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Oliver H Robak, Markus M Heimesaat, Andrey A Kruglov, Sandra Prepens, Justus Ninnemann, Birgitt Gutbier, Katrin Reppe, Hubertus Hochrein, Mark Suter, Carsten J Kirschning, Veena Marathe, Jan Buer, Mathias W Hornef, Markus Schnare, Pascal Schneider, Martin Witzenrath, Stefan Bereswill, Ulrich Steinhoff, Norbert Suttorp, Leif E Sander, Catherine Chaput, Bastian Opitz
Broad-spectrum antibiotics are widely used in patients on intensive care units (ICU), many of which develop hospital-acquired infections with Pseudomonas aeruginosa. Although preceding antimicrobial therapy is known as a major risk factor for P. aeruginosa-induced pneumonia, the underlying mechanisms remain incompletely understood. Here we demonstrate that depletion of the resident microbiota by broad-spectrum antibiotic treatment inhibited TLR-dependent production of a proliferation inducing ligand (APRIL), resulting in a secondary IgA deficiency in the lung in mice and human ICU patients...
May 17, 2018: Journal of Clinical Investigation
Abigail C Buenafe, Philip R Streeter
We describe here the humanisation of two mouse monoclonal antibodies that bind to surface markers on human pancreatic islet endocrine cells. Monoclonal antibodies produced by the HIC1-2B4 and HIC0-4F9 mouse hybridomas bind distinct surface molecules expressed on endocrine cells and have been validated for a number of experimental methods including immunohistochemistry and live cell sorting by flow cytometry. Variable region framework and first constant region domain sequences were replaced with that from compatible human antibody sequences, and the resulting recombinant antigen-binding fragments were cloned and expressed in mouse myeloma cells...
May 11, 2018: Journal of Immunological Methods
Rajesh Vij, Zhonghua Lin, Nancy Chiang, Jean-Michel Vernes, Kelly M Storek, Summer Park, Joyce Chan, Y Gloria Meng, Laetitia Comps-Agrar, Peng Luan, Sophia Lee, Kellen Schneider, Jack Bevers, Inna Zilberleyb, Christine Tam, Christopher M Koth, Min Xu, Avinash Gill, Marcy R Auerbach, Peter A Smith, Steven T Rutherford, Gerald Nakamura, Dhaya Seshasayee, Jian Payandeh, James T Koerber
Outer membrane proteins (OMPs) in Gram-negative bacteria are essential for a number of cellular functions including nutrient transport and drug efflux. Escherichia coli BamA is an essential component of the OMP β-barrel assembly machinery and a potential novel antibacterial target that has been proposed to undergo large (~15 Å) conformational changes. Here, we explored methods to isolate anti-BamA monoclonal antibodies (mAbs) that might alter the function of this OMP and ultimately lead to bacterial growth inhibition...
May 8, 2018: Scientific Reports
Frank A Engler, Joseph Ryan Polli, Tommy R Li, Bo An, Michael B Otteneder, Jun Qu, Joseph P Balthasar
This study examined the effects of target-expression, FcRn expression in tumors, and pH-dependent target binding on the disposition of monoclonal antibodies (mAb) in murine models of colorectal cancer. A panel of anti-carcinoembryonic antigen (CEA) mAb was developed via standard hybridoma technology, and then evaluated for pH-dependent CEA binding. Binding was assessed via immunoassay and radioligand binding assays. 10H6, a murine IgG1 mAb with high affinity for CEA at pH=7.4 (KD = 12.6±1.7 nM) and reduced affinity at pH=6...
May 7, 2018: Journal of Pharmacology and Experimental Therapeutics
Shiraazkhan Abdul, Miet Peeters, Els Brouwers, Joyce J M C Malfliet, Frank W G Leebeek, Paul J Declerck, Dingeman C Rijken, Shirley Uitte de Willige
Around 70% of circulating alpha-2-antiplasmin (α2AP), the main natural plasmin inhibitor, is N-terminally cleaved between residues Pro12 and Asn13 by antiplasmin-cleaving enzyme. This converts native Met-α2AP into the more potent fibrinolysis inhibitor Asn-α2AP. The Arg6Trp (R6W) polymorphism affects the N-terminal cleavage rate of Met-α2AP in a purified system, with ~8-fold faster conversion of Met(R6)-α2AP than Met(W6)-α2AP. To date, assays to determine N-terminally intact Met-α2AP in plasma have been limited to an ELISA that only measures Met(R6)-α2AP...
