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Antibody purification

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https://www.readbyqxmd.com/read/28646324/expression-purification-and-characterization-of-gmz2-10c-a-complex-disulphide-bonded-fusion-protein-vaccine-candidate-against-the-asexual-and-sexual-life-stages-of-the-malaria-causing-plasmodium-falciparum-parasite
#1
Ulrik H Mistarz, Susheel K Singh, Tam T T N Nguyen, Will Roeffen, Fen Yang, Casper Lissau, Søren M Madsen, Astrid Vrang, Régis W Tiendrebeogo, Ikhlaq H Kana, Robert W Sauerwein, Michael Theisen, Kasper D Rand
PURPOSE: Production and characterization of a chimeric fusion protein (GMZ2'.10C) which combines epitopes of key malaria parasite antigens: glutamate-rich protein (GLURP), merozoite surface protein 3 (MSP3), and the highly disulphide bonded Pfs48/45 (10C). GMZ2'.10C is a potential candidate for a multi-stage malaria vaccine that targets both transmission and asexual life-cycle stages of the parasite. METHODS: GMZ2'.10C was produced in Lactococcus lactis and purified using either an immunoaffinity purification (IP) or a conventional purification (CP) method...
June 23, 2017: Pharmaceutical Research
https://www.readbyqxmd.com/read/28644752/expression-and-purification-of-swine-rag2-in-e-coli-for-production-of-porcine-rag2-polyclonal-antibodies
#2
Yu-Bei Jin, Wen-Tao Yang, Ke-Yan Huang, Hong-Liang Chen, Seria-Masole Shonyela, Jing Liu, Qiong Liu, Bo Feng, You Zhou, Shu-Li Zhi, Yan-Long Jiang, Jian-Zhong Wang, Hai-Bin Huang, Chun-Wei Shi, Gui-Lian Yang, Chun-Feng Wang
Recombination activating gene 2 (RAG2) is necessary for immature B cell differentiation. Antibodies to human and rabbit RAG2 are currently commercially available, but antibodies to swine RAG remain unavailable to date. In this study, the swine RAG2 genes sequence was synthesized and then cloned into a pET-28a vector. The recombinant fusion protein was successfully expressed in E. coli, purified through nickel column chromatography, and further digested with Tobacco Etch Virus protease. The cleaved protein was purified by molecular-exclusion chromatography and named pRAG2...
June 23, 2017: Bioscience, Biotechnology, and Biochemistry
https://www.readbyqxmd.com/read/28640955/in-solution-antibody-harvesting-with-a-plant-produced-hydrophobin-protein-a-fusion
#3
Katri Kurppa, Lauri Reuter, Anneli Ritala, Markus Linder, Jussi Joensuu
Purification is a bottleneck and a major cost factor in the production of antibodies. We set out to engineer a bi-functional fusion protein from two building blocks, Protein A and a hydrophobin, aiming at low-cost and scalable antibody capturing in solutions. Immunoglobulin-binding Protein A is widely used in affinity-based purification. The hydrophobin fusion tag, on the other hand, has been shown to enable purification by two-phase separation. Protein A was fused to two different hydrophobin tags, HFBI or II, and expressed transiently in Nicotiana benthamiana...
June 22, 2017: Plant Biotechnology Journal
https://www.readbyqxmd.com/read/28635559/purification-of-antibody-against-ara-h-2-by-a-home-made-immunoaffinity-chromatography-column
#4
Zhihua Wu, Kun Li, Shaode Zhan, Ping Tong, Xin Li, Anshu Yang, Hongbing Chen
Antibodies are used extensively in numerous applications both in vivo and in vitro. To purify anti-Ara h 2 polyclonal antibody (PcaAb), a home-made immunoaffinity chromatography column (IAC) method was established. The properties of the home-made column were compared with those of the Mab Affinity Protein G Agarose High Flow (MPG), a commercially available column successfully used in capturing polyclonal antibodies. During antibody purification from rabbits antiserum against Ara h 2, the column capacity, recovery, and purification factor were characterized for IAC and MPG...
June 21, 2017: Preparative Biochemistry & Biotechnology
https://www.readbyqxmd.com/read/28630463/the-role-of-antibody-v%C3%AE%C2%BA-framework-3-region-towards-antigen-binding-effects-on-recombinant-production-and-protein-l-binding
#5
Chinh Tran-To Su, Wei-Li Ling, Wai-Heng Lua, Jun-Jie Poh, Samuel Ken-En Gan
Antibody research has traditionally focused on heavy chains, often neglecting the important complementary role of light chains in antibody formation and secretion. In the light chain, the complementarity-determining region 3 (VL-CDR3) is specifically implicated in disease states. By modulating VL-CDR3 exposure on the scaffold through deletions in the framework region 3 (VL-FWR3), we further investigated the effects on secretion in recombinant production and antigen binding kinetics. Our random deletions of two residues in the VL-FWR3 of a Trastuzumab model showed that the single deletions could impact recombinant production without significant effect on Her2 binding...
