A RAPID NONSELECTIVE METHOD TO GENERATE QUADROMAS BY MICROELECTROFUSION

A simple non-selective methodology was developed and standardized to generate desired hybrid-hybridoma or quadroma secreting bifunctional antibodies. This novel protocol is based on microelectrofusion on a meander chamber using a few hundred cells of each of the two parental hybridomas with no laborious drug selection procedures. Seeding similar to 10 cells per well in a 96-well microtitre plate after fusion in 200 mu l standard medium containing 20% FBS and 10% Origen growth factor generated positive quadromas secreting bispecific antibodies with good stability after the second redone. Compared to the conventional PEG fusion and other methods this simple protocol is both rapid and economical. Generally, conventional methods to make quadromas and triomas require the introduction of drug selection markers into one or both of the parental cells, a procedure that could take 3-6 months. Utilizing the non-selective microelectrofusion method described here, we have generated several quadromas in a very short time. Further, such a protocol could also be potentially adopted to generate human hybridomas with few B cells isolated from peripheral blood lymphocytes enriched by antigen specific panning or affinity microelectrofusions.