One-step method for establishing 8-azaguanine-resistant hybridomas suitable for the preparation of triomas

A simple and rapid one-step method for establishing azaguanine resistant (Agr) hybridomas, which can be used as a fusion partner for the construction of triomas (hybridoma x splenocyte), has been developed. The method relies on cloning the hybridoma cells in soft agar supplemented with 20 micrograms/ml 8-azaguanine. The drug-resistant subclones were isolated after 3-5 days, in comparison with 4-5 weeks reported for the conventional adaptation method. The high frequency (about 10(-3) of Agr-mutants achieved by the cloning method was demonstrated with five different hybridoma clones. One of the derived Agr-hybridomas was fused with mouse immune spleen cells in order to demonstrate its suitability for the generation of triomas secreting bispecific monoclonal antibodies.