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Antibody purification

Alessandra Altamirano, Andreas Naschberger, Barbara G Furnrohr, Radka Saldova, Weston B Struwe, Patrick M Jennings, Silvia Millán Martín, Suzana Malic, Immanuel Plangger, Stefan Lechner, Reina Pisano, Nicole Peretti, Bernd Linke, Mario M Aguiar, Friedrich Fresser, Andreas Ritsch, Tihana Lenac Rovis, Christina Goode, Pauline M Rudd, Klaus Scheffzek, Bernhard Rupp, Hans Dieplinger
Afamin is an 87 kDa glycoprotein with five predicted N-glycosylation sites. Afamin's glycan abundance contributes to conformational and chemical inhomogeneity presenting great challenges for molecular structure determination. For the purpose of studying the structure of afamin, various forms of recombinantly expressed human afamin (rhAFM) with different glycosylation patterns were thus created. Wild-type rhAFM and various hypo-glycosylated forms were expressed in CHO, CHO-Lec1 and HEK293T cells. Fully non-glycosylated rhAFM was obtained by transfection of point-mutated cDNA to delete all N-glycosylation sites of afamin...
February 14, 2018: Journal of Proteome Research
Nandana Das, Rajendra Kumar
Follicle-stimulating hormone (FSH) plays fundamental roles in male and female fertility. FSH is a heterodimeric glycoprotein expressed by gonadotrophs in the anterior pituitary. The hormone-specific FSH-subunit is non-covalently associated with the common -subunit that is also present in the luteinizing hormone (LH), another gonadotrophic hormone secreted by gonadotropes and thyroid-stimulating hormone (TSH) secreted by thyrotrophs. Several decades of research led to the purification, structural characterization and physiological regulation of FSH in a variety of species including humans...
February 7, 2018: Journal of Molecular Endocrinology
Fanfeng Meng, Qiuchen Li, Yawen Zhang, Zhihui Zhang, Sibao Tian, Zhizhong Cui, Shuang Chang, Peng Zhao
BACKGROUND: In spite of the purification of the laying hens and broilers of avian leukosis virus (ALV) has made remarkable achievements, the infection of ALV was still serious in Chinese indigenous chickens. METHODS: In order to assess the epidemic state of avian leukosis virus in indigenous chickens in China, 10 novel strains of ALV subgroup J (ALV-J), named JS16JH01 to JS16JH10, were isolated and identified by virus isolation and immunofluorescence antibody assays from a Chinese local breed farm with a sporadic incidence of tumors...
February 13, 2018: Virology Journal
Nanao Suzuki, Yuuki Takamuku, Tomohiro Asakawa, Makoto Inai, Tomoya Hino, So Iwata, Toshiyuki Kan, Takeshi Murata
Membrane proteins, such as G-protein coupled receptors, control communication between cells and their environments and are indispensable for many cellular functions. Nevertheless, structural studies on membrane proteins lag behind those on water-soluble proteins, due to their low structural stability, making it difficult to obtain crystals for X-ray crystallography. Optimizing conditions to improve the stability of membrane proteins is essential for successful crystallization. However, the optimization usually requires large amounts of purified samples, and it is a time-consuming and trial-and-error process...
February 9, 2018: Analytical Biochemistry
Mohammad Ali Saghiri, Armen Asatourian, Christine M Sorenson, Nader Sheibani
Dental pulp is a highly vascularized tissue with a high regenerative capacity. This is attributed to its unique blood supply and the presence of progenitor or postnatal dental pulp stem cells. Here we aimed to isolate and compare the angiogenic properties of endothelial cells (EC) prepared from mouse dental pulp and periodontal ligament (PDL). EC were isolated from 4-week-old wild type immorto mice. Mice were sacrificed and after mandible isolation, the molar and incisor teeth and the PDL from molar teeth were dissected...
February 2018: Tissue & Cell
Rachana Ohri, Sunil Bhakta, Aimee Fourie-O'Donohue, Josefa Dela Cruz-Chuh, Siao Ping Tsai, Ryan Cook, Binqing Wei, Carl Ng, Athena W Wong, Aaron B Bos, Farzam Farahi, Jiten Bhakta, Thomas H Pillow, Helga Raab, Richard Vandlen, Paul Polakis, Yichin Liu, Hans Erickson, Jagath R Junutula, Katherine R Kozak
THIOMAB antibody technology utilizes cysteine residues engineered onto an antibody to allow for site-specific conjugation. The technology has enabled the exploration of different attachment sites on the antibody in combination with small molecules, peptides, or proteins to yield antibody conjugates with unique properties. As reported previously ( Shen , B. Q. , et al. ( 2012 ) Nat. Biotechnol. 30 , 184 - 189 ; Pillow , T. H. , et al. ( 2017 ) Chem. Sci. 8 , 366 - 370 ), the specific location of the site of conjugation on an antibody can impact the stability of the linkage to the engineered cysteine for both thio-succinimide and disulfide bonds...
