Enzyme Immunoassays for Bacillus cereus Hbl Toxin

Indirect and sandwich enzyme immunoassays were performed as described before [20 (link),28 (link),32 (link),49 (link)].
For detection of rHbl components, established mAbs were used (1A12 and 8B12 [32 (link)] for Hbl L₂, 1E9 [29 (link)] for Hbl L₁ and 1B8 [29 (link)] for Hbl B), as well as the ones newly generated in this study (Table S1). To determine relative affinities of the newly generated mAbs, B. cereus culture supernatants and rHbl components were used in indirect EIAs. For that, dilution series of the antigens were applied, followed by the cell culture supernatants (constantly 1:20 in PBS). The relative affinity corresponds with the dilution that results in an absorbance value of 1.0. The productivity of the hybridoma cell lines was also determined in indirect EIAs. Supernatant of B. cereus MHI 1532 was applied (constantly 1:10 in PBS), followed by dilution series of the cell culture supernatants.
rHbl complex formation was investigated using indirect and sandwich EIAs. In the indirect assay, the microtiter plate was coated with a serial dilution of rHbl L₁ (120–0 pmol/mL) overnight. After washing, the second rHbl component was applied in constant concentration (60 pmol/mL) for 1 h. After blocking for 30 min with 3% sodium-caseinate in PBS, HblB-specific mAb 1B8 [29 (link)] and Hbl L₂-specific mAb 1H9 (this study) (2 μg/mL in PBS) were applied, respectively. After additional washing, rabbit-anti-mouse-HRP conjugate (1:2000 in 1% sodium-caseinate in PBS) was applied for detection. In the sandwich assay, the microtiter plate was coated with 10 μg/mL mAbs 1E9 [29 (link)] or 1G8 (this study), both Hbl L₁-specific, overnight. While the plate was blocked for 30 min with 3% sodium-caseinate in PBS, a mixture of rHbl components (L₁+B or L₁+L₂; 1.5 pmol/μL each, ratios 1:1, 5:1, 1:5, 10:1 or 1:10) was incubated at RT. The mixtures were applied to the microtiter plate as serial dilution from 75 to 0 pmol/mL. After washing, a specific mAb conjugate was applied for detection (1:2000 in 1% sodium-caseinate in PBS; 1B8-HRP [29 (link)] against Hbl B or 1H9-HRP (this study) against Hbl L₂). Analogously to the detection of the rHbl components, sandwich EIAs were used to detect Hbl complexes in the supernatant of B. cereus strain F837/76, which was applied to the microtiter plates as serial dilution. One-site-binding curves were applied to depict the absorption values compared to the sample dilutions.