Specific Noncompetitive Immunoassay for HT-2 Mycotoxin Detection

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• 作者：Henri O. Arola ; Antti Tullila ; Harri Kiljunen ; Katrina Campbell ; Harri Siitari ; Tarja K. Nevanen
• 刊名：Analytical Chemistry
• 出版年：2016
• 出版时间：February 16, 2016
• 年：2016
• 卷：88
• 期：4
• 页码：2446-2452
• 全文大小：316K
• ISSN：1520-6882

文摘

Here we demonstrate a novel homogeneous one-step immunoassay, utilizing a pair of recombinant antibody antigen-binding fragments (Fab), that is specific for HT-2 toxin and has a positive readout. Advantages over the conventional competitive immunoassay formats such as enzyme-linked immunosorbent assay (ELISA) are the specificity, speed, and simplicity of the assay. Recombinant antibody HT2-10 Fab recognizing both HT-2 and T-2 toxins was developed from a phage display antibody library containing 6 × 10⁷ different antibody clones. Specificity of the immunoassay was introduced by an anti-immune complex (IC) antibody binding the primary antibody–HT-2 toxin complex. When the noncompetitive immune complex assay was compared to the traditional competitive assay, an over 10-fold improvement in sensitivity was observed. Although the HT2-10 antibody has 100% cross-reactivity for HT-2 and T-2 toxins, the immune complex assay is highly specific for HT-2 alone. The assay performance with real samples was evaluated using naturally contaminated wheat reference material. The half-maximal effective concentration (EC50) value of the time-resolved fluorescence resonance energy transfer (TR-FRET) assay was 9.6 ng/mL, and the limit of detection (LOD) was 0.38 ng/mL (19 μg/kg). The labeled antibodies can be predried to the assay vials, e.g., microtiter plate wells, and readout is ready in 10 min after the sample application.