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Dai Zhang, Lifang Zhou, Yajing Lei, Zhan Zhou, Jie Zhou, Shuqing Chen
Diethylstilbestrol (DES) is used as a kind of animal feed additive and affects people's health through the food chain. The purpose of this study is to detect the residue level of DES in 576 human urine samples directly. DES-BSA was used to immunize Balb/c mice. The monoclonal antibody was produced by hybridoma that was screened through cell fusion techniques. Finally, we developed the indirect competitive ELISA method to analyze 576 human urine samples from Zhejiang Province, China. The IC50 of this method was 3...
January 14, 2017: Environmental Science and Pollution Research International
Anil Kumar Gupta, Saumya Srivastava, Amit Singh, Sarman Singh
AIMS AND OBJECTIVE: To develop novel immunodiagnostic test for tuberculosis. METHODS: The selected novel proteins named as Rv2145c (SS1), Rv1437 (SS2), Rv1827 (SS3), and Rv2970c (SS4) were cloned, expressed and purified under specific conditions. Additionally, monoclonal antibodies (mAbs) were developed using these recombinant antigens via hybridoma technology. Hybridoma clones were screened and positive clones were selected for further experiment. The mAbs were purified from cell culture supernatant using protein A/G column chromatography...
December 2016: International Journal of Mycobacteriology
Xujie Liu, Chengyan Dong, Jiyun Shi, Teng Ma, Zhongxia Jin, Bing Jia, Zhaofei Liu, Li Shen, Fan Wang
Epidermal growth factor receptor mutant III (EGFRvIII) is exclusively expressed in tumors, such as glioblastoma, breast cancer and hepatocellular carcinoma, but never in normal organs. Increasing evidence suggests that EGFRvIII has clinical significance in glioblastoma prognosis due to its enhanced tumorigenicity and chemo/radio resistance, thus the development of an imaging approach to early detect EGFRvIII expression with high specificity is urgently needed. To illustrate this point, we developed a novel anti-EGFRvIII monoclonal antibody 4G1 through mouse immunization, cell fusion and hybridoma screening and then confirmed its specificity and affinity by a serial of assays...
December 22, 2016: Oncotarget
He Huang, Xiaotong Wu, Lulu Cao, Lu Wang
CTRP4 (C1q/TNF-related protein 4) is a secreted cytokine homologous to adiponectin, which plays an important role in immunity and metabolism. This study was designed to generate CTRP4 monoclonal antibodies (MAbs). Splenocytes extracted from mice immunized with prokaryotic protein were fused with myeloma cells Sp2/0 to generate hybridoma cells. Three hybridoma strains (16, 33, and 35) were chosen and their MAbs were purified. The specificity and affinity were identified by Western blot and enzyme-linked immunosorbent assay, whereas CTRP4 endogenous expression was identified by immunofluorescence staining...
December 2016: Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
Madan Kumar Paudel, Seiichi Sakamoto, Le Van Huy, Hiroyuki Tanaka, Miyamoto Tomofumi, Takano Akihito, Satoshi Morimoto
Wogonin 7-O-β-D-glucuronide (Wgn) is a bioactive flavone present in the dried root of Scutellaria baicalensis Georgi. To generate a monoclonal antibody (MAb) against Wgn, BALB/c mice injected with Wgn-bovine serum albumin yielded splenocytes that we fused with SP2/0 myeloma cells using the polyethylene glycol method. We obtained a hybridoma designated 315A that produced a MAb reactive to Wgn. The anti-Wgn MAb 315A was applied to an indirect competitive enzyme-linked immunosorbent assay (icELISA) to quantify Wgn...
December 27, 2016: Journal of Immunoassay & Immunochemistry
Asiabanha Rezaee Majid, Rasaee Mohammad Javad, Paknejad Malihe, Mohammadnejad Javad
The Human Cardiac Troponin I (hcTnI) is a 210 amino acid protein, 23 kDa in molecular weight. This biomarker is commonly used to diagnose myocardial infarction, micro injury, and acute coronary syndrome (ACS) in patients referring to emergency departments. The American Heart Association (AHA) and European Society of Cardiology (ESC) proposed troponin I as the gold biomarker for early detection of heart attack, especially in myocardial infarction (MI). Therefore, developing monoclonal antibodies against this biomarker could help in for early detection of heart attack...
December 27, 2016: Journal of Immunoassay & Immunochemistry
Shin-Ichi Funahashi, Yasunori Suzuki, Kiyotaka Nakano, Shigeto Kawai, Masami Suzuki
Leucine-rich repeat-containing G protein-coupled receptor 6 (LGR6) is a seven-pass transmembrane protein known to be a marker of stem cells in several organs. To deepen our understanding of the cell biology of LGR6-positive cells, including stem cells, we generated monoclonal antibodies (mAbs) against human LGR6. DNA immunization followed by whole-cell immunization with LGR6-expressing transfectants was performed to obtain mAbs that recognized the native form of LGR6. Hybridomas were screened by flow cytometry using LGR6-transfected cells...
