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Immunoassays are commonly used methods for detection of small molecules that typically require numerous steps of the labeling between immune-recognition reagents and tracers, immobilization and recurrent washing, making them time consuming and difficult to adapt into point of care formats. Here we describe a "ready-to-use" homogeneous competitive immunosensor with an assay time of 10 min that is based exclusively on recombinant reagents. The signal is produced when the split fragments of the nano luciferase (Nluc) are brought together by the interaction of a heavy chain only variable domain (VHH) with a peptidomimetic of the target small molecule. A VHH to 2,4-dichlorophenoxyacetic acid (2,4-D) was used to isolated the peptidomimetic (NGFFEPWQVVYV) from phage display libraries using six panning conditions. Then the peptidomimetic and VHH were fused with the larger (LgN) and smaller piece (SmN) of split fragments of Nluc, respectively. In order to optimize the signal and sensitivity of the immunosensor, we explored the effects of the spacer between the peptidomimetic and LgN, the copy number of peptidomimetics, and the spacer between SmN and VHH, generating 24 combinations that allowed to conclude on their respective roles. Eventually, the developed "ready-to-use" immunosensor performed excellent signal-to-noise ratio and sensitivity, and could be applied to the detection of 2,4-D in real samples. Meanwhile, the immunosensor totally realizes labeling-free, immobilization-free and washing-free, also can be produced in a highly cost effective way.

Peptides isolated from phage display libraries are powerful reagents for small-molecule immunoassay; however, their application as phage-borne peptides is significantly limited by the biological nature of the phage. Here, we present the use of lysine scaffold to prepare a series of different valence peptides to serve as replacements for phage-borne peptides. Benzothiostrobin was selected as a model analyte, the cyclic benzothiostrobin-peptidomimetic in the form of monomer, dendrimer-like dimer, and tetramer were designed and synthesized. Compared with the monomer, the affinity of dendrimer-like dimer and tetramer increased 1.87 and 13.6 times, respectively, as determined by isothermal titration calorimetry (ITC). A novel inner filter effect immunoassay (IFE-IA) with positive readout was developed for benzothiostrobin detection utilizing the peptidomimetics attached to upconversion nanoparticles (UCNPs) as energy donor and monoclonal antibody (mAb)-labeled urchin-like gold nanoflowers (AuNFs) as energy absorber, respectively. The sensitivity of the assay based on dendrimer-like tetramer was approximately 6 and 3 times higher than monomer and dendrimer-like dimer, respectively. After optimization, 50% saturation of the signal (SC50) and detection range (SC10 to SC90) of the IFE-IA based on dendrimer-like tetramer were 11.81 ng mL-1 and 2.04-106.17 ng mL-1, respectively. The IFE-IA also shows good accuracy for the detection of benzothiostrobin in authentic samples.

Our group has previously developed immunoassays for noncompetitive detection of small molecules based on the use of phage borne anti-immunocomplex peptides. Recently, we substituted the phage particles by biotinylated synthetic anti-immunocomplex peptides complexed with streptavidin and named these constructs nanopeptamers. In this work, we report the results of combining AlphaLisa, a commercial luminescent oxygen channeling bead system, with nanopeptamers for the development of a noncompetitive homogeneous assay for the detection of small molecules. The signal generation of AlphaLisa assays relies on acceptor-donor bead proximity induced by the presence of the analyte (a macromolecule) simultaneously bound by antibodies immobilized on the surface of these beads. In the developed assay, termed as nanoAlphaLisa, bead proximity is sustained by the presence of a small model molecule (atrazine, MW = 215) using an antiatrazine antibody captured on the acceptor bead and an atrazine nanopeptamer on the donor bead. Atrazine is one of the most used pesticides worldwide, and its monitoring in water has relevant human health implications. NanoAlphaLisa allowed the homogeneous detection of atrazine down to 0.3 ng/mL in undiluted water samples in 1 h, which is 10-fold below the accepted limit in drinking water. NanoAlphaLisa has the intrinsic advantages for automation and high-throughput, simple, and fast homogeneous detection of target analytes that AlphaLisa assay provides.

