TY - JOUR
T1 - Homogenous 96-plex PEA immunoassay exhibiting high sensitivity, specificity, and excellent scalability
AU - Assarsson, Erika
AU - Lundberg, Martin
AU - Holmquist, Göran
AU - Björkesten, Johan
AU - Thorsen, Stine Buch
AU - Ekman, Daniel
AU - Eriksson, Anna
AU - Rennel Dickens, Emma
AU - Ohlsson, Sandra
AU - Edfeldt, Gabriella
AU - Andersson, Ann-Catrin
AU - Lindstedt, Patrik
AU - Stenvang, Jan
AU - Gullberg, Mats
AU - Fredriksson, Simon
N1 - OA
PY - 2014/4/22
Y1 - 2014/4/22
N2 - 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.
AB - 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.
U2 - 10.1371/journal.pone.0095192
DO - 10.1371/journal.pone.0095192
M3 - Journal article
C2 - 24755770
SN - 1932-6203
VL - 9
JO - PLoS Computational Biology
JF - PLoS Computational Biology
IS - 4
M1 - e95192
ER -