Immunoassay ciguatoxin

Assays of ciguatoxin. Determination of ciguatoxin levels in fish was carried out in many laboratories by mouse assays. Enzyme immunoassay to screen inedible fish has been proposed by Hokama (9). No specific chemical assay has been developed, as information on functional groups suitable for fluorescence labeling is not available. Analyses conducted in the authors laboratory on remnant fish retrieved from patients meals indicated that ciguatoxin content as low level as 1 ppb could cause intoxication in adults. An extremely high sensitivity and a sophisticated pretreatment method will be required for designing a fluorometric determination method for the toxin. 

Hokama, Y., An enzyme immunoassay for the detection of ciguatoxin and competive inhibition by related natural polyether toxins, in Seafood Toxins, Ragelis, E., ed., American Chemical Society, Washington, D.C., 1984. 

Enzyme-Immunoassay. Fish tissue samples for testing were cut into uniform 3mm thick slices with parallel razor blades mounted on a handle. Four discs were then punched out from each slice with a stainless steel borer, 3-mm in diameter, and each disc was placed in a well of a 96-well polystyrene microtiter plate (Flow Laboratories, Inc., Hamden, CT). Samples were washed once with 0.2 ml Tris buffer. After the wash solution was aspirated, each sample was fixed in 0.2 ml of 0.3% H O -methanol fixative for 30 min. at room temperature. Samples were then transferred to clean wells and 0.2 ml of a 1 100 dilution of sheep-anti-ciguatoxin-horseradish peroxidase conjugate in Tris buffer was added to each well. The plate was then incubated at room temperature for 1 hr. The sheep-anti-cigua-toxin-horseradish peroxidase was removed by aspiration, and the tissues were immersed for 5 min. in 0.2 ml Tris buffer. Each sample was transferred to clean wells and incubated for 5 min. at room temperature with 0.2 ml of 4-chloro-l-naphthol substrate. The final steps involved removal of the tissue and addition of 0.015 ml of 3 M sodium hydroxide to stop the enzymatic reaction. Absorbance readings at 405 nm of each well were obtained in the Titertek Multi-skan (Flow Laboratories, Inc., Hamden, CT). 

Competitive Inhibition in the Enzyme-Immunoassay with Purified Polyethers Including Ciguatoxin. Figure 2 represents an inhibition curve in the competitive test between a positive fish tissue and highly purified CTX (LD = 0.25 yg/kg mouse) in suspension. Approximately 0.002 ng of purified ciguatoxin gave 50% inhibition of the normal binding between sheep anti-CTX and the toxic fish tissue (Lutj anus sp.). 

The analysis of fish tissues for ciguatoxin by a newly developed enzyme-immunoassay procedure (26, 27) has been carried out in this study. Three areas of examinations have been attempted (1) the examination of clinically defined and documented and non-toxic consumed fish samples (2) the assessment of freshly caught fishes from the sites in the Leeward part of the island of Oahu where ciguatoxin is found and (3) competitive inhibition with suspension of purified ciguatoxin and closely related structurally similar polyether toxins. 

Tsumuraya, T., Fujii, L, Inoue, M., Tatami, A., Miyazaki, K., and Hirama, M., Production of monoclonal antibodies for sandwich immunoassay detection of ciguatoxin 51-hydroxyCTX3C. Toxicon, 48, 287-94, 2006. 

Hokama Y. A rapid, simplified enzyme immunoassay stick test for the detection of ciguatoxin and related polyethers from fish tissues. Toxicon 23 939-946, 1985. 

Immunoassays are also used to marine toxin detection. Hokama et al. first developed a test for detecting ciguatoxin directly from natmal somces by radioimmunoassay (RIA). In this assay. 

Hokama, Y, Abad, M.A. and Kimura, L.H., A rapid enzyme-immunoassay for the detection of ciguatoxin in contaminated fish tissues, Toxicon, 21, 817, 1983. 

Hokama, Y. Shirai, L. K. Iwamoto, L. M. Kobayashi, M. N. Goto, C. S. Nakagawa, L. K. Assessment of a rapid enzyme immunoassay stick test for the detection of ciguatoxin and related polyether toxins in fish tissues. Biol. Bull. (Woods Hole, Mass.), 172 144-53. 1987. 

Absolute configuration of ciguatoxin and development of immunoassay systems 07BCJ1870. 

Abbreviations UV, ultraviolet EIA, enzyme immunoassay S-PIA, solid-phase immunobead assay P-CTX-1, Pacific ciguatoxin-1 C-CTX-1, Caribbean ciguatoxin-1 EC50, half maximally effective concentration. 

Immunological detection of ciguatoxins and related polyethers has received particular attention compared to other marine toxins. The initial RIA and enzyme immunoassay employing a polyclonal sheep anti-ciguatoxin antibody revealed cross-reactivities between ciguatoxins and other polyether toxins, suggesting the need for monoclonal antibodies. The availability of monoclonal antibodies allowed for the development of stick enzyme immrmoassay methods and solid-phase immunobead techniques (known as the paddle test), which successfully recognized toxins attached to correction fluid-coated bamboo sticks or paddles previously exposed to toxic fish tissues. 

---