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Zhenxin Wang - One of the best experts on this subject based on the ideXlab platform.

  • profiling of multiple matrix metalloproteinases activities in the progression of osteosarcoma by Peptide Microarray based fluorescence assay on polymer brush coated zinc oxide nanorod substrate
    Sensors and Actuators B-chemical, 2021
    Co-Authors: Zhenxin Wang, Minghong Jian, Hua Zhang
    Abstract:

    Abstract Osteosarcoma (OS) is a primary malignant bone tumor with high rate of recurrence and lung metastasis. Matrix metalloproteinases (MMPs) are demonstrated as important biomarkers of tumor invasion and metastasis. Herein, a Peptide Microarray-based fluorescence assay is proposed to profile multiple MMPs (MMP-1, -2, -3, -7, -9 and -13) activities in the progression of OS by using the mouse-bearing xenograft U-2OS and Saos-2 human OSs. The Peptide Microarray containing different biotinylated Peptide substrate spots is fabricated on the poly(glycidyl methacrylate-co-2-hydroxyethyl methacrylate) brush coated zinc oxide nanorod (ZnONR@P(GMA-HEMA) decorated glass slides, and labeled by cyanine 3 (Cy3) modified avidin (Avidin-Cy3) to produce fluorescence signal. Special cleavage of Peptide substrate by MMP resulted the decrease of fluorescence signal. The MMPs activities are positively correlated with the change of fluorescence intensity. This method has excellent selectivity and sensitivity, which enables to detect the activities of cellular secreted MMP-1, -2, -3, -7, -9, and -13 with limit of detection downs to 10 pmol L−1, 30 pmol L−1, 113 pmol L−1, 13 pmol L−1, 93 pmol L−1 and 12 pmol L−1, respectively. Furthermore, it is demonstrated that the activity pattern of MMPs in serum closely relevant to the disease progression and type of tumor.

  • Peptide Microarray based fluorescence assay for quantitatively monitoring the tumor associated matrix metalloproteinase 2 activity
    Sensors and Actuators B-chemical, 2020
    Co-Authors: Zhenxin Wang, Minghong Jian, Jiaxue Gao
    Abstract:

    Abstract Matrix metalloproteinases (MMPs) are important biomarkers of various tumors. Herein, a Peptide Microarray-based fluorescence assay is developed for quantitatively profiling of MMP-2 activity in different matrices through the binding of the immobilized biotinylated Peptides on the Microarray with the fluorescein isothiocyanate (FITC) modified neutravidins. In the presence of MMP-2, the biotin moiety is released from Microarray by enzymatic cleavage of Peptide substrate, resulting in the decrease of fluorescence signal. The change of fluorescence intensity is correlated with MMP-2 activity. The detection limit down to 14 pg mL−1 in buffer solution is obtained by a selected Peptide substrate for MMP-2 from eleven Peptide substrate candidates. The cellular secreted MMP-2 activity levels of different living cells are further successfully quantitatively evaluated by the selected Peptide substrate. Using mouse-bearing MG-63 human osteosarcoma as a model system, we also demonstrate that the activity of MMP-2 in serum is closely related with the cancer progression.

  • evaluation of matrix metalloproteinase inhibition by Peptide Microarray based fluorescence assay on polymer brush substrate and in vivo assessment
    ACS Applied Materials & Interfaces, 2017
    Co-Authors: Zhen Lei, Yaoqi Wang, Xianying Meng, Hua Zhang, Hongda Chen, Zhenxin Wang
    Abstract:

    Matrix metalloproteinases (MMPs) are important biomarkers and potential therapeutic targets of tumor. In this report, a Peptide Microarray-based fluorescence assay is developed for MMPs inhibitors evaluation through immobilization of biotin-modified Peptides on the poly(glycidyl methacrylate-co-2-hydroxyethyl methacrylate) (P(GMA-HEMA)) brush-modified glass slides. After biotin is recognized with cyanine 3 (Cy3)-modified avidin (Cy3-avidin), the Microarrays can produce strong fluorescence signal. The biotin moieties detach from Microarray, when the biotin-modified Peptide substrates are specially cleaved by a MMP, resulting in decreased fluorescence intensity of the Microarray. The decreasing level of fluorescence intensity is correlated with the MMP inhibition. Nine known MMP inhibitors against MMP-2 and MMP-9 are evaluated by the assay, and the quantitative determination of inhibitory potencies (half maximal inhibitory concentration) are obtained, which are comparable with the literatures. Two biocompatible fluorogenic Peptides containing MMP-specific recognition sequences and FAM/Dabcyl fluorophore-quencher pair are designed as activatable reporter probes for sensing MMP-2 and MMP-9 activities in cell and in vivo. The Peptide Microarray-based results are well verified by the cell inhibition assay and in vitro fluorescence imaging, and further confirmed by the in vivo imaging of HT-1080 tumor-bearing mice.

