Purification and Production

Purification of monoclonal and polyclonal antibodies

Monoclonal antibodies are obtained from culture supernate or ascites fluid, while polyclonal antibodies are obtained from mouse antiserum. Purification of antibody from these sources may be performed using protein A or G affinity columns.

  • Protein A or G. Protein A or G affinity columns bind gamma chain-specific immunoglobulins, such as IgG2a, IgG2b, IgG1 or IgG3 isotypes. Purified antibody is then desalted with phosphate-buffered saline, after which the antibody concentration is determined.

In vitro monoclonal antibody production

  • Roller bottles. The in vitro roller bottle method used by the Antibody Hybridoma Core produces highly concentrated antibody supernate, which upon purification using a protein affinity column produces nearly 100 percent specific antibody.

    Antibody concentration is cell line-dependent and can vary, but investigators can expect an in vitro production run from one roller bottle with 1 liter of monoclonal antibody-containing supernate to produce a purified antibody concentration ranging from 20 to 50 mg, with an average of 20 mg.

    The Antibody Hybridoma Core can produce monoclonal antibodies using cell lines either manufactured in the core or developed elsewhere. Prior to their use for antibody production, all antibody-producing hybridoma cell lines not generated by the core are required to be certified as "mouse virus-free and mycoplasma-free." For more information, contact the core.

Polyclonal antibody production

Polyclonal antibodies are produced by immunizing and boosting antigen into Balb/c mice. Each mouse is pre-bled and post-bled, and serum is collected and processed. Antiserum may then be used neat (undiluted) or diluted, or it can be purified as described above.