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Abstract Using a modified proximity extension assay, total and immunoglobulin (Ig) class-specific anti-SARS-CoV-2 antibodies were sensitively and conveniently detected directly from ø1.2 mm discs cut from dried blood and saliva spots (DBS and DSS) without the need for elution. For total Ig detection, antigen probes were prepared by conjugating recombinant spike protein subunit 1 (S1-RBD) to a pair of oligonucleotides. To detect isotype-specific antibody reactivity, one antigen probe was replaced with oligonucleotide-conjugated antibodies specific for antibody isotypes. Binding of pairs of oligonucleotide-conjugated probes to antibodies in patient samples brings oligonucleotides in proximity. An added DNA polymerase uses a transient hybridization between the oligonucleotides to prime synthesis of a DNA strand, which serves as a DNA amplicon that is quantified by real-time PCR. The S1-RBD-specific IgG, IgM, and IgA antibodies in DBS samples collected over the course of a first and second vaccination exhibited kinetics consistent with previous reports. Both DBS and DSS collected from 42 individuals in the autumn of 2023 showed significant level of total S1-RBD antibodies with a correlation of R = 0.70. However, levels in DSS were generally 10 to 100-fold lower than in DBS. Anti-S1-RBD IgG and IgA in DSS demonstrated a correlation of R = 0.6.

Abstract Renowned for their role in haemostasis and thrombosis, platelets are also increasingly recognized for their contribution in innate immunity, immunothrombosis and inflammatory diseases. Platelets express a wide range of receptors, which allows them to reach a variety of activation endpoints and grants them immunomodulatory functions. Activated platelets release extracellular vesicles (PEVs), whose formation and molecular cargo has been shown to depend on receptor‐mediated activation and environmental cues. This study compared the immunomodulatory profiles of PEVs generated via activation of platelets by different receptors, glycoprotein VI, C‐type lectin‐like receptor 2 and combining all thrombin‐collagen receptors. Functional assays in vivo in zebrafish and in vitro in human macrophages highlighted distinct homing and secretory responses triggered by the PEVs. In contrast, omics analyses of protein and miRNA cargo combined with physicochemical particle characterization found only subtle differences between the activated PEV types, which were insufficient to predict their different immunomodulatory functions. In contrast, constitutively released PEVs, formed in the absence of an exogenous activator, displayed a distinct immunomodulatory profile from the receptor‐induced PEVs. Our findings underscore that PEVs are tunable through receptor‐mediated activation. To truly comprehend their role(s) in mediating platelet functions among immune cells, conducting functional assays is imperative.

Extracellular vesicles (EVs), whose main subtypes are exosomes, microparticles, and apoptotic bodies, are secreted by all cells and harbor biomolecules such as DNA, RNA, and proteins. They function as intercellular messengers and, depending on their cargo, may have multiple roles in cancer development. Thymidine kinase 1 (TK1) is a cell cycle-dependent enzyme used as a biomarker for cell proliferation. TK1 is usually elevated in cancer patients' serum, making the enzyme a valuable tumor proliferation biomarker that strongly correlates with cancer stage and metastatic capabilities. Here, we investigated the presence of TK1 in EVs derived from three prostate cancer cell lines with various p53 mutation statuses (LNCaP, PC3, and DU145), EVs from the normal prostate epithelial cell line RWPE-1 and EVs isolated from human seminal fluid (prostasomes). We measured the TK1 activity by a real-time assay for these EVs. We demonstrated that the TK1 enzyme activity is higher in EVs derived from the malignant cell lines, with the highest activity from cells deriving from the most aggressive cancer, compared to the prostasomes and RWPE-1 EVs. The measurement of TK1 activity in EVs may be essential in future prostate cancer studies.

