Epitope mapping of monoclonal antibodies to Escherichia coli ribosomal protein S3.
The antigenic structure of Escherichia coli ribosomal protein S3 has been investigated by use of monoclonal antibodies. Six S3-specific monoclonal antibodies secreted by mouse hybridomas have been identified by immunoblotting of two-dimensional ribosomal protein separation gels. By using a competitive enzyme-linked immunosorbent assay, we have divided these monoclonal antibodies into three mutual inhibition groups, members of which are directed to three distinct regions of the S3 molecule. The independence of these monoclonal antibody-defined regions was confirmed by the failure of pairs of monoclonal antibodies from two inhibition groups to block the binding of biotinylated monoclonal antibodies of the third group. To determine the regions recognized by these monoclonal antibodies, chemically cleaved S3 peptides were fractionated by gel filtration and reverse-phase high-performance liquid chromatography. The fractionated peptides were coated on plates and examined for specific interaction with monoclonal antibody by enzyme immunoassay. In this manner, two epitopes have been mapped at the ends of the S3 molecule: one, in the last 22 residues, is recognized by three monoclonal antibodies; and the second, in the first 21 residues, is defined by two monoclonal antibodies. The third S3 epitope, recognized by a single monoclonal antibody, has been localized in a central segment of about 90 residues by gel electrophoresis and immunoblotting. These epitope-mapped monoclonal antibodies are valuable probes for studying S3 structure in situ.
Department of Physiological Chemistry, University of Wisconsin, Madison 53706.
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D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D001426:Bacterial Proteins / Q000276:immunology*; D001665:Binding Sites; D002850:Chromatography, Gel; D000939:Epitopes / Q000276:immunology*; D004926:Escherichia coli / Q000276:immunology*; D006825:Hybridomas / Q000276:immunology; D015151:Immunoblotting; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D012269:Ribosomal Proteins / Q000276:immunology* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D000939:Epitopes; D010446:Peptide Fragments; D012269:Ribosomal Proteins; C044152:ribosomal protein S3