A Simple and Specific Noncompetitive ELISA Method for HT-2 Toxin Detection

Abstract

We developed an HT-2 toxin-specific simple ELISA format with a positive read-out. The assay is based on an anti-immune complex (IC) scFv antibody fragment, which is genetically fused with alkaline phosphatase (AP). The anti-IC antibody specifically recognizes the IC between a primary anti-HT-2 toxin Fab fragment and an HT-2 toxin molecule. In the IC ELISA format, the sample is added together with the scFv-AP antibody to the ELISA plate coated with the primary antibody. After 15 min of incubation and a washing step, the ELISA response is read. A competitive ELISA including only the primary antibody recognizes both HT-2 and T-2 toxins. The anti-IC antibody makes the assay specific for HT-2 toxin, and the IC ELISA is over 10 times more sensitive compared to the competitive assay. Three different naturally contaminated matrices: wheat, barley and oats, were used to evaluate the assay performance with real samples. The corresponding limits of detection were 0.3 ng/mL (13 µg/kg), 0.1 ng/mL (4 µg/kg) and 0.3 ng/mL (16 µg/kg), respectively. The IC ELISA can be used for screening HT-2 toxin specifically and in relevant concentration ranges from all three tested grain matrices.

Layered material platform for surface plasmon resonance biosensing

“…HT-2 is a fungal metabolite belonging to a family of mycotoxins, referred to as the trichothecenes, with a molecular weight ~424 Da, see ref. 12,13 . It is the main metabolite of T-2 mycotoxin 12 .…”

“…However, steps ii) and iii) were not previously systematically discussed in literature and are generic to any type of hybrid biosensing. As a demonstration, we fabricate ultrasensitive SPR sensor functionalized with an anti-HT-2 toxin Fab fragment (HT2-10) and detected HT-2 toxin (~424 Da) with an amplitude detection limit ~1 pg/mL, 3 orders of magnitude better than currently available methods 12,13 and a phase sensitivity limit ~0.5 fg/mL, which is comparable with label techniques. This paves the way to LM-based label-free SPR biosensing of small bio-objects at ultra-low concentrations.…”

Enhancing the detection of Toxoplasma gondii via an anti-SAG1 scFv-alkaline phosphatase immunoconjugate

“…Indeed, the presence of both the signal peptide and the first six amino acid residues of AP promote the efficient export of hybrids into the periplasm of E. coli , where their cysteines are oxidized into disulfide bonds and their native-like structure is likely to be formed. Other scFvs have been also fused successfully to AP and have been reported, using the same or a similar approach [16,17,42]. In all cases, the sequence encoding the signal peptide of AP precedes the inserted gene resulting in periplasmic localization and correct folding.…”

“…To address these problems, recombinant gene fusion techniques have provided new facilities and have opened a path for preparing fusion proteins as single molecular species with a definite molar ratio [13]. In this context, several attempts to produce recombinant bifunctional molecules in which the variable domains of an antibody were expressed as a single-chain antibody (scFv) genetically linked to a protein tracer have been successfully reported to develop robust immunoassays [14,15,17]. This approach presents several attractive advantageous.…”

Nanobody Technology for Mycotoxin Detection: Current Status and Prospects

“…A large number of antibodies against mycotoxins have been developed, including polyclonal antibodies (pAbs) [ 15 , 16 ], monoclonal antibodies (mAbs) [ 17 , 18 ], and recombinant antibodies (rAbs) [ 19 , 20 ]. Since heavy-chain-only antibodies (HcAbs) that are naturally devoid of light chain and lacking the CH1 domains were first discovered in the serum of the camel ( Camelus dromedarius ) [ 21 ], camelid antibodies have gained increasing attention due to their convenience in biotechnological applications.…”