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.

本研究中，采用来自多棘蜈蚣（Ethmostigmus rubripes）的萨克斯毒素结合蛋白（saxiphilin）同工型结合[³H]标记的石房蛤毒素（[³H]STX）构建微孔板结合测定法（microtitre plate binding assay），并通过改变盐浓度与种类、pH值，以及添加商业贝类的无毒提取物，对该测定体系开展干扰测试。
采用电感耦合等离子体光谱法（inductively coupled plasma spectroscopy）对贝类提取物中含有的元素（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、钴Co）进行定量检测，所涉贝类包括绿唇贻贝（Perna canalicularis）、悉尼岩牡蛎（Saccostrea glomerata）以及三角斧蛤（Donax deltoides）。
选取上述元素对应的四种最常见盐类：氯化钠（NaCl）、氯化钾（KCl）、氯化钙（CaCl₂）和氯化镁（MgCl₂），通过改变其浓度（0~800 mM），探究其对该测定体系的影响。
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）-2-(N-吗啉代)乙磺酸（Mes，10 mM）-乙酸（10 mM）混合缓冲液，并用四甲基氢氧化铵（tetramethylammonium hydroxide）或盐酸（HCl）将体系pH值调节至3.4~9.0的指定梯度，以此探究pH值对该测定体系的影响。
本研究同时旨在验证该微孔板萨克斯毒素结合蛋白测定法抵御贝类提取物中潜在干扰化合物的能力，并探究从贝类中提取麻痹性贝类毒素（PSTs）时，维持毒素稳定性所需的酸性pH环境对该测定体系的影响。