February 9, 2018: Bioconjugate Chemistry
Akihisa Matsuyama, Atsuko Shirai, Minoru Yoshida
Immunoprecipitation is one of the most important and widely used techniques for the detection and purification of a protein of interest. Thanks to highly specific interaction between antigen and antibody, a target protein is purified and concentrated effectively. To obtain reasonable amounts of a target protein, it is crucially important to prepare total cell lysates in which the target protein is present in a soluble form. Here, we describe methods to prepare total cell lysates of fission yeast, which are then used directly for immunoprecipitation...
2018: Methods in Molecular Biology
Mehran Makvandi, Edouard Dupis, Jonathan W Engle, F Meiring Nortier, Michael E Fassbender, Sam Simon, Eva R Birnbaum, Robert W Atcher, Kevin D John, Olivier Rixe, Jeffrey P Norenberg
Alpha-emitters are radionuclides that decay through the emission of high linear energy transfer α-particles and possess favorable pharmacologic profiles for cancer treatment. When coupled with monoclonal antibodies, peptides, small molecules, or nanoparticles, the excellent cytotoxic capability of α-particle emissions has generated a strong interest in exploring targeted α-therapy in the pre-clinical setting and more recently in clinical trials in oncology. Multiple obstacles have been overcome by researchers and clinicians to accelerate the development of targeted α-therapies, especially with the recent improvement in isotope production and purification, but also with the development of innovative strategies for optimized targeting...
February 8, 2018: Targeted Oncology
Pei-Pei Meng, Zhe Li, Sheng-Yu Wang, Wen-Wen Zhou, Malik Samiullah, Na Chen, Fang-Hong Luo, Ting Wu, Jiang-Hua Yan
Class three semaphorins were originally identified as mediators of axon guidance, which repelled axons and collapsed growth cones. As a member of class three semaphorins, semaphorin3F (Sema3F) has been found to have similar effects on tumor cells and endothelial cells and also is implicated in the signaling of tumor metastasis by forming a complex with neuropilins and plexins. In this study, our laboratory produced a monoclonal antibody against the C-terminal domain of Sema3F (Sema3Fc mAb) using the hybridoma method, expecting to explore the potential role of the antibody and its application in the detection of Sema3F...
February 8, 2018: Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
Toktam Abbasnia, Ahmad Asoodeh, Gholamreza Habibi, Alireza Haghparast
BACKGROUND: Tropical theileriosis is widely distributed from North Africa to East Asia. It is a tick-borne disease caused by Theileria annulata, an obligate two-host intracellular protozoan parasite of cattle. Theileria annulata use leukocytes and red blood cells for completion of the life-cycle in mammalian hosts. The stage of Theileria annulata in monocytes and B lymphocytes of cattle is an important step in pathogenicity and diagnosis of the disease. Glycosylphosphatidylinositols (GPIs) are a distinct class of glycolipid structures found in eukaryotic cells and are implicated in several biological functions...
February 6, 2018: Parasites & Vectors
Ritika Chauhan, Vinita Chauhan, Mula Kameshwar Rao, Dilip Chaudhary, Sameer Bhagyawant, Ram Kumar Dhaked
Botulinum neurotoxins (BoNTs) are the most toxic biological substances known. Their potential use as biological warfare agent results in their classification as category A biowarfare agent by Centers for Disease Control and Prevention (CDC), USA. Presently, there are no approved detection system and pharmacological treatments for BoNT intoxication. Although a toxoid vaccine is available for immuno-prophylaxis, vaccines cannot reverse the effect of pre-translocated toxin. Direct handling of the live BoNTs for developing detection and therapeutics may pose fatal danger...
January 30, 2018: Protein Expression and Purification
Hyunjoo Son, Jayoung Ku, Yoosik Kim, Sheng Li, Kookheon Char
Reactive pentafluorophenyl acrylate (PFPA) polymer brushes grafted on silica particles were prepared using surface initiated reversible addition and fragmentation chain transfer polymerization. The polymer brush was successfully immobilized with antibody, then used for protein separation. The immunoprecipitated proteins showed successful enrichment of target protein, with reduced non-specific background and less contamination from eluted antibody. To further improve protein recovery, the hydrophobic poly(PFPA) brush was modified with hydrophilic poly(ethylene glycol) (PEG)...
February 6, 2018: Biomacromolecules
Daniel Lotanna Ajaghaku, Peter Achunike Akah, Emmanuel Emeka Ilodigwe, Sunday Odunke Nduka, Uduma Eke Osonwa, Festus Basden Chinedu Okoye
BACKGROUND: Millettia aboensis (Hook. F.) Baker (Fabaceae) is popular in ethnomedicine for its acclaimed efficacy in a number of disease conditions. This study evaluated the immunomodulatory effect of the leaf extract as a possible mechanism of its ethnomedicinal uses. METHODS: Humoral and cellular immune responses of Balb/c mice to tetanus toxoid and cyclophosphamide, respectively, were used to monitor immunomodulatory activities of the ethanol leaf extract and fractions of M...
