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

To evaluate the Sepharose-unbinding ricin E as a preference sourer material for ricin A chain (RTA) in immunotoxin studies, RTA of ricin E (RTA(E)) wa s 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) sub units were determined to be 7.6 and 7.4, respectively, for RTA(E), while th ey 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 ioniza tion time-of-flight (MALDI-TOF) mass spectrometry were 31 059 and 32 266 Da , respectively, for RTA(E), while they were 30 892 and 32 179 Da, respectiv ely, 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) immuno toxin was prepared by conjugating RTA(E) with anti-CD4 monoclonal antibody using a heterobifunctional crosslinker, 4-succinimidyl-oxycarbonyl-alpha -m ethyl-alpha-(2-pyridyldithio) toluene. Anti-CD4-RTA(3) immunotoxin showed c omparable cytotoxic effects to anti-CD4-RTA(D) immunotoxin to antigen-posit ive 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 materia l of RTA in immunotoxin studies. (C) 2001 Elsevier Science B.V. All rights reserved.