Homogeneous antibody-based proximity extension assays provide sensitive and specific detection of low-abundant proteins in human blood
By Martin Lundberg, Anna Eriksson, Bonnie Tran, E. Assarsson, S. Fredriksson. Nucleic Acids Research, 2011
669 citations recorded by the source corpus.
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 proximity-based 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′Exonuclease-capable polymerases were found to be clearly superior in sensitivity over non-3′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 1 μL 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.
Subfields: Biomedical Engineering, Molecular Biology
View Paper: https://www.semanticscholar.org/paper/fc4e7698bec47eff4ea3d2b502891326ba2a2be1
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Citations in the corpus
- Homogenous 96-Plex PEA Immunoassay Exhibiting High Sensitivity, Specificity, and Excellent Scalability (2014, methodology): “Subsequent to the development of singleplex and 24-plex PEA [8], we chose to increase the multiplexing to a level of 96, and with a focus on cancer-related biomarkers.”
- Stereotypic Immune System Development in Newborn Children (2018, methodology): “Plasma protein analyses using ProSeek (Olink, Uppsala, Sweden), a sensitive dual-recognition immunoassay (Assarsson et al., 2014; Lundberg et al., 2011), allowed the quantification of 267 unique proteins in < 20 mL of plasma in (Figure 1A).”
- Bifidobacteria-mediated immune system imprinting early in life (2020, methodology): “In total, we profiled 110 immune cell populations from 270 blood samples and quantified 267 unique plasma proteins using using Olink assays (Lundberg et al., 2011) (Olink, Uppsala, Sweden), which elucidated immune responses and developmental changes (Olin et al., 2018) as well as infectious disease ”