  • Peptide Microarray based metal enhanced fluorescence assay for multiple profiling of matrix metalloproteinases activities
    Analytical Chemistry, 2017
    Co-Authors: Zhen Lei, Xianying Meng, Yaoqi Wang, Hua Zhang, Zhenxin Wang
    Abstract:

    Matrix metalloproteinases (MMPs) are closely associated with cancer cell invasion and metastasis. Herein, a fluorescence resonance energy transfer (FRET)-Peptide Microarray-based metal enhanced fluorescence (MEF) assay is proposed for multiple and sensitive profiling of MMPs activities on a novel Au/Ag@SiO2 substrate. The Au/Ag@SiO2 substrate is prepared by electroless deposition of silver on gold nanoparticle (GNP) seeds, followed by SiO2 shell coating and surface functionalization. The specific FRET Peptides are spotted on the Au/Ag@SiO2 substrate to sensitively detect MMPs (MMP-2, -3, -7, -9, -14) via fluorescence recovery by the MMP cleavage of quenched Peptide motifs and further enhanced by MEF. Under the optimal conditions, the limits of detection are 12.2 fg mL–1 for MMP-2, 60 pg mL–1 for MMP-3, 0.22 pg mL–1 for MMP-7, 102 fg mL–1 for MMP-9, and 0.68 ng mL–1 for MMP-14, respectively. The practicability of the FRET-Peptide Microarray-based MEF assay is demonstrated by profiling of multiplexed MMPs a...

  • poly glycidyl methacrylate co 2 hydroxyethyl methacrylate brushes as Peptide protein Microarray substrate for improving protein binding and functionality
    ACS Applied Materials & Interfaces, 2016
    Co-Authors: Zhen Lei, Xia Liu, Dianjun Liu, Jiaxue Gao, Zhenxin Wang
    Abstract:

    We developed a three-dimensional (3D) polymer-brush substrate for protein and Peptide Microarray fabrication, and this substrate was facilely prepared by copolymerization of glycidyl methacrylate (GMA) and 2-hydroxyethyl methacrylate (HEMA) monomers via surface-initiated atom transfer radical polymerization (SI-ATRP) on a glass slide. The performance of obtained poly(glycidyl methacrylate-co-2-hydroxyethyl methacrylate) (P(GMA-HEMA)) brush substrate was assessed by binding of human IgG with rabbit antihuman IgG antibodies on a protein Microarray and by the determination of matrix metalloproteinase (MMP) activities on a Peptide Microarray. The P(GMA-HEMA) brush substrate exhibited higher immobilization capacities for proteins and Peptides than those of a two-dimensional (2D) planar epoxy slide. Furthermore, the sensitivity of the P(GMA-HEMA) brush-based Microarray on rabbit antihuman IgG antibody detection was much higher than that of its 2D counterpart. The enzyme activities of MMPs were determined specif...

Davide Valentini - One of the best experts on this subject based on the ideXlab platform.

  • Peptide Microarray based characterization of antibody responses to host proteins after bacille calmette guerin vaccination
    International Journal of Infectious Diseases, 2017
    Co-Authors: Davide Valentini, Daniel F Hoft, Lalit Rane, Sayma Rahman, Rebecca Axelssonrobertson, Rainer Heuchel, Matthias Lohr, Susanna Brighenti
    Abstract:

