Antibodies

Antibodies

Antibodies are produced by the immune system of an animal in a specific response to a challenge by an immunogen.

Because of the potential for pain and distress associated with the production of antibodies, along with the high level of competency required and the actual costs of in-house production, consideration should be given to procuring commercially available antibodies from sources that comply with CCAC guidelines.

Polyclonal Antibodies

A polyclonal humoral response is comprised of antibodies derived from various clonal populations having varying specificities (for different 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 of antibodies made in the polyclonal response. The level and quality of the antibodies produced will vary from animal to animal, and from a single animal over time. Therefore, polyclonal antibodies (pAbs) have a finite availability and are subject to possible character change during the period of production.

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 by subsequent booster injections of the antigen, with or without adjuvant. Blood samples are obtained from the animal to assess the level of antibodies produced. Once a sufficiently high titre has been reached, the antiserum is prepared by blood collection, followed by serum preparation with subsequent purification of antibodies from the serum if required.

Animal welfare concerns relate to:

  • choice of adjuvant
  • injection of antigen and adjuvant
  • blood collection

To minimize pain and distress the adjuvant chosen should be the least reactogenic possible that will stimulate sufficient response. Procedures used for injection of the antigen and antibody should ensure that the smallest possible volumes are used, and that they are injected in locations where the reaction has the least potential to cause distress to the animal.

For best practices in blood collection, see Dosing and Blood Sampling.

Monoclonal Antibodies

Monoclonal antibodies (mAbs) are derived from a single clone, and hence, are specific for a single epitope and have a defined affinity for that epitope. Thus, if the right mAb is obtained, it can be extremely specific for the relevant immunogen, and under appropriate conditions, an almost limitless production of a constant product is possible. Monoclonal antibodies can be produced in vitro and in vivo.

Ascites fluid production and tumour growth have been reported to cause pain in human patients. Therefore, the production of mAbs in mice by the ascites method raises several animal welfare concerns:

  • Intraperitoneal (IP) injection of primer (e.g., Pristane or Freund’s Incomplete Adjuvant)
  • The effect of primer after administration (a chemical irritation leading to peritonitis and the secretion of serous fluid)
  • IP inoculation of hybridoma cells
  • Growth of tumour cells in the abdominal cavity (primary effects) as solid peritoneal plaques that infiltrate the abdominal wall and/or abdominal organs
  • Complex pathophysiological and pathological changes
  • Immobilization and administration of anesthetic prior to ascitic fluid harvesting

A number of in vitro replacements for the mice ascites method of mAb production have been developed. It is now generally accepted that the in vitro techniques of mAb production have progressed to the point where these techniques can be used for more than 90% of mAb production. In addition, a number of countries have instituted a ban on routine in vivo production of mAbs.

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