Proximity Ligation Assay

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Chenggang Zhu - One of the best experts on this subject based on the ideXlab platform.

  • a novel method for the sensitive detection of mutant proteins using a covalent bonding tube based Proximity Ligation Assay
    2014
    Co-Authors: Xuecheng Jiang, Feng Shi, Lixiao Zhou, Jie Cheng, Hua Zhang, Huixia Wang, Zhixiong Chen, Chenggang Zhu
    Abstract:

    Abstract Tumorigenesis is the cumulative result of multiple gene mutations. The mutant proteins that are expressed by mutant genes in cancer cells are secreted into the blood and are useful biomarkers for the early diagnosis of cancer. However, some difficulties exist; for example, the same gene will express different protein mutants in different patients, and early tumors secrete only small amounts of mutant protein. Thus, the presence of mutant proteins in plasma has not previously been exploited for the early diagnosis of cancer. Proximity Ligation Assay is a protein-detection method that has been developed in recent years and has been widely used because of its high sensitivity. However, this approach still suffers from some shortcomings that should be addressed. In this paper, we develop a covalent-bonding tube-based Proximity Ligation Assay (TB-PLA). The limit of detection of TB-PLA for 0.001 pM, and the method exhibited a broad dynamic range of up to seven orders of magnitude. Furthermore, we coupled the conformation-specific antibody PAb240 of p53 mutants to PCR tubes for TB-PLA. The Assay was capable of detecting an approximately 500-fold lower concentration of mutant p53 in serum compared with sandwich ELISA. Thus, we demonstrate TB-PLA to be a highly sensitive and effective approach that is suitable for the early clinical diagnosis of cancer using the conformation-specific antibodies of protein mutants.

  • the development of an indirect competitive immunomagnetic Proximity Ligation Assay for small molecule detection
    2013
    Co-Authors: Xuecheng Jiang, Feng Shi, Luming Wang, Zhenhong Zhu, Zhihao Sun, Lixiao Zhou, Hanqiang Miao, Zhengting Zhang, Chenggang Zhu
    Abstract:

    The development of an indirect competitive immunomagnetic-Proximity Ligation Assay (ICIPLA), which is a novel method for detecting small molecules, is described in this report. Free small molecules in samples can be detected using a Proximity Ligation Assay (PLA); the detection is based on the Proximity effect caused by a high concentration of small molecule–BSA conjugates bound to streptavidin magnetic beads. As an indirect format competitive immunoAssay, the ICIPLA method has the advantage in that the quantity of monoclonal antibody (mAb) used for small-molecule detection is 8-fold lower than that required for the competitive immunomagnetic-Proximity Ligation Assay (CIPLA) described in our previous work. Small molecules can be detected using a single monoclonal antibody, and the PLA method can be used to amplify high-performance signals. In this work, the small molecular compound ractopamine (RAC) was selected as a target for ICIPLA. The limit of detection (LOD) was 0.01 ng ml−1, and the method exhibited a broad dynamic range of up to six orders of magnitude. We also employed the ICIPLA method to detect RAC in serum, urine, and muscle extracts; the results indicated that the LOD and dynamic range were not altered. The cross-reactivity studies showed that the cross-reactivity values for all RAC analogs were below 0.01%. These results suggest that ICIPLA is a sensitive, specific and practical method for small-molecule detection. This is the first report of the improved PLA technology for small-molecule detection by indirect competitive formats in the biological samples.

  • sensitive detection of small molecules by competitive immunomagnetic Proximity Ligation Assay
    2012
    Co-Authors: Shuyan Cheng, Feng Shi, Xuecheng Jiang, Luming Wang, Weiqing Chen, Chenggang Zhu
    Abstract:

    A novel detection method of small molecules, competitive immunomagnetic-Proximity Ligation Assay (CIPLA), was developed and described in this report. Through the Proximity effect caused by special Proximity probes we prepared, small molecules can be detected using only one monoclonal antibody. CIPLA overcomes the obstacle that the Proximity Ligation Assay (PLA) cannot be used in small molecular detection, as two antibodies are unable to combine to one small molecule due to its small molecular structure. Two small molecular compounds, clenbuterol (CLE) and ractopamine (RAC), were selected as targets for CIPLA. The limit of detection (LOD) reached 0.01 ng mL(-1), which was 10-50-fold lower than ELISA. With 5 orders of magnitude of the dynamic range achieved, the excellent sensitivity and broad dynamic range of CIPLA are noted. It can be applied widely in the sensitive detection of many other small molecular materials such as pesticides, additives in food, drugs, and biological samples, which have great significance in both theoretical and practical aspects.

