptically
prepared. A sterile hypodermic needle witha cotton plug is inserted into the dorsal halfof the chamber through one of the holes
and the fluid withdrawn through a second
needle connected to a syringe (Whary, etal., 1998).

10. Disposition of the Animals
Disposition of the animals must be carriedout in accordance with CCAC guidelines
and local ACC regulations. It is the investigators’ responsibility to inform the animalcare staff when the collection of antibody
has been completed so that animals canbe disposed of as previously agreed in
the protocol. This can include euthanasia,

13

ccac guidelines

or re-homing where relevant adoption
programs are in place. It should be notedthat if FCA is used in meat producing
animals, they must not be resold for humanconsumption. The Canadian Food Inspection Agency2 should be contacted for advice
on disposition of animals where otheradjuvants have been used.

D. MONOCLONAL ANTIBODYPRODUCTION
Monoclonal antibodies (mAb) are antibodies
which have a single, selected specificity andwhich, usually, are secreted continuously by"immortalized" hybridoma cells. A hybridoma is a biologically constructed hybrid ofan antibody-producing lymphoid cell and amalignant (immortal) myeloma cell. The
development of hybridoma technology by
Köhler & Milstein in 1975, provided a meansfor obtaining large quantities of highly
specific antibodies which has had aprofound impact on disease diagnosis,
therapy, and on biomedical research ingeneral.
There are essentially two major stages in
the production of mAbs: 1) the inductionof antibody producing lymphoid cells invivo (immunization) and the selectionof antibody-producing hybridoma cells
in vitro; and 2) the propagation of selectedhybridoma clones, either in vitro or in vivo.
Appendix C contains a graphical representation of the stages of mAb production.
Newer methods for production of mAbs,

2

14

Canadian Food Inspection Agency, National
Manager, Veterinary Biologics Section, Ottawa
ON (tel.: 613-225-2342).

for example phage display techniques andexpression as recombinant proteins, do not
involve the use of animals and are therefore
not discussed here.
Despite the original comments on thegeneral methodology (in vitro) of hybridoma
technology by Köhler and Milstein – "themanufacture of predefined specific antibodies by means of permanent tissue culture cell lines is of general interest" and"such cells can be grown in vitro in massive
cultures" – the propagation of mAbs has,for almost all purposes, been carried out invivo. It was apparent that mAbs could be
produced by injecting the hybridoma cells
into the abdominal cavities of different
species of rodents. The subsequent propagation of the hybridoma cells in ascitic fluid
offered an easy, economical route to the
production of mAbs.
An alternative method of mAb production,although not routinely used, is the production of recombinant antibodies in themilk of genetically modified animals. As theneed for larger quantities of antibodies
increases, this approach may be increasingly
adopted as it would appear that the expression of recombinant mAbs in the milk oftransgenic mice and goats may be muchgreater than in cell culture systems (Young,et al., 1998). The creation and use ofanimals as bioreactors for mAb production
must be carried out in accordance with the
CCAC guidelines on: transgenic animals(1997b).
General guideline: Every attempt should
be made to obtain material alreadyavailable or to use an in vitro method
for production of mAbs. Therefore, any
proposed production of monoclonal
antibodies using the ascites methodrequires justification by the investigatorto the animal care committee.

The production of mAbs in mice by the
ascites method raises several issues ofconcern regarding the potential for severe
and unnecessary pain and suffering for the
animals (Anon., 1989). In recognition of thisfact, a number of in vitro replacements forthe rodent ascites method of mAb production have been developed. A number ofcountries have instituted a ban on routine invivo production of mAbs (Shavlev, 1998). A
recent report prepared by a US National
Research Council Committee, in response
to a request from the National Institutes of
Health, recommended that in vitro mAb
production methods should be adopted as
routine whenever practical, and that if themouse ascites method is used, it must be
justified to an institutional animal care anduse committee (Peterson, 1999). It is now
generally accepted that the in vitro techniques of mAb production have progressedto the point where these techniques can beused for more than 90% of mAb production(ILAR, 1999).
In this context, it should be noted that
the CCAC Ethics of Animal Investigation(1989) requires investigators to follow the"Three Rs" of Russell & Burch (1959):
Replacement (of animals with other, nonsentient material or with animals of lower
sentience); Reduction (of numbers of animals used); and Refinement (of technique,

The potential for severe pain and/or distressin the animals relates to: intraperitoneal (IP)injection of primer (e.g., Pristane or FIA);the effect of primer after administration; theIP inoculation of hybridoma cells; and thegrowth of the tumor cells in the abdominal
cavity (primary effects) as solid peritoneal
plaques that infiltrate the abdominal wall
and/or abdominal organs. Specifically, IP
injection is believed to cause moderate
distress; ascites fluid production and tumor
growth have been reported to cause suffering in human patients (Kuhlmann, et al.,1989); and, in addition, the complex pathophysiological and pathological changes may
cause severe distress for the animal
involved (Hendriksen, 1998). While ascites
fluid tapping is believed to relieve some ofthe pain and/or distress associated withabdominal distension, the immobilization
and administration of anesthetic prior to
ascitic fluid harvesting is an additionalsource of distress.

antibody production, 2002

Commercially produced mAbs should
be obtained from sources that comply
with CCAC guidelines, or if international
are accredited by AAALAC. For lists ofsuppliers of in vitro produced mAbs, see
http://www.frame.org.uk/Monoclonal_
Suppliers.htm.

to "reduce to an absolute minimum theamount of distress imposed on thoseanimals that are still used").

The CCAC recognizes that it has a responsibility to support ACCs in requiring thatinvestigators provide evidence that the useof replacement methods are not appropriate(CCAC guidelines on: animal use protocol
review, 1997a). Therefore, the CCAC willdevelop and maintain a repository of
information on suitable in vitro techniques
for the production of mAbs (Appendix D). It
is also recognized that, in countries where aban on the ascites method has been
implemented, the use of in vitro methodology has been stimulated by the creation ofCentres of Excellence, centralizing the
production of mAbs in vitro, and providing
training, expertise and resources for the in15

vitro techniques. A number of organizations
also offer to produce material by in vitro
means on a contract basis.

ccac guidelines

Ideally, a replacement in vitro systemshould be equally or less expensive, notrequire special culture conditions, produce
high concentrations of mAbs, be free fromcontamination, be re-usable, and require a
reasonably short period of time to produce
relatively pure mAbs in adequate quantities(Falkenberg, 1993; 1995). Currently, there
are no in vitro systems that meet all ofthese criteria, but depending on thequality and quantity of mAbs required,
it is possible for investigators to select anappropriate in vitro system (for references
to various mAb production methods
see Appendix D). If the required mAb
is not commercially available from a
reputable source, investigators should makeall reasonable efforts to produce therequired mAbs using an in vitro approach.
In line with recommendations madein the US and elsewhere, prior to approvalof a protocol for ascites production,
the investigator should show that he or
she has made a substantial effort toadapt the hybridoma to a culture system
suitable for scale of mAb production
required (e.g., tissue culture flasks,
simple membrane systems, hollow fibre,
fermentation systems) (Saxby, 1999;
Smith & De Tolla, 1999). The productionof mAbs by growth of hybridomas asascites tumors should be avoided unlessabsolutely necessary.

