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Antibody purification

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https://www.readbyqxmd.com/read/28534915/quantum-dot-based-sensitive-detection-of-disease-specific-exosome-in-serum
#1
Kseniia Boriachek, Md Nazmul Islam, Vinod Gopalan, Alfred K Lam, Nam-Trung Nguyen, Muhammad J A Shiddiky
Tumor-derived exosomes have emerged as promising cancer biomarkers due to their unique composition and functions. Herein, we report a stripping voltammetric immunoassay for the electrochemical detection of disease-specific exosomes using quantum dots as signal amplifiers. The assay involves three subsequent steps where bulk exosome populations are initially magnetically captured on magnetic beads by a generic tetraspanin antibody (e.g., CD9 or CD63) followed by the identification of disease-specific exosomes using cancer-related...
May 23, 2017: Analyst
https://www.readbyqxmd.com/read/28533942/hla-a24-ligandome-analysis-of-colon-and-lung-cancer-cells-identifies-a-novel-cancer-testis-antigen-and-a-neoantigen-that-elicits-specific-and-strong-ctl-responses
#2
Vitaly Kochin, Takayuki Kanaseki, Serina Tokita, Sho Miyamoto, Yosuke Shionoya, Yasuhiro Kikuchi, Daichi Morooka, Yoshihiko Hirohashi, Tomohide Tsukahara, Kazue Watanabe, Shingo Toji, Yasuo Kokai, Noriyuki Sato, Toshihiko Torigoe
This study focused on HLA-A24 and comprehensively analyzed the ligandome of colon and lung cancer cells without the use of MHC-binding in silico prediction algorithms. Affinity purification using the antibody specific to HLA-A24 followed by LC-MS/MS sequencing was used to detect peptides, which harbored the known characteristics of HLA-A24 peptides in terms of length and anchor motifs. Ligandome analysis demonstrated the natural presentation of two different types of novel tumor-associated antigens. The ligandome contained a peptide derived from SUV39H2, a gene found to be expressed in a variety of cancers but not in normal tissues (except for the testis)...
2017: Oncoimmunology
https://www.readbyqxmd.com/read/28508217/bac-based-recovery-of-recombinant-respiratory-syncytial-virus-rsv
#3
Christopher C Stobart, Anne L Hotard, Jia Meng, Martin L Moore
Respiratory syncytial virus (RSV) is an enveloped, nonsegmented negative-strand RNA virus, which causes lower respiratory tract infections and is a leading cause of mortality in young infants. There is no available RSV vaccine and currently administered prophylactic antibodies are limited to high-risk populations. Current efforts to develop vaccines include development of live-attenuated RSV candidates. We describe here methods for preparation and recovery of recombinant RSV using an efficient bacterial artificial chromosome (BAC)-based system, expansion and plaque purification of recovered virus, and generation of master and working stocks...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28506354/-immunoprotective-effect-of-combined-pneumococcal-endopeptidase-o-and-pneumococcal-surface-adhesin-a-vaccines-against-streptococcus-pneumoniae-infection
#4
Jing Zhang, Ya-Li Cui, Yong-Mei Jiang
OBJECTIVE: To investigate the prokaryotic expression of proteins pneumococcal endopeptidase O (PepO) and pneumococcal surface adhesin A (PsaA) in Streptococcus pneumoniae and their immunoprotective effect as vaccine candidate proteins. METHODS: Specific primers of target gene fragments were designed, and then PCR amplification was performed to establish recombinant plasmids pET28a(+)-pepO and pET28a(+)-psaA, which were transformed into host cells, Escherichia coli BL21 and DE3, respectively, to induce expression...
May 2017: Zhongguo Dang Dai Er Ke za Zhi, Chinese Journal of Contemporary Pediatrics
https://www.readbyqxmd.com/read/28496951/can-aptameric-ligands-specific-to-plasma-coagulation-factor-vii-bind-the-recombinant-form-with-high-affinity-affinity-measurement-by-fluorescence-method
#5
Maryam Tabarzad, Marzieh Jafari, Nastaran Nafissi-Varcheh
BACKGROUND: Among diverse protein purification systems, affinity chromatography is the most attractive one in the purification process of coagulation factors. Coagulation factor VII is a plasma serine protease that has a significant role in natural human hemostasis and its recombinant form such as AryoSeven(™), has been applied in clinical treatment of bleeding disorders. Immunoaffinity chromatography is the purification method of choice that is currently applied in the development of coagulation factor VIIa products...
