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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.

To date, there are a few technologies for the development of noncompetitive immunoassays for small molecules, the most common of which relies on the use of anti-immunocomplex antibodies. This approach is laborious, case specific, and relies upon monoclonal antibody technology for its implementation. We recently demonstrated that, in the case of monoclonal antibody-based immunoassays, short peptide loops isolated from phage display libraries can be used as substitutes of the anti-immunocomplex antibodies for noncompetitive immunodetection of small molecules. The aim of this work was to demonstrate that such phage ligands can be isolated even when the selector antibodies are polyclonal in nature. Using phenoxybenzoic acid (PBA), a major pyrethroid metabolite, as a model system, we isolated the CFNGKDWLYC peptide after panning a cyclic peptide library on the PBA/anti-PBA immunocomplex. The sensitivity of the noncompetitive enzyme-linked immunosorbent assay (ELISA) setup with this peptide was 5-fold (heterologous) or 400-fold (homologous) higher than that of the competitive assay setup with the same antibody. Phage anti-inmunocomplex assay (PHAIA) was also easily adapted into a rapid and highly sensitive dipstick assay. The method not only provides a positive readout but also constitutes a major shortcut in the development of sensitive polyclonal-based assays, avoiding the need of synthesizing heterologous competing haptens.

Due to their size, small molecules cannot be simultaneously bound by two antibodies, precluding their detection by noncompetitive two-site immunoassays, which are superior to competitive ones in terms of sensitivity, kinetics, and working range. This has prompted the development of anti-immune complex antibodies, but these are difficult to produce, and often exhibit high cross-reactivity with the unliganded primary antibody. This work demonstrates that anti-immune complex antibodies can be substituted by phage particles isolated from phage display peptide libraries. Phages bearing specific small peptide loops allowed to focus the recognition to changes in the binding area of the immune complex. The concept was tested using environmental and drug analytes; with improved sensitivity and ready adaptation into on-site formats. Peptides specific for different immune complexes can be isolated from different peptide libraries in a simple and systematic fashion allowing the rapid development of noncompetitive assays for small molecules

There is a great demand for rapid tests that can be used on-site for the detection of small analytes, such as pesticides, persistent organic pollutants, explosives, toxins, medicinal and abused drugs, hormones, etc. Dipsticks and lateral flow devices, which are simple and provide a visual readout, may be the answer, but the available technology for these compounds requires a competitive format that loses sensitivity and produces readings inversely proportional to the analyte concentration, which is counterintuitive and may lead to potential misinterpretation of the result. In this work, protein-mu1tipept~de constructs composed of anti-unmunocomplex peptides selected from phage libraries and streptavidin/avidin as core protein were used for direct detection of small compounds in a noncompetitive two-site immunoassay format that performs with increased sensitivity and positive readout. These constructs that we termed "nanopeptamers" allow the development of rapid point-of-use tests with a positive visual end point of easy interpretation. As proof of concept, lateral flow assays for the herbicides molinate and domazone were developed and their performance was characterized with field samples. [ABSTRACT FROM AUTHOR]

Competitive immunoassays for the detection of small analytes, such as pesticides and their metabolites, use haptens that compete with the target compounds for binding to the antibody. This cOmpeting hapten can be either the same as the immunizing hapten (homologous assay), or structurally modified mimics: of the immunizing hapten (heterologous assay). Polyclonal antibody-based heterologous immunoassays have shown superiorsensftivitiesto homologous ones, butthe synthesis of heterologous haptens may be time-consuming, requiring expertise in synthetic chemistry. In this work we demonstrate that phage display peptide libraries can be used as a source of phage-borne peptidomimetics to facilitate the development of sensitive heterologous assays. Different strategies for the isolation of these peptides were explored using two metabolites of pyrethroid insecticides. The sensitivities of the best competitive phage heterologous enzyme-linked immunosorbent assays were 13 fold and 100 fold better than the homologous assay, for the glycine conjugate of trans-3-(2,2-dichlorovinyl)- 2,2-dimethylcyclopropane-1 -carboxylic acid and 3-phenoxy- benzoic acid, respectively. The phage particles were highly versatile as tracer reagents, allowing the use of enzymatic, chemiluminescent, or immuno-polymerase chain reaction detection. The data presented here shows a new systematic procedure that enables the fast generation of several competing haptens for the rapid development of sensitive heterologous immunoassays. [ABSTRACT FROM AUTHOR]

Due to their simplicity, speed, low cost and specificity, immunoassays have become a useful tool for the analysis of environmental pollutants. Once the anti-hapten antibodies are produced, the same hapten or a related molecule is conjugated to a tracer enzyme or coating protein to set up the assay. Here we report the use of peptides that mimic the analyte as advantageous substitutes of competing haptens. These peptides, which open opportunities for innovation in the development of tracer reagents, can be selected from phage display libraries in a straightforward systematic manner. The concept was proven using assays for the herbicides molinate and atrazine as model systems. Several characteristics of the selection process that may affect the final assay were analyzed, such as the phage coat proteins fused to the peptide, the use of linear or constrained peptide libraries, the effect of the concentration of analyte used during the selection process, and the use of monoclonal or polyclonal antibodies as selector molecules. In all cases we found that the selected peptides performed with improved sensitivity as compared with the chemical hapten conventional assays, showing an analogous cross-reactivity pattern. Interestingly, the phage particles perform as robust and highly standardized assay reagents, and due to their filamentous repetitive structure, they function as sensitive multienzymatic reporters. [ABSTRACT FROM AUTHOR]