A competitive direct enzyme-linked immunofiltration assay for the detection of saxitoxin was developed, using polyclonal antibodies against saxitoxin and a saxitoxin-horseradish peroxidase conjugate. The test was performed in an eight-well plastic test device, in which antibody-coated nylon membranes were pressed tightly to an absorbent cellulose layer. Saxitoxin standard or sample extract solution, saxitoxin-conjugate, and enzyme substrate/chromogen solution were sequentially added on to the membrane. The test was evaluated visually by comparing the intensity of the resulting coloured (blue) dot with that of a negative control. The detection limits for saxitoxin in buffer solution and in shellfish tissue were 4 ng/ml and 80 ng/g respectively, with an assay time of less than 15 min. Under the conditions of the immunofiltration assay, decarbamoyl-saxitoxin, gonyautoxin 2/3, and neosaxitoxin standards (in buffer) gave a positive response at concentrations of about 10 ng/ml, 40 ng/ml, and 80 ng/ml, respectively. The relative cross-reactivity of the antibody to these PSPs was similar when determined using both direct and indirect (using a saxitoxin-bovine serum albumin conjugate) competitive enzyme immunoassays in microtitre plate format. In competitive direct microtitre plate assays, the 50% binding values found for saxitoxin, decarbamoyl-saxitoxin, gonyautoxin 2/3 and neosaxitoxin were 15 pg/ml, 47.5 pg/ml, 163.5 pg/ml, and 510 pg/ml respectively. In competitive indirect microtitre assay, the respective values were 138 pg/ml, 404 pg/ml, 1582 pg/ml, and 6982 pg/ml.
Animals Bivalvia