Indirect Enzyme-Linked Immunosorbent Assay for Saxitoxin in Shellfish

Abstract

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of saxitoxin (STX). Antibodies against STX were demonstrated in rabbits 5 weeks after immunizing with STX-bovine serum albumin (STX-HCHO-BSA). In the ELISA, STX-HCHO-BSA or polylysine-STX was coated onto the microtiter plate, followed by incubation with standard toxin and anti-STX antibody. The amount of antibody bound to the solid phase was determined by incubation with goat anti-rabbit IgG peroxidase conjugate and a reaction with chromogenic substrate. Competitive indirect ELISA revealed that the antiserum did not cross-react with either carbamoyl-neo-STX-suIfate or tetrodotoxin. The antibodies for STX cross-reacted with decarbamoyl- STX and neo-STX about 56% and 16% as much as they did with STX, respectively. The lower detection limits for STX, decarbamoyl-STX, and neo-STX in this sytem were about 25, 45, and 156 pg per assay, respectively. When STX added to clams or mussels was assayed, the detection limit for STX was about 50-100 ppb, and recoveries were in the range of 86.8-107%.

Effect of heterologous paralytic shellfish poisoning toxin-enzyme conjugates on the cross-reactivity of a saxitoxin enzyme immunoassay

“…The indirect assay had the least overall sensitivity, particularly for neoSTX. Similar results were also found by Chu and Fan (1985). By contrast, all three direct assays were very sensitive, with 50% inhibition binding values ranging from 15-20 pg ml-' for STX to 270-510 pg ml-' for neoSTX.…”

“…An STX-bovine serum albumin conjugate (STX-BSA) was prepared by formaldehyde treatment (Johnson et al 1964) according to the method of Chu and Fan (1985) and Renz and Terplan (1988). To prepare toxin-HRP conjugates, HRP (4 mg) was activated with periodate according to Wilson and Nakane (1978).…”

“…Because of the variable composition of structurally different toxins, which may be present in a single sample, assay specificity is a major problem in immunochemical approaches to the detection of PSP toxins. Enzyme immunoassay (EIA) methods have been described for saxitoxin (Chu and Fan 1985) and neosaxitoxin (Chu et al 1992). These assays, and those we have described (Renz and Terplan 1988;Usleber et al 1991), have high sensitivity for the parent toxin but cross-react extensively with other PSP toxins.…”

Paralytic Shellfish Toxins: Their Origin, Characteristics and Methods of Detection: A Review

“…The antibody failed to cross-react with a mixture of N-21 sulfocarbamoyl derivatives of STX, which suggests that the sulfonate group at the N-2 1 position effectively blocks antigen attachment to STX-Ab. It is possible that substitution of a sulfonate group at the C-1 1 position plus the substitution of an OH group at the N-1 position may significantly alter the conformation of the STX derivative so as not to react with STX-Ab as observed with carbamoyl-neo-STX-SO, by Chu and Fan (1985). It may also be possible that conjugation of STX to polyalanyllysine may preclude resulting antibodies from reacting with N-2 1 sulfocarbamoyl derivatives.…”

Development of a Tropical Fish Assay for Saxitoxin

“…About 0.2 pg of STX is required to kill a 20 g mouse in 15 min (AOAC 1990). Other more sensitive methods such as high performance liquid chromatography (Sullivan and Iwaoka 1983), enzyme-linked immunosorption assay (Chu and Fan 1985) and tissue culture assay (Kogure et al 1988) are costly to establish or maintain. and G. monilutu.…”