Institute of Agricultural Chemistry and Food Technology (IATA), Spanish Scientific Research Council (CSIC), Av. Agustí Escardino 7, Paterna 46980, Valencia, Spain.
Department of Organic Chemistry, University of Valencia, Doctor Moliner 50, Burjassot 46100, Valencia, Spain.
Anal Chem. 2022 Aug 2;94(30):10857-10864. doi: 10.1021/acs.analchem.2c01939. Epub 2022 Jul 19.
Algal blooms that contaminate freshwater resources with cyanotoxins constitute, nowadays, a global concern. To deal with this problem, a variety of analytical methods, including immunochemical assays, are available for the main algal toxins, for example, microcystins, nodularins, and saxitoxins, with the remarkable exception of anatoxin-a. Now, for the first time, highly sensitive, enantioselective immunoassays for anatoxin-a have been validated using homemade monoclonal antibodies. Two competitive enzyme-linked immunosorbent assays were developed in different formats, with detection limits for (+)-anatoxin-a of 0.1 ng/mL. Excellent recovery values between 82 and 117%, and coefficients of variation below 20%, were observed using environmental water samples fortified between 0.5 and 500 ng/mL. In addition, a lateral-flow immunochromatographic assay was optimized for visual and instrumental reading of results. This test showed a visual detection limit for (+)-anatoxin-a of 4 ng/mL. Performance with a reader was validated in accordance with the European guidelines for semiquantitative rapid methods for small chemical contaminants. Thus, at a screening target concentration of 2 ng/mL, the probability of a blank sample to be classified as "suspect" was as low as 0.2%. Finally, the optimized direct enzyme immunoassay was validated by comparison with high-performance liquid chromatography-tandem mass spectroscopy data and showed a good correlation ( = 0.995) with a slope of 0.94. Moreover, environmental water samples containing more than 2 ng/mL of anatoxin-a were detected by the developed dipstick assay. These results provide supplementary and complementary strategies for monitoring the presence of anatoxin-a in water.
藻类水华会使淡水中的蓝藻毒素污染，这是当前全球关注的问题。为了解决这个问题，已经有多种分析方法，包括免疫化学检测法，可用于检测主要的藻类毒素，如微囊藻毒素、节球藻毒素和石房蛤毒素，只有鱼腥藻毒素-a 是个例外。现在，首次使用自制的单克隆抗体对鱼腥藻毒素-a 进行了高灵敏度、对映选择性的免疫分析验证。在不同的格式中开发了两种竞争性酶联免疫吸附测定法，检测 (+)-鱼腥藻毒素-a 的检出限为 0.1ng/mL。使用环境水样进行加标，浓度在 0.5 到 500ng/mL 之间，观察到回收率在 82%到 117%之间，变异系数低于 20%。此外，还优化了侧向流动免疫层析测定法，用于结果的目视和仪器读取。该测试对 (+)-鱼腥藻毒素-a 的目视检测限为 4ng/mL。按照欧洲小型化学污染物半定量快速方法指南对仪器读数进行了验证。因此，在筛查目标浓度为 2ng/mL 的情况下，空白样本被错误分类为“可疑”的概率低至 0.2%。最后，通过与高效液相色谱-串联质谱数据进行比较，对优化的直接酶免疫测定法进行了验证，相关性良好(=0.995)，斜率为 0.94。此外，还可以用开发的试条检测到含鱼腥藻毒素-a 超过 2ng/mL 的环境水样。这些结果为监测水中鱼腥藻毒素-a 的存在提供了补充和互补的策略。
