Sepharose-unbinding ricin E as a source for ricin A chain immunotoxin

To evaluate the Sepharose-unbinding ricin E as a preference source material for ricin A chain (RTA) in immunotoxin studies, RTA of ricin E (RTA(E)) was characterized and compared with RTA of the Sepharose-binding ricin D (RTA(D)). RTA(E) and RTA(D) were separated into two subunits of A(1) and A(2) by capillary electrophoresis. The isoelectric points of A(1) and A(2) subunits were determined to be 7.6 and 7.4, respectively, for RTA(E), while they were 7.4 and 7.3, respectively, for RTA(D). The molecular masses of A(1) and A(2) isomers determined by the matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry were 31059 and 32266 Da, respectively, for RTA(E), while they were 30892 and 32179 Da, respectively, for RTA(D). There were no significant differences in the cell surface affinity and cytotoxicity between RTA(E) and RTA(D). Anti-CD4-RTA(E) immunotoxin was prepared by conjugating RTA(E) with anti-CD4 monoclonal antibody using a heterobifunctional crosslinker, 4-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyldithio) toluene. Anti-CD4-RTA(E) immunotoxin showed comparable cytotoxic effects to anti-CD4-RTA(D) immunotoxin to antigen-positive CEM cells in vitro. It is concluded that RTA(E) from ricin E is one of different variants of RTA(D) and may be used as a preference source material of RTA in immunotoxin studies.