Journal Article

Monoclonal antibodies recognizing the surface autolysin IspC of Listeria monocytogenes Serotype 4b: epitope localization, kinetic characterization, and cross-reaction studies

Lin, M, Ronholm, J, Cao, X, van Faassen, H, MacKenzie, R, Zhang, Z

2013

Overview

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 associated autolysin. Epitope localization experiments revealed that each of the 15 MAbs recognized the C-terminal cell-wall binding domain of IspC. The presence of IspC was shown to be highly conserved within L. monocytogenes serotype 4b, as evidenced by a strong reaction between anti-IspC MAbs and all 4b isolates. To determine the range of cross-reactivity with other L. monocytogenes serotypes ELISA was used to test each MAb against multiple isolates from each of the L. monocytogenes serotypes. Of the 15 MAbs, five: M2774, M2775, M2780, M2790 and M2797, showed specificity for L. monocytogenes serotype 4b and only cross reacted with serotype 4ab isolates. The kinetics of the interaction between each of the MAbs and IspC was measured using surface plasmon resonance. The MAbs M2773, M2792, M2775, M2797 and M2781 each had very low dissociation constants (4.5 × 10-9 to 1.2 × 10-8 M). While several of these antibodies have properties which could be useful in diagnostic tests, the combined high fidelity and affinity of M2775 for the IspC protein and serotype 4b isolates, makes it a particularly promising candidate for use in the development of a specific L. monocytogenes serotype 4b diagnostic test. © 2013 Ronholm et al.

• Publisher: PLOS,Public Library of Science,Public Library of Science (PLoS)
• Subject: Amino Acid Sequence / amino terminal sequence / Antibodies, Monoclonal / Antibodies, Monoclonal - immunology / antibody affinity / Antibody Specificity / Antigens / Antigens, Bacterial / Antigens, Bacterial - analysis / Antigens, Bacterial - immunology / autolysin / Bacteria / Bacterial Proteins / Bacterial Proteins - analysis / Bacterial Proteins - immunology / bacterium isolate / Binding Sites / Biochemistry / Biology / C terminal cell wall binding domain / Care and treatment / Causes of / Cell surface / Cell Wall / Cell Wall - chemistry / Cell Wall - immunology / Cell walls / Councils / Cross Reactions / Cross-reaction / Cross-reactivity / Diagnosis / Diagnostic systems / Dissociation / dissociation constant / E coli / Enzyme-Linked Immunosorbent Assay / epitope / epitope mapping / Epitopes / Epitopes - chemistry / Epitopes - immunology / Escherichia coli / Food processing industry / Foodborne diseases / Genetic aspects / Gram-positive bacteria / Health aspects / immunogenic surface protein C / Immunoglobulins / Immunology / Infections / Kinetics / Listeria / Listeria monocytogenes / Listeria monocytogenes - chemistry / Listeria monocytogenes - immunology / Listeriosis / Localization / Mass Spectrometry / Mass spectroscopy / Medicine / Molecular Sequence Data / molecular weight / Monoclonal antibodies / monoclonal antibody / monoclonal antibody M2773 / monoclonal antibody M2774 / monoclonal antibody M2775 / monoclonal antibody M2777 / monoclonal antibody M2778 / monoclonal antibody M2779 / monoclonal antibody M2780 / monoclonal antibody M2781 / monoclonal antibody M2785 / monoclonal antibody M2787 / monoclonal antibody M2790 / monoclonal antibody M2791 / monoclonal antibody M2792 / monoclonal antibody M2795 / monoclonal antibody M2797 / monoclonal antibody M2799 / monoclonal antibody M2800 / N-Acetylmuramoyl-L-alanine Amidase / N-Acetylmuramoyl-L-alanine Amidase - analysis / N-Acetylmuramoyl-L-alanine Amidase - immunology / nucleotide sequence / Pathogens / Physiological aspects / Protein Binding / protein domain / protein localization / Proteins / Reaction kinetics / Reagents / Sequence Alignment / serotype / Serotypes / Serotyping / Surface Plasmon Resonance / Target recognition / Testing laboratories / unclassified drug