Modification of HAT Medium and Hybridoma Formation

Abstract

Among the major challenges in the field of hybridoma formation is the need to find ways of increasing the efficiency of myeloma-spleen cell hybridization. HAT medium originally developed by Littlefield in 1964 (1) has been one of the key factors that has made hybridoma generation practical. The value of this medium is generally considered from the viewpoint of its ability to inhibit unfused myeloma cell proliferation. We have recently explored modifications of this selective medium to determine whether an augmented medium designed to further support hybridoma growth would be capable of enhancing and stabilizing the newly formed hybrid clones at their earliest stage, closest to their formation. Our approach was based on the assumption that the first hours and days after cell fusion are the most critical for the outcome of a given fusion, and therefore enhancement efforts should be concentrated on these early phases (2). Our strategy consisted of adding known biologically active molecules to HAT medium immediately following the PEG-mediated cell fusion, anticipating that the successful agent would enhance hybridoma formation (3). It was further assumed that an effective agent would ultimately cause an increase in the number of the relevant hybridomas produced per fusion, increase the average size of the clones formed, and consequently be associated with higher production of monoclonal antibodies, as compared to yields of fusions using conventional HAT medium alone.