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Antibody purification

David Falck, Bas C Jansen, Noortje de Haan, Manfred Wuhrer
This chapter contains a nanoscale liquid chromatography-mass spectrometry method for the glycoform profiling of the conserved Fc N-glycosylation site of monoclonal and polyclonal immunoglobulin G (IgG). It describes in detail LaCyTools, a program for automated data (pre-)processing of the obtained LC-MS data. The minimal sample preparation necessary is explained as well as an optional method for affinity purification of (polyclonal) antibodies from serum or plasma.After (optional) affinity purification, the pure IgG is cleaved with trypsin...
2017: Methods in Molecular Biology
Bumpei Samata, Daisuke Doi, Kaneyasu Nishimura, Tetsuhiro Kikuchi, Akira Watanabe, Yoshimasa Sakamoto, Jungo Kakuta, Yuichi Ono, Jun Takahashi
Human induced pluripotent stem cells (iPSCs) can provide a promising source of midbrain dopaminergic (mDA) neurons for cell replacement therapy for Parkinson's disease (PD). However, iPSC-derived donor cells inevitably contain tumorigenic or inappropriate cells. To eliminate these unwanted cells, cell sorting using antibodies for specific markers such as CORIN or ALCAM has been developed, but neither marker is specific for ventral midbrain. Here we employ a double selection strategy for cells expressing both CORIN and LMX1A::GFP, and report a cell surface marker to enrich mDA progenitors, LRTM1...
October 14, 2016: Nature Communications
Saloumeh Kadkhodayan Fischer, Melissa Cheu, Kun Peng, John Lowe, James Araujo, Elaine Murray, Dana McClintock, John Matthews, Patricia Siguenza, An Song
Host cell proteins are manufacturing process-related impurities that may co-purify with the product despite extensive efforts to optimize the purification process. The risks associated with these impurities can vary and may be patient and/or therapeutic dependent. Therefore, it is critical to monitor and control the levels of these impurities in products and their potential impact on safety and efficacy. Lebrikizumab is a humanized immunoglobulin G4 monoclonal antibody (mAb) that binds specifically to soluble interleukin 13...
October 13, 2016: AAPS Journal
Shalom D Goldberg, Rosa M F Cardoso, Tricia Lin, Tracy Spinka-Doms, Donna Klein, Steven A Jacobs, Vadim Dudkin, Gary Gilliland, Karyn T O'Neil
Targeted delivery of therapeutic payloads to specific tissues and cell types is an important component of modern pharmaceutical development. Antibodies or other scaffold proteins can provide the cellular address for delivering a covalently linked therapeutic via specific binding to cell-surface receptors. Optimization of the conjugation site on the targeting protein, linker chemistry and intracellular trafficking pathways can all influence the efficiency of delivery and potency of the drug candidate. In this study, we describe a comprehensive engineering experiment for an EGFR binding Centyrin, a highly stable fibronectin type III (FN3) domain, wherein all possible single-cysteine replacements were evaluated for expression, purification, conjugation efficiency, retention of target binding, biophysical properties and delivery of a cytotoxic small molecule payload...
October 13, 2016: Protein Engineering, Design & Selection: PEDS
O K Savushkina, E B Tereshkina, T A Prokhorova, E A Vorob'eva, I S Boksha, G Sh Burbaeva
AIM: To compare patterns of brain isoform creatine phosphokinase (CPK B) distributions in post-mortem brain from patients with schizophrenia (Sch) and patients with somatic diseases (controls). MATERIAL AND METHODS: Extracts of readily soluble and membrane-associated proteins were prepared from post-mortem samples of prefrontal cortex (Brodmann area 10), anterior (area 24) and posterior (area 23) cingulate cortex, hippocampus and cerebellum cortex from patients with Sch and control group (the samples were matched by age and postmortem interval)...
2016: Zhurnal Nevrologii i Psikhiatrii Imeni S.S. Korsakova
Paul Leonard, Stephen Hearty, Hui Ma, Richard O'Kennedy
The use of optical biosensors for studying macromolecular interactions is gaining increasing popularity. In one study, 1514 papers that involved the application of biosensor data were identified for the year 2009 alone (Rich and Myszka, J Mol Recognit 24:892-914, 2011), the sheer volume and variety of which present a daunting task for the burgeoning biosensor user to accumulate and decipher. This chapter is designed to provide the reader with the tools necessary to prepare, design, and efficiently execute a kinetic experiment on Biacore...
