Related Experiment Video
Updated: Oct 29, 2026
Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope
Published on: March 24, 2017
Monoclonal antibodies against Streptococcus pneumoniae detect epitopes on eubacterial ribosomal proteins L7/L12 and
Jan Kolberg1, E Arne Høiby2, Rodrigo Lopez3
1National Institute of Public Health, Departments of Vaccinology, PO Box 4404 Torshov, N-0403 Oslo, Norway.
Abstract:
Two monoclonal antibodies (mAbs) designated 144,H-3 (IgG2a) and 218,C-5 (IgM) were produced after immunization of mice with two different heat-treated and sonicated pneumococcal strains. Western blotting, with solubilized proteins from different bacterial genera and from mammalian lymphocytes, showed that both mAbs reacted with a protein of approximately 12 kDa in all 66 strains of eubacteria examined, representing 27 different species. The 12 kDa protein was isolated by immunoaffinity chromatography. Subsequent preparative Western blotting enabled N-terminal amino acid sequence analysis by microsequencing. A high degree of amino acid sequence similarity with eubacterial ribosomal proteins L7/L12 was demonstrated. One of the mAbs (144,H-3) also cross-reacted in Western blotting with a 43 kDa protein, but only from streptococci. The 43 kDa protein carrying the common streptococcal epitope was isolated and sequenced in the N-terminal region. A high degree of amino acid sequence identity was found to elongation factor Ts from Escherichia coli.
Insights
Monoclonal antibodies identified a conserved 12 kDa eubacterial protein, similar to ribosomal protein L7/L12. Another antibody recognized a 43 kDa streptococcal protein, homologous to elongation factor Ts.
Area of Science:
- Immunology
- Microbiology
- Molecular Biology
Background:
- Monoclonal antibodies (mAbs) are crucial tools for identifying specific antigens.
- Pneumococcal strains are significant pathogens requiring precise diagnostic markers.
Purpose of the Study:
- To develop and characterize monoclonal antibodies against pneumococcal antigens.
- To identify conserved bacterial proteins and specific streptococcal epitopes.
Main Methods:
- Production of monoclonal antibodies (mAbs) 144,H-3 (IgG2a) and 218,C-5 (IgM) via mouse immunization.
- Western blotting to detect antigen-antibody reactions across diverse bacterial and mammalian samples.
- Immunoaffinity chromatography and preparative Western blotting for protein isolation.
- N-terminal amino acid sequencing for protein identification.
Main Results:
- Both mAbs recognized a conserved 12 kDa protein in 66 eubacterial strains (27 species).
- The 12 kDa protein showed high sequence similarity to eubacterial ribosomal proteins L7/L12.
- mAb 144,H-3 cross-reacted with a 43 kDa protein exclusively from streptococci.
- The 43 kDa streptococcal protein exhibited sequence identity to elongation factor Ts.
Conclusions:
- A conserved 12 kDa eubacterial protein, likely ribosomal protein L7/L12, is recognized by specific mAbs.
- A distinct 43 kDa streptococcal protein, homologous to elongation factor Ts, can serve as a target for streptococcal identification.
