Immunization and serum collection
Mice are acclimated for one week prior to immunization and then housed in a pathogen-free facility. Immunogens commonly used are purified proteins, synthetic peptides, cells or cell membranes. Most material for immunization is mixed with adjuvant (Ribi or TiterMax). Immunizations (boosts) are done in two- to four-week intervals. Mice are then bled and sera is collected for screening to determine the presence of the specific antibody desired and its titer. This screening can be performed by the investigator or the Immunology Core.
When the titers to the immunogen in the injected mice are elevated, one or two mice will be selected for spleen harvest and fusion to a myeloma fusion partner cell line. The fusion of the spleen cells with a HAT-sensitive myeloma fusion partner will be done using a 50 percent polyethylene glycol-electrolyte solution. Following the core's standard protocol, the fusion is plated in 96-well plates at a total cell concentration of ~1 x 105 cells per well in the HAT selection media.
Fusion plates are screened 10 to14 days after the fusion is performed. Initial screening of the fusion plates by ELISA is performed by the core. ELISA positive samples (100 to 150 microliters) are given to the investigator’s lab for screening on other assays (immunoprecipitation, immunofluorescence, Western blot and others) At this stage there are likely many wells of cells that test positive for antibodies of interest. To maximize the chances of success the core has the capability to grow and cryostore many of these promising heterogeneous cultures to provide some insurance in case the top candidates do not end up working out.
Following the fusion and drug selection, the next step is to isolate the hybridomas producing antibodies of interest through the screening process. The choice of the appropriate screening strategy is one of the most important parts of hybridoma production. This screening strategy should be determined prior to the fusion. Each assay in the screening strategy should be fully developed and robust, as there will not be enough time to eliminate problems with the assay during the screening of the fusion.
The initial assays used to screen the fusion must be able to screen a large number of samples. Many hundreds of samples will have to be assayed. Unless the initial screening assay can be done on a large number of samples at a reasonable cost and effort, the isolation of interesting hybridomas is likely to fail. In addition, the assay must be sensitive enough to detect low levels of specific antibodies present in the fusion supernatants.
Since a monoclonal antibody that works in one assay may not function in another, the eventual use of the antibody must be considered when designing the screening strategy. As soon as possible, the antibodies should be tested in the assay in which they are intended. Such as through the use of Western blot, FACS or immunohistochemistry (an assay specifically designed for its antibody application).
The core screens fusions by ELISA using a 96-well microtiter plate. The facility may assist in other assays (for example, flow cytometry, immunohistochemistry and others) depending on the specific requirements of the screening strategy that the core works out with the investigator.
Cloning and subcloning
The purpose of cloning is to isolate a single cell that produces the desired antibody from the hundreds of other cells in a culture so that a monoclonal cell line can be established. Immediately after the fusion of spleen cells to myeloma cells, several hybrid cells, all producing different antibodies, are often growing in the same microtiter well (using the core's standard fusion protocol).
The fusion process also leads to the formation of unstable hybrids, which lose chromosomes to become stable. This process can lead to the formation of mutants that do not produce or secrete antibody. These mutants often overgrow the desired hybrids that are producing antibody. For these reasons, it is essential that cultures producing interesting antibodies are either subcloned as soon as possible to isolate the single cells that are producing the desired antibody, or cryostored for future subcloning. To maximize success the core can grow and cryostore up to 50 of these heterogeneous cultures.
The most common method of cloning hybridoma cells is to dilute the cell culture out, so that when plated in a microtiter plate, there is only one cell deposited in each well. This cell then divides and gives rise to a culture where all cells are producing antibody that is of the same specificity. Because each cell line has a different cloning efficiency, it is necessary to plate out the cells using several different cell concentrations.
The standard limiting dilution cloning that the Immunology Core performs on each selected culture, is to plate out the cells in 96-well microtiter plates using the following calculated cell concentrations: 2.0, 1.0 and 0.5 cells per well. An enriched hybridoma media with growth factors is used to support the growth of single-cell hybridomas. When colonies begin to appear, each well is looked at microscopically to determine the wells where it is most likely that only one cell gave rise to the growth of the new culture.