16

little or no mAb in vivo, where in vitro
methods cannot be used, investigators
should be asked to test new hybridomas ina small pilot study involving 2-3 animals.
General guideline: Clearly defined endpoints and close monitoring of thecondition of the animals are required tominimize the potential for pain and/or
distress.
In accordance with CCAC guidelines on:choosing an appropriate endpoint inexperiments using animals for research,teaching and testing (1998), clear endpoints
must be developed which minimize pain
and/or distress for the animal. A clear
schedule for monitoring and reportingmust be established, and SOPs must be
established for each new immunogen/adjuvant combination.

1. Animal Selection and Care
Guideline: When selecting species andstrain of animals for mAb production,
the overall consideration must be tominimize pain and/or distress.

The following guidelines have been
elaborated to safeguard the well-being ofanimals (principally mice) that continue tobe used in mAb production.

The mice (or rats) used should be the samestrain (syngenic) both for immunization to
produce the hybridoma clone and for subsequent mAb production, so that the tissue
used is histocompatible. BALB/c mice areoften the animal of choice, as many of theparental myeloma cells used in the fusion
process are derived from BALB/c mice.
Retired breeders may offer some advantages for ascites production due to thepreviously stretched abdominal musculature(Falkenburg, 1998).

As up to 15% of hybridomas may produce

SCID mice, although expensive, have been

ACCs should ensure that the appropriate
numbers of animals are used. This can be
estimated by presuming that mice produce
approximately 0.5-10mg/ml of mAb in
ascites fluid, and that on average it ispossible to harvest 2ml ascites fluid pertap (Smith & De Tolla, 1999). These are
generalized estimates, and each hybridoma
must be considered as a unique cell line.

2. Production of Hybridoma
Clones
SOPs for immunization should be developed based on guidelines for choice ofadjuvant, route of injection and volume andnumber of injection sites (see Sections C.5C.7). Immunization procedures that do not
require adjuvant and that can be performedin short time periods have been shown tobe effective for hybridoma derivation(Chase, et al., 2001). The procedures,
which appear to cause less stress to themice, require multiple boosts in a short timeframe, eliminating the requirement for
adjuvant. However, rapid immunization may
not always yield mAbs of high affinity, afactor which should be considered in choiceof immunization regimen.
As an alternative to in vivo immunization, in
vitro immunization with antigen has beensuccessfully used (Borrebaeck, 1989). This
procedure is particularly useful when an

antigen is in limited supply and previous
attempts at in vivo immunization haveyielded negative results due to similarity toself antigens or to the weak immunogenicity
of antigen (Bunse & Henz, 1994). Other
advantages of this approach are: fewer
animals are required; the immunization
period is 4-5 days, compared to several
weeks for traditional in vivo immunization(Grimaldi & French, 1995); it is possible tomonitor and control the immune response
without the influence of the immune regulatory mechanisms that occur in vivo; andmAbs can be generated against agents thatare toxic to animals.

antibody production, 2002

reported to produce fewer non-specificmurine antibodies with equivalent yields ofspecific mAb, thus minimizing interference
in immunoassays, etc., (Pistillo, et al.,1992). It should be noted that SCID micerequire barrier facilities for housing andextensive (and expensive) procedures forappropriate care.

For further details concerning production of
hybridoma clones, see Grimaldi & French(1995). For a single-step selection andcloning of hybridomas, see Stebeck, et al.
(1996); and Chase, et al. (2001).
It is important that prior to immunization, themethodology for detecting the specificantibody of interest in the mouse sera
and tissue culture supernatant has beendeveloped, otherwise significant time andresources may be wasted later in the mAbdevelopment phase. In addition, the use forthe mAb should be carefully considered, sothat mAbs that are effective in the final functional assay can be selected by appropriate
screening.

3. Ascites Production
3.1 Priming
Guideline: Freund’s Incomplete Adjuvant(FIA) or other less invasive adjuvant
should be used as the intraperitoneal
priming agent, with a maximum volumeof 0.3ml (FIA) administered in one injec-

17

tion. If the use of Pristane is proposed, itshould be justified to the institutional
animal care committee and the volume
should be limited to 0.2ml.

ccac guidelines

As originally described, the ascites method
of mAb production included the use of
Pristane (0.5ml injected intraperitoneally) as
the chemical used to "prime" the peritoneal
cavity of the mouse. The effect of the primer
is two-fold; it suppresses the immune
system so that the growth of the hybridoma
cells in the abdominal cavity is not (strongly)
impaired, and it causes a chemical irritation,
which leads to peritonitis and the secretionof serous fluid (Kuhlmann, et al., 1989). A
hybridoma cell suspension is injected
7-10 days later.
Preliminary results indicate that the use ofanalgesics prior to priming with Pristane
may lessen the pain and distress experience by the mouse (Fletch, A. & Delaney,K., pers. comm.).
Studies comparing Pristane (at several
volumes) and other priming agents, haveshown that the use of FIA has several
advantages over Pristane, including: fewer
indications of stress to the animals;
injection of hybridoma cells can be
performed as early as one day after
priming; and fewer animals are required(Gillette, 1987; Mueller, et al., 1986; andJones, et al., 1990). Trypan blue has also
been used as a priming agent (Wu &Kearney, 1980). It should be noted that,prior to use, Trypan blue must be dialyzed
in fresh glass-distilled water for 48 hours,
replacing the water twice per day, toreduce low molecular weight impurities.
Priming with Trypan blue is carried out
24 hours and again one hour prior to
injection of the hybridoma cells.

