3% BSA for 30 min. The bound antibodies were detected by incubation with alkaline phosphatase-labeled rabbit anti-goat IgG (2 ��g/ml; Zymed) for 30 min. The sections were stained with a BCIP/NBT Alkaline sellckchem Phosphatase Substrate Kit IV (Vector, Burlingame, CA, USA). Cell viability assay To test toxin neutralization, Stx1-sensitive Vero cells were employed [31]. Vero cells (American Type Culture Collection; Rockville, MD, USA) were incubated with 20 pg/ml of Stx1 holotoxin that had been pre-treated with or without the plantibody for 1 h. After 48 h incubation at 37��C under a humidified atmosphere of 5% CO2/95% air, cell viability was measured by means of a colorimetric assay using a Cell Counting Kit-8 (DOJINDO, Kumamoto, Japan). Absorbance readings were made at 450 nm with reference at 650 nm.

Viability was defined as the percentages of absorbance readings for the Stx1-treated cells relative to those for untreated cells. DNA fragmentation assay The DNA fragmentation assay was performed as described previously [31]. Vero cells were placed in the wells of a 6-well culture plate (Falcon 3046; BD, Franklin Lakes, NJ, USA) at 1.5��106 cells per well and cultured for 16 h. Stx1 that had been pre-incubated with the plantibody for 1 h at 37��C was added to Vero cells to a final concentration of 10 pg/ml. After 48 h incubation, the cell monolayer was washed with 0.02% EDTA�CPBS, and then cells were recovered by treatment with 0.25% Trypsin�C0.02% EDTA�CPBS. After washing in PBS, the cells were lysed with 0.5% Triton X-100 containing 10 mM Tris�CHCl (pH 7.4) and 10 mM EDTA for 10 min on ice.

The cell lysates were centrifuged and the supernatants were collected. The supernatants were incubated in 0.2 mg/ml of RNase A (QIAGEN) for 1 h at 37��C, followed by incubation with 0.2 mg/ml of proteinase K (Roche) for 30 min at 50��C, and then the DNA fragments were precipitated with 50% isopropanol containing 0.4 M NaCl at �C30��C overnight. The DNA fragments were dissolved in 10 mM Tris�CHCl containing 1 mM EDTA, analysed by agarose gel electrophoresis, and then stained with ethidium bromide. Activated caspase-3 detection assay Stx1 holotoxin and appropriately diluted plantibody were mixed and incubated for 1 h at 37��C.

The mixture containing 10 pg/ml of Stx1 was added to 2��105 of Ramos cells (American Type Culture Collection; Rockville, MD, USA), followed by incubation for 5 h at 37��C in RPMI 1640 (Nissui Pharmaceuticals, Tokyo, Japan) containing 60 ��g/ml kanamycin with 10% fetal bovine serum (Hyclone, South Logan, UT, USA) under a humidified Drug_discovery atmosphere of 5% CO2/95% air. Activated caspase-3 within the cells was detected with a fluorescent substrate using an APOPCYTO? Intracellular Caspase-3 Activity Detection Kit (MBL, Nagoya, Japan), and analyzed with an FACSCanto? II (BD).