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Antibody purification

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https://www.readbyqxmd.com/read/29216587/quantification-of-immobilized-protein-in-pharmaceutical-production-by-bio-assisted-potentiometric-multisensor-system
#1
Edita Voitechovič, Anton Korepanov, Dmitry Kirsanov, Andrey Legin
Quantification of proteins is a key biochemical assay in molecular biology, biotechnology, medicine and pharmacology. Protein quantification protocols can be based on spectrophotometry, enzyme-linked immunosorbent assay, mass spectrometry or quantitative immunoblotting depending on analyte. In case of immobilized protein these methods require suitable sample preparation. Thus, sophisticated analysis becomes even more complex, expensive and time-consuming. Such drawbacks are highly undesirable in industry. In this study we propose a new approach for evaluation of immobilized protein concentration based on application of bio-assisted potentiometric multisensor system...
December 4, 2017: Journal of Pharmaceutical and Biomedical Analysis
https://www.readbyqxmd.com/read/29213997/cloning-optimization-of-induction-conditions-and-purification-of-mycobacterium-tuberculosis-rv1733c-protein-expressed-in-escherichia-coli
#2
Mitra Ashayeri-Panah, Fereshteh Eftekhar, Bahram Kazemi, Joan Joseph
Background and Objectives: Rv1733c is a latency antigen from Mycobacterium tuberculosis, a probable integral-membrane protein with promiscuous T-cell and B-cell epitopes, making it a potential vaccine candidate against tuberculosis. This study aimed to clone and optimize the expression of recombinant Rv1733c in Escherichia coli for purification. Materials and Methods: Chemically synthesized rv1733c coding sequence was cloned in pET-23a(+) followed by transforming E...
April 2017: Iranian Journal of Microbiology
https://www.readbyqxmd.com/read/29211630/production-of-monoclonal-antibody-that-recognizes-zika-virus-and-other-flaviviruses-in-serum-free-conditions
#3
Fernando de Paiva Conte, Raquel de Souza Martins, Ana Carolina Dos Reis Albuquerque Cajaraville, Hilton Jorge Nascimento, Patricia Barbosa Jurgilas, Sheila Maria Barbosa de Lima, Sotiris Missailidis, Márcia Arissawa
With the recent outbreaks of Zika and Dengue virus infections in various countries worldwide, production of vaccines or diagnostic kits is an urgent public health demand. Production of a monoclonal antibody (mAb) that specifically binds to a common antigen shared by the Flavivirus genus will be necessary for new diagnostic kits or characterization and viral identity tests during vaccine development. This study aimed to cultivate, in serum-free conditions, the 4G2 hybridoma that produces an mAb, which recognizes a shared epitope from the Flavivirus genus...
December 6, 2017: Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
https://www.readbyqxmd.com/read/29206861/preparative-two-step-purification-of-recombinant-h1-0-linker-histone-and-its-domains
#4
Nives Ivic, Silvija Bilokapic, Mario Halic
H1 linker histones are small basic proteins that have a key role in the formation and maintenance of higher-order chromatin structures. Additionally, many examples have shown that linker histones play an important role in gene regulation, modulated by their various subtypes and posttranslational modifications. Obtaining high amounts of very pure linker histones, especially for efficient antibody production, remains a demanding and challenging procedure. Here we present an easy and fast method to purify human linker histone H1...
2017: PloS One
https://www.readbyqxmd.com/read/29202520/-expression-and-purification-of-rabies-virus-glycoprotein-and-analysis-of-its-specific-binding-capacity-to-memory-b-cells
#5
Liwei Yan, Wei Gong, Wenbing Zhu, Xuemei Zhang, Jingwen Xu, Zhongxiang Wu, Kongjie Lu, Ming Sun, Shaozhong Dong
We aimed to express and purify three rabies virus glycoproteins with different tags and sizes. After analyzing their binding function, we wish to obtain a rabies virus glycoprotein with higher affinity and ability to specifically bind memory B cells. Experiments were carried out to express full length, as well as the ectodomain RVG by gene engineering method. Combined with the antibody of CD19 and CD27, the candidate protein labeling with fluorescence was used to analyze its binding function. Flow cytometry was used to detect the anti-rabies virus specific memory B cells in PBMCs, and confirm the binding ability between the candidate proteins and anti-rabies virus-specific memory B cells...
