title: Homogenous 96-plex PEA immunoassay exhibiting high sensitivity, specificity, and excellent scalability.
abstract: Medical research is developing an ever greater need for comprehensive high-quality data generation to realize the promises of personalized health care based on molecular biomarkers. The nucleic acid proximity-based methods proximity ligation and proximity extension assays have, with their dual reporters, shown potential to relieve the shortcomings of antibodies and their inherent cross-reactivity in multiplex protein quantification applications. 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. This was enabled by: (1) a modified design leading to a reduced number of pipetting steps compared to the existing PEA protocol, as well as improved intra-assay precision; (2) a new enzymatic system that uses a hyper-thermostabile enzyme, Pwo, for uniting the two probes allowing for room temperature addition of all reagents and improved the sensitivity; (3) introduction of an inter-plate control and a new normalization procedure leading to improved inter-assay precision (reproducibility). The multiplex proximity extension assay was found to perform well in complex samples, such as serum and plasma, and also in xenografted mice and resuspended dried blood spots, consuming only 1 µL sample per test. All-in-all, the development of the current multiplex technique is a step toward robust high throughput protein marker discovery and research.
authors.affiliation: Olink Bioscience, Uppsala, Sweden.
authors.forename: Erika
authors.initials: E
authors.lastname: Assarsson
authors.affiliation: Olink Bioscience, Uppsala, Sweden.
authors.forename: Martin
authors.initials: M
authors.lastname: Lundberg
authors.affiliation: Olink Bioscience, Uppsala, Sweden.
authors.forename: Göran
authors.initials: G
authors.lastname: Holmquist
authors.affiliation: Olink Bioscience, Uppsala, Sweden.
authors.forename: Johan
authors.initials: J
authors.lastname: Björkesten
authors.affiliation: Section for Molecular Disease Biology and Sino-Danish Breast Cancer Research Centre, Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Stine Bucht
authors.initials: SB
authors.lastname: Thorsen
authors.affiliation: Olink Bioscience, Uppsala, Sweden.
authors.forename: Daniel
authors.initials: D
authors.lastname: Ekman
authors.affiliation: Olink Bioscience, Uppsala, Sweden.
authors.forename: Anna
authors.initials: A
authors.lastname: Eriksson
authors.affiliation: Olink Bioscience, Uppsala, Sweden.
authors.forename: Emma
authors.initials: E
authors.lastname: Rennel Dickens
authors.affiliation: Olink Bioscience, Uppsala, Sweden.
authors.forename: Sandra
authors.initials: S
authors.lastname: Ohlsson
authors.affiliation: Olink Bioscience, Uppsala, Sweden.
authors.forename: Gabriella
authors.initials: G
authors.lastname: Edfeldt
authors.affiliation: Olink Bioscience, Uppsala, Sweden.
authors.forename: Ann-Catrin
authors.initials: AC
authors.lastname: Andersson
authors.affiliation: Olink Bioscience, Uppsala, Sweden.
authors.forename: Patrik
authors.initials: P
authors.lastname: Lindstedt
authors.affiliation: Section for Molecular Disease Biology and Sino-Danish Breast Cancer Research Centre, Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Jan
authors.initials: J
authors.lastname: Stenvang
authors.affiliation: Olink Bioscience, Uppsala, Sweden.
authors.forename: Mats
authors.initials: M
authors.lastname: Gullberg
authors.affiliation: Olink Bioscience, Uppsala, Sweden.
authors.forename: Simon
authors.initials: S
authors.lastname: Fredriksson
mesh_terms: D000818:Animals; D001798:Blood Proteins / Q000378:metabolism; D003429:Cross Reactions; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D059788:Dried Blood Spot Testing; D004795:Enzyme Stability; D005260:Female; D064593:Heterografts; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D008819:Mice, Nude; D009841:Oligonucleotides / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013696:Temperature
chemical_list: D001798:Blood Proteins; D009841:Oligonucleotides; D004259:DNA-Directed DNA Polymerase
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