Homogeneous antibody-based proximity extension assays provide sensitive and specific detection of low-abundant proteins in human blood
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TLDR
A well-characterized nucleic acid proximity-based assay using antibodies, called Proximity Extension Assay (PEA), showing good performance in plasma samples, and the use of a macro-molecular crowding matrix in combination with this homogeneous assay to drive target binding for low-affinity antibodies, thereby improving the sensitivity and increasing affinity reagent availability by lowering assay development dependency on high-Affinity antibodies.Abstract:
Convenient and well-performing protein detection methods for a wide range of targets are in great demand for biomedical research and future diagnostics. Assays without the need for washing steps while still unaffected when analyzing complex biological samples are difficult to develop. Herein, we report a well-characterized nucleic acid proximitybased assay using antibodies, called Proximity Extension Assay (PEA), showing good performance in plasma samples. Target-specific antibody pairs are linked to DNA strands that upon simultaneous binding to the target analyte create a real-time PCR amplicon in a proximity-dependent manner enabled by the action of a DNA polymerase. 3 0 Exonucleasecapable polymerases were found to be clearly superior in sensitivity over non-3 0 exonuclease ones. A PEA was set up for IL-8 and GDNF in a user-friendly, homogenous assay displaying femtomolar detection sensitivity, good recovery in human plasma, high specificity and up to 5-log dynamic range in 1kL samples. Furthermore, we have illustrated the use of a macro-molecular crowding matrix in combination with this homogeneous assay to drive target binding for low-affinity antibodies, thereby improving the sensitivity and increasing affinity reagent availability by lowering assay development dependency on high-affinity antibodies. Assay performance was also confirmed for a multiplex version of PEA.read more
Citations
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Journal ArticleDOI
Homogenous 96-plex PEA immunoassay exhibiting high sensitivity, specificity, and excellent scalability.
Erika Assarsson,Martin Lundberg,Göran Holmquist,Johan Björkesten,Stine Bucht Thorsen,Daniel Ekman,Anna Eriksson,Emma Rennel Dickens,Sandra Ohlsson,Gabriella Edfeldt,Ann-Catrin Andersson,Patrik Lindstedt,Jan Stenvang,Mats Gullberg,Simon Fredriksson +14 more
TL;DR: The aim of the present study was to develop a robust 96-plex immunoassay based on the proximity extension assay (PEA) for improved high throughput detection of protein biomarkers and the development of the current multiplex technique is a step toward robust high throughput protein marker discovery and research.
Journal ArticleDOI
Genomic atlas of the human plasma proteome.
Benjamin B. Sun,Joseph C. Maranville,James E. Peters,James E. Peters,David Stacey,James R Staley,James A. Blackshaw,Stephen Burgess,Tao Jiang,Ellie Paige,Ellie Paige,Praveen Surendran,Clare Oliver-Williams,Mihir A Kamat,Bram P. Prins,Sheri K. Wilcox,Erik S Zimmerman,An Chi,Narinder Bansal,Narinder Bansal,Sarah L. Spain,Angela M. Wood,Nicholas W. Morrell,Nicholas W. Morrell,John Bradley,Nebojsa Janjic,David J. Roberts,David J. Roberts,Willem H. Ouwehand,John A. Todd,Nicole Soranzo,Karsten Suhre,Dirk S. Paul,Caroline S. Fox,Robert M. Plenge,John Danesh,John Danesh,Heiko Runz,Adam S. Butterworth +38 more
TL;DR: The genetic architecture of the human plasma proteome in healthy blood donors from the INTERVAL study is characterized, and it is shown that protein quantitative trait loci overlap with gene expression quantitative traits, as well as with disease-associated loci, and evidence that protein biomarkers have causal roles in disease is found.
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TL;DR: The inflammatory response in MIS-C differs from the cytokine storm of severe acute COVID-19, shares several features with Kawasaki disease, but also differs from this condition with respect to T cell subsets, interleukin (IL)-17A, and biomarkers associated with arterial damage.
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References
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Protein detection using proximity-dependent DNA ligation assays
Simon Fredriksson,Mats Gullberg,Jonas Jarvius,Charlotta Olsson,Kristian Pietras,Sigrun M. Gustafsdottir,Arne Östman,Ulf Landegren +7 more
TL;DR: This proximity ligation assay detects zeptomole amounts of the cytokine platelet-derived growth factor without washes or separations, and the mechanism can be generalized to other forms of protein analysis.
Journal ArticleDOI
Molecular beacon sequence analysis for detecting drug resistance in Mycobacterium tuberculosis
Amy S. Piatek,Sanjay Tyagi,Arno C. Pol,Amalio Telenti,Lincoln P. Miller,Fred Russell Kramer,David Alland +6 more
TL;DR: A new approach to DNA sequence analysis that uses fluorogenic reporter molecules—molecular beacons—and demonstrated their ability to discriminate alleles in real-time PCR assays of genomic DNA is developed.
Journal ArticleDOI
Cytokine detection by antibody-based proximity ligation
Mats Gullberg,Sigrun M. Gustafsdottir,Edith Schallmeiner,Jonas Jarvius,Mattias Bjarnegård,Christer Betsholtz,Ulf Landegren,Simon Fredriksson +7 more
TL;DR: This work generalizes the recently established proximity ligation mechanism by providing simple and convenient protocols to convert any polyclonal antibodies or matched pair of monoclonal antibodies to proximity probe sets through the attachment of oligonucleotide sequences.
Journal ArticleDOI
Multiplexed protein detection by proximity ligation for cancer biomarker validation
Simon Fredriksson,William Dixon,Hanlee P. Ji,Albert C. Koong,Michael N. Mindrinos,Ronald W. Davis +5 more
TL;DR: A proximity ligation–based multiplexed protein detection procedure in which several selected proteins can be detected via unique nucleic-acid identifiers and subsequently quantified by real-time PCR is presented.
Journal ArticleDOI
Proximity extension of circular DNA aptamers with real-time protein detection
Daniel A. Di Giusto,Wjatschesslaw A. Wlassoff,J. Justin Gooding,Barbara A. Messerle,Garry C. King +4 more
TL;DR: This application elaborates the pleiotropic properties of circular DNA aptamers beyond the stability, potency and multitargeting characteristics described earlier: their intrinsic compatibility with a highly sensitive protein detection method termed the ‘proximity extension’ assay.