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https://www.readbyqxmd.com/read/28928083/preparation-and-characterization-of-a-high-affinity-monoclonal-antibody-against-human-epididymis-protein-4
#1
Yunlong Shen, Yuxi Wang, Xiaohua Jiang, Liang Lu, Chengdi Wang, Wenxin Luo, Yongxia Zhang, Pei Li, Zhengwei Du, Tengfei Dai, Congcong Wu, Aiping Fang, Yuqin Yao, Qian Peng, Jinliang Yang
Human epididymis protein-4 (HE4) may serve as a putative biomarker for the early diagnosis, therapy and especially prognosis of ovarian, lung and breast cancer. Detection and targeting of HE4 using the anti-HE4 antibody could be one of the effective strategies for the cancer diagnosis and treatment in clinical practice. In this study, a high-efficiency expression system was established to purify recombinant HE4. We obtained high purity HE4 in 400 mg quantity from 1 L culture supernatant of HEK293F cells. CCK-8 and cell cycle assays indicated that the purified recombinant HE4 protein could promote SKOV3 cell cycle and proliferation at the concentration of 0...
September 18, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28926954/evidence-for-complex-formation-of-the-bacillus-cereus-haemolysin-bl-components-in-solution
#2
Franziska Tausch, Richard Dietrich, Kristina Schauer, Robert Janowski, Dierk Niessing, Erwin Märtlbauer, Nadja Jessberger
Haemolysin BL is an important virulence factor regarding the diarrheal type of food poisoning caused by Bacillus cereus. However, the pathogenic importance of this three-component enterotoxin is difficult to access, as nearly all natural B. cereus culture supernatants additionally contain the highly cytotoxic Nhe, the second three-component toxin involved in the aetiology of B. cereus-induced food-borne diseases. To better address the toxic properties of the Hbl complex, a system for overexpression and purification of functional, cytotoxic, recombinant (r)Hbl components L₂, L₁ and B from E...
September 16, 2017: Toxins
https://www.readbyqxmd.com/read/28922396/biochemical-and-immunological-characterization-of-a-novel-monoclonal-antibody-against-mouse-leukotriene-b4-receptor-1
#3
Fumiyuki Sasaki, Tomoaki Koga, Kazuko Saeki, Toshiaki Okuno, Saiko Kazuno, Tsutomu Fujimura, Yasuyuki Ohkawa, Takehiko Yokomizo
Leukotriene B4 (LTB4) receptor 1 (BLT1) is a G protein-coupled receptor expressed in various leukocyte subsets; however, the precise expression of mouse BLT1 (mBLT1) has not been reported because a mBLT1 monoclonal antibody (mAb) has not been available. In this study, we present the successful establishment of a hybridoma cell line (clone 7A8) that produces a high-affinity mAb for mBLT1 by direct immunization of BLT1-deficient mice with mBLT1-overexpressing cells. The specificity of clone 7A8 was confirmed using mBLT1-overexpressing cells and mouse peripheral blood leukocytes that endogenously express BLT1...
2017: PloS One
https://www.readbyqxmd.com/read/28919331/production-and-characterization-of-monoclonal-antibodies-against-cathepsin-b-and-cathepsin-b-like-proteins-of-naegleria-fowleri
#4
Gi-Sang Seong, Hae-Jin Sohn, Heekyoung Kang, Ga-Eun Seo, Jong-Hyun Kim, Ho-Joon Shin
Naegleria fowleri causes fatal primary amoebic meningoencephalitis (PAM) in humans and experimental animals. In previous studies, cathepsin B (nfcpb) and cathepsin B-like (nfcpb-L) genes of N. fowleri were cloned, and it was suggested that refolding rNfCPB and rNfCPB-L proteins could play important roles in host tissue invasion, immune response evasion and nutrient uptake. In this study, we produced anti-NfCPB and anti-NfCPB-L monoclonal antibodies (McAb) using a cell fusion technique, and observed their immunological characteristics...
September 14, 2017: Experimental Parasitology
https://www.readbyqxmd.com/read/28898398/integrating-high-throughput-screening-and-sequencing-for-monoclonal-antibody-discovery-and-engineering
#5
REVIEW
Cristina Parola, Daniel Neumeier, Sai T Reddy
Monoclonal antibody discovery and engineering is a field that has traditionally been dominated by high-throughput screening platforms (e.g., hybridomas and surface display). In recent years the emergence of high-throughput sequencing has made it possible to obtain large-scale information on antibody repertoire diversity. Additionally, it has now become more routine to perform high-throughput sequencing on antibody repertoires to also directly discover antibodies. In this review, we provide an overview of the progress in this field to date and show how high-throughput screening and sequencing are converging to deliver powerful new workflows for monoclonal antibody discovery and engineering...
