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scfv phage display

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
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
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
Anna Säll, Maria Walle, Christer Wingren, Susanne Müller, Tomas Nyman, Andrea Vala, Mats Ohlin, Carl A K Borrebaeck, Helena Persson
Antibody-based proteomics offers distinct advantages in the analysis of complex samples for discovery and validation of biomarkers associated with disease. However, its large-scale implementation requires tools and technologies that allow development of suitable antibody or antibody fragments in a high-throughput manner. To address this we designed and constructed two human synthetic antibody fragment (scFv) libraries denoted HelL-11 and HelL-13. By the use of phage display technology, in total 466 unique scFv antibodies specific for 114 different antigens were generated...
October 2016: Protein Engineering, Design & Selection: PEDS
Shin-Ichi Tsunoda
No abstract text is available yet for this article.
September 2016: Nihon Yakurigaku Zasshi. Folia Pharmacologica Japonica
Yi-Hsing Hsiao, Chao-Yang Huang, Chih-Yung Hu, Yen-Yu Wu, Chung-Hsiun Wu, Chia-Hsien Hsu, Chihchen Chen
Finding an interactive ligand-receptor pair is crucial to many applications, including the development of monoclonal antibodies. Biopanning, a commonly used technique for affinity screening, involves a series of washing steps and is lengthy and tedious. Here we present an approach termed continuous microfluidic assortment of interactive ligands, or CMAIL, for the screening and sorting of antigen-binding single-chain variable antibody fragments (scFv) displayed on bacteriophages (phages). Phages carrying native negative charges on their coat proteins were electrophoresed through a hydrogel matrix functionalized with target antigens under two alternating orthogonal electric fields...
2016: Scientific Reports
Shuai Dong, Hongxi Shi, Donghui Cao, Yicun Wang, Xintong Zhang, Yan Li, Xiang Gao, Li Wang
Candida albicans (C. albicans) is an important human commensal and opportunistic fungal pathogen. Secreted aspartyl proteinases (Saps) are a major virulence trait of C. albicans, and among these proteases Sap2 has the highest expression levels. It is possible that antibodies against Sap2 could provide an antifungal effect. In this study, two phages displaying anti-rSap2 single chain variable fragments (scFvs) were screened from human single fold scFv libraries, and their potential therapeutic roles were evaluated using a murine model infected by C...
2016: Scientific Reports
Shin-Chen Hou, Hong-Sen Chen, Hung-Wei Lin, Wei-Ting Chao, Yao-Sheng Chen, Chi-Yu Fu, Chung-Ming Yu, Kai-Fa Huang, Andrew H-J Wang, An-Suei Yang
Immunotoxins are an important class of antibody-based therapeutics. The potency of the immunotoxins depends on the antibody fragments as the guiding modules targeting designated molecules on cell surfaces. Phage-displayed synthetic antibody scFv libraries provide abundant antibody fragment candidates as targeting modules for the immunoconjugates, but the discovery of optimally functional immunoconjugates is limited by the scFv-payload conjugation procedure. In this work, cytotoxicity screening of non-covalently assembled immunotoxins was developed in high throughput format to discover highly functional synthetic antibody fragments for delivering toxin payloads...
2016: Scientific Reports
Leila Rahbarnia, Safar Farajnia, Hossein Babaei, Jafar Majidi, Kamal Veisi, Asghar Tanomand, Bahman Akbari
Phage display is a prominent screening technique for development of novel high affinity antibodies against almost any antigen. However, removing false positive clones in screening process remains a challenge. The aim of this study was to develop an efficient and rapid method for isolation of high affinity scFvs by removing NSBs without losing rare specific clones. Therefore, a novel two rounds strategy called invert biopanning was developed for isolating high affinity scFvs against EGFRvIII antigen from human scFv library...
