Editing Hybridoma production (section)

=== Discussion ===

The generation of monoclonal antibodies by hybridoma technology requires an intense and detailed epitope analysis, especially with regard to the final application, when the antibody should recognize the target. This is often underestimated by users and leads to antibodies with weak performances. The fusion process is always random, which means that the outcome of specific hybridomas is highly dependent upon the cell ratio and the vitality at this point. After limited dilution, the cells are very unstable and require stringent monitoring of cell growth and antibody production. Those parameters should be carefully analyzed when troubleshooting the method.

Further limitations of the technique include the time needed for cell line generation and the selection of the desired antibody-producing cell. Due to the tetraploid set of chromosomes after fusion, the cells are very unstable and afterwards reduce the set to haploid again. This could lead to the loss of antibody-related genes, causing the cells to not produce antibodies anymore. Another limitation is that the selection of the desired antibody-producing cells is based on the culture supernatant and not on the purified antibody solutions. Therefore, a final characterization of the antibody is, in most cases, only possible after mass culture of the selected hybridoma clone. Thus, the risk that the antibody is not able to work in the final application is very high.

Several technological modifications, such as laser-enabled analysis and processing technology (LEAP)<sup>16</sup>, affinity capture surface display (ACSD)<sup>17</sup>, or gel microdrop technology<sup>18</sup>, were described in order to circumvent the drawbacks during the selection of suitable antibodies. All these methods are able to improve different aspects of hybridoma generation and selection, but most of them require costly equipment or optimization for every single cell line<sup>16</sup>.

Another improvement or modification that was developed in our lab is a novel immunization strategy with chimeric viral-like particles (VLPs), as described in Messerschmidt ''et al''<sup>19</sup>. The particles can be modified by the desired epitope sequences embedded in the viral coat structure and presented on the surface of those particles. This leads to very fast and specific immunization after 4 weeks in the corresponding animals. The generated monoclonal antibodies have IgG2 or IgG1 isotypes and are dependent upon the epitope selection highly specific for the native antigen. We also developed a specific selection step for hybridoma cells by using artificial cell surface markers. The used transgenic myeloma cells display a stably inserted surface marker. This surface marker can be used to antigen- or isotype-specifically sort hybridoma cells directly after fusion, without limiting the dilution and without excessive ELISA screening, as is actually necessary in conventional hybridoma technology<sup>20</sup>.

As mentioned above, there are some promising alternatives available in monoclonal antibody generation that, in the future, could improve the handling and reduce the drawbacks of the current hybridoma technology. Nevertheless, the procedure will always require the detailed identification of the desired epitope in combination with a stringent and carefully planned application so that the generated monoclonal antibody works successfully. The generation of monoclonal antibodies by hybridoma technology will be an important and necessary step in the future due to the enormous demand in science, diagnostics, and therapy.