title: Phage-display selection of antibodies to the left end of CTX3C using synthetic fragments.
abstract: Ciguatoxins (CTXs) are a family of toxins that contaminate a variety of fish and cause ciguatera seafood poisoning. The limited availability of CTXs from natural sources has hampered preparation of antibodies and, thus, the development of immunoassays for these toxins. In the current studies, we utilized synthetic fragments as haptens to prepare antibodies against CTX3C, a congener of CTXs, thereby avoiding the problem of its scarcity. Synthetic ABC-ring fragment (ABC) of CTX3C was conjugated with keyhole limpet hemocyanin (KLH) and immunized on mice. Phage-displayed antibodies were first screened based on affinity to a soluble biotin-linked ABC-ring fragment that was captured on streptavidin-linked magnetic beads. The beads were then eluted with the ABCD-ring fragment (ABCD), and recovered phages were amplified. This elution with a synthetic fragment allowed the preparation of antibodies to ABCD as well as to ABC. Three antibodies with affinities of K(d) approximately 10(-5) M for ABCD were selected and prepared as soluble recombinant Fabs (rFabs). One rFab, 1C49, bound to CTX3C itself, although the binding affinity for CTX3C was weaker than for ABCD.
authors.affiliation: Department of Chemistry, Graduate School of Science, Tohoku University, Sendai, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Nagumo
authors.forename: Hiroki
authors.initials: H
authors.lastname: Oguri
authors.forename: Kouhei
authors.initials: K
authors.lastname: Tsumoto
authors.forename: Yumi
authors.initials: Y
authors.lastname: Shindo
authors.forename: Masahiro
authors.initials: M
authors.lastname: Hirama
authors.forename: Takeshi
authors.initials: T
authors.lastname: Tsumuraya
authors.forename: Ikuo
authors.initials: I
authors.lastname: Fujii
authors.forename: Yoshihisa
authors.initials: Y
authors.lastname: Tomioka
authors.forename: Michinao
authors.initials: M
authors.lastname: Mizugaki
authors.forename: Izumi
authors.initials: I
authors.lastname: Kumagai
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000378:metabolism; D000915:Antibody Affinity; D002922:Ciguatoxins / Q000737:chemistry / Q000276:immunology* / Q000633:toxicity; D006241:Haptens / Q000737:chemistry / Q000276:immunology; D006433:Hemocyanins / Q000737:chemistry; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D051379:Mice; D008863:Microspheres; D008969:Molecular Sequence Data; D019151:Peptide Library; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D019809:Streptavidin / Q000737:chemistry
chemical_list: D000906:Antibodies; D006241:Haptens; D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library; D011994:Recombinant Proteins; D002922:Ciguatoxins; C082191:ciguatoxin 2; D019809:Streptavidin; D006433:Hemocyanins