August 9, 2016: Biologicals: Journal of the International Association of Biological Standardization
Sanam Sadreddini, Mehrnosh Seifi-Najmi, Babollah Ghasemi, Hossein Samadi Kafil, Vahideh Alinejad, Sevil Sadreddini, Vahid Younesi, Farhad Jadidi-Niaragh, Mehdi Yousefi
BACKGROUND: Tetanus neurotoxin (TeNT) is composed of a light (LC) and heavy chain (HC) polypeptides, released by anaerobic bacterium Clostridium tetani and can cause fatal life-threatening infectious disease. Toxin HC and LC modules represents receptor binding and zinc metalloprotease activity, respectively. The passive administration of animal-derived antibodies against tetanus toxin has been considered as the mainstay therapy for years. However, this treatment is associated with several adverse effects due to the presence of anti-isotype antibodies...
December 23, 2015: Human Antibodies
Janita Lövgren, Juha-Pekka Pursiheimo, Mikko Pyykkö, Jussi Salmi, Urpo Lamminmäki
Next generation sequencing (NGS) can be applied to monitoring antibody phage display library selection processes to follow the enrichment of each individual antibody clone. Utilising the recent development of the Illumina sequencing platform enabling sequencing up to 2×300bp, we have developed a method to deep sequence all complementarity determining regions (CDRs) in the clones obtained from a synthetic single framework antibody library. This was complemented by an in-house bioinformatics pipeline for efficient analysis of the sequencing results...
July 19, 2016: New Biotechnology
Fengchun Zhao, Yuan Tian, Huimin Wang, Jiye Liu, Xiao Han, Zhengyou Yang
Organophosphorus pesticides (OPs) are the most widely used pesticides in agriculture, and OP residues have been broadly reported in food and environmental samples. The aim of this study is to develop a recombinant antibody-based broad-specificity immunoassay for OPs. A phage display library was prepared from a mouse pre-immunized with a generic immunogen of OPs, and a single-chain variable fragment (scFv) antibody was selected. The selected scFv antibody was fused with biotin acceptor domain (BAD) and overexpressed as an inclusion body in Escherichia coli BL21 (DE3)...
September 2016: Analytical and Bioanalytical Chemistry
Guangyong Yang, Haitao Guo, Ximing Liu, Guangzhi He, Weiyi Tian, Kun Cai, Ping Wang, Wenjia Wang
Objective To construct the recombinant human interleukin 4 receptor (rhIL-4R) single-chain Fv (scFv) antibody library by phage display technique to obtain the anti-IL-4R scFv clones selected from the library. Methods Total RNA was extracted from splenocytes of the BALB/c mice immunized with rhIL-4R. Complementary DNA fragments of variable heavy (VH) and variable light (VL) chains of the antibodies were prepared by reverse transcription PCR and assembled into scFv by splice overlap extension PCR (SOE-PCR). Both scFv and the pCANTAB5E vector were respectively double-digested with restriction endonuclease Sfi I and Not I, connected with T4 ligase, and then transformed into the competent cells E...
June 2016: Xi Bao Yu Fen Zi Mian Yi Xue za Zhi, Chinese Journal of Cellular and Molecular Immunology
Wen-Si Zhao, Yi Luo, Bo-Yi Li, Han-Jing Zhou, Tao Zhang
ABCG2 is a multidrug resistance efflux pump expressed in many diverse tumors. The overexpression of ABCG2 is associated with resistance to a wide variety of anticancer agents, providing a noticeable setback to successful cancer therapy. Therapies targeting ABCG2 may therefore be a promising candidate for reversal of chemoresistance. The anti-ABCG2 single-chain variable fragment (scFv) antibody was constructed by phage display peptide library technology. Immunoblotting, ELISA and immunocytochemistry were used to evaluate the soluble expression and immunoreactivity of the scFv...
