Departamento de Farmacología, Facultad de Veterinaria, Universidad de Santiago de Compostela, Campus Universitario s/n, 27002 Lugo, Spain.
Anal Chim Acta. 2011 Sep 9;701(2):200-8. doi: 10.1016/j.aca.2011.05.034. Epub 2011 May 31.
In 2009, we achieve the first inhibition FP assay to detect imine cyclic toxins. In the present paper we propose a new FP assay for direct quantify spirolides. This new method has resulted in significant improvement of sensitivity, rapidity and accessibility. In the method design, nicotinic acetylcholine receptor from Torpedo marmorata membranes labelled with a derivative of fluorescein was used. Spirolides, 13-desmethyl spirolide C (13-desMeC) and 13,19-didesmethyl spirolide C (13,19-didesMeC) were extracted and purified from cultures of the Alexandrium ostenfeldii dinoflagellate. Data showed the decrease of FP when toxin concentration was increased. Thus, a relationship between the FP units and the spirolides amount present in a sample was obtained. This direct assay is a reproducible, simple and very sensitive method with a detection limit about 25 nM for 13-desMeC and 150 nM for 13,19-didesMeC. The procedure was used to measure spirolides in mussel samples using an extraction and clean up protocol suitable for the FP assay. Results obtained show that this method is able to quantify 13-desMeC in the range of 50-350 μg kg(-1) meat. Other liposoluble toxins did not interfere with the assay, proving a specific method. Moreover, the matrix do not affect in the range of toxin concentrations that involving risk of spirolides intoxication.
2009 年，我们首次实现了抑制荧光偏振（FP）分析来检测亚氨基环酸毒素。在本研究中，我们提出了一种新的 FP 分析方法，用于直接定量检测麻痹性贝类毒素。这种新方法在灵敏度、速度和可及性方面取得了显著的提高。在方法设计中，我们使用了从蓝斑马鲛鱼毒蕈碱型乙酰胆碱受体膜标记的荧光素衍生物。从亚历山大藻培养物中提取和纯化了去甲二氢石房蛤毒素（13-desMeC）和 13,19-二去甲二氢石房蛤毒素（13,19-didesMeC）。结果表明，随着毒素浓度的增加，FP 会减少。因此，我们得到了 FP 单位与样品中存在的麻痹性贝类毒素数量之间的关系。这种直接检测法是一种可重现、简单且非常灵敏的方法，13-desMeC 的检测限约为 25 nM，13,19-didesMeC 的检测限约为 150 nM。我们使用适合 FP 分析的提取和净化方案，对贻贝样品中的麻痹性贝类毒素进行了检测。结果表明，该方法能够在 50-350μgkg(-1) 肉的范围内定量检测 13-desMeC。其他脂溶性毒素不会干扰该分析方法，证明了其特异性。此外，在涉及麻痹性贝类毒素中毒风险的毒素浓度范围内，基质不会影响分析结果。