September 12, 2017: Immunology
https://www.readbyqxmd.com/read/28881357/a-novel-inhibitory-anti-invasive-mab-isolated-using-phenotypic-screening-highlights-anxa6-as-a-functionally-relevant-target-protein-in-pancreatic-cancer
#6
Dermot O'Sullivan, Paul Dowling, Helena Joyce, Edel McAuley, Andrew McCann, Michael Henry, Brianan McGovern, Paul Barham, Fergal C Kelleher, Jean Murphy, Susan Kennedy, Niall Swan, Michael Moriarty, Martin Clynes, Annemarie Larkin
BACKGROUND: Discovery and validation of new antibody tractable targets is critical for the development of new antibody therapeutics to address unmet needs in oncology. METHODS: A highly invasive clonal variant of the MDA-MB-435S cell line was used to generate monoclonal antibodies (MAbs), which were screened for anti-invasive activity against aggressive cancer cells in vitro. The molecular target of selected inhibitory MAb 9E1 was identified using immunoprecipitation/liquid chromatography-tandem mass spectrometry...
September 7, 2017: British Journal of Cancer
https://www.readbyqxmd.com/read/28870527/identification-and-phenotyping-of-circulating-autoreactive-proteinase-3-specific-b-cells-in-patients-with-pr3-anca-associated-vasculitis-and-healthy-controls
#7
Divi Cornec, Alvise Berti, Amber Hummel, Tobias Peikert, Jacques-Olivier Pers, Ulrich Specks
OBJECTIVES: To develop a method to detect and phenotype circulating proteinase 3 (PR3)-specific B-cells in patients with PR3-ANCA associated vasculitis (AAV). METHODS: Recombinant human PR3 (rPR3) was tagged with FITC or biotin, and its binding characteristics were studied by flow cytometry using three hybridoma cell lines secreting antibodies (Ab) against human PR3, mouse PR3 (no cross-reactivity with human PR3), and human neutrophil elastase. We measured the proportion of PR3-specific B-cells and studied their surface phenotype in patients with PR3-AAV and healthy controls (HC)...
September 1, 2017: Journal of Autoimmunity
https://www.readbyqxmd.com/read/28865256/identification-and-verification-of-hybridoma-derived-monoclonal-antibody-variable-region-sequences-using-recombinant-dna-technology-and-mass-spectrometry
#8
Lmar Babrak, Jeffery A McGarvey, Larry H Stanker, Robert Hnasko
Antibody engineering requires the identification of antigen binding domains or variable regions (VR) unique to each antibody. It is the VR that define the unique antigen binding properties and proper sequence identification is essential for functional evaluation and performance of recombinant antibodies (rAb). This determination can be achieved by sequence analysis of immunoglobulin (Ig) transcripts obtained from a monoclonal antibody (MAb) producing hybridoma and subsequent expression of a rAb. However the polyploidy nature of a hybridoma cell often results in the added expression of aberrant immunoglobulin-like transcripts or even production of anomalous antibodies which can confound production of rAb...
October 2017: Molecular Immunology
https://www.readbyqxmd.com/read/28864569/modification-of-antibody-function-by-mutagenesis
#9
James R Dasch, Amy L Dasch
The ability to "fine-tune" recombinant antibodies by mutagenesis separates recombinant antibodies from hybridoma-derived antibodies because the latter are locked with respect to their properties. Recombinant antibodies can be modified to suit the application: Changes in isotype, format (e.g., scFv, Fab, bispecific antibodies), and specificity can be made once the heavy- and light-chain sequences are available. After immunoglobulin heavy and light chains for a particular antibody have been cloned, the binding site-namely, the complementarity determining regions (CDR)-can be manipulated by mutagenesis to obtain antibody variants with improved properties...
September 1, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/28861555/cranberry-proanthocyanidins-protein-complexes-for-macrophage-activation
#10
Sergio M Carballo, Linda Haas, Christian G Krueger, Jess D Reed
In this work we characterize the interaction of cranberry (Vaccinium macrocarpon) proanthocyanidins (PAC) with bovine serum albumin (BSA) and hen egg-white lysozyme (HEL) and determine the effects of these complexes on macrophage activation and antigen presentation. We isolated PAC from cranberry and complexed the isolated PAC with BSA and HEL. The properties of the PAC-protein complexes were studied by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), gel electrophoresis and zeta-potential...
