2013
DOI: 10.1371/journal.pone.0055098
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Monoclonal Antibodies Recognizing the Surface Autolysin IspC of Listeria monocytogenes Serotype 4b: Epitope Localization, Kinetic Characterization, and Cross-Reaction Studies

Abstract: Listeria monocytogenes serotype 4b is responsible for a high percentage of fatal cases of food-borne infection. In a previous study, we created 15 monoclonal antibodies (MAbs) against a ∼77 kDa antigen that is associated with the cell surface of live L. monocytogenes serotype 4b cells. Here we report an extensive characterization of these MAbs to further their development as diagnostic reagents. The ∼77 kDa target antigen was identified by mass spectrometry and N-terminal sequencing to be IspC, a novel surface… Show more

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Cited by 15 publications
(11 citation statements)
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“…A total of 48 stable hybridoma clones secreting MAbs to rLapB were obtained. Of these MAbs, 41 were Ig subclass G1 (M3478 to M3491, M3493 to M3495, M3497 to M3511, M3514 to M3519, M3521, M3524, and M3525) and 7 were Ig subclass G2a (M3492, M3496, M3512 and M3513, M3520, M3522, and M3523). All 48 MAbs, with the exception of M3511, reacted to rLapB in ELISA with an OD 414 of Ͼ2.0, and all but one MAb (M3511) did not react with soluble proteins of E. coli BL21(DE3)/pLysS (with no pLapB expression vector) (see Fig.…”
Section: Resultsmentioning
confidence: 99%
“…A total of 48 stable hybridoma clones secreting MAbs to rLapB were obtained. Of these MAbs, 41 were Ig subclass G1 (M3478 to M3491, M3493 to M3495, M3497 to M3511, M3514 to M3519, M3521, M3524, and M3525) and 7 were Ig subclass G2a (M3492, M3496, M3512 and M3513, M3520, M3522, and M3523). All 48 MAbs, with the exception of M3511, reacted to rLapB in ELISA with an OD 414 of Ͼ2.0, and all but one MAb (M3511) did not react with soluble proteins of E. coli BL21(DE3)/pLysS (with no pLapB expression vector) (see Fig.…”
Section: Resultsmentioning
confidence: 99%
“…In addition, while the epitope recognized by M3643 is identical between sequenced L. monocytogenes isolates and other Listeria species isolates, M3643 was highly specific to L. monocytogenes cultured in BHI. This observation should not be surprising, as other L. monocytogenes serotype-specific MAbs have been shown to recognize antigens that are not serotype specific (14). A panel of MAbs against the GW modules of L. monocytogenes autolysin, IspC, reacted specifically to serotype 4b and 4ab isolates even though the targeted GW modules are encoded in the genomic sequences of other L. monocytogenes serotypes and other Listeria species (14).…”
Section: Discussionmentioning
confidence: 99%
“…For assessment of antigen expression in standard enrichment culture, selected Listeria isolates of 12 serotypes, representative of all three lineages, were grown according to methods MFHPB-07 (25) and MFHPB-30 (26), as published in the Health Canada Compendium of Analytical Methods. For the MFHPB-07 method, Listeria isolates were cultured in 10 ml Palcam broth (medium base and selective supplement from Oxoid, Basingstoke, England) at 35°C for 26 to 28 h, followed by 1:10-fold dilution culture in UVM2 broth (medium base and selective supplement from Oxoid) at 30°C for 26 to 28 h. For the MFHPB-30 method, bacterial isolates were cultured in 10 ml Listeria enrichment broth (LEB; UVM1 base and supplement from Oxoid, Basingstoke, England) at 30°C for 48 h, followed by 1:10 dilution cultures in modified Fraser broth (MFB; medium base from EMD, Gibbstown, NJ, USA; supplement from Oxoid) at 35°C for 24 h. Cell concentrations were estimated by measuring the optical density of the culture at 620 nm (OD 620 ) and confirmed by plating, as previously described (14). Escherichia coli strains [DH5␣ and Rosetta DE3/(pLysS)] used for cloning and recombinant protein expression, respectively, were cultured in Luria-Bertani (LB) broth (BD Biosciences, Sparks, MD, USA) supplemented with 50 g/ml kanamycin.…”
Section: Methodsmentioning
confidence: 99%
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