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Aiming to accommodate the unmet need for easily accessible biomarkers with a focus on biological differences between haematological diseases, the diagnostic value of plasma proteins in acute leukaemias and lymphomas was investigated. A multiplex proximity extension assay (PEA) was used to analyze 183 proteins in diagnostic plasma samples from 251 acute leukaemia and lymphoma patients and compared with samples from 60 healthy controls. Multivariate modelling using partial least square discriminant analysis revealed highly significant differences between distinct disease subgroups and controls. The model allowed explicit distinction between leukaemia and lymphoma, with few patients misclassified. Acute leukaemia samples had higher levels of proteins associated with haemostasis, inflammation, cell differentiation and cell-matrix integration, whereas lymphoma samples demonstrated higher levels of proteins known to be associated with tumour microenvironment and lymphoma dissemination. PEA technology can be used to screen for large number of plasma protein biomarkers in low mu L sample volumes, enabling the distinction between controls, acute leukaemias and lymphomas. Plasma protein profiling could help gain insights into the pathophysiology of acute leukaemia and lymphoma and the technique may be a valuable tool in the diagnosis of these diseases.

Circulating nucleic acids, encapsulated within small extracellular vesicles (EVs), provide a remote cellular snapshot of biomarkers derived from diseased tissues, however selective isolation is critical. Current laboratory‐based purification techniques rely on the physical properties of small‐EVs rather than their inherited cellular fingerprints. We established a highly‐selective purification assay, termed EV‐CATCHER, initially designed for high‐throughput analysis of low‐abundance small‐RNA cargos by next‐generation sequencing. We demonstrated its selectivity by specifically isolating and sequencing small‐RNAs from mouse small‐EVs spiked into human plasma. Western blotting, nanoparticle tracking, and transmission electron microscopy were used to validate and quantify the capture and release of intact small‐EVs. As proof‐of‐principle for sensitive detection of circulating miRNAs, we compared small‐RNA sequencing data from a subset of small‐EVs serum‐purified with EV‐CATCHER to data from whole serum, using samples from a small cohort of recently hospitalized Covid‐19 patients. We identified and validated, only in small‐EVs, hsa‐miR‐146a and hsa‐miR‐126‐3p to be significantly downregulated with disease severity. Separately, using convalescent sera from recovered Covid‐19 patients with high anti‐spike IgG titers, we confirmed the neutralizing properties, against SARS‐CoV‐2 in vitro, of a subset of small‐EVs serum‐purified by EV‐CATCHER, as initially observed with ultracentrifuged small‐EVs. Altogether our data highlight the sensitivity and versatility of EV‐CATCHER., ‘A customizable, low background, high‐affinity assay for specific immuno‐capture and release of circulating small extracellular vesicles prior to small‐RNA sequencing for identification of miRNA biomarkers or in‐vitro evaluation: The EV‐CATCHER assay (Extracellular Vesicle Capture by AnTibody of CHoice and Enzymatic Release)’.

In this study, levels of inflammatory protein biomarkers in venous plasma, plasma derived from capillary blood from the earlobe, and capillary plasma stored as dried plasma spots (DPS) were compared. Samples from 12 male individuals were assessed with a panel of 92 inflammation-related proteins using multiplex proximity extension assay. Correlations between sample types varied greatly between analytes. A high correlation of rho > 0.8 was observed between capillary plasma and DPS for 32 analytes. At this level of correlation, 13 analytes correlated between venous and capillary plasma and 5 analytes in the comparison of venous blood with DPS.

There are increasing demands for informative cancer biomarkers, accessible via minimally invasive procedures, both for initial diagnostics and for follow‐up of personalized cancer therapy, including immunotherapy. Fine‐needle aspiration (FNA) biopsy provides ready access to relevant tissue samples; however, the minute amounts of sample require sensitive multiplex molecular analysis to be of clinical biomarker utility. We have applied proximity extension assays (PEA) to analyze 167 proteins in FNA samples from patients with breast cancer (BC; n = 25) and benign lesions (n = 32). We demonstrate that the FNA BC samples could be divided into two main clusters, characterized by differences in expression levels of the estrogen receptor (ER) and the proliferation marker Ki67. This clustering corresponded to some extent to established BC subtypes. Our analysis also revealed several proteins whose expression levels differed between BC and benign lesions (e.g., CA9, GZMB, IL‐6, VEGFA, CXCL11, PDL1, and PCD1), as well as several chemokines correlating with ER and Ki67 status (e.g., CCL4, CCL8, CCL20, CXCL8, CXCL9, and CXCL17). Finally, we also identified three signatures that could predict Ki67 status, ER status, and tumor grade, respectively, based on a small subset of proteins, which was dominated by chemokines. To our knowledge, expression profiles of CCL13 in benign lesions and BC have not previously been described but were shown herein to correlate with proliferation (P = 0.00095), suggesting a role in advanced BC. Given the broad functional range of the proteins analyzed, immune‐related proteins were overrepresented among the observed alterations. Our pilot study supports the emerging role of chemokines in BC progression. Due to the minimally traumatic sampling and clinically important molecular information for therapeutic decisions, this methodology is promising for future immunoscoring and monitoring of treatment efficacy in BC.

Protein interactions and posttranslational modifications orchestrate cellular responses to e.g. cytokines and drugs, but it has been difficult to monitor these dynamic events in high-throughput. Here, we describe a semi-automated system for large-scale in situ proximity ligation assays (isPLA), combining isPLA in microtiter wells with automated microscopy and computer-based image analysis. Phosphorylations and interactions are digitally recorded along with subcellular morphological features. We investigated TGF-β-responsive Smad2 linker phosphorylations and complex formations over time and across millions of individual cells, and we relate these events to cell cycle progression and local cell crowding via measurements of DNA content and nuclear size of individual cells, and of their relative positions. We illustrate the suitability of this protocol to screen for drug effects using phosphatase inhibitors. Our approach expands the scope for image-based single cell analyses by combining observations of protein interactions and modifications with morphological details of individual cells at high throughput., To improve our ability to monitor cellular responses to e.g. cytokines or drugs, Lönn et al have developed a semi-automated system for large-scale in situ proximity ligation assays (isPLA) in HaCAT keratinocyte cells. Their approach expands the scope for image-based single cell analyses by combining observations of protein interactions and modifications with morphological details of individual cells at high throughput.

