Antibody Mapping of the Linear Epitopes of CMY-2 and SHV-1 β-Lactamases

There are multiple ways to identify the linear epitopes of a protein antigen. Protease digestion of antigen proteins, chemical cleavage at specific residues within a protein, and modification of specific amino acids are all methods to identify epitopes and study antigenicity (2, 3, 9, 19). Two of the more widely used and sophisticated methods employed today are the phage display technique and the SPOT synthesis (SPOTs) method. In phage display, peptide libraries are screened by displaying a vast array of peptides on the surface of bacteriophage (18). Complementary binding peptides are then assayed by affinity binding of the antibody. Through sequence comparison, the reactive peptides displayed by the phage can identify essential binding regions. However, disadvantages to this method include limitation of library size and the number of clones that must be processed by sequencing. This technique is used primarily when few details are known about the protein-protein (e.g., antibody-antigen) binding interaction. In the SPOTs method, antibody-reactive epitopes are mapped on an antigen with known amino acid sequence. SPOTs screens the entire sequence by displaying it as overlapping peptides that are usually 8 to 15 amino acids in length. The simultaneous chemical preparation of these peptides on a membrane support (immobilization) is what is known as SPOT synthesis (6, 7, 15, 17). The term actually refers to the individual peptide spots that are synthesized. These overlapping peptides are assayed for binding reactivity, and the sequence common to the reactive peptides, the consensus sequence, is the epitope (8). We have used the SPOT peptide synthesis method to map the linear epitopes recognized by anti-SHV-1 and anti-CMY-2 polyclonal antibodies to their respective antigens. From the sequence of immunoreactive peptides, we determined which amino acid residues play a role in the binding of the polyclonal rabbit antibodies. Using alanine substitutional analysis, we also identified the amino acids that defined the important epitopes in both β-lactamases. Hujer et al. used this anti-SHV-1 polyclonal antibody in an enzyme-linked immunosorbent assay (ELISA) format for identification and quantification of SHV β-lactamases produced by clinical isolates and to assess levels of SHV protein expression in SHV mutants created in the laboratory setting (14). In the present study we wanted to discern whether SHV-1 variants are recognized as well as SHV-1 by the anti-SHV-1 antibody or whether SHV recognition due to mutations would be altered. This information could potentially affect screening and quantitation of SHV β-lactamases in clinical isolates and laboratory variants (10, 12-14). We also wanted to explain observed differences between the polyclonal anti-CMY-2 and anti-SHV-1 antibodies. It was previously reported that the anti-SHV-1 antibody recognized SHV-1 but not TEM-1 β-lactamase, whereas the anti-CMY-2 antibody recognized many AmpC enzymes (14). Also, the anti-CMY-2 antibody could effectively neutralize multiple class C β-lactamases. To understand these observations as well as to further define the selectivity and specificity of both ELISAs, we performed antibody epitope mapping for SHV-1 and CMY-2 β-lactamases via SPOT synthesis analysis. These results validate our use of the SHV ELISA to assess the effects of amino acid substitutions on SHV expression and to screen for quantitate SHV β-lactamase expression in clinical isolates, and they also explain why the anti-CMY-2 antibody recognizes multiple class C β-lactamases (10, 12-14).