Rapid and Sensitive ELISA Screening Assay for Several Paralytic Shellfish Toxins in Human Urine

Abstract

Paralytic shellfish poisoning is caused by a group of paralytic shellfish toxins that are produced by dinoflagellates. Toxins in this group include saxitoxin, neosaxitoxin and gonyautoxins. A rapid diagnostic test to identify poisoning by these toxins can be helpful in guiding the appropriate treatment of victims. Additionally, quick receipt of diagnostic results can provide timely proof that shellfish harvesting should be stopped in a given area, thereby preventing additional exposures. We have developed and validated a rapid urinary enzyme-linked immunosorbent assay-based screening test to diagnose exposure to several major paralytic shellfish toxins. The lower limit of detection (LLOD) for multiple paralytic shellfish toxins was characterized as 0.02, 0.10, 0.10, 1.0, 1.0 and 15 ng/mL for saxitoxin, gonyautoxin 2,3, decarbamoyl gonyautoxin 2,3, decarbamoyl saxitoxin, neosaxitoxin and gonyautoxin 1,4, respectively. No interferences were identified in unspiked pooled urine or in specimens collected from unexposed individuals indicating that this method is specific for the paralytic shellfish toxins tested. The accuracy of this test was demonstrated in 10 individual urine specimens with osmolalities ranging from 217 to 1,063 mOsmol/kg and pHs ranging between 5.06 and 7.45. These specimens were spiked with toxins at their LLODs and the presence of toxins at these concentrations was accurately identified in all cases. These results indicate that this diagnostic test can be used to rapidly and accurately screen urine for paralytic shellfish toxins.

Novel Polyclonal Antibody Raised against Tetrodotoxin Using Its Haptenic Antigen Prepared from 4,9-anhydrotetrodotoxin Reacted with 1,2-Ethaneditiol and Further Reacted with Keyhole Limpet Hemocyanin

“…ELISA technique for summed PSTs concentration has been reported [36,39]. In comparison to them, sensitivity for TTX on ELISA in the present study seems to be insufficient.…”

Saxitoxin Exposure Confirmed by Human Urine and Food Analysis

“…The current regulatory limit for total PST toxicity in commercially harvested shellfish is 80 μg STX equivalents/100 g shellfish (16). Methods such as the mouse bioassay (MBA, AOAC Method 959.08), receptor binding assay and enzyme-linked immunosorbent assays do not differentiate the various STX congeners, but still provide a measure of toxicity that has been comparable across laboratories (7, 17–19). Currently, there is one pre-column oxidation HPLC method (AOAC Method 2005.06) approved for use on the US commercial shellfish that can identify individual congeners, but not all regulatory labs have implemented this testing (20).…”