MUC5AC has been indicated to be a marker for mucinous ovarian cancer (OC). We investigated the use of in situ proximity ligation assay (PLA) for blood group ABH expressing MUC5AC to differentiate between serous and mucinous OC, to validate preceding observations that also MUC5AC ABH expression is increased in mucinous OC. We developed PLA for anti-A, B, and H/anti-MUC5AC and a PLA using a combined lectin Ulex europaeus agglutinin I (UEA I)/anti-MUC5AC assay. The PLAs were verified with mass spectrometry, where mucinous OC secretor positive patients’ cysts fluids containing ABH O-linked oligosaccharides also showed positive OC tissue PLA staining. A nonsecretor mucinous OC cyst fluid was negative for ABH and displayed negative PLA staining of the matched tissue. Using the UEA I/MUC5AC PLA, we screened a tissue micro array of 410 ovarian tissue samples from patients with various stages of mucinous or serous OC, 32 samples with metastasis to the ovaries and 34 controls. The PLA allowed differentiating mucinous tumors with a sensitivity of 84% and a specificity of 97% both against serous cancer but also compared to tissues from controls. This sensitivity is close to the expected incidence of secretor individuals in a population. The recorded sensitivity was also found to be higher compared to mucinous type cancer with metastasis to the ovaries, where only 32% were positive. We conclude that UEA 1/MUC5AC PLA allows glycospecific differentiation between serous and mucinous OC in patients with positive secretor status and will not identify secretor negative individuals with mucinous OC.

Host proteins incorporated into virus particles have been reported to contribute to infectivity and tissue-tropism. This incorporation of host proteins is expected to be variable among viral particles, however, protein analysis at single-virus levels has been challenging. We have developed a method to detect host proteins incorporated on the surface of virions using the in situ proximity ligation assay (isPLA) with rolling circle amplification (RCA), employing oligonucleotide-conjugated antibody pairs. The technique allows highly selective and sensitive antibody-based detection of viral and host proteins on the surface of individual virions. We detected recombinant noninfectious sub-viral particles (SVPs) of tick-borne encephalitis virus (TBEV) immobilized in microtiter wells as fluorescent particles detected by regular fluorescence microscopy. Counting the particles in the images enabled us to estimate individual TBEVSVP counts in different samples. Using isPLA we detected individual calnexin-, CD9-, CD81-, CD29-and CD59-positive SVPs among the viral particles. Our data suggests that a diversity of host proteins may be incorporated into TEBV, illustrating that isPLA with digital counting enables single-virus analysis of host protein incorporation. (C) 2020 The Authors. Published by Elsevier Inc.

Post-translational modifications (PTMs) regulate protein activities to help orchestrate and fine-tune cellular processes. Dysregulation of PTMs is often related with disorders and malignancies, and may serve as a precise biomarker of disease. Developing sensitive tools to measure and monitor low-abundant PTMs in tissue lysates or serum will be instrumental for opening up new PTM-based diagnostic avenues. Here, we investigate the use of solid-phase proximity ligation assay (SP-PLA) for detection of different PTMs. The assay depends on the recognition of the target protein molecule and its modification by three affinity binders. Using antibodies and lectins, we applied the method for detection of glycosylated CD44 and E-Cadherin, and phosphorylated p53 and EGFR. The assay was found to have superior dynamic range and limit of detection compared to standard ELISAs. In summary, we have established the use of SP-PLA as an appropriate method for sensitive detection of PTMs in lysates and sera, which may provide a basis for future PTM-based diagnostic and prognostic biomarkers.

Postpartum depression (PPD) is a devastating disorder affecting not only more than 10% of all women giving birth, but also the baby, the family, and the society. Compiling evidence suggests the involvement of the immune system in the pathophysiology of major depression; yet, the immune response in perinatal depression is not as well studied. The aim of this study was to investigate the alterations in peripheral levels of inflammatory biomarkers in 169 Swedish women with and without depressive symptoms according to the Edinburgh postnatal depression scale or the M.I.N.I neuropsychiatric interview at eight weeks postpartum. Among the 70 markers analyzed with multiplex proximity extension assay, five were significantly elevated in women with postpartum depressive symptoms in the adjusted LASSO logistic regression analysis: Tumor necrosis factor ligand superfamily member (TRANCE) (OR-per 1 SD increase = 1.20), Hepatocyte growth factor (HGF) (OR = 1.17) Interleukin (IL)-18 (OR = 1.06), Fibroblast growth factor 23 (FGF-23) (OR = 1.25), and C-X-C motif chemokine 1 (CXCL1) (OR 1.11). These results indicate that women with PPD have elevated levels of some inflammatory biomarkers. It is, therefore, plausible that PPD is associated with a compromised adaptability of the immune system.

Soluble triggering receptor expressed on myeloid cells 2 (sTREM2) is derived from cleavage of TREM2, which is expressed on the cell surface of microlgia and other tissue-specific macrophages. In the present study, the changes in the sTREM2 levels after ischemic stroke (IS) and their association with clinical outcomes were evaluated. A total of 43 patients diagnosed with non-cardioembolic IS between June 2011 and May 2014 were consecutively included in this study. Patients treated with intravenous thrombolysis or intra-arterial thrombectomy were excluded. Plasma samples were collected three times (days 1, 7, and 90) after ictus. The sTREM2 level was measured in the samples using the highly sensitive solid-phase proximity ligation assay (SP-PLA). Among the 43 subjects, higher initial NIH stroke scale (NIHSS) score (P = 0.005), early increment of sTREM2 (P 0.001), and late decrement of sTREM2 (P = 0.002), were more common in patients with poor outcome. Based on multivariate analysis, initial NIHSS score (P = 0.015) and early increment of sTREM2 (P = 0.032) were independently associated with poor outcome. The results from the present study indicate that increment of sTREM2 level at the early phase was a predictor of poor outcome. Serial follow-up of sTREM2 may aid prognosis after stroke.

In chronic lymphocytic leukaemia (CLL) patients, treatment with the Bruton tyrosine kinase inhibitor ibrutinib induces a rapid shift of tumour cells from lymph nodes (LN) to peripheral blood (PB). Here, we characterized in depth the dynamics of ibrutinib-induced inflammatory, transcriptional and cellular changes in different compartments immediately after treatment initiation in seven relapsed/refractory CLL patients. Serial PB and LN samples were taken before start and during the first 29 days of treatment. Changes in plasma inflammation-related biomarkers, CLL cell RNA expression, B-cell activation and migration markers expression, and PB mononuclear cell populations were assessed. A significant reduction of 10 plasma inflammation markers, the majority of which were chemokines and not CLL-derived, was observed within hours, and was paralleled by very early increase of CD19+ circulating cells. At the RNA level, significant and continuous changes in transcription factors and signalling molecules linked to B-cell receptor signalling and CLL biology was observed in both PB and LN CLL cells already after 2 days of treatment. In conclusion, ibrutinib seems to instantly shut off an ongoing inflammatory response and interfere with diverse sensitive pathways in the LN.