November 25, 2017: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
https://www.readbyqxmd.com/read/29196601/immunoprecipitation
#6
James DeCaprio, Thomas O Kohl
This immunoprecipitation protocol details individual steps for the enrichment and purification process of specific proteins from a complex cell lysate using an antibody bound to a solid matrix. Purified antigen(s) can be eluted by various methods, and the resultant protein target can be analyzed and/or identified by numerous assays, including the enzyme-linked immunosorbent assay (ELISA), western blotting, or mass spectrometry.
December 1, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29193902/bispecific-antibody-process-development-assembly-and-purification-of-knob-and-hole-bispecific-antibodies
#7
Glen Giese, Ambrose Williams, Maricel Rodriguez, Josefine Persson
Production of knob and hole dual light chain bispecific antibodies poses several unique challenges for development of a feasible industrial scale manufacturing process. We developed an efficient process for the assembly and purification of knob and hole dual light chain bispecific antibodies. Two distinct half-antibodies targeting two different antigens were expressed separately in E. coli cells and captured independently using Protein A chromatography. When combined, the knob and hole mutations in the CH3 domains promoted heterodimer formation...
November 29, 2017: Biotechnology Progress
https://www.readbyqxmd.com/read/29192002/ms-western-a-method-of-multiplexed-absolute-protein-quantification-is-a-practical-alternative-to-western-blotting
#8
Mukesh Kumar, Shai R Joseph, Martina Augsburg, Aliona Bogdanova, David Drechsel, Nadine L Vastenhouw, Frank Buchholz, Marc Gentzel, Andrej Shevchenko
Absolute quantification of proteins elucidates the molecular composition, regulation and dynamics of multiprotein assemblies and networks. Here we report on a method termed MS Western that accurately determines the molar abundance of dozens of user-selected proteins at the sub-femtomole level in whole cell or tissue lysates without metabolic or chemical labelling and without using specific antibodies. MS Western relies upon GeLC-MS/MS and quantifies proteins by in-gel co-digestion with an isotopically labelled QconCAT protein chimera composed of concatenated proteotypic peptides...
November 30, 2017: Molecular & Cellular Proteomics: MCP
https://www.readbyqxmd.com/read/29187229/optimal-rna-isolation-method-and-primer-design-to-detect-gene-knockdown-by-qpcr-when-validating-drosophila-transgenic-rnai-lines
#9
Roslyn L Mainland, Taylor A Lyons, Mike M Ruth, Jamie M Kramer
OBJECTIVE: RNA interference is employed extensively in Drosophila research to study gene function within a specific cell-type or tissue. Thousands of transgenic Drosophila lines have been generated to express double stranded RNA for gene knockdown; however, no standardized method exists for quantifying their knockdown efficiency. Since antibodies are not available for many proteins, quantitative real-time PCR is often used. Here, we explore how primer design and RNA isolation method can influence detection of gene knockdown using qPCR...
November 29, 2017: BMC Research Notes
https://www.readbyqxmd.com/read/29178403/n-glycan-engineering-of-a-plant-produced-anti-cd20-hil-2-immunocytokine-significantly-enhances-its-effector-functions
#10
Carla Marusic, Claudio Pioli, Szymon Stelter, Flavia Novelli, Chiara Lonoce, Elena Morrocchi, Eugenio Benvenuto, Anna Maria Salzano, Andrea Scaloni, Marcello Donini
Anti-CD20 recombinant antibodies are among the most promising therapeutics for the treatment of B-cell malignancies such as non-Hodgkin lymphomas. We recently demonstrated that an immunocytokine (2B8-Fc-hIL2), obtained by fusing an anti-CD20 scFv-Fc antibody derived from C2B8 mAb (rituximab) to the human interleukin 2 (hIL-2), can be efficiently produced in Nicotiana benthamiana plants. The purified immunocytokine (IC) bearing a typical plant protein N-glycosylation profile showed a CD20 binding activity comparable to that of rituximab and was efficient in eliciting antibody-dependent cell-mediated cytotoxicity (ADCC) of human PBMC against Daudi cells, indicating its fuctional integrity...
November 27, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/29172900/development-of-a-high-yield-expression-and-purification-system-for-platelet-factor-4
#11
Angela Huynh, Donald M Arnold, Jane C Moore, James W Smith, John G Kelton, Ishac Nazy
Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction characterized by IgG antibodies bound to complexes of platelet factor 4 (PF4) and heparin. The majority of diagnostic tests for HIT rely on an exogenous source of PF4 to identify anti-PF4/heparin antibodies. These include the PF4-dependent enhanced serotonin release assay (PF4-SRA) among others. Using a bacterial expression system, we developed a novel and efficient method of producing recombinant human PF4 (rhPF4) that is biochemically and antigenically similar to platelet-derived human PF4...