18

3.2 Contamination
Guideline: Hybridomas should be tested
for the presence of adventitious viraland mycoplasma agents prior to use
for in vivo propagation of monoclonal
antibodies.
Viral contamination is commonly found inmurine specimens inoculated with a murinederived hybridoma. This type of contamination may result in infection of the hostanimal(s) and in spread of the disease
throughout an animal facility. In addition,
lymphocytic choriomeningitis virus, which
can lead to disease in humans, has also
been isolated from murine tumor cell lines(Dykewicz, et al., 1992). Mouse hepatitis
virus (MHV) can provoke an immunomodulatory response, influencing thesubsequent production of mAbs. It also
spreads rapidly through an animal facility,
affecting other work in progress. The typicaltesting method is the Mouse Antibody
Production (MAP) test, which involves
injecting a sample of tissue in question
into mice, and, after an incubation period of
3-4 weeks, euthanizing the mice to run
serological tests for a panel of known
adventitious agents (Jackson, et al., 1999).
Molecular tests using polymerase chain
reaction (PCR) and other related techniques
will improve testing capabilities and turnaround times.

3.3 Hybridoma implantation
6

Guideline: Up to 3 x 10 hybridoma cells
in a maximum volume of 1.0ml may be
injected into the peritoneal cavity of aprimed mouse.
Tumor cell lines prone to forming solid

3.4 Monitoring the animals andendpoints
Following injection of hybridoma cells,routine care should include daily observations by appropriately trained staff for the
first week (approximately) and before
ascites fluid accumulation is evident (asindicated by the swelling of the abdomen).
Any observations of unusual behavior orsymptoms during this time should be
addressed promptly. The CCAC guidelines
on: choosing an appropriate endpoint inexperiments using animals for research,teaching and testing (1998) should be used
to develop monitoring procedures and an
appropriate endpoint for the animal.
Pertinent signs of distress include: decreasein activity; hunched appearance; ruffled haircoat; respiratory distress; and weightloss (which may be masked by theaccumulating fluid in the abdomen). Oncethe ascites fluid accumulation has resulted
in obvious abdominal swelling, usually
7-10 days, the condition of the animal must
be assessed at least twice every 24 hours
at regularly spaced intervals. It must be
remembered that the rate of hybridoma cell
propagation in the mouse can be quite
variable.

antibody production, 2002

tumors produce less ascitic fluid. They canbe optimized for ascites production by
performing serial passages of non-attachedtumor cells that have been selected frommice with solid tumors by peritoneal lavage.
Selection of hybridoma cell-lines which do
not adhere to the tissue culture flask
has been shown to reduce the likelihoodof formation of solid tumors. For many6
hybridomas, 3 x 10 cells have been shown
to be the maximum necessary to give gooddevelopment of mAbs and ascitic fluid. In
some circumstances (e.g., hybridomas
produced in 1970s-1980s using first generation myeloma cells), a higher concentrationof cells may be needed to produce any
ascitic fluid. Some rapid proliferating,aggressive cell lines may require a lower
concentration for inoculation as a rapidly
developing tumor can lead to severe
distress and death.

3.5 Ascites tumor growth
Guideline: The increase in body weight
due to the accumulation of ascitic fluidin the abdomen and/or tumor growth
should not produce pain and/or distressto the animal; in no case should theincrease in body weight exceed 20% of
the normal body weight of age and sex
matched animals of the same strain.
In accordance with the CCAC guidelines on:choosing an appropriate endpoint in experiments using animals for research, teaching
and testing (1998), a chart for monitoring
the animals is required with clearly defined
endpoints and reporting lines. Once theendpoint has been reached, abdominalpressure must be relieved by harvesting of
the ascitic fluid – or by humane killing
followed by recovery of the ascitic fluid.
Mice should be weighed daily, beginning
four days after inoculation, to monitor
progression of the ascites tumor by weightgain and to monitor the general health of
the animals. A baseline weight measurement of the mouse should be taken on
the day of inoculation and this value canthen be used to assist in determining
when the mouse reaches an increase of
20% body weight (Workman, et al., 1998).
Caution should be used when relying on
weight, as mice are likely to lose body mass19

ccac guidelines

at the same time as the tumor is growing.
Body condition scores can also be used as
an indicator of well-being (Ullman-Culleré &Foltz, 1999) and can assist in deciding
when to terminate the procedure.

3.6 Ascites fluid collection
Guideline: Depending on the conditionof the mouse, a maximum of two taps ofthe ascitic fluid are permitted, with thesecond tap being a terminal procedure.
Training and experience in tapping or
draining ascitic fluid is essential.
It should be noted that fluid removal carries
the risk of hemorrhage, oedema and death.
For more aggressive cell lines, known tocause significant morbidity, the number oftaps should be limited to one terminal
procedure under general anesthesia.
Anesthesia, as recommended by current
CCAC guidelines may be used for the first(non-terminal) tap. However, anesthesia
leads to a decrease in blood pressure andrespiratory suppression, which may be
detrimental to the already compromisedmouse. Therefore, it is recommended thatmice are placed in an oxygen filled
induction chamber for 5-10 minutes,
followed by mild gaseous anesthesia(isoflurane) to give good immobilizationof the animal for the procedure with rapid
recovery. To minimize any bacterial contamination, the site of paracentesis should be
aseptically prepared for the first tap.
It is recommended that a large gauge
needle (21-22 gauge) be used for paracentesis. A large gauge needle permits rapid
collection of the viscous ascitic fluid, reducing the period of restraint/anesthesia
required; however, too large a needle cancause tissue damage. A maximum of 4-5ml

20

of ascitic fluid may be collected at the first(survival) tap. The abdomen of the mouseshould be palpated to determine whether an
intra-abdominal solid tumor is present,
the presence of a tumor being grounds
for humane euthanasia of the animal.
Administration of replacement fluids (1-2ml
subcutaneous) should be considered whenlarge volumes of ascitic fluid are harvested
and the animals are not terminated(Jackson & Fox, 1995).
The animals should be closely monitored forthe first 60 minutes and regularly for several
hours following the first tap. Any signs of
distress should result in euthanasia of theanimal, according to current CCAC guidelines.
ACCs must ensure that the personnel
responsible for carrying out these procedures (listed on the animal use protocol)
have obtained the necessary training
prior to conducting the procedure. CCAC
guidelines on: institutional animal user
training (1999) mandates the training of any
personnel prior to the performance ofanimal-based procedures.