There are increasing demands for informative cancer biomarkers, accessible via minimally invasive procedures, both for initial diagnostics and to follow‐up personalized cancer therapy. Fine‐needle aspiration (FNA) biopsy provides ready access to relevant tissues; however, the minute sample amounts require sensitive multiplex molecular analysis to achieve clinical utility. We have applied proximity extension assays (PEA) and NanoString (NS) technology for analyses of proteins and of RNA, respectively, in FNA samples. Using samples from patients with breast cancer (BC, n = 25) or benign lesions (n = 33), we demonstrate that these FNA‐based molecular analyses (a) can offer high sensitivity and reproducibility, (b) may provide correct diagnosis in shorter time and at a lower cost than current practice, (c) correlate with results from routine analysis (i.e., benchmarking against immunohistochemistry tests for ER, PR, HER2, and Ki67), and (d) may also help identify new markers related to immunotherapy. A specific 11‐protein signature, including FGF binding protein 1, decorin, and furin, distinguished all cancer patient samples from all benign lesions in our main cohort and in smaller replication cohort. Due to the minimally traumatic sampling and rich molecular information, this combined proteomics and transcriptomic methodology is promising for diagnostics and evaluation of treatment efficacy in BC.

We analysed and compared MUC7 O-glycosylation and inflammatory biomarkers in saliva from female patients with burning mouth syndrome (BMS) and gender/age-matched controls. Oligosaccharides from salivary MUC7 from BMS and controls were released. Inflammatory mediators were measured by multiplex proximity extension assay. Presence of sialyl-Lewisx (Si-Lex) epitope on MUC7 was confirmed using Western blot. MUC7 O-glycans and measured inflammatory biomarkers were found to be similar between BMS and controls. However, oligosaccharides sialyl-Lewisx (Si-Lex) was found to be reduced in samples from BMS patients. Positive correlation (combined patients and controls) was found between levels of C-C motif chemokine 19 (CCL-19) and the amount of core-2 oligosaccharides on MUC7 as well as fractalkine (CX3CL1) and level of sialylation. Patients with BMS were shown to represent a heterogeneous group in terms of inflammatory biomarkers. This indicates that BMS patients could be further stratified on the basis of low-level inflammation. The results furthermore indicate that reduced sialylation of MUC7, particularly Si-Lex, may be an important feature in patients with BMS. However, the functional aspects and potential involvement in immune regulation of Si-Lex remains unclear. Our data suggests a chemokine driven alteration of MUC7 glycosylation.

Traumatic brain injury (TBI) is followed by secondary injury mechanisms strongly involving neuroinflammation. To monitor the complex inflammatory cascade in human TBI, we used cerebral microdialysis (MD) and multiplex proximity extension assay (PEA) technology and simultaneously measured levels of 92 protein biomarkers of inflammation in MD samples every three hours for five days in 10 patients with severe TBI under neurointensive care. One μL MD samples were incubated with paired oligonucleotide-conjugated antibodies binding to each protein, allowing quantification by real-time quantitative polymerase chain reaction. Sixty-nine proteins were suitable for statistical analysis. We found five different patterns with either early (96 h; e.g., CD40, MCP2, MCP3), biphasic peaks (e.g., CXCL1, CXCL5, IL8) or stable (e.g., CCL4, DNER, VEGFA)/low trends. High protein levels were observed for e.g., CXCL1, CXCL10, MCP1, MCP2, IL8, while e.g., CCL28 and MCP4 were detected at low levels. Several proteins (CCL8, -19, -20, -23, CXCL1, -5, -6, -9, -11, CST5, DNER, Flt3L, and SIRT2) have not been studied previously in human TBI. Cross-correlation analysis revealed that LIF and CXCL5 may play a central role in the inflammatory cascade. This study provides a unique data set with individual temporal trends for potential inflammatory biomarkers in patients with TBI. We conclude that the combination of MD and PEA is a powerful tool to map the complex inflammatory cascade in the injured human brain. The technique offers new possibilities of protein profiling of complex secondary injury pathways.

Exosomes have been implicated in numerous biological processes, and they may serve as important disease markers. Surface proteins on exosomes carry information about their tissues of origin. Because of the heterogeneity of exosomes it is desirable to investigate them individually, but this has so far remained impractical. Here, we demonstrate a proximity-dependent barcoding assay to profile surface proteins of individual exosomes using antibody-DNA conjugates and next-generation sequencing. We first validate the method using artificial streptavidin-oligonucleotide complexes, followed by analysis of the variable composition of surface proteins on individual exosomes, derived from human body fluids or cell culture media. Exosomes from different sources are characterized by the presence of specific combinations of surface proteins and their abundance, allowing exosomes to be separately quantified in mixed samples to serve as markers for tissue-specific engagement in disease., The use of antibodies to capture and profile exosomes limits the number of target proteins that can be detected. Here the authors develop a proximity-dependent barcoding assay that allows profiling of 38 surface proteins on individual exosomes from heterogeneous samples such as serum and seminal fluid.

Background: Gastric cancer (GC) is the third leading cause of cancer death. Early detection is a key factor to reduce its mortality. Methods: We retrospectively collected pre- and postoperative serum samples as well as tumor tissues and adjacent normal tissues from 100 GC patients. Serum samples from non-cancerous patients were served as controls (n=50). A high-throughput protein detection technology, multiplex proximity extension assays (PEA), was applied to measure levels of over 300 proteins. Alteration of each protein was analyzed by univariate analysis. Elastic-net logistic regression was performed to select serum proteins into the diagnostic model. Findings: We identified 19 serum proteins (CEA, CA9, MSLN, CCL20, SCF, TGF-alpha, MMP-1, MMP-10, IGF-1, CDCP1, PPIA, DDAH-1, HMOX-1, FLI1, IL-7, ZBTB-17, APBB1IP, KAZALD-1, and ADAMTS-15) that together distinguish the GC cases from controls with a diagnostic sensitivity of 93%, specificity of 100%, and area under receiver operating characteristic curve (AUC) of 0·99 (95% CI: 0·98-1). Moreover, the 19-serum protein signature provided a satisfactory diagnostic capacity in patients at TNM I-II stage (sensitivity 89%, specificity 100%, AUC 0·988) and in patients with high microsatellite instability (91%, 98%, and 0·990). A large-scale independent cohort of cases and controls will be necessary for validating the proposed protein signature. Interpretation: Based on targeted proteomics and elastic-net logistic regression, we identified a 19-serum protein signature which could contribute to clinical GC diagnosis. Funding Statement: This study was supported by a Marie Curie ITN grant (316929, GastricGlycoExplorer). Funder had no influence on trial design, data evaluation, and interpretation. Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: This study was conducted following the Declaration of Helsinki. All patients were provided with the informed consent and ethical approval was granted by the Institutional Ethics Committee of University of Siena.