November 27, 2017: Platelets
https://www.readbyqxmd.com/read/29172059/hydroxamate-column-based-purification-of-zirconium-89-89zr-using-an-automated-fluidic-platform
#12
Matthew J O'Hara, Nathaniel J Murray, Jennifer C Carter, Cynthia M Kellogg, Jeanne M Link
Zirconium-89 (89Zr) is a long-lived (t1/2 = 78.4h) positron-emitting isotope that is useful for positron emission tomography (PET) based diagnostic imaging using radiolabeled antibodies. Hydroxamate resin columns are predominantly used for the purification of 89Zr from cyclotron bombarded natY targets dissolved in strong HCl. 89Zr is conventionally eluted from the resin in 1M oxalic acid (H2C2O4), a complexant that is conducive to follow-on binding of 89Zr through a transchelation process to the deferoxamine siderophore...
November 21, 2017: Applied Radiation and Isotopes
https://www.readbyqxmd.com/read/29169803/expression-purification-and-characterization-of-two-leucine-aminopeptidases-of-the-blood-fluke-schistosoma-mansoni
#13
Gabriela Maggioli, Gabriel Rinaldi, Ines Giaudrone, Patricia Berasain, José F Tort, Paul J Brindley, Carlos Carmona
Schistosomiasis is a major neglected tropical disease (NTD) and considered the most important of the human helminthiases in terms of morbidity and mortality. Whereas treatment with praziquantel has been effective since the 1980s, the potential for the emergence of drug resistance has propelled the search for new interventions. Studies have revealed key roles of proteases in parasitic helminths during establishment of infection, tissue invasion, immune evasion, parasite feeding and development throughout the different developmental stages, pinpointing them as possible candidates...
November 20, 2017: Molecular and Biochemical Parasitology
https://www.readbyqxmd.com/read/29161226/development-of-competitive-enzyme-linked-immunosorbent-assays-for-antibody-detection-based-on-bluetongue-virus-monoclonal-antibodies
#14
Zhou Han, Zang Mingxin, Li Xuechun, Xu Yigang, Qiao Xinyuan, Wang Li, Cui Wen, Jiang Yanping, Li Yijing, Tang Lijie
Bluetongue is a ruminant infectious disease that is characterized by hyperpyrexia, leukocyte decrease and mucosal ulcerative inflammation changes. In this study, three segments of Bluetongue virus (BTV)-8 VP2 protein (BTV-8A, 8B, and 8C) were cloned into pET-28a (+) and pMAl-c5X vectors and expressed in Escherichia coli BL21 (DE3) as histidine (His)-tagged (His-8A/8B/8C) and maltose-binding protein (MBP)-tagged (MBP-8A/8B/8C) fusion proteins. After purification, His-8A/8B/8C were used to immunize BALB/mice and MBP-8A/8B/8C were used to screen for monoclonal antibody (mAb)-secreting hybridomas...
November 21, 2017: Viral Immunology
https://www.readbyqxmd.com/read/29159220/data-on-atherosclerosis-specific-antibody-conjugation-to-nanoemulsions
#15
Geoffrey Prévot, Martine Duonor-Cérutti, Mélusine Larivière, Jeanny Laroche-Traineau, Marie Josée Jacobin-Valat, Philippe Barthélémy, Gisèle Clofent-Sanchez, Sylvie Crauste-Manciet
This article present data related to the publication entitled "Iron oxide core oil-in-water nanoemulsion as tracer for atherosclerosis MPI and MRI imaging" (Prévot et al., 2017) [1]. Herein we describe the engineering in the baculovirus-insect cell system and purification processes of the human scFv-Fc TEG4-2C antibody, specific of platelets within the atheroma plaque. For molecular targeting purpose, atheroma specific antibody was conjugated to nanoemulsions (NEs) using a heterobifunctional linker (DSPE-PEG-maleimide)...
December 2017: Data in Brief
https://www.readbyqxmd.com/read/29146152/improved-antifungal-activity-of-barley-derived-chitinase-i-gene-that-overexpress-a-32kda-recombinant-chitinase-in-escherichia-coli-host
#16
Nida Toufiq, Bushra Tabassum, Muhammad Umar Bhatti, Anwar Khan, Muhammad Tariq, Naila Shahid, Idrees Ahmad Nasir, Tayyab Husnain
Agricultural crops suffer many diseases, including fungal and bacterial infections, causing significant yield losses. The identification and characterisation of pathogenesis-related protein genes, such as chitinases, can lead to reduction in pathogen growth, thereby increasing tolerance against fungal pathogens. In the present study, the chitinase I gene was isolated from the genomic DNA of Barley (Hordeum vulgare L.) cultivar, Haider-93. The isolated DNA was used as template for the amplification of the ∼935bp full-length chitinase I gene...
