Gauging the effects of shellfish extract matrices, salts and pH upon performance of an assay for paralytic shellfish toxins using saxiphilin

In this study, a microtitre plate binding assay using a saxiphilin isoform [3H]STX from the centipede Ethmostigmus rubripes, was challenged with differing conditions of salt concentration and identity, pH and the addition of non-toxic extracts of commercial shellfish.Elements present (Na, K, Mg, Ca, Zn, Ni, Mn, Cr, Sr, Mo, Al, V, Fe, Cu, Pb, Co) in the shellfish extracts (green lipped mussel, Perna canalicularis; Sydney rock oyster, Saccostrea glomerata; and pipis, Donax deltoides) were measured by inductively coupled plasma spectroscopy. The effects upon the assay by the four most common salts of these elements, NaCl, KCl, CaCl2 and MgCl2 were studied by varying their concentrations (0-800 mM).The effect of pH upon the assay was measured by replacing the assay buffer in standard conditions with a buffer mixture of Tris (20 mM) -Mes (10 mM) -acetic acid (10 mM), adjusted with tetramethylammonium hydroxide or HCl to indicated pH which ranged from 3.4 to 9.0.
To investigate the microtitre plate saxiphilin assay's ability to deal with potential interfering compounds from shellfish extracts, and the effect of acidic pH necessary for toxin stability when extracting PSTs from shellfish.

本研究采用源自蜈蚣（Ethmostigmus rubripes）的saxiphilin（Saxiphilin）亚型结合[3H]STX的微孔板结合测定法，分别通过调整盐浓度与种类、pH值，以及添加商用贝类无毒提取物的方式，对该测定法进行了干扰性能考察。
通过电感耦合等离子体光谱法，对所使用的贝类提取物（包括青口贝Perna canalicularis、悉尼岩牡蛎Saccostrea glomerata以及斧蛤Donax deltoides）中的下述元素进行了定量检测：钠（Na）、钾（K）、镁（Mg）、钙（Ca）、锌（Zn）、镍（Ni）、锰（Mn）、铬（Cr）、锶（Sr）、钼（Mo）、铝（Al）、钒（V）、铁（Fe）、铜（Cu）、铅（Pb）与钴（Co）。
选取上述元素中四种最常见的盐类（氯化钠NaCl、氯化钾KCl、氯化钙CaCl₂与氯化镁MgCl₂），通过改变其浓度范围（0~800 mM），考察其对该测定法的影响。
通过将标准条件下的测定缓冲液替换为三羟甲基氨基甲烷（Tris，20 mM）-吗啉乙磺酸（Mes，10 mM）-乙酸（10 mM）的混合缓冲体系，使用四甲基氢氧化铵或盐酸将体系pH值调节至3.4~9.0的指定区间，以此考察pH值对该测定法的影响。
本研究同时旨在探究该微孔板saxiphilin测定法应对贝类提取物中潜在干扰化合物的能力，以及从贝类中提取麻痹性贝类毒素（PSTs, paralytic shellfish toxins）时，维持毒素稳定性所需的酸性pH值所产生的影响。