Affinity purification and characterization of Shiga-like toxin II and production of toxin-specific monoclonal antibodies.
AUTOR(ES)
Downes, F P
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=259503Documentos Relacionados
- Purification of Shiga toxin and Shiga-like toxins I and II by receptor analog affinity chromatography with immobilized P1 glycoprotein and production of cross-reactive monoclonal antibodies.
- Characterization of monoclonal antibodies against Shiga-like toxin from Escherichia coli.
- Hybridization of Escherichia coli producing Shiga-like toxin I, Shiga-like toxin II, and a variant of Shiga-like toxin II with synthetic oligonucleotide probes.
- Purification and characterization of an Escherichia coli Shiga-like toxin II variant.
- Expression and purification of Shiga-like toxin II B subunits.