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The mutation Glu151Asp in the B-component of the Bacillus cereus non-hemolytic enterotoxin (Nhe) leads to a diverging reactivity in antibody-based detection systems.

Didier A, Jeßberger N, Krey V, Dietrich R, Scherer S, Märtlbauer E - Toxins (Basel) (2015)

Bottom Line: The ability of Bacillus cereus to cause foodborne toxicoinfections leads to increasing concerns regarding consumer protection.Sequence analysis revealed the point-mutation (Glu)151(Asp) in the potential binding region of the capture antibody.Application of this antibody also results in low titers in an indirect EIA format and shows variable detection intensities in Western-immunoblots.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Science, Faculty of Veterinary Medicine, Ludwig-Maximilians Universität München, 85763 Oberschleißheim, Germany. a.didier@mh.vetmed.uni-muenchen.de.

ABSTRACT
The ability of Bacillus cereus to cause foodborne toxicoinfections leads to increasing concerns regarding consumer protection. For the diarrhea-associated enterotoxins, the assessment of the non-hemolytic enterotoxin B (NheB) titer determined by a sandwich enzyme immunoassay (EIA) correlates best with in vitro cytotoxicity. In general, the regulation of enterotoxin expression of B. cereus is a coordinately-regulated process influenced by environmental, and probably also by host factors. As long as these factors are not completely understood, the currently-applied diagnostic procedures are based on indirect approaches to assess the potential virulence of an isolate. To date, sandwich EIA results serve as a surrogate marker to categorize isolates as either potentially low or highly toxic. Here, we report on a single amino acid exchange in the NheB sequence leading to an underestimation of the cytotoxic potential in a limited number of strains. During the screening of a large panel of B. cereus isolates, six showed uncommon features with low sandwich EIA titers despite high cytotoxicity. Sequence analysis revealed the point-mutation (Glu)151(Asp) in the potential binding region of the capture antibody. Application of this antibody also results in low titers in an indirect EIA format and shows variable detection intensities in Western-immunoblots. A commercially-available assay based on a lateral flow device detects all strains correctly as NheB producers in a qualitative manner. In conclusion, isolates showing low NheB titers should additionally be assayed in an indirect EIA or for their in vitro cytotoxicity to ensure a correct classification as either low or highly toxic.

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Related in: MedlinePlus

Western blot results of the membranes probed with mAB 2B11 (upper panel) and 1E11 (lower panel). Except for the low-producer, for which an adjustment of the NheB amounts was impossible, all strains reacted similarly with mAb 1E11. Loaded amounts were adjusted to contain similar NheB levels as calculated based on the indirect EIA results. Under denaturing conditions mAb 2B11 is capable of detecting NheB in all supernatants, with varying intensities. Mean signal for the probed membrane with mAb 1E11 was 219 RLU (±18.5), and 145 RLU (±70.2) for mAb 2B11, thus indicating a lower reactivity, as well as a higher degree of variability for this antibody.
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toxins-07-04655-f003: Western blot results of the membranes probed with mAB 2B11 (upper panel) and 1E11 (lower panel). Except for the low-producer, for which an adjustment of the NheB amounts was impossible, all strains reacted similarly with mAb 1E11. Loaded amounts were adjusted to contain similar NheB levels as calculated based on the indirect EIA results. Under denaturing conditions mAb 2B11 is capable of detecting NheB in all supernatants, with varying intensities. Mean signal for the probed membrane with mAb 1E11 was 219 RLU (±18.5), and 145 RLU (±70.2) for mAb 2B11, thus indicating a lower reactivity, as well as a higher degree of variability for this antibody.

Mentions: To further assay the antibody performance under denaturing conditions, Western immunoblots were carried out on samples adjusted to equal amounts of NheB according to the results of the indirect EIA (mAb 1E11). One membrane was probed with mAb 1E11 to control the loading of equal protein amounts. A second membrane was probed with mAb 2B11. Results depicted in Figure 3 show that, except for the low NheB producer MHI, 3178 mAb 1E11 reacts equally well with all strains under study. Interestingly, NheB is detectable in all strains to varying degrees irrespective of the weak performance in the 2B11-based EIAs wherein—under non-denaturing conditions—the protein is targeted by the antibody.


