Affimers and nanobodies as molecular probes and their applications in imaging

Cordell, Paul; Carrington, Glenn; Curd, Alistair; Parker, Francine; Tomlinson, Darren C.; Peckham, Michelle

doi:10.1242/jcs.259168

Cited by 8 publications

(4 citation statements)

References 78 publications

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“…The structure in VNARs and the characteristics of the composition of the individual Loop loops allow them to recognize more hidden epitopes, and therefore VNARs may play an unexpected role in development studies of OGT and O-GlcNAcylation. Conventional monoclonal antibodies are limited in their in-depth application by their large size, complex structure and sensitivity to extreme environmental temperatures ( 42 ). Shark VNAR is better able to penetrate tissues and dense structures and penetrate deeper into the target protein.…”

Section: Discussionmentioning

confidence: 99%

A novel shark single-domain antibody targeting OGT as a tool for detection and intracellular localization

Xiao²,

An³

et al. 2023

Front. Immunol.

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IntroductionO-GlcNAcylation is a type of reversible post-translational modification on Ser/Thr residues of intracellular proteins in eukaryotic cells, which is generated by the sole O-GlcNAc transferase (OGT) and removed by O-GlcNAcase (OGA). Thousands of proteins, that are involved in various physiological and pathological processes, have been found to be O-GlcNAcylated. However, due to the lack of favorable tools, studies of the O-GlcNAcylation and OGT were impeded. Immunoglobulin new antigen receptor (IgNAR) derived from shark is attractive to research tools, diagnosis and therapeutics. The variable domain of IgNARs (VNARs) have several advantages, such as small size, good stability, low-cost manufacture, and peculiar paratope structure.MethodsWe obtained shark single domain antibodies targeting OGT by shark immunization, phage display library construction and panning. ELISA and BIACORE were used to assess the affinity of the antibodies to the antigen and three shark single-domain antibodies with high affinity were successfully screened. The three antibodies were assessed for intracellular function by flow cytometry and immunofluorescence co-localization.ResultsIn this study, three anti-OGT VNARs (2D9, 3F7 and 4G2) were obtained by phage display panning. The affinity values were measured using surface plasmon resonance (SPR) that 2D9, 3F7 and 4G2 bound to OGT with KD values of 35.5 nM, 53.4 nM and 89.7 nM, respectively. Then, the VNARs were biotinylated and used for the detection and localization of OGT by ELISA, flow cytometry and immunofluorescence. 2D9, 3F7 and 4G2 were exhibited the EC50 values of 102.1 nM, 40.75 nM and 120.7 nM respectively. VNAR 3F7 was predicted to bind the amino acid residues of Ser375, Phe377, Cys379 and Tyr 380 on OGT.DiscussionOur results show that shark single-domain antibodies targeting OGT can be used for in vitro detection and intracellular co-localization of OGT, providing a powerful tool for the study of OGT and O-GlcNAcylation.

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Section: Discussionmentioning

confidence: 99%

A novel shark single-domain antibody targeting OGT as a tool for detection and intracellular localization

Xiao²,

An³

et al. 2023

Front. Immunol.

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“…Affimers could act as molecular probes to interrogate the interactions between receptor and ligand, or could themselves act as template biopharmaceuticals with beneficial effects on atherosclerotic plaque formation. To date, the majority of applications for Affimers have been as probes to support high-resolution imaging ( 51 , 52 ) or as antibody replacements in diagnostics and biosensor development ( 53 , 54 , 55 ). However, they have also proven to be powerful tools in the study of protein-protein interactions ( 56 , 57 ), with the capability to discriminate between very closely related proteins ( 58 ).…”

Section: Discussionmentioning

confidence: 99%

“Affimer” synthetic protein scaffolds block oxidized LDL binding to the LOX-1 scavenger receptor and inhibit ERK1/2 activation

Roper,

Tiede,

Abdul-Zani

et al. 2023

Journal of Biological Chemistry

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“…Affibodies can be Affimers. Affimers are scaffolding proteins that are not antibodies but are capable of binding to other molecules with high affinity and specificity [238]. Affimers are obtained using the phage display method, in which those that have the highest affinity for the analyte of interest are selected from a library of random protein sequences [239].…”

Section: Other Types Of Biorecognition Elements Used In Impedimetric ...mentioning

confidence: 99%

Hybrid Impedimetric Biosensors for Express Protein Markers Detection

Sitkov,

Ryabko,

Moshnikov

et al. 2024

Micromachines

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Impedimetric biosensors represent a powerful and promising tool for studying and monitoring biological processes associated with proteins and can contribute to the development of new approaches in the diagnosis and treatment of diseases. The basic principles, analytical methods, and applications of hybrid impedimetric biosensors for express protein detection in biological fluids are described. The advantages of this type of biosensors, such as simplicity and speed of operation, sensitivity and selectivity of analysis, cost-effectiveness, and an ability to be integrated into hybrid microfluidic systems, are demonstrated. Current challenges and development prospects in this area are analyzed. They include (a) the selection of materials for electrodes and formation of nanostructures on their surface; (b) the development of efficient methods for biorecognition elements’ deposition on the electrodes’ surface, providing the specificity and sensitivity of biosensing; (c) the reducing of nonspecific binding and interference, which could affect specificity; (d) adapting biosensors to real samples and conditions of operation; (e) expanding the range of detected proteins; and, finally, (f) the development of biosensor integration into large microanalytical system technologies. This review could be useful for researchers working in the field of impedimetric biosensors for protein detection, as well as for those interested in the application of this type of biosensor in biomedical diagnostics.

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Affimers and nanobodies as molecular probes and their applications in imaging

Cited by 8 publications

References 78 publications

A novel shark single-domain antibody targeting OGT as a tool for detection and intracellular localization

A novel shark single-domain antibody targeting OGT as a tool for detection and intracellular localization

“Affimer” synthetic protein scaffolds block oxidized LDL binding to the LOX-1 scavenger receptor and inhibit ERK1/2 activation

Hybrid Impedimetric Biosensors for Express Protein Markers Detection

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