Development of microfusion techniques to generate human hybridomas.
The rarity of antigen-specific B cells in peripheral blood and lymphoid tissues is a major limitation in the production of human monoclonal antibodies. This has led to a requirement for techniques capable of fusing small numbers of cells and achieving a higher hybridoma formation efficiency than currently is possible. The approach used in these studies to generate human hybridomas is based on the observation that under hypo-osmolar conditions electric field induced cell fusion or electrofusion is facilitated. Electrofusion parameters have been defined in strongly hypo-osmolar solutions which have resulted in a hybridoma formation efficiency greater than 5 X 10(-3) under optimal conditions. Furthermore, this has been accomplished with total input B cells of 1-2 X 10(5). This is a ten-fold reduction in the required number of input B cells and is associated with a hybridoma formation efficiency at least equal to that achieved with a higher input B cell number. An important factor in the development of this microfusion technique appears to be the duration of exposure to the hypo-osmolar solution by B cells to be immortalized. Other parameters which may affect hybridoma yield include the electrical field strength used for cell alignment and membrane breakdown, ratio of human B cells to fusion partner, washing procedure, post-fusion incubation time, and the elimination of toxic molecules.
Foung
S
S
Department of Pathology, Stanford University School of Medicine, CA 94305.
Perkins
S
S
Kafadar
K
K
Gessner
P
P
Zimmermann
U
U
D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D001402:B-Lymphocytes / Q000276:immunology; D002459:Cell Fusion; D002461:Cell Line, Transformed / Q000276:immunology; D002469:Cell Separation; D004558:Electric Stimulation; D004854:Herpesvirus 4, Human / Q000254:growth & development; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D008722:Methods; D009994:Osmolar Concentration
D000911:Antibodies, Monoclonal