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 ammonium sulfate or protein A or G affinity columns.

  • Ammonium sulfate. Ammonium sulfate precipitation of antibody proteins is a crude step in which purification is based upon the molecular weight of the antibody. Some antibody preparations are adequately cleaned up with ammonium sulfate alone, as it eliminates low and high molecular weight proteins, depending upon the weight of the desired antibody isotype.
  • Protein A or G. Protein A or G affinity column purification may be performed on ammonium sulfate precipitated antibody or be used in place of ammonium sulfate precipitation. Protein A affinity columns bind gamma chain-specific immunoglobulins, such as IgG2a or IgG2b, while protein G affinity columns more efficiently bind 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 10 to 50 mg, with an average of 14 to 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. Mayo investigators can also visit the Antibody Hybridoma Core intranet site (must be logged in to the Mayo network).

  • Membrane chambers. Concentrated culture supernate can also be generated using in vitro membrane chambers. Yield will vary based on chamber size and cell line. Contact the core for more information.
  • Ascites fluid. In vitro methods, which use roller bottles or membrane chambers, have replaced the generation of ascites fluids as the core's primary method for antibody production. Only in instances where in vitro methods prove inadequate — such as when there are poor yields or if the hybridoma will not grow — will ascites fluid methods be utilized.

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 be purified as described above.