Synthetic peptides derived from the Listeria monocytogenes p60 protein as antigens for the generation of polyclonal antibodies specific for secreted cell-free L. monocytogenes p60 proteins

Abstract

All species of the genus Listeria secrete a major extracellular protein called p60. A comparison of the deduced amino acid sequences of all listerial p60 proteins previously indicated there were only a few regions which were unique to the pathogenic, food-borne species Listeria monocytogenes. Two of these p60 regions were chosen for the development of antibodies specific for the facultative intracellular species L. monocytogenes. Initially, these regions were characterized via epitope mapping, and this led to the development of two different synthetic peptides. Rabbits immunized with these synthetic peptides generated polyclonal antibodies that were then used in Western blot (immunoblot) analyses. Antiserum against peptide A (PepA) recognized the p60 protein in the supernatants collected from most L. monocytogenes serotypes except for several strains belonging to serotypes 4a and 4c. No p60-related protein was detected in the supernatants from other Listeria species with this anti-PepA antiserum. Antibodies raised against peptide D (PepD) reacted with p60 from all L. monocytogenes serotypes, including all 4a and 4c strains that were tested, and also showed no cross-reactivity with supernatant proteins from other Listeria species. Both antisera also detected p60 in supernatants of a large number of environmental isolates of L. monocytogenes. Besides Western blot analyses, these antisera to PepA and PepD reacted with secreted p60 in an enzyme-linked immunosorbent assay, indicating recognition of the native antigen in addition to the denatured form. These data suggest that synthetic peptides derived from the variable region of the L. monocytogenes p60 protein may be useful for the development of an immunological diagnostic assay.