December 24, 2016: Journal of Biochemistry
Amanda Sudworth, Ke-Zheng Dai, John T Vaage, Lise Kveberg
Measuring degranulation through CD107a expression has become an advantageous tool for testing the functional capacity of cytotoxic cells. Such functional studies have been hampered in the rat by the lack of a suitable anti-rat CD107a antibody. In this study, we report a novel hybridoma generated by immunizing Armenian inbred hamsters with transfected Chinese hamster ovary cells expressing CD107a. The SIM1 clone exhibited specific reactivity with CD107a and measured degranulation from natural killer (NK) cells stimulated with target cells or mAb crosslinking of their activating receptors...
2016: Frontiers in Immunology
Lichun Chen, Liangliang Wang, Jianghua Yan, Chao Ma, Jing Lu, Guoqiang Chen, Shengyou Chen, Fu Su, Weixing Wang, Xinhui Su
As a co-receptor for vascular endothelial growth factor‑3 (VEGF‑3), neuropilin receptor type‑2 (NRP‑2) plays a central role in lymphangiogenesis and angiogenesis. Recently, mounting data of evidence show that NRP‑2 is overexpressed in several human cancers, and its overexpression is often associated with poor prognosis. Therefore, it is necessary for us to develop an affinity reagent for noninvasive imaging of NRP‑2 expression because it may be possible to provide early cancer diagnosis, more accurate prognosis, and better treatment planning...
February 2017: International Journal of Oncology
Shinji Iyama, Masao Ono, Hitomi Kawai-Nakahara, Ryan Edbert Husni, Tomoko Dai, Toshihiro Shiozawa, Akiko Sakata, Hirotsugu Kohrogi, Masayuki Noguchi
OBJECTIVES: With the aim of searching for novel oncofetal tumor biomarkers of lung adenocarcinoma other than carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP), we developed a strategy involving monoclonal antibodies generated from embryonic tissue of miniature swine. MATERIALS AND METHODS: Using immunohistochemistry, we selected suitable hybridoma clones that were reactive against swine fetal lung but not adult lung using tissue microarray loading of human normal lung, lung cancer, and fetal and adult swine tissues...
December 2016: Lung Cancer: Journal of the International Association for the Study of Lung Cancer
Fang Chen, Yanqing Li, Junli Zhao, Qinwen Mao, Haibin Xia
The nuclear receptor Rev-erbβ is a potent transcriptional factor whose functional study has been limited by the lack of suitable antibodies against it. To better understand Rev-erbβ's biological roles, we generated five hybridoma cell lines secreting antibodies against human Rev-erbβ in mice immunized with the purified, prokaryotically expressed recombinant Rev-erbβ-6His fusion protein. Using Western blotting and immunofluorescence analyses, all the five monoclonal antibodies (MAbs) showed strong immunoreactivity to both prokaryotically and eukaryotically expressed recombinant Rev-erbβ...
December 16, 2016: Applied Biochemistry and Biotechnology
Hiroyuki Oyama, Izumi Morita, Yuki Kiguchi, Erika Banzono, Kasumi Ishii, Satoshi Kubo, Yoshiro Watanabe, Anna Hirai, Chiaki Kaede, Mitsuhiro Ohta, Norihiro Kobayashi
Immunoassays for cotinine, a major nicotine metabolite, in the urine are useful for monitoring the degree of tobacco smoke exposure. However, hybridoma-based anti-cotinine antibodies lack sufficient binding affinity to perform practically sensitive measurements, and thus most cotinine assays still rely on polyclonal antibodies. Here, we describe the generation of a mutant single-chain Fv fragment (scFv) that was used in an enzyme-linked immunosorbent assay (ELISA) to determine urinary cotinine levels in passive smokers...
December 16, 2016: Analytical Chemistry
Michael Fehlings, Lea Drobbe, Macarena Beigier-Bompadre, Pablo Renner Viveros, Verena Moos, Thomas Schneider, Thomas F Meyer, Toni Aebischer, Ralf Ignatius
Direct effects of Helicobacter pylori (H. pylori) on human CD4(+) T-cells hamper disentangling a possible bacterial-mediated interference with major histocompatibility complex class II (MHC-II)-dependent antigen presentation to these cells. To overcome this limitation, we employed a previously described assay, which enables assessing human antigen-processing cell function by using murine T-cell hybridoma cells restricted by human leukocyte antigen (HLA) alleles. HLA-DR1(+) monocyte-derived dendritic cells were exposed to H...
December 1, 2016: European Journal of Microbiology & Immunology
Chun San Tai, Yi Yun Chen, Wen Liang Chen
β-Lactoglobulin (LG) is suspected to enhance or modulate human immune responses. Moreover, LG is also hypothesized to increase human cell proliferation. However, these potential functions of LG have not been directly or thoroughly addressed. In this study, we demonstrated that LG is a potent stimulator of cell proliferation using a hybridoma cell (a splenocyte fused with a myeloma cell) model. LG's ability to promote cell proliferation was lost when the protein is denatured. To further investigate the influence of LG's conformation on cell proliferation, we chemically modified LG by either carboxymethylation (CM) or acetylation and observed significantly reduced cell proliferation when the protein structure was altered...
