The subcloning of a hybridoma has two purposes. Firstly, once we have identified the cells containing the desired antibody, the specific cell line producing this antibody must be isolated from others in the culture and established as a monoclonal cell line. The separation of this cell line can be achieved by limiting dilution or using single cell deposition by the sorters in Monash FlowCore. The second reason is because the fusion process also leads to the formation of hybrids which lose chromosomes in order to become stable. Loss of genes involved in the production of antibodies will result in Ig non-producers which often out perform the antibody-producing hybridoma cells and ultimately take over the culture.
Because each cell line has a different cloning efficiency, we plate the cells at different cell densities. We view each well microscopically and screen the all wells containing subclones by ELISA. The top 2 positive wells are expanded for the next round of subcloning. Once all wells are positive the culture is monoclonal and we will then proceed to antibody purification. At all stages we cryopreserve vials of cells for safe storage.