title: Sensitive plasma protein analysis by microparticle-based proximity ligation assays.
abstract: Detection of proteins released in the bloodstream from tissues damaged by disease can promote early detection of pathological conditions, differential diagnostics, and follow-up of therapy. Despite these prospects and a plethora of candidate biomarkers, efforts in recent years to establish new protein diagnostic assays have met with limited success. One important limiting factor has been the challenge of detecting proteins present at trace levels in complex bodily fluids. To achieve robust, sensitive, and specific detection, we have developed a microparticle-based solid-phase proximity ligation assay, dependent on simultaneous recognition of target proteins by three antibody molecules for added specificity. After capture on a microparticle, solid-phase pairs of proximity probes are added followed by washes, enabling detection and identification of rare protein molecules in blood while consuming small amounts of sample. We demonstrate that single polyclonal antibody preparations raised against target proteins of interest can be readily used to establish assays where detection depends on target recognition by three individual antibody molecules, recognizing separate epitopes. The assay was compared with state-of-the-art sandwich ELISAs for detection of vascular endothelial growth factor, interleukin-8 and interleukin-6, and it was found to be superior both with regard to dynamic range and minimal numbers of molecules detected. Furthermore, the assays exhibited excellent performance in undiluted plasma and serum as well as in whole blood, producing comparable results for nine different antigens. We thus show that solid-phase proximity ligation assay is suitable for validation of a variety of protein biomarkers over broad dynamic ranges in clinical samples.
authors: Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden.
authors: Spyros
authors: S
authors: Darmanis
authors: Rachel Yuan
authors: RY
authors: Nong
authors: Maria
authors: M
authors: Hammond
authors: Jijuan
authors: J
authors: Gu
authors: Anders
authors: A
authors: Alderborn
authors: Johan
authors: J
authors: Vänelid
authors: Agneta
authors: A
authors: Siegbahn
authors: Sigrun
authors: S
authors: Gustafsdottir
authors: Olle
authors: O
authors: Ericsson
authors: Ulf
authors: U
authors: Landegren
authors: Masood
authors: M
authors: Kamali-Moghaddam
mesh_terms: D001798:Blood Proteins / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay; D055436:Growth Differentiation Factor 15 / Q000097:blood; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D008863:Microspheres
chemical_list: D001798:Blood Proteins; C526140:GDF15 protein, human; D055436:Growth Differentiation Factor 15