title: Recombinant streptavidin nanopeptamer anti-immunocomplex assay for noncompetitive detection of small analytes.
abstract: Short peptide loops selected from phage libraries can specifically recognize the formation of hapten-antibody immunocomplexes and can thus be used to develop phage anti-immunocomplex assays (PHAIA) for noncompetitive detection of small molecules. In this study, we generated recombinant chimeras by fusing anti-immunocomplex peptides selected from phage libraries to the N- or C-termini of core streptavidin and used them to setup phage-free noncompetitive assays for the herbicide clomazone (MW 240 Da). The best conditions for refolding were optimized by a high throughput screening allowing to obtain tens of mg of purified protein per liter of culture. The noncompetitive assay developed with these chimeras performed with a 50% saturating concentration (SC50) of 2.2 ± 0.3 ng/mL and limit of detection (LOD) of 0.48 ng/mL. Values that are 13- and 8-fold better that those obtained for the SC50 and LOD of the competitive assay setup with the same antibody. Apart from the first demonstration that recombinant peptide-streptavidin chimeras can be used for sensitive immunodetection of small molecules with a positive readout, this new assay component is a highly standardized reagent with a defined stoichiometry, which can be used in combination with the broad option of existing biotinylated reagents offering a great versatility for the development of conventional immunoassay and biosensors. The utility of the test was demonstrated analyzing the clomazone runoff during the rice growing season in northern Uruguay.
authors.affiliation: Cátedra de Inmunología, DEPBIO, Facultad de Química, Instituto de Higiene, UDELAR , Avinguda A. Navarro 3051, piso 2, Montevideo 11600, Uruguay.
authors.forename: Mariana
authors.initials: M
authors.lastname: Carlomagno
authors.forename: Gabriel
authors.initials: G
authors.lastname: Lassabe
authors.forename: Martín
authors.initials: M
authors.lastname: Rossotti
authors.forename: Andrés
authors.initials: A
authors.lastname: González-Techera
authors.forename: Lucía
authors.initials: L
authors.lastname: Vanrell
authors.forename: Gualberto
authors.initials: G
authors.lastname: González-Sapienza
mesh_terms: D052158:Aptamers, Peptide / Q000737:chemistry* / Q000235:genetics; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D006540:Herbicides / Q000737:chemistry*; D007118:Immunoassay; D007555:Isoxazoles / Q000737:chemistry; D057230:Limit of Detection; D012275:Oryza / Q000737:chemistry; D023303:Oxazolidinones / Q000737:chemistry; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics; D019809:Streptavidin / Q000737:chemistry*
chemical_list: D052158:Aptamers, Peptide; D006540:Herbicides; D007555:Isoxazoles; D023303:Oxazolidinones; D011994:Recombinant Proteins; C095255:clomazone; D019809:Streptavidin