采用电感耦合等离子体质谱法评价不同竞争免疫分析法测定牛奶中黄曲霉毒素 M 的结果。
Evaluation of different competitive immunoassays for aflatoxin M determination in milk samples by means of inductively coupled plasma mass spectrometry.
机构信息
Department of Analytical Chemistry, Nutrition and Food Sciences, University of Alicante, PO Box 99, 03080, Alicante, Spain.
Department of Biotechnology, University of Alicante, PO Box 99, 03080, Alicante, Spain.
出版信息
Anal Chim Acta. 2019 Feb 21;1049:10-19. doi: 10.1016/j.aca.2018.11.024. Epub 2018 Nov 13.
Haptens (i.e. biomolecules which molecular weight is lower than 10 kDa) determination by inductively coupled plasma mass spectrometry (ICP-MS) is usually performed by means of competitive immunoassays. In these immunoassays, analyte quantification is indirectly carried out using two different tracer species (i.e. antibodies or antigen-protein conjugates). However, the benefits (and drawbacks) derived from using a given tracer species have not been systematically investigated so far. The goal of this work is to evaluate the influence of the tracer species employed in competitive immunoassays on the analytical figures of merit for aflatoxin M (AFM) determination in milk samples. To this end, three different strategies have been developed and evaluated, namely: (i) antibody binding inhibition assay (ABIA); (ii) capture inhibition assay (CIA); and (iii) capture bridge inhibition assay (CBIA). Experimental results show that the use of the antibody as tracer species (as in the ABIA approach) affords better analytical figures of merit for AFM determination than using the antigen-protein conjugate as the tracer one (as in the CIA and CBIA strategies). The limit of detection afforded by ABIA strategy (i.e. 0.1 ng kg) for AFM determination was 1000-fold and 50-fold lower regarding the CIA and CBIA strategies, respectively. In the case of the ABIA approach, the characteristics of the metal nanoparticle label employed to detect the tracer species is critical on the analytical figures of merit. However, when the hapten-protein conjugates are used as tracer species, immunocomplex formation is severely hampered by steric effects caused by the protein moiety and, consequently, the characteristics of the metal nanoparticle label is not critical in the immunoassay performance. The different immunoassay strategies were successfully validated for AFM determination in milk samples using a certified reference material of whole milk powder (ERM-BD283) according to European Conformity guidelines for analytical methods of food contaminants and mycotoxins. Compared to ELISA, the immunoassay developed for AFM determination in milk samples improve limits of detection up to 10-fold.
通过电感耦合等离子体质谱法 (ICP-MS) 测定半抗原（即分子量低于 10 kDa 的生物分子）通常通过竞争性免疫测定来完成。在这些免疫测定中，使用两种不同的示踪物（即抗体或抗原-蛋白缀合物）间接进行分析物定量。然而，到目前为止，还没有系统地研究使用给定示踪物的好处（和缺点）。本工作的目的是评估在竞争免疫测定中使用的示踪物种类对牛奶样品中黄曲霉毒素 M（AFM）测定的分析性能的影响。为此，开发并评估了三种不同的策略，即：（i）抗体结合抑制测定（ABIA）；（ii）捕获抑制测定（CIA）；和（iii）捕获桥抑制测定（CBIA）。实验结果表明，与使用抗原-蛋白缀合物作为示踪物（如 CIA 和 CBIA 策略）相比，使用抗体作为示踪物（如 ABIA 方法）可获得更好的 AFM 测定分析性能。ABIA 策略（即 0.1ngkg）对 AFM 测定的检出限比 CIA 和 CBIA 策略分别低 1000 倍和 50 倍。在 ABIA 方法中，用于检测示踪物的金属纳米粒子标记的特性对分析性能至关重要。然而，当使用半抗原-蛋白缀合物作为示踪物时，免疫复合物的形成受到蛋白部分引起的空间位阻的严重阻碍，因此，金属纳米粒子标记的特性在免疫测定性能中并不关键。根据欧洲合格评定关于食品污染物和真菌毒素分析方法的准则，使用全脂奶粉的认证参考物质（ERM-BD283）对牛奶样品中 AFM 测定的不同免疫测定策略进行了成功验证。与 ELISA 相比，开发的用于牛奶样品中 AFM 测定的免疫测定将检出限提高了 10 倍。
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