2018: PloS One
Tapas Makar, Vamshi K Nimmagadda, Poornachander R Guda, Brian Hampton, Weiliang Huang, Maureen A Kane, Paul S Fishman, Bernard Pessac, Christopher T Bever, David Trisler
BACKGROUND: Adult bone marrow contains stem cells that replenish the myeloid and lymphoid lineages. A subset of human and mouse CD34+ bone marrow stem cells can be propagated in culture to autonomously express embryonic stem cell genes and embryonic germ layer lineage genes. The current study was undertaken to determine whether these CD34+ stem cells could be obtained from human blood, whether gene expression could be modulated by culture conditions and whether the cells produce insulin...
May 2, 2018: Stem Cell Research & Therapy
Masoud Reza Seyfi Abad Shapouri, Mohammad Rashno, Pezhman Mahmoodi, Mahshid Aria
Monoclonal antibodies (MAbs) are invaluable molecules which have several advantages over polyclonal immunoglobulins (Igs) including consistency and higher specificity and hence can be used in biological researches, diagnosis and treatment of diseases. The present study was conducted to produce monoclonal antibody against chicken IgG. The IgG molecules were purified from chicken serum and used as antigens to immunize several mice. Thereafter, a well-immunized mouse was chosen and used for fusion process. After production of hybridoma cells, several rounds of cloning were carried out and produced MAbs were examined by various immunological assays including enzyme-linked immunosorbent assay (ELISA) and western and dot blotting...
2018: Veterinary Research Forum
Liwei Li, Fei Gao, Yifeng Jiang, Wu Tong, Hao Zheng, Tongling Shan, Ning Kong, Hai Yu, Deqiang Yang, Kuan Zhao, Yujiao Zhang, Guangzhi Tong
Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important viral pathogens that has caused tremendous economic losses to the swine industry worldwide. Although extensive research has been focused on PRRSV, little is known about the structure and biological functions of individual nonstructural viral proteins, especially the nonstructural protein 12 (Nsp12). In this study, we generated and identified the monoclonal antibody (mAb) against PRRSV Nsp12. Six strains of hybridoma cells named 2B10, 2B12, 5E1, 5G6, 5E7, and 8B2 secreting anti-Nsp12 mAbs were obtained by the hybridoma technique...
April 2018: Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
Praneeth Reddy Devulapally, Jörg Bürger, Thorsten Mielke, Zoltán Konthur, Hans Lehrach, Marie-Laure Yaspo, Jörn Glökler, Hans-Jörg Warnatz
Existing methods for paired antibody heavy- and light-chain repertoire sequencing rely on specialized equipment and are limited by their commercial availability and high costs. Here, we report a novel simple and cost-effective emulsion-based single-cell paired antibody repertoire sequencing method that employs only basic laboratory equipment. We performed a proof-of-concept using mixed mouse hybridoma cells and we also showed that our method can be used for discovery of novel antigen-specific monoclonal antibodies by sequencing human CD19+ B cell IgM and IgG repertoires isolated from peripheral whole blood before and seven days after Td (Tetanus toxoid/Diphtheria toxoid) booster immunization...
April 27, 2018: Genome Medicine
Peng Wang, Suli Zhang, Jie Ren, Li Yan, Lina Bai, Li Wang, Pengli Wang, Jingwei Bian, Xiaochen Yin, Huirong Liu
Autoantibodies against angiotensin II Type 1 receptor (AT1-AA) are routinely detected in the serum of preeclampsia patients, which results in an increase in vascular tone and an elevation in intracellular calcium concentration of rat vascular smooth muscle (VSM). The big conductance calcium-activated potassium channels (BKCa channels) account for the dominant outward currents in VSMCs, contributing to membrane hyperpolarization and vasodilation. In the present study, we investigated the effect of AT1-AA on BKCa channels...
