The purpose of subcloning is twofold. Firstly, after the successful identification of the primary supernatant containing the desired antibody, the hybridoma cell line producing this antibody has to be established as monoclonal cell line. The separation of this cell line from other cell clones also present in the same well is achieved by limiting dilution. The second reason is of genetic nature. 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 grow faster than the antibody-producing hybridoma cells.

We perform single-cell subcloning by the limiting dilution method. Because each cell line has a different cloning efficiency, we plate the cells at different ccell densities. We employ an enriched hybridoma medium to support the growth of single cell cultures. We view each well microscopically and screen the subclones by ELISA. The cells from positively scored wells are expanded for the next round of subcloning.

To ensure that a hybridoma clone is stable and monoclonal we continue the cloning rounds until every well that contains cells scores positive on antigen-coated plates by ELISA.

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