Production, characterisation and potential application of a novel monoclonal antibody for rapid identification of virulent Listeria monocytogenes.
A panel of hybridomas was produced using intact Listeria monocytogenes serotype 1/2a cells as the immunogen. An IgG2a monoclonal antibody (mAb) 'mAb2B3' was isolated that reacted with L. monocytogenes but not with a representative panel of related Listeria spp. and non-Listeria spp. Binding activity was greatest against L. monocytogenes serotype 1/2a and was significantly enhanced when cells were prepared in Listeria enrichment broth (LEB). The reactive epitope was deduced, by immunoblot analysis, to be a surface localised protein of approximately 80 kilodaltons (kDa), putatively assumed to be internalin A (InlA). Recombinant InlA protein was subsequently expressed in Escherischia coli. When crude E. coli cell lysates were subjected to immunoblot analysis, it was demonstrated that the mAb bound specifically to the heterologously expressed recombinant InlA protein, thus confirming the specificity of the mAb. The mAb was further evaluated in a series of enzyme-linked-immunosorbent assay (ELISA)-based formats and in a surface plasmon resonance (SPR)-based biosensor platform. Both configurations were capable of differential identification of virulent L. monocytogenes at concentrations greater than or equal to 1x10(7) cells/ml. Notwithstanding the apparent insensitivity, the results indicate that InlA could be exploited as a marker for highly specific confirmatory identification of pathogenic L. monocytogenes following primary enrichment of suspect food samples, using the anti-InlA antibody 'mAb2B3', described herein.
Hearty
Stephen
S
School of Biotechnology, Dublin City University, Dublin 9, Ireland.
Leonard
Paul
P
Quinn
John
J
O'Kennedy
Richard
R
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000737:chemistry* / Q000276:immunology; D000942:Antigens, Bacterial / Q000737:chemistry / Q000276:immunology; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D015374:Biosensing Techniques; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D005260:Female; D005516:Food Microbiology; D008089:Listeria monocytogenes / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D008088:Listeriosis / Q000517:prevention & control*; D051379:Mice; D008807:Mice, Inbred BALB C; D018625:Microscopy, Atomic Force; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D012680:Sensitivity and Specificity; D020349:Surface Plasmon Resonance
D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D000939:Epitopes; D011994:Recombinant Proteins; C069761:internalin protein, Bacteria