“Ready-to-use” immunosensor for the detection of small molecules with fast readout

Yuan, Ding; Cui, Pengcheng; Chen, He; Li, Jiao; Huang, Lianrun; González‐Sapienza, Gualberto; Hammock, Bruce D.; Wang, Minghua; Hua, Xiude

doi:10.1016/j.bios.2022.113968

Cited by 14 publications

(4 citation statements)

References 27 publications

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“…As our group and others have previously reported ( 37 – 39 ), coating ELISA plates directly with nanobodies often results in inefficient antigen capture, presumably because their small size means that their structure and therefore also their antigen binding capability is compromised when they are adsorbed to the plate. To overcome this problem, biotinylated nanobodies were immobilized in streptavidin-coated plates, ensuring in addition a more favorable spatial orientation of capture nanobodies.…”

Section: Resultsmentioning

confidence: 93%

A highly sensitive nanobody-based immunoassay detecting SARS-CoV-2 nucleocapsid protein using all-recombinant reagents

Segovia-de los Santos,

Padula-Roca,

Simon

et al. 2023

Front. Immunol.

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Antigen tests have been crucial for managing the COVID-19 pandemic by identifying individuals infected with SARS-CoV-2. This remains true even after immunity has been widely attained through natural infection and vaccination, since it only provides moderate protection against transmission and is highly permeable to the emergence of new virus variants. For this reason, the widespread availability of diagnostic methods is essential for health systems to manage outbreaks effectively. In this work, we generated nanobodies to the virus nucleocapsid protein (NP) and after an affinity-guided selection identified a nanobody pair that allowed the detection of NP at sub-ng/mL levels in a colorimetric two-site ELISA, demonstrating high diagnostic value with clinical samples. We further modified the assay by using a nanobody-NanoLuc luciferase chimeric tracer, resulting in increased sensitivity (detection limit = 61 pg/mL) and remarkable improvement in diagnostic performance. The luminescent assay was finally evaluated using 115 nasopharyngeal swab samples. Receiver Operating Characteristic (ROC) curve analysis revealed a sensitivity of 78.7% (95% confidence interval: 64.3%-89.3%) and specificity of 100.0% (95% confidence interval: 94.7%-100.0%). The test allows the parallel analysis of a large number of untreated samples, and fulfills our goal of producing a recombinant reagent-based test that can be reproduced at low cost by other laboratories with recombinant expression capabilities, aiding to build diagnostic capacity.

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Section: Resultsmentioning

confidence: 93%

A highly sensitive nanobody-based immunoassay detecting SARS-CoV-2 nucleocapsid protein using all-recombinant reagents

Segovia-de los Santos,

Padula-Roca,

Simon

et al. 2023

Front. Immunol.

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“…The G8‐GFP is a fusion protein of G8 and green fluorescent protein (GFP) (Shuai et al, 2022). A competitive screening protocol was conducted to isolate Nbs binding to the G8 and G8‐GFP, as described in previous work (Ding et al, 2022). Briefly, the antigen was diluted with 0.01 M PBS and coated on a 96‐well microtiter plate overnight at 4°C (each round was coated at a concentration of 100, 75, 50, 25, and 10 μg/mL).…”

Section: Experimental Methodsmentioning

confidence: 99%

Sequence‐based design and construction of synthetic nanobody library

Liu,

Li,

et al. 2024

Biotech & Bioengineering

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Nanobody (Nb), the smallest antibody fragments known to bind antigens, is now widely applied to various studies, including protein structure analysis, bioassay, diagnosis, and biomedicine. The traditional approach to generating specific nanobodies involves animal immunization which is time‐consuming and expensive. As the understanding of the antibody repertoire accumulation, the synthetic library, which is devoid of animals, has attracted attention widely in recent years. Here, we describe a synthetic phage display library (S‐Library), designed based on the systematic analysis of the next‐generation sequencing (NGS) of nanobody repertoire. The library consists of a single highly conserved scaffold (IGHV3S65*01‐IGHJ4*01) and complementary determining regions of constrained diversity. The S‐Library containing 2.19 × 108 independent clones was constructed by the one‐step assembly and rapid electro‐transformation. The S‐Library was screened against various targets (Nb G8, fusion protein of Nb G8 and green fluorescent protein, bovine serum albumin, ovalbumin, and acetylcholinesterase). In comparison, a naïve library (N‐Library) from the source of 13 healthy animals was constructed and screened against the same targets as the S‐Library. Binders were isolated from both S‐Library and N‐Library. The dynamic affinity was evaluated by the biolayer interferometry. The data confirms that the feature of the Nb repertoire is conducive to reducing the complexity of library design, thus allowing the S‐Library to be built on conventional reagents and primers.

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“…For example, references [9,10] described that only 17-34% and 23% of antibodies retain their antigen-binding ability for widely used adsorptive immobilization of antibodies on gold nanoparticles (GNPs), respectively, the most common immunochromatographic marker. Some kinds of biosensors realize immobilization-free approaches (see [11][12][13] as examples). However, in the case of LFIA, to create detectable complexes in the analytical zone, we should use direct (before assay) or indirect (before assay or in the course of the assay) binding of immunoreactants with membrane surface and nanoparticle surface.…”

Section: Introductionmentioning

confidence: 99%

Modular Set of Reagents in Lateral Flow Immunoassay: Application for Antibiotic Neomycin Detection in Honey

et al. 2023

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A scheme of modular competitive immunochromatography with an analyte-independent test strip and changeable specific immunoreactants has been proposed. Native (detected) and biotinylated antigens interact with specific antibodies during their preincubation in solution, that is, without the immobilization of reagents. After this, the detectable complexes on the test strip are formed by the use of streptavidin (which binds biotin with high affinity), anti-species antibodies, and immunoglobulin-binding streptococcal protein G. The technique was successfully applied for the detection of neomycin in honey. The visual and instrumental detection limits were 0.3 and 0.014 mg/kg, respectively, and the degree of neomycin revealed in honey samples varied from 85% to 113%. The efficiency of the modular technique with the use of the same test strip for different analytes was confirmed for streptomycin detection. The proposed approach excludes the necessity of finding the condition of immobilization for each new specific immunoreactant and transferring the assay to other analytes by a simple choice of concentrations for preincubated specific antibodies and the hapten–biotin conjugate.

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“Ready-to-use” immunosensor for the detection of small molecules with fast readout

Cited by 14 publications

References 27 publications

A highly sensitive nanobody-based immunoassay detecting SARS-CoV-2 nucleocapsid protein using all-recombinant reagents

A highly sensitive nanobody-based immunoassay detecting SARS-CoV-2 nucleocapsid protein using all-recombinant reagents

Sequence‐based design and construction of synthetic nanobody library

Modular Set of Reagents in Lateral Flow Immunoassay: Application for Antibiotic Neomycin Detection in Honey

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