Surgery on the arch or descending aorta is associated with significant risk of neurological complications. As a consequence of intubation and sedation, early neurologic injury may remain unnoticed. Biomarkers to aid in the initial diagnostics could prove of great value as immediate intervention is critical. Twenty-three patients operated in the thoracic aorta with significant risk of perioperative neurological injury were included. Cerebrospinal fluid (CSF) and serum were obtained preoperatively and in the first and second postoperative days and assessed with a panel of 92 neurological-related proteins. Three patients suffered spinal cord injury (SCI), eight delirium, and nine hallucinations. There were markers in both serum and CSF that differed between the affected and non-affected patients (SCI; IL6, GFAP, CSPG4, delirium; TR4, EZH2, hallucinations; NF1). The study identifies markers in serum and CSF that reflect the occurrence of neurologic insults following aortic surgery, which may aid in the care of these patients. Electronic supplementary material The online version of this article (10.1007/s12265-018-9835-8) contains supplementary material, which is available to authorized users.

The neurotransmitter γ-aminobutyric acid (GABA) is an extracellular signaling molecule in the brain and in pancreatic islets. Here, we demonstrate that GABA regulates cytokine secretion from human peripheral blood mononuclear cells (PBMCs) and CD4+ T cells. In anti-CD3 stimulated PBMCs, GABA (100 nM) inhibited release of 47 cytokines in cells from patients with type 1 diabetes (T1D), but only 16 cytokines in cells from nondiabetic (ND) individuals. CD4+ T cells from ND individuals were grouped into responder or non-responder T cells according to effects of GABA (100 nM, 500 nM) on the cell proliferation. In the responder T cells, GABA decreased proliferation, and inhibited secretion of 37 cytokines in a concentration-dependent manner. In the non-responder T cells, GABA modulated release of 8 cytokines. GABA concentrations in plasma from T1D patients and ND individuals were correlated with 10 cytokines where 7 were increased in plasma of T1D patients. GABA inhibited secretion of 5 of these cytokines from both T1D PBMCs and ND responder T cells. The results identify GABA as a potent regulator of both Th1- and Th2-type cytokine secretion from human PBMCs and CD4+ T cells where GABA generally decreases the secretion., Highlights • GABA regulates cytokine secretion in anti-CD3-stimulated peripheral blood mononuclear cells (PBMCs) and CD4+ T cells. • GABA inhibits secretion of 47 cytokines in PBMCs from type 1 diabetes patients. • GABA regulates secretion of pro- and anti-inflammatory cytokines in a concentration-dependent manner. GABA is a signal molecule in the brain, blood and pancreatic islets where it is secreted by the insulin-producing β cells. GABA has many roles in human islets including optimizing function and survival of β cells. Bhandage et al. now show that GABA is a potent regulator of secretion of both pro- and anti-inflammatory cytokines in stimulated immune cells. In type 1 diabetes the β-cell mass is diminished and thus the protective effect of GABA in the islets although not in blood. Targeting GABA signaling in diabetes mellitus is likely to be a part of the solution when curing diabetes.

Cytokines play important roles as regulators of cell functions, and over the last decades a number of cytokine assays have been developed. The aim of the present study was to investigate the effects of age and gender on a large number of cytokines. Plasma samples were collected from 33 healthy blood donors. The samples were analyzed using a multiplex proximity extension assay (PEA) allowing simultaneous measurement of 92 cytokines and four technical controls. Biomarkers with less than 80% quantitative results were excluded leaving 63 cytokines that were analyzed for the effects of gender and age. The plasma level of three of the investigated biomarkers (DNER, MCP-4 and MMP-10) were found to be significantly different for the two genders (adjusted p-value0.05), and 15 of the biomarkers (CCL11, CCL25, CDCP1, CSF-1, CXCL11, CXCL9, FGF-23, Flt3L, HGF, IL-10RB, MCP-3, MCP-4, MMP-10, OPG, VEGF-A) were significantly associated with age. This study reveals the effects of age and gender on a large number of cytokine assays. CXCL5 and TNFB were significantly higher in females, while the other markers with significant gender-dependent differences were higher in males. For the markers that were significantly associated with age, only CXCL6 was found to decrease with age, while the other biomarkers increased with age.

Extracellular vesicles (EVs) are membrane-coated objects such as exosomes and microvesicles, released by many cell-types. Their presence in body fluids and the variable surface composition and content render them attractive potential biomarkers. The ability to determine their cellular origin could greatly move the field forward. We used multiplex proximity extension assays (PEA) to identify with high specificity and sensitivity the protein profiles of exosomes of different origins, including seven cell lines and two different body fluids. By comparing cells and exosomes, we successfully identified the cells originating the exosomes. Furthermore, by principal component analysis of protein patterns human milk EVs and prostasomes released from prostate acinar cells clustered with cell lines from breast and prostate tissues, respectively. Milk exosomes uniquely expressed CXCL5, MIA, and KLK6, whereas prostasomes carried NKX31, GSTP1, and SRC, highlighting that EVs originating from different origins express distinct proteins. In conclusion, PEA provides a powerful protein screening tool in exosome research, for purposes of identifying the cell source of exosomes, or new biomarkers in diseases such as cancer and inflammation.

Background A number of factors regarding blood collection, handling and storage may affect sample quality. The purpose of this study was to assess the impact on plasma protein profiles by delayed centrifugation and plasma separation and multiple freeze-thaw cycles. Methods Blood samples drawn from 16 healthy individuals were collected into ethylenediaminetetraacetic acid tubes and kept either at 4 °C or 22 °C for 1–36 h prior to centrifugation. Plasma samples prepared 1 h after venipuncture were also subjected to two to eight cycles of freezing at −80 °C and thawing at 22 °C. Multiplex proximity extension assay, an antibody-based protein assay, was used to investigate the influence on plasma proteins. Results Up to 36 h delay before blood centrifugation resulted in significant increases of 16 and 40 out of 139 detectable proteins in samples kept at 4 °C or 22 °C, respectively. Some increases became noticeable after 8 h delay at 4 °C but already after 1 h at 22 °C. For samples stored at 4 °C, epidermal growth factor (EGF), NF-kappa-B essential modulator, SRC, interleukin 16 and CD6 increased the most, whereas the five most significantly increased proteins after storage at 22 °C were CD40 antigen ligand (CD40-L), EGF, platelet-derived growth factor subunit B, C-X-C motif chemokine ligand 5 and matrix metallopeptidase 1 (MMP1). Only matrix metallopeptidase 7 (MMP7) decreased significantly over time and only after storage at 22 °C. No protein levels were found to be significantly affected by up to eight freeze-thaw cycles. Conclusions Plasma should be prepared from blood after a limited precentrifugation delay at a refrigerated temperature. By contrast, the influence by several freeze-thaw cycles on detectable protein levels in plasma was negligible.

