Read by QxMD icon Read


Kristine M Ravelo, Natalia D Andersen, Paula V Monje
To date, magnetic-activated cell sorting (MACS) remains a powerful method to isolate distinct cell populations based on differential cell surface labeling. Optimized direct and indirect MACS protocols for cell immunolabeling are presented here as methods to divest Schwann cell (SC) cultures of contaminating cells (specifically, fibroblast cells) and isolate SC populations at different stages of differentiation. This chapter describes (1) the preparation of single-cell suspensions from established human and rat SC cultures, (2) the design and application of cell selection strategies using SC-specific (p75NGFR , O4, and O1) and fibroblast-specific (Thy-1) markers, and (3) the characterization of both the pre- and post-sorting cell populations...
2018: Methods in Molecular Biology
Alina Suzann Fichtner, Mohindar Murugesh Karunakaran, Lisa Starick, Richard W Truman, Thomas Herrmann
1-5% of human blood T cells are Vγ9Vδ2 T cells whose T cell receptor (TCR) contain a TRGV9/TRGJP rearrangement and a TRDV2 comprising Vδ2-chain. They respond to phosphoantigens (PAgs) like isopentenyl pyrophosphate or (E)-4-hydroxy-3-methyl-but-2-enyl-pyrophosphate (HMBPP) in a butyrophilin 3 (BTN3)-dependent manner and may contribute to the control of mycobacterial infections. These cells were thought to be restricted to primates, but we demonstrated by analysis of genomic databases that TRGV9, TRDV2 , and BTN3 genes coevolved and emerged together with placental mammals...
2018: Frontiers in Immunology
Andrey L Matveev, Vadim B Krylov, Ljudmila A Emelyanova, Arsenii S Solovev, Yana A Khlusevich, Ivan K Baykov, Thierry Fontaine, Jean-Paul Latgé, Nina V Tikunova, Nikolay E Nifantiev
A panel of specific monoclonal antibodies (mAbs) against synthetic pentasaccharide β-D-Galf-(1→5)-[β-D-Galf-(1→5)]3-α-D-Manp, structurally related to Aspergillus fumigatus galactomannan, was generated using mice immunized with synthetic pentasaccharide-BSA conjugate and by hybridoma technology. Two selected mAbs, 7B8 and 8G4, could bind with the initial pentasaccharide with affinity constants of approximately 5.3 nM and 6.4 nM, respectively, based on surface plasmon resonance-based biosensor assay. The glycoarray, built from a series of synthetic oligosaccharide derivatives representing different galactomannan fragments, demonstrated that mAb 8G4 could effectively recognize the parental pentasaccharide while mAb 7B8 recognizes its constituting trisaccharide parts...
2018: PloS One
Edward A Greenfield
Once a good immune response has developed in an animal and an appropriate screening procedure has been developed, the construction of hybridomas is ready to begin. Polyethylene glycol (PEG) is the fusing agent of choice for hybridoma production, allowing the rapid and manageable fusion of mammalian cells. PEG fuses the plasma membranes of adjacent myeloma and/or antibody-secreting cells, forming a single cell with two or more nuclei. This heterokaryon retains these nuclei until the nuclear membranes dissolve before mitosis...
March 1, 2018: Cold Spring Harbor Protocols
Edward A Greenfield
Not all lots of fetal bovine serum (FBS) are good at supporting hybridoma growth at low density. The key constituents that distinguish good batches of serum from bad are not known. It is necessary to order test batches from several suppliers and screen them as described here or purchase prescreened serum directly from the distributor.
March 1, 2018: Cold Spring Harbor Protocols
Peng Luan, Sophia Lee, Tia A Arena, Maciej Paluch, Joe Kansopon, Sharon Viajar, Zahira Begum, Nancy Chiang, Gerald Nakamura, Philip E Hass, Athena W Wong, Greg A Lazar, Avinash Gill
To rapidly find "best-in-class" antibody therapeutics, it has become essential to develop high throughput (HTP) processes that allow rapid assessment of antibodies for functional and molecular properties. Consequently, it is critical to have access to sufficient amounts of high quality antibody, to carry out accurate and quantitative characterization. We have developed automated workflows using liquid handling systems to conduct affinity-based purification either in batch or tip column mode. Here, we demonstrate the capability to purify >2000 antibodies per day from microscale (1 mL) cultures...
March 1, 2018: MAbs
Rogier M Schoeman, Wesley T E van den Beld, Evelien W M Kemna, Floor Wolbers, Jan C T Eijkel, Albert van den Berg
We present a microfluidic chip that enables electrofusion of cells in microdroplets, with exchange of nuclear components. It is shown, to our knowledge for the first time, electrofusion of two HL60 cells, inside a microdroplet. This is the crucial intermediate step for controlled hybridoma formation where a B cell is electrofused with a myeloma cell. We use a microfluidic device consisting of a microchannel structure in PDMS bonded to a glass substrate through which droplets with two differently stained HL60 cells are transported...