    Summary Background Bacille Calmette–Guerin (BCG) is the world's most widely distributed vaccine, used against tuberculosis (TB), in cancer immunotherapy, and in autoimmune diseases due to its immunomodulatory properties. To date, the effect of BCG vaccination on antibody responses to host proteins has not been reported. High-content Peptide Microarrays (HCPM) offer a unique opportunity to gauge specific humoral immune responses. Methods The sera of BCG-vaccinated healthy adults were tested on a human HCPM platform (4953 randomly selected epitopes of human proteins) to detect specific immunoglobulin gamma (IgG) responses. Samples were obtained at 56, 112, and 252 days after vaccination. Immunohistology was performed on lymph node tissue from patients with TB lymphadenitis. Results were analysed with a combination of existing and novel statistical methods. Results IgG recognition of host Peptides exhibited a peak at day 56 post BCG vaccination in all study subjects tested, which diminished over time. Primarily, IgG responses exhibited increased reactivity to ion transporters (sodium, calcium channels), cytokine receptors (interleukin 2 receptor β (IL2Rβ), fibroblast growth factor receptor 1 (FGFR1)), other cell surface receptors (inositol, somatostatin, angiopoeitin), ribonucleoprotein, and enzymes (tyrosine kinases, phospholipase) on day 56. There was decreased IgG reactivity to transforming growth factor-beta type 1 receptor (TGFβR1) and, in agreement with the Peptide Microarray findings, immunohistochemical analysis of TB-infected lymph node samples revealed an overexpression of TGFβR in granulomatous lesions. Moreover, the vesicular monoamine transporter (VMAT2) showed increased reactivity on days 112 and 252, but not on day 56 post-vaccination. IgG to interleukin 4 receptor (IL4R) showed increased reactivity at 112 days post-vaccination, while IgG to IL2Rβ and FGFR1 showed decreased reactivity on days 112 and 252 as compared to day 56 post BCG vaccination. Conclusions BCG vaccination modifies the host's immune landscape after 56 days, but this imprint changes over time. This may influence the establishment of immunological memory in BCG-vaccinated individuals.

  • h1n1 viral proteome Peptide Microarray predicts individuals at risk for h1n1 infection and segregates infection versus pandemrix vaccination
    Immunology, 2015
    Co-Authors: Davide Valentini, Isabelle Magalhaes, Aditya Ambati, Emanuele Montomoli, Guilia Lapini, Fabrizio Biuso, Holger Wenschuh
    Abstract:

    A high content Peptide Microarray containing the entire influenza A virus [A/California/08/2009(H1N1)] proteome and haemagglutinin proteins from 12 other influenza A subtypes, including the haemagglutinin from the [A/South Carolina/1/1918(H1N1)] strain, was used to gauge serum IgG epitope signatures before and after Pandemrix(®) vaccination or H1N1 infection in a Swedish cohort during the pandemic influenza season 2009. A very narrow pattern of pandemic flu-specific IgG epitope recognition was observed in the serum from individuals who later contracted H1N1 infection. Moreover, the pandemic influenza infection generated IgG reactivity to two adjacent epitopes of the neuraminidase protein. The differential serum IgG recognition was focused on haemagglutinin 1 (H1) and restricted to classical antigenic sites (Cb) in both the vaccinated controls and individuals with flu infections. We further identified a novel epitope VEPGDKITFEATGNL on the Ca antigenic site (251-265) of the pandemic flu haemagglutinin, which was exclusively recognized in serum from individuals with previous vaccinations and never in serum from individuals with H1N1 infection (confirmed by RNA PCR analysis from nasal swabs). This epitope was mapped to the receptor-binding domain of the influenza haemagglutinin and could serve as a correlate of immune protection in the context of pandemic flu. The study shows that unbiased epitope mapping using Peptide Microarray technology leads to the identification of biologically and clinically relevant target structures. Most significantly an H1N1 infection induced a different footprint of IgG epitope recognition patterns compared with the pandemic H1N1 vaccine.

  • Humoral ‘reactome’ profiles using Peptide Microarray chips
    Trends in Immunology, 2010
    Co-Authors: Davide Valentini, Simani Gaseitsiwe, Markus Maeurer
    Abstract:

    In their recent article, Prechl and coworkers discussed the use of protein antigen Microarrays as a viable platform to gauge the humoral ‘immunome’ or ‘reactome’ against target proteins [1]; an approach which has successfully been used to gauge autoimmune responses [2]. Peptide Microarrays might exhibit a certain advantage over protein antigen Microarrays in that they are able to identify the immunoglobulin recognition hotspots on the nominal target protein. Our knowledge of epitope-specific recognition patterns is limited to small regions of the proteome [3] of pathogens with large genomes.

  • Peptide Microarray based identification of mycobacterium tuberculosis epitope binding to hla drb1 0101 drb1 1501 and drb1 0401
    Clinical and Vaccine Immunology, 2010
    Co-Authors: Davide Valentini, Shahnaz Mahdavifar, Anneka Ehrnst, Simani Gaseitsiwe, Markus Maeurer, Marie Reilly
    Abstract:

    A more effective vaccine against Mycobacterium tuberculosis is needed, and a number of M. tuberculosis vaccine candidates are currently in preclinical or clinical phase I and II studies. One of the strategies to select M. tuberculosis (protein) targets to elicit a CD8+ or CD4+ T-cell response is to gauge the binding of candidate Peptides to major histocompatibility complex (MHC) class I or class II molecules, a prerequisite for successful Peptide presentation and to expand antigen-specific T cells. We scanned 61 proteins from the M. tuberculosis proteome for potential MHC class II-presented epitopes that could serve as targets for CD4+ T-cell responses. We constructed a Peptide Microarray consisting of 7,466 unique Peptides derived from 61 M. tuberculosis proteins. The Peptides were 15-mers overlapping by 12 amino acids. Soluble recombinant DRB1*0101 (DR1), DRB1*1501 (DR2), and DRB1*0401 (DR4) monomers were used to gauge binding to individual Peptide species. Out of 7,466 Peptides, 1,282, 674, and 1,854 Peptides formed stable complexes with HLA-DR1, -DR2, and -DR4, respectively. Five hundred forty-four Peptides bound to all three MHC class II molecules, 609 bound to only two, and 756 bound to only a single MHC class II molecule. This allowed us to rank M. tuberculosis proteins by epitope density. M. tuberculosis proteins contained “hot spots,” i.e., regions with enriched MHC class II binding epitopes. Two hundred twenty-two Peptides that formed MHC class II-Peptide complexes had previously been described as exclusively recognized by IgG in sera from patients with active pulmonary tuberculosis, but not in sera from healthy individuals, suggesting that these Peptides serve as B-cell and CD4+ T-cell epitopes. This work helps to identify not only M. tuberculosis Peptides with immunogenic potential, but also the most immunogenic proteins. This information is useful for vaccine design and the development of future tools to explore immune responses to M. tuberculosis.

  • major histocompatibility complex class ii molecule human immunodeficiency virus Peptide analysis using a Microarray chip
    Clinical and Vaccine Immunology, 2009
    Co-Authors: Simani Gaseitsiwe, Davide Valentini, Raija K Ahmed, Shahnaz Mahdavifar, Isabelle Magalhaes, Emmanuel Gautherot, Felix Montero, Johannes Zerweck, Mike Schutkowski, Anneka Ehrnst
    Abstract:

    Identification of major histocompatibility complex (MHC) class II binding Peptides is a crucial step in rational vaccine design and immune monitoring. We designed a novel MHC class II molecule-Peptide Microarray binding assay and evaluated 346 Peptides from already identified human immunodeficiency virus (HIV) epitopes and an additional set (n = 206) of 20-mer Peptides, overlapping by 15 amino acid residues, from HIV type 1B (HIV-1B) gp160 and Nef as a paradigm. Peptides were attached via the N-terminal part to a linker that covalently binds to the epoxy glass slide. The 552 Peptides were printed in triplicate on a single Peptide Microarray chip and tested for stable formation of MHC class II molecule-Peptide complexes using recombinant soluble DRB1*0101(DR1), DRB1*1501(DR2), and DRB1*0401(DR4) molecules. Cluster analysis revealed unique patterns of Peptide binding to all three, two, or a single MHC class II molecule. MHC class II binding Peptides reside within previously described immunogenic regions of HIV gp160 and Nef, yet we could also identify new MHC class II binding Peptides from gp160 and Nef. Peptide Microarray chips allow the comprehensive and simultaneous screening of a high number of candidate Peptide epitopes for MHC class II binding, guided by subsequent quality data extraction and binding pattern cluster analysis.

Ole Lund - One of the best experts on this subject based on the ideXlab platform.

  • high density Peptide Microarray exploration of the antibody response in a rabbit immunized with a neurotoxic venom fraction
    Toxicon, 2017
    Co-Authors: Mikael Engmark, Bruno Lomonte, Martin Closter Jespersen, Ole Lund, Andreas Hougaard Laustsen
    Abstract:

    Abstract Polyvalent snakebite antivenoms derive their therapeutic success from the ability of their antibodies to neutralize venom toxins across multiple snake species. This ability results from a production process involving immunization of large mammals with a broad suite of toxins present in venoms. As a result of immunization with this wide range of toxins, many polyvalent antivenoms have a high degree of cross-reactivity to similar toxins in other snake venoms – a cross-reactivity which cannot easily be deconvoluted. As a proof of concept, we aimed at exploring the opposite scenario by performing a high-throughput evaluation of the extent of cross-reactivity of a polyclonal mixture of antibodies that was raised against only a single snake venom fraction. For this purpose, a venom fraction containing short neurotoxin 1 (SN-1; Uniprot accession number P01416 , three-finger toxin (3FTx) family), which is the medically most important toxin from the notorious black mamba ( Dendroaspis polylepis ), was employed. Following immunization of a rabbit, a specific polyclonal antibody response was confirmed by ELISA and immunodiffusion. Subsequently, these antibodies were investigated by high-density Peptide Microarray to reveal linear elements of recognized epitopes across 742 3FTxs and 10 dendrotoxins. This exploratory study demonstrates in a single immunized animal that cross-reactivity between toxins of high similarity may be difficult to obtain when immunizing with a single 3FTx containing venom fraction. Additionally, this study explored the influence of employing different lengths of Peptides in high-density Peptide Microarray experiments for identification of toxin epitopes. Using 8-mer, 12-mer, and 15-mer Peptides, a single linear epitope element was identified in SN-1 with high precision.

