The role of valency in the selection of anti-carbohydrate single-chain Fvs from phage display libraries

The aggregation of the amyloid β peptide (Aβ) into amyloid fibrils is a defining characteristic of Alzheimer’s disease. Because of the complexity of this aggregation process, effective therapeutic inhibitors will need to target the specific microscopic steps that lead to the production of neurotoxic species. We introduce a strategy for generating fibril-specific antibodies that selectively suppress fibril-dependent secondary nucleation of the 42-residue form of Aβ (Aβ42). We target this step because it has been shown to produce the majority of neurotoxic species during aggregation of Aβ42. Starting from large phage display libraries of single-chain antibody fragments (scFvs), the three-stage approach that we describe includes (i) selection of scFvs with high affinity for Aβ42 fibrils after removal of scFvs that bind Aβ42 in its monomeric form; (ii) ranking, by surface plasmon resonance affinity measurements, of the resulting candidate scFvs that bind to the Aβ42 fibrils; and (iii) kinetic screening and analysis to find the scFvs that inhibit selectively the fibril-catalyzed secondary nucleation process in Aβ42 aggregation. By applying this approach, we have identified four scFvs that inhibit specifically the fibril-dependent secondary nucleation process. Our method also makes it possible to discard antibodies that inhibit elongation, an important factor because the suppression of elongation does not target directly the production of toxic oligomers and may even lead to its increase. On the basis of our results, we suggest that the method described here could form the basis for rationally designed immunotherapy strategies to combat Alzheimer’s and related neurodegenerative diseases.

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SELECTION OF SPECIFIC SINGLE CHAIN VARIABLE FRAGMENTS (SCFV) AGAINST POLYMYXA BETAE FROM PHAGE DISPLAY LIBRARIES

Journal of Plant Protection Research ◽

10.2478/jppr-2013-0054 ◽

2013 ◽

Vol 53 (4) ◽

pp. 357-363 ◽

Cited By ~ 2

Author(s):

Mohammad Reza Safarnejad ◽

Hossein Safarpour ◽

Fatemeh Shahryari ◽

Marzieh Basirat ◽

Meisam Tabatabaei ◽

...

Keyword(s):

Sugar Beet ◽

Phage Display ◽

Recombinant Antibodies ◽

Diagnostic Methods ◽

Polymyxa Betae ◽

Single Chain ◽

Glutathione S Transferase ◽

Phage Display Libraries ◽

Single Chain Variable Fragments ◽

Selection Of

Abstract Sugar beet is one of the most important industrial crops in Iran. For the last two decades it has been mainly affected by a destructive virus, beet necrotic yellow vein virus (BNYVV). The Polymyxa betae is the only natural transmitting agent of the disease among the plants. Developing accurate diagnostic methods may have a major impact on the rising of resistant germplasms. In the present study, specific monoclonal recombinant antibodies in the form of single chain variable fragments (scFv) were obtained from naïve phage display libraries. The fungus specific glutathione-S-transferase (GST) protein was chosen as an antigen for developing antibodies and diagnostic purposes. To generate specific scFv, screening of Tomlinson phage display libraries was performed by applying both recombinant and native fungal GST. Using the recombinant GST in the panning process resulted in the isolation of an antibody only bound to recombinant GST but it failed to detect native GST in the infected plants. Alternatively, the process of panning was carried out by applying native fungal GST trapped to immunotubes through specific polyclonal antibody intermediate. The recent approach resulted in the selection of a specific scFv binding to native GST which was able to detect the presence of the fungi within infected plants. To the best of our knowledge, this is the first report on the generation of recombinant antibodies against Polymyxa betae, fungal vector of sugar beet rhizomania disease.

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Selection of antibody single-chain variable fragments with improved carbohydrate binding by phage display

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)36914-4 ◽

1994 ◽

Vol 269 (13) ◽

pp. 9533-9538

Author(s):

S.J. Deng ◽

C.R. MacKenzie ◽

J. Sadowska ◽

J. Michniewicz ◽

N.M. Young ◽

...