2016: BioMed Research International
Róża Sawicka, Paweł Siedlecki, Barbara Kalenik, Jan P Radomski, Violetta Sączyńska, Anna Porębska, Bogusław Szewczyk, Agnieszka Sirko, Anna Góra-Sochacka
Hemagglutinin (HA), as a major surface antigen of influenza virus, is widely used as a target for production of neutralizing antibodies. Monoclonal antibody, mAb6-9-1, directed against HA of highly pathogenic avian influenza virus A/swan/Poland/305-135V08/2006(H5N1) was purified from mouse hybridoma cells culture and characterized. The antigenic specificity of mAb6-9-1 was verified by testing its cross-reactivity with several variants of HA. The mimotopes recognized by mAb6-9-1 were selected from two types of phage display peptide libraries...
December 7, 2016: Acta Biochimica Polonica
Hideyuki Arimitsu, Keiko Sasaki, Takao Tsuji
Here, we report the development of an immunochromatographic test strip that can detect heat-labile enterotoxin (LT) produced by enterotoxigenic Escherichia coli. Five types of monoclonal antibody (mAb)-producing hybridomas were isolated: three mAbs were A subunit specific and two were B subunit specific. Four mAbs also cross-reacted with both LT proteins derived from swine and human E. coli strains, but only one mAb 57B9 additionally cross-reacted with cholera toxin. Thus, mAb 57B9 was used to form a gold colloid-conjugated antibody for the immunochromatographic test by combination with polyclonal anti-LT rabbit IgG...
January 2017: Journal of Microbiological Methods
Fatemeh Khademi, Ali Mostafaie, Shahram Parvaneh, Farah Gholami Rad, Pantea Mohammadi, Gholamreza Bahrami
To date, several new anti-CD20 monoclonal antibodies (mAbs) have been developed for potential efficacies compared with familiar mAb rituximab. Despite the recent advances in development of anti-CD20 mAbs for the treatment of B cell malignancies, the efforts should be continued to develop novel antibodies with improved properties. However, the development of mAbs against CD20 as a multi-transmembrane protein is challenging due to the difficulty of providing a lipid environment that can maintain native epitopes...
February 2017: International Immunopharmacology
Jacalyn Rosenblatt, Richard M Stone, Lynne Uhl, Donna Neuberg, Robin Joyce, James D Levine, Jon Arnason, Malgorzata McMasters, Katarina Luptakova, Salvia Jain, Jeffrey I Zwicker, Ayad Hamdan, Vassiliki Boussiotis, David P Steensma, Daniel J DeAngelo, Ilene Galinsky, Poorvi Somaiya Dutt, Emma Logan, Mary Paty Bryant, Dina Stroopinsky, Lillian Werner, Kristen Palmer, Max Coll, Abigail Washington, Leandra Cole, Donald Kufe, David Avigan
We developed a personalized cancer vaccine in which patient-derived acute myeloid leukemia (AML) cells are fused with autologous dendritic cells, generating a hybridoma that potently stimulates broad antitumor responses. We report results obtained from the first 17 AML patients, who achieved remission after chemotherapy and were then serially vaccinated to target minimal residual disease and prevent relapse. Vaccination was well tolerated and induced inflammatory responses at the site of administration, characterized by the dense infiltration of T cells...
December 7, 2016: Science Translational Medicine
Andreas Naschberger, Barbara G Fürnrohr, Tihana Lenac Rovis, Suzana Malic, Klaus Scheffzek, Hans Dieplinger, Bernhard Rupp
The monoclonal antibody N14 is used as a detection antibody in ELISA kits for the human glycoprotein afamin, a member of the albumin family, which has recently gained interest in the capture and stabilization of Wnt signalling proteins, and for its role in metabolic syndrome and papillary thyroid carcinoma. As a rare occurrence, the N14 Fab is N-glycosylated at Asn26L at the onset of the VL1 antigen-binding loop, with the α-1-6 core fucosylated complex glycan facing out of the L1 complementarity-determining region...
December 1, 2016: Acta Crystallographica. Section D, Structural Biology
Zehra Elgundi, Mouhamad Reslan, Esteban Cruz, Vicki Sifniotis, Veysel Kayser
It has been over four decades since the development of monoclonal antibodies (mAbs) using a hybridoma cell line was first reported. Since then more than thirty therapeutic antibodies have been marketed, mostly as oncology, autoimmune and inflammatory therapeutics. While antibodies are very efficient, their cost-effectiveness has always been discussed owing to their high costs, accumulating to more than one billion dollars from preclinical development through to market approval. Because of this, therapeutic antibodies are inaccessible to some patients in both developed and developing countries...
December 2, 2016: Advanced Drug Delivery Reviews
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