February 27, 2018: Scientific Reports
Andrew R M Bradbury, Nathan D Trinklein, Holger Thie, Ian C Wilkinson, Atul K Tandon, Stephen Anderson, Catherine L Bladen, Brittany Jones, Shelley Force Aldred, Marco Bestagno, Oscar Burrone, Jennifer Maynard, Fortunato Ferrara, James S Trimmer, Janina Görnemann, Jacob Glanville, Philipp Wolf, Andre Frenzel, Julin Wong, Xin Yu Koh, Hui-Yan Eng, David Lane, Marie-Paule Lefranc, Mike Clark, Stefan Dübel
Monoclonal antibodies are commonly assumed to be monospecific, but anecdotal studies have reported genetic diversity in antibody heavy chain and light chain genes found within individual hybridomas. As the prevalence of such diversity has never been explored, we analyzed 185 random hybridomas, in a large multicenter dataset. The hybridomas analyzed were not biased towards those with cloning difficulties or known to have additional chains. Of the hybridomas we evaluated, 126 (68.1%) contained no additional productive chains, while the remaining 59 (31...
February 27, 2018: MAbs
Thomas P Conway, Karla J Daniels, Yang-Nim Park, David R Soll
Firefly luciferase (FLuc) is commonly used as a reporter gene PpyLuc1 in bioanalytical assays. We have produced five mouse-derived monoclonal antibodies (mAbs) that recognize FLuc. The mAbs, DSHB-LUC-2, DSHB-LUC-3, DSHB-LUC-9, DSHB-LUC-16, and DSHB-LUC-24, were generated by immunizing mice with purified 6xHIS-tagged FLuc (6xHis-FLuc) in suspension with an adjuvant. All five were validated by dot blots. Four of the mAbs provided strong signals in western blot analysis, and one a weak signal. All five were validated for immunostaining in fixed cell culture...
February 2018: Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
Saskia Meyer, Mitchell Evers, Johannes H M Jansen, Jos Buijs, Blanca Broek, Stephanie E Reitsma, Petra Moerer, Mojtaba Amini, Anna Kretschmer, Toine Ten Broeke, Marcel T den Hartog, Mark Rijke, Christian Klein, Thomas Valerius, Peter Boross, Jeanette H W Leusen
Based on their mechanisms-of-action, CD20 monoclonal antibodies (mAbs) are grouped into Type I [complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)] and Type II [programmed cell death (PCD) and ADCC] mAbs. We generated 17 new hybridomas producing CD20 mAbs of different isotypes and determined unique heavy and light chain sequence pairs for 13 of them. We studied their epitope binding, binding kinetics and structural properties and investigated their predictive value for effector functions, i...
March 2018: British Journal of Haematology
Nachiket Shembekar, Hongxing Hu, David Eustace, Christoph A Merten
Monoclonal antibodies are a main player in modern drug discovery. Many antibody screening formats exist, each with specific advantages and limitations. Nonetheless, it remains challenging to screen antibodies for the binding of cell-surface receptors (the most important class of all drug targets) or for the binding to target cells rather than purified proteins. Here, we present a high-throughput droplet microfluidics approach employing dual-color normalized fluorescence readout to detect antibody binding. This enables us to obtain quantitative data on target cell recognition, using as little as 33 fg of IgG per assay...
February 20, 2018: Cell Reports
Yumiko Hayashi, Mie Okutani, Shohei Ogawa, Takamitsu Tsukahara, Ryo Inoue
T cell-mediated cellular immunity and humoral immunity are equally important for the prevention of diseases. To assess activation of human and mouse cellular immunity, early activation markers of lymphocytes are often used in flow cytometry targeting expression of CD69 molecules. Response of humoral immunity against infection or vaccination has been well investigated in pigs, but that of cellular immunity has been largely neglected due to lack of direct evaluation tools. Thus, in pig research a proper assay of antibody reacted with porcine CD69 is still unavailable...
February 19, 2018: Animal Science Journal, Nihon Chikusan Gakkaihō
Jinzhu Ma, Yuhua Wei, Limeng Zhang, Xintong Wang, Di Yao, Daolong Liu, Wei Liu, Simiao Yu, Yongzhong Yu, Zhijun Wu, Liquan Yu, Zhanbo Zhu, Yudong Cui
PURPOSE: To explore an epitope-based vaccine against Staphylococcus aureus, we screened the epitopes in the N2N3 subdomain of fibronectin-binding protein A (FnBPA) as a surface component of S. aureus. METHODOLOGY: We expressed N2N3 proteins and prepared monoclonal antibodies (mAbs) against N2N3 by the hybridoma technique, before screening the B-cell epitopes in N2N3 using a phage-displayed random 12-mer peptide library with these mAbs against N2N3. Finally, we analysed the characters of the screened epitopes using immunofluorescence and an S...
January 18, 2018: Journal of Medical Microbiology
Ichiro Takada, Megumi Tsuchiya, Kaori Yanaka, Shinya Hidano, Sayuri Takahashi, Takashi Kobayashi, Hidesato Ogawa, Sinichi Nakagawa, Makoto Makishima
