Transfer of monoclonal antibodies into mammalian cells by electroporation.
A simple rapid and reproducible procedure for transferring monoclonal antibodies into mammalian cells by electroporation is described. Two functionally different monoclonal antibodies (Mab 3F3 and Mab 2B4) specific for asparagine synthetase (EC 6.3.1.1) were used for electroporation into HeLa, HT-5, and L5178Y D10/R (L-asparaginase-resistant) cells. The conditions were optimized so that the viability of the electroporated cells was very high (80-90%), and 90% of the viable cells had antibody incorporated. Electropermeabilized cells were structurally intact, and the high voltage electric pulse had no inhibitory effect on overall cellular DNA and protein synthesis. Incorporated immunoglobulins showed unaltered structural integrity and were functionally active. L5178Y D10/R cells incorporated with an antibody (Mab 3F3) known to be a potent inhibitor of tumor asparagine synthetase showed increased dependence on an exogenous source of asparagine in the culture medium, while the growth of cells incorporated with a control (noninhibitory) antibody (Mab 2B4) remained unaffected. These studies demonstrate that electroporation can be employed successfully for large scale transfer of antibodies into cultured mammalian cells for the study of cellular metabolism.
Chakrabarti
R
R
Department of Biochemistry and Molecular Biology, University of Florida, Gainesville 32610.
Wylie
D E
DE
Schuster
S M
SM
D000818:Animals; D000911:Antibodies, Monoclonal / Q000008:administration & dosage*; D002455:Cell Division; D002460:Cell Line; D004273:DNA, Neoplasm / Q000096:biosynthesis; D004558:Electric Stimulation / Q000379:methods; D005434:Flow Cytometry; D006367:HeLa Cells / Q000502:physiology; D006801:Humans; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009363:Neoplasm Proteins / Q000096:biosynthesis; D013936:Thymidine / Q000378:metabolism
D000911:Antibodies, Monoclonal; D004273:DNA, Neoplasm; D009363:Neoplasm Proteins; D008715:Methionine; D013936:Thymidine