April 25, 2018: Acta Biochimica et Biophysica Sinica
Fadel Sayes, Catherine Blanc, Louis S Ates, Nathalie Deboosere, Mickael Orgeur, Fabien Le Chevalier, Matthias I Gröschel, Wafa Frigui, Ok-Ryul Song, Richard Lo-Man, Florence Brossier, Wladimir Sougakoff, Daria Bottai, Priscille Brodin, Pierre Charneau, Roland Brosch, Laleh Majlessi
The pathogenic potential of Mycobacterium tuberculosis largely depends on ESX secretion systems exporting members of the multigenic Esx, Esp, and PE/PPE protein families. To study the secretion and regulation patterns of these proteins while circumventing immune cross-reactions due to their extensive sequence homologies, we developed an approach that relies on the recognition of their MHC class II epitopes by highly discriminative T cell receptors (TCRs) of a panel of T cell hybridomas. The latter were engineered so that each expresses a unique fluorescent reporter linked to specific antigen recognition...
April 24, 2018: Cell Reports
Shun Yang, Xiaoqian Tang, Xiuzhen Sheng, Jing Xing, Wenbin Zhan
B cells in some fish were recently found to have potent phagocytic activities. Sea bass (Lateolabrax japonicus) as an important economical marine fish species, it could be used as an appropriate model to study the functions of B cells in phagocytosis. In the paper, three positive hybridomas designated as 1E11, 2H4 and 3F3 secreting monoclonal antibodies (MAbs) against sea bass immunoglobulin M (IgM) were produced and used as research tools. Indirect enzyme-linked immunosorbent assay showed that all the three MAbs had a high binding capacity with sea bass serum IgM...
April 20, 2018: Fish & Shellfish Immunology
Mohsen Rokni, Ali Reza Razavi, Fazel Shokri, Kazem Ahmadi Kia, Farid Solaymani-Mohammadi, Reza Chahardoli, Ali Akbar Saboor-Yaraghi
Murine hybridoma cells can produce monoclonal antibody (MAb) and the production of these antibodies in culture and peritoneum can be affected by different factors, including stimulants, inhibitors and supplements. Among these factors, the impact of micronutrients on the production of MAbs by mouse hybridoma cells has not fully been explored. In this study the murine hybridoma cells, M3C5, were cultured and treated with different concentrations of ATRA and DHA, alone, in combinations, and at different time of exposure...
April 17, 2018: International Immunopharmacology
Ronghong Zhang, Tiewen Jia, Fang Lian, Jielin Liu, Meng Zhou
Objective To prepare monoclonal antibodies (mAb) against RecQ helicase, characterize their biological properties, and then investigate their activity against RecQ helicase in tumor cells. Methods BALB/c mice were immunized with purified recombinant RecQ helicase from E.coli. Hybridoma technique was used to generate the mAb against RecQ helicase. The chromosome karyotype analysis of the hybridoma cells was performed by colchicine blocking. The Ig subtype and titer of mAb in ascitic fluid were determined by ELISA...
February 2018: Xi Bao Yu Fen Zi Mian Yi Xue za Zhi, Chinese Journal of Cellular and Molecular Immunology
David A Rhodes, Hung-Chang Chen, James C Williamson, Alfred Hill, Jack Yuan, Sam Smith, Harriet Rhodes, John Trowsdale, Paul J Lehner, Thomas Herrmann, Matthias Eberl
Activation of human Vγ9/Vδ2 T cells by "phosphoantigens" (pAg), the microbial metabolite ( E )-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP) and the endogenous isoprenoid intermediate isopentenyl pyrophosphate, requires expression of butyrophilin BTN3A molecules by presenting cells. However, the precise mechanism of activation of Vγ9/Vδ2 T cells by BTN3A molecules remains elusive. It is not clear what conformation of the three BTN3A isoforms transmits activation signals nor how externally delivered pAg accesses the cytosolic B30...