April 2017: Avicenna Journal of Medical Biotechnology
https://www.readbyqxmd.com/read/28492063/vaccination-with-killed-but-metabolically-active-e-coli-over-expressing-hemagglutinin-elicits-neutralizing-antibodies-to-h1n1-swine-origin-influenza-a-virus
#6
Pei-Feng Liu, Yanhan Wang, Yu-Tsueng Liu, Chun-Ming Huang
There is a need for a fast and simple method for vaccine production to keep up with the pace of a rapidly spreading virus in the early phases of the influenza pandemic. The use of whole viruses produced in chicken eggs or recombinant antigens purified from various expression systems has presented considerable challenges, especially with lengthy processing times. Here, we use the killed but metabolically active (KBMA) Escherichia coli (E. coli) to harbor the hemagglutinin (HA) of swine origin influenza A (H1N1) virus (S-OIV) San Diego/01/09 (SD/H1N1-S-OIV)...
February 2017: Journal of Nature and Science
https://www.readbyqxmd.com/read/28487989/purification-of-a-polyclonal-antibody-against-cd147-for-elisa-using-antigen%C3%A2-immunoaffinity-chromatography
#7
Shuangshuang Liu, Shasha Li, Yang Zhang, Ye Wang, Yumeng Zhu, Bin Wang, Zhi-Nan Chen
The immunoglobulin superfamily member CD147 is a widely expressed glycoprotein that occurs in both a membrane‑spanning and soluble form. Sandwich ELISA is a powerful tool for analyzing soluble antigens. The aim of the present study was to obtain a highly specific polyclonal antibody against human CD147 that can be used for sandwich ELISA analysis. Expression of recombinant CD147 by a eukaryotic expression system was used to immunize rabbits to obtain antiserum. A highly specific polyclonal antibody that was able to detect soluble CD147 in sandwich ELISA was obtained by antigen‑immunoaffinity chromatography purification...
June 2017: Molecular Medicine Reports
https://www.readbyqxmd.com/read/28477201/isolation-and-purification-of-th9-cells-for-the-study-of-inflammatory-diseases-in-research-and-clinical-settings
#8
Patricia Keating, James X Hartmann
Th9 cells are associated with atopic and inflammatory diseases, and their increased levels and function correlate with the severity of symptoms in various inflammatory disorders including asthma, food allergy, atopic dermatitis, ulcerative colitis, and psoriatic arthritis. Thus, clinical trials are warranted to evaluate the role of Th9 cells in allergic diseases with the goal of controlling these ailments.Circulating T cells (naïve or memory CD4+ T cells) purified from human blood and expanded using anti-CD3 and anti-CD28 antibodies can be treated with appropriate cytokines in order to polarize them to the Th9 phenotype as evidenced by their production of IL-9...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28475929/production-of-pure-fractions-of-immunoglobulin-g-subclass-autoantibodies-against-tumor-necrosis-factor
#9
Fedor D Kireev, Julia A Lopatnikova, Sergey V Sennikov
Autoantibodies directed against cytokines are important effector molecules regulating the biological activity of cytokines. There is experimental evidence indicating that autoantibodies belonging to different immunoglobulin G (IgG) subclasses may have different functional activity. The purpose of this work was to develop a protocol for the purification of fractions of IgG subclass antibodies directed against tumor necrosis factor (TNF). We developed a series of steps, including gel filtration, positive and negative affinity chromatography, and ultrafiltration, to achieve this goal...
April 24, 2017: Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
https://www.readbyqxmd.com/read/28473885/prospects-and-limitations-of-antibody-mediated-clearing-of-lipoproteins-from-blood-plasma-prior-to-nanoparticle-tracking-analysis-of-extracellular-vesicles
#10
Morten Mørk, Aase Handberg, Shona Pedersen, Malene M Jørgensen, Rikke Bæk, Morten K Nielsen, Søren R Kristensen
Introduction: Nanoparticle tracking analysis (NTA) enables measurement of extracellular vesicles (EVs) but lacks the ability to distinct between EVs and lipoproteins which are abundantly present in blood plasma. Limitations in ultracentrifugation and size exclusion chromatography applied for EV isolation may result in inadequate EV purification and preservation. In this proof of concept study, we aimed to evaluate the potential of antibody-mediated removal of lipoproteins from plasma prior to extracellular vesicle (EV) analysis by nanoparticle tracking analysis (NTA)...
