



Antibody production using animals
Antibody production using animals is a matter of ethical concern primarily because it involves varying degrees of suffering for the animals involved.
Animals used for antibody production are typically mice, rats, hamsters, guinea pigs, rabbits and sheep, but can also be goats, horses, llamas, monkeys and other species. Production by the ascites method generally uses mice but can also use rats.
Animals are first immunized by injection with an antigen, to which the animals’ immune systems will produce antibodies. The antigen injection includes an adjuvant to help promote a stronger immune system reaction. To obtain good levels of usable antibodies, an animal will be immunized several times. Physical reactions to immunisation range from moderate to severe inflammatory response at the site of administration and each animal reacts slightly differently to immunisation with the same substance. The adjuvants used to enhance the immune response can as mentioned have severely unpleasant effects on the animals. After immunization, blood samples are taken at intervals to assess the antibody concentration. Finally, animals used for monoclonal antibody production will normally be killed, blood will be collected, and the spleen will be removed. Subsequently the lymphoid cells, including progenitor antibody-producing cells, are isolated from the spleen. These lymphoid cells are fused with myeloma cells which have been cultivated in vitro. The newly formed hybrid cells are cultivated in vitro and special cloning procedures are used so that the cultivated hybridomas should have only a single antibody specificity. In production they can then continue to be cultivated in vitro.
For production of monoclonal antibodies (mAbs) by the ascites method, the immune systems of the animals are suppressed by intraperitoneal (i.p.) injection of a primer, such as pristane or Freund’s incomplete adjuvant, about two weeks before the animals are injected i.p. with the hybridoma cells prepared as described in the previous paragraph. The hybridoma cells then multiply in the peritoneal cavity and the ascitic fluid which forms is a very rich source of the secreted antibody. When sufficient ascites fluid has formed, the animal is killed, and the ascites fluid is collected. Sometimes the animal may be anaesthetized, tapped for ascites fluid and allowed to wake up. When the ascites fluid has reformed, the animal will then be killed, and a second harvest of ascites fluid is taken. The ascites method is extremely painful for the animals due to injection of the primer, which causes peritonitis, abdominal tension, and invasive tumours. The ascitic mAbs tend to have lower immunoreactivity than those produced in vitro and greater batch to batch variability due to contamination with immunoglobulins and cross-reacting cytokines.
In contrast to mAb production, animals used to produce polyclonal antibodies are tapped many times over their lifetime for blood containing antibodies. Rabbits are typically used and are immunized with either complete or incomplete Freund’s adjuvant and are tapped for blood every 14 days. Freund’s adjuvant can result in significant pain or distress and is known to cause inflammation. For that reason it may only be used at the initial immunization. Incomplete Freund’s adjuvant may be used at subsequent booster immunizations. The amount of blood to be taken is supposed to be calculated according to the animal’s weight. Blood is generally tapped from the rabbit’s ear. As withdrawing blood through a needle is inclined to cause lump formation at the withdrawal site, instead a small puncture hole is made near the edge of the ear and blood is allowed to drip into a container before bleeding is stopped and the animal is returned to its cage. Rabbits used for polyclonal antibody production are tapped regularly for between two and six years. The annual statistics on animal use will only register these animals when they are first taken into use.