  • cross recognition of a pit viper crotalinae polyspecific antivenom explored through high density Peptide Microarray epitope mapping
    PLOS Neglected Tropical Diseases, 2017
    Co-Authors: Mikael Engmark, Andreas Hougaard Laustsen, Bruno Lomonte, Jose Maria Gutierrez, Federico De Masi, Mikael Rordam Andersen, Ole Lund
    Abstract:

    Snakebite antivenom is a 120 years old invention based on polyclonal mixtures of antibodies purified from the blood of hyper-immunized animals. Knowledge on antibody recognition sites (epitopes) on snake venom proteins is limited, but may be used to provide molecular level explanations for antivenom cross-reactivity. In turn, this may help guide antivenom development by elucidating immunological biases in existing antivenoms. In this study, we have identified and characterized linear elements of B-cell epitopes from 870 pit viper venom protein sequences by employing a high-throughput methodology based on custom designed high-density Peptide Microarrays. By combining data on antibody-Peptide interactions with multiple sequence alignments of homologous toxin sequences and protein modelling, we have determined linear elements of antibody binding sites for snake venom metalloproteases (SVMPs), phospholipases A2s (PLA2s), and snake venom serine proteases (SVSPs). The studied antivenom antibodies were found to recognize linear elements in each of the three enzymatic toxin families. In contrast to a similar study of elapid (non-enzymatic) neurotoxins, these enzymatic toxins were generally not recognized at the catalytic active site responsible for toxicity, but instead at other sites, of which some are known for allosteric inhibition or for interaction with the tissue target. Antibody recognition was found to be preserved for several minor variations in the protein sequences, although the antibody-toxin interactions could often be eliminated completely by substitution of a single residue. This finding is likely to have large implications for the cross-reactivity of the antivenom and indicate that multiple different antibodies are likely to be needed for targeting an entire group of toxins in these recognized sites.

  • arraypitope automated analysis of amino acid substitutions for Peptide Microarray based antibody epitope mapping
    PLOS ONE, 2017
    Co-Authors: Christian Skjodt Hansen, Ole Lund, Thomas Osterbye, Paolo Marcatili, Soren Buus, Morten Nielsen
    Abstract:

    : Identification of epitopes targeted by antibodies (B cell epitopes) is of critical importance for the development of many diagnostic and therapeutic tools. For clinical usage, such epitopes must be extensively characterized in order to validate specificity and to document potential cross-reactivity. B cell epitopes are typically classified as either linear epitopes, i.e. short consecutive segments from the protein sequence or conformational epitopes adapted through native protein folding. Recent advances in high-density Peptide Microarrays enable high-throughput, high-resolution identification and characterization of linear B cell epitopes. Using exhaustive amino acid substitution analysis of Peptides originating from target antigens, these Microarrays can be used to address the specificity of polyclonal antibodies raised against such antigens containing hundreds of epitopes. However, the interpretation of the data provided in such large-scale screenings is far from trivial and in most cases it requires advanced computational and statistical skills. Here, we present an online application for automated identification of linear B cell epitopes, allowing the non-expert user to analyse Peptide Microarray data. The application takes as input quantitative Peptide data of fully or partially substituted overlapping Peptides from a given antigen sequence and identifies epitope residues (residues that are significantly affected by substitutions) and visualize the selectivity towards each residue by sequence logo plots. Demonstrating utility, the application was used to identify and address the antibody specificity of 18 linear epitope regions in Human Serum Albumin (HSA), using Peptide Microarray data consisting of fully substituted Peptides spanning the entire sequence of HSA and incubated with polyclonal rabbit anti-HSA (and mouse anti-rabbit-Cy3). The application is made available at: www.cbs.dtu.dk/services/ArrayPitope.

Zhen Lei - One of the best experts on this subject based on the ideXlab platform.

