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Sensitive Protein Detection Using Site-Specifically Oligonucleotide-Conjugated Nanobodies
[Image: see text] High-quality affinity probes are critical for sensitive and specific protein detection, in particular for detection of protein biomarkers in the early phases of disease development. Proximity extension assays (PEAs) have been used for high-throughput multiplexed protein detection o...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Chemical Society
2022
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9310004/ https://www.ncbi.nlm.nih.gov/pubmed/35786874 http://dx.doi.org/10.1021/acs.analchem.2c00584 |
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author | Al-Amin, Rasel A. Muthelo, Phathutshedzo M. Abdurakhmanov, Eldar Vincke, Cécile Amin, Shahnaz P. Muyldermans, Serge Danielson, U. Helena Landegren, Ulf |
author_facet | Al-Amin, Rasel A. Muthelo, Phathutshedzo M. Abdurakhmanov, Eldar Vincke, Cécile Amin, Shahnaz P. Muyldermans, Serge Danielson, U. Helena Landegren, Ulf |
author_sort | Al-Amin, Rasel A. |
collection | PubMed |
description | [Image: see text] High-quality affinity probes are critical for sensitive and specific protein detection, in particular for detection of protein biomarkers in the early phases of disease development. Proximity extension assays (PEAs) have been used for high-throughput multiplexed protein detection of up to a few thousand different proteins in one or a few microliters of plasma. Clonal affinity reagents can offer advantages over the commonly used polyclonal antibodies (pAbs) in terms of reproducibility and standardization of such assays. Here, we explore nanobodies (Nbs) as an alternative to pAbs as affinity reagents for PEA. We describe an efficient site-specific approach for preparing high-quality oligo-conjugated Nb probes via enzyme coupling using Sortase A (SrtA). The procedure allows convenient removal of unconjugated affinity reagents after conjugation. The purified high-grade Nb probes were used in PEA, and the reactions provided an efficient means to select optimal pairs of binding reagents from a group of affinity reagents. We demonstrate that Nb-based PEA (nano-PEA) for interleukin-6 (IL6) detection can augment assay performance, compared to the use of pAb probes. We identify and validate Nb combinations capable of binding in pairs without competition for IL6 antigen detection by PEA. |
format | Online Article Text |
id | pubmed-9310004 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | American Chemical Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-93100042022-07-26 Sensitive Protein Detection Using Site-Specifically Oligonucleotide-Conjugated Nanobodies Al-Amin, Rasel A. Muthelo, Phathutshedzo M. Abdurakhmanov, Eldar Vincke, Cécile Amin, Shahnaz P. Muyldermans, Serge Danielson, U. Helena Landegren, Ulf Anal Chem [Image: see text] High-quality affinity probes are critical for sensitive and specific protein detection, in particular for detection of protein biomarkers in the early phases of disease development. Proximity extension assays (PEAs) have been used for high-throughput multiplexed protein detection of up to a few thousand different proteins in one or a few microliters of plasma. Clonal affinity reagents can offer advantages over the commonly used polyclonal antibodies (pAbs) in terms of reproducibility and standardization of such assays. Here, we explore nanobodies (Nbs) as an alternative to pAbs as affinity reagents for PEA. We describe an efficient site-specific approach for preparing high-quality oligo-conjugated Nb probes via enzyme coupling using Sortase A (SrtA). The procedure allows convenient removal of unconjugated affinity reagents after conjugation. The purified high-grade Nb probes were used in PEA, and the reactions provided an efficient means to select optimal pairs of binding reagents from a group of affinity reagents. We demonstrate that Nb-based PEA (nano-PEA) for interleukin-6 (IL6) detection can augment assay performance, compared to the use of pAb probes. We identify and validate Nb combinations capable of binding in pairs without competition for IL6 antigen detection by PEA. American Chemical Society 2022-07-05 2022-07-19 /pmc/articles/PMC9310004/ /pubmed/35786874 http://dx.doi.org/10.1021/acs.analchem.2c00584 Text en © 2022 The Authors. Published by American Chemical Society https://creativecommons.org/licenses/by/4.0/Permits the broadest form of re-use including for commercial purposes, provided that author attribution and integrity are maintained (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Al-Amin, Rasel A. Muthelo, Phathutshedzo M. Abdurakhmanov, Eldar Vincke, Cécile Amin, Shahnaz P. Muyldermans, Serge Danielson, U. Helena Landegren, Ulf Sensitive Protein Detection Using Site-Specifically Oligonucleotide-Conjugated Nanobodies |
title | Sensitive Protein Detection Using Site-Specifically
Oligonucleotide-Conjugated Nanobodies |
title_full | Sensitive Protein Detection Using Site-Specifically
Oligonucleotide-Conjugated Nanobodies |
title_fullStr | Sensitive Protein Detection Using Site-Specifically
Oligonucleotide-Conjugated Nanobodies |
title_full_unstemmed | Sensitive Protein Detection Using Site-Specifically
Oligonucleotide-Conjugated Nanobodies |
title_short | Sensitive Protein Detection Using Site-Specifically
Oligonucleotide-Conjugated Nanobodies |
title_sort | sensitive protein detection using site-specifically
oligonucleotide-conjugated nanobodies |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9310004/ https://www.ncbi.nlm.nih.gov/pubmed/35786874 http://dx.doi.org/10.1021/acs.analchem.2c00584 |
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