2017: Methods in Molecular Biology
Elaine Darcy, Paul Leonard, Jenny Fitzgerald, Martin Danaher, Hui Ma, Richard O'Kennedy
Affinity chromatography permits the isolation of a target analyte from a complex mixture and can be utilized to purify proteins, carbohydrates, drugs, haptens, or any analyte of interest once an affinity pair is available. It involves the exploitation of specific interactions between a binding affinity pair, such as those between an antibody and its associated antigen, or between any ligand and its associated binding receptor/protein. With the discovery of protein A in 1970, and, subsequently protein G and L, immuno-affinity chromatography has grown in popularity and is now the standard methodology for the purification of antibodies which may be implemented for a selection of different applications such as immunodiagnostics...
2017: Methods in Molecular Biology
Jenny Fitzgerald, Paul Leonard, Elaine Darcy, Shikha Sharma, Richard O'Kennedy
Antibody-based separation methods, such as immunoaffinity chromatography (IAC), are powerful purification and isolation techniques. Antibodies isolated using these techniques have proven highly efficient in applications ranging from clinical diagnostics to environmental monitoring. Immunoaffinity chromatography is an efficient antibody separation method which exploits the binding efficiency of a ligand to an antibody. Essential to the successful design of any IAC platform is the optimization of critical experimental parameters such as (a) the biological affinity pair, (b) the matrix support, (c) the immobilization coupling chemistry, and (d) the effective elution conditions...
2017: Methods in Molecular Biology
Helieh S Oz
Microbiomic flora in digestive tract is pivotal to the state of our health and disease. Antibiotics affect GI, control composition of microbiome, and shift equilibrium from health into disease status. Coccidiosis causes gastrointestinal inflammation. Antibiotic additives contaminate animal products and enter food chain, consumed by humans with possible allergic, antibiotic resistance and enigmatic side effects. Purposed study induced nonpathogenic, immunogenic organisms to protect against disease and abolish antibiotics' use in food animals and side effects in man...
2016: Gastroenterology Research and Practice
Anna Sroka-Bartnicka, Isabella Karlsson, Lorena Ndreu, Alessandro Quaranta, Matthijs Pijnappel, Gunnar Thorsén
Glycosylation is one of the most common and important post-translational modifications, influencing both the chemical and the biological properties of proteins. Studying the glycosylation of the entire protein population of a sample can be challenging because variations in the concentrations of certain proteins can enhance or obscure changes in glycosylation. Furthermore, alterations in the glycosylation pattern of individual proteins, exhibiting larger variability in disease states, have been suggested as biomarkers for different types of cancer, as well as inflammatory and neurodegenerative diseases...
October 3, 2016: Journal of Pharmaceutical and Biomedical Analysis
Livia Brunner, Christophe Barnier-Quer, Nicolas Collin
QS-21, a saponin extracted from the tree Quillaja saponaria Molina, is a vaccine adjuvant which has been shown to elicit robust antibody and cell-mediated immune responses in a variety of preclinical and clinical studies [1]. Its purification from the natural source is a lengthy and difficult process. The commercially available saponin mixture Quil-A(®) is a fraction of the bark extract containing a variety of saponins, including QS-21. In order to facilitate access to QS-21 at laboratory-scale amounts, we propose here a method of purification of QS-21 starting from Quil-A(®)...
2017: Methods in Molecular Biology
Raghav Joshi, Edi Goihberg, Wenying Ren, Monika Pilichowska, Paul Mathew
: The haematopoietic niche is contributed to by bone marrow-resident mesenchymal stromal cells (BM-MSCs) and subverted by prostate cancer cells. To study mechanisms by which BM-MSCs and prostate cancer cells may interact, we assessed the migration, invasion, adhesion and proliferation of bone-derived prostate cancer cells (PC-3) in co-culture with pluripotent human BM-MSCs. We observed a strong adhesive, migratory and invasive phenotype of PC-3 cells with BM- MSC-co-culture and set out to isolate and characterize the bioactive principle...
August 11, 2016: Cell Adhesion & Migration
Steen Sørensen, Vibeke Myrhøj, Thanh Ha Nguyen, Per Aaslo, Young Bae Hansen
INTRODUCTION: Glycodelin is a glycoprotein with different oligosaccharides that are responsible for its diverse biological functions in contraception and immunosuppression. Therefore, it is necessary to have access to adequate amounts of glycodelin with retained carbohydrate structure for functional studies because the carbohydrate part can be lacking or be insufficient in recombinant glycodelin from prokaryotic and eukaryotic cell systems. METHODS AND RESULTS: Native glycodelin was purified from amniotic fluid by a series of affinity chromatography steps and had many glycosylated forms verified by mass spectrometry...