Summary: Background: γ-aminobutyric acid (GABA), known as the main inhibitory neurotransmitter in the brain, exerts immunomodulatory functions by interaction with immune cells, including T cells. Metabolic programs of T cells are closely linked to their effector functions including proliferation, differentiation, and cytokine production. The physiological molecules glucose and insulin may provide environmental cues and guidance, but whether they coordinate to regulate GABA-mediated T cell immunomodulation is still being examined. Methods: CD4+ T cells that were isolated from blood samples from healthy individuals and from patients with type 1 diabetes (T1D) were activated in vitro. We carried out metabolic assays, multiple proximity extension assay (PEA), ELISA, qPCR, immunoblotting, immunofluorescence staining, flow cytometry analysis, MS-based proteomics, as well as electrophysiology and live-cell Ca2+ imaging. Findings: We demonstrate that GABA-mediated reduction of metabolic activity and the release of inflammatory proteins, including IFNγ and IL-10, were abolished in human CD4+ T cells from healthy individuals and patients with T1D when the glucose concentration was elevated above levels typically observed in healthy people. Insulin increased GABAA receptor-subunit ρ2 expression, enhanced the GABAA receptors-mediated currents and Ca2+ influx. GABA decreased, whereas insulin sustained, hexokinase activity and glycolysis in a glucose concentration-dependent manner. Interpretation: These findings support that metabolic factors, such as glucose and insulin, influence the GABA-mediated immunomodulation of human primary T cells effector functions. Funding: The Swedish Children’s Diabetes Foundation, The Swedish Diabetes Foundation, The Swedish Research Council 2018-02952, EXODIAB, The Ernfors Foundation, The Thurings Foundation and the Science for Life Laboratory.

Surface biotinylation, proximity ligation assays and proteomics are used to identify surface markers of prostate-derived small extracellular vesicles.

Platelet extracellular vesicles (PEVs) generated upon platelet activation may play a role in inflammatory pathologies such as atherosclerosis. Oxidized low-density lipoprotein (oxLDL), a well-known contributor to atherogenesis, activates platelets and presensitizes them for activation by other agonists. We studied the effect of oxLDL on the secretion, composition, and inflammatory functions of PEVs using contemporary EV analytics. Platelets were activated by co-stimulation with thrombin (T) and collagen (C) ± oxLDL and characterized by high-resolution flow cytometry, nanoparticle tracking analysis, proximity extension assay, western blot, and electron microscopy. The effect of PEVs on macrophage differentiation and functionality was examined by analyzing macrophage surface markers, cytokine secretion, and transcriptome. OxLDL upregulated TC-induced formation of CD61+, P-selectin+ and phosphatidylserine+ PEVs. Blocking the scavenger receptor CD36 significantly suppressed the oxLDL+TC-induced PEV formation, and HDL caused a slight but detectable suppression. The inflammatory protein cargo differed between the PEVs from stimulated and unstimulated platelets. Both oxLDL+TC- and TC-induced PEVs enhanced macrophage HLA-DR and CD86 expression and decreased CD11c expression as well as secretion of several cytokines. Pathways related to cell cycle and regulation of gene expression, and immune system signaling were overrepresented in the differentially expressed genes between TC PEV -treated vs. control macrophages and oxLDL+TC PEV -treated vs. control macrophages, respectively. In conclusion, we speculate that oxLDL and activated platelets contribute to proatherogenic processes by increasing the number of PEVs that provide an adhesive and procoagulant surface, contain inflammatory mediators, and subtly finetune the macrophage gene expression.

