Molecular super-gluing: a straightforward tool for antibody labelling and its application to mycotoxin biosensing.

Mycotoxins are low molecular weight toxic compounds, which can cause severe health problems in animals and humans. Immunoassays allow rapid, simple and cost-effective screening of mycotoxins. Sandwich assays with a direct readout provide great improvement in terms of selectivity and sensitivity, compared to the widely used competitive assay formats, for the analysis of low molecular weight molecules. In this work, we report a non-competitive fluorescence anti-immune complex (IC) immunoassay, based on the specific recognition of HT-2 toxin with a pair of recombinant antibody fragments, namely antigen-binding fragment (Fab) (anti-HT-2 (10) Fab) and single-chain variable fragment (scFv) (anti-IC HT-2 (10) scFv). The SpyTag and SpyCatcher glue proteins were applied for the first time as a bioconjugation tool for the analysis of mycotoxins. To this aim, a SpyTag-mScarlet-I (fluorescent protein) and scFv-SpyCatcher fusion proteins were constructed, produced and fused in situ during the assay by spontaneous Tag-Catcher binding. The assay showed an excellent sensitivity with an EC50 of 4.8 ± 0.4 ng mL-1 and a dynamic range from 1.7 ± 0.3 to 13 ± 2 ng mL-1, an inter-day reproducibility of 8.5% and a high selectivity towards HT-2 toxin without cross-reactivity with other Fusarium toxins. The bioassay was applied to the analysis of the toxin in an oat reference material and in oat samples, with a LOD of 0.6 µg kg-1, and the results were validated by analysing a certificate reference material and by HPLC-MS/MS.

Recombinant Peptide Mimetic NanoLuc Tracer for Sensitive Immunodetection of Mycophenolic Acid.

Mycophenolic acid (MPA) is an immunosuppressant drug commonly used to prevent organ rejection in transplanted patients. MPA monitoring is of great interest due to its small therapeutic window. In this work, a phage-displayed peptide library was used to select cyclic peptides that bind to the MPA-specific recombinant antibody fragment (Fab) and mimic the behavior of MPA. After biopanning, several phage-displayed peptides were isolated and tested to confirm their epitope-mimicking nature in phage-based competitive immunoassays. After identifying the best MPA mimetic (ACEGLYAHWC with a disulfide constrained loop), several immunoassay approaches were tested, and a recombinant fusion protein containing the peptide sequence with a bioluminescent enzyme, NanoLuc, was developed. The recombinant fusion enabled its direct use as the tracer in competitive immunoassays without the need for secondary antibodies or further labeling. A bioluminescent sensor, using streptavidin-coupled magnetic beads for the immobilization of the biotinylated Fab antibody, enabled the detection of MPA with a detection limit of 0.26 ng mL-1 and an IC50 of 2.9 ± 0.5 ng mL-1. The biosensor showed good selectivity toward MPA and was applied to the analysis of the immunosuppressive drug in clinical samples, of both healthy and MPA-treated patients, followed by validation by liquid chromatography coupled to diode array detection.

Inkjet-printed electrochemically reduced graphene oxide microelectrode as a platform for HT-2 mycotoxin immunoenzymatic biosensing.

The design and application of an inkjet-printed electrochemically reduced graphene oxide microelectrode for HT-2 mycotoxin immunoenzymatic biosensing is reported. A water-based graphene oxide ink was first formulated and single-drop line working microelectrodes were inkjet-printed onto poly(ethylene 2,6-naphthalate) substrates, with dimensions of 78 µm in width and 30 nm in height after solvent evaporation. The printed graphene oxide microelectrodes were electrochemically reduced and characterized by Raman and X-ray photoelectron spectroscopies in addition to microscopies. Through optimization of the electrochemical reduction parameters, differential pulse voltammetry were performed to examine the sensing of 1-naphthol (1-N), where it was revealed that reduction times had significant effects on electrode performance. The developed microelectrodes were then used as an immunoenzymatic biosensor for the detection of HT-2 mycotoxin based on carbodiimide linking of the microelectrode surface and HT-2 toxin antigen binding fragment of antibody (anti-HT2 (10) Fab). The HT-2 toxin and anti-HT2 (10) Fab reaction was reported by anti-HT2 immune complex single-chain variable fragment of antibody fused with alkaline phosphatase (anti-IC-HT2 scFv-ALP) which is able to produce an electroactive reporter - 1-N. The biosensor showed detection limit of 1.6 ng ∙ mL-1 and a linear dynamic range of 6.3 - 100.0 ng ∙ mL-1 within a 5 min incubation with 1-naphthyl phosphate (1-NP) substrate.

Quantitation of Thyroid Hormone Binding to Anti-Thyroxine Antibody Fab Fragment by Native Mass Spectrometry.

Thyroid hormones are important regulatory hormones, acting on nearly every cell in the body. The two main thyroid hormones are l-thyroxine (tetraiodo-l-thyronine, T4) and 3,3',5-triiodo-l-thyronine (T3), which are produced in the thyroid gland and secreted into the blood stream. Other important thyroid hormone metabolites are 3,3'-diiodo-l-thyronine (T2) and l-thyronine (T0), which may show increased levels in circulation due to dietary iodine deficiency or other medical disorders. Owing to their central role in cellular functions, sensitive and specific detection methods for thyroid hormones are needed. In this work, native mass spectrometry (MS) was used to quantitate thyroid hormone binding to the anti-T4 antibody Fab fragment. First, the binding affinity for T2 was determined via direct ligand titration experiments. Then, the affinities for the other ligands were determined by competition experiments using T2 as the "low-affinity" reference ligand. The highest affinity was measured for T3, followed by T4, T2, and T0 (K d = 29, 3.4, and 260 nM and 130 µM, respectively). Thus, it is evident that the number and positions of the iodine substituents within the thyronine rings are important for the ligand binding affinity of anti-T4 Fab. Surprisingly, structurally related tetrahalogen bisphenols were also able to bind to anti-T4 Fab with nanomolar affinities.

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

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.

Specific Noncompetitive Immunoassay for HT-2 Mycotoxin Detection.

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.