The signal is propagated back up to the fiber and is detected in real time by a fluorometer. This format has been successfully applied to many foodborne microorganisms and toxins, however, the limit of PXD101 mouse detection largely depends on the antibody and the reagents used [31, 44, 46–48]. In the present study, monoclonal selleck chemicals antibodies (MAbs) against L. monocytogenes and Listeria spp. were generated, characterized, and employed to concentrate L. monocytogenes using PMBs. Finally, MAbs were used on the fiber optic sensor to detect
L. monocytogenes from inoculated food products (soft cheese and hotdogs). In parallel, qPCR and light-scattering sensor methods were performed to confirm the results. Results MAb production and characterization by ELISA and Western blotting We selected 11 stable hybridomas, of which 7 (2F2, 2A2, 3B3, 3B7, 4E8, 2D12, and 4E4) reacted with both rInlA and L. monocytogenes cells, and 4 (4E5, 4C1, 2A12, and 3F8) reacted with
L. monocytogenes, L. innocua, and L. seeligeri. After another round of Selleck APO866 screening of MAbs-2D12, -3B7, -4E4, and -3F8 against rInlA or L. monocytogenes cells (serotypes 4b, 4a, 1/2a, and 1/2b) by ELISA, we chose MAb-2D12 (subclass IgG2a) and MAb-3F8 (subclass IgM) for future use. An ELISA (Figure 1a) revealed that, among the anti-InlA antibodies, MAbs-2D12 and -3B7 strongly reacted (A 450 = 1.0 or higher) with L. monocytogenes 4b cells, while MAb-4E4 gave slightly lower reaction values (A 450 = 0.75–0.9). The Listeria genus-specific MAb-3F8 gave strong ELISA values (A 450 = 0.8–1.5) when tested against other Listeria spp., without producing significant cross-reactions with other bacterial species (Figure 1b). Figure 1 Indirect ELISA using (a) MAbs 2D12, 3B7, 4E4, and 3F8 or (b) MAb-3F8 against different bacterial strains and purified rInlA. Several 96-well microtiter plates were coated with live bacteria (~1 × 109 CFU/mL) for 16 h at 4 °C. Data are the mean ± SD of 3 independent assays performed in duplicate.
In the Western blot, MAb-2D12 reacted with an 80-kDa protein band (InlA) from L. monocytogenes and L. ivanovii, but it did not react with other Listeria spp., including L. marthii or L. rocourtiae buy Regorafenib (Figure 2a). MAb-2D12 was reactive with all 13 serotypes; however, a relatively weak reaction with 2 strains of serotype 1/2c (ATCC 19112 and ATCC 7644) was observed. MAb-2D12 also reacted with a 66-kDa band from serotype 3c (SLCC 2479), which is presumably a truncated InlA-protein variant (Figure 2b) . MAb-2D12-reactive InlA was distributed in the secreted, cell wall, and intracellular protein fractions of bacteria (Figure 2c). Immunofluorescence microscopy confirmed the specific binding of anti-InlA antibody (MAb-2D12) to the surface of L. monocytogenes cells, but it did not react with L. innocua (Additional file 1: Figure S1).