Extracellular vesicles (EVs) are continuously released by most cells, and they carry surface markers of their cells of origin. Found in all body fluids, EVs function as conveyers of cellular information, and evidence implicates them as markers of disease. These characteristics make EVs attractive diagnostic targets. However, detection and characterization of EVs is challenging due to their small size. We've established a method, called ExoPLA, that allows individual EVs to be detected and characterized at high specificity and sensitivity. Based on the in situ proximity ligation assay (in situ PLA), proximal oligonucleotide-conjugated antibodies bound to their targets on the surfaces of the EVs allow formation of circular products that can be fluorescently labeled by rolling circle amplification. The intense fluorescent signals produced in this assay allow detection and enumeration of individual EVs by flow cytometry. We describe the procedures for ExoPLA, along with expected results and troubleshooting. © 2017 by John Wiley & Sons, Inc. Keywords: ExoPLA; exosomes; extracellular vesicles; flow cytometry; proximity ligation assay

BACKGROUND Detecting proteins at low concentrations in plasma is crucial for early diagnosis. Current techniques in clinical routine, such as sandwich ELISA, provide sensitive protein detection because of a dependence on target recognition by pairs of antibodies, but detection of still lower protein concentrations is often called for. Proximity ligation assay with rolling circle amplification (PLARCA) is a modified proximity ligation assay (PLA) for analytically specific and sensitive protein detection via binding of target proteins by 3 antibodies, and signal amplification via rolling circle amplification (RCA) in microtiter wells, easily adapted to instrumentation in use in hospitals. METHODS Proteins captured by immobilized antibodies were detected using a pair of oligonucleotide-conjugated antibodies. Upon target recognition these PLA probes guided oligonucleotide ligation, followed by amplification via RCA of circular DNA strands that formed in the reaction. The RCA products were detected by horseradish peroxidase-labeled oligonucleotides to generate colorimetric reaction products with readout in an absorbance microplate reader. RESULTS We compared detection of interleukin (IL)-4, IL-6, IL-8, p53, and growth differentiation factor 15 (GDF-15) by PLARCA and conventional sandwich ELISA or immuno-RCA. PLARCA detected lower concentrations of proteins and exhibited a broader dynamic range compared to ELISA and iRCA using the same antibodies. IL-4 and IL-6 were detected in clinical samples at femtomolar concentrations, considerably lower than for ELISA. CONCLUSIONS PLARCA offers detection of lower protein levels and increased dynamic ranges compared to ELISA. The PLARCA procedure may be adapted to routine instrumentation available in hospitals and research laboratories.

An important motivation for the construction of biobanks is to discover biomarkers that identify diseases at early, potentially curable stages. This will require biobanks from large numbers of individuals, preferably sampled repeatedly, where the samples are collected and stored under conditions that preserve potential biomarkers. Dried blood samples are attractive for biobanking because of the ease and low cost of collection and storage. Here we have investigated their suitability for protein measurements. 92 proteins with relevance for oncology were analyzed using multiplex proximity extension assays (PEA) in dried blood spots collected on paper and stored for up to 30 years at either +4°C or -24°C. Our main findings were that 1) the act of drying only slightly influenced detection of blood proteins (average correlation of 0.970), and in a reproducible manner (correlation of 0.999), 2) detection of some proteins was not significantly affected by storage over the full range of three decades (34% and 76% of the analyzed proteins at +4°C and -24°C, respectively), while levels of others decreased slowly during storage with half-lives in the range of 10 to 50 years, and 3) detectability of proteins was less affected in dried samples stored at -24°C compared to at +4°C, as the median protein abundance had decreased to 80% and 93% of starting levels after 10 years of storage at +4°C or -24°C, respectively. The results of our study are encouraging as they suggest an inexpensive means to collect large numbers of blood samples, even by the donors themselves, and to transport, and store biobanked samples as spots of whole blood dried on paper. Combined with emerging means to measure hundreds or thousands of protein, such biobanks could prove of great medical value by greatly enhancing discovery as well as routine analysis of blood biomarkers.

The appearance of neurofibrillary tangles (NFT), one of the major hallmarks of Alzheimer's disease (AD), is most likely caused by inappropriate phosphorylation and/or dephosphorylation of tau, eventually leading to the accumulation of NFTs. Enhanced phosphorylation of tau on Ser(262) is detected early in the course of the disease and may have a role in the formation of tangles. Several kinases such as microtubule-affinity regulating kinase (MARK), protein kinase A, calcium calmodulin kinase II, and checkpoint kinase 2 are known to phosphorylate tau on Ser(262) in vitro. In this study, we took advantage of the in situ proximity ligation assay to investigate the role of MARK2, one of the four MARK isoforms, in AD. We demonstrate that MARK2 interacts with tau and phosphorylates tau at Ser(262) in stably transfected NIH/3T3 cells expressing human recombinant tau. Staurosporine, a protein kinase inhibitor, significantly reduced the interaction between MARK2 and tau, and also phosphorylation of tau at Ser(262). Furthermore, we observed elevated interactions between MARK2 and tau in post-mortem human AD brains, compared to samples from non-demented elderly controls. Our results from transfected cells demonstrate a specific interaction between MARK2 and tau, as well as MARK2-dependent phosphorylation of tau at Ser(262). Furthermore, the elevated interactions between MARK2 and tau in AD brain sections suggests that MARK2 may play an important role in early phosphorylation of tau in AD, possibly qualifying as a therapeutic target for intervention to prevent disease progression.

Recent studies indicate that the immune system adaptation during pregnancy could play a significant role in the pathophysiology of perinatal depression. The aim of this study was to investigate if inflammation markers in a late pregnancy plasma sample can predict the presence of depressive symptoms at eight weeks postpartum. Blood samples from 291 pregnant women (median and IQR for days to delivery, 13 and 7-23days respectively) comprising 63 individuals with postpartum depressive symptoms, as assessed by the Edinburgh postnatal depression scale (EPDS≥12) and/or the Mini International Neuropsychiatric Interview (M.I.N.I.) and 228 controls were analyzed with an inflammation protein panel using multiplex proximity extension assay technology, comprising of 92 inflammation-associated markers. A summary inflammation variable was also calculated. Logistic regression, LASSO and Elastic net analyses were implemented. Forty markers were lower in late pregnancy among women with depressive symptoms postpartum. The difference remained statistically significant for STAM-BP (or otherwise AMSH), AXIN-1, ADA, ST1A1 and IL-10, after Bonferroni correction. The summary inflammation variable was ranked as the second best variable, following personal history of depression, in predicting depressive symptoms postpartum. The protein-level findings for STAM-BP and ST1A1 were validated in relation to methylation status of loci in the respective genes in a different population, using openly available data. This explorative approach revealed differences in late pregnancy levels of inflammation markers between women presenting with depressive symptoms postpartum and controls, previously not described in the literature. Despite the fact that the results do not support the use of a single inflammation marker in late pregnancy for assessing risk of postpartum depression, the use of STAM-BP or the novel notion of a summary inflammation variable developed in this work might be used in combination with other biological markers in the future.

