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Sensitive protein d...
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Al-Amin, Rasel Abdullah,Researcher,1983-Uppsala universitet,Science for Life Laboratory, SciLifeLab,Molekylära verktyg,Ulf Landegren
(author)
Sensitive protein detection using site-specifically oligonucleotide-conjugated nanobody reagents
- Article/chapterEnglish2022
Publisher, publication year, extent ...
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2022-07-05
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American Chemical Society (ACS),2022
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electronicrdacarrier
Numbers
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LIBRIS-ID:oai:DiVA.org:uu-461424
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https://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-461424URI
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https://doi.org/10.1021/acs.analchem.2c00584DOI
Supplementary language notes
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Language:English
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Summary in:English
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Subject category:ref swepub-contenttype
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Subject category:art swepub-publicationtype
Notes
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We thank Dr. Junhong Yan for discussions about Nbs oligo conjugation. The Single Cell Proteomic Facility at SciLifeLab, Uppsala for supports to setup PEA assay. This work was supported by a grant (2012-5852) from the Swedish Research Council’s special call for future therapy, Swedish Research Council under grant agreement No. 2020-02258, a Torsten Söderbergs Stiftelse (M130/16) and the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013)/ ERC Grant Agreement No. 294409, ProteinSeq and from The Swedish Collegium for Advanced Studies (SCAS).
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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.
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Added entries (persons, corporate bodies, meetings, titles ...)
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Muthelo, Phathutshedzo M.Uppsala universitet,Science for Life Laboratory, SciLifeLab,Molekylära verktyg,Ulf Landegren(Swepub:uu)phamu375
(author)
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Abdurakhmanov, Eldar,1977-Uppsala universitet,Biokemi,Science for Life Laboratory, SciLifeLab,Helena U. Danielson(Swepub:uu)eldab338
(author)
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Vincke, CecileStructural Biology Research Center, Vrije Universiteit Brussel, Belgium.,Serge Muyldermans
(author)
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Muyldermans, SergeStructural Biology Research Center, Vrije Universiteit Brussel, Belgium,Serge Muyldermans
(author)
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Danielson, U. Helena,Professor,1959-Uppsala universitet,Biokemi,Science for Life Laboratory, SciLifeLab,Helena Danielson(Swepub:uu)helenads
(author)
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Landegren, UlfUppsala universitet,Molekylära verktyg,Science for Life Laboratory, SciLifeLab,Ulf Landegren(Swepub:uu)ulfland
(author)
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Uppsala universitetScience for Life Laboratory, SciLifeLab
(creator_code:org_t)
Related titles
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In:Analytical Chemistry: American Chemical Society (ACS)98:28, s. 10054-100610003-27001520-6882
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