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antibody library

Mieko Kato, Yoshiro Hanyu
We present a simple colony assay method for screening antibody libraries based on autoinduction of antibody fragment expression. This protocol eliminates the need for colony size monitoring and a separate induction step for single-chain Fv (scFv) antibody fragment expression. Here, scFvs are expressed in an automatic and timely fashion during the assay, resulting in high yields of positive clones and substantial time savings. The method was used successfully to establish monoclonal scFvs with high affinity and specificity against human IgG...
February 20, 2019: BioTechniques
Chongxin Xu, Xiaoqin Liu, Yuan Liu, Xiao Zhang, Cunzheng Zhang, Jianhong Li, Xianjin Liu
Microcystin-LR (MC-LR) is one of common high-toxic biotoxins produced by cyanobacteria in waterbody. A high sensitive and convenient detection method is necessary for monitoring for MC-LR. To establish a high sensitive indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) based on single chain variable fragment (scFv) for detecting MC-LR, 16 positive anti-MC-LR phage scFv particles were screened out from a MC-LR-immunized mouse phage scFv library, which was successfully constructed with the capacity of 8...
May 15, 2019: Talanta
Wonil Chae, Paul Kim, Beom Jeung Hwang, Baik Lin Seong
Seasonal and pandemic influenza infections remain a serious public health concern. Many health authorities recommend annual vaccination as the most effective way to control influenza infection. Accordingly, regulatory guidelines ask vaccine manufacturers to determine vaccine potency at the time of release and throughout shelf-life to ensure vaccine quality. The potency of inactivated influenza vaccine is related to the quantity of hemagglutinin (HA). Since 1970s, single radial immunodiffusion (SRID) assay has been standardly used for the quantitation of HA in influenza vaccine...
February 11, 2019: Vaccine
Luke J Norbury, Katarzyna Basałaj, Piotr Bąska, Alicja Sielicka, Anna Zawistowska-Deniziak, Huan Yong Yap, Przemysław Wilkowski, Agnieszka Wesołowska, Halina Wędrychowicz
Phage display technology to produce recombinant monoclonal antibodies or antibody fragments permits the identification of sought after antibodies in short time frames at low cost along with direct and rapid selection for antibody characteristics. Monoclonal antibodies can facilitate the identification and characterisation of parasite molecules that function at the host-parasite interface to help understand at the molecular level the biology of the parasite and disease progression, which often leads to new drug targets, diagnostic antigens or vaccine candidates...
February 11, 2019: Experimental Parasitology
Andrew M Wollacott, Luke N Robinson, Boopathy Ramakrishnan, Hamid Tissire, Karthik Viswanathan, Zachary Shriver, Gregory J Babcock
IgA nephropathy (IgAN) is the most prevalent cause of primary glomerular disease worldwide, and the cytokine A PRoliferation-Inducing Ligand (APRIL) is emerging as a key player in IgAN pathogenesis and disease progression. For a panel of anti-human APRIL antibodies with known antagonistic activity, we sought to define their structural mode of engagement to understand molecular mechanisms of action and aid rational antibody engineering. Reliable computational prediction of antibody-antigen complexes remains challenging, and experimental methods such as X-ray co-crystallography and cryoEM have considerable technical, resource, and throughput barriers...
February 13, 2019: Journal of Molecular Recognition: JMR
Thomas Wilkop, Sivakumar Pattathil, Guangxi Ren, Destiny Jade Davis, Wenlong Bao, Dechao Duan, Angelo Gabriel Peralta, David Domozych, Michael G Hahn, Georgia Drakakaki
The plant endomembrane system facilitates transport of polysaccharides, associated enzymes and glycoproteins through its dynamic pathways. Although enzymes involved in cell wall biosynthesis have been identified, little is known about the endomembrane-based transport of glycan components. This is partially attributed to technical challenges in biochemically determining polysaccharide cargo in specific vesicles. Here we introduce a hybrid approach addressing this limitation. By combining vesicle isolation with a large-scale carbohydrate antibody arraying technique, we charted an initial large-scale map describing the glycome profile of the Syntaxin of Plants 61 (SYP61) trans-Golgi network (TGN) compartment...
February 13, 2019: Plant Cell
Anna Davydova, Mariya Vorobyeva, Eugenia Bashmakova, Pavel Vorobjev, Olga Krasheninina, Alexey Tupikin, Marsel Kabilov, Vasilisa Krasitskaya, Ludmila Frank, Alya Venyaminova
Aptamers are short DNA and RNA fragments which bind their molecular targets with affinity and specificity comparable to those of antibodies. Here, we describe the selection of novel 2'-F-RNA aptamers against total human hemoglobin or its glycated form HbA1c. After SELEX and high-throughput sequencing of the enriched libraries, affinities and specificities of candidate aptamers and their truncated variants were examined by the solid-phase bioluminescent assay. As a result, we identified aptamers specific to both hemoglobins or only glycated HbA1c...