BACKGROUND: Antenatal depression and use of serotonin reuptake inhibitors (SSRI) in pregnancy have both been associated with an increased risk of poor pregnancy outcomes, such as preterm birth and impaired fetal growth. While the underlying biological pathways for these complications are poorly understood, it has been hypothesized that inflammation may be a common physiological pathway. The aim of the present study was to assess peripheral inflammatory markers in healthy women, women with antenatal depression, and in women using SSRI during pregnancy.METHODS: 160 healthy pregnant controls, 59 women with antenatal depression and 39 women on treatment with SSRIs were included. The relative levels of 92 inflammatory proteins were analyzed by proximity extension assay technology.RESULTS: Overall, 23 of the inflammatory markers were significantly lower in women with antenatal depression and in women on treatment with SSRIs in comparison with the healthy controls. No difference in any of the inflammatory markers was observed between women with antenatal depression and those who were using SSRI. Top three inflammatory markers that were down-regulated in women with antenatal depression were TNF-related apoptosis-inducing ligand (TRAIL), p=0.000001, macrophage colony-stimulating factor 1 (CSF-1), p=0.000004, and fractalkine (CX3CL1), p=0.000005. Corresponding inflammatory markers in SSRI users were CSF-1, p=0.000011, vascular endothelial growth factor A (VEGF-A), p=0.000016, and IL-15 receptor subunit alpha (IL-15RA), p=0.000027. The inflammatory markers were negatively correlated with cortisone serum concentrations in controls, but not in the cases. Differential DNA methylation of was found for seven of these inflammatory markers in an independent epigenetics cohort.CONCLUSION: Women with antenatal depression or on SSRI treatment have lower levels of a number of peripheral inflammatory markers than healthy pregnant controls. Hypothetically, this could be due to dysregulated switch to the pro-M2 milieu that characterizes normal third trimester pregnancy. However, longitudinal blood sampling is needed to elucidate whether the presumably dysregulated M2 shift is driving the development of antenatal depression or is a result of the depression.

The objective of this study was to develop and apply a novel multiplex panel of solid-phase proximity ligation assays (SP-PLA) requiring only 20 μL of samples, as a tool for discovering protein biomarkers for neurological disease and treatment thereof in cerebrospinal fluid (CSF). We applied the SP-PLA to samples from two sets of patients with poorly understood nervous system pathologies amyotrophic lateral sclerosis (ALS) and neuropathic pain, where patients were treated with spinal cord stimulation (SCS). Forty-seven inflammatory and neurotrophic proteins were measured in samples from 20 ALS patients and 15 neuropathic pain patients, and compared to normal concentrations in CSF from control individuals. Nineteen of the 47 proteins were detectable in more than 95% of the 72 controls. None of the 21 proteins detectable in CSF from neuropathic pain patients were significantly altered by SCS. The levels of the three proteins, follistatin, interleukin-1 alpha, and kallikrein-5 were all significantly reduced in the ALS group compared to age-matched controls. These results demonstrate the utility of purpose designed multiplex SP-PLA panels in CSF biomarker research for understanding neuropathological and neurotherapeutic mechanisms. The protein changes found in the CSF of ALS patients may be of diagnostic interest.

Background The diagnosis of malignant melanoma currently relies on clinical inspection of the skin surface and on the histopathological status of the excised tumor. The serum marker S100B is used for prognostic estimates at later stages of the disease, but analyses are marred by false positives and inadequate sensitivity in predicting relapsing disorder. Objectives To investigate SOX10 as a potential biomarker for melanoma and vitiligo. Methods In this study we have applied proximity ligation assay (PLA) to detect the transcription factor SOX10 as a possible serum marker for melanoma. We studied a cohort of 110 melanoma patients. We further investigated a second cohort of 85 patients with vitiligo, which is a disease that also affects melanocytes. Results The specificity of the SOX10 assay in serum was high, with only 1% of healthy blood donors being positive. In contrast, elevated serum SOX10 was found with high frequency among vitiligo and melanoma patients. In patients with metastases, lack of SOX10 detection was associated with treatment benefit. In two responding patients, a change from SOX10 positivity to undetectable levels was seen before the response was evident clinically. Conclusions We show for the first time that SOX10 represents a promising new serum melanoma marker for detection of early stage disease, complementing the established S100B marker. Our findings imply that SOX10 can be used to monitor responses to treatment and to assess if the treatment is of benefit at stages earlier than what is possible radiologically.

Despite great interest, investments, and efforts, the ongoing search for plasma protein biomarkers for disease so far has come up surprisingly empty-handed. Although discovery programs have revealed large numbers of biomarker candidates, the clinical utility has been validated for only a very small number of these. While this disappointing state of affairs may suggest that plasma protein biomarkers have little more to offer for diagnostics, we take the perspective that experimental conditions might not have been optimal and that analyses will be required that offer far greater sensitivity than currently available, in terms of numbers of molecules needed for unambiguous detection. Accordingly, techniques are needed to search deep and wide for protein biomarker candidates. The requirements and feasibility of such assays will be discussed.

Prostasomes are microvesicles (mean diameter, 150 nm) that are produced and secreted by normal and malignant prostate acinar cells. It has been hypothesized that invasive growth of malignant prostate cells may cause these microvesicles, normally released into seminal fluid, to appear in interstitial space and therewith into peripheral circulation. The suitability of prostasomes as blood biomarkers in patients with prostate cancer was tested by using an expanded variant of the proximity ligation assay (PLA). We developed an extremely sensitive and specific assay (4PLA) for detection of complex target structures such as microvesicles in which the target is first captured via an immobilized antibody and subsequently detected by using four other antibodies with attached DNA strands. The requirement for coincident binding by five antibodies to generate an amplifiable reporter results in both increased specificity and sensitivity. The assay successfully detected significantly elevated levels of prostasomes in blood samples from patients with prostate cancer before radical prostatectomy, compared with controls and men with benign biopsy results. The medians for prostasome levels in blood plasma of patients with prostate cancer were 2.5 to sevenfold higher compared with control samples in two independent studies, and the assay also distinguished patients with high and medium prostatectomy Gleason scores (8/9 and 7, respectively) from those with low score (≤6), thus reflecting disease aggressiveness. This approach that enables detection of prostasomes in peripheral blood may be useful for early diagnosis and assessment of prognosis in organ-confined prostate cancer.