February 6, 2018: Immunological Investigations
Seongyeon Cho, Sang Min Lee, Ho Yun Shin, Min Su Kim, Yiel Hea Seo, Yong Kyun Cho, Jinwoo Lee, Sang Pyo Lee, Moon Il Kim
Nanomaterials that exhibit enzyme-like characteristics, which are called nanozymes, have recently attracted significant attention due to their potential to overcome the intrinsic limitations of natural enzymes, such as low stability and relatively high cost for preparation and purification. In this study, we report a highly efficient colorimetric allergy detection system based on an immunoassay utilizing the peroxidase-mimicking activity of hierarchically structured platinum nanoparticles (H-Pt NPs). The H-Pt NPs had a diameter of 30 nm, and were synthesized by a seed-mediated growth method, which led to a significant amount of peroxidase-like activity...
February 6, 2018: Analyst
Francis J Eng, Ahmed El-Shamy, Erin H Doyle, Arielle Klepper, A Scott Muerhoff, Andrea D Branch
Hepatitis C virus (HCV) is one of the most prevalent causes of chronic blood-borne infections worldwide. Despite developments of highly effective treatments, most infected individuals are unaware of their infection. Approximately 75% of infections are in low- and middle-income countries; therefore, continuing research in HCV molecular virology and the development of vaccines and affordable diagnostics is required to reduce the global burden. Various intracellular forms of the HCV nucleocapsid (core) protein are produced in cell culture; these comprise the conventional p21 core and the newly discovered shorter isoforms (minicores)...
January 2018: Hepatology Communications
Naoya Shinozaki, Ryuji Hashimoto, Masanori Noda, Susumu Uchiyama
Recently, we showed that immunized rabbit heavy chain variable regions (rVHs) can have strong antigen binding activity comparable to that of the camelid variable domain of the heavy chain of heavy chain antibody (VHH). These rVHs lack the light chain variable regions (rVLs), which exist in the authentic Fab format; thus, molecular surfaces at the interface region of rVHs are exposed to solvent. This physical feature may change physicochemical properties, such as causing reduced stability. By overcoming potential physicochemical issues through engineering the interface region, rVHs could become more useful as single-domain antibodies...
February 1, 2018: Journal of Bioscience and Bioengineering
Julio Inocencio, Joshua D Frenster, Dimitris G Placantonakis
This chapter describes a straightforward method for isolating glioblastoma stem cells (GSCs) from in vitro tissue cultures via fluorescence-activated cell sorting (FACS) using CD133 as a surface marker. The use of a directly conjugated antibody to an APC fluorophore against the CD133 molecule provides sufficient and clear detection of positive cells from the rest of the population. This strategy avoids an unnecessary secondary antibody incubation step thereby minimizing loss and increasing yield. The same protocol can be applied to other GSC surface markers...
2018: Methods in Molecular Biology
Zhi Wan, Heather P Ostendorff, Ziying Liu, Lynda C Schneider, Kenneth J Rothschild, Mark J Lim
Multiplex serological immunoassays, such as implemented on microarray or microsphere-based platforms, provide greater information content and higher throughput, while lowering the cost and blood volume required. These features are particularly attractive in pediatric food allergy testing to facilitate high throughput multi-allergen analysis from finger- or heel-stick collected blood. However, the miniaturization and microfluidics necessary for creating multiplex assays make them highly susceptible to the "matrix effect" caused by interference from non-target agents in serum and other biofluids...
2018: PloS One
Daan van der Es, Francesca Berni, Wouter F J Hogendorf, Nico Meeuwenoord, Diana Laverde, Angela van Diepen, Herman S Overkleeft, Dmitri V Filippov, Cornelis H Hokke, Johannes Huebner, Gijsbert A van der Marel, Jeroen Codée
Teichoic acids (TAs) are key components of the Gram-positive bacterial cell wall that are composed of alditol phosphate repeating units, decorated with alanine or carbohydrate appendages. Because of their microhetereogeneity, pure well-defined TAs for biological or immunological evaluation cannot be obtained from natural sources. We here present a streamlined automated solid phase synthesis approach for the rapid generation of well-defined glycosylated, glycerol-based TA oligomers. Building on the use of a "universal" linker system and fluorous tag purification strategy, a library of glycerolphosphate pentadecamers, decorated with various carbohydrate appendages, is generated...
February 1, 2018: Chemistry: a European Journal
Cher Hui Goey, David Bell, Cleo Kontoravdi
Host cell proteins (HCPs) are endogenous impurities, and their proteolytic and binding properties can compromise the integrity, and, hence, the stability and efficacy of recombinant therapeutic proteins such as monoclonal antibodies (mAbs). Nonetheless, purification of mAbs currently presents a challenge because they often co-elute with certain HCP species during the capture step of protein A affinity chromatography. A Quality-by-Design (QbD) strategy to overcome this challenge involves identifying residual HCPs and tracing their source to the harvested cell culture fluid (HCCF) and the corresponding cell culture operating parameters...
January 30, 2018: MAbs
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