2017: Journal of Extracellular Vesicles
https://www.readbyqxmd.com/read/28471227/from-in-silico-immunogenicity-verification-to-in-vitro-expression-of-recombinant-core-ns3-fusion-protein-of-hcv
#11
S Hekmat, S D Siadat, M R Aghasadeghi, S M Sadat, G Bahramali, M M Aslani, M Mahdavi, S Shahbazi
BACKGROUND AND OBJECTIVE: Hepatitis C virus (HCV) is a serious global health burden. There is no effective vaccine against HCV and new direct acting antivirals (DAAs) are so expensive and virtually unavailable to the public. Therefore, seeking for therapeutic or prophylactic vaccines is exigent and reliever. METHODS: The secondary and tertiary structures of the recombinant Core-NS3 (rC-N) fusion protein of HCV and its B and T-cells epitopes were evaluated with bioinformatics software...
2017: Bratislavské Lekárske Listy
https://www.readbyqxmd.com/read/28470603/a-generic-protocol-for-purifying-disulfide-bonded-domains-and-random-protein-fragments-using-fusion-proteins-with-sumo3-and-cleavage-by-senp2-protease
#12
Hüseyin Besir
Recombinant expression of heterologous proteins in E. coli is well established for a wide range of proteins, although in many cases, purifying soluble and properly folded proteins remains challenging (Sorensen and Mortensen, J Biotechnol 115:113-128, 2005; Correa and Oppezzo, Methods Mol Biol 1258:27-44, 2015). Proteins that contain disulfide bonds (e.g., cytokines, growth factors) are often particularly difficult to purify in soluble form and still need optimizing of protocols in almost every step of the process (Berkmen, Protein Expr Purif 82:240-251, 2012; de Marco, Microb Cell Fact 11:129, 2012)...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28465147/immunoprotection-induced-by-cpg-odn-poly-i-c-combined-with-recombinant-gp90-protein-in-chickens-against-reticuloendotheliosis-virus-infection
#13
Fei Yuan, Ying Chu, Lihong Qi, Hongmei Li, Shuhong Sun, Peng Zhao, Shuang Chang, Huijun Guo
The present study is focused on investigating the immunoprotective effects of CpG-ODN/Poly(I:C) combined with the viral glycoprotein gp90 protein against reticuloendotheliosis virus (REV) infection in chickens. REV's gp90 gene was amplified from the REV-infected cells and expressed in Escherichia coli (E.coli). The expressed products, upon purification, were inoculated into 7-day-old chickens with PBS, CpG-ODN or Poly(I:C) adjuvant; Two booster inoculations were then conducted, and then each chicken was challenged...
April 29, 2017: Antiviral Research
https://www.readbyqxmd.com/read/28464293/amb-a-1-isoforms-unequal-siblings-with-distinct-immunological-features
#14
Martin Wolf, Teresa E Twaroch, Sara Huber, Manuel Reithofer, Markus Steiner, Lorenz Aglas, Michael Hauser, Iris Aloisi, Claudia Asam, Heidi Hofer, Maria A Parigiani, Christof Ebner, Barbara Bohle, Peter Briza, Neubauer Angela, Frank Stolz, Beatrice Jahn-Schmid, Michael Wallner, Fatima Ferreira
BACKGROUND: Ragweed pollen represents a major allergy risk factor. Ragweed extracts contain five different isoforms of the major allergen Amb a 1. However, the immunologic characteristics of Amb a 1 isoforms are not fully investigated. Here we compared the physicochemical and immunological properties of three most important Amb a 1 isoforms. METHODS: After purification, the isoforms were physicochemically characterized, tested for antibody-binding and induction of human T cell proliferative responses...
May 2, 2017: Allergy
https://www.readbyqxmd.com/read/28458052/production-of-monoclonal-antibodies-and-development-of-a-quantitative-immuno-polymerase-chain-reaction-assay-to-detect-and-quantify-recombinant-glutathione-s-transferase
#15
Abud Je, Luque Eh, Ramos Jg, Rodriguez Ha
GST-tagged proteins are important tools for the production of recombinant proteins. Removal of GST tag from its fusion protein, frequently by harsh chemical treatments or proteolytic methods, is often required. Thus, the monitoring of the proteins in tag-free form requires a significant effort to determine the remnants of GST during purification process. In the present study, we developed both a conventional enzyme-linked immunosorbent assay (ELISA) and an immuno-polymerase chain reaction (IPCR) assay, both specific for detection of recombinant GST (rGST)...
