Phage-display selection of antibodies to the left end of CTX3C using synthetic fragments.
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.
Nagumo
Yoko
Y
Department of Chemistry, Graduate School of Science, Tohoku University, Sendai, Japan.
Oguri
Hiroki
H
Tsumoto
Kouhei
K
Shindo
Yumi
Y
Hirama
Masahiro
M
Tsumuraya
Takeshi
T
Fujii
Ikuo
I
Tomioka
Yoshihisa
Y
Mizugaki
Michinao
M
Kumagai
Izumi
I
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
D000906:Antibodies; D006241:Haptens; D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library; D011994:Recombinant Proteins; D002922:Ciguatoxins; C082191:ciguatoxin 2; D019809:Streptavidin; D006433:Hemocyanins