Quantification of saxitoxin in human blood by ELISA

Abstract

Saxitoxin (STX) is a potent marine toxin that causes paralytic shellfish poisoning (PSP) which can result in significant morbidity and mortality in humans. Low lethal doses, rapid onset of PSP symptoms, and brief STX half-life in vivo require sensitive and rapid diagnostic techniques to monitor human exposures. Our laboratory has validated an enzyme-linked immunosorbent assay (ELISA) for quantitative detection of STX from 0.020 to 0.80 ng/mL in human whole blood and from 0.06 to 2.0 ng/mL in dried human blood which is simple, sensitive, rapid, and cost-effective. To our knowledge, this is the first validated method for the quantitation of saxitoxin in whole blood. Microsampling devices were used in sample collection which allows for standardized collection of blood, stable storage, and cost-efficient shipping. Quality control precision and accuracy were evaluated over the course of validation and were within 20% of theoretical concentrations. No detectable background concentrations of STX were found among fifty whole blood and dried blood convenience samples. Additionally, ten spiked individual whole blood and dried blood samples were tested for accuracy and precision and were within 20% of theoretical concentrations. Gonyautoxins 2&3 (GTX2&3) cross-reacted with this ELISA by 21%, but all other structurally related PSP toxins tested cross-reacted less than two percent. While clinical diagnosis or treatment of PSP would be unaffected by GTX2&3 cross-reactivity by ELISA, to accurately quantify individual PSP toxins, these results should be coupled with high performance liquid chromatography mass spectrometry measurements.

Keywords: Dried blood; ELISA; Microsampling devices; Saxitoxin.

Fig. 1.

(A) Calibration curve for STX. Each point on the curve represents the mean of duplicate samples and is representative of two independent experiments. (B) The working range was assessed by performing linear regression on a plot of theoretical spiked STX concentrations against STX concentrations interpolated by the assay (n = 2) and is representative of two independent experiments. Solid line, spiked pooled blood; Dashed line, synthetic blood.

Fig. 2.

Accuracy and precision for 10 individual whole blood convenience samples each spiked at 2 concentrations with STX and interpolated from a (A) pooled blood standard curve or a (B) synthetic blood standard curve. 0.3 ng/mL STX, black bar; 0.080 ng/mL STX, gray bar. Error bars reflect the standard deviations from 2 replicate experiments. Spiked convenience sample mean, average % accuracy, and average RSD interpolated from (C) whole blood calibrators or (D) synthetic blood calibrators from 2 replicate experiments. ¹Standard deviation/average × 100%. ²Measured concentration/theoretical concentration × 100%.

Fig. 3.

(A) Accuracy and precision for ten individual whole blood convenience samples each spiked at two concentrations with STX. 0.16 ng/mL STX, black bar; 1.2 ng/mL STX, gray bar. Error bars reflect the standard deviations from three replicate experiments. (B) Mean, % accuracy, and RSD averages from three replicate experiments. ¹Standard deviation/average × 100%. ²Measured concentration/theoretical concentration × 100%.

Fig. 4.

(A) Standard ELISA curves for STX and structurally related PSP toxins. Each point on the curve represents the mean of duplicate samples ± the standard deviation and is representative of 2 independent experiments. (B) Cross-reactivities of PSP toxins based on the standard curves. ¹Midpoint STX/midpoint PSP analyte × 100%.