| 1. |
- Bendes, Annika, et al.
(författare)
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Bead-Based Assays for Validating Proteomic Profiles in Body Fluids
- 2021
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Ingår i: Protein Microarrays for Disease Analysis. - New York, NY : Springer Nature. ; , s. 65-78
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Bokkapitel (refereegranskat)abstract
- Protein biomarkers in biological fluids represent an important resource for improving the clinical management of diseases. Current proteomics technologies are capable of performing high-throughput and multiplex profiling in different types of fluids, often leading to the shortlisting of tens of candidate biomarkers per study. However, before reaching any clinical setting, these discoveries require thorough validation and an assay that would be suitable for routine analyses. In the path from biomarker discovery to validation, the performance of the assay implemented for the intended protein quantification is extremely critical toward achieving reliable and reproducible results. Development of robust sandwich immunoassays for individual candidates is challenging and labor and resource intensive, and multiplies when evaluating a panel of interesting candidates at the same time. Here we describe a versatile pipeline that facilitates the systematic and parallel development of multiple sandwich immunoassays using a bead-based technology.
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| 2. |
- Björkesten, Johan, et al.
(författare)
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A multiplex platform for digital measurement of circular DNA reaction products
- 2020
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Ingår i: Nucleic Acids Research. - : OXFORD UNIV PRESS. - 0305-1048 .- 1362-4962. ; 48:13
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Tidskriftsartikel (refereegranskat)abstract
- Digital PCR provides high sensitivity and unprecedented accuracy in DNA quantification, but current approaches require dedicated instrumentation and have limited opportunities for multiplexing. Here, we present an isothermal platform for digital enumeration of DNA reaction products in multiplex via standard fluorescence microscopy. Circular DNA strands, which may result from a wide range of molecular detection reactions, are captured on streptavidin-coated surfaces via hybridized biotinylated primers, followed by rolling circle amplification (RCA). The addition of 15% polyethylene glycol 4000 during RCA resulted in uniform, easily recorded reaction products. Immobilized DNA circles were visualized as RCA products with 100% efficiency, as determined by droplet digital PCR. We confirmed previous reports about the influence on RCA by sequence composition and size of RCA templates, and we developed an efficient one-step restaining procedure for sequential multiplexing using toehold-triggered DNA strand displacement. Finally, we exemplify applications of this digital readout platform by demonstrating more than three orders of magnitude improved sensitivity by digital measurement of prostate specific antigen (PSA) (detection threshold similar to 100 pg/l), compared to a commercial enzyme-linked immunosorbent assay (ELISA) with analogue readout (detection threshold similar to 500 ng/l), using the same antibody pair.
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| 3. |
- Fredolini, Claudia, et al.
(författare)
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Proteome profiling of home-sampled dried blood spots reveals proteins of SARS-CoV-2 infections
- 2024
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Ingår i: Communications Medicine. - : Springer Nature. - 2730-664X. ; 4:1
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Tidskriftsartikel (refereegranskat)abstract
- Background Self-sampling of dried blood spots (DBS) offers new routes to gather valuable health-related information from the general population. Yet, the utility of using deep proteome profiling from home-sampled DBS to obtain clinically relevant insights about SARS-CoV-2 infections remains largely unexplored.Methods Our study involved 228 individuals from the general Swedish population who used a volumetric DBS sampling device and completed questionnaires at home during spring 2020 and summer 2021. Using multi-analyte COVID-19 serology, we stratified the donors by their response phenotypes, divided them into three study sets, and analyzed 276 proteins by proximity extension assays (PEA). After normalizing the data to account for variances in layman-collected samples, we investigated the association of DBS proteomes with serology and self-reported information.Results Our three studies display highly consistent variance of protein levels and share associations of proteins with sex (e.g., MMP3) and age (e.g., GDF-15). Studying seropositive (IgG+) and seronegative (IgG-) donors from the first pandemic wave reveals a network of proteins reflecting immunity, inflammation, coagulation, and stress response. A comparison of the early-infection