2018: Frontiers in Immunology
Priyanka Soni, Atsuhiro Yasuhara, Toru Takenaga, Kiyoko Iwatsuki-Horimoto, Ryuta Uraki, Mutsumi Ito, Tadahiro Sasaki, Kazuyoshi Ikuta, Seiya Yamayoshi, Yoshihiro Kawaoka
Influenza B virus has been known to infect humans and other animals, including seals. Vaccination efficacy varies across seasons. Human monoclonal antibodies (mAbs) can be useful for developing novel vaccines, guided by epitope analysis, and can be used therapeutically. Hybridoma technology has been used to make mAbs. Here we evaluated SPYMEG as a fusion partner cell line for human mAb generation specific to influenza B hemagglutinin (HA). SPYMEG is a human/murine myeloma partner cell line that has previously been used to generate human mAbs that recognize the HA of influenza A and B viruses...
April 17, 2018: Journal of Veterinary Medical Science
Katharina Röltgen, Natalie Rose, Alessia Ruggieri, Louisa Warryn, Nicole Scherr, Carlos Augusto Pinho-Nascimento, Marco Tamborrini, Thomas Jaenisch, Gerd Pluschke
Dengue fever can be caused by one of four distinct dengue virus (DENV) serotypes that cocirculate in many parts of the world. Point of care serotype-specific nonstructural protein-1 (NS1) capture assays for the rapid serotyping of DENV in human sera would greatly support epidemiological surveillance and potentially also prognosis in individual patients. To ensure both serotype specificity and broad coverage of variants within serotypes, we have applied an innovative approach for the generation and selection of serotype-specific anti-NS1 mAbs...
April 16, 2018: Journal of Immunology: Official Journal of the American Association of Immunologists
Ruramayi M Nzuma, Fuquan Liu, Irene R Grant
Campylobacter jejuni is a leading cause of foodborne illness worldwide, mainly due to consumption and handling of contaminated raw chicken. Rapid detection methods for C. jejuni are vital for monitoring contamination levels in chicken products and reducing human Campylobacteriosis cases. The 'gold standard' culture-based method of Campylobacter detection takes 3-5 days and is too slow to permit effective intervention. Immuno-based methods are faster, but usually necessitate use of animals or hybridoma technology to produce antibodies; making them difficult and expensive to produce...
April 7, 2018: Applied Microbiology and Biotechnology
Claudia Martínez-García, Yolanda Medina-Flores, José Manuel de la Rosa-Vázquez, Eva Elda Soriano-Pérez, Juan Ramón Villalobos-Hernández, Eva Ramón-Gallegos
BACKGROUND: In this work, a drug product composed of an IgM antibody derived from a hybridoma subclone 4C1F6D5G7B8 was prepared and further labeled with PpIX to be used in cell lines A-549 and MRC-5. The aim of this work was to evaluate the potential theranostic activity of the obtained product together with photodynamic therapy (PDT). METHODS: The IgM antibody labeled with PpIX was used in different concentrations to perform theranostics with PDT in cell lines A-549 and MRC-5 in order to identify the specificity of IgM antibody in lung cancer cells by means of a LED-irradiation system set at 630 nm...
March 27, 2018: Photodiagnosis and Photodynamic Therapy
Natalia Vasilyevna Rudenko, Anna Petrovna Karatovskaya, Anatolyi Nikolaevich Noskov, Anna Olegovna Shepelyakovskaya, Margarita Pavlovna Shchannikova, Irina Vladimirovna Loskutova, Olga Anatolievna Artyemieva, Daria Alexandrovna Nikanova, Elena Alexandrovna Gladyr, Fedor Alexandrovich Brovko
Staphylococcal enterotoxins cause food poisoning of various degrees of severity. For milk and meat products, there is a high probability of contamination with staphylococcal enterotoxin H (SEH). In this regard specific and sensitive methods are required to be developed for its detection and monitoring. In this work, the gene seh was expressed and a preparation of recombinant toxin was obtained. Using hybridoma technology, a panel of high-affinity monoclonal antibodies (mAbs) to SEH was produced. The antibodies were characterized and shown to have no cross-reactivity towards the main staphylococcal enterotoxins (A, B, C1, D, E, G and I)...
April 2018: Journal of Food and Drug Analysis
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