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.

Hypoxia and inflammation conditions differentially affect the expression of tissue transglutaminase spliced variants and functional properties of extravillous trophoblast cells

“…Goat antimouse Alexa Fluor 488 (1:100, A11029 , Thermo Fisher Scientific, USA) and goat antirabbit Alexa Fluor 594 (1:1500, ab150080 , Abcam, UK) were used for detection and 0.1 µg/mL DAPI was used for staining the nuclei. Swan‐71 cells without primary antibodies and with irrelevant primary antibodies 52,53 were used as negative controls. LSM 800 confocal laser scanning microscope (Zeiss, Germany) was used to acquire fluorescence images.…”

“…Swan-71 cells without primary antibodies and with irrelevant primary antibodies 52,53 were used as negative controls. LSM 800 confocal laser scanning microscope (Zeiss, Germany) was used to acquire fluorescence images.…”

Mimetic peptide and special antibody: promising agents for optimizing hapten-analyzing systems

“…The LODs were 1.2 and 3.2 ng mL À1 for molinate and clomazone, respectively. Based on this nding, Carlomagno et al 77 further developed the SA nanopeptamer technology, generating recombinant peptide-SA chimeras by fusing anti-immunocomplex peptides to the N-or C-termini of core SA to optimize their production, as well as established phage-free noncompetitive assays for clomazone (Fig. 6).…”

Development of a noncompetitive idiometric nanobodies phage immumoassay for the determination of fumonisin B 1

“…Several attempts have been made to overcome this difficulty, and several novel immunoassays that can noncompetitively detect haptens have been developed. These methodologies are based on chemical modification to the analytes (Carlomagno et al, 2014;Haasnoot & Verheijen, 2001), idiometric assays with anti-idiotype antibodies (Niwa et al, 2009), the anti-metatype antibody (Omi et al, 2015;Tsutsumi, Nagata, Yoshida, Harada, & Ueno, 2000), the split recombinant variable region fragments of the antibody (Chung, Makino, Dong, & Ueda, 2015) or other principles (Saha, Roy, & Dhar, 2013). Although these approaches have been successfully used to measure several haptens, preparing the reagents and these unconventional antibodies is complicated and time consuming.…”