phase (IgM+IgG-) with the post-infection phase (IgM-IgG+) indicates several proteins from the respiratory system. In DBS from the later pandemic wave, we find that levels of a virus receptor on B-cells differ between seropositive (IgG+) and seronegative (IgG-) donors.Conclusions Proteome analysis of volumetric self-sampled DBS facilitates precise analysis of clinically relevant proteins, including those secreted into the circulation or found on blood cells, augmenting previous COVID-19 reports with clinical blood collections. Our population surveys support the usefulness of DBS, underscoring the role of timing the sample collection to complement clinical and precision health monitoring initiatives. The COVID-19 pandemic has posed multiple challenges to healthcare systems. A significant gap that remains is a lack of understanding of the impact of SARS-CoV-2 on individuals who did not seek or require hospitalization. To address this, we distribute self-sampling devices to random citizens, aiming to analyze how blood protein levels are affected in people who have had COVID-19 but had no or mild symptoms. Conducting multiple molecular measurements in dried blood, our study confirms clinically known markers and their relationship to infection stages, even if the donors themselves collect the sample. Our work highlights the potential of combining self-sampling with laboratory methods to provide useful information on human health. This convenient patient-centric sampling approach may potentially be useful when studying other diseases. Fredolini et al. present a proteomics analysis of home-sampled dried blood spots taken from the general population in Stockholm during the COVID-19 pandemic. The study provides insights into the molecular effects of SARS-CoV-2 infection in non-hospitalized individuals and demonstrates the compatibility of self-sampled blood spots with proteomics.
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| 4. |
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| 5. |
- Hauser, Janosch, et al.
(författare)
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Microfluidic Device for Patient-Centric Multiplexed Assays with Readout in Centralized Laboratories
- 2023
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 95:2, s. 1350-1358
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Tidskriftsartikel (refereegranskat)abstract
- Patient-centric sampling strategies, where the patient performs self-sampling and ships the sample to a centralized laboratory for readout, are on the verge of widespread adaptation. However, the key to a successful patient-centric workflow is user-friendliness, with few noncritical user interactions, and simple, ideally biohazard-free shipment. Here, we present a capillary-driven microfluidic device designed to perform the critical biomarker capturing step of a multiplexed immunoassay at the time of sample collection. On-chip sample drying enables biohazard-free shipment and allows us to make use of advanced analytics of specialized laboratories that offer the needed analytical sensitivity, reliability, and affordability. Using C-Reactive Protein, MCP1, S100B, IGFBP1, and IL6 as model blood biomarkers, we demonstrate the multiplexing capability and applicability of the device to a patient-centric workflow. The presented quantification of a biomarker panel opens up new possibilities for e-doctor and e-health applications.
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| 6. |
- Hauser, Janosch, et al.
(författare)
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On-Chip Assay for Home-Sampling, Mail-Based Shipping and Centralized Laboratory Readout
- 2021
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Ingår i: MicroTAS 2021. - : Chemical and Biological Microsystems Society. ; , s. 807-808
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Konferensbidrag (refereegranskat)abstract
- On-chip immunoassays have great potential to accelerate point-of-care (POC) testing. However, most attempts require either impractical readout equipment or are outperformed by the sensitivity, reliability and affordability offered by centralized laboratories. Here, we present a capillary-driven microfluidic device that unites the best of two worlds: Bead-based analyte isolation at the POC, where analyte drying enables simple mail-based shipping, combined with the powerful capabilities of centralized laboratories. Using C-reactive protein (CRP) as model biomarker we demonstrate on-chip assay performance similar to conventional assays, with a LOD of 10 pg/ml and average CV of 9.7 % (48 microfluidic devices).
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| 7. |
- Ikebuchi, Ryoyo, et al.
(författare)
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Human proteins incorporated into tick-borne encephalitis virus revealed by in situ proximity ligation
- 2020
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : ACADEMIC PRESS INC ELSEVIER SCIENCE. - 0006-291X .- 1090-2104. ; 525:3, s. 714-719
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 8. |
- Liotta, Lance A., et al.
