Monoclonal Antibody Core Facility Description of Services
- Project Planning
- Immunization Strategies
- Maximizing Fusion Outcomes
- Monoclonal Antibody Screening Options
Project planning is critical to the successful identification of desired monoclonal antibodies and is an integral part of the services offered by the core facility. Production of monoclonal antibodies can be time consuming, labor intense and expensive. To most efficiently use investigator laboratory and core facility resources, planning discussions involving personnel from both laboratories are conducted prior to project initiation. These discussions allow a comprehensive understanding of the target(s) for monoclonal antibody production, early identification and trouble shooting of potential barriers, and the identification of optimal immunization and antibody screening strategies.
Immunization strategies employed by the core facility are tailored to meet distinct project needs. The methods available within the core facility range from standard immunizations to subtractive or decoy immunization strategies. Standard immunization methods are routinely used when the target immunogen is abundant. Subtractive or decoy immunization methods are used to facilitate generation of monoclonal antibodies directed against rare molecules or cell subsets present within complex protein or cell mixtures.
The core facility has optimized the early handling and selection of hybridomas to maximize fusion outcomes. Spleen or lymph node cells recovered from immunized mice or rats are fused with the myeloma SP2/0. Following cell fusion, cells are plated in a semisolid medium that supports expansion of cell hybrids. Cells grow in this medium as discrete clones that are easily visualize after approximately 8-12 days of incubation. Individual clones are then transferred to liquid culture in 96-well plates to facilitate further expansion and antibody production. On the fourth day following cell transfer, supernatants are harvested for antibody screening, and replica 96-well cultures are prepared and cryopreserved.
This early cell handling strategy offers investigators multiple advantages over standard liquid culture expansion systems. Advantages include:
- The ability to perform primary antibody screens using supernatants from cloned hybrids. This strategy eliminates the potential for loss of hybridomas when cloning is performed after primary screening is completed. The strategy also enhances accuracy in that evaluated supernatants do not contain multiple antibodies as can occur with standard liquid culture expansion methods;
- Screening can be performed on larger numbers of hybrids. By avoiding screens of supernatants from mixed clones, a larger number of candidate supernatants from discrete clones are available to screen;
- The transfer of clones from semisolid medium to liquid culture results in uniform cell expansion rates. Uniform expansion rates simplify coordination of large-scale supernatant screening and increase cryopreservation efficiency by allowing cultures to reach optimal cell densities prior to being frozen; and
- The early cryopreservation of all hybrids provides investigators with the ability to retrieve hybridomas of potential interest long after fusions are performed. This service provides investigators with the opportunity to re-evaluate and retrieve desired hybridomas that may not have been identified during the early stages of hybridoma screening.
The core facility offers investigators multiple antibody screening options. Antibody screening is perhaps the most important element associated with identifying desired hybridomas. To that end, core facility personnel have extensive experience in identifying and developing screening strategies designed to best meet investigator need. For all projects, the screening strategies will be established before or during the immunization phase of the project. Personnel from the investigator laboratory or the core facility can conduct screening of hybridoma supernatants for desired antibody. Screening options offered by core facility personnel include: ELISA, immunoblots, flow cytometric analyses, and tissue section analyses.
The core facility offers a full range of services to both members and non-members. For further information regarding these services or other aspects of the core facility please inquire here.