Inoue, and T

Inoue, and T. tag sequence was produced in 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 Atrimustine determination of SEB by enzyme-linked immunosorbent assay and Western blot analysis. Staphylococcal enterotoxins (SEs) are extracellular toxic proteins with a molecular size range of 25 to 28 kDa that cause food poisoning (19). They are known as superantigens, releasing excessive amounts of cytokines by cross-linking with major histocompatibility complex II molecules and T-cell receptors (30, 35). Staphylococcal enterotoxin B (SEB) is one of six SEs antigenetically classified as antigen types A, B, C, D, E, and G (2). The SEB gene has been cloned from chromosomal DNA, and the crystal structure of SEB has been elucidated (15, 33). SEB is a thermostable protein that can withstand heating at 100C for several minutes (14). Due to its structural stability and toxicity, SEB is listed as a potential biological warfare agent by the Centers for Disease Control and Prevention and the World Health Organization. Because a small amount of SEs (0.1 mg) is sufficient to cause intoxication in humans, sensitive and rapid detection of SEs is therefore critical for successful medical treatment. Detection of SEs are commonly done by immunoassays, including enzyme-linked immunosorbent assay (ELISA) (5, 6), surface plasmon resonance (SPR) assay (27), and biomolecular interaction mass spectrometry (23). The antibodies used in these assays have been prepared by hybridoma technology Atrimustine or purified from antisera of animals immunized with enterotoxins, but there are several problems in the production of anti-toxic protein antibodies: (i) identical antisera cannot be prepared constantly, (ii) maintaining hybridoma involves high costs, and (iii) the preparation of antibodies against toxic proteins is dangerous. As an alternative strategy, phage display technology has been widely used to generate the molecular recognition peptides and Atrimustine proteins (10). Compared to antibodies, smaller monovalent antibody fragments such as the fragment antigen-binding (Fab fragment) and single-chain variable fragment (scFv) may be favorable because of protein stability due to the small molecular size. An scFv is a fusion molecule of the variable regions of heavy and light antibody chains linked together with a short linker peptide (18). In phage display technology, the DNA regions encoding antibody fragments or short peptides are cloned into phagemid vectors and subsequently expressed as fusion proteins with phage coat proteins in Rabbit Polyclonal to SMUG1 selection process called panning (20, 21). Unlike conventional antibodies, recombinant antibodylike proteins can be permanently produced in large quantities at low cost. The affinity and specificity of recombinant antibodylike proteins can be improved by random or site-specific mutagenesis (4, 9). Several investigators reported the production of recombinant antibodylike proteins that bound selectively to biological warfare agents such as Shiga toxin, ricin, and the spore forms of (7, 11, 22). Hexamer and dodecamer peptide ligands that bind to SEB have been isolated from a combinatorial peptide library (8, 32, 34), but the affinity of these small-molecule ligands to SEB does not seem high. However, production of a SEB-specific recombinant antibodylike protein has not been reported. The aim of the present study was to generate Fab fragments and scFv proteins binding to SEB using phage display technology. A unique method for preparing an anti-SEB Fab fragment library was developed. The SEB epitope was first elucidated by phage display screening from the Ph.D-7 and Ph.D-12 library against commercial anti-SEB antibody. Mice were immunized with carrier maltose-binding protein (MBP) fused directly with a SEB epitope peptide instead of toxic SEB directly. Positive clones were obtained from a phage display Fab library prepared from spleen cells, and soluble anti-SEB Fab fragment protein was isolated from one of the three clones. The obtained Fab fragment was converted into scFv, and both proteins were produced in and characterized for SEB binding affinity. The usefulness of these agents as molecular recognition tools was ascertained by successful application to the SEB determinants from serum by Western blotting. This is the first report associated with the anti-SEB Fab fragment and scFv. MATERIALS AND METHODS Materials. SEB and other biochemicals were purchased from Sigma Chemical Company (St. Louis, MO). Anti-SEB monoclonal antibody (ab53981) was purchased form Abcam (Cambridge, United Kingdom). Anti-M13 monoclonal antibody, restriction endonucleases, Ph.D.-12 and Ph.D-7 phage display peptide libraries, and the pMal-PIII vector were purchased from New England Biolabs (Ipswich, MA). KOD-Plus DNA polymerase was purchased from Toyobo (Osaka, Japan). The pET3a vector.