Jonathan A Javitch - One of the best experts on this subject based on the ideXlab platform.

  • Detecting G protein-coupled receptor complexes in postmortem human brain with Proximity Ligation Assay and a Bayesian classifier
    2020
    Co-Authors: Ying Zhu, Pierre Trifilieff, József Mészáros, Roman Walle, Rongxi Fan, Ziyi Sun, Andrew Dwork, Jonathan A Javitch
    Abstract:

    Despite the controversy regarding the existence and physiological relevance of class A G protein-coupled receptor dimerization, there is substantial evidence for functional interactions between the dopamine D2 receptor (D2R) and the adenosine A2A receptor (A2AR). A2AR-D2R complexes have been detected in rodent brains by Proximity Ligation Assay; however, their existence in the human brain has not been demonstrated. In this study, we used Brightfield Proximity Ligation Assay, combined with a systematic sampling and a parameter-free naive Bayesian classifier, and demonstrated Proximity between the D2R and the A2AR in the adult human ventral striatum, consistent with their colocalization within complexes and the possible existence of D2R-A2AR heteromers. These methods are applicable to the relative quantification of Proximity of two proteins, as well as the expression levels of individual proteins. METHOD SUMMARY : Brightfield Proximity Ligation Assay was used to assess the expression of G protein-coupled receptors and their Proximity in postmortem adult human brains. A novel automated machine learning method (Bayesian optimized PLA signal sorting) was developed to automatically quantify Brightfield Proximity Ligation Assay data.

  • detection of g protein coupled receptor complexes in postmortem human brain by Proximity Ligation Assay
    2020
    Co-Authors: Ying Zhu, Pierre Trifilieff, Andrew Dwork, Jonathan A Javitch
    Abstract:

    Combining immunological and molecular biological methods, the antibody-based Proximity Ligation Assay (PLA) has been used for more than a decade to detect and quantify protein-protein interactions, protein modification, and protein expression in situ, including in brain tissue. However, the transfer of this technology to human brain samples requires a number of precautions due to the nature of the specimens and their specific processing. Here, we used the PLA brightfield detection technique to assess the expression of dopamine D2 receptor and adenosine A2A receptor and their Proximity in human postmortem brains, and we developed a systematic random sampling method to help quantify the PLA signals. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Sample preparation and sectioning for PLA_BF Basic Protocol 2: PLA_BF staining of brain tissue Basic Protocol 3: Image acquisition and result analysis Support Protocol: Luxol fast blue/cresyl violet staining.

  • detecting gpcr complexes in postmortem human brain with Proximity Ligation Assay and a bayesian classifier
    2019
    Co-Authors: Ying Zhu, Pierre Trifilieff, József Mészáros, Roman Walle, Rongxi Fan, Ziyi Sun, Andrew Dwork, Jonathan A Javitch
    Abstract:

    Abstract Despite the general controversy regarding the existence and physiological relevance of Class A GPCR dimerization, there is substantial evidence for functional interactions between dopamine D2 receptor (D2R) and adenosine A2A receptor (A2AR). A2AR-D2R complexes have been detected in rodent brains by Proximity Ligation Assay (PLA), but their existence in the human brain is yet to be demonstrated. In this study, we used brightfield PLA, combined with a systematic sampling and a parameter-free naive Bayesian classifier, and demonstrated Proximity between D2R and A2AR in the adult human ventral striatum, consistent with their colocalization within complexes and the possible existence of D2R-A2AR heteromers. These methods are applicable to the quantitative analysis of Proximity of two proteins and the expression of individual proteins. Method Summary Brightfield Proximity Ligation Assay (PLA) was used to assess the expression of G protein-coupled receptors and their Proximity in postmortem adult human brains. A novel automated machine learning method (Bayesian Optimized PLA Signal Sorting) was developed to automatically quantify brightfield PLA data.