Transcription and pre-mRNA splicing are complex, coupled processes that involve transcriptional co-regulators. Ess2 (also termed Dgcr14) is a nuclear protein that enhances the transcriptional activity of retinoic acid receptor-related orphan receptor gamma/gamma-t (Rorγ/γt). Ess2 is also a component of the spliceosomal C complex (containing U2, U5 and U6 snRNAs). However, the domains in Ess2 that function in splicing and transcription have not been identified. To elucidate the roles of Ess2 in splicing and transcription, we performed RNA immunoprecipitation (RIP) assays to detect Ess2-interacting snRNAs...
February 15, 2018: Biochemical and Biophysical Research Communications
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
Sumei Ling, Shiwei Xiao, Chengjie Xie, Rongzhi Wang, Linmao Zeng, Ke Wang, Danping Zhang, Xiulan Li, Shihua Wang
Brevetoxin-1 (BTX-1), a marine toxin mostly produced by the dinoflagellatae Karenia brevis, has caused the death of marine organisms and has had numerous toxicological effects on human health. Hence, it is very necessary to develop a rapid, economical, and reliable immunoassay method for BTX-1 detection. In this study, two kinds of complete antigen were synthesized using the succinic anhydride and isobutyl chloroformate two-step methods. Conjugate BTX-1-OVA was used as an antigen for mice immunization, and BTX-1-BSA for measuring the titer of the produced antibodies...
February 8, 2018: Toxins
Yinan Wang, Shaodong Meng, Jialin Zhang, Jun Ding, Qiang Li
Sea urchin is one of marine animals with high economic and great scientific research values. Axial organ is a glandular organ that has been presumed as coelomocytes origin site. In this paper, two monoclonal antibodies (3G10 and 6B3) against coelomocytes of sea urchin Strongylocentrotus intermedius were developed by hybridoma technique. The mAbs were characterized by indirect immunofluorescence assay test (IIFAT), flow cytometry (FCM) and western blot assay. Results showed that mAb 3G10 recognized a protein of a molecular weight of 17 kDa in the spherule cells, while mAb 6B3 reacted with a protein of a molecular weight of 35 kDa in the phagocytes...
January 30, 2018: Fish & Shellfish Immunology
Sergey E Sedykh, Victor V Prinz, Valentina N Buneva, Georgy A Nevinsky
Antibodies (Abs) containing two different antigen-binding sites in one molecule are called bispecific. Bispecific Abs (BsAbs) were first described in the 1960s, the first monoclonal BsAbs were generated in the 1980s by hybridoma technology, and the first article describing the therapeutic use of BsAbs was published in 1992, but the number of papers devoted to BsAbs has increased significantly in the last 10 years. Particular interest in BsAbs is due to their therapeutic use. In the last decade, two BsAbs - catumaxomab in 2009 and blinatumomab in 2014, were approved for therapeutic use...
2018: Drug Design, Development and Therapy
Xiaoran Liu, Ya Li, Zhixin Li, Xiaomin Wei, Yanjie Ma, Peng Cheng, Rui Jiao, Jinzhi Fang, Yun Xing, Jiali Tang, Min Wang, Taiming Li
Xanthine oxidase (XOD) is a key enzyme that catalyzes xanthine to uric acid. Most of the urate-lowering medicines targeting XOD have a limited effect on alleviating inflammation in spite of significant effects on decreasing serum uric acid level. In this study, we produced and characterized a novel monoclonal antibody (Anti-XOD mAb) using hybridoma technology based on a novel peptide OI5P-1(O-IA2(5)-P2-1),which containing a B-cell epitope of XOD and a novel Th2 built-in adjuvant I5P-1(IA2(5)-P2-1). Results of western blotting and cross-reactivity assay indicated that the mAb binds specifically to XOD and the affinity was 2...
January 27, 2018: International Journal of Biological Macromolecules
Girja S Shukla, Stephanie C Pero, Yu-Jing Sun, Chelsea L Carman, Seth Harlow, David N Krag
Autoantibodies to breast and other cancers are commonly present in cancer patients. A method to rapidly produce these anti-cancer autoantibodies in the lab would be valuable for understanding immune events and to generate candidate reagents for therapy and diagnostics. The purpose of this report is to evaluate sentinel nodes (SNs) of breast cancer patients as a source of anti-cancer antibodies. Radiotracer lymphatic mapping in 29 patients with breast cancer confirmed the identity of the SNs which provided source cells for this study...
January 27, 2018: Journal of Immunological Methods
Fetch more papers »
Fetching more papers... Fetching...
Read by QxMD. Sign in or create an account to discover new knowledge that matter to you.
Remove bar
Read by QxMD icon Read

Search Tips

Use Boolean operators: AND/OR

diabetic AND foot
diabetes OR diabetic

Exclude a word using the 'minus' sign

Virchow -triad

Use Parentheses

water AND (cup OR glass)

Add an asterisk (*) at end of a word to include word stems

Neuro* will search for Neurology, Neuroscientist, Neurological, and so on

Use quotes to search for an exact phrase

"primary prevention of cancer"
(heart or cardiac or cardio*) AND arrest -"American Heart Association"