E. REFERENCES
1. General
ABBAS, A.K. & LICHTMAN, A.H. (2001).
Basic Immunology. 309 pp. Philadelphia PA:
W.B. Saunders.
ABBAS, A.K., LICHTMAN, A. & POBER, J.
(2000). Cellular and Molecular Immunology,
4th Edn. 553 pp. Philadelphia PA: W.B.
Saunders.
ANDERSON, W.L. (1999). Immunology.
200 pp. Madison CT: Fence Creek Publishing(Blackwell).

COLIGAN, J.E., KRUISBEEK, A.M.,
MARGULIES, D.H., et al. (eds.) (1997).
Current Protocols in Immunology. Baltimore
MD: John Wiley & Sons Inc.

glycoprotein E2 induces an immune responsein cotton rats. Virology 278(1):234-243.

JANEWAY, C. Jr. & TRAVERS, P. (1999).
Immunobiology: The Immune System in
Health and Disease, 4th Edn. 633 pp. New
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21

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23

ccac guidelines

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24

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WHARY, M.T., CAREY, D.D. & FERGUSON,
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ccac guidelines

INSTITUTE FOR LABORATORY ANIMALRESEARCH (ILAR) (1999). Monoclonal
Antibody Production: A Report of theCommittee on Methods of Producing
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26

PETERSON, N.C. (1999). In vitro antibodies
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edu/publications/humane_exp/het-toc.htm).
SAXBY, S.J.Y. (1999). Perspective on in vitro
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SHAVLEV, M. (1998). European and US regulation of monoclonal antibodies. Lab Animal
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IACUC guide to reviewing protocols calling formonoclonal antibody production by mouse
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(1994). Some ascites monoclonal antibody
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KUHLMANN, I., KURTH, W. & RUHDEL, I.
(1989). Monoclonal antibodies in vivo and invitro production on a laboratory scale, with aconsideration of the legal aspects of animal
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STEBECK, C.E., FREVERT, U., MOMMSEN,
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MUELLER, U.W., HAWES, C.S. & JONES,
W.R. (1986). Monoclonal antibody production
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ULLMAN-CULLERÉ, M.H. & FOLTZ, C.J.
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WORKMAN, P., TWENTYMAN, P.,
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ascites production of monoclonal antibodies.
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General information on mAbs (http://altweb.
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et al. (1998). Production of recombinant
antibodies in the milk of transgenic antibodies.
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Nature Biotechnology Guide (http://www.
guide.nature.com/).

Other useful references

APPELMELK, B.J., VERWEIJ-VAN VUGHT,
A.M., MAASKANT, J.J., et al. (1992). Murine
ascites fluids contain varying amounts ofan inhibitor that interferes with complement mediated effector functions of monoclonal antibodies. Immunological Letters 33:
135-138.
FROMER, M.J. (1997). NIH denies petition to
ban in vivo mAb production: Lawsuit threatened. Oncology Times 19:37-40.
MARX, U. & MERZ, W. (1995). In Vivo and In
Vitro Production of Monoclonal Antibodies.
Bioreactors versus Immune Ascites. In:
Methods in Molecular Biology, Vol. 45,
Monoclonal Antibody Protocols (ed. W.C.
Davis). Pp. 169-176. Totowa NJ: Humana
Press.
MARX, U., EMBLETON, J.M., FISCHER, R.,
et al. (1997). Monoclonal antibody production:
The report and recommendations of ECVAM
Workshop 23. Alternatives to Laboratory
Animals 25(2):121-137 (http://altweb.jhsph.
edu/publications/ECVAM/ecvam23.htm).
MCARDLE, J. (1997/98). Alternatives toAdditional useful information
antibody production, 2002

3.1

3.2

Lists of suppliers of in vitro produced mAbs
(http://www.frame.org.uk/Monoclonal_
Suppliers.htm).

University of California, Davis CA - Readings
and Resources for mAbs (http://www.vetmed.
ucdavis.edu/Animal_Alternatives/biblio~1.
htm).

F. GLOSSARY
Adjuvant: A substance which increases
the immune response to an antigen when
it is administered at the same time andat the same site as the antigen. When
adjuvants are used, smaller doses of theantigen are required and the antibody
response persists for a longer period oftime.
Affinity: The strength and stability ofthe bond between an epitope and antibody
as measured by the amount of antibodyantigen complex found at equilibrium.
Antibody: A protein which is produced in
response to a particular antigen and whose
unique structure gives it the capacity to
combine specifically with that antigen.
Antibodies are secreted by lymphocytes
and are divided into five classes: IgG, IgM,
IgA, IgE, and IgD.
Monoclonal Antibody: antibody molecules which are produced by cells of one

27

clone and recognize only one epitope onan antigen.

ccac guidelines

Polyclonal Antibody: antibody molecules produced by different families of B
lymphocytes and consequently recognize more than one epitope, even on asingle antigen.
Antigen: Any substance which can bind toan antibody raised against it.
Antiserum: The serum, or non-cellularcomponents of blood which remain after
clotting, which results from the injection of
antigens and contains the antibodies
produced in response to those antigens.
Ascitic Fluid (also called ascites): An
intraperitoneal fluid extracted from animals
that have developed a peritoneal tumor.
Avidity: The strength of the bond between
an antigen and antibody. Avidity depends
on the affinity as well as the valences of theantigen and antibody.
Clone: Genetically identical cells derived
from the same cell.
Monoclonal: derived from the sameclone and therefore all genetically
identical.
Polyclonal: derived from different
clones.
Complement: A set of plasma proteins that
act together to attack extracellular forms ofpathogens. Complement can be activatedspontaneously on certain pathogens, or by
antibody binding to the pathogen.
ELISA (Enzyme Linked Immunosorbent
Assay): Immunological analysis using
antigens or antibodies marked by an
enzyme.
Epitope: The part of an antigen which isrecognized by an antibody. An epitope28

is capable of combining with only one
antibody molecule, but a single antigen may
have more than one epitope.
Granuloma: Chronic inflammatory
response maintained by persistent stimulation.
Histocompatibility: The degree of genetic
similarity between the donor and receiver ofa graft.
Major Histocompatibility Complex: thegenes responsible for the rejection ofgrafts between individuals.
Hybridoma: A biologically constructedhybrid of an antibody-producing lymphoidcell and a malignant (immortal) myeloma
cell.
Immune Response: A humoral or cellmediated response of the immune system
to an antigen.
Humoral Response: the generation of
antibodies in response to the presenceof an antigen.
Cell-Mediated Response: the binding ofa particular kind of T lymphocyte, known
as a cytotoxic T lymphocytes, withforeign or infected cells, followed by lysisof these cells.
Immunoblotting: A technique used to
identify characteristics of protein antigens. A
Western blot is a blot of protein onto nitrocellulose paper followed by detection of
specific proteins using labeled antibodies.
Immunogen: A substance which canstimulate the immune system.
Immunogen/Adjuvant Formulation:
Emulsion: stable oil-in-water or waterin-oil mixture.
Liposome Formation: formation of a

spherical particulate in an aqueous
medium by a lipid bilayer enclosing anaqueous compartment.

