Antibodies are considered essential components of many research projects.

1. Statutory requirements
2. Polyclonal antibody production
3. Monoclonal antibody production

1. Statutory requirements

If you are going to use animals in your research, you must be familiar with the Australian code for the care and use of animals for scientific purposes 8th edition 2013, (The Code), Sections 3.2.1, 3.3.69 to 3.3.71. Failure to comply with the code may incur hefty penalties and imprisonment.

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2. Polyclonal antibody production

The in vivo production of antibodies is greatly enhanced by the use of adjuvants – substances that increase the immune response of the body to an antigen.

In the past, serious concerns have been raised regarding the effect of adjuvants on the welfare of individual animals.

The Animal Ethics Committee (AEC) requires research personnel to seriously consider using the least invasive or non-animal alternatives of antibody production, as detailed below, in preference to traditional methods. The AEC also recognises that under limited circumstances the use of an in vivo system is unavoidable.

Preferred Methods (in order of preference)

1. Egg yolk

The use of chickens where the egg yolk is the antibody source is a relatively non-invasive technique. The amount of purified antibody obtained from one laying hen is 10 times greater than the amount that can be purified from the serum of a rabbit over the same time frame.

This system is appealing on both animal welfare and cost grounds. (Svendsen et al, 1995).

2. Non-ulcerative adjuvants

The selection and use of non-ulcerative adjuvants depends on experimental requirements. Information on the following can be obtained from the Animal Welfare Officer:

• TiterMax
• TiterMax Gold
• Ribi system
• Montanide/Marcol
• Aluminium Salts
  
• Muramyl dipeptide.

The use of Freund’s Adjuvant - specific policies

Freund’s Adjuvants (FA) are not recommended unless all alternatives have been excluded. FA are still perceived as the "gold standard" for the production of polyclonal antibodies. Freund’s Complete Adjuvant (FCA) is a water-in-oil emulsion containing mycobacteria.

Freund’s Incomplete Adjuvant (FIA) is the same emulsion without added mycobacteria. The addition of mycobacteria is thought to enhance the immune response by stimulation of the inflammatory process.

FCA can cause chronic ulceration with inflammation and formation of granulomatous lesions present up to six months after immunisation. The AEC is aware of the considerable controversy surrounding the use of FCA and therefore will only consider its use under the following circumstances, which must be fully justified by the Chief Investigator:

• where the antigen being used is extremely valuable and only available in small quantities
• for the induction of cell-mediated immunity
• for the development of specific animal models.

Only one priming dose of FCA is permitted. This must be given in an appropriate maximum volume; for example for rabbits, antigen of 1 ml in total, sub-cutaneously and divided among six to eight sites, ensuring due care is taken in preparation of the site prior to administration – that is, the rabbit must be shaved and skin swabbed to ensure aseptic technique observed at all times.

The AEC endorses The Canadian Council of Animal Care Guidelines on Acceptable Immunological Procedures with some reservations. The overall policy is detailed in the following points.

Sites of inoculation - for all adjuvants

• Intradermal: The AEC has banned the use of intradermal injections of all adjuvants. Intradermal injections are painful and difficult to perform. Intradermal injection of FCA almost always causes ulceration and infection. Any researcher wishing to appeal this decision should contact the Animal Welfare Officer.
• Subcutaneous injections: Subcutaneous injections are less painful and easier to perform. Due care must be given to aseptic technique.
• Intramuscular route: The AEC does not recommend intramuscular injection of adjuvants as the injection is painful, and detection of inflammation and/or abscesses is difficult. Any investigator wishing to use this method must submit full justification to the AEC.
• Intraperitoneal: This procedure is not recommended, but where the case can be justified this route will be permitted in mice only at a maximum total volume of 50 µl.
• Intravenous route: The AEC does not recommend intravenous injection of any adjuvant. Any investigator wishing to use this method must submit a full justification to the AEC.
• Footpad injections: The AEC will consider fully justified requests for footpad injections with the following provisions:
  • only one footpad to be injected
  • a maximum volume of antigen/adjuvant, eg: 50 µl in rats
  • animals must be maintained on soft bedding
  • animals must be checked twice-daily
  • analgesia should normally be administered.

Monitoring

Injection sites must be monitored regularly by investigators. Appropriate monitoring is a minimum of once daily for four weeks after each injection with FCA, and at least three times weekly for four weeks after each injection with other adjuvants.

Any inflammation/lesions that develop must be recorded and checked daily. Any animal showing a degree of pain/distress not anticipated must be euthanased.

Should research or animal care staff have difficulties in establishing whether an animal is suffering pain or distress as a result of the procedures, please contact the Animal Welfare Officer for advice on euthanasia criteria. Please promptly notify the Animal Welfare Officer of any animal welfare problems.

