Following is a summary of the many of services that can be performed by the Monoclonal Antibody Facility. If you don’t see the sort of monoclonal or polyclonal project you have in mind, please contact us directly via email.
Female Balb/c mice are immunized with the antigen of choice to provide a source of antibody-secreting splenic B-cells. Complete and Incomplete Freund's Adjuvant is routinely used to maximize the chances of a successful result. Mouse serum titers are analyzed to evaluate the success of the immunization series and the effectiveness of the screening assay. Serial dilutions of serum from the immunized mice are tested by ELISA to determine which individual is the best candidate to provide spleen cells for the fusion.
All needed materials (syringes, common adjuvants, diluents, etc.) are stocked in the facility. Routine immunizations require from three to six months. Some immunogens will require alternative and possibly longer immunization series.
Antibody-secreting splenic B-cells from the selected immunized mouse are fused with immortalized myeloma cells to produce the hybridoma. The SP2/0 myeloma cell line is routinely used.
Proper planning for the fusions is critical.
A fusion is performed only when the immunized mice have an adequate antibody titer. If IgG antibody is preferred, a comparison ELISA to determine the relative levels of IgG and IgM may be required.
The screening assay for the hybridomas to be developed is perfected during serum testing. A suitable assay must be developed in advance of the fusion in order to quickly screen the hybridomas produced.
Because the spleen contains cells that produce a variety of antibodies, the hybridomas resulting from the fusion must be carefully screened to select those producing the antibody of choice. An ELISA test is the most commonly used test for screening hybridomas for antibody production. The Monoclonal Antibody Facility provides this service, but screening using other assays (fluorescence, Western blot, neutralization assay, etc.) may be performed by the investigator's laboratory using in-process samples provided by the MAF. This must be prearranged in order to ensure quick and unambiguous communication of results.
Maintenance of large numbers of hybridomas, once they begin to require individual attention, is laborious and time-consuming. Each of the several hundred cell lines must be cultured independently so that cross-contamination of cell lines does not occur.
Isotype testing is an integral part of the screening process. In some cases, an IgM antibody is acceptable, in others only an IgG will do. Let the MAF know if there are any restrictions on the isotype to expedite selection.
Cloning, Expansion, and Preservation
Once a useful hybridoma cell line is identified, it is sub-cloned and expanded. Eight vials of frozen cells will be prepared for long term storage in a liquid nitrogen Dewar. These frozen cells are maintained by the facility. One vial of the frozen cells will be thawed and checked for viability and antibody production. The others will be stored until needed. The facility will typically provide one liter of spent hybridoma medium containing from 10 to 20 mg per ml of antibody from each clone selected by the client.
Concentration and Purification
Spent tissue culture fluid from the hybridomas contains the antibody. These fluids can be further concentrated by precipitating with saturated ammonium sulfate (SAS) and purified by affinity column chromatography. The facility is equipped to perform both SAS precipitation and affinity chromatography with Protein A or G columns. All concentrated and purified antibody is tested for specific binding, titered, and concentration determined by UV spectrophotometry.
The UGA Bioexpression and Fermentation Facility (UGA BFF) is currently implementing a pharmaceutical Q-10 based quality system. As a part of the UGA BFF, the MAF now falls under this quality directive. Standard operating procedures are used for all laboratory processes and standard reporting forms and procedures are in place to ensure that your product receives the precise attention to every detail that is necessary for reproducible research results.
We can provide release documentation certifying product performance and purity as may be required for your product. Please let us know of any special quality documentation needs.