Adsorption: attraction and retention ofother material on the surface.
Immunogenicity: The ability to elicit an
immune response.
Immunoglobulin: A member of a group ofproteins having antibody properties, regardless of whether or not its binding target is
known. (To be considered an antibody, theantigen with which it binds must be known.)
In mammalian species there are antibodies
to one of five immunoglobulin (Ig) classes:
IgM; IgG; IgA; IgD; or IgE. In avian species
they belong to one of three classes: IgM;
IgY or IgA.
Immunohistochemistry, Immunoprecipitation, Immunofluorescence, Immunoelectron Microscopy: Techniques for
detection or separation of molecules relying
on binding of the molecules with specificantibodies, often labeled with fluorescentdyes, etc., in order to visualize the locationof the molecules.
Lymphocytes: Cells which circulate
through the lymph and blood and play a rolein immunity. They are subdivided into B
lymphocytes and T lymphocytes.
B Lymphocytes: produce antibodies
and their precursors.
T Lymphocytes: act as the basis of cellmediated immunity and assist B lymphocytes in the production of antibodies.

Plasma Cells: Specialized cells that arederived from B lymphocytes and synthesize
antibodies.

antibody production, 2002

Immunostimulatory Complex (iscom)
Formation: complex of antigen held
within a lipid matrix that acts as an
adjuvant and enables the antigen to betaken up into the cytoplasm of a cell
after fusion of the lipid within the plasma
membrane.

Memory Cells: Lymphocytes that are able
to quickly and intensively respond to a new
antigen due to previous exposure to theantigen.

SCID: Severe Combined Immunodeficient.

Based on definitions from:
BACH, J.-F. (1993). Traité d'immunologie.
Collection Médecine-Sciences. 1205 pp.
Paris: Flammarion.
GENETET, N. (1997). Immunologie, 3 e éd.
Collection Biologie Médicale. 604 pp.
Rennes: Ministère de l'éducation nationale,
Éditions Médicales Internationales.
GODING, J.W. (1986). Monoclonal Antibodies: Principles and Practice. 2nd Edn.
315 pp. London UK: Academic Press.
HARLOW, E. & LANE, D. (1988). Antibodies:
A Laboratory Manual. 726 pp. Cold Spring
Harbor NY: Cold Spring Harbor Laboratory.
REGNAULT, J.-P. (1988). Immunologie
générale. 469 pp. Montréal: Descarie Éditeur.

February 15, 2002

For more information on these andother guidelines contact:
Canadian Council on Animal Care
315-350 Albert Street
Ottawa ON CANADA K1R 1B1(Website: www.ccac.ca)

29

30

ccac guidelines

APPENDIX ACOMMON ADJUVANTS

Freund’s Complete Adjuvant (FCA) is awater-in-oil emulsion of mineral oil, mannide
mono-oleate, and heat-killed Mycobacterium tuberculosis (or M. butyricum) orcomponents of the organism. FCA is apotent adjuvant that stimulates both
humoral and cell-mediated immunity. FCA
is often strongly reactive as the mineraloil cannot be metabolized and the mycobacterial components can elicit severe granulomatous (inflammatory) reactions. The
concentration of mycobacteria varies greatly
among commercial preparations of FCA. In
this regard, it should be noted that if theconcentration of mycobacteria is less than
0.5mg/ml (0.1mg/ml in Broderson [1989]),less severe inflammatory reactions result.
Freund’s Incomplete Adjuvant (FIA) is thesame as FCA minus the mycobacterial cells
or cellular components. FIA is less effective
than FCA for inducing high antibody titresand enhancing cell-mediated immunity.
TM

Ribi Adjuvants are oil-in-water emulsions
in which the antigen is blended with aminimal volume of oil and then emulsified ina saline solution containing the surfactant
Tween 80 (Ribi, et al., 1975). Althoughoil-in-water emulsions are less viscous andeasier to inject than water-in-oil emulsions,they are poor adjuvants alone, requiring
immunostimulants to increase the immunogenicity. The immunostimulants used in theTM
Ribi system include two refined mycobac-

antibody production, 2002

This is a list of the more commonly-usedadjuvants and their basic properties. It
is not an endorsement of any specificproduct.

terial products (trehalose 6,6'-dimycolate
and cell wall skeleton) and a purified gramnegative bacterial product (monophosphoryl
lipid A). For a further explanation ofTM
the biological activities of Ribi adjuvantsTM
see Jennings (1995). Ribi adjuvants areusually less potent than FCA, but they arealso less toxic and have provided satisfactory adjuvant activity for many purposes.
Monophosphoryl lipid A (one of theTMcomponents of the Ribi system) is byitself a potential adjuvant option. As withaluminum hydroxide, the safety profile
of MPL is well documented and provides
excellent adjuvant activity for someimmunogens.
TM

TiterMax Adjuvant relies on a microparticulate water-in-oil emulsion formed witha non-ionic block copolymer (CRL-8941)
and squalene, a metabolizable oil. CRL8941 is coated with silica particles which
stabilize the emulsion. Stability is a key
TMproperty of TiterMax as it enables theemulsion to contain a wide variety ofantigens without the use of large amounts
of toxic emulsifying agents. The copolymer
enables more antigen to be carried on its
surface than if the antigen were in solution.
It also activates complement, assisting in
retention of the antigen in the lymphoidtissues and activation of immunoreactive
cells. TiterMaxTM induces increased expression of class II (Ia) major histocompatibility
complex molecules (MHC) on macrophages, and increases the presentation of
Ag to T cells. When TiterMaxTM works well, it
can induce antibody titers as high or higher
than FCA and exhibits less toxicity.
However, adjuvantation with TiterMax TM
is not always successful. In addition,
TiterMaxTM contains small quantities of anemulsifying agent and silica particles which31

have been implicated in delayed-type
vaccine reactions.