Safety

Investigators must take due care in the preparation and use of FCA and FIA.

The success of the immunisation is partially dependant on appropriate mixing, but the high viscosity of the oil-in-water emulsions makes this difficult and must only be performed by trained personnel with appropriate equipment and protective clothing.

Should the mixture spray out under pressure, or inadvertently be injected into personnel, then they are at high risk of developing a severe reaction at the site, and must immediately seek medical attention.

The incident must be reported as per Risk Management Guidelines.

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3. Monoclonal antibody production

The production of monoclonal antibodies (MABs) in vivo has been a common technique in research and diagnostic and therapeutic procedures. MABs are of single, selected specificity and are secreted continuously by a hybridoma line. The technique for production occurs in two phases:

1. Phase 1: the initial immunisation with a target antigen to obtain the antibody producing cells, and the selection of antibody-producing hybridoma cells in vitro.
2. Phase 2: the propagation of the cloned hybridoma cells either by continued growth in vitro or through the propagation of ascites tumours in vivo.

In many European countries the use of animals for ascites production, that is, phase 2, has been banned.

In the 1980s, serious doubts were raised regarding the necessity of such a painful procedure. ECVAM (the European Centre for the Validation of Alternative Methods) have recognised the urgent need for experts to disseminate information about antibody production, taking animal welfare issues into consideration by publishing the Report and Recommendations of ECVAM Workshop 23 – Monoclonal Antibody Production.

The AEC requires the highest possible standard of animal welfare to be maximised at all stages during the production of MABs. Investigators must give serious consideration to the non-animal alternatives available during phase 2 of production. This should be detailed at the time of application.

Phase 1: The primary immunisation - factors for consideration

• Traditionally FCA has been used as the adjuvant at this stage of production. A maximum total volume (antigen plus adjuvant) of 50 µl is to be given intraperitoneally.
• The mouse must be euthanased as soon as a sufficient response has been obtained.
• A minimum number of secondary boosts with FIA may occur, but test bleeds to assess titre must occur after each boost. A maximum of two boosts is permitted.
• Animals must be regularly monitored and in the event of unexpected pain/distress, must be euthanased.
  
• Should research personnel have difficulties establishing criteria for euthanasia, the Animal Ethics Officer should be promptly contacted.

Phase 2: In vitro production of MABs

It is during the various steps in Phase 2 of the traditional MABs production method that significant pain and distress occurs. For this reason, alternative non-animal technologies have been developed.

A wide range of in vitro MAB production systems are available to suit the needs of all potential users. In vitro methods have the additional advantage of producing antibodies with very high immunoactivity compared to those produced in vivo. The three main methods of in vitro production are:

• suspension cultures
• membrane culture systems
• high cell density bioreactors.

As stated in the ECVAM, "continuing efforts to produce the MAB in vitro would be expected, in themselves, convenience, 'custom and practice', lack of equipment, and/or lack of familiarity with cell-culture methods are not justifications for new or continued use of the ascites method."

The AEC requires all reseachers planning the production of MABs to fully justify the use of animals, and to consult the ECVAM document prior to application.

In Vivo production of MABs

Where the AEC is willing to approve the use of mice, each phase of the in vivo procedure must be fully detailed with monitoring schedules, criteria for euthanasia and so on, and limited in terms of time and the number of animals required.

Substantial pain and distress is associated with all stages of in vivo production and thus the AEC requires a high level of detail to be provided in the proposal.

• Pristane priming: Pristane is an irritant. Usually 0.2 ml is given to "prime" the abdomen 14 days before hybridoma is injectd. Pristane can cause a significant granulomatous reaction and peritonitis resulting in weight loss, hunching and so on. Should this occur, the mouse must be euthanased before the hybridoma is injected. Monitoring must occur at least once daily. An alternative to Pristane is FIA.
• Injection of hybridoma: Injection is given under aseptic conditions. The hybridoma can produce both solid tumours and fluid within four to eight days of the injections. The rapidly growing tumour and ascites causes pain and distress, including abdominal distension, inability to breathe and move easily, and/or indigestion/heartburn. Mice must be euthanased without delay when they show more than mild distress, obvious tumours, significant dehydration or general ill-health.
• Ascites Production: In the absence of the above signs, the volume of ascites should not exceed 20 per cent of the initial body weight. This percentage increase in body weight is indicative of an almost imperceptible swelling of the abdomen. (UKCCCR Guidelines).
• In the absence of ascites production, mice must be euthanased 18 – 20 days after hybridoma injection, or before, should ill-health occur. Mice must be checked twice daily by trained staff and all observations recorded.
• Harvesting of Ascites: Only one abdominal tap is permitted and this must occur under anaesthesia as part of a terminal procedure.

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