The RAS-MAPK syndromes are a group of clinically and genetically related disorders caused by dysregulation of the RAS-MAPK pathway. A member of this group of disorders, Noonan syndrome (NS), is associated with several different genes within the RAS-MAPK pathway. To date, mutations in PTPN11, SOS1, KRAS, RAF1 and SHOC2 are known to cause NS and a small group of patients harbour mutations in BRAF, MEK1 or NRAS. The majority of the mutations are predicted to cause an up-regulation of the pathway; hence they are gain-of-function mutations. Despite recent advances in gene identification in NS, the genetic aetiology is still unknown in about ¼ of patients. To investigate the contribution of gene dosage imbalances of RAS-MAPK-related genes to the pathogenesis of NS, a multiplex ligation-dependent probe amplification (MLPA) assay was developed. Two probe sets were designed for seven RAS-MAPK-syndrome-related candidate genes: PTPN11, SOS1, RAF1, KRAS, BRAF, MEK1 and MEK2. The probe sets were validated in 15 healthy control individuals and in glioma tumour cell lines. Subsequently, 44 NS patients negative for mutations in known NS-associated genes were screened using the two probe sets. The MLPA results for the patients revealed no gene dosage imbalances. In conclusion, the present results exclude copy number variation of PTPN11, SOS1, RAF1, KRAS, BRAF, MEK1 and MEK2 as a common pathogenic mechanism of NS. The validated and optimised RAS-MAPK probe sets presented here enable rapid high throughput screening of further patients with RAS-MAPK syndromes.

Cytokines and chemokines regulate many functions in the body including the brain. The interactions between adipose tissue and the central nervous system (CNS) are important for the regulation of energy balance. CNS function is also influenced by age. The aim of the present study was to investigate the effects of body mass index (BMI) and age on cytokine and chemokine levels in cerebrospinal fluid. Cerebrospinal fluid samples (n=89) were collected from patients undergoing routine surgical procedures. The samples were analyzed using the multiplex proximity extension assay (PEA) in which 92 different cytokines are measured simultaneously using minute sample volume. We found no significant correlations between age and cytokine levels for any of the studied markers. In contrast, at a false discovery rate of 10%, 19 markers were significantly associated with BMI (in decreasing significance: FGF-5, ADA, Beta-NGF, CD40, IL-10RB, CCL19, TGF-alpha, SIRT2, TWEAK, SCF, CSF-1, 4E-BP1, DNER, LIF-R, STAMPB, CXCL10, CXCL6, VEGF-A and CX3CL1). This study reveals a clear effect of BMI on cytokine and chemokine levels in cerebrospinal fluid.

Glutamic acid decarboxylase 65 (GAD65) and autoantibodies specific for GAD65 (GADA) are associated with autoimmune diseases including Stiff Person Syndrome (SPS) and Type 1 diabetes (T1D). GADA is recognized as a biomarker of value for clinical diagnosis and prognostication in these diseases. Nonetheless, it remains medically interesting to develop sensitive and specific assays to detect GAD65 preceding GADA emergence and to monitor GADA-GAD65 immune complexes in blood samples. In the present study, we developed a highly sensitive proximity ligation assay to measure serum GAD65. This novel assay allowed detection of as little as 0.65 pg/ml GAD65. We were also able to detect immune complexes involving GAD65 and GADA. Both free GAD65 and GAD65-GADA levels were significantly higher in serum samples from SPS patients compared to healthy controls. The proximity ligation assays applied for detection of GAD65 and its immune complexes may thus enable improved diagnosis and better understanding of SPS.

The quality of DNA-labeled affinity probes is critical in DNA-assisted protein analyses, such as proximity ligation and extension assays, immuno-PCR, and immuno-rolling circle amplification reactions. Efficient, high-performance methods are therefore required for isolation of pure conjugates from reactions where DNA strands have been coupled to antibodies or recombinant affinity reagents. Here we describe a universal, scalable approach for preparing high-quality oligonucleotide-protein conjugates by sequentially removing any unconjugated affinity reagents and remaining free oligonucleotides from conjugation reactions. We applied the approach to generate high-quality probes using either antibodies or recombinant affinity reagents. The purified high-grade probes were used in proximity ligation assays in solution and in situ, demonstrating both augmented assay sensitivity and improved signal-to-noise ratios.

The ability to detect minute amounts of specific proteins or protein modifications in blood as biomarkers for a plethora of human pathological conditions holds great promise for future medicine. Despite a large number of plausible candidate protein biomarkers published annually, the translation to clinical use is impeded by factors such as the required size of the initial studies, and limitations of the technologies used. The proximity ligation assay (PLA) is a versatile molecular tool that has the potential to address some obstacles, both in validation of biomarkers previously discovered using other techniques, and for future routine clinical diagnostic needs. The enhanced specificity of PLA extends the opportunities for large-scale, high-performance analyses of proteins. Besides advantages in the form of minimal sample consumption and an extended dynamic range, the PLA technique allows flexible assay reconfiguration. The technology can be adapted for detecting protein complexes, proximity between proteins in extracellular vesicles or in circulating tumor cells, and to address multiple post-translational modifications in the same protein molecule. We discuss herein requirements for biomarker validation, and how PLA may play an increasing role in this regard. We describe some recent developments of the technology, including proximity extension assays, the use of recombinant affinity reagents suitable for use in proximity assays, and the potential for single cell proteomics. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.

While antibodies currently play a dominant role as affinity reagents in biological research and for diagnostics, a broad range of recombinant proteins are emerging as promising alternative affinity reagents in detection assays and quantification. DNA-mediated affinity-based assays, such as immuno-PCR and proximity ligation assays (PLA), use oligonucleotides attached to affinity reagents as reporter molecules. Conjugation of oligonucleotides to affinity reagents generally employs chemistries that target primary amines or cysteines. Because of the random nature of these processes neither the number of oligonucleotides conjugated per molecule nor their sites of attachment can be accurately controlled for affinity reagents with several available amines and cysteines. Here, we present a straightforward and convenient approach to functionalize recombinant affinity reagents for PLA by expressing the reagents as fusion partners with SNAP protein tags. This allowed us to conjugate oligonucleotides in a site-specific fashion, yielding precisely one oligonucleotide per affinity reagent. We demonstrate this method using designed ankyrin repeat proteins (DARPins) recognizing the tumor antigen HER2 and we apply the conjugates in different assay formats. We also show that SNAP or CLIP tags, expressed as fusion partners of transfected genes, allow oligonucleotide conjugations to be performed in fixed cells, with no need for specific affinity reagents. The approach is used to demonstrate induced interactions between the fusion proteins FKBP and FRB by allowing the in situ conjugated oligonucleotides to direct the production of templates for localized rolling circle amplification reactions.

The microtubule-affinity regulating kinase (MARK) family consists of four highly conserved members that have been implicated in phosphorylation of tau protein, causing formation of neurofibrillary tangles in Alzheimer's disease (AD). Understanding of roles by individual MARK isoform in phosphorylating tau has been limited due to lack of antibodies selective for each MARK isoform. In this study, we first applied the proximity ligation assay on cells to select antibodies specific for each MARK isoform. In cells, a CagA peptide specifically and significantly inhibited tau phosphorylation at Ser²⁶² mediated by MARK4 but not other MARK isoforms. We then used these antibodies to study expression levels of MARK isoforms and interactions between tau and individual MARK isoforms in postmortem human brains. We found a strong and significant elevation of MARK4 expression and MARK4-tau interactions in AD brains, correlating with the Braak stages of the disease. These results suggest the MARK4-tau interactions are of functional importance in the progression of AD and the results also identify MARK4 as a promising target for AD therapy.