February 8, 2019: Analytical Biochemistry
K Martin Chow, Sidney W Whiteheart, Jeffrey R Smiley, Savita Sharma, Kathy Boaz, Meggie J Coleman, Alvina Maynard, Louis B Hersh, Craig W Vander Kooi
In this manuscript, a method for the immunization of alpaca and the use of molecular biology methods to produce antigen-specific single domain antibodies is described and demonstrated. Camelids, such as alpacas and llamas, have become a valuable resource for biomedical research since they produce a novel type of heavy chain-only antibody which can be used to produce single domain antibodies. Because the immune system is highly flexible, single domain antibodies can be made to many different protein antigens, and even different conformations of the antigen, with a very high degree of specificity...
January 26, 2019: Journal of Visualized Experiments: JoVE
Gaetano Gallo, Giuseppe Sena, Giuseppina Vescio, Matteo Papandrea, Rosario Sacco, Mario Trompetto, Giuseppe Sammarco
BACKGROUND: Colorectal cancer (CRC) is one of the leading cause of cancer deaths worldwide. The aetiology of CRC is complex and involves interaction on environmental and genetic factors. The two most important pathways are the EGFR (Epidermal Grow Factor Receptor) signaling pathway, with the involvement of KRAS and BRAF, and the DNA mismatch repair (MMR). Generally, KRAS and BRAF mutations are mutually exclusive. They are both able to cause RAS/RAF/MAPK signaling pathway upregulation and are necessary for CRC development...
February 4, 2019: Annali Italiani di Chirurgia
Flávio Sádio, Gerhard Stadlmayr, Katharina Stadlbauer, Florian Rüker, Gordana Wozniak-Knopp
Since the introduction of the yeast display platform, this method has increasingly gained popularity for the discovery and affinity maturation of antibodies and other protein scaffolds intended for antigen recognition. Yeast display is particularly well suited for the selection of antigen-binding Fc fragments (Fcabs) as it allows rapid combinatorial library construction via gap repair-driven homologous recombination and an efficient display of a glycosylated Fc able to interact with Fcγ receptors. Apart from expression-related normalization, isolation of properly folded Fcabs can be guided efficiently by simultaneous staining with ligands such as protein A, FcγRI, or the conformation-sensitive anti-FigCH 2 antibody, whose binding is critically dependent on the integrity of the Fc structure...
2019: Methods in Molecular Biology
He Zhu, Lin Zhao, Zemin Li, Biyan Wen, Chuangnan Qiu, Mengmiao Liu, Zhimin Xu, Shuzhuang Hu, Huangjin Li
Epidermal growth factor receptor (EGFR) is an effective target for the treatment of many epithelial cancers. However, EGFR inhibitors have low clinical response rates and are prone to drug resistance arising from mutations and heterodimerization of EGFR. Therefore, targeting the highly conserved dimer interface of EGFR may be an effective strategy for improving the clinical response of anti-EGFR therapies. Nanobodies have significant advantages over conventional antibodies in terms of size, solubility, stability and cost-effectiveness...
February 5, 2019: Protein Expression and Purification
Jinsung Noh, Okju Kim, Yushin Jung, Haejun Han, Jung-Eun Kim, Soohyun Kim, Sanghyub Lee, Jaeseong Park, Rae Hyuck Jung, Sang Il Kim, Jaejun Park, Jerome Han, Hyunho Lee, Duck Kyun Yoo, Amos C Lee, Euijin Kwon, Taehoon Ryu, Junho Chung, Sunghoon Kwon
In antibody discovery, in-depth analysis of an antibody library and high-throughput retrieval of clones in the library are crucial to identifying and exploiting rare clones with different properties. However, existing methods have technical limitations, such as low process throughput from the laborious cloning process and waste of the phenotypic screening capacity from unnecessary repetitive tests on the dominant clones. To overcome the limitations, we developed a new high-throughput platform for the identification and retrieval of clones in the library, TrueRepertoire™...
February 8, 2019: MAbs
Janine Heinrich, Chris Drewniok, Eva Neugebauer, Harald Kellner, Thomas Wiegert
BACKGROUND: Heterologous gene expression is well established for various prokaryotic model systems. However, low yield, incorrect folding and instability still impede the production of soluble, bioactive proteins. To improve protein production with the Gram-positive host Bacillus subtilis, a secretory expression system was designed that enhances translocation, folding and stability of heterologous proteins, and simplifies purification. Based on the theta-replication plasmid pHT01, a B...
February 7, 2019: Microbial Cell Factories
Anthony Siau, Ximei Huang, Han Ping Loh, Neng Zhang, Wei Meng, Siu Kwan Sze, Laurent Renia, Peter R Preiser
Efforts to develop vaccines against malaria represent a major research target. The observations that 1) sterile protection can be obtained when the host is exposed to live parasites and 2) the immunity against blood stage parasite is principally mediated by protective antibodies suggest that a protective vaccine is feasible. However, only a small number of proteins have been investigated so far and most of the Plasmodium proteome has yet to be explored. To date, only few immunodominant antigens have emerged for testing in clinical trials but no formulation has led to substantial protection in humans...