2016: American Journal of Cancer Research
Qian Wu, Xiaodong Wang, Yong Gu, Xiao Zhang, Yao Qin, Heng Chen, Xinyu Xu, Tao Yang, Mei Zhang
Zinc transporter 8 (ZnT8) is a major autoantigen and a predictive marker in type 1 diabetes (T1D). To investigate ZnT8-specific antibodies, a phage display library from T1D was constructed and single-chain antibodies against ZnT8 were screened and identified. Human T1D single-chain variable fragment (scFv) phage display library consists of approximately 1×10(8) clones. After four rounds of bio-panning, seven unique clones were positive by phage ELISA. Among them, C27 and C22, which demonstrated the highest affinity to ZnT8, were expressed in Escherichia coli Top10F' and then purified by affinity chromatography...
July 2016: Science China. Life Sciences
Frank Camacho, María E Sarmiento, Fatima Reyes, Louise Kim, Jim Huggett, Marco Lepore, Oscar Otero, Martine Gilleron, Germain Puzo, Mohd Nor Norazmi, Graham Rook, Lucia Mori, Gennaro De Libero, Armando Acosta
OBJECTIVE/BACKGROUND: The development of new tools capable of targeting Mycobacterium tuberculosis (Mtb)-infected cells have potential applications in diagnosis, treatment, and prevention of tuberculosis. In Mtb-infected cells, CD1b molecules present Mtb lipids to the immune system (Mtb lipid-CD1b complexes). Because of the lack of CD1b polymorphism, specific Mtb lipid-CD1b complexes could be considered as universal Mtb infection markers. 2-Stearoyl-3-hydroxyphthioceranoyl-2'-sulfate-α-α'-d-trehalose (Ac2SGL) is specific for Mtb, and is not present in other mycobacterial species...
June 2016: International Journal of Mycobacteriology
Bas van der Woning, Gitte De Boeck, Christophe Blanchetot, Vladimir Bobkov, Alex Klarenbeek, Michael Saunders, Magali Waelbroeck, Toon Laeremans, Jan Steyaert, Anna Hultberg, Hans De Haard
The identification of functional monoclonal antibodies directed against G-protein coupled receptors (GPCRs) is challenging because of the membrane-embedded topology of these molecules. Here, we report the successful combination of llama DNA immunization with scFv-phage display and selections using virus-like particles (VLP) and the recombinant extracellular domain of the GPCR glucagon receptor (GCGR), resulting in glucagon receptor-specific antagonistic antibodies. By immunizing outbred llamas with plasmid DNA containing the human GCGR gene, we sought to provoke their immune system, which generated a high IgG1 response...
August 2016: MAbs
Jing Liu, Qing Zhang, Hang Chen, Zhihui Gao, Yao Li, Zhongyuan Sun, Rong Xiang, Sihe Zhang
: Hypoxia, which is frequently observed in liver cancer and metastasis, influences tumor progression and resistance to therapy. Although hypoxia-associated biomarkers are of use in other cancers, none is recognized as a surrogate for hypoxia in liver cancer. In this study, we generated seven unique human single-chain Fv (scFv) antibodies (Abs) specific to hypoxic liver cancer cells, using normoxia-depleted vs hypoxia-selected phage library panning technology. By developing the scFv immunoprecipitation-based mass spectrometry method, the antigen that bound with one of the Abs (H103) was identified as the M2 splice isoform of pyruvate kinase (PKM2), an enzyme that is a key regulator of aerobic glycolysis in cancer cells...
May 18, 2016: Oncotarget
Martina L Jones, Mohamed A Alfaleh, Sumukh Kumble, Shuo Zhang, Geoffrey W Osborne, Michael Yeh, Neetika Arora, Jeff Jia Cheng Hou, Christopher B Howard, David Y Chin, Stephen M Mahler
A critical factor in the successful isolation of new antibodies by phage display is the presentation of a correctly folded antigen. While this is relatively simple for soluble proteins which can be purified and immobilized onto a plastic surface, membrane proteins offer significant challenges for antibody discovery. Whole cell panning allows presentation of the membrane protein in its native conformation, but is complicated by a low target antigen density, high background of irrelevant antigens and non-specific binding of phage particles to cell surfaces...
2016: Scientific Reports
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