April 27, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28456726/optimization-of-the-production-process-and-characterization-of-the-yeast-expressed-sars-cov-recombinant-receptor-binding-domain-rbd219-n1-a-sars-vaccine-candidate
#16
Wen-Hsiang Chen, Shivali M Chag, Mohan V Poongavanam, Amadeo B Biter, Ebe A Ewere, Wanderson Rezende, Christopher A Seid, Elissa M Hudspeth, Jeroen Pollet, C Patrick McAtee, Ulrich Strych, Maria Elena Bottazzi, Peter J Hotez
From 2002 to 2003, a global pandemic of severe acute respiratory syndrome (SARS) spread to five continents and caused 8,000 respiratory infections and 800 deaths. To ameliorate the effects of future outbreaks as well as to prepare for biodefense, a process for the production of a recombinant protein vaccine candidate is under development. Previously, we reported the 5-L scale expression and purification of a promising recombinant SARS vaccine candidate, RBD219-N1, the 218-amino acid residue receptor-binding domain (RBD) of SARS coronavirus expressed in yeast- Pichia pastoris X-33...
April 26, 2017: Journal of Pharmaceutical Sciences
https://www.readbyqxmd.com/read/28450393/immunoglobulin-domain-interface-exchange-as-a-platform-technology-for-the-generation-of-fc-heterodimers-and-bispecific-antibodies
#17
Darko Skegro, Cian Stutz, Romain Ollier, Emelie Svensson, Paul Wassmann, Bourquin Florence, Thierry Monney, Sunitha Gn, Stanislas Blein
Bispecific antibodies (bsAbs) are of significant importance to the development of novel antibody-based therapies, and heavy chain (Hc) heterodimers represent a major class of bispecific drug candidates. Current technologies for the generation of Hc heterodimers are suboptimal and often suffer from contamination by homodimers posing purification challenges. Here, we introduce a new technology based on biomimicry wherein the protein-protein interfaces of two different immunoglobulin (Ig) constant domain pairs are exchanged in part or fully to design new heterodimeric domains...
April 27, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28447981/isolation-characterization-and-purification-of-macrophages-from-tissues-affected-by-obesity-related-inflammation
#18
Joselyn N Allen, Adwitia Dey, Ruth Nissly, James Fraser, Shan Yu, Gayathri Balandaram, Jeffrey M Peters, Pamela A Hankey-Giblin
Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention...
April 3, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28445010/detection-of-serum-antibodies-to-hepatitis-e-virus-based-on-hev-genotype-3-orf2-capsid-protein-expressed-in-nicotiana-benthamiana
#19
Milena Mazalovska, Nikola Varadinov, Tsvetoslav Koynarski, Ivan Minkov, Pavel Teoharov, George P Lomonossoff, Gergana Zahmanova
BACKGROUND: Hepatitis E virus (HEV) causes epidemics in developing countries and is primarily transmitted through the fecal-oral route. There have been recent reports on the zoonotic spread of the virus, and several animal species, primarily pigs, have been recognized as reservoirs of HEV. Because of its possible spread, there is an urgent need of a method for the cost-effective production of HEV proteins that can be used as diagnostic antigens for the serological detection of anti-HEV antibodies...
July 2017: Annals of Laboratory Medicine
https://www.readbyqxmd.com/read/28439867/mapping-protein-protein-interactions-using-affinity-purification-and-mass-spectrometry
#20
Chin-Mei Lee, Christopher Adamchek, Ann Feke, Dmitri A Nusinow, Joshua M Gendron
The mapping of protein-protein interaction (PPI) networks and their dynamics are crucial steps to deciphering the function of a protein and its role in cellular pathways, making it critical to have comprehensive knowledge of a protein's interactome. Advances in affinity purification and mass spectrometry technology (AP-MS) have provided a powerful and unbiased method to capture higher-order protein complexes and decipher dynamic PPIs. However, the unbiased calling of nonspecific interactions and the ability to detect transient interactions remains challenging when using AP-MS, thereby hampering the detection of biologically meaningful complexes...
2017: Methods in Molecular Biology
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