September 20, 2017: Food & Function
https://www.readbyqxmd.com/read/28852189/production-of-monoclonal-antibodies-to-pathologic-%C3%AE-sheet-oligomeric-conformers-in-neurodegenerative-diseases
#11
Fernando Goñi, Mitchell Martá-Ariza, Daniel Peyser, Krystal Herline, Thomas Wisniewski
We describe a novel approach to produce conformational monoclonal antibodies selected to specifically react with the β-sheet secondary structure of pathological oligomeric conformers, characteristic of many neurodegenerative diseases. Contrary to past and current efforts, we utilize a mammalian non-self-antigen as an immunogen. The small, non-self peptide selected was covalently polymerized with glutaraldehyde until it reached a high β-sheet secondary structure content, and species between 10-100kDa that are immunogenic, stable and soluble (p13Bri)...
August 29, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28851380/vaccine-draining-lymph-nodes-of-cancer-patients-for-generating-anti-cancer-antibodies
#12
Girja S Shukla, Walter C Olson, Stephanie C Pero, Yu-Jing Sun, Chelsea L Carman, Craig L Slingluff, David N Krag
BACKGROUND: Our research is focused on using the vaccine draining lymph node to better understand the immune response to cancer vaccines and as a possible source of anti-cancer reagents. We evaluated vaccine draining lymph nodes archived from a clinical study in melanoma patients and determined the reaction of B cells to the vaccine peptides. METHODS: Mononuclear cells (MNCs) were recovered from cryopreserved lymph nodes that were directly receiving drainage from multi-peptide melanoma vaccine...
August 29, 2017: Journal of Translational Medicine
https://www.readbyqxmd.com/read/28846506/rare-high-affinity-mouse-anti-pd-1-antibodies-that-function-in-checkpoint-blockade-discovered-using-microfluidics-and-molecular-genomics
#13
Adam S Adler, Rena A Mizrahi, Matthew J Spindler, Matthew S Adams, Michael A Asensio, Robert C Edgar, Jackson Leong, Renee Leong, David S Johnson
Conventionally, mouse hybridomas or well-plate screening are used to identify therapeutic monoclonal antibody candidates. In this study, we present an alternative to hybridoma-based discovery that combines microfluidics, yeast single-chain variable fragment (scFv) display, and deep sequencing to rapidly interrogate and screen mouse antibody repertoires. We used our approach on six wild-type mice to identify 269 molecules that bind to programmed cell death protein 1 (PD-1), which were present at an average of 1 in 2,000 in the pre-sort scFv libraries...
August 28, 2017: MAbs
https://www.readbyqxmd.com/read/28843853/the-development-of-monoclonal-anti-adamts18-antibodies-with-precise-validation-of-adamts18-post-translational-modification-status-in-living-organisms
#14
Rui Zhu, Mengting Cheng, Shuai Ye, Meng Liu, Lijie Sun, Tiantian Lu, Ning Yang, Tao Hong, Suying Dang, Wei Zhang
ADAMTS18 is a member of a secreted Zn-metalloproteinase ADAMTS family, and has been implicated in development, hemostasis, and various malignancies. It has thus far proven difficult to resolve its post-translational modification status, cleaved forms, and splice variants in living organisms due to the lack of specific antibodies available to characterize this enzyme. In this study, we develop six murine monoclonal antibodies (mAbs) against different functional regions of ADAMTS18 using hybridoma technology...
August 24, 2017: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/28833498/generation-and-selection-of-na%C3%A3-ve-fab-library-for-parasitic-antigen-anti-bmsxp-antibodies-for-lymphatic-filariasis
#15
Noorsharmimi Omar, Nurul Hamizah Hamidon, Muhammad Hafiznur Yunus, Rahmah Noordin, Yee Siew Choong, Theam Soon Lim
Phage display has been applied successfully as a tool for the generation of monoclonal antibodies (mAbs). Naive antibody libraries are unique as they are able to overcome several limitations associated with conventional mAb generation methods like the hybridoma technology. Here, we performed an in vitro selection and generation of Fab antibodies against Brugia malayi SXP protein (BmSXP), a recombinant antigen for the detection of lymphatic filariasis. We developed a naïve multi ethnic Fab antibody library with an estimated diversity of 2...
