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Antibody purification

Sajid Mushtaq, Jong Kook Rho, Jung Ae Kang, Joong-Jae Lee, Jung Young Kim, You Ree Nam, Seong-Jae Yun, Gyeong Hee Lee, Sang Hyun Park, Dong-Eun Lee, Hak-Sung Kim
Antibody-mimetic proteins are intensively being developed for biomedical applications including tumor imaging and therapy. Among them, repebody is a new class of protein that consists of highly diverse leucine-rich repeat (LRR) modules. Although all possible biomedical applications with repebody are ongoing, it's in vivo biodistribution and excretion pathway has not yet been explored. In this study, hexahistidine (His6)-tag bearing repebody (rEgH9) was labeled with [(99m)Tc]-tricarbonyl, and biodistribution was performed following intravenous (I...
September 25, 2017: Bioorganic & Medicinal Chemistry Letters
Namrata D Udeshi, Kayvon Pedram, Tanya Svinkina, Shaunt Fereshetian, Samuel A Myers, Ozan Aygun, Karsten Krug, Karl Clauser, Dominic Ryan, Tslil Ast, Vamsi K Mootha, Alice Y Ting, Steven A Carr
Although purification of biotinylated molecules is highly efficient, identifying specific sites of biotinylation remains challenging. We show that anti-biotin antibodies enable unprecedented enrichment of biotinylated peptides from complex peptide mixtures. Live-cell proximity labeling using APEX peroxidase followed by anti-biotin enrichment and mass spectrometry yielded over 1,600 biotinylation sites on hundreds of proteins, an increase of more than 30-fold in the number of biotinylation sites identified compared to streptavidin-based enrichment of proteins...
October 16, 2017: Nature Methods
Xiaolin Wang, Xiangyang Chi, Ju Liu, Weicen Liu, Shuling Liu, Shunfang Qiu, Zhonghua Wen, Pengfei Fan, Kun Liu, Xiaohong Song, Ling Fu, Jun Zhang, Changming Yu
Anthrax is a highly lethal infectious disease caused by the spore-forming bacterium Bacillus anthracis. The major virulence factor of B. anthracis consists of protective antigen (PA), lethal factor (LF) and edema factor (EF). PA binds with LF to form lethal toxin (LT), and PA binds with EF to form edema toxin (ET). Antibiotics is hard to work in advanced anthrax infections, because injuries and deaths of the infected are mainly caused by lethal toxin (LT). Thus, the therapeutic neutralizing antibody is the most effective treatment of anthrax...
November 25, 2016: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
R Vijayakumar, Timir Tripathi
We report the molecular cloning, expression, and single-step homogeneous purification of a full-length asparaginyl tRNA synthetase (NRS) from Fasciola gigantica (FgNRS). Fasciola gigantica is a parasitic liver fluke of the class Trematoda. It causes fascioliasis that infects the livers of various mammals, including humans. Aminoacyl tRNA synthetases (AARS) catalyze the first step of protein synthesis. They attach an amino acid to its cognate tRNA, forming an amino acid-tRNA complex. The gene that codes for FgNRS was generated by amplification by polymerase chain reaction...
October 12, 2017: Protein Expression and Purification
Luca Palmieri, Filipe Elvas, Christel Vangestel, Koon Pak, Brian Gray, Sigrid Stroobants, Steven Staelens, Leonie Wyffels
INTRODUCTION: [(99m)Tc]duramycin is a SPECT tracer for cell death imaging. We evaluated the impact of kit formulation, purification and species difference on the pharmacokinetic profile and cell death targeting properties of [(99m)Tc]duramycin in order to define the optimal conditions for (pre-)clinical use. METHODS: Three kits were prepared (A: traditional formulation, B: containing 1/3 of ingredients, C: containing HYNIC-PEG12-duramycin). Following labeling, the kits were used without purification, or with SPE or HPLC purification...
September 14, 2017: Nuclear Medicine and Biology
Tilman Läppchen, Raffaella Rossin, Tiemen R van Mourik, Guillaume Gruntz, Freek J M Hoeben, Ron M Versteegen, Henk M Janssen, Johan Lub, Marc S Robillard
INTRODUCTION: Pretargeted radioimmunoimaging and -therapy approaches building on the bioorthogonal inverse-electron-demand Diels-Alder (IEDDA) reaction between strained trans-cyclooctenes (TCO) and electron-deficient tetrazines (Tz) have yielded impressive results in recent years and have proven a vital alternative to biological pretargeting systems. After improvement of the TCO-antibody conjugates, we here report on our evaluation of a new series of radiolabeled Tz-probes. METHODS: Four new Tz-probes were synthesized, radiolabeled with lutetium-177, and characterized in vitro in terms of lipophilicity, reactivity, and stability in PBS and mouse serum...
