Hybridoma-free generation of monoclonal antibodies

Abstract

Production of monoclonal antibodies requires immortalization of splenocytes by somatic fusion to a myeloma cell line partner (hybridomas). Although hybridomas can be immortal, they may depend on a feeder cell layer and may be genetically unstable. Since the inception of hybridoma technology, efforts to improve efficiency and stability of monoclonal antibody-producing cell lines have not brought about substantial progress. Moreover, suitable human multiple myeloma-derived cell lines for the production of human antibodies have been very difficult to develop. Here we report a strategy that greatly simplifies the generation of antibodies and eliminates the need for hybridomas. We show that splenocytes derived from transgenic mice harboring a mutant temperature-sensitive simian virus 40 large tumor antigen under the control of a mouse major histocompatibility promoter are conditionally immortal at permissive temperatures and produce monoclonal antibodies. This simple approach may become a method of choice for generation and production of both polyclonal and monoclonal antibodies with advantages in high-throughput discovery and antibody-based immunotherapy.

Fig. 1.

Screening and validation of antibodies obtained from H-2K^b-tsA58 transgenic mouse-derived immortal splenocytes. Cells were released and resuspended in hybridoma medium (see Materials and Methods for details). ELISA against filamentous phage (fd-tet) and against recombinant phage capsid pIII protein was performed as described (2). BSA, hybridoma medium alone, preimmune serum, and secondary antibody served as negative controls. Immune polyclonal serum and anti-phage antibody served as positive controls. Antibodies were plated directly from culture supernatants and tested as follows: 1, preimmune serum at 1:400 dilution; 2, postimmune serum at 1:3,200 dilution; 3–10, supernatants derived different 6-, 24-, and 96-well plates, 2 weeks after plating of the spleen; 11, cultured medium alone as a negative control. Bars correspond to the mean. Standard errors of the mean were <1% of the mean.

Fig. 2.

Characterization of H-2K^b-tsA58 transgenic mouse-derived immortal splenocytes producing anti-phage antibodies. Clones 1–3 correspond to clones that underwent freeze–thaw. All thawed clones recovered and remained positive. Clones 4–9 correspond to different wells expanded from 96-well plates to 24-well plates and cultured for 6 weeks; 10 indicates cultured medium alone as a negative control. Other controls included were pre- and postimmune sera. Bars correspond to the mean. Standard errors of the mean were <1% of the mean.

Fig. 3.

Evaluation of H-2K^b-tsA58 transgenic mouse-derived immortal splenocytes producing antibodies against intact phage particles and against the pIII phage capsid protein. 1, culture medium, negative control; 2, preimmune serum; 3–7, supernatants derived different monoclonal lines after 8 weeks in culture. 4, 5, and 7 react strongly with the pIII protein and, at this concentration, also react with intact filamentous phage particles. Bars correspond to the mean. Standard errors of the mean were <1% of the mean.

Fig. 4.

Reactivity of supernatants from H-2K^b-tsA58 transgenic mouse-derived immortal splenocytes producing antibodies against phage proteins. Reactivity was evaluated after incubation with preimmune serum, postimmune serum, an anti-phage antibody, or supernatants containing anti-phage IgGs secreted from immortal splenocyte clones, as indicated. Cell culture medium alone served as an additional negative control. Antibodies reacting specifically against pIII and pVIII phage capsid proteins (arrows) were detected in supernatants from H-2K^b-tsA58 transgenic mouse-derived immortal splenocytes.