ABSTRACT Infectious bronchitis virus (IBV) is a coronavirus responsible for major health problems in the poultry industry. New virus strains continue to appear, causing large economic losses. To develop a rapid and accurate new quantitative assay for diagnosis of the virus without DNA extraction, we selected highly specific single-stranded DNA (ssDNA) aptamers with a high affinity to IBV, using the systematic evolution of ligands by exponential enrichment (SELEX) technology for aptamer screening, followed by high-throughput sequencing technology. Two of these aptamers, AptIBV5 and AptIBV2, were used to establish homogenous and solid-phase proximity ligation assays (PLAs). The developed assays were evaluated for their sensitivity and specificity using collected field samples and then compared to the newly developed sandwich enzyme-linked aptamer assay (ELAA) and reverse transcription-quantitative PCR (qRT-PCR), as the gold-standard method. The solid-phase PLA showed a lower limit of detection and a broader dynamic range than the two other assays. The developed technique may serve as an alternative assay for the diagnosis of IBV, with the potential to be extended to the detection of other important animal or human viruses. IMPORTANCE Infectious bronchitis virus (IBV) causes high morbidity and mortality and large economic losses in the poultry industry. The virus has the ability to genetically mutate into new IBV strains, causing devastating disease and outbreaks. To better monitor the emergence of this virus, the development of a rapid and highly sensitive diagnostic method should be implemented. For this, we generated aptamers with high affinity and specificity to the IBV in an ssDNA library. Using two high-affinity aptamers, we developed a sandwich ELAA and a very sensitive aptamer-based proximity ligation assay (PLA). The new assay showed high sensitivity and specificity and was used to detect IBV in farm samples. The PLA was compared to the newly developed sandwich ELAA and qRT-PCR, as the gold-standard technique.

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.

Detecting viral antigens at low concentrations in field samples can be crucial for early veterinary diagnostics. Proximity ligation assays (PLAs) in both solution and solid‐phase formats are widely used for high‐performance protein detection in medical research. However, the affinity reagents used, which are mainly poly‐ and monoclonal antibodies, play an important role in the performance of PLAs. Here, we have established the first homogeneous and solid‐phase proximity‐dependent DNA aptamer ligation assays for rapid and accurate detection of Newcastle disease virus (NDV). NDV is detected by a pair of extended DNA aptamers that, upon binding in proximity to proteins on the envelope of the virus, are joined by enzymatic ligation to form a unique amplicon that can be sensitively detected using real‐time PCR. The sensitivity, specificity, and reproducibility of the assays were validated using 40 farm samples. The results demonstrated that the developed homogeneous and solid‐phase PLAs, which use NDV‐selective DNA aptamers, are more sensitive than the sandwich enzymatic‐linked aptamer assay (ELAA), and have a comparable sensitivity to real‐time reverse transcription PCR (rRT‐PCR) as the gold standard detection method. In addition, the solid‐phase PLA was shown to have a greater dynamic range with improved lower limit of detection, upper‐ and lower limit of quantification, and minimal detectable dose as compared with those of ELAA and rRT‐PCR. The specificity of PLA is shown to be concordant with rRT‐PCR.

Abstract Background Cardiovascular toxicity is a notorious complication of doxorubicin (DXR) therapy for diffuse large B-cell lymphoma (DLBCL). Although surveillance of well-known biological markers for cardiovascular disease (CVD) as NTproBNP and Troponins may be helpful, there are no established markers to monitor for evolving CVD during treatment. New possibilities have arisen with the emergence of newer techniques allowing for analysis of plasma proteins that can be associated with cardiovascular disease. Proximity Extension Assay is one of them. Objectives We aimed to illustrate the incidence of CVD in DLBCL patients treated with DXR and to establish whether there are plasma proteins associated with pre-existing or emerging CVD. Methods In 95 patients, 182 different proteins from OLINK panels, NTproBNP, Troponin I and CRP were assessed prior to, during and after treatment. For comparison, samples from controls were analyzed. Results In the DLBCL cohort, 33.3% had pre-treatment CVD compared to 5.0% in the controls and 23.2% developed new CVD. Of the 32.6% who died during follow up, CVD was the cause in 4 patients. Spondin-1 (SPON-1) correlated to pre-treatment CVD (1.22 fold change, 95% CI 1.10–1.35, p = 0.00025, q = 0.045). Interleukin-1 receptor type 1 (IL-1RT1) was associated to emerging CVD (1.24 fold change, 95% CI 1.10–1.39, p = 0.00044, q = 0.082). Conclusion We observed a higher prevalence of CVD in DLBCL patients compared to controls prior to DXR therapy. Two proteins, SPON-1 and IL-1RT1, were related to pre-existing and emerging CVD in DXR treated patients. If confirmed in larger cohorts, IL-1RT1 may emerge as a reliable biomarker for unfolding CVD in DLBCL.

