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Paul Leonard, Stephen Hearty, Hui Ma, Richard O'Kennedy
The use of optical biosensors for studying macromolecular interactions is gaining increasing popularity. In one study, 1514 papers that involved the application of biosensor data were identified for the year 2009 alone (Rich and Myszka, J Mol Recognit 24:892-914, 2011), the sheer volume and variety of which present a daunting task for the burgeoning biosensor user to accumulate and decipher. This chapter is designed to provide the reader with the tools necessary to prepare, design, and efficiently execute a kinetic experiment on Biacore...
2017: Methods in Molecular Biology
William J J Finlay, Laird Bloom, Sreeja Varghese, Bénédicte Autin, Orla Cunningham
High-affinity, highly specific binding proteins are a key class of molecules used in the development of new affinity chromatography methods. Traditionally, antibody-based methods have relied on the use of immunoglobulins purified from immune animal sera, from egg yolks, or from murine monoclonal hybridoma supernatants. To accelerate and refine the reagent antibody generation process, we have developed optimized methods that allow the rapid assembly of scFv libraries from chickens immunized with pools of immunogens...
2017: Methods in Molecular Biology
Suguru Shigemori, Masaki Ihara, Takashi Sato, Yoshinari Yamamoto, Shireen Nigar, Tasuku Ogita, Takeshi Shimosato
Interleukin 6 (IL-6) is an important pathogenic factor in development of various inflammatory and autoimmune diseases and cancer. Blocking antibodies against molecules associated with IL-6/IL-6 receptor signaling are an attractive candidate for the prevention or therapy of these diseases. In this study, we developed a genetically modified strain of Lactococcus lactis secreting a single-chain variable fragment antibody against mouse IL-6 (IL6scFv). An IL6scFv-secretion vector was constructed by cloning an IL6scFv gene fragment into a lactococcal secretion plasmid and was electroporated into L...
October 8, 2016: Applied Microbiology and Biotechnology
Jian Ping Wang, Jun Dong, Chang Fei Duan, Hui Cai Zhang, Xin He, Geng Nan Wang, Guo Xian Zhao, Jing Liu
A recombinant anti-sarafloxacin ScFv antibody was produced by direct transformation of its gene into Rosetta-gami(DE3) for expression, and then its recognition mechanisms for 12 fluoroquinolones were studied by using molecular docking method. Based on the results of virtual mutation, the ScFv antibody was evolved by directional mutagenesis of contact amino acid residue Tyr99 to His. The ScFv mutant showed highly increased affinity for the 12 drugs with up to 7-folds improved sensitivity. Finally, the mutant was used to develop an indirect competitive enzyme linked immunosorbent assay for determination of the 12 drugs in milk...
October 10, 2016: Journal of Agricultural and Food Chemistry
Karen Manoutcharian, Roxanna Perez-Garmendia, Goar Gevorkian
Recombinant antibody fragments are promising alternatives to full-length immunoglobulins and offer important advantages compared with conventional monoclonal antibodies: extreme specificity, higher affinity, superior stability and solubility, reduced immunogenicity as well as easy and inexpensive large-scale production. Different antibody formats such as single-chain fragment variable (scFv), single-domain antibody fragments (VHHs or sdAbs), bispecific antibodies (bsAbs), intrabodies and nanobodies, are currently being studied in pre-clinical models of cancer as well as infectious and autoimmune diseases and many of them are being tested as therapeutics in clinical trials...
September 30, 2016: Current Neuropharmacology
Hui-Yun Liu, Qi Zhao, Tian-Peng Zhang, Yue Wu, Yun-Xia Xiong, Shi-Ke Wang, Yuan-Long Ge, Jin-Hui He, Peng Lv, Tian-Miao Ou, Jia-Heng Tan, Ding Li, Lian-Quan Gu, Jian Ren, Yong Zhao, Zhi-Shu Huang
G-quadruplexes are specialized secondary structures in nucleic acids that possess significant conformational polymorphisms. The precise G-quadruplex conformations in vivo and their relevance to biological functions remain controversial and unclear, especially for telomeric G-quadruplexes. Here, we report a novel single-chain variable fragment (scFv) antibody, D1, with high binding selectivity for parallel G-quadruplexes in vitro and in vivo. Genome-wide chromatin immunoprecipitation using D1 and deep-sequencing revealed the consensus sequence for parallel G-quadruplex formation, which is characterized by G-rich sequence with a short loop size (<3 nt)...
October 20, 2016: Cell Chemical Biology
Xiaochen Guo, Hongxue Cao, Yunxin Wang, Yang Liu, Yanmin Chen, Nan Wang, Shan Jiang, Shengqi Zhang, Qiang Wu, Tianhe Li, Yingjie Zhang, Bing Zhou, Jiechao Yin, Deshan Li, Guiping Ren
We have previously reported an antigen and antibody co-expression (AAC) technology to demonstrate the interaction between a known antigen and antibody. To validate the co-expression system for screening antibody libraries, a single chain fragment variable(scFv)antibody library was constructed from chickens immunized with the VP2 protein of infectious bursal disease virus (IBDV). The genes of both VP2 and scFv antibodies were inserted into the pBFD-Ab-Ag vector. The co-expression library was subjected to three rounds of screening by flow cytometry (FCM) using a polyclonal antibody against VP2 through a bacteria display system...