The mutation Glu151Asp in the B-component of the Bacillus cereus non-hemolytic enterotoxin (Nhe) leads to a diverging reactivity in antibody-based detection systems.

Didier A, Jeßberger N, Krey V, Dietrich R, Scherer S, Märtlbauer E - Toxins (Basel) (2015)

Western blot results of the membranes probed with mAB 2B11 (upper panel) and 1E11 (lower panel). Except for the low-producer, for which an adjustment of the NheB amounts was impossible, all strains reacted similarly with mAb 1E11. Loaded amounts were adjusted to contain similar NheB levels as calculated based on the indirect EIA results. Under denaturing conditions mAb 2B11 is capable of detecting NheB in all supernatants, with varying intensities. Mean signal for the probed membrane with mAb 1E11 was 219 RLU (±18.5), and 145 RLU (±70.2) for mAb 2B11, thus indicating a lower reactivity, as well as a higher degree of variability for this antibody.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4663526&req=5

toxins-07-04655-f003: Western blot results of the membranes probed with mAB 2B11 (upper panel) and 1E11 (lower panel). Except for the low-producer, for which an adjustment of the NheB amounts was impossible, all strains reacted similarly with mAb 1E11. Loaded amounts were adjusted to contain similar NheB levels as calculated based on the indirect EIA results. Under denaturing conditions mAb 2B11 is capable of detecting NheB in all supernatants, with varying intensities. Mean signal for the probed membrane with mAb 1E11 was 219 RLU (±18.5), and 145 RLU (±70.2) for mAb 2B11, thus indicating a lower reactivity, as well as a higher degree of variability for this antibody.
Mentions: To further assay the antibody performance under denaturing conditions, Western immunoblots were carried out on samples adjusted to equal amounts of NheB according to the results of the indirect EIA (mAb 1E11). One membrane was probed with mAb 1E11 to control the loading of equal protein amounts. A second membrane was probed with mAb 2B11. Results depicted in Figure 3 show that, except for the low NheB producer MHI, 3178 mAb 1E11 reacts equally well with all strains under study. Interestingly, NheB is detectable in all strains to varying degrees irrespective of the weak performance in the 2B11-based EIAs wherein—under non-denaturing conditions—the protein is targeted by the antibody.

Bottom Line: The ability of Bacillus cereus to cause foodborne toxicoinfections leads to increasing concerns regarding consumer protection.Sequence analysis revealed the point-mutation (Glu)151(Asp) in the potential binding region of the capture antibody.Application of this antibody also results in low titers in an indirect EIA format and shows variable detection intensities in Western-immunoblots.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Science, Faculty of Veterinary Medicine, Ludwig-Maximilians Universität München, 85763 Oberschleißheim, Germany. a.didier@mh.vetmed.uni-muenchen.de.

ABSTRACT
The ability of Bacillus cereus to cause foodborne toxicoinfections leads to increasing concerns regarding consumer protection. For the diarrhea-associated enterotoxins, the assessment of the non-hemolytic enterotoxin B (NheB) titer determined by a sandwich enzyme immunoassay (EIA) correlates best with in vitro cytotoxicity. In general, the regulation of enterotoxin expression of B. cereus is a coordinately-regulated process influenced by environmental, and probably also by host factors. As long as these factors are not completely understood, the currently-applied diagnostic procedures are based on indirect approaches to assess the potential virulence of an isolate. To date, sandwich EIA results serve as a surrogate marker to categorize isolates as either potentially low or highly toxic. Here, we report on a single amino acid exchange in the NheB sequence leading to an underestimation of the cytotoxic potential in a limited number of strains. During the screening of a large panel of B. cereus isolates, six showed uncommon features with low sandwich EIA titers despite high cytotoxicity. Sequence analysis revealed the point-mutation (Glu)151(Asp) in the potential binding region of the capture antibody. Application of this antibody also results in low titers in an indirect EIA format and shows variable detection intensities in Western-immunoblots. A commercially-available assay based on a lateral flow device detects all strains correctly as NheB producers in a qualitative manner. In conclusion, isolates showing low NheB titers should additionally be assayed in an indirect EIA or for their in vitro cytotoxicity to ensure a correct classification as either low or highly toxic.

Show MeSH
Related in: MedlinePlus