  • evaluation of matrix metalloproteinase inhibition by Peptide Microarray based fluorescence assay on polymer brush substrate and in vivo assessment
    ACS Applied Materials & Interfaces, 2017
    Co-Authors: Zhen Lei, Yaoqi Wang, Xianying Meng, Hua Zhang, Hongda Chen, Zhenxin Wang
    Abstract:

    Matrix metalloproteinases (MMPs) are important biomarkers and potential therapeutic targets of tumor. In this report, a Peptide Microarray-based fluorescence assay is developed for MMPs inhibitors evaluation through immobilization of biotin-modified Peptides on the poly(glycidyl methacrylate-co-2-hydroxyethyl methacrylate) (P(GMA-HEMA)) brush-modified glass slides. After biotin is recognized with cyanine 3 (Cy3)-modified avidin (Cy3-avidin), the Microarrays can produce strong fluorescence signal. The biotin moieties detach from Microarray, when the biotin-modified Peptide substrates are specially cleaved by a MMP, resulting in decreased fluorescence intensity of the Microarray. The decreasing level of fluorescence intensity is correlated with the MMP inhibition. Nine known MMP inhibitors against MMP-2 and MMP-9 are evaluated by the assay, and the quantitative determination of inhibitory potencies (half maximal inhibitory concentration) are obtained, which are comparable with the literatures. Two biocompatible fluorogenic Peptides containing MMP-specific recognition sequences and FAM/Dabcyl fluorophore-quencher pair are designed as activatable reporter probes for sensing MMP-2 and MMP-9 activities in cell and in vivo. The Peptide Microarray-based results are well verified by the cell inhibition assay and in vitro fluorescence imaging, and further confirmed by the in vivo imaging of HT-1080 tumor-bearing mice.

  • Peptide Microarray based metal enhanced fluorescence assay for multiple profiling of matrix metalloproteinases activities
    Analytical Chemistry, 2017
    Co-Authors: Zhen Lei, Xianying Meng, Yaoqi Wang, Hua Zhang, Zhenxin Wang
    Abstract:

    Matrix metalloproteinases (MMPs) are closely associated with cancer cell invasion and metastasis. Herein, a fluorescence resonance energy transfer (FRET)-Peptide Microarray-based metal enhanced fluorescence (MEF) assay is proposed for multiple and sensitive profiling of MMPs activities on a novel Au/Ag@SiO2 substrate. The Au/Ag@SiO2 substrate is prepared by electroless deposition of silver on gold nanoparticle (GNP) seeds, followed by SiO2 shell coating and surface functionalization. The specific FRET Peptides are spotted on the Au/Ag@SiO2 substrate to sensitively detect MMPs (MMP-2, -3, -7, -9, -14) via fluorescence recovery by the MMP cleavage of quenched Peptide motifs and further enhanced by MEF. Under the optimal conditions, the limits of detection are 12.2 fg mL–1 for MMP-2, 60 pg mL–1 for MMP-3, 0.22 pg mL–1 for MMP-7, 102 fg mL–1 for MMP-9, and 0.68 ng mL–1 for MMP-14, respectively. The practicability of the FRET-Peptide Microarray-based MEF assay is demonstrated by profiling of multiplexed MMPs a...

  • poly glycidyl methacrylate co 2 hydroxyethyl methacrylate brushes as Peptide protein Microarray substrate for improving protein binding and functionality
    ACS Applied Materials & Interfaces, 2016
    Co-Authors: Zhen Lei, Xia Liu, Dianjun Liu, Jiaxue Gao, Zhenxin Wang
    Abstract:

    We developed a three-dimensional (3D) polymer-brush substrate for protein and Peptide Microarray fabrication, and this substrate was facilely prepared by copolymerization of glycidyl methacrylate (GMA) and 2-hydroxyethyl methacrylate (HEMA) monomers via surface-initiated atom transfer radical polymerization (SI-ATRP) on a glass slide. The performance of obtained poly(glycidyl methacrylate-co-2-hydroxyethyl methacrylate) (P(GMA-HEMA)) brush substrate was assessed by binding of human IgG with rabbit antihuman IgG antibodies on a protein Microarray and by the determination of matrix metalloproteinase (MMP) activities on a Peptide Microarray. The P(GMA-HEMA) brush substrate exhibited higher immobilization capacities for proteins and Peptides than those of a two-dimensional (2D) planar epoxy slide. Furthermore, the sensitivity of the P(GMA-HEMA) brush-based Microarray on rabbit antihuman IgG antibody detection was much higher than that of its 2D counterpart. The enzyme activities of MMPs were determined specif...

