Production and Characterization of Monoclonal Antibodies against the Hemolysin BL Enterotoxin Complex Produced by Bacillus cereus

Abstract

A total of five hybridoma cell lines that produced monoclonal antibodies against the components of the hemolysin BL (HBL) enterotoxin complex and sphingomyelinase produced by Bacillus cereuswere established and characterized. Monoclonal antibody 2A3 was specific for the B component, antibodies 1A12 and 8B12 were specific for the L2 component, and antibody 1C2 was specific for the L1 protein of the HBL enterotoxin complex. No cross-reactivity with other proteins produced by different strains ofB. cereus was observed for monoclonal antibodies 2A3, 1A12, and 8B12, whereas antibody 1C2 cross-reacted with an uncharacterized protein of approximately 93 kDa and with a 39-kDa protein, which possibly represents one component of the nonhemolytic enterotoxin complex. Antibody 2A12 finally showed a distinct reactivity withB. cereus sphingomyelinase. The monoclonal antibodies developed in this study were also successfully applied in indirect enzyme immunoassays for the characterization of the enterotoxic activity of B. cereus strains. About 50% of the strains tested were capable of producing the HBL enterotoxin complex, and it could be demonstrated that all strains producing HBL were also highly cytotoxic.

Colony immunoblot assay for the detection of hemolysin BL enterotoxin producing

“…Monoclonal antibodies 2A3, specific for B protein of HBL [13] and monoclonal antibody 1A8, specific for nhe A (details will be published elsewhere) are used. Specificity of the antibody against nhe A was proved by immunoblot analyses of recombinant nheA, toxin producing B. cereus (DSM 4384 and NVH 0075/95) and the NHE negative strain 391/98 [6].…”

“…The HBL titer of cell-free culture supernatants of B. cereus strains was determined using an indirect EIA as described recently [13] with slight modifications. Briefly, plates were coated with serial dilutions (in carbonate-bicarbonate buffer) of cell-free supernatants of the strains grown in CGY medium.…”

“…Cytotoxicity of the supernatants was determined as described recently [13]. Briefly, growth medium consisted of Eagle minimum essential medium (Biochrom KG, Berlin, Germany) with Earle salts supplemented with 1% fetal calf serum and 2 mmol l À1 glutamine.…”

“…Both types of assays utilize an enrichment step of 24-48 h in addition to the detection procedure and are therefore time consuming and laborious. In order to facilitate the differentiation between HBL producing and HBL negative colonies we describe here a colony immunoblot assay (CIA), based on monoclonal antibodies recently developed in our laboratory [13]. The applicability and reliability of the colony immunoblot assay were tested by analyzing B. cereus strains, which had been characterized by enzyme immunoassay [13], cytotoxicity assay as well as PCR for HBL and NHE genes.…”

The enterotoxin T (BcET) from Bacillus cereus can probably not contribute to food poisoning

“…After centrifugation at 12 000 rpm at 4 ‡C for 30 min, the supernatants were transferred into Eppendorf tubes and stored at 320 ‡C. Monoclonal anti- body 1C2, reacting with HblD and NheB from Hbl and Nhe toxins, respectively [9], was used after a 10-fold dilution in saline solution.…”