In Vitro Assessment of a Chemically Synthesized Shiga Toxin Receptor Analog Attached to Chromosorb P (Synsorb Pk) as a Specific Absorbing Agent of Shiga Toxin 1 and 2

Abstract

A synthetic analog of Shiga toxin (Stx) receptor (Synsorb Pk) was quantitatively assessed to determine whether it can protect human renal adenocarcinoma cells (ACHN cells) from the cytotoxicity of Stx1 and Stx2 by coincubation experiments. Coincubation of 100 and 20ng of Stx1 and Stx2 with 50mg of Synsorb Pk for 1hr at 37C in 1ml of Eagle's Minimum Essential Medium supplemented with 1% (v/v) nonessential amino acid and 10% (v/v) fetal calf serum protected 50% of the cells from the cytotoxic effect. Chromosorb P, an inert matrix control, did not absorb the Stxs at all. Heat-treatment (boiled for 10min) to Synsorb Pk caused a 50% decrease in Stx2-binding activity, but did not effect the Stx1 binding. Further, Stxs bound to Synsorb Pk could be demonstrated. When 20mg of Synsorb Pk was coincubated for 30min at 37C in 1ml of phosphate-buffered saline with 1 and 10ng or more of Stx1 or Stx2, respectively, the toxins could be detected on the surface when the bound toxins on Synsorb Pk were used as the solid phase in enzyme immunoassay. The amount of 100ng/ml of both Stx1 and Stx2 appeared to saturate 20mg/ml of Synsorb Pk after coincubating for 30min at 37C. While assessing the Stxs' binding activity to Synsorb Pk, it was demonstrated that Stx1 had a higher affinity to Pk trisaccharide than Stx2. These observations provide useful information on the effectiveness of Synsorb Pk to trap and eliminate free Stxs produced in the gut of patients infected by Stx-producing Escherichia coli, and to prevent the progression of hemorrhagic colitis to hemolytic uremic syndrome.