  • Poly(glycidyl methacrylate-co-2-hydroxyethyl methacrylate) Brushes as Peptide/Protein Microarray Substrate for Improving Protein Binding and Functionality
    2016
    Co-Authors: Zhen Lei, Xia Liu, Dianjun Liu, Jiaxue Gao, Zhenxin Wang
    Abstract:

    We developed a three-dimensional (3D) polymer-brush substrate for protein and Peptide Microarray fabrication, and this substrate was facilely prepared by copolymerization of glycidyl methacrylate (GMA) and 2-hydroxyethyl methacrylate (HEMA) monomers via surface-initiated atom transfer radical polymerization (SI-ATRP) on a glass slide. The performance of obtained poly­(glycidyl methacrylate-co-2-hydroxyethyl methacrylate) (P­(GMA-HEMA)) brush substrate was assessed by binding of human IgG with rabbit antihuman IgG antibodies on a protein Microarray and by the determination of matrix metalloproteinase (MMP) activities on a Peptide Microarray. The P­(GMA-HEMA) brush substrate exhibited higher immobilization capacities for proteins and Peptides than those of a two-dimensional (2D) planar epoxy slide. Furthermore, the sensitivity of the P­(GMA-HEMA) brush-based Microarray on rabbit antihuman IgG antibody detection was much higher than that of its 2D counterpart. The enzyme activities of MMPs were determined specifically with a low detection limit of 6.0 pg mL–1 for MMP-2 and 5.7 pg mL–1 for MMP-9. By taking advantage of the biocompatibility of PHEMA, the P­(GMA-HEMA) brush-based Peptide Microarray was also employed to evaluate the secretion of MMP-2 and MMP-9 by cells cultured off the chip or directly on the chip, and satisfactory results were obtained

  • Peptide Microarray based fluorescence assay for simultaneously detecting matrix metalloproteinases
    Analytical Methods, 2016
    Co-Authors: Zhen Lei, Xia Liu, Dianjun Liu, Jiaxue Gao, Zhenxin Wang
    Abstract:

    Matrix metalloproteinases (MMPs) are believed to play an important role in tumor invasion. Herein, a Peptide Microarray-based fluorescence assay has been proposed for simultaneously determining the activities of MMP-2 and MMP-9 through the strong binding affinity of fluorescein isothiocyanate modified avidin (avidin-FITC) with the immobilized biotinylated Peptide substrate on the Microarray. In the presence of MMPs, the biotin moiety is released from the Microarray by enzymatic cleavage of the Peptide substrate, resulting in a significant decrease of the fluorescence signal. Under the optimal experimental conditions, the fluorescence intensity changes (ΔF%) are proportional to the concentrations of MMP-2 and MMP-9 within the ranges of 50 pg mL−1 to 50 ng mL−1 and 50 pg mL−1 to 100 ng mL−1 in the enzyme mixture, respectively. The detection limits are 45 pg mL−1 for MMP-2, and 60 pg mL−1 for MMP-9. In particular, the activities of extracellular MMP-2 and MMP-9 are determined by the Peptide Microarray-based fluorescence assay, and satisfactory results are obtained.

Soren Buus - One of the best experts on this subject based on the ideXlab platform.

  • hla class ii specificity assessed by high density Peptide Microarray interactions
    Journal of Immunology, 2020
    Co-Authors: Thomas Osterbye, Morten Nielsen, Nadine L Dudek, Sri H Ramarathinam, Anthony W Purcell, Claus Schafernielsen, Soren Buus
    Abstract:

    The ability to predict and/or identify MHC binding Peptides is an essential component of T cell epitope discovery, something that ultimately should benefit the development of vaccines and immunotherapies. In particular, MHC class I prediction tools have matured to a point where accurate selection of optimal Peptide epitopes is possible for virtually all MHC class I allotypes; in comparison, current MHC class II (MHC-II) predictors are less mature. Because MHC-II restricted CD4+ T cells control and orchestrated most immune responses, this shortcoming severely hampers the development of effective immunotherapies. The ability to generate large panels of Peptides and subsequently large bodies of Peptide-MHC-II interaction data are key to the solution of this problem, a solution that also will support the improvement of bioinformatics predictors, which critically relies on the availability of large amounts of accurate, diverse, and representative data. In this study, we have used rHLA-DRB1*01:01 and HLA-DRB1*03:01 molecules to interrogate high-density Peptide arrays, in casu containing 70,000 random Peptides in triplicates. We demonstrate that the binding data acquired contains systematic and interpretable information reflecting the specificity of the HLA-DR molecules investigated, suitable of training predictors able to predict T cell epitopes and Peptides eluted from human EBV-transformed B cells. Collectively, with a cost per Peptide reduced to a few cents, combined with the flexibility of rHLA technology, this poses an attractive strategy to generate vast bodies of MHC-II binding data at an unprecedented speed and for the benefit of generating Peptide-MHC-II binding data as well as improving MHC-II prediction tools.

