Recombinant Streptavidin Nanopeptamer Anti-Immunocomplex Assay for Noncompetitive Detection of Small Analytes

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 f...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 2014-10, Vol.86 (20), p.10467-10473
Main Authors: Carlomagno, Mariana, Lassabe, Gabriel, Rossotti, Martín, González-Techera, Andrés, Vanrell, Lucía, González-Sapienza, Gualberto
Format: Article
Language:English
Subjects:
Aptamers, Peptide - chemistry
Aptamers, Peptide - genetics
Assaying
Biosensors
Chemistry Techniques, Analytical - methods
Herbicides
Herbicides - chemistry
Immunoassay
Isoxazoles - chemistry
Libraries
Limit of Detection
Molecules
Nanostructure
Oryza - chemistry
Oxazolidinones - chemistry
Peptides
Phage
Protein Folding
Proteins
Recombinant
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
Streptavidin - chemistry
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Summary: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.
ISSN:0003-2700
1520-6882
DOI:10.1021/ac503130v