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∼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.

Introduction

Ciguatera is widespread human seafood poisoning caused by ingestion of reef fish, and is estimated to affect more than 20,000 people annually Scheuer, 1994, Lewis, 2001. The causative toxins of ciguatera, known as ciguatoxins (CTXs) Yasumoto, 2001, Yasumoto and Murata, 1993, are produced by the marine dinoflagellate Gambierdiscus toxicus and accumulate in various reef fish through the food chain. Ciguatoxin (Murata et al., 1990) and its congeners, CTX3C (Satake et al., 1993), 51-hydroxy-CTX3C (Satake et al., 1998), and CTX4B (Murata et al., 1990) are structurally classified as ladder-like polyethers, in which structural difference between the congeners mainly arises from the terminal substituents at A and L-ring (Fig. 1). Pharmacological studies revealed that CTXs exert potent toxicities to mammals [acute toxicity on mice median lethal dose (LD₅₀) 0.27–4.0 μg/kg] by binding to the voltage-sensitive sodium channels. Despite the negative impacts of ciguatera in tropical and subtropical regions, there is currently no rapid and reliable method for detecting these toxins at fisheries. Development of a practical immunochemical test to assess contamination prior to consumption of the seafood has been a longstanding problem (Hokama, 1993) mostly because CTXs are available in only extremely limited amounts, making antibody development difficult (Hokama et al., 1998).

To solve the problems of antibody development, we have focused on the use of synthetic fragments as haptens in place of the natural CTXs Oguri et al., 1999, Oguri et al., 2003, Pauillac et al., 2000. In the synthesis-based approach toward CTX3C Satake et al., 1993, Hirama et al., 2001, Inoue et al., 2002, antibodies that bind to targeted positions of CTX3C were prepared by immunization of protein conjugates of rationally designed haptens. X-ray analyses of the complexes of antibodies and low-molecular-weight haptens showed that the buried surface area of small haptens in the antigen-binding site is approximately 200–400 Ų Arevalo et al., 1993, Jeffrey et al., 1995, Tanaka et al., 1996, Romesberg et al., 1998, Xu et al., 1999. This suggested that the tricyclic ABC-ring skeleton (calculated water accessible surface area (Conolly surface) of approximately 250 Ų) may be an effective epitope for the molecular recognition of CTX3C. We initially designed ABC-ring fragment (ABC) (Nagumo et al., 2001) as a hapten for production of antibodies that bind to the left side of CTX3C (Fig. 1). The ABC-ring fragment was conjugated with carrier proteins and used to immunize mice. Using hybridoma technology (Goding, 1980), positive clones were screened against both a bovine serum albumin (BSA) conjugate (ABC–BSA) and the free ABC. After considerable effort, we prepared more than 20 mAbs that bind to either ABC–BSA or ABC. However, none of the mAbs bound to CTX3C itself. These results led us to believe that the possible epitope for the left side of CTX3C should be larger than the tricyclic ABC-ring skeleton. Therefore, we envisioned the tetracyclic ABCD-ring skeleton as the possible epitope. In this report, we describe the preparation of antibodies that bind to ABCD-ring fragment (ABCD) of CTX3C based on phage display technology Clackson et al., 1991, Hawkins et al., 1992, Scholthof et al., 1997, Lee et al., 2002. Selected positive clones were expressed as soluble recombinant Fabs (rFabs), and binding properties of the rFabs to the synthetic fragments (ABC, ABCD) and CTX3C were characterized.