The transmembrane protease complex γ-secretase is responsible for the generation of the neurotoxic amyloid β-peptide (Aβ) from its precursor (APP). Aβ has a causative role in Alzheimer disease, and thus, γ-secretase is a therapeutic target. However, since there are more than 70 γ-secretase substrates besides APP, selective inhibition of APP processing is required. Recent data indicates the existence of several γ-secretase associated proteins (GSAPs) that affect the selection and processing of substrates. Here, we use a γ-secretase inhibitor for affinity purification of γ-secretase and associated proteins from microsomes and detergent resistant membranes (DRMs) prepared from rat or human brain. By tandem mass spectrometry we identified a novel brain GSAP; erlin-2. This protein was recently reported to reside in DRMs in the ER. A proximity ligation assay, as well as co-immunoprecipitation, confirmed the association of erlin-2 with γ-secretase. We found that a higher proportion of erlin-2 was associated with γ-secretase in DRMs than in soluble membranes. siRNA experiments indicated that reduced levels of erlin-2 resulted in a decreased Aβ production, whereas the effect on Notch processing was limited. In summary, we have found a novel brain GSAP, erlin-2, that resides in DRMs and affects Aβ production.

An essential skill for every researcher is to learn how to select and apply the most appropriate methods for the questions they are trying to answer. With the extensive variety of methods available, it is increasingly important to scrutinize the advantages and disadvantages of these techniques prior to making a decision on which to use. In this article, we describe an approach to evaluate methods by reducing them into subcomponents. This is exemplified by a brief description of some commonly used proteomics methods. The same approach can also be used in method development by rearranging subcomponents in order to create new methods, as demonstrated with the development of proximity ligation assays (PLAs). PLA is a method as designed in our laboratory for detection of proteins, protein-protein interactions and post-translational modifications. Fundamentally, protein-recognition events are converted into detectable DNA molecules. The technique uses protein-DNA conjugates as binders for the targets of interest. Binding of two or more conjugates to the target results in assembly of an assay-specific DNA molecule. Subsequent amplification of the DNA molecule generates a signal that can be detected using PCR, for detection of minute amounts of proteins in serum, or standard fluorescence microscopy for detection of protein-protein interactions in tissue sections. Lastly, we apply the approach of recombining subcomponents to develop a few novel hypothetical methods hoping this might stimulate the readers to utilize this approach themselves.

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.

Knowledge about the total human genome sequence now provides opportunities to study its myriad gene products. However, the presence of alternative splicing, post-translational modifications, and innumerable protein-protein interactions among proteins occurring at widely different concentrations, all combine to place extreme demands on the specificity and sensitivity of assays. The choice of method also depends on matters such as whether proteins will be analyzed in body fluids and lysates, or localized inside single cells. In this review we discuss commonly used detection methods and compare these to the recently-developed proximity ligation technique.

Despite intense interest, methods that provide enhanced sensitivity and specificity in parallel measurements of candidate protein biomarkers in numerous samples have been lacking. We present herein a multiplex proximity ligation assay with readout via realtime PCR or DNA sequencing (ProteinSeq). We demonstrate improved sensitivity over conventional sandwich assays for simultaneous analysis of sets of 35 proteins in 5 µl of blood plasma. Importantly, we observe a minimal tendency to increased background with multiplexing, compared to a sandwich assay, suggesting that higher levels of multiplexing are possible. We used ProteinSeq to analyze proteins in plasma samples from cardiovascular disease (CVD) patient cohorts and matched controls. Three proteins, namely P-selectin, Cystatin-B and Kallikrein-6, were identified as putative diagnostic biomarkers for CVD. The latter two have not been previously reported in the literature and their potential roles must be validated in larger patient cohorts. We conclude that ProteinSeq is promising for screening large numbers of proteins and samples while the technology can provide a much-needed platform for validation of diagnostic markers in biobank samples and in clinical use.

Background Protein aggregation plays important roles in several neurodegenerative disorders. For instance, insoluble aggregates of phosphorylated tau and of Aβ peptides are cornerstones in the pathology of Alzheimer's disease. Soluble protein aggregates are therefore potential diagnostic and prognostic biomarkers for their cognate disorders. Detection of the aggregated species requires sensitive tools that efficiently discriminate them from monomers of the same proteins. Here we have established a proximity ligation assay (PLA) for specific and sensitive detection of Aβ protofibrils via simultaneous recognition of three identical determinants present in the aggregates. PLA is a versatile technology in which the requirement for multiple target recognitions is combined with the ability to translate signals from detected target molecules to amplifiable DNA strands, providing very high specificity and sensitivity. Results For specific detection of Aβ protofibrils we have used a monoclonal antibody, mAb158, selective for Aβ protofibrils in a modified PLA, where the same monoclonal antibody was used for the three classes of affinity reagents required in the assay. These reagents were used for detection of soluble Aβ aggregates in solid-phase reactions, allowing detection of just 0.1 pg/ml Aβ protofibrils, and with a dynamic range greater than six orders of magnitude. Compared to a sandwich ELISA setup of the same antibody the PLA increases the sensitivity of the Aβ protofibril detection by up to 25-fold. The assay was used to measure soluble Aβ aggregates in brain homogenates from mice transgenic for a human allele predisposing to Aβ aggregation. Conclusions The proximity ligation assay is a versatile analytical technology for proteins, which can provide highly sensitive and specific detection of Aβ aggregates - and by implication other protein aggregates of relevance in Alzheimer's disease and other neurodegenerative disorders.

Background: Tyrosine phosphorylation (pTyr) is an important cancer relevant posttranslational modification since it regulates protein activity and cellular localization. By controlling cell growth and differentiation it plays an important role in tumor development. This paper describes a novel approach for detection and visualization of a panel of pTyr proteins in tumors using in situ proximity ligation assay. Methods: K562 leukemia cells were treated with tyrosine kinase and/or phosphatase inhibitors to induce differences in pTyr levels and mimic cells with different malignant properties. Cells were then probed with one antibody against the pTyr modification and another probe against the detected protein, resulting in a detectable fluorescent signal once the probes were in proximity. Results: Total and protein specific pTyr levels on ABL, SHC, ERK2 and PI3K proteins were detected and samples of control and treated cells were distinguished at the pTyr level using this novel approach. Promising results were also detected for formalin fixed and paraffin embedded cells in the micro array format. Conclusions: This application of in situ proximity ligation assay is valuable in order to study the pTyr modification of a panel of proteins in large data sets to validate mass spectrometric data and to be combined with tissue microarrays. The approach offers new opportunities to reveal the pTyr signatures in cells of different malignant properties that can be used as biomarker of disease in the future.