ccac guidelines

Quil A is a partially purified form of saponinor triterpene glycoside derived from the barkof the Quila saponara tree and purified toreduce the presence of components whichcause adverse local reactions. In general,saponins should not be injected intraperitoneally or intravenously due to theirhemolytic activity. As Quil A is only
semi-purified, it can still induce significant
pyrogenic and local reactions. Therefore,prior to using a formulation containing QuilA, it should be evaluated in vitro (i.e., forcell culture toxicity). The adjuvant propertiesof Quil A rely on the formation of iscoms(immune stimulating complexes) which areespecially useful in inducing Abs tomembrane antigens. The iscoms are 35nm
clathrate bodies like micelles made of QuilA, cholesterol, phosphatidyl choline andantigen. The purified saponin component
which has the highest adjuvant/toxicity ratio
is called QS21. Saponin containing formulations should never be administered via
mucosal surfaces, such as intranasal wheninduction of IgA is desired.
Mineral-Based Adjuvants. Three mineralcompounds are generally used in adjuvants:
aluminum hydroxide; aluminum phosphate;and calcium phosphate. Aluminium salts
are claimed to be superior to all other adjuvants in their ability to increase immune
responses against weak immunogens,including those for which FCA does notwork. Alhydrogels are sterile aluminiumhydroxide gels that are pyrogen-free andstable, they have a high adsorptive capacity. At pH <9 alhydrogels have a positive
charge so they readily adsorb negatively
charged molecules (e.g., proteins at neutral
pH).

32

CpG DNA is a non-specific immune activator that can be used alone or in combination
with other adjuvant components to augment
immune responses. Commercial preparations of CpG DNA comprise short pieces
of DNA (oligonucleotides) that contain
unmethylated cytosine-guanine dinucleotides within a certain base contact. The
mammalian immune system has evolved torecognize these sequences, which arefound naturally in bacterial DNA, as a signof infection. Different CpG DNA sequences
activate the immune systems of different
species, and the commercial preparations of this adjuvant are therefore speciesspecific.

Additional Information on Adjuvants
The Adjuvant Guide: CEDARLANE Laboratories Limited (http://www.cedarlanelabs.
com).
Animal Welfare Information Center, Information Resources for Adjuvant and Antibody
Production: Comparisons and Alternative
Technologies (1990-97) (http://www.nal.
usda.gov/awic/pubs/antibody/).
LEENARS, P.P.A.M. (1997). Adjuvants inLaboratory Animals. 207 pp. Rotterdam, The
Netherlands: Erasmus University.
RIBI, E.T., MEYER, J., AZUMA R., et al.
(1975). Biologically active components
from mycobacterial cell walls IV. Protectionof Mice Against Aerosol Infection with
Virulent Mycobacterium tuberculosis. Cellular
Immunology 16:1-10.
STEWART-TULL, D.E.S. (ed.) (1995). The
Theory and Practical Application of Adjuvants.
392 pp. New York NY: Wiley.
WEERATNA, R.D., McCLUSKIE, M.J., XU, Y.
et al. (2000). CpG DNA induces stronger
immune responses with less toxicity thanother adjuvants. Vaccine 18:1755-1762.

APPENDIX B
IMMUNIZATION – RECOMMENDED ROUTES AND VOLUMES(adapted from Leenars, et al., 1999)

SUGGESTED ROUTES OF INJECTION WITH OR WITH0UTADJUVANT

Primary Injection
with adjuvant
without adjuvant
SC
IM+
ID‡
IP*

*
+
‡

Booster Injection(s)with adjuvant
without adjuvant

IV
SC
IM
IP
ID‡

SC
IM+
ID‡

SC
IM
IV
IP
ID‡

Only in mice, and not recommended in general.
Not to be used for viscous adjuvants in small animals.
Not to be used in small animals, and not recommended in general.

TABLE IIMAXIMUM VOLUMES FOR INJECTION OF IMMUNOGEN/
DEPOT FORMING ADJUVANT MIXTURES PER SITE OFINJECTION FOR DIFFERENT ANIMAL SPECIESSpeciesMice, hamsters
Mice, hamsters
Mice
Guinea pigs, rats
Rabbits
Sheep, goats, donkeys, pigs
Chickens*+

antibody production, 2002

TABLE I

Maximum Volume
Per Site

Primary
Injection

Subsequent
Injections

100µl (0.1ml)
50µl (0.05ml)
500µl (0.5ml)
200µl (0.2ml)
250µl (0.25ml)
25µl (0.025ml)
500µl (0.5ml) (if in multiple
sites, 250µl [0.25ml]/site)
500µl

SC
IM+
IP*
SC, IM+SC, IM
ID
SC, IMSC
IM+
SC, IM+SC, IM+SC, IMSC, IMSC, IMNot recommended in general for pAb production.
Not recommended in general, in particular not for viscous adjuvants.

33

TABLE III

RECOMMENDED MAXIMUM VOLUME OF INJECTION USEDFOR ANTIGEN WITHOUT ADJUVANT FOR DIFFERENTANIMAL SPECIES

ccac guidelines

Maximum injection volume in mls

Mice
Hamsters
Guinea pigs
Rats
Rabbits
Sheep or goats
Pigs (<50kg)
Chickens

SC

IM

0.5ml
1.0ml
1.0ml
1.0ml
1.5ml
5.0ml
3.0ml
4.0ml

0.05ml
0.1ml
0.1ml
0.1ml
0.5ml
2.0ml
0.5ml

These volumes are suggested maximum
volumes, additional information can befound in the following texts:
BAUMANS, V., TENBERG, R.G.M.,
BERTENS, A.P.M.G., et al. (1993). Experimental Techniques. In: Principles of Laboratory Animal Science (eds. L.F.M. van Zutphen,
V. Baumans & A.C. Beynen). Pp. 299-318.
Amsterdam, The Netherlands: Elsevier.
DIEHL, K.-H., HULL, R., MORTON, D., et al.
(2001). A good practice guide to the administration of substances and removal of blood,
including routes and volumes. Journal ofApplied Toxicology 21:15-23.
HARLOW, E. & LANE, D. (1988). Adjuvants.
In: Antibodies: A Laboratory Manual. Pp. 96-

34

ID

0.05ml0.05ml
0.1ml

IP

IV

1ml
2-3ml
10ml
5ml
20ml
10ml
250ml
10ml

0.2ml
0.3ml
0.5ml
0.5ml
1-5ml
30ml
20ml
0.5ml

124. Cold Spring Harbour NY: Cold Spring
Harbor Laboratory.
HERBERT, W.J. & KRISTENSEN, F. (1986).
Laboratory animal techniques for immunology.
In: Handbook of Experimental Immunology,
Vol. 1, Immunology – Laboratory Manuals (ed.
D.M. Weir). Pp. 133.1-133.13. Oxford UK:
Blackwell.
LEENARS, M.P.P.A., HENDRIKSEN, C.F.M.,
DE LEEUW, W.A., et al. (1999). The production of polyclonal antibodies in laboratory
animals: The report and recommendations of
ECVAM Workshop 35. Alternatives to Laboratory Animals 27(1):79-102 (http://altweb.
jhsph.edu/publications/ECVAM/ecvam35.
htm).