November 1, 2016: Veterinary Immunology and Immunopathology
Chunbao Liu, Xiao Zhang, Yiling Song, Yichun Wang, Fengzhen Zhang, Yingying Zhang, Yongxue Zhang, Xiaoli Lan
BACKGROUND AND AIMS: Early detection and evaluation of vulnerable atherosclerotic plaque are important for risk stratification and timely intervention, and vascular cell adhesion molecule 1 (VCAM1) assists in adhesion and recruitment of inflammatory cells to vulnerable lesions. We labeled a single-chain variable fragment (scFv) of VCAM1 with (99m)technetium ((99m)Tc) and fluorescent markers to investigate its potential utility in detecting vulnerable plaques in animal models of atherosclerosis...
September 13, 2016: Atherosclerosis
Li-Ming Xu, Jing-Zhuang Zhao, Miao Liu, Yong-Sheng Cao, Jia-Sheng Yin, Hong-Bai Liu, Tongyan Lu
Infectious pancreatic necrosis is a significant disease of farmed salmonids in China. In this study, a single chain variable fragment (scFv) antibody library derived from rainbow trout (Oncorhynchus mykiss) and viral protein VP2 of a Chinese infectious pancreatic necrosis virus (IPNV) isolate ChRtm213 were co-expressed by a bacterial display technology. The library was subjected to three rounds of screening by flow cytometry (FCM) to select IPNV specific antibodies. Six antibody clones with different mean fluorescence intensities (MFI) were obtained by picking colonies at random...
November 2016: Journal of Virological Methods
Laila Ismail Sakhnini, Anja Kallesøe Pedersen, Haleh Ahmadian, Jens-Jacob Hansen, Leif Bülow, Maria Dainiak
Monoclonal antibody (mAb) based affinity resins usually suffer from low binding capacity, most probably as a result of steric hindrance by the large 150kDa size of the mAb and a random immobilisation approach. The present work investigates the influence of a variety of factors on dynamic binding capacity (DBC) such as pore/ligand size ratio, accessibility of ligand and ligand density. The effect of pore/ligand size ratio was investigated using Fab and scFv fragments on various resins with different pore sizes...
October 14, 2016: Journal of Chromatography. A
Chi-Hsin Lee, Yu-Ching Lee, Sy-Jye Leu, Liang-Tzung Lin, Jen-Ron Chiang, Wei-Jane Hsu, Yi-Yuan Yang
: Banded krait (Bungarus multicinctus; BM), one of the major envenomation species in Taiwan, contains neurotoxic venom proteins (BM proteins), that poses a serious medical problem in tropical and sub-tropical countries. Even though horse-derived serum is an efficient therapy against snake venom, it is associated with high cost and side effects. Therefore, developing a more cost-effective alternative treatment option is highly envisaged. In this study, chickens were immunized with BM proteins, and polyclonal immunoglobulin Y (IgY) antibodies were purified from eggs...
September 23, 2016: Applied and Environmental Microbiology
Joerg U Schmohl, Martin Felices, Deborah Todhunter, Elizabeth Taras, Jeffrey S Miller, Daniel A Vallera
BACKGROUND: The design of a highly effective anti-cancer immune-engager would include targeting of highly drug refractory cancer stem cells (CSC). The design would promote effective antibody-dependent cell-mediated cytotoxicity (ADCC) and simultaneously promote costimulation to expand and self-sustain the effector NK cell population. Based on our bispecific NK cell engager platform we constructed a tetraspecific killer engager (TetraKE) comprising single-chain variable fragments (scFvs) binding FcγRIII (CD16) on NK cells, EpCAM on carcinoma cells and CD133 on cancer stem cells in order to promote ADCC...
September 16, 2016: Oncotarget
Hiroyuki Ohashi, Takashi Matsumoto, Hee-Jin Jeong, Jinhua Dong, Ryoji Abe, Hiroshi Ueda
Recently, we reported a novel immunoassay reagent Quenchbody (Q-body): a single chain antibody variable region (scFv) fragment labeled with fluorescent dye, whose fluorescence intensity increases when it binds to the antigen. Here we analyze its working mechanism by immuno- and fluorescence polarization (FP) assays. In an enzyme-linked immunosorbent assay, we found that in the presence of antigen osteocalcin peptide (BGP-C7), more TAMRA-labeled Q-bodies bound to anti-TAMRA antibody than in its absence. Moreover, we found that anti-BGP Q-body with the shortest linker that exhibits the largest antigen-dependency in fluorescence showed the highest binding signal...
