Electroinsertion of full length recombinant CD4 into red blood cell membrane

Electroinsertion is a novel technique of protein implantation in cell membranes using electrical pulses, of field strength between 1.3 kV/cm and 2.1 kV/cm and up to 1 ms duration. The full length recombinant CD4 receptor could thus be inserted in human and murine red blood cell (RBC) membranes. 100% of the RBC subjected to this procedure were shown to expose different CD4 epitopes after electroinsertion. An average of 5000 epitopes per cell has been detected by immunofluorescence assay using flow cytometry and whole cell ELISA. CD4 electroinserted in red blood cell membranes showed upon reaction with monoclonal antibody significant patching similar to that observed in T4 cells expressing CD4. Furthermore, the fluorescent enhancement coming from accumulation of immune complex phycoerythrin-antiphycoerythrin was similar for both native CD4 on T4 cells or CD4 electroinserted into erythrocyte membrane. Attempts to electroinsert proteins without a membrane spanning sequence have consistently failed, suggesting that adsorption is not responsible for the observed phenomena.