Homogenous 96-Plex PEA Immunoassay Exhibiting High Sensitivity, Specificity, and Excellent Scalability
Open accessAbstract
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.
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Proximity extension assays have been shown to be sensitive and specific.
“We mainly used proximity extension assays (PEA), which have been shown to be sensitive and specific (Assarsson et al, 2014; Katz et al, 2022)”
From The genetic regulation of protein expression in cerebrospinal fluid (2023) · cites this paper as Assarsson et al, 2014
The GigaMUGA contains 143,259 genetic markers specifically designed for genetic mapping in the DO mouse population.
“The GigaMUGA contains 143,259 genetic markers that were speci cally designed for genetic mapping in the DO mouse population [19,33].”
From Identification of Genetic Variants Regulating the Abundance of Clinically Relevant Plasma Proteins using the Diversity Outbred Mouse Model (2021) · cites this paper as [33]
The Materials and Methods section describes xenograft experiments in NMRI-nude mice, while the Results and Discussion describes a protein immunoassay. Searches for “GigaMUGA 143,259 genetic markers” and “Diversity Outbred mouse genotyping array” found no passage addressing the marker count or the array’s design for DO mouse mapping. The claim cannot be verified from our copy. (LLM, conf=0.76)
Experimental — accuracy not yet measuredOlink Explore 3072 uses 2941 antibody-nucleic acid assay probes to quantify 2172 human protein targets.
“Olink Explore 3072 uses 2941 antibody-nucleic acid assay probes to quantify 2172 human protein targets59,60.”
From Cross-ancestry comparison of aptamer and antibody protein measures (2026) · cites this paper as [59]
The Results and Discussion section describes a 96-plex PEA panel with 94 probe pairs, including controls. Our copy does not address Olink Explore 3072 or state that it uses 2,941 probes to quantify 2,172 human protein targets (searched for the product name and both counts). (LLM, conf=0.77)
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“Proteins were quanti ed by real-time quantitative PCR of the ampli ed oligonucleotide tags [35].”
Method
- Authors
- Assarsson, E., Lundberg, M., Holmquist, G., Björkesten, J., Bucht Thorsen, S., Ekman, D., Eriksson, A., Rennel Dickens, E., Ohlsson, S., Edfeldt, G., Andersson, A.-C., Lindstedt, P., Stenvang, J., Gullberg, M., & Fredriksson, S.
- Affiliations
- Olink Bioscience (Sweden) (SE)
- University of Copenhagen (DK)
- Corresponding author
- Assarsson, Erika
- ORCID
- Venue
- PLoS ONE
- Publisher
- Public Library of Science (PLoS)
- Published
- 2014-04-22
- Volume
- 9
- Issue
- 4
- Pages
- e95192
- ISSN
- 1932-6203 (ISSN-L)
- PMC
- PMC3995906
- OpenAlex
- W2010084870
- Cited by
- 1,874
- References
- 0
- Citations by year
- 2026: 215 · 2025: 216 · 2024: 245 · 2023: 271 · 2022: 242 · 2021: 224 · 2020: 156 · 2019: 111 · 2018: 84 · 2017: 52 · 2016: 21 · 2015: 23 · 2014: 7 · 2013: 1 · 2012: 3
- FWCI
- 14.09
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- PLoS ONE (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Research at the University of Copenhagen (University of Copenhagen) (publishedVersion, other-oa)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Figshare (submittedVersion, cc-by)
- Funders
- VINNOVA
- FP7 Health (EU FP7)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Advanced Biosensing Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Advanced biosensing and bioanalysis techniques
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Keywords
- proximity extension assay, multiplex immunoassay, protein biomarkers, proximity ligation, normalization, dried blood spots, serum and plasma samples
- MeSH terms
- Animals, Blood Proteins, Cross Reactions, DNA-Directed DNA Polymerase, Enzyme Stability, Female, Humans, Immunoassay, Mice, Nude, Oligonucleotides, Sensitivity and Specificity, Temperature, Polymerase Chain Reaction, Dried Blood Spot Testing, Heterografts
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