Adenine nucleotide translocases (ANTs) and uncoupling proteins (UCPs) are known to facilitate proton leak across the inner mitochondrial membrane. However, it remains to be unravelled whether UCP2/3 contribute to significant amount of proton leak in vivo. Reports are indicative of UCP2 dependent proton-coupled efflux of C4 metabolites from the mitochondrial matrix. Previous studies have suggested that UCP2/3 knockdown (KD) contributes to increased ANT-dependent proton leak. Here we investigated the hypothesis that interaction exists between the UCP2 and ANT2 proteins, and that such interaction is regulated by the cellular metabolic demand. Protein-protein interaction was evaluated using reciprocal co-immunoprecipitation and in situ proximity ligation assay. KD of ANT2 and UCP2 was performed by siRNA in human embryonic kidney cells 293A (HEK293A) cells. Mitochondrial and cellular respiration was measured by high-resolution respirometry. ANT2-UCP2 interaction was demonstrated, and this was dependent on cellular metabolism. Inhibition of ATP synthase promoted ANT2-UCP2 interaction whereas high cellular respiration, induced by adding the mitochondrial uncoupler FCCP, prevented interaction. UCP2 KD contributed to increased carboxyatractyloside (CATR) sensitive proton leak, whereas ANT2 and UCP2 double KD reduced CATR sensitive proton leak, compared to UCP2 KD. Furthermore, proton leak was reduced in double KD compared to UCP2 KD. In conclusion, our results show that there is an interaction between ANT2-UCP2, which appears to be dynamically regulated by mitochondrial respiratory activity. This may have implications in the regulation of mitochondrial efficiency or cellular substrate utilization as increased activity of UCP2 may promote a switch from glucose to fatty acid metabolism.

Abstract 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.

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.

Abstract Background The overall prognosis of non-small cell lung cancer (NSCLC) is poor, and currently only patients with localized disease are potentially curable. Therefore, preferably non-invasively determined biomarkers that detect NSCLC patients at early stages of the disease are of high clinical relevance. The aim of this study was to identify and validate novel protein markers in plasma using the highly sensitive DNA-assisted multiplex proximity extension assay (PEA) to discriminate NSCLC from other lung diseases. Methods Plasma samples were collected from a total of 343 patients who underwent surgical resection for different lung diseases, including 144 patients with lung adenocarcinoma (LAC), 68 patients with non-malignant lung disease, 83 patients with lung metastasis of colorectal cancers and 48 patients with typical carcinoid. One microliter of plasma was analyzed using PEA, allowing detection and quantification of 92 established cancer related proteins. The concentrations of the plasma proteins were compared between disease groups. Results The comparison between LAC and benign samples revealed significantly different plasma levels for four proteins; CXCL17, CEACAM5, VEGFR2 and ERBB3 (adjusted p-value

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 tumour 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 analysed by univariate analysis. Elastic-net logistic regression was performed to select serum proteins into the diagnostic model. Findings: We identified 19 serum proteins (CEACAM5, 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 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 an increased diagnostic capacity in patients at TNM I-II stage (sensitivity 89%, specificity 100%, AUC 0·99) and in patients with high microsatellite instability (MSI) (91%, 98%, and 0·99) compared to individual proteins. These promising results will inspire a large-scale independent cohort study to be pursued 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, especially for patients at early stage and those with high MSI. Fund: This study was supported by a European H2020-Marie Skłodowska-Curie Innovative Training Networks grant (316,929, GastricGlycoExplorer). Funder had no influence on trial design, data evaluation, and interpretation. Keywords: Gastric cancer, Diagnosis, Biomarker, PEA, Proteomics

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.

