Production and characterization of antibodies against each of the three subunits of the Bacillus cereus nonhemolytic enterotoxin complex

Abstract

The nonhemolytic enterotoxin (Nhe) is one of the two three-component enterotoxins which are responsible for diarrheal food poisoning syndrome caused by Bacillus cereus. To facilitate the detection of this toxin, consisting of the subunits NheA, NheB, and NheC, a complete set of high-affinity antibodies against each of the three components was established and characterized. A rabbit antiserum specific for the C-terminal part (15 amino acids) of NheC was produced using a respective synthetic peptide coupled to a protein carrier for immunization. Using purified B. cereus exoprotein preparations as immunogens, one monoclonal antibody against NheA and several antibodies against NheB were obtained. No cross-reactivity with other proteins produced by different strains of B. cereus was observed. Antibodies against the NheB component were able to neutralize the cytotoxic activity (up to 98%) of Nhe. Based on indirect enzyme immunoassays, the antibodies developed in this study were successfully used in the characterization of the enterotoxic activity of several B. cereus strains. For the first time, it could be shown that strains carrying the nhe genes usually express the complete set of the three components, including NheC. However, the amount of toxin produced varies considerably between the different strains.

FIG. 1.

Immunoblot reactivity of the monoclonal antibodies 1A8, anti-NheA (A), 1E11, anti-NheB (C), and 3F1, anti-NheB (D), and anti-NheC rabbit antiserum (B) with exoprotein preparations from B. cereus strain B-4ac (lanes 1), B. cereus strain NVH 0075/95 (lanes 2), B. cereus strain NVH 391/98 (lanes 3), and recombinant NheA (lanes 4), NheB (lanes 5), and NheC (lanes 6).

FIG. 2.

SDS-PAGE documentation of the NheB purification by IAC using monoclonal antibody 1E11: lane 1, crude culture supernatant of the B. cereus strain NVH 0075/95; lane 2, flowthrough of the IAC column; lane 3, rinse fraction of the IAC column; lane 4, eluted NheB.