September 14, 2017: Nuclear Medicine and Biology
Yuzu Zhao, Kui Zhang, Mei Tang, Man Xu, Chongyang Li, Guangzhao Pan, Li Shen, Hongjuan Cui, Liqun Yang
Scavenger receptor class B is involved in various indispensable physiological processes, like the formation and inhibition of atherosclerosis or other cardiovascular diseases, innate immune defense and the removal of apoptotic cells. Here, we cloned BmSCRB8, a member of scavenger receptor class B in silkworm. We obtained the full-length cDNA sequence of BmSCRB8 by rapid amplification of cDNA ends (RACE), including 2 668 bp. The ORF of BmSCRB8 is 1 704 bp, encoding 567 amino acids. Online software prediction indicated that the molecular weight of BmSCRB8 is 63...
October 25, 2016: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
Marc Bayliss, Matthew I Donaldson, Sergey A Nepogodiev, Giulia Pergolizzi, Andrew E Scott, Nicholas J Harmer, Robert A Field, Joann L Prior
Burkholderia pseudomallei and its close relative B. mallei are human pathogens that are classified as Tier 1 bio-threat agents. Both organisms have previously been shown to constitutively produce a capsular polysaccharide (CPS) that is both a virulence determinant and protective antigen. Extraction and purification of CPS for use as a potential vaccine candidate requires containment level 3 laboratories which is expensive and time-consuming. B. thailandensis strain E555 is closely related to B. pseudomallei and B...
September 21, 2017: Carbohydrate Research
Philippe Desharnais, Jean-Francois Naud, Christiane Ayotte
According to World Anti-Doping Agency (WADA) technical document for erythropoiesis stimulating agents (ESA) analysis (TD2014EPO), double- blotting of serum/plasma samples is mandatory for all analysis by isoelectric focusing (IEF) and for the confirmation procedures (CP) performed by SDS-PAGE or SAR-PAGE. The goal is to prevent potential cross-reactions of the secondary antibody with remaining proteins in the purified samples. To this end, we have developed an immunopurification method of ESA in serum/plasma samples using a combination of streptavidin-coated immunomagnetic beads and biotinylated anti-EPO polyclonal antibodies...
October 9, 2017: Drug Testing and Analysis
Fuxin Lu, Guo Shao, Yongqiang Wang, Shenheng Guan, Alma L Burlingame, Xuemei Liu, Xiao Liang, Renatta Knox, Donna M Ferriero, Xiangning Jiang
The N-methyl-d-aspartate-type glutamate receptor (NMDAR)-associated multiprotein complexes are indispensable for synaptic plasticity and cognitive functions. While purification and proteomic analyses of these signaling complexes have been performed in adult rodent and human brain, much less is known about the protein composition of NMDAR complexes in the developing brain and their modifications by neonatal hypoxic-ischemic (HI) brain injury. In this study, the postsynaptic density proteins were prepared from postnatal day 9 naïve, sham-operated and HI-injured mouse cortex...
October 6, 2017: Experimental Neurology
Andrew Swartz, Xuankuo Xu, Steven Traylor, Zheng Jian Li, Wilfred Chen
Protein A chromatography has been identified as a potential bottleneck in the monoclonal antibody production platform, leading to increased interest in non-chromatographic capture technologies. Affinity precipitation using environmentally responsive, Z-domain-elastin-like polypeptide (Z-ELP) fusion proteins has been shown to be a promising alternative. However, elevated temperature and salt concentrations necessary for precipitation resulted in decreased antibody monomer content and reduced purification capacity...
October 7, 2017: Biotechnology and Bioengineering
Feny Gunawan, Julie Nishihara, Peter Liu, Wendy Sandoval, Marty Vanderlaan, Heidi Zhang, Denise Krawitz
During expression of biotherapeutic proteins, complex mixtures of additional proteins are also produced by normal expression machinery of the host cell (termed "host cell proteins", or HCP). HCPs pose a potential impact to patient safety and product efficacy, and therefore must be well-characterized and the ability of the process to clear these proteins must be demonstrated. Due to the complexity of HCP, the method(s) used for monitoring must be demonstrated to provide sufficient information about relevant proteins...
October 7, 2017: Biotechnology and Bioengineering
Maximilian Bönisch, Carolin Sellmann, Daniel Maresch, Claudia Halbig, Stefan Becker, Lars Toleikis, Björn Hock, Florian Rüker
Targeting two unique antigens with a single bispecific antibody is an attractive approach with potential broad therapeutic applicability. However, the production of heterodimeric bispecific antibodies (bsAbs) presents a challenge, requiring the co-expression and accurate pairing of two distinct heavy and light chain units. Several undesirable by-products can be formed in the production process, including heavy chain homodimers and non-cognate light chain pairings. Although additional downstream purification methods exist, they are often time consuming and restrict practical large-scale production...
