Generation of Fab fragment-like molecular recognition proteins against staphylococcal enterotoxin B by phage display technology.
Antigen-binding fragments (Fab fragments) and single-chain variable fragments (scFv) against staphylococcal enterotoxin B (SEB) were produced by phage display technology. SEB epitopes were first identified by phage display approach using the commercial anti-SEB monoclonal antibody ab53981 as the target. Heptamer and dodecamer mimotope peptides recognized by ab53981 were screened from Ph.D-7 or Ph.D-12 random peptide phage libraries expressed in Escherichia coli. The isolated 7-mer and 12-mer mimotopes were shown to share a sequence homologous to ⁸PDELHK¹⁴S in the amino acid sequence of SEB. The N-terminal 15-mer peptide of SEB was determined to be an epitope of ab53981. After immunization of mice with maltose-binding protein-tagged N-terminal 15-mer peptide, a phage display Fab library was constructed using cDNA prepared from the mRNAs of spleen cells. Three phage clones displaying the Fab molecule which recognized SEB were isolated through three rounds of panning. Only one of them produced a soluble Fab fragment from the transformed cells, and the fragment fused with a histidine tag sequence was produced in E. coli cells and converted into scFv. Surface plasmon resonance analysis showed that the dissociation constants of these proteins with SEB were (4.1 ± 1.1) × 10⁻⁹ M and (8.4 ± 2.3) × 10⁻¹⁰ M, respectively. The produced molecule was applied to the determination of SEB by enzyme-linked immunosorbent assay and Western blot analysis.
Urushibata
Yuji
Y
Third Department of Forensic Science, National Research Institute of Police Science, Kashiwanoha, Kashiwa 277-0882, Japan.
Itoh
Kunihiko
K
Ohshima
Motohiro
M
Seto
Yasuo
Y
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D004768:Enterotoxins / Q000276:immunology / Q000378:metabolism*; D018604:Epitope Mapping / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D007140:Immunoglobulin Fab Fragments / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D019151:Peptide Library; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000276:immunology / Q000378:metabolism*; D020349:Surface Plasmon Resonance
D000911:Antibodies, Monoclonal; D004768:Enterotoxins; D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; C031818:enterotoxin B, staphylococcal