  • detection of antigen interactions ex vivo by Proximity Ligation Assay endogenous dopamine d2 adenosine a2a receptor complexes in the striatum
    2011
    Co-Authors: Pierre Trifilieff, Marielaure Rives, Eneko Urizar, Rebecca A Piskorowski, Harshad D Vishwasrao, John Castrillon, Claudia Schmauss, Maria Slattman, Mats Gullberg, Jonathan A Javitch
    Abstract:

    The existence of G protein-coupled receptor (GPCR) dimers and/or oligomers has been demonstrated in heterologous systems using a variety of biochemical and biophysical Assays. While these interactions are the subject of intense research because of their potential role in modulating signaling and altering pharmacology, evidence for the existence of receptor interactions in vivo is still elusive because of a lack of appropriate methods to detect them. Here, we adapted and optimized a Proximity Ligation Assay (PLA) for the detection in brain slices of molecular Proximity of two antigens located on either the same or two different GPCRs. Using this approach, we were able to confirm the existence of dopamine D2 and adenosine A2A receptor complexes in the striatum of mice ex vivo.

Javier Alegreabarrategui - One of the best experts on this subject based on the ideXlab platform.

  • tau Proximity Ligation Assay reveals extensive previously undetected pathology prior to neurofibrillary tangles in preclinical alzheimer s disease
    2021
    Co-Authors: Nora Bengoavergniory, Richard Wademartins, Elisavet Velentzaalmpani, Ana Maria Silva, Connor Scott, Mariana Vargascaballero, Magdalena Sastre, Javier Alegreabarrategui
    Abstract:

    Multimerization is a key process in prion-like disorders such as Alzheimer’s disease (AD), since it is a requirement for self-templating tau and beta-amyloid amyloidogenesis. AT8-immunohistochemistry for hyperphosphorylated tau is currently used for the diagnosis and staging of tau pathology. Given that tau–tau interactions can occur in the absence of hyperphosphorylation or other post-translational modifications (PTMs), the direct visualization of tau multimerization could uncover early pathological tau multimers. Here, we used bimolecular fluorescent complementation, rapamycin-dependent FKBP/FRB-tau interaction and transmission electron microscopy to prove the in vitro specificity of tau-Proximity Ligation Assay (tau-PLA). We then analyzed MAPT KO and P301S transgenic mice, and human hippocampus and temporal isocortex of all Braak stages with tau-PLA and compared it with immunohistochemistry for the diagnostic antibody AT8, the early phosphorylation-dependent AT180, and the conformational-dependent antibody MC1. Finally, we performed proteinase-K treatment to infer the content of amyloidogenic beta-sheet fold. Our novel tau-Proximity Ligation Assay (tau-PLA) directly visualized tau–tau interactions in situ, and exclusively recognized tau multimers but not monomers. It elicited no signal in MAPT KO mouse brains, but extensively labelled P301S transgenic mice and AD brain. Two groups of structures were detected, a previously unreported widespread small-sized diffuse pathology and large, neurofibrillary-like lesions. Tau-PLA-labelled diffuse pathology appeared from the earliest Braak stages, mostly unaccompanied by tangle-like tau-immunohistochemistry, being significantly more sensitive than any small-sized dot-/thread-like pathology labelled by AT180-, AT8- and MC1-immunohistochemistry in most regions quantified at stages 0-II. Tau-PLA-labelled diffuse pathology was extremely sensitive to Proteinase-K, in contrast to large lesions. Tau-PLA is the first method to directly visualize tau multimers both in vitro and in situ with high specificity. We find that tau multimerization appears extensively from the earliest presymptomatic Braak stages as a previously unreported type of diffuse pathology. Importantly, in our study multimerization is the earliest detectable molecular event of AD tau pathology. Our findings open a new window to the study of early tau pathology, with potential implications in early diagnosis and the design of therapeutic strategies.