Aptamers are short single-stranded DNA (ssDNA), RNA or synthetic XNA molecules, which are used as a class of affinity binders recognizing target molecules with a very high affinity and specificity. The aim of this study was to generate and characterize ssDNA aptamers for the detection of Newcastle disease virus (NDV). These aptamers were selected using systematic evolution of ligands by exponential enrichment (SELEX) in combination with quantitative high-throughput DNA sequencing. After three rounds of selections, a highly enriched ssDNA pool was sequenced, and the results were analyzed using FASTAptamer Toolkit. Sequencing reads were sorted by copy numbers and clustered into groups, according to their sequence homology. Top aptameric sequences were used to develop a sandwich enzymatic linked aptamer assay (ELAA) for rapid and sensitive detection of NDV in farm samples. The selected aptamers have an affinity within the nanomolar range, and a high specificity with no cross-reactivity towards other avian viruses. Following optimization of the sandwich ELAA method, the results demonstrated that both selected aptamers Apt_NDV01 and Apt_NDV03 with dissociation constant values of 31 nM and 78.1 nM, respectively, showed the highest specificity and affinity for NDV detection. The ELAA results were verified by quantitative real-time PCR, demonstrating strong concordance, and showing outstanding accuracy for detection of NDV in field sample. In summary, combination of SELEX with high-throughput DNA sequencing allowed rapid screening and selection of aptamers. The selected aptamers allowed recognition of NDV with high affinities. This is the first report that uses a validated sandwich ELAA for rapid and specific detection of NDV in poultry samples.

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.

BACKGROUND: Due to difficulties in predicting recurrences in colorectal cancer stages II and III, reliable prognostic biomarkers could be a breakthrough for individualized treatment and follow-up. OBJECTIVE: To find potential prognostic protein biomarkers in colorectal cancer, using the proximity extension assays. METHODS: A panel of 92 oncology-related proteins was analyzed with proximity extension assays, in plasma from a cohort of 261 colorectal cancer patients with stage II-IV. The survival analyses were corrected for disease stage and age, and the recurrence analyses were corrected for disease stage. The significance threshold was adjusted for multiple comparisons. RESULTS: The plasma proteins expression levels had a greater prognostic relevance in disease stage III colorectal cancer than in disease stage II, and for overall survival than for time to recurrence. Osteoprotegerin was the only biomarker candidate in the protein panel that had a statistical significant association with overall survival (P = .00029). None of the proteins were statistically significantly associated with time to recurrence. CONCLUSIONS: Of the 92 analyzed plasma proteins, osteoprotegerin showed the strongest prognostic impact in patients with colorectal cancer, and therefore osteoprotegerin is a potential predictive marker, and it also could be a target for treatments.

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. Keywords: PBMCs, Immune cells, Proliferation, Cytokine, GABAA receptor, Diabetes, T1D, Autoimmune disease, T cell

Abstract Detection and quantification of proteins and their post-translational modifications are crucial to decipher functions of complex protein networks in cell biology and medicine. Capillary isoelectric focusing together with antibody-based detection can resolve and identify proteins and their isoforms with modest sample input. However, insufficient sensitivity prevents detection of proteins present at low concentrations and antibody cross-reactivity results in unspecific detection that cannot be distinguished from bona fide protein isoforms. By using DNA-conjugated antibodies enhanced signals can be obtained via rolling circle amplification (RCA). Both sensitivity and specificity can be greatly improved in assays dependent on target recognition by pairs of antibodies using in situ proximity ligation assays (PLA). Here we applied these DNA-assisted RCA techniques in capillary isoelectric focusing to resolve endogenous signaling transducers and isoforms along vascular endothelial growth factor (VEGF) signaling pathways at concentrations too low to be detected in standard assays. We also demonstrate background rejection and enhanced specificity when protein detection depended on binding by pairs of antibodies using in situ PLA, compared to assays where each antibody preparation was used on its own.

