The Nhe enterotoxin from Bacillus cereus is known to induce cytotoxicity on Vero and CaCo-2 cells by ordered binding of its single components NheA, NheB, and NheC. This study aimed to elucidate functional sites on NheB by identifying the epitopes of the neutralizing monoclonal antibodies 1E11 and 2B11. The binding regions of both antibodies were determined by using recombinant NheB fragments and synthetic peptides. The antigenic site of antibody 1E11 was located within the amino acids 321 to 341 of NheB, whereas reactivity of antibody 2B11 was dependent on the presence of amino acids 122 to 150 and on conformation. Both antibodies were able to bind simultaneously to NheB and did not interfere with target cell binding as shown by immunofluorescence microscopy. A set of neutralization assays revealed that antibody 2B11 most likely interfered with the interaction between NheB and NheC both on the epithelium cell surface and in solution. In contrast, antibody 1E11 inhibited association between NheA and cell-bound NheB in a competitive manner, and effectively neutralized Nhe cytotoxicity on a variety of human cell lines. This distinct mechanism further supports that NheA is the key component during the Nhe mode of action and the C-terminal epitope recognized by antibody 1E11 points to an important functional region of NheB. Bacillus cereus is a major food-borne pathogen known to produce a range of cytotoxins (for reviews, see references 27 and 28). There are four major toxins involved in food poisoning cases, namely, the emetic toxin (cereulide), a dodecadepsipeptide (1), and the three-component diarrheal toxins hemolysin BL (Hbl) (4, 5) and nonhemolytic enterotoxin (Nhe) (23). In addition, a single-component protein toxin (cytotoxin K) causing severe necrotic enteritis was identified in a rare B. cereus strain (22), for which the name "Bacillus cytotoxis" has been proposed (19). Studies on the prevalence of the nhe and hbl genes (10,15,16,26,31) in B. cereus indicate that all strains of B. cereus possess the genes of at least one of the diarrheal enterotoxins, and Nhe is the most prevalent enterotoxin harbored by B. cereus. In addition, the overall B. cereus-associated cytotoxic activity is correlated with the Nhe expression level (24).Nhe was first identified in strain NVH 0075/95, which was isolated following a large food-poisoning outbreak in Norway (23). It is a three-component toxin and consists of the exoproteins NheA (41.0 kDa), NheB (39.8 kDa), and NheC (36.5 kDa) (14). Studies using cell-based tests to assay Nhe-specific cytotoxicity demonstrated toxic effects in Vero, GH4, and CaCo-2 cells (17,20,21). The susceptibility of other cell lines has not yet been tested. It is known that Nhe has intrinsic pore-forming capacity (11) and that maximum toxicity will be reached when the ratio of the individual components is 10:10:1 for NheA, NheB, and NheC, respectively (20). NheB and NheC are mostly ␣-helical molecules with a predicted ␤-tongue, showing structural similarities to ClyA (11). The region of the predicte...