Antibodies Against Recombinant Shiga Toxin Subunit B Neutralize Shiga Toxin Toxicity in HeLa Cells

Abstract

Shigella dysenteriae type 1 and Escherichia coli O157:H7 produce Shiga toxin 1 (Stx) and Shiga toxin1 (Stx1), respectively and these two toxins are almost identical. E. coli O157:H7 is the major cause of diarrhea-associated hemolytic uremic syndrome. Stx and Stx1 are AB5 type of toxin with a molecular weight of 70 kDa, comprising an enzymaticaly-active A subunit (32 kDa) and five receptor-binding B subunits (7.7 kDa). In this study DNA fragment (289 bp, Gene Bank Accn No. EF685161) coding for B chain of Stx was amplified from S. dysenteriae type1 and cloned. Shiga toxin-binding subunit was expressed and purified in native conditions by affinity and gel permeation chromatography with the yield of 5.1 mg/L in shake flask culture. For the purpose of immunization, the polypeptide was polymerized with glutaraldehyde. Hyper immune serum produced in mice reacted with the purified polypeptide and intact Shiga toxin. The anti-StxB antiserum effectively neutralized the cytotoxicity of Shiga toxin towards HeLa cells.

Immunological detection assays for recombinant Shiga toxin & Shigella dysenteriae

“…Specificity and titre of anti-Shiga toxin B (StxB) serum: The rStxB was purified using affinity and gel filtration chromatography. After cross-linking rStxB was found to produce high-titre protective antibodies in mice as reported in our previous study11. The rabbit and mouse antisera were analyzed for the cross-reactivity with other toxins, i.e .…”

“…In our previous study10, the stxB gene (289 bp from 967 to 1255, accession number EF685161) coding for B chain of Stx was amplified. After cloning, StxB was expressed and purified in the native conditions11. BALB/c mice and New Zealand female white rabbits were maintained in animal care facility of Defence Research & Development Establishment (DRDE), Gwalior, India, at temperature 25°C±2°C and humidity 30-70 per cent with 12:12-h light:dark period.…”

“…Two-fold serial dilutions of antiserum were prepared starting with 1:1000 up to 1:512,000 in phosphate-buffered saline (PBS). Standard Western blot protocol was performed as described elsewhere11. ELISA was performed in which antigen solution (500 ng) was prepared in coating buffer.…”

Food Forensics: Using Mass Spectrometry To Detect Foodborne Protein Contaminants, as Exemplified by Shiga Toxin Variants and Prion Strains

“…Shiga toxin production is also inferred by the presence of specific virulence genes . Antibody-based methods have been widely used to detect and distinguish among the Shiga toxins. − To detect any new Shiga toxins using antibodies, new antibodies need to be developed. Top-down mass spectrometry is also used to detect Shiga toxins. − A MRM-based approach has advantages over these other methods in that it can be used to both distinguish among types and variants of Shiga toxins and quantitate them ( vide infra ). , …”