June 19, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28627567/dissecting-binding-of-a-%C3%AE-barrel-membrane-protein-by-phage-display
#6
Luz M Meneghini, Sarvind Tripathi, Marcus A Woodworth, Sudipta Majumdar, Thomas L Poulos, Gregory A Weiss
Membrane proteins (MPs) constitute a third of all proteomes, and contribute to a myriad of cellular functions including intercellular communication, nutrient transport and energy generation. For example, TonB-dependent transporters (TBDTs) in the outer membrane of Gram-negative bacteria play an essential role transporting iron and other nutrients into the bacterial cell. The inherently hydrophobic surfaces of MPs complicates protein expression, purification, and characterization. Thus, dissecting the functional contributions of individual amino acids or structural features through mutagenesis can be a challenging ordeal...
June 19, 2017: Molecular BioSystems
https://www.readbyqxmd.com/read/28623675/development-of-a-prototype-immunochromatographic-test-for-rapid-diagnosis-of-respiratory-adenovirus-infection
#7
Inarei Paulini, Joselma Siqueira-Silva, Luciana Thomaz, Leticia Rocha, Charlotte Harsi, Nancy Bellei, Celso Granato
Human adenoviruses comprise an important group of etiologic agents that are responsible for various diseases in adults and children, such as respiratory, ocular, gastroenteric, and urinary infections. In immunocompromised and organ-transplanted individuals, these agents can cause generalized infections. Rapid diagnostic methods for detecting these infectious agents are not widely available. The aim of this work was to produce monoclonal and polyclonal anti-adenovirus antibodies to be used in a rapid diagnostic test for respiratory infections...
June 14, 2017: Brazilian Journal of Infectious Diseases
https://www.readbyqxmd.com/read/28600648/secretory-expression-of-a-novel-human-spermatozoa-antigen-in-e-coli-and-its-application-to-a-protein-chip
#8
Ming-Zhu Wang, Zhuo-Lin Qiu, Xiang-Sheng Cai, Jing-Jing Li, Miao-Qin She, Yuan-Feng Xu, Ying-Song Wu
OBJECTIVE: To produce a recombinant spermatozoa antigen peptide using the E. coli: PhoA system on a protein chip for screening anti-sperm antibodies (ASA). RESULTS: The purity of the recombinant spermatozoa antigen exceeded 95% after two-step purification, as assessed using SDS-PAGE and HPLC. The diagnostic performance of a protein chip coated with the recombinant antigen peptide was evaluated by examining ASA in 51 infertile patients in comparison with a commercial ELISA kit...
June 9, 2017: Biotechnology Letters
https://www.readbyqxmd.com/read/28600127/detection-and-purification-of-backbone-cyclized-proteins-using-a-bacterially-expressed-anti-myc-tag-single-chain-antibody
#9
Sushma Chauhan, Chen Yuan Hou, Sang Taek Jung, Taek Jin Kang
A myc-tag and of which recognition by an antibody 9E10 has long been used for the detection and purification of recombinant proteins. We have previously expanded the application of the tag to the specific detection and purification of backbone-cyclized proteins. Here we sought a more practical way to using the 9E10 antibody by expressing its single chain antibody (scAb) form in Escherichia coli. The combined use of a strong T7 promoter and auto-induction strategy rather than early to mid-log induction of a Lac promoter resulted in the soluble over-expression of 9E10 scAb...
June 7, 2017: Analytical Biochemistry
https://www.readbyqxmd.com/read/28598898/a-99mtc-labelled-scfv-antibody-fragment-that-binds-to-prostate-specific-membrane-antigen
#10
Saima Nawaz, Gregory E D Mullen, Philip J Blower, James R Ballinger
INTRODUCTION: Prostate-specific membrane antigen (PSMA) is an extensively studied antigen for imaging prostate cancer. We prepared a single-chain variable fragment (scFv) of J591, a monoclonal antibody that recognises an external epitope of PSMA, incorporating a His-tag for labelling with Tc tricarbonyl, and evaluated its binding using human PCa cell lines. METHODS: J591(scFv) was expressed in HEK-293T cells and purified by metal ion affinity chromatography, followed by size exclusion chromatography...
