title: Specific Noncompetitive Immunoassay for HT-2 Mycotoxin Detection.
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 × 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 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.
authors.affiliation: VTT Technical Research Centre of Finland, Tietotie 2, FI-02150 Espoo, Finland.
authors.forename: Henri O
authors.initials: HO
authors.lastname: Arola
authors.affiliation: VTT Technical Research Centre of Finland, Tietotie 2, FI-02150 Espoo, Finland.
authors.forename: Antti
authors.initials: A
authors.lastname: Tullila
authors.affiliation: VERIFIN, Finnish Institute for Chemical Weapons Convention, Department of Chemistry, University of Helsinki , A. I. Virtasen aukio 1, FI-00014 Helsinki, Finland.
authors.forename: Harri
authors.initials: H
authors.lastname: Kiljunen
authors.affiliation: Institute for Global Food Security, Queen's University Belfast, School of Biological Sciences , 8 Cloreen Park, Malone Road, Belfast BT7 1NN, United Kingdom.
authors.forename: Katrina
authors.initials: K
authors.lastname: Campbell
authors.affiliation: Faculty of Pharmacy, University of Helsinki , Viikinkaari 5 E, FI-00014 Helsinki, Finland.
authors.forename: Harri
authors.initials: H
authors.lastname: Siitari
authors.affiliation: VTT Technical Research Centre of Finland, Tietotie 2, FI-02150 Espoo, Finland.
authors.forename: Tarja K
authors.initials: TK
authors.lastname: Nevanen
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D007118:Immunoassay; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D008968:Molecular Conformation; D013605:T-2 Toxin / Q000031:analogs & derivatives* / Q000032:analysis / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D013605:T-2 Toxin; C012351:HT-2 toxin