[Expression, purification and specific monoclonal antibodies preparation of diphtheria toxin A fragment].
Diphtheria toxin A fragment (DTA) is an essential catalytic domain of diphtheria toxin (DT)-based immunotoxin. DTA protein and its antibodies play an important role in the studies on toxicology, purification and identification of DT-based immunotoxins. In this paper, DTA was expressed and purified from E. coli. After Q-Sepharose FF chromatography and (Ni+)-Sepharose affinity chromatography, 6 x His-DTA fusion protein with 90% purity was achieved. Using the purified DTA as antigen to immunize BalB/c mice, 2 hybridoma cell lines (designated as 3B6 and 3B9, respectively) secreting monoclonal antibodies (McAbs) against DTA were established. Investigations showed that both McAbs were characterized as IgG1 with titers of 1: 10(6). The binding of the McAbs to DTA was competitively inhibited by horse sera against DT. The fact that anti-DTA McAbs could be used in western blot analysis and affinity chromatography purification of DT-based immunotoxins implied that they will be useful agents in the studies on DT-based immunotoxins.
Ouyang
Jing
J
Institute for Viral Disease Control and Prevention, Chinese Center of Disease Control and Prevention, Beijing 100052, China.
Wang
Jian-Wei
JW
Wang
Chun-Xiao
CX
Guo
Li
L
Tuo
Hou-Zhen
HZ
Cui
Ting
T
Hong
Tao
T
D000818:Animals; D000907:Antibodies, Bacterial / Q000235:genetics*; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics*; D002846:Chromatography, Affinity; D004167:Diphtheria Toxin / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D000922:Immunotoxins / Q000302:isolation & purification; D051379:Mice; D008807:Mice, Inbred BALB C; D010446:Peptide Fragments / Q000276:immunology*; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D004167:Diphtheria Toxin; D000922:Immunotoxins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C029795:diphtheria toxin fragment A