Analytically Sensitive Protein Detection in Microtiter Plates by Proximity Ligation with Rolling Circle Amplification

Ebai, Tonge; Oliveira, Felipe Marques Souza de; Löf, Liza; Wik, Lotta; Schweiger, Caroline; Larsson, Anders; Keilholtz, Ulrich; Haybaeck, Johannes; Landegren, Ulf; Kamali‐Moghaddam, Masood

doi:10.1373/clinchem.2017.271833

Cited by 25 publications

(16 citation statements)

References 28 publications

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“…Moreover, the requirement for target binding by sets of three rather than two antibodies to generate an amplifiable DNA signal reduces the risk for cross-reactive detection of irrelevant proteins 26 , 27 . The DNA products that form may be amplified by PCR or alternatively, using the in situ PLA mechanism, by RCA for isothermal amplification 17 , 18 .…”

Section: Resultsmentioning

confidence: 99%

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Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes

Klaesson

Grannas

Ebai

et al. 2018

Sci Rep

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We have redesigned probes for in situ proximity ligation assay (PLA), resulting in more efficient localized detection of target proteins. In situ PLA depends on recognition of target proteins by pairs of antibody-oligonucleotide conjugates (PLA probes), which jointly give rise to DNA circles that template localized rolling circle amplification reactions. The requirement for dual recognition of the target proteins improves selectivity by ignoring any cross-reactivity not shared by the antibodies, and it allows detection of protein-protein interactions and post-translational modifications. We herein describe an improved design of the PLA probes –UnFold probes – where all elements required for formation of circular DNA strands are incorporated in the probes. Premature interactions between the UnFold probes are prevented by including an enzymatic “unfolding” step in the detection reactions. This allows DNA circles to form by pairs of reagents only after excess reagents have been removed. We demonstrate the performance of UnFold probes for detection of protein-protein interactions and post-translational modifications in fixed cells and tissues, revealing considerably more efficient signal generation. We also apply the UnFold probes to detect IL-6 in solution phase after capture on solid supports, demonstrating increased sensitivity over both normal sandwich enzyme-linked immunosorbent assays and conventional PLA assays.

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Section: Resultsmentioning

confidence: 99%

“…In situ PLA has also been applied to improve sensitivity, specificity, and target range in other methods for localized protein detection, for example, western blotting 14 , flow cytometry 15 , 16 , and sandwich enzyme-linked immunosorbent assay (ELISA) 17 , 18 .…”

Section: Introductionmentioning

confidence: 99%

Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes

Klaesson

Grannas

Ebai

et al. 2018

Sci Rep

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“…RCA products can be detected using gel electrophoresis [120][121][122] or visually using fluorescence spectroscopy, microscopy and flow cytometry. [123][124][125][126][127] Furthermore, incorporating nanoparticles such as gold nanoparticles (AuNPs), magnetic beads, and quantum dots into RCA products [128][129][130] can aid the visualisation of products. Primers designed for PCR can be used for RCA.…”

Section: Dovepressmentioning

confidence: 99%

<p>Current and Future Perspectives on Isothermal Nucleic Acid Amplification Technologies for Diagnosing Infections</p>

Obande

Singh

2020

IDR

120

115

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Nucleic acid amplification technology (NAAT) has assumed a critical position in disease diagnosis in recent times and contributed significantly to healthcare. Application of these methods has resulted in a more sensitive, accurate and rapid diagnosis of infectious diseases than older traditional methods like culture-based identification. NAAT such as the polymerase chain reaction (PCR) is widely applied but seldom available to resource-limited settings. Isothermal amplification (IA) methods provide a rapid, sensitive, specific, simpler and less expensive procedure for detecting nucleic acid from samples. However, not all of these IA techniques find regular applications in infectious diseases diagnosis. Disease diagnosis and treatment could be improved, and the rapidly increasing problem of antimicrobial resistance reduced, with improvement, adaptation, and application of isothermal amplification methods in clinical settings, especially in developing countries. This review centres on some isothermal techniques that have found documented applications in infectious diseases diagnosis, highlighting their principles, development, strengths, setbacks and imminent potentials for use at points of care.

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“…The high potential of this technique for clinical diagnostics has been demonstrated for tumour profiling and cell type mapping . Similarly, a method named proximity ligation assay (PLA), based on similar principles of ligation mediated target recognition and RCA, but for the spatial analysis of proteins has been reported potentially useful for in situ and in vitro diagnostics .…”

Section: Diagnostic Tests In Clinical Routinementioning

confidence: 99%

Smartphone‐based clinical diagnostics: towards democratization of evidence‐based health care

et al. 2018

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Recent advancements in bioanalytical techniques have led to the development of novel and robust diagnostic approaches that hold promise for providing optimal patient treatment, guiding prevention programs and widening the scope of personalized medicine. However, these advanced diagnostic techniques are still complex, expensive and limited to centralized healthcare facilities or research laboratories. This significantly hinders the use of evidence-based diagnostics for resource-limited settings and the primary care, thus creating a gap between healthcare providers and patients, leaving these populations without access to precision and quality medicine. Smartphone-based imaging and sensing platforms are emerging as promising alternatives for bridging this gap and decentralizing diagnostic tests offering practical features such as portability, cost-effectiveness and connectivity. Moreover, towards simplifying and automating bioanalytical techniques, biosensors and lab-on-a-chip technologies have become essential to interface and integrate these assays, bringing together the high precision and sensitivity of diagnostic techniques with the connectivity and computational power of smartphones. Here, we provide an overview of the emerging field of clinical smartphone diagnostics and its contributing technologies, as well as their wide range of areas of application, which span from haematology to digital pathology and rapid infectious disease diagnostics.

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Analytically Sensitive Protein Detection in Microtiter Plates by Proximity Ligation with Rolling Circle Amplification

Abstract: 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.

Cited by 25 publications

References 28 publications

Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes

Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes

<p>Current and Future Perspectives on Isothermal Nucleic Acid Amplification Technologies for Diagnosing Infections</p>

Smartphone‐based clinical diagnostics: towards democratization of evidence‐based health care

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