APPENDIX CSTAGES OF MONOCLONALANTIBODY PRODUCTION

The first stage (formation and selection of
hybridoma clones) generally involves theuse of one or more mice or rats. Whenusing mice, it is recommended that they be
6-8 weeks of age and free of any concurrent
infections.

antibody production, 2002

Formation and Selection of the Hybridoma Clone

1. The immunogen is injected into theanimals, often, but not always, in combination with an adjuvant to enhance theimmune response (see Sections C.2C.7 – Immunization Protocol, Standard
Operating Procedures, Immunogen
Preparation, Choice of Adjuvant, Routeof Injection, and Volume and Number of
Injection Sites).
2. In general, the interval between booster
doses of immunogen should be aminimum of 7-10 days, unless a rapid
immunization protocol without adjuvant
is used.

Production of monoclonal antibodies on a laboratory scale –in vivo (mouse) and in vitro (cell culture) methods

Reproduced with permission from Kuhlmann, I., Kurth, W. & Ruhdel, I. (1989).
Monoclonal antibodies: In vivo and in vitro production on a laboratory scale,with a consideration of the legal aspects of animal protection. Alternatives toLaboratory Animals 17:73-82.

35

ccac guidelines

3. Test bleeds should be performed threedays after the last booster to ensurethat there is an appropriate response to
the antigen and that specific antibodies
are being produced. Most immunologically-based assays for determining
whether the desired Abs are being produced require less than 10µl of mouseserum. Once an appropriate response
has been confirmed, the mouse should
be boosted again; three days after theboost, the mouse should be euthanized
and the spleen harvested. Lymphoidcells are then isolated from the spleen,
and in some cases from the lymph
nodes.
4. The lymphoid cells are fused withparental myeloma cells grown in vitro,by the addition of polyethylene glycol topromote membrane fusion. Only a small
proportion of these cells fuse successfully.
5. The mixture of the two unfused cell
types and the newly formed hybrids
is cultured in a selective cell culture
medium containing HAT. HAT is a
mixture of hypoxanthine aminopterin
and thymidine. One of the components
of HAT, aminopterin, is a powerful toxin
which blocks a metabolic pathway. This
pathway can be bypassed if the cellis provided with the intermediate
metabolites hypoxanthine and thymidine. While spleen cells can use thisbypass pathway and grow in HAT
medium, myeloma cells have a meta-

36

bolic defect which prevents them fromusing the bypass pathway. Consequently, myeloma cells die in HAT
medium. The spleen cells will later die
naturally in culture after 1-2 weeks, but
the fused cells will continue to survive
as they have the immortality of the
myeloma cells and the metabolic
bypass of the spleen cells. Some of the
fused cells will also have the antibody
producing capacity of the spleen cells.
6. Immunoassay procedures are used toscreen for hybridomas secreting thedesired antibody. If positive, thecultures are cloned. This is accomplished by plating out the cells so that
only one cell is in each well. This
produces a clone of cells derived from asingle progenitor which is both immortal
and produces antibodies.
7. The selected hybridoma cells are often
cloned a second time or third time invitro to ensure cultures of truly monoclonal hybridomas with a single
antibody specificity are produced (seeSection D.3.2 – Contamination). At thisstage, cells are grown to larger
numbers in order to prepare for cryopreservation.
8. The propagation of cloned hybridoma
cells can be accomplished either bycontinuing to grow the cells in vitro, orby propagating them in vivo. For
information on in vitro production
systems see Appendix D.

the production of monoclonal antibodies
from hollow fibre bioreactors. 576-587.

INFORMATION ON IN VITROTECHNIQUES FOR
MONOCLONAL ANTIBODYPRODUCTION

DE GEUS, B. The next generation of
antibodies: Production of recombinant
antibodies or fragments derived thereof.
587-589.

74th Forum in Immunology. Research
Immunology 149:533-620, 1998.
DE GEUS, B. & HENDRIKSEN, C.F.M. In
vivo and in vitro production of monoclonal
antibodies: Current possibilities and future
perspectives. 533-534.
FALKENBERG, F.W. Monoclonal antibody
production: Problems and solutions. 542547.
LIPSKI, L.A., WITZLEB, M.P. & REDDINGTON, G.M. Evaluation of small to moderate scale in vitro monoclonal antibody
via the use of the i-mabTM gas-permeable
bag system. 547-552.
PETERSON, N.C. Considerations for invitro monoclonal antibody production. 553557.
MARX, U. Membrane-based cell culture
technologies: A scientifically and economically satisfactory alternative to malignant
ascites production for monoclonal antibodies. 557-559.
FALKENBERG, F.W. Production of monoclonal antibodies in the minipermTM bioreactor: Comparison with other hybridoma
culture methods. 560-570.
LIPMAN, N.S. & JACKSON, L.R. Hollow
fibre bioreactors: An alternative to murine
ascites for small scale (<1g) monoclonal
antibody production. 571-576.
SHI, Y., SARDONNI, C.A. & GOFFE, R.A.
The use of oxygen carriers for increasing

antibody production, 2002

APPENDIX DFRENKEN, L.G.J., HESSING, J.G.M.,
VAN DEN HONDEL, C.A.M.J.J., et al.
Recent advances in the large-scale
production of antibody fragments using
lower eukaryotic microorganisms. 589-599.
PENNELL, C.A. & ELDIN, P. In vitro
production of recombinant antibody
fragments in Pichia Pastoris. 599-603.
LARRICK, J.W., YU, J., CHEN, S., et al.
Production of antibodies in transgenic
plants. 603-608.

