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 microparticlebased 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-ofthe-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. Molecular & Cellular Proteomics 9:327-335, 2010.Analyses of the plasma proteome, its protein content, their modifications, and interactions, hold great promise to improve detection, classification, and prognostication of pathological conditions such as cancer (1). The attraction of serum or plasma biomarkers lies in their potential to reveal disease processes throughout the body and to guide selection of therapy and follow-up using minimally invasive blood sampling.This optimism is tempered by the molecular complexity of plasma and the fact that the abundance of known plasma proteins varies over at least 12 orders of magnitude (1), posing great challenges for immunoassays used to investigate the plasma proteome. Thus, new assay formats are needed that can offer improved sensitivity and specificity over a broad dynamic range with good precision to assess new protein biomarkers for analysis in plasma, serum, or whole blood.The proximity ligation assay (PLA), 1 first described by Fredriksson et al. (2) in 2002, is an immunoassay for detection of protein molecules via DNA ligation and amplification, offering high specificity and sensitivity. In PLA, pairs of affinity probes directed against the same target molecule are modified by attaching short single-stranded DNA m...

Multiple recognition assay reveals prostasomes as promising plasma biomarkers for prostate cancer

“…Sequences for all oligonucleotides are shown in Table S1. All oligonucleotide-streptavidin conjugates used to prepare PLA probes for 4PLA and solid-phase PLA tests were combined with free streptavidin and briefly heated to obtain streptavidin tetramers containing reduced number of oligonucleotides as described (49). Oligonucleotide-streptavidin conjugates (100 nM) were incubated with 100 nM biotinylated antibodies in 1× PBS solution for 1 h at 21°C.…”

Sensitive detection of aggregated prion protein via proximity ligation

“…Prior to use the streptavidinoligonucleotide conjugates were treated with a 3-fold molar excess of free streptavidin (S4762, Sigma-Aldrich) at 65 C for 30 min as previously described. 24,33 The connector oligonucleotide (5 0 -TACTTAGACACGA CACGATTTAGTTT-3 0 ) serving as template for the ligation, the forward (5 0 -CATCGCCCTTGGACTACGA-3 0 ) and reverse (5 0 -GGGAATCAAGGTAACGGACTTTAG-3 0 ) primers were all purchased from Integrated DNA Technology. The TaqMan probe (FAM-5 0 -TGACGAACCGCTTTGCCTGA-3 0 -MGB [quencher]) was purchased from Applied Biosystems.…”

“…The samples were incubated at room temperature (RT) for ten minutes before they were further diluted at least 10-fold in PLA buffer (1 mM D-bio- Solid-phase proximity ligation assay SP-PLA was performed as previously described. 24,34 Briefly, 1.5 mg of the biotinylated antibody -either 3F4 or 6H4 -was immobilized on 1 mg streptavidin-coated magnetic beads (Dynabeads MyOne Streptavidin T1; 65602, Life Technologies). Five ml of denatured brain homogenate sample was diluted ten times in PLA buffer and thereafter mixed with the beads to allow capture of the target molecules.…”

“…The specific detection of aggregated PrP is based on the solid-phase proximity ligation assay (SP-PLA). 23,24 In SP-PLA targeted proteins are first captured on a solid support via immobilized antibodies before addition of 2 PLA probes, that is antibodies with conjugated oligonucleotides, followed by washes, ligation of oligonucleotides on pairs of antibodies that have bound in proximity, and amplified detection by quantitative real-time PCR (qPCR). If all 3 affinity reagents required for detection are directed against the same epitope, then the assay can be used to detect aggregated forms of a protein recognized by the antibody.…”

“…25 We have previously shown that SP-PLA shows similar assay performance in whole blood as in buffer or plasma. 24 The only instrumentation required for the assay is a qPCR machine, which represents standard laboratory instrumentation. We therefore believe that the assay given further optimization will be suitable for routine analyses of samples from body fluids such as CSF or blood, allowing ante mortem testing.…”