August 21, 2017: Biotechnology and Applied Biochemistry
https://www.readbyqxmd.com/read/28817087/the-preparation-and-identification-of-a-monoclonal-antibody-against-domoic-acid-and-establishment-of-detection-by-indirect-competitive-elisa
#16
Abdullah F U H Saeed, Sumei Ling, Jun Yuan, Shihua Wang
Domoic acid (DA) is a potent toxin, marine biotoxin, and primarily produced by Pseudo-nitzschia. The DA hapten was coupled with bovine serum albumin (BSA), and ovalbumin (OVA) as carrier proteins. DA-BSA conjugate was used as immunogen and DA-OVA as coating antigen. Cell fusion between spleen cells and sp2/0 myeloma cells developed 1C3 hybridoma clone producing 1C3 monoclonal antibody (mAb). Hybridoma was injected into the mice to produce ascites, and further purified by caprylic acid/ammonium sulfate method...
August 17, 2017: Toxins
https://www.readbyqxmd.com/read/28806153/expression-purification-and-refolding-of-human-lipocalin-6-and-production-of-a-monoclonal-antibody-against-this-protein
#17
Jiong Chen, Wei Feng, Yue Zhao, Yaqin Li, Fei Zhan
Human lipocalin 6 (hLCN6) is a member of the lipocalin family, which is a group of structurally conserved hydrophobic ligand binding proteins, and widely distributed in animal, plant, and bacteria. Specific expression of hLCN6 in the epididymis and localization of this protein on the surface of spermatozoa suggest a role played by hLCN6, which may function as a transporter to carry ligands in the epididymal channel. However, the role of hLCN6 in sperm maturation has been largely unknown due to the lack of effective antibodies...
August 2017: Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
https://www.readbyqxmd.com/read/28806151/establishment-of-a-monoclonal-antibody-against-chgilz
#18
Zhiyuan He, Yongqiang Wang
The chicken glucocorticoid-induced leucine zipper (chGILZ) participates in the inflammation of avian immunosuppressive diseases. We aimed to establish a monoclonal antibody (MAb) against chGILZ and to investigate its distribution in clinical diagnosis. A gene cloned from chicken embryo fibroblast (CEF) cell was inserted into the expression vector pET-28a and pGEX-6p-1. The recombinant expression vectors were transformed into Escherichia coli BL21 (DE3). Then the recombinant proteins His-chGILZ and GST-chGILZ were successfully expressed and purified...
August 2017: Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
https://www.readbyqxmd.com/read/28804489/normalized-synergy-predicts-that-cd8-co-receptor-contribution-to-t-cell-receptor-tcr-and-pmhc-binding-decreases-as-tcr-affinity-increases-in-human-viral-specific-t-cells
#19
Chad M Williams, Alexandra A Schonnesen, Shu-Qi Zhang, Ke-Yue Ma, Chenfeng He, Tori Yamamoto, S Gail Eckhardt, Christopher A Klebanoff, Ning Jiang
The discovery of naturally occurring T cell receptors (TCRs) that confer specific, high-affinity recognition of pathogen and cancer-associated antigens remains a major goal in cellular immunotherapies. The contribution of the CD8 co-receptor to the interaction between the TCR and peptide-bound major histocompatibility complex (pMHC) has previously been correlated with the activation and responsiveness of CD8(+) T cells. However, these studies have been limited to model systems of genetically engineered hybridoma TCRs or transgenic mouse TCRs against either a single epitope or an array of altered peptide ligands...
2017: Frontiers in Immunology
https://www.readbyqxmd.com/read/28803843/high-throughput-screening-of-hybridoma-supernatants-using-multiplexed-fluorescent-cell-barcoding-on-live-cells
#20
Mei Lu, Brian M Chan, Peter W Schow, Wesley S Chang, Chadwick T King
With current available assay formats using either immobilized protein (ELISA, enzyme-linked immunosorbent assay) or immunostaining of fixed cells for primary monoclonal antibody (mAb) screening, researchers often fail to identify and characterize antibodies that recognize the native conformation of cell-surface antigens. Therefore, screening using live cells has become an integral and important step contributing to the successful identification of therapeutic antibody candidates. Thus the need for developing high-throughput screening (HTS) technologies using live cells has become a major priority for therapeutic mAb discovery and development...
August 10, 2017: Journal of Immunological Methods
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