Abstract Chronic myeloid leukemia (CML) is characterized in the majority of cases by a t(9;22)(q34;q11) translocation, also called the Philadelphia chromosome, giving rise to the BCR-ABL1 fusion protein. Current treatment with tyrosine kinase inhibitors is directed against the constitutively active ABL1 domain of the fusion protein, and minimal residual disease (MRD) after therapy is monitored by real-time quantitative PCR (RQ-PCR) of the fusion transcript. Here, we describe a novel approach to detect and enumerate cells positive for the BCR-ABL1 fusion protein by combining the in situ proximity ligation assay with flow cytometry as readout (PLA-flow). By targeting of the BCR and ABL1 parts of the fusion protein with one antibody each, and creating strong fluorescent signals through rolling circle amplification, PLA-flow allowed sensitive detection of cells positive for the BCR-ABL1 fusion at frequencies as low as one in 10,000. Importantly, the flow cytometric results correlated strongly to those of RQ-PCR, both in diagnostic testing and for MRD measurements over time. In summary, we believe this flow cytometry-based method can serve as an attractive approach for routine measurement of cells harboring BCR-ABL1 fusions, also allowing simultaneously assessment of other cell surface markers as well as sensitive longitudinal follow-up.

Extracellular vesicles (EVs) derived from blood cells are promising biomarkers for various diseases. However, they are difficult to measure accurately in plasma due to their small size. Here, we demonstrate that platelet-derived EVs in plasma can be measured using solid-phase proximity ligation assay with high sensitivity and specificity using very small sample volume of biological materials. The results correlate well with high-sensitivity flow cytometry with the difference that the smallest EVs are detected. Briefly, the EVs are first captured on a solid phase, using lactadherin binding, and detection requires recognition with two antibodies followed by qPCR. The assay, using cholera toxin subunit-B or lactadherin as capture agents, also allowed detection of the more rare population of tissue factor (TF)-positive EVs at a concentration similar to sensitive TF activity assays. Thus, this assay can detect different types of EVs with high specificity and sensitivity, and has the potential to be an attractive alternative to flow cytometric analysis of preclinical and clinical samples. Improved techniques for measuring EVs in plasma will hopefully contribute to the understanding of their role in several diseases.

Earlier studies suggest that lumbar radicular pain following disc herniation may be associated with a local or systemic inflammatory process. In the present study, we investigated the serum inflammatory protein profile of such patients. All 45 patients were recruited from Oslo University Hospital, Ullevål, Norway, during the period 2007–2009. The new multiplex proximity extension assay (PEA) technology was used to analyze the levels of 92 proteins. Interestingly, the present data showed that patients with radicular pain 12 months after disc herniation may be different from other patients with regard to many measurable serum cytokines. Given a false discovery rate (FDR) of 0.10 and 0.05, we identified 41 and 13 proteins, respectively, which were significantly upregulated in the patients with severe pain one year after disc herniation. On the top of the list ranked by estimated increase we found C-X-C motif chemokine 5 (CXCM5; 217% increase), epidermal growth factor (EGF; 142% increase), and monocyte chemotactic protein 4 (MCP-4; 70% increase). Moreover, a clear overall difference in the serum cytokine profile between the chronic and the recovered patients was demonstrated. Thus, the present results may be important for future protein serum profiling of lumbar radicular pain patients with regard to prognosis and choice of treatment. We conclude that serum proteins may be measurable molecular markers of persistent pain after disc herniation.

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.To investigate SOX10 as a potential biomarker for melanoma and vitiligo.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.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.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.

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.

Proximity ligation assay (PLA) is a recently developed strategy for protein analysis in which antibody-based detection of a target protein via a DNA ligation reaction of oligonucleotides linked to the antibodies results in the formation of an amplifiable DNA strand suitable for analysis. Here we describe a faster and more cost-effective strategy to construct the antibody-based proximity ligation probes used in PLA that is based on the noncovalent interaction of biotinylated oligonucleotides with streptavidin followed by the interaction of this complex with biotinylated antibodies.

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.

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.