Production of Monoclonal Antibodies
Monoclonal antibodies can be made in animals immunized with purified protein, peptides, or bacterial or mammalian fusion proteins. The majority of the mAbs are made using fusion proteins since they can be produced in large quantities and most closely resemble, or are identical to, native protein. The first step in the production of monoclonal antibodies is the expression of the purified antigen. This often underappreciated step is critical to the success of the project.
Protein Expression and Purification
The Center for Therapeutic Antibody Development (CTAD) has expert assistance available to prepare, purify and express proteins using methods that maximize native protein folding and stability. In most instances proteins can be produced when given only the genetic sequence of the protein.
Immunization and Fusion
The CTAD has IACUC approval for the generation of hybridomas in mice, hamster and rat. Immunization protocols are selected according to the target immunogen and intended use of the antibody by the investigator. After fusion of B cells to an appropriate immortalized cell line, clones are selected and grown in semi-solid media. Clones are picked and transferred to 96 well plates for screening and expansion of cells.
The screening method will be done through consultation with the investigator. Generally, the screening method is selected according to the assay of interest. If the priority for a successful antibody is use in flow cytometry, then flow is the most appropriate screening method. Other screens could include ELISA and functional screens. Screening can be done using a proxy antigen, which is a recombinant protein typically similar to the immunogen, or using the naturally expressed protein. All clones generated will be cryopreserved for storage for possible future applications.
Antibodies that pass the screening criteria are isotyped and may or may not go through a second type of screening. When the desired antibodies are identified, CTAD will expand the clone and purify the antibody preparations.
Center for Therapeutic Antibody Development (CTAD)
One Gustave L. Levy Place
Annenberg Building, 17th Floor
New York, NY 10029