Production of monoclonal antibodies: strategy and tactics

Abstract

A myeloma line has been developed which produces no globulin chains of its own, has a duplication of 8.7 h, fuses effectively with B-lymphoblasts and produces stable hybrids. An enhancing effect of macrophages on hybridoma yields has been observed. Among the fusing agents tested, PEG of mol.wt. 4000 gave the best results, 20 degrees C being the optimum working temperature. The maintenance medium of choice has been found to be Iscove's with 10% FCS. Direct exposure of fusion cultures to a selective medium with hypoxanthine, aminopterine and thymidine reduced the labor involved and increased the yield. A mechanical device for changing the medium has been designed. The replacement of standard trays by microtrays resulted in a higher frequency of surviving hybrids. By using a feeder layer, the spleen cell input can be reduced 50-fold. At such low multiplicities the positive cultures arise predominantly from single hybrids, eliminating the need for subsequent cloning. The hybrids can be labelled and will yield in serum-free medium. Since at least a third of them inherit the fast growth rate of their myeloma parent and keep producing over 2000 antibody molecules per second, readaptation to ascitic growth is also superfluous. A simplified technique of producing monoclonal antibodies is given in detail, together with the experimental evidence prompting modifications of the classical method of Köhler and Milstein (1975, 1976).