Single domain heavychain binders (nanobodies) obtained from camelid antibody libraries hold a great promise for immunoassay development. However, there is no simple method to select the most valuable nanobodies from the crowd of positive clones obtained after the initial screening. In this paper, we describe a novel nanobody-based platform that allows comparison of the reactivity of hundreds of clones with the labeled antigen, and identifies the best nanobody pairs for two-site immunoassay development. The output clones are biotinylated in vivo in 96-well culture blocks and then used to saturate the biotin binding capacity of avidin coated wells. This standardizes the amount of captured antibody allowing their sorting by ranking their reactivity with the labeled antigen. Using human soluble epoxide hydrolase (sEH) as a model antigen, we were able to classify 96 clones in four families and confirm this classification by sequencing. This provided a criterion to select a restricted panel of five capturing antibodies and to test each of them against the rest of the 96 clones. The method constitutes a powerful tool for epitope binning, and in our case allowed development of a sandwich ELISA for sEH with a detection limit of 63 pg/mL and four log dynamic range, which performed with excellent recovery in different tissue extracts. This strategy provides a systematic way to test nanobody pairwise combinations and would have a broad utility for the development of highly sensitive sandwich immunoassays.

The soluble epoxide hydrolase (sEH) is a potential pharmacological target for treating hypertension, vascular inflammation, pain, cancer, and other diseases. However, there is not a simple, inexpensive, and reliable method to estimate levels of active sEH in tissues. Toward developing such an assay, a polyclonal variable domain of heavy chain antibody (VHH) sandwich immunoassay was developed. Ten VHHs, which are highly selective for native human sEH, were isolated from a phage-displayed library. The ten VHHs have no significant cross-reactivity with human microsomal epoxide hydrolase, rat and mouse sEH, and denatured human sEH. There is a high correlation between protein levels of the sEH determined by the enzyme-linked immunosorbent assay (ELISA) and the catalytic activity of the enzyme in S9 fractions of human tissues (liver, kidney, and lung). The VHH-based ELISA appears to be a new reliable method for monitoring the sEH and may be useful as a diagnostic tool for diseases influenced by sEH. This study also demonstrates the broad utility of VHH in biochemical and pharmacological research.

Cestodes use own lipid-binding proteins to capture and transport hydrophobic ligands, including lipids that they cannot synthesise as fatty acids and cholesterol. In E. granulosus s.l., one of these lipoproteins is antigen B (EgAgB), codified by a multigenic and polymorphic family that gives rise to five gene products (EgAgB8/1-5 subunits) assembled as a 230 kDa macromolecule. EgAgB has a diagnostic value for cystic echinococcosis, but its putative role in the immunobiology of this infection is still poorly understood. Accumulating research suggests that EgAgB has immunomodulatory properties, but previous studies employed denatured antigen preparations that might exert different effects than the native form, thereby limiting data interpretation. This work analysed the modulatory actions on macrophages of native EgAgB (nEgAgB) and the recombinant form of EgAg8/1, which is the most abundant subunit in the larva and was expressed in insect S2 cells (rEgAgB8/1). Both EgAgB preparations were purified to homogeneity by immunoaffinity chromatography using a novel nanobody anti-EgAgB8/1. nEgAgB and rEgAgB8/1 exhibited differences in size and lipid composition. The rEgAgB8/1 generates mildly larger lipoproteins with a less diverse lipid composition than nEgAgB. Assays using human and murine macrophages showed that both nEgAgB and rEgAgB8/1 interfered with in vitro LPS-driven macrophage activation, decreasing cytokine (IL-1β, IL-6, IL-12p40, IFN-β) secretion and ·NO generation. Furthermore, nEgAgB and rEgAgB8/1 modulated in vivo LPS-induced cytokine production (IL-6, IL-10) and activation of large (measured as MHC-II level) and small (measured as CD86 and CD40 levels) macrophages in the peritoneum, although rEgAgB8/1 effects were less robust. Overall, this work reinforced the notion that EgAgB is an immunomodulatory component of E. granulosus s.l. Although nEgAgB lipid’s effects cannot be ruled out, our data suggest that the EgAgB8/1 subunit contributes to EgAgB´s ability to regulate the inflammatory activation of macrophages. [ABSTRACT FROM AUTHOR]