June 8, 2017: Nuclear Medicine Communications
https://www.readbyqxmd.com/read/28579231/optimized-production-and-purification-of-coxsackievirus-b1-vaccine-and-its-preclinical-evaluation-in-a-mouse-model
#11
Minna M Hankaniemi, Olli H Laitinen, Virginia M Stone, Amirbabak Sioofy-Khojine, Juha A E Määttä, Pär G Larsson, Varpu Marjomäki, Heikki Hyöty, Malin Flodström-Tullberg, Vesa P Hytönen
Coxsackie B viruses are among the most common enteroviruses, causing a wide range of diseases. Recent studies have also suggested that they may contribute to the development of type 1 diabetes. Vaccination would provide an effective way to prevent CVB infections, and the objective of this study was to develop an efficient vaccine production protocol for the generation of novel CVB vaccines. Various steps in the production of a formalin-inactivated Coxsackievirus B1 (CVB1) vaccine were optimized including the Multiplicity Of Infection (MOI) used for virus amplification, virus cultivation time, type of cell growth medium, virus purification method and formulation of the purified virus...
June 1, 2017: Vaccine
https://www.readbyqxmd.com/read/28575911/rapid-transient-production-of-a-monoclonal-antibody-neutralizing-the-porcine-epidemic-diarrhea-virus-pedv-in-nicotiana-benthamiana-and-lactuca-sativa
#12
Kaewta Rattanapisit, Anchalee Srijangwad, Taksina Chuanasa, Suchada Sukrong, Angkana Tantituvanont, Hugh S Mason, Dachrit Nilubol, Waranyoo Phoolcharoen
Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea, vomiting, dehydration, weight loss, and high mortality rate in neonatal piglets. Porcine epidemic diarrhea (PED) has been reported in Europe, America, and Asia including Thailand. The disease causes substantial losses to the swine industry in many countries. Presently, there is no effective PEDV vaccine available. In this study, we developed a plant-produced monoclonal antibody (mAb) 2C10 as a prophylactic candidate to prevent the PEDV infection...
June 2, 2017: Planta Medica
https://www.readbyqxmd.com/read/28575696/regiospecific-radiolabelling-of-nanofitin-on-ni-magnetic-beads-with-18-f-fbem-and-in-vivo-pet-studies
#13
Sylvestre Dammicco, Marine Goux, Christian Lemaire, Guillaume Becker, Mohamed Ali Bahri, Alain Plenevaux, Mathieu Cinier, André Luxen
INTRODUCTION: Nanofitins are low molecular weight, single chain and cysteine-free protein scaffolds able to selectively bind a defined biological target. They derive from Sac7d bacterial protein family and are highly stable over a wide range of pH (0-13) and temperature (Tm ~80°C). Their extreme stability, low cost of production and high tolerability for chemical coupling make Nanofitins a very interesting alternative to antibodies and their fragments. Here, a hexahistidine tagged model Nanofitin (H4) directed against hen egg white lysozyme was radiolabelled and injected in mice to provide a baseline biodistribution and pharmacokinetic profiles to support future Nanofitin development programs...
April 27, 2017: Nuclear Medicine and Biology
https://www.readbyqxmd.com/read/28570837/high-throughput-screening-and-analysis-of-charge-variants-of-monoclonal-antibodies-in-multiple-formulations
#14
Larysa Alekseychyk, Cheng Su, Gerald W Becker, Michael J Treuheit, Vladimir I Razinkov
Among different biopharmaceutical products, monoclonal antibodies (mAbs) show a high level of complexity, including heterogeneity due to differences in size, hydrophobicity, charge, and so forth. Such heterogeneity can be related to both cell-based production and any of the stages of purification, storage, and delivery that the mAb is subjected to. Choosing the right formulation composition providing both physical and chemical stabilities can be a very challenging process, especially when done in the limited time frame required for a typical drug development cycle...
June 1, 2017: SLAS Discovery
https://www.readbyqxmd.com/read/28570826/specific-monoclonal-antibodies-recognizing-the-endogenous-chicken-high-mobility-group-box-1-protein
#15
Yurong Qu, Yuan Zhan, Shen Yang, Xusheng Qiu, Lei Tan, Yingjie Sun, Chunchun Meng, Cuiping Song, Shengqing Yu, Chan Ding
High mobility group box 1 (HMGB1) is a key member of the "danger associated molecular patterns" (DAMPs), which can localize in various compartments of the cell, and plays important roles in systemic inflammation. In the present study, monoclonal antibodies (MAbs) specifically against chicken HMGB1 were generated. The open reading frame of chicken HMGB1 was amplified by RT-PCR and cloned into the prokaryotic expression vector pET-28a to construct a recombinant plasmid pET-chHMGB1. The recombinant chicken HMGB1 protein was expressed in Escherichia coli Rosetta under IPTG induction and then purified by Ni-NTA Purification System...