October 3, 2016: Protein Expression and Purification
Qiong Liu, Qing Liu, Jie Yi, Kang Liang, Bo Hu, Xiangmin Zhang, Roy Curtiss, Qingke Kong
Outer membrane vesicles (OMVs) isolated from Salmonella Typhimurium are potentially useful for developing subunit vaccines because of high immunogenicity and protective efficacy. However, flagella might remain in OMV pellets following OMV purification, resulting in non-essential immune responses and counteraction of bacterial protective immune responses when developing a vaccine against infection of multiple serotypes Salmonella. In this study, a flagellin-deficient S. Typhimurium mutant was constructed. Lipopolysaccharide profiles, protein profiles and cryo-electron microscopy revealed that there were no significant differences between the wild-type and mutant OMVs, with the exception of a large amount of flagellin in the wild-type OMVs...
October 4, 2016: Scientific Reports
Sujina Mali, Wilna J Moree, Morgan Mitchell, William Widger, Steven J Bark
Co-affinity purification mass spectrometry (CoAP-MS) is a highly effective method for identifying protein complexes from a biological sample and inferring important interactions, but the impact of the solid support is usually not considered in design of such experiments. Affinity purification (AP) experiments typically utilize a bait protein expressing a peptide tag such as FLAG, c-Myc, HA or V5 and high affinity antibodies to these peptide sequences to facilitate isolation of a bait protein to co-purify interacting proteins...
September 29, 2016: Analytical Biochemistry
Sandra M Walser, Bernhard Brenner, Anika Wunderlich, Christian Tuschak, Stefanie Huber, Stefanie Kolb, Reinhard Niessner, Michael Seidel, Christiane Höller, Caroline E W Herr
The urbanization of agricultural areas results in a reduction of distances between residential buildings and livestock farms. In the public debate, livestock farming is increasingly criticized due to environmental disturbance and odor nuisance originating from such facilities. One method to reduce odor and ammonia is by exhaust air treatment, for example, by biological exhaust air purification processes with bio-trickling filters filled with tap water. Higher temperatures in the summer time and the generation of biofilms are ideal growth conditions for Legionella...
September 28, 2016: Science of the Total Environment
Geert A Martens, Veerle De Punt, Geert Stangé
We quantified rat and human beta cell proteomes by LC-MS/MS searching for cell surface markers. In human beta cells, CD99 ranked among the plasma membrane proteins that associate a high molar abundance, to a relative degree of selectivity to the endocrine cells of the islets of Langerhans. Therefore, we investigated CD99's applicability as anchor for islet endocrine cell purification. We studied CD99 gene and protein expression by microarray, LC-MS/MS, Western blotting, flow cytometry and immunofluorescence and developed a protocol for magnetic bead-mediated beta cell enrichment from human pancreas digests using available anti-CD99 antibodies...
September 29, 2016: Journal of Tissue Engineering and Regenerative Medicine
Ali Ansari, Reema Patel, Kinsey Schultheis, Vesna Naumovski, P I Imoukhuede
One of the limiting factors to the adoption and advancement of personalized medicine is the inability to develop diagnostic tools to probe individual nuances in expression from patient to patient. Current methodologies that try to separate cells to fill this niche result in disruption of physiological expression, making the separation technique useless as a diagnostic tool. In this protocol, we describe the functionalization and optimization of a surface for the cellular capture and release. This functionalized surface integrates biotinylated antibodies with a glass surface functionalized with an aminosilane (APTES), desthiobiotin and streptavidin...
2016: Journal of Visualized Experiments: JoVE
Xiu-Li Cao, Xing Zhang, Yu-Fei Zhang, Yi-Zhe Zhang, Chang-Geng Song, Fan Liu, Yi-Yang Hu, Min-Hua Zheng, Hua Han
Ttyh1 is a murine homolog of the Drosophila Tweety and is predicted as a five-pass transmembrane protein. The Ttyh1 mRNA is expressed in mouse brain tissues with a restricted pattern and in human glioma cells. Ttyh1 protein may function as a large-conductance chloride channel, however, the role of Ttyh1 in normal neural development and tumorigenesis has been largely unknown, at least partially due to the lack of effective antibodies. Here we report the expression in E. coli and purification of two recombinant Ttyh1 protein fragments corresponding to one of the predicted extracellular domains and the carboxyl terminus of the mouse Ttyh1...
September 24, 2016: Protein Expression and Purification
Timothy M Pabst, Michaela Wendeler, Xiangyang Wang, Sandra Bezemer, Pim Hermans, Alan K Hunter
Interest in new and diverse classes of molecules such as recombinant toxins, enzymes, and blood factors continues to grow for use a biotherapeutics. Compared to monoclonal antibodies, these novel drugs typically lack a commercially available affinity chromatography option, which leads to greater process complexity, longer development timelines, and poor platformability. To date, for both monoclonal antibodies and novel molecules, affinity chromatography has been mostly reserved for separation of process-related impurities such as host cell proteins and DNA...
September 27, 2016: Biotechnology Journal
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