Specific Noncompetitive Immunoassay for HT-2 Mycotoxin Detection

Henri O. Arola, Antti Tullila, Harri Kiljunen, Katrina Campbell, Harri Siitari, Tarja K. Nevanen

Research output: Contribution to journalArticleScientificpeer-review

21 Citations (Scopus)

Abstract

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.
Original languageEnglish
Pages (from-to)2446-2452
JournalAnalytical Chemistry
Volume88
Issue number4
DOIs
Publication statusPublished - 2016
MoE publication typeA1 Journal article-refereed

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Mycotoxins
Assays
Antibodies
Antigen-Antibody Complex
T-2 Toxin
Immunosorbents
Immunoglobulin Fab Fragments
Bacteriophages
Display devices
Antigens
Enzymes

Cite this

Arola, Henri O. ; Tullila, Antti ; Kiljunen, Harri ; Campbell, Katrina ; Siitari, Harri ; Nevanen, Tarja K. / Specific Noncompetitive Immunoassay for HT-2 Mycotoxin Detection. In: Analytical Chemistry. 2016 ; Vol. 88, No. 4. pp. 2446-2452.
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Specific Noncompetitive Immunoassay for HT-2 Mycotoxin Detection. / Arola, Henri O.; Tullila, Antti; Kiljunen, Harri; Campbell, Katrina; Siitari, Harri; Nevanen, Tarja K.

In: Analytical Chemistry, Vol. 88, No. 4, 2016, p. 2446-2452.

Research output: Contribution to journalArticleScientificpeer-review

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AB - 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.

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