August 31, 2017: Protein Engineering, Design & Selection: PEDS
Bhumit A Patel, Nuno D S Pinto, Adrian Gospodarek, Bruce Kilgore, Kudrat Goswami, William N Napoli, Jayesh Desai, Jun H Heo, Dominick Panzera, David Pollard, Daisy Richardson, Mark Brower, Douglas D Richardson
Combining process analytical technology (PAT) with continuous production provides a powerful tool to observe and control monoclonal antibody (mAb) fermentation and purification processes. This work demonstrates on-line liquid chromatography (on-line LC) as a PAT tool for monitoring a continuous biologics process and forced degradation studies. Specifically, this work focused on ion exchange chromatography (IEX), which is a critical separation technique to detect charge variants. Product-related impurities, including charge variants, that impact function are classified as critical quality attributes (CQAs)...
October 18, 2017: Analytical Chemistry
Juan Calvo-Iglesias, Daniel Pérez-Estévez, África González-Fernández
The mussel shell protein 22.8 (MSP22.8) is recognized by a monoclonal antibody (M22.8) directed against larvae of the mussel Mytilus galloprovincialis. After being secreted by cells of the mantle-edge epithelium into the extrapallial (EP) space (the gap between the mantle and the shell), the protein is detected in the extrapallial fluid (EPF) and EP hemocytes and finally becomes part of the shell matrix framework in adult specimens of M. galloprovincialis. In the work described here, we show how MSP22.8 is detected in EPF samples from different species of mussels (M...
October 2017: FEBS Open Bio
Hossein Vahidi, Nastaran Nafissi-Varcheh, Bahram Kazemi, Reza Aboofazeli, Soraya Shahhosseini, Maryam Tabarzad
Protein specific aptamers are highly applicable affinity ligands in different fields of research and clinical applications. They have been developed against various targets, in particular, bio-macromolecules such as proteins. Among human proteins, the coagulation factors are the most attractive targets for aptamer selection and their specific aptamers have valuable characteristics in therapeutic and analytical applications. In this study, a plasma derived coagulation factor VIII was considered as the protein target for DNA aptamer selection using size exclusion chromatography-SELEX...
2017: Iranian Journal of Pharmaceutical Research: IJPR
Manish Mishra, Shuchita Tiwari, Aldrin V Gomes
Western blotting is one of the most commonly used techniques in molecular biology and proteomics. Since western blotting is a multistep protocol, variations and errors can occur at any step reducing the reliability and reproducibility of this technique. Recent reports suggest that a few key steps, such as the sample preparation method, the amount and source of primary antibody used, as well as the normalization method utilized, are critical for reproducible western blot results. Areas covered: In this review, improvements in different areas of western blotting, including protein transfer and antibody validation, are summarized...
October 3, 2017: Expert Review of Proteomics
Gorawit Yusakul, Poomraphie Nuntawong, Seiichi Sakamoto, Pahweenvaj Ratnatilaka Na Bhuket, Toshitaka Kohno, Nao Kikkawa, Pornchai Rojsitthisak, Kuniyoshi Shimizu, Hiroyuki Tanaka, Satoshi Morimoto
Due to the highly specific binding between an antibody and its target, superior analytical performances was obtained by immunoassays for phytochemical analysis over conventional chromatographic techniques. Here, we describe a simple method for producing a functional single-chain variable fragment (scFv) antibody against ganoderic acid A (GAA), a pharmacologically active metabolite from Ganoderma lingzhi. The Escherichia coli BL21(DE3) strain produced a large amount of anti-GAA scFv. However, in vitro refolding steps, which partially recovered the reactivity of the scFv, were required...
2017: Biological & Pharmaceutical Bulletin
Ioannis Karageorgos, Elyssia S Gallagher, Connor Galvin, D Travis Gallagher, Jeffrey W Hudgens
Monoclonal antibody pharmaceuticals are the fastest-growing class of therapeutics, with a wide range of clinical applications. To assure their safety, these protein drugs must demonstrate highly consistent purity and stability. Key to these objectives is higher order structure measurements validated by calibration to reference materials. We describe preparation, characterization, and crystal structure of the Fab fragment prepared from the NIST Reference Antibody RM 8671 (NISTmAb). NISTmAb is a humanized IgG1κ antibody, produced in murine cell culture and purified by standard biopharmaceutical production methods, developed at the National Institute of Standards and Technology (NIST) to serve as a reference material...
September 28, 2017: Biologicals: Journal of the International Association of Biological Standardization
F Guérard, L Navarro, Y-S Lee, A Roumesy, C Alliot, M Chérel, M W Brechbiel, J-F Gestin
In this report we describe the development of an alternative approach to arylstannane chemistry for radiolabeling antibodies with radioiodine or astatine based on aryliodonium salts precursors. Bifunctional aryliodonium salts were designed and tested for the synthesis of (125)I and (211)At labeled prosthetic groups for bioconjugation. The nature of the electron rich aryl group was varied and its impact on the regioselectivity of radiohalogenation was evaluated. Unexpectedly, whereas the 2-thienyl group provided the best regioselectivity towards the radioiodination of the aryl moiety of interest (98:2), it was less selective for astatination (87:13); the anisyl group providing the best regioselectivity of astatination (94:6)...
September 19, 2017: Bioorganic & Medicinal Chemistry
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