  • hla class ii specificity assessed by high density Peptide Microarray interactions
    bioRxiv, 2020
    Co-Authors: Thomas Osterbye, Morten Nielsen, Nadine L Dudek, Sri H Ramarathinam, Anthony W Purcell, Claus Schafernielsen, Soren Buus
    Abstract:

    The ability to predict and/or identify MHC binding Peptides is an essential component of T cell epitope discovery; something that ultimately should benefit the development of vaccines and immunotherapies. In particular, MHC class I (MHC-I) prediction tools have matured to a point where accurate selection of optimal Peptide epitopes is possible for virtually all MHC-I allotypes; in comparison, current MHC class II (MHC-II) predictors are less mature. Since MHC-II restricted CD4+ T cells control and orchestrate most immune responses, this shortcoming severely hampers the development of effective immunotherapies. The ability to generate large panels of Peptides and subsequently large bodies of Peptide-MHC-II interaction data is key to the solution of this problem; a solution that also will support the improvement of bioinformatics predictors, which critically relies on the availability of large amounts of accurate, diverse and representative data. Here, we have used recombinant HLA-DRB1*01:01 and HLA-DRB1*03:01 molecules to interrogate high-density Peptide arrays, in casu containing 70,000 random Peptides in triplicates. We demonstrate that the binding data acquired contains systematic and interpretable information reflecting the specificity of the HLA-DR molecules investigated. Collectively, with a cost per Peptide reduced to a few cents combined with the flexibility of recombinant HLA technology, this poses an attractive strategy to generate vast bodies of MHC-II binding data at an unprecedented speed and for the benefit of generating Peptide-MHC-II binding data as well as improving MHC-II prediction tools.

  • arraypitope automated analysis of amino acid substitutions for Peptide Microarray based antibody epitope mapping
    PLOS ONE, 2017
    Co-Authors: Christian Skjodt Hansen, Ole Lund, Thomas Osterbye, Paolo Marcatili, Soren Buus, Morten Nielsen
    Abstract:

    : Identification of epitopes targeted by antibodies (B cell epitopes) is of critical importance for the development of many diagnostic and therapeutic tools. For clinical usage, such epitopes must be extensively characterized in order to validate specificity and to document potential cross-reactivity. B cell epitopes are typically classified as either linear epitopes, i.e. short consecutive segments from the protein sequence or conformational epitopes adapted through native protein folding. Recent advances in high-density Peptide Microarrays enable high-throughput, high-resolution identification and characterization of linear B cell epitopes. Using exhaustive amino acid substitution analysis of Peptides originating from target antigens, these Microarrays can be used to address the specificity of polyclonal antibodies raised against such antigens containing hundreds of epitopes. However, the interpretation of the data provided in such large-scale screenings is far from trivial and in most cases it requires advanced computational and statistical skills. Here, we present an online application for automated identification of linear B cell epitopes, allowing the non-expert user to analyse Peptide Microarray data. The application takes as input quantitative Peptide data of fully or partially substituted overlapping Peptides from a given antigen sequence and identifies epitope residues (residues that are significantly affected by substitutions) and visualize the selectivity towards each residue by sequence logo plots. Demonstrating utility, the application was used to identify and address the antibody specificity of 18 linear epitope regions in Human Serum Albumin (HSA), using Peptide Microarray data consisting of fully substituted Peptides spanning the entire sequence of HSA and incubated with polyclonal rabbit anti-HSA (and mouse anti-rabbit-Cy3). The application is made available at: www.cbs.dtu.dk/services/ArrayPitope.

  • automated high throughput mapping of linear b cell epitopes using a statistical analysis of high density Peptide Microarray data
    Methods of Molecular Biology, 2015
    Co-Authors: Thomas Osterbye, Soren Buus
    Abstract:

    Detailed information of antibodies' specificity is often missing or inadequate even for continuous (i.e., linear) epitopes. Recent developments in Peptide Microarray technology has enabled the synthesis of up to two million Peptides per array thereby allowing linear Peptide epitopes to be examined by a systematic amino acid substitution and positional scanning approach. This kind of analysis generates a very large body of data, which needs to be analyzed and interpreted in a robust and automated manner. Here, we describe a rational systematic approach to define linear antibody epitopes using ANOVA statistics to identify not only significant but also important residues involved in antibody recognition. This statistical approach can be used to perform a comprehensive linear epitope discovery. For polyclonal antibodies, this could be extended to entire proteins pinpointing critical residues for each epitope. We argue that the ANOVA analysis levels out issues of unknown Peptide concentration/quality and unknown antibody titers leading to identification of epitopes that otherwise would be neglected if the evaluation was based merely on signal strength.