Characterisation of mouse monoclonal antibodies for pneumolysin: fine epitope mapping and V gene usage

Immunol Lett. 2003 Sep 8;88(3):227-39. doi: 10.1016/s0165-2478(03)00081-6.


Pneumolysin (PLY) is a cholesterol-dependent cytolysin (CDC) produced by Streptococcus pneumoniae, the main cause of community-acquired pneumonia. We have applied a set of diverse molecular methodologies (PCR-derived PLY peptides, biopanning of a library of phage-displayed random nonapeptides, indirect ELISA and competition tests with soluble peptides) to achieve concordant complementary observations in order to obtain a fine epitope mapping of three mouse monoclonal antibodies (PLY-4, PLY-7 and PLY-8) for PLY. PLY-4 seems to recognise a conformation-dependent epitope with a core reactivity involving R232. The epitopes recognised by PLY-7 and PLY-8 are within the sequences (401)GQDLTAH(407) and (450)KRTISIWGT(458), respectively. PLY-7 also recognises suilysin (SLY), in which the homologous reactive amino acid stretch is (429)GVNLTSH(435). In a homology model of PLY with the crystal structure of perfringolysin O (PFO), R232 is part of a well-exposed contorted loop on the edge of the concave and convex faces of domain 1. The sequences reactive with PLY-7 and PLY-8 would conform one of the loops at the bottom of domain 4 and a beta strand of one of the two beta sheets of this domain, respectively. Western blot analyses carried out with anti-PLY rabbit IgG and polyclonal mouse serum identified stretches comprising residues 40-98, 199-248, 352-414 and 415-471 of PLY as immunogenic and antigenic; altogether with their recognition by the monoclonal antibodies herein considered, these results stress the immunological significance of domains 1 and 4 of the PLY molecule. PLY-4, PLY-7 and PLY-8 share the same Vkappa chain; this chain and that of the PLY-5 monoclonal antibody are essentially in germline configuration, whereas the VH regions of these monoclonals come from diverse gene segments and are mutated.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibodies, Monoclonal / chemistry
  • Antibodies, Monoclonal / genetics*
  • Antibodies, Monoclonal / immunology*
  • Bacterial Proteins
  • Blotting, Western
  • Enzyme-Linked Immunosorbent Assay
  • Epitope Mapping
  • Epitopes / chemistry
  • Epitopes / immunology*
  • Humans
  • Immune Sera / immunology
  • Immunoglobulin Variable Region / genetics*
  • Mice
  • Models, Molecular
  • Neutralization Tests
  • Peptide Library
  • Protein Conformation
  • Sequence Alignment
  • Streptococcus pneumoniae / immunology
  • Streptolysins / chemistry
  • Streptolysins / immunology*


  • Antibodies, Monoclonal
  • Bacterial Proteins
  • Epitopes
  • Immune Sera
  • Immunoglobulin Variable Region
  • Peptide Library
  • Streptolysins
  • plY protein, Streptococcus pneumoniae