ALTSHULER, G.L., DZIEWULSKI, D.M.,
SOWEK, J.A., et al. (1986). Continuous
hybridoma growth and monoclonal antibody production in hollow fiber reactorsseparators. Biotechnology and Bioengineering
28:646-658.
AMOS, B., AL-RUBEAI, M. & EMERY, A.N.
(1994). Hybridoma growth and monoclonal
antibody production in a dialysis perfusion
system. Enzyme and Microbial Technology
16(8):688-695.
BLASEY, H.D. & WINZER, U. (1989). Low
protein serum-free medium for antibody production in stirred bioreactors.
Biotechnology Letters 11(7):455-460.
BLIEM, R., OAKLEY, R., MATSUOKA, K., etal. (1990). Antibody production in packed bed
reactors using serum-free and protein-free
medium. Cytotechnology 4(3):279-283.
BOYD, J.E. & JAMES, K. (1989). Human
Monoclonal Antibodies: Their Potential,
Problems and Prospects. In: Monoclonal
Antibodies: Production and Application (ed. A.
Mizrahi). Pp. 1-43. New York NY: Alan R. Liss.

37

BRENNAN, A., DENOMME, L., VELEZ, D., etal. (1987). A novel perfusion system for
growth of shear-sensitive hybridoma cells andantibody production in a stirred reactor.
Abstracts from the Annual Meeting of theAmerican Society of Microbiologists 87:268.

ccac guidelines

BUGARSKI, B., KING, G.A., JAVANOVIC, G.,
et al. (1989). Performance of an external loop
airlift bioreactor for the production of monoclonal antibodies by immobilized hybridoma
cells. Applied Microbiology and Biotechnology
30:264-269.
FALKENBERG, F.W., HENGELAGE, F.W.T.,
KRANE, M., et al. (1993). A simple andinexpensive high density dialysis tubing cell
culture system for the in vitro production of
monoclonal antibodies in high concentration.
Journal of Immunological Methods 165:
193-206.
FALKENBERG, F.W., WEICHERT, W.H.,
KRANE, M., et al. (1995). In vitro production of
monoclonal antibodies in high concentration ina new and easy to handle modular minifermenter. Journal of Immunological Methods
179:13-29.
GOODALL, M. (1997). Production of monoclonal antibodies: In vivo versus in vitro
methods. In: Animal Alternatives, Welfare andEthics: Proceedings of the 2nd World
Congress on Alternatives and Animal Use in
the Life Sciences. (eds. L.F.M. van Zutphen &M. Balls) Pp. 965-972. Amsterdam: Elsevier.
GORTER, A., VAN DE GRIEND, R.J., VAN
EENDENBURG, J.D., et al. (1993). Productionof bi-specific monoclonal antibodies in ahollow fibre bioreactor. Journal of Immunological Methods 161(2):145-150.
HEIDEL, J.R. & STANG, B.V. (1997).
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38

HEIDEMANN, R., RIESE, U., LUTKEMEYER,
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HEWISH, D. (1996). Ascites and alternatives.
Australian and New Zealand Council for theCare of Animals in Research and Teaching
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HOFMANN, F., WRASIDLO, W., DEWINTER,
D., et al. (1989). Fully Integrated, Compact
Membrane Reactor System for the Large
Scale Production of Monoclonal Antibodies.
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HOLLEY, M.C. (1992). A simple in vitro
method for raising monoclonal antibodies
to cochlear proteins. Tissue Cell 24(5):613624.
HOPKINSON, J. (1985). Hollow fiber cell
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INSTITUTE FOR LABORATORY ANIMALRESEARCH (ILAR) (1999). Monoclonal
Antibody Production: A Report of theCommittee on Methods of Producing
Monoclonal Antibodies. ILAR, NRC.
Washington DC: National Academy Press(http://grants.nih.gov/grants/policy/
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JACKSON, L.R. & FOX, J.G. (1995).
Institutional policies and guidelines on
adjuvants and antibody production. Institutefor Laboratory Animal Research Journal 37(3):141-152.
JACKSON, L.R., TRUDEL, L.J., FOX, J.G., etal. (1996). Evaluation of hollow fiber bioreactors as an alternative to murine ascites
production for small scale monoclonal
antibody production. Journal of Immunological
Methods 189(2):217-231.
JACKSON, L.R., TRUDEL, L.J. & LIPMAN,

N.S. (1999). Small-scale monoclonal antibody
production in vitro: Methods and resources.
Lab Animal Autumn special edition:20-30.

JENSEN, D'A. & PETRIE SMITH, C. (1999).
Selected list of company and instituteresources providing new technologies inmonoclonal antibody production. Lab Animal
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MARX, U., EMBLETON, M.J., FISCHER, R.,
et al. (1997). Monoclonal antibody production.
The report and recommendations of ECVAM
Workshop 23. Alternatives to Laboratory
Animals 25(2):121-137 (http://altweb.jhsph.
edu/publications/ECVAM/ecvam23.htm).

KAGAN, E., VIERA, E. & PETRIE, H.T.
(1997). Comparison of hollow fiber bioreactors
and modular minifermentors for the productionof MABs in vitro. In: CAAT Technical Report#8: Alternatives in Monoclonal Antibody
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MCARDLE, J. (1997/98). Alternatives toascites production of monoclonal antibodies.
National Agricultural Library, Beltsville MD.
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KARU, A.E. (1993). Monoclonal Antibodies
and Their Use in Detection of Hazardous
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MORO, A.M., RODRIGUES, M.T.A.,
GOUVEA, M.N., et al. (1994). Multiparametricanalyses of hybridoma growth on cylinders ina packed-bed bioreactor system with internal
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Journal of Immunological Methods 176:
67-77.

KUHLMANN, I., KURTH, W. & RUHDEL, I.
(1989). Monoclonal antibodies: In vitro and invivo production on a laboratory scale, with aconsideration of the legal aspects of animal
protection. Alternatives to Laboratory Animals
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OH, D.J., CHOI, S.K. & CHANG, H.N. (1994).
High-density continuous cultures of hybridoma
cells in a depth filter perfusion system.
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KURKELA, R., FRAUNE, E. & VIHKO, P.
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Biotechniques 15(4):674-683.

PANNELL, R. & MILSTEIN, C. (1992). An
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LERNER, R.A., KANG, A.S., BAIN, J.D., et al.
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PERSSON, B. & EMBORG, C. (1992). A
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LOWREY, D.M., MESLOVICH, K., NGUYEN,
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JASPERT, R., GESKE, T., TEICHMANN, A.,et al. (1995). Laboratory scale production of
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MARX, U. & MERZ, W. (1995). In Vivo and In
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ccac guidelines

RAMIREZ, O.T., MUTHARASAN, R. &MAGEE, W.E. (1987). A novel immobilized
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