February 4, 2019: Molecular & Cellular Proteomics: MCP
Francis Jingxin Hu, Magnus Lundqvist, Mathias Uhlén, Johan Rockberg
Mutagenesis libraries are essential for combinatorial protein engineering. Despite improvements in gene synthesis and directed mutagenesis, current methodologies still have limitations regarding the synthesis of complete antibody single-chain variable fragment (scFv) genes and simultaneous diversification of all six CDRs. Here, we describe the generation of mutagenesis libraries for antibody affinity maturation using a cell-free solid-phase technique for annealing of single-strand mutagenic oligonucleotides...
January 31, 2019: Nucleic Acids Research
Daniel Newsted, Sunandan Banerjee, Kathleen Watt, Sarah Nersesian, Peter Truesdell, Levi L Blazer, Lia Cardarelli, Jarrett J Adams, Sachdev S Sidhu, Andrew W Craig
Epithelial ovarian cancer (EOC) is a leading cause of cancer-related death in women. EOC is often diagnosed at late stages, with peritoneal metastases and ascites production. Current surgery and platinum-based chemotherapy regimes fail to prevent recurrence in most patients. High levels of Transforming growth factor-β (TGF-β) within ascites has been linked to poor prognosis. TGF-β signaling promotes epithelial-mesenchymal transition (EMT) in EOC tumor cells, and immune suppression within the tumor microenvironment, with both contributing to chemotherapy resistance and metastasis...
2019: Oncoimmunology
Anthony Tanoto Tan, Ninghan Yang, Thinesh Lee Krishnamoorthy, Vincent Oei, Alicia Chua, Zhao Xinyuan, Tan Hui Si, Adeline Chia, Nina Le Bert, Diana Low, Hiang Keat Tan, Rajneesh Kumar, Farah Gillan Irani, Ho Zi Zong, Qi Zhang, Ernesto Guccione, Wai Lu-En, Sarene Koh, William Hwang, Wan Cheng Chow, Antonio Bertoletti
BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is often associated with hepatitis B virus (HBV) infection. Cells of most HBV-related HCCs contain HBV DNA fragments that do not encode entire HBV antigens. We investigated whether these integrated HBV DNA fragments encode epitopes that are recognized by T cells and whether their presence in HCCs can be used to select HBV-specific T-cell receptors (TCRs) for immunotherapy. METHODS: HCC cells negative for HBV antigens, based on immunohistochemistry, were analyzed for the presence of HBV mRNAs by real-time PCR, sequencing, and Nanostring approaches...
January 31, 2019: Gastroenterology
Ki Hyun Kim, Jinhee Kim, Meehyun Ko, June Young Chun, Hyori Kim, Seungtaek Kim, Ji-Young Min, Wan Beom Park, Myoung-Don Oh, Junho Chung
Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease localized to China, Japan, and Korea that is characterized by severe hemorrhage and a high fatality rate. Currently, no specific vaccine or treatment has been approved for this disease. To develop a therapeutic agent for SFTS, we isolated antibodies from a phage-displayed antibody library that was constructed from a patient who recovered from SFTS virus (SFTSV) infection. One antibody, designated as Ab10, was reactive to the Gn envelope glycoprotein of SFTSV and protected host cells and A129 mice from infection in both in vitro and in vivo experiments...
February 1, 2019: PLoS Pathogens
Kai Wang, Zhiping Liu, Guochun Ding, Ji Li, Natalia Vasylieva, Qing X Li, Dongyang Li, Shirley J Gee, Bruce D Hammock, Ting Xu
Triazophos is mainly used in Asian and African countries for the control of insects in agricultural production. Camelid variable domains of heavy-chain antibodies (VHHs) show great promise in monitoring environmental chemicals such as pesticides. To improve the rate of success in the generation of VHHs against triazophos, genes specifically encoding VHH fragments from the unique allotype IgG3a of an immunized Camelus bactrianus were amplified by using a pair of novel primers and introduced to construct a diverse VHH library...
February 1, 2019: Analytical and Bioanalytical Chemistry
Kang-Chih Fan, Ting-Hua Yang, Yu-Chen Huang
Vitiligo is associated with (autoimmune) thyroid disease. However, confounding factors, including type and onset of vitiligo, require elucidation. We conducted a meta-analysis to identify vitiligo patients with increased risk of (autoimmune) thyroid disease. Studies were identified based on searches in PubMed, MEDLINE, Embase, and the Cochrane Library from inception of these databases to August 31st, 2017. Odds ratios (ORs) for the prevalence of (autoimmune) thyroid disease and thyroid antibodies in vitiligo patients were pooled, and subgroup analysis was performed...
December 1, 2018: European Journal of Dermatology: EJD
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