Reactivities of Genus-Specific Monoclonal Antibody EM-6E11 against Listeria Species and Serotypes of Listeria monocytogenes Grown in Nonselective and Selective Enrichment Broth Media

Abstract

Depending on the growth medium used for enrichment of bacterial cells prior to assay, the monoclonal antibody (MAb) EM-6E11 recognizing Listeria genus-specific epitope on 43 and 94 to 97 kDa cell-surface antigens (A. K. Bhunia and M. G. Johnson, Appl. Environ. Microbiol. 58:1924-1929, 1992) exhibited extensive variability in the detection of Listeria species. MAb EM-6E11 strongly detected live cells of all Listeria species and all serotypes of L. monocytogenes by ELISA when cells were grown in nonselective brain heart infusion (BHI) broth, in selective Listeria enrichment broth (LEB), or in Listeria repair broth (LRB). In contrast, EM-6E11 detected only four of the thirteen serotypes of L. monocytogenes (serotypes 1/2c, 3b, 4ab, and 7) when cells were grown in the UVM1 formulation of Listeria enrichment broth (UVM1) or Fraser broth (FRB). This MAb failed to react with live cells of four other Listeria species, including L. ivanovii, L. welshimeri, L. grayi, and L. murrayi cells grown in UVM1 or FRB. Heating of Listeria cells at 100 degrees C for 20 min, irrespective of the enrichment media used, led to large losses of MAb EM-6E11 reactivity in ELISA, suggesting that the specific cell-surface epitopes involved may not be heat stable. Our results confirm that MAb EM-6E11 is suitable for detection of live cells but not heat-killed cells of Listeria spp. and can be used in conjunction with an enrichment step in BHI, LEB, or LRB but not in UVM1 or FRB.

Expression of Surface Protein LapB by a Wide Spectrum of Listeria monocytogenes Serotypes as Demonstrated with Anti-LapB Monoclonal Antibodies

“…Expression of LapB in L. monocytogenes grown under these culture enrichment conditions points to the feasibility of using anti-LapB MAbs to isolate and detect L. monocytogenes from food samples following culture enrichment. This is in contrast to the few previous studies performed with two MAbs (EM-6E11 and C11E9) that do not detect L. monocytogenes cells grown in UVM broth (42,43). Expression of the lapB gene is presumably directed by a B -dependent promoter predicted 67 bp upstream of its translational start codon (44) and was reported to be positively regulated by transcriptional activator PrfA (18,45).…”

Unveiling the Expression Characteristics of IspC, a Cell Wall-Associated Peptidoglycan Hydrolase in Listeria monocytogenes, during Growth under Stress Conditions

“…The problem with growing cells for a limited time in the stress condition followed by transfer to enrichment broth is that residual protein from the original culture does not get degraded quickly enough to disappear after the short incubation time and therefore is still detected (Ronholm et al, unpublished). Our findings that IspC was detectable with this specific MAb in all growth conditions tested makes this protein antigen a novel potential diagnostic marker for diagnostics for L. monocytogenes serotype 4b, since studies which have used a methodology similar to the one presented here have found that their antigens were not expressed at detectable levels under all tested conditions (4,11,19,20). Since the primary objective of this study was to validate IspC as a marker for diagnostic use in pre-enrichment bacterial detection, possibly in a biosensor in a microfludics-based flow cytometer, our methodology, where cells were grown entirely in the stress condition and detected by immunofluorescence signal, allowed us to examine IspC expression in cells from selected environments under detection conditions simulating those that would be present in a biosensor applied to the detection of L. monocytogenes directly from food or environmental samples.…”

Actin Polymerization Drives Septation of Listeria monocytogenes namA Hydrolase Mutants, Demonstrating Host Correction of a Bacterial Defect

“…Several previously reported studies classified NamA with primary functions closely associated with the bacterial surface based on the presence of its LysM peptidoglycan binding domains (3,6,7,10), its detection at the bacterial surface (8,52), and its ability to serve as an immunoreactive epitope in L. monocytogenes detection studies (19,20,44). Other studies, however, have detected NamA in supernatant fractions derived from L. monocytogenes strains containing a mutationally activated form of the virulence regulator PrfA [PrfA(L140F)] as well as in wild-type supernatants in comparison to secA2 mutants (33,50), although those studies did not determine if secreted NamA was functionally active.…”

“…NamA was initially identified as a variably expressed immunogenic epitope of L. monocytogenes recognized by monoclonal antibodies at the bacterial surface (19,20,44). NamA contributes to cell division, as mutants lacking namA form bacterial chains during exponential-phase growth in broth culture (10).…”