October 31, 2017: Brazilian Journal of Microbiology: [publication of the Brazilian Society for Microbiology]
https://www.readbyqxmd.com/read/29144195/extraction-purification-of-prostate-specific-antigen-psa-and-establishment-of-radioimmunoassay-system-as-a-diagnostic-tool-for-prostate-disorders
#17
Ahmed Sami Abu-Bakr El-Bayoumy, Akaber Tarek Hessien Keshta, Khaled Mohamed Sallam, Nahed Hassan Ebeid, Hatem Mohamed Elsheikh, Bsheer El-Sayed Bayoumy
This study aimed to provide an easy and effective method for extraction and purification of prostate specific antigen (PSA) from human seminal fluid with high quantity (14 mg) and high purity (98%). The obtained PSA was injected into rabbits for production of anti-PSA polyclonal antibody (titer 1/1000), labeled with radioactive iodine-125 for preparation of radioactive PSA tracer (purity 98 ± 1.8% and specific activity 64 ± 1.9 µCi/µg), and used in preparation of PSA standards. All prepared components can be used in PSA immunoassays specially radioimmunoassay (RIA) kit preparation as a diagnostic tool for prostatic diseases...
November 16, 2017: Journal of Immunoassay & Immunochemistry
https://www.readbyqxmd.com/read/29138497/rapid-purification-of-human-bispecific-antibodies-via-selective-modulation-of-protein-a-binding
#18
Adam Zwolak, Catherine N Leettola, Susan H Tam, Dennis R Goulet, Mehabaw G Derebe, Jose R Pardinas, Songmao Zheng, Rose Decker, Eva Emmell, Mark L Chiu
Methods to rapidly generate high quality bispecific antibodies (BsAb) having normal half-lives are critical for therapeutic programs. Here, we identify 3 mutations (T307P, L309Q, and Q311R or "TLQ") in the Fc region of human IgG1 which disrupt interaction with protein A while enhancing interaction with FcRn. The mutations are shown to incrementally alter the pH at which a mAb elutes from protein A affinity resin. A BsAb comprised of a TLQ mutant and a wild-type IgG1 can be efficiently separated from contaminating parental mAbs by differential protein A elution starting from either a) purified parental mAbs, b) in-supernatant crossed parental mAbs, or c) co-transfected mAbs...
November 14, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29132905/purification-and-biochemical-characterization-of-a-22-kda-stable-cysteine-like-protease-from-the-excretory-secretory-product-of-the-liver-fluke-fasciola-hepatica-by-using-conventional-techniques
#19
Ahmed Hemici, Roumaila Sabrina Benerbaiha, Dalila Bendjeddou
This study describes the purification and characterization of a stable protease activity isolated from Fasciola hepatica adult worms maintained in vitro by employing acetone precipitation (40-60%) followed by a gel filtration through Sephadex G-100 and DEAE- cellulose ion exchange column. Through this three-step purification, the enzyme was purified 11-fold with a specific activity of 1893.9U/mg and 31.5% recovery. After the final ultrafiltration step, the purification fold was increased up to 13.1 and the overall activity yield reached a rate of 18...
October 28, 2017: Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
https://www.readbyqxmd.com/read/29126399/production-and-purification-of-chimeric-hbc-virus-like-particles-carrying-influenza-virus-lah-domain-as-vaccine-candidates
#20
Andris Kazaks, I-Na Lu, Sophie Farinelle, Alex Ramirez, Vincenzo Crescente, Benjamin Blaha, Olotu Ogonah, Tarit Mukhopadhyay, Mapi Perez de Obanos, Alejandro Krimer, Inara Akopjana, Janis Bogans, Velta Ose, Anna Kirsteina, Tatjana Kazaka, Nicola J Stonehouse, David J Rowlands, Claude P Muller, Kaspars Tars, William M Rosenberg
BACKGROUND: The lack of a universal influenza vaccine is a global health problem. Interest is now focused on structurally conserved protein domains capable of eliciting protection against a broad range of influenza virus strains. The long alpha helix (LAH) is an attractive vaccine component since it is one of the most conserved influenza hemagglutinin (HA) stalk regions. For an improved immune response, the LAH domain from H3N2 strain has been incorporated into virus-like particles (VLPs) derived from hepatitis B virus core protein (HBc) using recently developed tandem core technology...
November 10, 2017: BMC Biotechnology
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