Sensitive detection of aggregated prion protein via proximity ligation

Hammond, Maria; Wik, Lotta; Deslys, Jean‐Philippe; Comoy, Emmanuel; Linné, Tommy; Landegren, Ulf; Kamali‐Moghaddam, Masood

doi:10.4161/pri.32231

Cited by 5 publications

(3 citation statements)

References 34 publications

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“…This method may be conducted in both homogeneous‐ and solid‐phase formats, allowing accurate visualization of single protein molecules or complexes with higher precision. These properties underline the potential of PLA in research, diagnosis, pharmacology, and a wide variety of applications that require high specificity and sensitivity, and accurate protein expression assessments such as cancer biomarkers [22,23], prions [24], exosomes [25,26], and personalized medicine [24]. Furthermore, PLA has been reported to detect a range of viral disease agents such as foot‐and‐mouth disease virus [14].…”

Section: Discussionmentioning

confidence: 99%

Accurate detection of Newcastle disease virus using proximity‐dependent DNA aptamer ligation assays

et al. 2021

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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.

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Section: Discussionmentioning

confidence: 99%

Accurate detection of Newcastle disease virus using proximity‐dependent DNA aptamer ligation assays

et al. 2021

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“…Indeed, PLA has been used for a wide variety of applications that range from detection of proteins in single cells [22], cancer biomarkers [23] and prions [24] to proposals for its use with personalised medicine [25]. However, apart from a report detailing the proof of principle for the detection of bacteria, [4], there is only one other report of its use to detect pathogenic Escherichia coli [5].…”

Section: Discussionmentioning

confidence: 99%

Homogeneous and digital proximity ligation assays for the detection of Clostridium difficile toxins A and B

Dhillon

Johnson

Shannon

et al. 2016

Biomolecular Detection and Quantification

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BackgroundThe proximity ligation assay (PLA) detects proteins via their interaction with pairs of proximity probes, which are antibodies coupled to noncomplementary DNA oligonucleotides. The binding of both proximity probes to their epitopes on the target protein brings the oligonucleotides together, allowing them to be bridged by a third oligonucleotide with complementarity to the other two. This enables their ligation and the detection of the resulting amplicon by real-time quantitative PCR (qPCR), which acts as a surrogate marker for the protein of interest. Hence PLA has potential as a clinically relevant diagnostic tool for the detection of pathogens where nucleic acid based tests are inconclusive proof of infection.MethodsWe prepared monoclonal and polyclonal proximity probes targeting Clostridium difficile toxins A (TcdA) and B (TcdB) and used hydrolysis probe-based qPCR and digital PCR (dPCR) assays to detect antibody/antigen interactions.ResultsThe performance of the PLA assays was antibody-dependent but both TcdA and TcdB assays were more sensitive than comparable ELISAs in either single- or dualplex formats. Both PLAs could be performed using single monoclonal antibodies coupled to different oligonucleotides. Finally, we used dPCR to demonstrate its potential for accurate and reliable quantification of TcdA.ConclusionsPLA with either qPCR or dPCR readout have potential as new diagnostic applications for the detection of pathogens where nucleic acid based tests do not indicate viability or expression of toxins. Importantly, since it is not always necessary to use two different antibodies, the pool of potential antibodies useful for PLA diagnostic assays is usefully enhanced.

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“…While not being pathogens, prion proteins are infectious particles made up of misfolded proteins that act as a template for the conversion of normal brain proteins to abnormal prion proteins. In contrast to immune-PCR which was not more sensitive than an ELISA [8], a solid-phase PLA using either of two monoclonal antibodies was very sensitive at detecting prion protein aggregates in brain homogenates from infected hamsters [22], opening up the possibility of proximity assays becoming useful tools in the development of diagnostic approaches of prion diseases.…”

Section: Pathogen Detection By Proximity Assaysmentioning

confidence: 99%

Recent progress in developing proximity ligation assays for pathogen detection

Greenwood

Johnson

Dhillon

et al. 2015

Expert Review of Molecular Diagnostics

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The effective management of infectious diseases depends on the early detection of the microbes responsible, since pathogens are most effectively eliminated in the initial stages of infection. Current immunodiagnostic methods lack the sensitivity for earliest possible diagnosis. Nucleic acid-based tests (NATs) are more sensitive, but the detection of microbial DNA does not definitively prove the presence of a viable microorganism capable of causing a given infection. Proximity assays combine the specificity of antibody-based detection of proteins with the sensitivity and dynamic range of NATs, and their use may allow earlier as well as more clinically relevant detection than is possible with current NATs or immunoassays. However, the full potential of proximity assays for pathogen detection remains to be fulfilled, mainly due to the challenges associated with identifying suitable antibodies and antibody combinations, sensitivity issues arising from non-specific interactions of proximity probes and the longer incubation times required to carry out the assays.

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Sensitive detection of aggregated prion protein via proximity ligation

Cited by 5 publications

References 34 publications

Accurate detection of Newcastle disease virus using proximity‐dependent DNA aptamer ligation assays

Accurate detection of Newcastle disease virus using proximity‐dependent DNA aptamer ligation assays

Homogeneous and digital proximity ligation assays for the detection of Clostridium difficile toxins A and B

Recent progress in developing proximity ligation assays for pathogen detection

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