(författare)
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Clinical proteomics and molecular pathology
- 2020
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Ingår i: Essential Concepts in Molecular Pathology. - : Elsevier BV. ; , s. 149-163
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Genomic and proteomic research is launching the next era of cancer molecular medicine. Molecular expression profiles can uncover clues to functionally important molecules in the development of human disease and generate information to subclassify human tumors and tailor a treatment to the individual patient. The next revolution is the synthesis of proteomic information into functional pathways and circuits in cells and tissues. Such synthesis must take into account the dynamic state of protein post-translational modifications; protein-protein or protein-DNA/RNA interactions; cross-talk between signal pathways; and feedback regulation within cells, between cells, and between tissues. This full set of information may be required before we can fully dissect the specific dysregulated pathways driving tumorigenesis. This higher level of functional understanding will be the basis for true rational therapeutic design that specifically targets the molecular lesions underlying human disease.
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| 9. |
- Martinson, Neil, et al.
(författare)
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Proteomic Analysis of Mucosal and Systemic Responses to SARS-CoV-2 Antigen
- 2023
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Ingår i: Vaccines. - : MDPI AG. - 2076-393X. ; 11:2
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Tidskriftsartikel (refereegranskat)abstract
- The mucosal environment of the upper respiratory tract is the first barrier of protection against SARS-CoV-2 transmission. However, the mucosal factors involved in viral transmission and potentially modulating the capacity to prevent such transmission have not fully been identified. In this pilot proteomics study, we compared mucosal and systemic compartments in a South African cohort of vaccinated and unvaccinated individuals undergoing maxillofacial surgery with previous history of COVID-19 or not. Inflammatory profiles were analyzed in plasma, nasopharyngeal swabs, and nasal and oral tissue explant cultures, using Olink and Luminex technologies. SARS-CoV-2-specific antibody levels were measured in serum and tissue explants. An increased pro-inflammatory proteomic profile was measured in the nasal compartment compared to plasma. However, IP-10 and MIG levels were higher in secretions than in nasal tissue, and the opposite was observed for TGF-beta. Nasal anti-SARS-CoV-2 spike IgG correlated with mucosal MIG expression for all participants. A further positive correlation was found with IP-10 in BioNTech/Pfizer-vaccinated individuals. Systemic levels of anti-SARS-CoV-2 spike IgG elicited by this vaccine correlated with plasma IL-10, IL-6 and HBD4. Proteomic profiles measured in mucosal tissues and secretions using combined technologies could reveal correlates of protection at the mucosal portals of viral entry.
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| 10. |
- Ribet, Federico, et al.
(författare)
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Microneedle Patch for Painless Intradermal Collection of Interstitial Fluid Enabling Multianalyte Measurement of Small Molecules, SARS‐CoV‐2 Antibodies, and Protein Profiling
- 2023
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Ingår i: Advanced Healthcare Materials. - : Wiley. - 2192-2640 .- 2192-2659. ; 12:13
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Tidskriftsartikel (refereegranskat)abstract
- Blood sampling is a common practice to monitor health, but it entails a series of drawbacks for patients including pain and discomfort. Thus, there is a demand for more convenient ways to obtain samples. Modern analytical techniques enable monitoring of multiple bioanalytes in smaller samples, opening possibilities for new matrices, and microsampling technologies to be adopted. Interstitial fluid (ISF) is an attractive alternative matrix that shows good correlation with plasma concentration dynamics for several analytes and can be sampled in a minimally invasive and painless manner from the skin at the point-of-care. However, there is currently a lack of sampling devices compatible with clinical translation. Here, to tackle state-of-the-art limitations, a cost-effective and compact single-microneedle-based device designed to painlessly collect precisely 1.1 µL of dermal ISF within minutes is presented. The fluid is volume-metered, dried, and stably stored into analytical-grade paper within the microfluidic device. The obtained sample can be mailed to a laboratory, quantitatively analyzed, and provide molecular insights comparable to blood testing. In a human study, the possibility to monitor various classes of molecular analytes is demonstrated in ISF microsamples, including caffeine, hundreds of proteins, and SARS-CoV-2 antibodies, some being detected in ISF for the first time.
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