With regard to the Nhe productivity, marked differences between the particular isolates could be observed; for example, NheB antigen titers ranged from 1:180 to 1 1:6,000. of the enterotoxic activity of severalB. cereusstrains. For the first time, it could be shown that strains carrying thenhegenes usually express the complete set of the three components, including NheC. However, the amount of toxin produced varies considerably between the different strains. Bacillus cereusis known to cause two different types of food poisoning (for reviews, see references9,15, and26), which are characterized by either emesis or diarrhea. At present, two different protein complexes, each consisting of three exoproteins, as well as a single protein (cytotoxin K) (19), are discussed as causative brokers (9,13). The enterotoxin described by Beecher and Wong (3,5), consisting of the components B, L1, and L2, showed hemolytic activity and was therefore named hemolysin BL (HBL). NBP35 HBL has been characterized intensively in view of the biological activity (5) as well as genetically (14,25). The nonhemolytic enterotoxin (Nhe) described by Lund and Granum (17) contains the protein components NheA (41.0 kDa), NheB (39.8 kDa), and NheC (36.5 kDa). The genes encoding the components of Nhe have been cloned and characterized, and it has been shown that they are transcribed as one operon (10,16). Specific monoclonal antibodies (MAbs) for immunochemical studies around the protein level are available only for HBL (6). Due to this limitation, the detection of theB. cereusenterotoxins is still not satisfactory, and a range of in vivo and in vitro assessments is used to estimate the toxicity ofB. cereusisolates, e.g., the mouse lethality test, the rabbit ileal loop test, the vascular permeability reaction, and cell culture assays (1,3,5,27). These assays, however, do not allow differentiation between the specific activities of the individual toxins. On the other hand, several studies published during recent years showed that nearly all strains ofB. cereusharbornhe, whereashblgenes MPEP were detected in MPEP only about 50% of the tested isolates (7,11,12,21,22,28). Although there is usually some evidence that both complexes are coexpressed frequently (1,7,11,24), quantitative data on the amount of toxin produced byB. cereusisolates carrying bothhblandnhegenes are not available. To provide tools for such detailed studies and to improve the detection of Nhe, we describe here the production of specific antibodies against NheA, NheB, and NheC. == MATERIALS AND METHODS == == B. cereus strains, culture medium, and culture conditions. == Enterotoxic strains ofB. cereusused in this study were B-4ac (DSM 4384; DSM, Germany) and NVH 0075/95 and NVH 391/98 (nhedeficient) (17,19). AB. subtilisstrain expressing recombinant NheB andEscherichia colistrains producing either NheA or NheC (16) were also used. All otherB. cereusstrains (prefix MHI) were isolated from infant food or dried milk products (2). For cytotoxicity testing and indirect enzyme immunoassay (EIA) analyses, cells were produced in casein hydrolysate-yeast extract broth (3) supplemented with 1% glucose (CGY medium) for 6 h at 32C with shaking. To inhibit proteolytic cleavage of the toxins by metalloproteases, EDTA (1 mM) was added at MPEP the time of harvesting. Cell-free supernatants obtained by centrifugation (10,000 gat 4C for 20 min) and filtration through 0.2-m Millipore filters were used for purification of proteins and as coating antigens in the EIA. For the production of recombinant NheB and NheC,B. subtilisandE. colistrains were grown in brain heart infusion supplemented with 50 g kanamycin per ml for 24 h. == Production of MAbs. == Purified NheA was prepared according to the method of Lund and Granum (17,18) and used as an immunogen. Additionally, an exoprotein preparation of strain B-4ac was produced MPEP as described by Dietrich et al. (6), and the fraction B2 obtained by gel filtration on Sephadex G-75sf was used for the immunization of mice. Two groups of 12-week-old female mice (three BALB/c strain mice.