Production of antibodies and development of highly sensitive formats of enzyme immunoassay for saxitoxin analysis

In this paper the production of antibodies against saxitoxin (STX) is described, as is the optimization and comparison of two competitive ELISA formats (direct and indirect) for the detection of this toxin. Tests were performed in a 96-well microplate using the toxin-specific polyclonal antibodies produced in our laboratory, obtained from rabbits immunized with saxitoxin-keyhole limpet hemocyanin (STX–KLH). In indirect ELISA format saxitoxin, conjugated to bovine serum albumin (STX– BSA) was coated onto the microtitre plate and incubated with standard toxin and anti-STX antibody. A goat antirabbit IgG Peroxidase conjugate was used to enable detection. In the direct ELISA format, STX standard, STX conjugate to horseradish peroxidase (STX–HRP), and enzyme substrate/chromogen solution were sequentially added to the microplate after antibody coating. Results showed the saxitoxin detection limit to be 3 and 10 pg mL–1 for direct and indirect ELISA formats, respectively. The suitability of the assay for quantification of saxitoxin in mussels was also studied. Samples were spiked with saxitoxin before and after sample treatment to study the extraction efficiency and matrix effect, respectively. After treatment, samples were analysed at 1:1000 v/v dilution in PBS to minimize the matrix effect and to detect the regulatory limit of 40–80 μg saxitoxin per 100 g mussels as stipulated by the Food and Drug Administration. The efficiency of extraction of saxitoxin was from 72 to 102%. These data were confirmed by liquid chromatography coupled with fluorimetric detection, the technique currently used for quantitative determination of toxins in seafood.