Monoclonal antibodies production in vivo

A few words about controversy

The European legislation has recently restricted the use of living animals for scientific purposes (see the directive 2010/63/EU published in the Official Journal of the European Union), and especially for the production of monoclonal antibodies within ascites. Indeed, animals can feel the stress and pain caused by such methods, and must be used only as a last resort.

For that reason, we will always first propose you to produce your monoclonal antibody in vitro and carry out an adapted feasibility study. If the feasibility study demonstrates that the antibody-producing hybridoma is not suitable for a production in vitro (and in this case only), we will consider producing your antibody in vivo.

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Why produce in vivo?

The environment in which the hybridoma cells are implanted is more adapted to development, for cells can get all required nutrients and development factors for the production of monoclonal antibodies directly from the metabolism of the animal. However, ascitic fluids can contain proteolytic enzymes, which might damage immunoglobulins produced by hybridomas, as well as endogenous immunoglobulins which can reduce the specificity of the antibody solution after purification by protein A or G.

Although new production methods have been developed, and despite the legislation restraining the use of animals to produce monoclonal antibodies in vivo, we are able to carry out this widely used technique to yield high amounts of antibodies within a relatively short period of time.

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Hybridoma cells

You can require us to use a hybridoma clone we just developed for you within a monoclonal antibody development project. You can also provide us with your own clone in cell culture medium shipped at 4°C. Depending on the expected quantity of antibodies to be produced, the number of cells to provide must be adapted. Please to have more information, our scientific staff will advise you accordingly. To be accepted for culture in our laboratory, please note that each hybridoma must be accompanied by a material transfert sheet as well as a certificate testifying the absence of mycoplasma.

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The first step consists in preparing a minimum of four BALB/c or NUDE (e.g. for rat hybridomas) mice with an injection of adjuvant into the abdominal cavity. The adjuvant causes an irritation of the peritoneum and subsequently the secretion of fluid into the peritoneal cavity which allows the growth of ascites.

A concentrated hybridoma cell suspension is then injected into the peritoneal cavity. After adaptation to their new environment, hybridoma cells grow in a constant manner and continuously secrete monoclonal antibodies in the ascitic fluid.

Ascites are collected upon animal sacrifice and ascitic fluid can then be purified on protein A or G column on request. Ascitic fluid usually contains high concentrations of monoclonal antibodies, which can reach up to 1 mg/mL depending on the nature of the hybridoma.

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Yielding 10 to 20 mg of antibody in ascitic fluid takes about one month, and is in most cases less expensive as compared to in vitro production. However, antibody production is greatly dependent on the hybridoma and can require an optimisation of the protocol prior to in vivo production. Several additional weeks might then be necessary to reach the expected quantity of antibodies.

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