Screening and Cloning
In order to obtain clonal lines of hybridomas secreting the monoclonal antibody of interest, the hybridoma corresponding to the positive supernatant screened by ELISA (primary screening) is cloned by limiting dilution (or flow cytometry). Each clone is then tested by ELISA (secondary screening) to verify its ability to bind the target antigen or hapten used in the immunization step.
Finally, the specificity of the antibodies secreted by selected clones is controlled to eliminate those that induce cross-reactivity with other negative control antigens.
The hybridomas are obtained at the end of this step.