Polyclonal Antibody-Based Noncompetitive Immunoassay for Small Analytes Developed with Short Peptide Loops Isolated from Phage Libraries

Abstract

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-immunocomplex 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.

Nanopeptamers for the Development of Small-Analyte Lateral Flow Tests with a Positive Readout

“…8 Using M13 phage particles displaying these peptides, we developed two-site phage anti-IC assays (PHAIA) (figure 1A) for the herbicides molinate, atrazine and clomazone, 8,9 the drugs digoxin and cyclosporine, 8 the flame-retardant brominated diphenyl ether, 10 and the pyrethroid metabolite phenoxybenzoic acid. 11 Recently, other groups have also isolated anti-IC peptides for gibberellins 12 and a 1,3-diketone hapten derivative 13 further showing the general applicability of the concept. The PHAIA method provides higher sensitivity than the conventional format (one or more orders of magnitude) and the two-site recognition also accounts for a higher specificity.…”

Immunoassays and Biosensors for Monitoring Environmental and Human Exposure to Pyrethroid Insecticides

“…To develop a sensitive non-competitive assay for 3-PBA, we have recently introduced a novel non-competitive two-site assay termed phage anti-immune complex assay (PHAIA). The phage-borne peptides selected by biopanning a randomly produced peptide library are capable of forming a trivalent complex of antibody, 3-PBA, and peptide as the peptide recognizes the conformational change of an antibody binding pocket caused upon binding to 3-PBA, resulting in a sandwich type two-ligand complex (60, 62). …”

“…The resulting dose-response curve of the 3-PBA PHAIA showed significantly improved assay sensitivity (60), compared to that of the homologous competitive hapten-based microplate ELISA. We further demonstrated the application of the PHAIA to a magnetic bead-based assay (62).…”

“…Moreover, unlike the typical competitive ELISAs, where low concentrations produce a high signal against a high background, the PHAIA is unique in that low concentrations generate a positive signal that is easily distinguishable from the signal intensity at zero concentration. We took advantage of this technology by adapting the PHAIA into a dipstick format which is useful for a rapid on-site monitoring of human exposure to pyrethroid insecticides (60). The difference in signals developed on a nitrocellulose strip on which the immobilized antibody captures 3-PBA and phage peptide could be read by the naked eye giving a very high assay sensitivity.…”

Competitive and noncompetitive phage immunoassays for the determination of benzothiostrobin

“…Anti-immunocomplex phage peptides can specifically recognize an analyte-bound antibody to enhance the affinity and selectivity of the primary antibody because of the formation of a ternary complex, which translates into an improved noncompetitive immunoassay for a low-molecular-weight compound [8]. Although only eight low-molecular-weight compounds (brominated diphenyl ether 47, clomazone, malachite green, leucomalachite green, gibberellin, 2,2′,4,4′-tetrabromodiphenyl ether, phenoxybenzoic acid, molinate and atrazine) have been successfully detected by noncompetitive immunoassays based on anti-immunocomplex phage peptides, each presented better performance than the corresponding conventional competitive immunoassays [8, 9, 19–24]. Additionally, real-time polymerase chain reaction and loop-mediated isothermal amplification have been successfully used to detect low-molecular-weight chemicals by using phage-displayed peptides because of the unique characteristic that phage-displayed peptides can be connected to nucleic acids (single stranded DNA) [3, 25, 26].…”