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.

Developments in mycotoxin analysis: an update for 2016-2017

“…The authors concluded that the DON http://www.wageningenacademic.com/doi/pdf/10.3920/WMJ2016.2138 -Friday, May 11, 2018 11:37:55 PM -IP Address:18.236.120.13 concentrations quantitated by LC-MS/MS correlated nicely with those obtained by the Quenchbody method (r 2 =0.98). The group of Arola et al (2016) demonstrated a novel rapid one-step immunoassay for determination of HT-2, utilising a pair of recombinant antibody antigen-binding fragments (Fab). The primary Fab recognises HT-2 and the secondary Fab binds specifically to the immune complex formed by the primary antibody and HT-2.…”

Developments in mycotoxin analysis: an update for 2015-2016

“…The group of Arola et al (2016) demonstrated a novel rapid one-step immunoassay for determination of HT-2, utilising a pair of recombinant antibody antigen-binding fragments (Fab). The primary Fab recognises HT-2 and the secondary Fab binds specifically to the immune complex formed by the primary antibody and HT-2.…”

Developments in mycotoxin analysis: an update for 2013-2014

“…The authors concluded that the DON concentrations quantitated by LC-MS/MS correlated nicely with those obtained by the Quenchbody method (r 2 =0.98). The group of Arola et al (2016) demonstrated a novel rapid one-step immunoassay for determination of HT-2, utilising a pair of recombinant antibody antigen-binding fragments (Fab). The primary Fab recognises HT-2 and the secondary Fab binds specifically to the immune complex formed by the primary antibody and HT-2.…”