Development of a Rapid, Sensitive and Reliable Clinical Diagnostic Test to Measure Paralytic Shellfish Toxins in Human Matrices

Abstract

Paralytic shellfish poisoning is a lethal poisoning caused by the consumption of shellfish like clams, oysters, scallops and mussels that are contaminated with paralytic shellfish toxins. These toxins block sodium channel receptors on respiratory smooth muscle and can cause respiratory paralysis in severe exposures. A cure has not been discovered for this poisoning yet, so supportive treatment is the only treatment available for these victims. A quick clinical diagnostic test is necessary to identify poisoning so that patients can be treated properly. A rapid screening ELISA was developed to detect saxitoxin, gonyautoxin (GTX) 2,3, decarbamoyl gonyautoxin 2,3, decarbamoyl saxitoxin, neosaxitoxin and gonyautoxin 1,4 in human urine with a sensitivity of 0.02, 0.10, 0.10, 1.0, 1.0 and 15.0 ng/mL, respectively. I have also validated a rapid screening ELISA assay to detect these toxins in blood with a sensitivity of 0.02, 0.12, 1.20, 0.08, 5.0 and 25.0 ng/mL, respectively. Though these assays can be performed quickly and are easily accessible to most laboratories, they are semi-quantitative and cannot be used to determine total toxicity. High performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) can overcome the deficiencies of an ELISA method by providing identification of toxins present in a sample and quantification of each toxin. Therefore, I developed an HPLC-MS/MS to quantitate GTXs 1,4 and GTXs 2,3 in urine. An Oasis mixed mode strong cationic exchange cartridge was used for solid phase extraction and melamine was used as a surrogate internal standard in the HPLC-MS/MS method. Linear dynamic ranges of 4.25-544, 4.07-521, 1.55-199 and 1.37-176 ng/mL were achieved for GTX1, 2, 3 and 4, respectively, in pooled urine. In the future, the ELISA method can be further refined to increase the sensitivity for neosaxitoxin and GTX 1,4 using anti-neosaxitoxin antibody kits. Validation of the HPLC-MS/MS method will provide specificity for paralytic shellfish toxins in individual urine specimens.