Samenvatting
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.
Originele taal-2 | English |
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Pagina's (van-tot) | 10054-10061 |
Aantal pagina's | 8 |
Tijdschrift | Analytical Chemistry |
Volume | 94 |
Nummer van het tijdschrift | 28 |
Vroegere onlinedatum | 5 jul 2022 |
DOI's | |
Status | Published - 19 jul 2022 |
Bibliografische nota
Funding Information:The authors thank Dr. Junhong Yan for discussions on Nb–oligo conjugation and the Single Cell Proteomics Facility at SciLifeLab, Uppsala for support in establishing PEA assays. The Swedish Research Council [2012-5852, 2020-02258 to U.L.], the Swedish Foundation for Strategic Research [SB16-0046], Torsten Söderbergs Stiftelse [M130/16 to U.L.], the European Research Council under the European Union’s Seventh Framework Programme [FP/2007-2013/ERC Grant Agreement No. 294409 to U.L.], and the Swedish Collegium for Advanced Studies (SCAS) are acknowledged.
Funding Information:
The authors thank Dr. Junhong Yan for discussions on Nb–oligo conjugation and the Single Cell Proteomics Facility at SciLifeLab Uppsala for support in establishing PEA assays. The Swedish Research Council [2012-5852, 2020-02258 to U.L.], the Swedish Foundation for Strategic Research [SB16-0046], Torsten Söderbergs Stiftelse [M130/16 to U.L.], the European Research Council under the European Union’s Seventh Framework Programme [FP/2007-2013/ERC Grant Agreement No. 294409 to U.L.], and the Swedish Collegium for Advanced Studies (SCAS) are acknowledged.
Publisher Copyright:
© 2022 American Chemical Society. All rights reserved.
Copyright:
Copyright 2022 Elsevier B.V., All rights reserved.