  • direct visualization of alpha synuclein oligomers reveals previously undetected pathology in parkinson s disease brain
    2015
    Co-Authors: Rosalind F Roberts, Richard Wademartins, Javier Alegreabarrategui
    Abstract:

    Oligomeric forms of alpha-synuclein are emerging as key mediators of pathogenesis in Parkinson's disease. Our understanding of the exact contribution of alpha-synuclein oligomers to disease is limited by the lack of a technique for their specific detection. We describe a novel method, the alpha-synuclein Proximity Ligation Assay, which specifically recognizes alpha-synuclein oligomers. In a blinded study with post-mortem brain tissue from patients with Parkinson's disease (n = 8, age range 73-92 years, four males and four females) and age- and sex-matched controls (n = 8), we show that the alpha-synuclein Proximity Ligation Assay reveals previously unrecognized pathology in the form of extensive diffuse deposition of alpha-synuclein oligomers. These oligomers are often localized, in the absence of Lewy bodies, to neuroanatomical regions mildly affected in Parkinson's disease. Diffuse alpha-synuclein Proximity Ligation Assay signal is significantly more abundant in patients compared to controls in regions including the cingulate cortex (1.6-fold increase) and the reticular formation of the medulla (6.5-fold increase). In addition, the alpha-synuclein Proximity Ligation Assay labels very early perikaryal aggregates in morphologically intact neurons that may precede the development of classical Parkinson's disease lesions, such as pale bodies or Lewy bodies. Furthermore, the alpha-synuclein Proximity Ligation Assay preferentially detects early-stage, loosely compacted lesions such as pale bodies in patient tissue, whereas Lewy bodies, considered heavily compacted late lesions are only very exceptionally stained. The alpha-synuclein Proximity Ligation Assay preferentially labels alpha-synuclein oligomers produced in vitro compared to monomers and fibrils, while stained oligomers in human brain display a distinct intermediate proteinase K resistance, suggesting the detection of a conformer that is different from both physiological, presynaptic alpha-synuclein (proteinase K-sensitive) and highly aggregated alpha-synuclein within Lewy bodies (proteinase K-resistant). These disease-associated conformers represent previously undetected Parkinson's disease pathology uncovered by the alpha-synuclein Proximity Ligation Assay.

Masood Kamalimoghaddam - One of the best experts on this subject based on the ideXlab platform.

  • sensitive and specific detection of platelet derived and tissue factor positive extracellular vesicles in plasma using solid phase Proximity Ligation Assay
    2018
    Co-Authors: Asa Thulin, Masood Kamalimoghaddam, Junhong Yan, Mikael Aberg, Christina Christersson, Agneta Siegbahn
    Abstract:

    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.

  • sensitive detection of platelet derived and tissue factor positive extracellular vesicles in plasma using solid phase Proximity Ligation Assay
    2018
    Co-Authors: Asa Thulin, Masood Kamalimoghaddam, Junhong Yan, Mikael Aberg, Christina Christersson, Agneta Siegbahn
    Abstract:

    Sensitive detection of platelet-derived and tissue factor positive extracellular vesicles in plasma using solid-phase Proximity Ligation Assay

  • detection of extracellular vesicles using Proximity Ligation Assay with flow cytometry readout exopla
    2017
    Co-Authors: Liza Lof, Ulf Landegren, Linda Arngarden, Tonge Ebai, Ola Soderberg, Masood Kamalimoghaddam
    Abstract:

    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

  • tyrosine phosphorylation profiling via in situ Proximity Ligation Assay
    2014
    Co-Authors: Lioudmila Elfineh, Christina Classon, Anna Asplund, Ulf Pettersson, Masood Kamalimoghaddam, Sara Bergstrom Lind
    Abstract:

    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.

Agneta Siegbahn - One of the best experts on this subject based on the ideXlab platform.

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