期刊
ANALYTICAL CHEMISTRY
卷 88, 期 4, 页码 2446-2452出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.5b04591
关键词
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资金
- VTT
- Finnish Funding Agency for Technology and Innovation (TEKES) [40305/2011]
- Thermo Fisher Scientific
- Labmaster Ltd.
- Pribori Oy
- PBL Brewing Laboratory
- Graduate School of Advanced Diagnostic Technologies and Applications (DIA-NET)
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 x 10(7) 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 mu g/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.
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