Multidetection of Paralytic, Diarrheic, and Amnesic Shellfish Toxins by an Inhibition Immunoassay Using a Microsphere-Flow Cytometry System
Multidetection of Paralytic, Diarrheic, and Amnesic Shellfish Toxins by an Inhibition Immunoassay Using a Microsphere-Flow Cytometry System
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DOI：
10.1021/ac401146m
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发表时间：
2013-08-20
影响因子：
7.4
通讯作者：
Botana, Luis M.
中科院分区：
文献类型：
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作者：
Fraga, Maria;Vilarino, Natalia;Botana, Luis M.
麻痹性贝毒(PSP)、腹泻性贝毒(DSP)和健忘性贝毒(ASP)毒素在海产品中的存在对人类健康构成了严重且日益严重的威胁。为了将人类暴露的风险降到最低，世界各地的法律法规都对海鲜中这些毒素的最高含量进行了限制。规定的限量相当于PSP、DSP和ASP的主要代表基团：岩藻毒素(STX)、冈田酸(OA)和软骨藻酸(DA)。在这项研究中，建立了一种同时筛查贝类样品中这些毒素存在的多重检测方法。基于Luminex xMAP技术，使用固相微球分析和流动荧光检测相结合的方法实现了多路复用。多重检测方法由三种同时竞争免疫分析方法组成。溶液中的游离毒素与固定在三种不同类型微球表面的STX、OA或DA竞争结合特定的单抗。在缓冲液中获得的IC50在单次和多次检测中相似：STX为5.6+/-1.1 ng/mL，OA为1.1+/-0.03 ng/mL，DA为1.9+/-0.1 ng/mL。对样品前处理方法进行了优化，建立了甲醇-醋酸盐缓冲液同时萃取STX、OA和DA的方法。这三种免疫分析在贻贝和扇贝基质上表现良好，表现出足够的动态范围和回收率(STX约90%，OA约80%，DA约100%)。这种基于微球的多重检测免疫分析提供了一种简单快速的筛选方法，能够在同一样本中同时检测三组受管制的海洋毒素。
The presence of paralytic shellfish poisoning (PSP), diarrheic shellfish poisoning (DSP), and amnesic shellfish poisoning (ASP) toxins in seafood is a severe and growing threat to human health. In order to minimize the risks of human exposure, the maximum content of these toxins in seafood has been limited by legal regulations worldwide. The regulated limits are established in equivalents of the main representatives of the groups: saxitoxin (STX), okadaic acid (OA), and domoic acid (DA), for PSP, DSP, and ASP, respectively. In this study a multidetection method to screen shellfish samples for the presence of these toxins simultaneously was developed. Multiplexing was achieved using a solid-phase microsphere assay coupled to flow-fluorimetry detection, based on the Luminex xMap technology. The multidetection method consists of three simultaneous competition immunoassays. Free toxins in solution compete with STX, OA, or DA immobilized on the surface of three different classes of microspheres for binding to specific monoclonal antibodies. The IC50 obtained in the buffer was similar in single- and multidetection: 5.6 +/- 1.1 ng/mL for STX, 1.1 +/- 0.03 ng/mL for OA, and 1.9 +/- 0.1 ng/mL for DA. The sample preparation protocol was optimized for the simultaneous extraction of STX, OA, and DA with a mixture of methanol and acetate buffer. The three immunoassays performed well with mussel and scallop matrixes displaying adequate dynamic ranges and recovery rates (around 90% for STX, 80% for OA, and 100% for DA). This microsphere-based multidetection immunoassay provides an easy and rapid screening method capable of detecting simultaneously in the same sample three regulated groups of marine toxins.