Receptor Binding Assay for Paralytic Shellfish Poisoning Toxins: Optimization and Interlaboratory Comparison

Abstract

A receptor binding assay (RBA) for detection of paralytic shellfish poisoning (PSP) toxins was formatted for use in a high throughput detection system using microplate scintillation counting. The RBA technology was transferred from the National Ocean Service, which uses a Wallac TriLux 1450 MicroBeta microplate scintillation counter, to the California Department of Health Services, which uses a Packard TopCount scintillation counter. Due to differences in the detector arrangement between these 2 counters, markedly different counting efficiencies were exhibited, requiring optimization of the RBA protocol for the TopCount instrument. Precision, accuracy, and sensitivity [limit of detection = 0.2 μg saxitoxin (STX) equiv/100 g shellfish tissue] of the modified protocol were equivalent to those of the original protocol. The RBA robustness and adaptability were demonstrated by an interlaboratory study, in which STX concentrations in shellfish generated by the TopCount were consistent with MicroBeta-derived values. Comparison of STX reference standards obtained from the U.S. Food and Drug Administration and the National Research Council, Canada, showed no observable differences. This study confirms the RBA's value as a rapid, high throughput screen prior to testing by the conventional mouse bioassay (MBA) and its suitability for providing an early warning of increasing PSP toxicity when toxin levels are below the MBA limit of detection.

Receptor binding assay for the detection of paralytic shellfish poisoning toxins: comparison to the mouse bioassay and applicability under regulatory use

“…A Deming Regression of the RBA and MBA data for 190 samples indicates a slight positive bias to the RBA, with a slope of 1.2 (Figure 3). This is consistent with our previous work (Ruberu et al 2003) and deemed acceptable considering the inherent variability or uncertainty of each method (~ +/-20%) (Doucette et al 1997).…”

“…Our effort has been to eliminate the reliance on live animals while improving our analytical capacity with a method that is more sensitive and allows for some automation, thereby increasing sample throughput. We have been exploring the RBA as an alternate test method for the MBA (Ruberu et al 2003 andRuberu et al 2012) in joint studies with the Center for Coastal Environmental Health and Biomolecular Research, National Oceanographic and Atmospheric Administration (NOAA) and the USFDA. Our initial work demonstrated PSP toxin concentrations measured by the RBA to be generally higher than those measured by the MBA (Ruberu et al 2003).…”

“…The methodology of Doucette (Doucette et al 1997) as modified by Ruberu (Ruberu et al 2003) was followed. The RBA procedure involved the initial addition of 35 μL of buffer (MOPS/choline Cl or HEPES/NaCl) to a 96-well microtiter filtration plate (EMD Millipore, part # MAIBN0B50) to 'wet' the wells, followed by the addition of 35 μL of unknown sample (or STX diHCl standard), 35 μL of 3 H -STX, and 105 μL of a 1:6 diluted rat membrane homogenate, in this order.…”

Differential upregulation of Nox homologues of NADPH oxidase by tumor necrosis factor‐α in human aortic smooth muscle and embryonic kidney cells

“…Cells were pretreated with 3 mM DETCA for 1h to inactivate endogenous Cu 2+ /Zn 2+ SOD activity. Chemiluminescence was detected by dark-adapted lucigenin (5 μM) with Wallac TriLux MicroBeta scintillation counter (PerkinElmer) at different time intervals [26], and expressed as luminescence counts per second (LCPS).…”

Marine biotoxins in shellfish – Saxitoxin group

“…The method of Vieytes et al (1993) was further developed by Doucette et al (1997) and made available in a radioactivity-labelled isotope [ 3 H] microplate format (Ruberu et al, 2003). The use of the microtiter plate format, in conjunction with microplate scintillation counting, makes the assay suitable for use in a high throughput regulatory setting.…”