Aims:  This study aims to develop and characterize monoclonal antibodies (Mabs) with high specificity and affinity for surface antigens of an epidemiologically important serotype 4b of Listeria monocytogenes. Methods and Results:  Hybridoma clones were derived from B lymphocytes of mice immunized with L. monocytogenes serotype 4b and screened against this strain by an enzyme‐linked immunosorbent assay. Twenty‐nine clones secreting Mabs reactive with formalin‐killed bacteria were obtained; 15, 8, 5 and 1 Mabs were immunoglobulin subclasses IgG2a, IgG2b, IgM and IgG1, respectively. Immuofluoresence or immunogold labelling demonstrated all except five IgM and one IgG2a Mabs bound to the surface of a live L. monocytogenes serotype 4b. The majority of the 23 surface‐binding Mabs recognized linear epitopes on a 77‐kDa protein. These surface‐binding Mabs exhibited little or no cross‐reactivity with non‐4b serotypes (1/2a, 1/2b, 3a, etc.) of L. monocytogenes, five other Listeria species, Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium. Conclusions:  The Mabs recognizing a 77‐kDa surface protein are novel antibodies with specificity and affinity for L. monocytogenes serotype 4b. Significance and Impact of the Study:  These anti‐77 kDa surface protein Mabs may be explored as reagents for the development of Mabs‐based diagnostic immunoassays for L. monocytogenes serotype 4b strains.

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