June 1, 2017: Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
https://www.readbyqxmd.com/read/28569168/construct-design-production-and-characterization-of-plasmodium-falciparum-48-45-r0-6c-subunit-protein-produced-in-lactococcus-lactis-as-candidate-vaccine
#16
Susheel K Singh, Will Roeffen, Ulrik H Mistarz, Bishwanath Kumar Chourasia, Fen Yang, Kasper D Rand, Robert W Sauerwein, Michael Theisen
BACKGROUND: The sexual stages of Plasmodium falciparum are responsible for the spread of the parasite in malaria endemic areas. The cysteine-rich Pfs48/45 protein, exposed on the surface of sexual stages, is one of the most advanced antigens for inclusion into a vaccine that will block transmission. However, clinical Pfs48/45 sub-unit vaccine development has been hampered by the inability to produce high yields of recombinant protein as the native structure is required for the induction of functional transmission-blocking (TB) antibodies...
May 31, 2017: Microbial Cell Factories
https://www.readbyqxmd.com/read/28565780/comparison-of-the-purity-and-vitality-of-natural-killer-cells-with-different-isolation-kits
#17
Guangchuan Wang, Guang Yu, Dongmei Wang, Shengnan Guo, Fengping Shan
Natural killer (NK) cells are innate lymphocytes that aid in the protection of the host from infectious diseases and cancer. In vitro studies of NK cells have provided a foundation for developing clinical adoptive NK-cell transferred immunotherapy against human tumors. To elucidate the functions and mechanisms of NK cell populations, it is important to develop an optimal, highly reproducible and reliable isolation method. The present comparative study was performed with four different NK cell isolation kits of magnetic bead labeling made by Miltenyi and Stemcell companies, including positive selection kits [cluster of differentiation (CD)-49b, using the monoclonal antibody DX5) MicroBeads] and negative selection kits...
May 2017: Experimental and Therapeutic Medicine
https://www.readbyqxmd.com/read/28564596/the-histone-variant-macroh2a1-is-a-brca1%C3%A2-ubiquitin-ligase-substrate
#18
Beom-Jun Kim, Doug W Chan, Sung Yun Jung, Yue Chen, Jun Qin, Yi Wang
The breast- and ovarian-cancer-specific tumor suppressor BRCA1 and its heterodimeric partner BARD1 contain RING domains that implicate them as E3 ubiquitin ligases. Despite extensive efforts, the bona fide substrates of BRCA1/BARD1 remain elusive. Here, we used recombinant GST fused to four UBA domains to enrich ubiquitinated proteins followed by a Lys-ε-Gly-Gly (diGly) antibody to enrich ubiquitinated tryptic peptides. This tandem affinity purification method coupled with mass spectrometry identified 101 putative BRCA1/BARD1 E3 substrates...
May 30, 2017: Cell Reports
https://www.readbyqxmd.com/read/28561305/genetic-fusion-protein-3%C3%A3-sta-ovalbumin-is-an-effective-coating-antigen-in-elisa-to-titrate-anti-sta-antibodies
#19
Qiangde Duan, Weiping Zhang
Heat-stable toxin type I (STa)-ovalbumin chemical conjugates are currently used as the only coating antigen in enzyme-links immune sorbent assay (ELISA) to titrate anti-STa antibodies for enterotoxigenic Escherichia coli (ETEC) vaccine candidates. STa-ovalbumin chemical conjugation requires STa toxin purification, a process can be carried out by only a couple of laboratories and often with a low yield. Alternative ELISA coating antigens are needed for anti-STa antibody titration for ETEC vaccine development...
May 31, 2017: Microbiology and Immunology
https://www.readbyqxmd.com/read/28560668/aptamer-based-methods-for-detection-of-circulating-tumor-cells-and-their-potential-for-personalized-diagnostics
#20
Anna S Zamay, Galina S Zamay, Olga S Kolovskaya, Tatiana N Zamay, Maxim V Berezovski
Cancer diagnostics and treatment monitoring rely on sensing and counting of rare cells such as cancer circulating tumor cells (CTCs) in blood. Many analytical techniques have been developed to reliably detect and quantify CTCs using unique physical shape and size of tumor cells and/or distinctive patterns of cell surface biomarkers. Main problems of CTC bioanalysis are in the small number of cells that are present in the circulation and heterogeneity of CTCs. In this chapter, we describe recent progress towards the selection and application of synthetic DNA or RNA aptamers to capture and detect CTCs in blood...
2017: Advances in Experimental Medicine and Biology
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