Llamas Nuria M, Stewart Linda, Fodey Terry, Higgins H Cowan, Velasco María Luisa R, Botana Luis M, Elliott Christopher T
Institute of Agri-Food and Land Use, Queen's University of Belfast, Stranmillis Road, BT9 5AG Belfast, Northern Ireland, UK.
Anal Bioanal Chem. 2007 Sep;389(2):581-7. doi: 10.1007/s00216-007-1444-3. Epub 2007 Jul 24.
The mouse bioassay is the methodology that is most widely used to detect okadaic acid (OA) in shellfish samples. This is one of the best-known toxins, and it belongs to the family of marine biotoxins referred to as the diarrhetic shellfish poisons (DSP). Due to animal welfare concerns, alternative methods of toxin detection are being sought. A rapid and specific biosensor immunoassay method was developed and validated for the detection of OA. An optical sensor instrument based on the surface plasmon resonance (SPR) phenomenon was utilised. A polyclonal antibody to OA was raised against OA-bovine thyroglobulin conjugate and OA-N-hydroxy succinimide ester was immobilised onto an amine sensor chip surface. The assay parameters selected for the analysis of the samples were: antibody dilution, 1/750; ratio of antibody to standard, 1:1; volume of sample injected, 25 microl min(-1); flow rate, 25 microl min(-1). An assay action limit of 126 ng g(-1) was established by analysing of 20 shellfish samples spiked with OA at the critical concentration of 160 ng g(-1), which is the action limit established by the European Union (EU). At this concentration of OA, the assay delivered coefficient of variations (CVs) of <10%. The chip surface developed was shown to be highly stable, allowing more than 50 analyses per channel. When the concentrations of OA determined with the biosensor method were compared with the values obtained by LC-MS in contaminated shellfish samples, the correlation between the two analytical methods was found to be highly satisfactory (r(2) = 0.991).
小鼠生物测定法是检测贝类样品中冈田酸（OA）最广泛使用的方法。这是最知名的毒素之一，属于被称为腹泻性贝类毒素（DSP）的海洋生物毒素家族。由于动物福利问题，人们正在寻找毒素检测的替代方法。开发并验证了一种用于检测OA的快速、特异性生物传感器免疫测定方法。使用了一种基于表面等离子体共振（SPR）现象的光学传感器仪器。针对OA-牛甲状腺球蛋白偶联物制备了抗OA的多克隆抗体，并将OA-N-羟基琥珀酰亚胺酯固定在胺传感器芯片表面。选择用于样品分析的测定参数为：抗体稀释度1/750；抗体与标准品的比例1:1；进样体积25微升/分钟（-1）；流速25微升/分钟（-1）。通过分析20个添加了临界浓度为160纳克/克（-1）的OA（这是欧盟设定的行动限值）的贝类样品，确定了126纳克/克（-1）的测定行动限值。在此OA浓度下，该测定的变异系数（CV）<10%。所开发的芯片表面显示出高度稳定性，每个通道可进行50多次分析。当将生物传感器方法测定的OA浓度与受污染贝类样品中通过液相色谱-质谱法获得的值进行比较时，发现两种分析方法之间的相关性非常令人满意（r（2）=0.991）。