September 20, 2016: Bioconjugate Chemistry
Morteza Motallebnezhad, Vahid Younesi, Leili Aghebati-Maleki, Hamid Nickho, Elham Safarzadeh, Majid Ahmadi, Ali Akbar Movassaghpour, Ahmad Hosseini, Mehdi Yousefi
Insulin-like growth factor I receptor (IGF-IR) is expressed on breast cancer cells and involves in metastasis, survival, and proliferation. Currently, application of IGF-IR-targeting monoclonal antibodies (mAbs), alone or in combination with other drugs, is a promising strategy for breast cancer therapy. Single-chain fragment variable (scFv) antibodies have been introduced as appropriate tools for tumor-targeting purposes because of their advantages over whole antibodies. In the present study, we employed a naïve phage library and isolated scFvs against a specific epitope from extracellular domain of IGF-IR by panning process...
September 17, 2016: Tumour Biology: the Journal of the International Society for Oncodevelopmental Biology and Medicine
Yağmur Derman, Katja Selby, Sebastian Miethe, André Frenzel, Yvonne Liu, Christine Rasetti-Escargueil, Arnaud Avril, Thibaut Pelat, Remi Urbain, Alexandre Fontayne, Philippe Thullier, Dorothea Sesardic, Miia Lindström, Michael Hust, Hannu Korkeala
Botulinum neurotoxins (BoNTs) cause botulism and are the deadliest naturally-occurring substances known to humans. BoNTs have been classified as one of the category A agents by the Centers for Disease Control and Prevention, indicating their potential use as bioweapons. To counter bio-threat and naturally-occurring botulism cases, well-tolerated antibodies by humans that neutralize BoNTs are relevant. In our previous work, we showed the neutralizing potential of macaque (Macaca fascicularis)-derived scFv-Fc (scFv-Fc ELC18) by in vitro endopeptidase immunoassay and ex vivo mouse phrenic nerve-hemidiaphragm assay by targeting the light chain of the botulinum neurotoxin type E (BoNT/E)...
2016: Toxins
Han Wang, Rui Yu, Ting Fang, Ting Yu, Xiangyang Chi, Xiaopeng Zhang, Shuling Liu, Ling Fu, Changming Yu, Wei Chen
Tetanus neurotoxin (TeNT) produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc) as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H) can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro...
2016: Toxins
Rongzhi Wang, Xiaosong Gu, Zhenghong Zhuang, Yanfang Zhong, Hang Yang, Shihua Wang
Citreoviridin (CIT), a small food-borne mycotoxin produced by Penicillium citreonigrum, is generally distributed in various cereal grains and farm crop products around the world and has caused cytotoxicity as an uncompetitive inhibitor of ATP hydrolysis. A high affinity single chain variable fragment (scFv) antibody that can detect the citreoviridin in samples is still not available; therefore, it is very urgent to prepare an antibody for CIT detection and therapy. In this study, an amplified and assembled scFv from hybridoma was used to construct the mutant phage library by error-prone PCR, generating a 2 × 10(8) capacity mutated phage display library...
October 12, 2016: Journal of Agricultural and Food Chemistry
Emily E Reichard, Nisha Nanaware-Kharade, Guillermo A Gonzalez, Shraddha Thakkar, S Michael Owens, Eric C Peterson
PURPOSE: Methamphetamine (METH) abuse is a worldwide drug problem, yet no FDA-approved pharmacological treatments are available for METH abuse. Therefore, we produced an anti-METH single chain antibody fragment (scFv7F9Cys) as a pharmacological treatment for METH abuse. ScFv's have a short half-life due to their small size, limiting their clinical use. Thus, we examined the pharmacokinetic effects of conjugating poly(ethylene) glycol (-PEG) to scFv7F9Cys to extend its functional half-life...
September 12, 2016: Pharmaceutical Research
Alexander S Jones, James I Austerberry, Rana Dajani, Jim Warwicker, Robin Curtis, Jeremy P Derrick, Colin Robinson
The Tat system transports folded proteins across the bacterial plasma membrane, and in Escherichia coli preferentially transports correctly-folded proteins. Little is known of the mechanism by which Tat proofreads a substrate's conformational state, and in this study we have addressed this question using a heterologous single-chain variable fragment (scFv) with a defined structure. We introduced mutations to surface residues while leaving the folded structure intact, and also tested the importance of conformational flexibility...
September 13, 2016: Biochimica et Biophysica Acta
Yanling Wu, Shibo Jiang, Tianlei Ying
INTRODUCTION: A variety of approaches are being pursued to improve the safety and antitumor potency of chimeric antigen receptor (CAR) T-cell therapy. However, most engineering efforts have thus far been focused on its intracellular signaling domain, while its extracellular antigen-binding domain has received less attention. AREAS COVERED: Herein, the authors summarize the current knowledge of CAR T-cell therapy. Accordingly, they focus on its antigen-binding domain, discuss key considerations for selecting an optimal single-chain variable fragment (scFv) when designing a CAR, and suggest potential directions aimed at developing the next-generation CARs...
September 19, 2016: Expert Opinion on Biological Therapy
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