The University of Georgia College of Veterinary Medicine

Monoclonal Antibody Facility

Services

The following is a summary of the procedures performed by the Monoclonal Antibody Facility. More detailed information is available upon request.

Immunization

Mice are immunized with the antigen of choice to provide a source of antibody-secreting spleen cells. Two different adjuvants are 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 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 approximately six weeks. Some immunogens will require alternative and possibly longer immunization series.

Fusions

Antibody-secreting spleen cells from the selected immunized mouse are fused with immortalized myeloma cells to produce the hybridoma. The SP2/0 myeloma cell line is used. Proper planning for the fusions is critical. A fusion is performed when the immunized mice have an adequate antibody titer. The screening assay is perfected during serum testing, as it must be developed in advance of the fusion in order to screen the hybridomas produced.

Screening

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 may also be performed by the investigator's laboratory.

As hybridoma screening must be done quickly and unambiguously, the screening protocol must be established and agreed upon before the fusion. Hybridoma medium to be tested will be provided early in the morning for those laboratories performing their own screening. In the case of an ELISA assay, the test samples can be added directly to wells pre-coated with antigen. If an investigator is performing his or her own screening, the test results must be available to the facility by the afternoon following obtaining the samples.

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. It is therefore essential that screening be completed before this situation arises. Consequently, screening assays must be performed in a timely fashion. Laboratories performing their own screening assume additional responsibilities since any delays will require assessment of additional fees on an ad hoc basis. Moreover, the longer it takes to identify useful cell lines, the greater the probability that one or more such lines will be lost.

Cloning, Expansion, and Preservation

Once a useful cell line is identified, it is cloned and expanded. During the expansion stage, the cell lines are screened regularly for antigen binding properties. 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 g per ml of antibody. A second culture method, continuous culture in CL350 flasks, is now available. This technique generates about 30 ml of concentrated antibody, eliminating the need for ammonium sulfate precipitation.

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 G columns. Isotyping assays and testing of purified antibody can also be performed, for an additional fee.

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