Affinity purification and characterization of Shiga-like toxin II and production of toxin-specific monoclonal antibodies.
Shiga-like toxin (SLT-II) was purified to apparent homogeneity from Escherichia coli K-12 strain NM522 containing the cloned toxin genes on recombinant plasmid pEB1. Purification was accomplished by a series of column chromatography techniques: anion-exchange, chromatofocusing, cation-exchange, and monoclonal antibody affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the pure toxin showed that SLT-II consisted of A and B subunits with apparent molecular weights of 32,000 and 10,200 +/- 800, respectively. A band of molecular weight 25,000 was also observed after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified as the A1 subunit by Western immunoblot analysis with toxin-specific monoclonal antibodies (MAbs). The pI of the purified toxin was 5.2. Approximately 1 pg of pure SLT-II, but was not neutralized by polyclonal antibodies or MAbs to SLT-I. Five hybridomas against SLT-II were produced (BC5 BB12, DC1 EH5, EA5 BA3, ED5 DF3, and GB6 BA4). Culture supernatant fluids containing MAbs from these hybridomas did not neutralize the cytotoxicity of SLT-I or Shiga toxin. Western blot analysis showed that two MAbs (MAb DC1 EH5 and MAb GB6 BA4) recognized the A and A1 subunits of SLT-II and three MAbs (MAb BC5 BB12, MAb EA5 BA3, and MAb ED5 DF3) recognized the B subunit of SLT-II. MAb BC5 BB12 was used to prepare an affinity column for toxin purification.
Downes
F P
FP
Department of Parasitology and Laboratory Practice, School of Public Health, University of North Carolina, Chapel Hill 27514.
Barrett
T J
TJ
Green
J H
JH
Aloisio
C H
CH
Spika
J S
JS
Strockbine
N A
NA
Wachsmuth
I K
IK
D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D000942:Antigens, Bacterial / Q000276:immunology*; D001427:Bacterial Toxins / Q000302:isolation & purification*; D002470:Cell Survival / Q000187:drug effects; D002845:Chromatography; D004768:Enterotoxins / Q000276:immunology / Q000302:isolation & purification* / Q000633:toxicity; D004926:Escherichia coli / Q000276:immunology*; D006367:HeLa Cells / Q000187:drug effects; D007163:Immunosorbent Techniques; D066298:In Vitro Techniques; D007526:Isoelectric Point; D008970:Molecular Weight; D009500:Neutralization Tests; D022641:Shiga Toxin 2; D014709:Vero Cells / Q000187:drug effects
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D004768:Enterotoxins; D022641:Shiga Toxin 2