Keyword(s):

Phage Display ◽

Carbohydrate Binding ◽

Single Chain ◽

Single Chain Variable Fragments ◽

Selection Of

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Selection of Peptides Targeting Helix 31 of Bacterial 16S Ribosomal RNA by Screening M13 Phage-Display Libraries

Molecules ◽

10.3390/molecules16021211 ◽

2011 ◽

Vol 16 (2) ◽

pp. 1211-1239 ◽

Cited By ~ 18

Author(s):

Tek N. Lamichhane ◽

N. Dinuka Abeydeera ◽

Anne-Cécile E. Duc ◽

Philip R. Cunningham ◽

Christine S. Chow

Keyword(s):

Phage Display ◽

Ribosomal Rna ◽

16S Ribosomal Rna ◽

Phage Display Libraries ◽

M13 Phage ◽

Selection Of

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Human Single-Chain Fv Fragments Specific for Natural Killer Cell Receptors from Phage Display Libraries

Natural Killer Cell Protocols ◽

10.1385/1-59259-044-6:219 ◽

2003 ◽

pp. 219-237

Author(s):

Barbara Uchanska-Ziegler ◽

Qiao Xugang ◽

Armin Volz ◽

Ahuva Nissim ◽

Andreas Ziegler

Keyword(s):

Natural Killer Cell ◽

Phage Display ◽

Natural Killer ◽

Killer Cell ◽

Single Chain ◽

Cell Receptors ◽

Natural Killer Cell Receptors ◽

Single Chain Fv ◽

Phage Display Libraries

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[29] Design and use of phage display libraries for the selection of antibodies and enzymes

Methods in Enzymology - Applications of Chimeric Genes and Hybrid Proteins Part A: Gene Expression and Protein Purification ◽

10.1016/s0076-6879(00)26071-0 ◽

2000 ◽

pp. 480-505 ◽

Cited By ~ 58

Author(s):

Francesca Viti ◽

Fredrik Nilsson ◽

Salvatore Demartis ◽

Adrian Huber ◽

Dario Neri

Keyword(s):

Phage Display ◽

Phage Display Libraries ◽

Selection Of

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Preparation of Functional Single-Chain Antibodies against Bioactive Gibberellins by Utilizing Randomly Mutagenized Phage-Display Libraries

Bioscience Biotechnology and Biochemistry ◽

10.1271/bbb.69.610 ◽

2005 ◽

Vol 69 (3) ◽

pp. 610-619 ◽

Cited By ~ 12

Author(s):

Yoshihito SUZUKI ◽

Shinsaku ITO ◽

Kaori OTSUKA ◽

Eriko IWASAWA ◽

Masatoshi NAKAJIMA ◽

...

Keyword(s):

Phage Display ◽

Single Chain ◽

Single Chain Antibodies ◽

Phage Display Libraries

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87 Selection of pre-invasive lung cancer binding peptides using random phage display libraries

European Journal of Cancer Supplements ◽

10.1016/s1359-6349(04)80095-8 ◽

2004 ◽

Vol 2 (8) ◽

pp. 29

Author(s):

J.Y. Hung ◽

A.J. Rebello ◽

S. Lam ◽

J.C. Ieriche

Keyword(s):

Lung Cancer ◽

Phage Display ◽

Phage Display Libraries ◽

Binding Peptides ◽

Selection Of

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Selection of single chain antibody fragments binding to the extracellular domain of 4-1BB receptor by phage display technology

Tumor Biology ◽

10.1177/1010428317695924 ◽

2017 ◽

Vol 39 (3) ◽

pp. 101042831769592 ◽

Cited By ~ 5

Author(s):

Salman Bagheri ◽

Mehdi Yousefi ◽

Elmira Safaie Qamsari ◽

Farhad Riazi-Rad ◽

Mohsen Abolhassani ◽

...

Keyword(s):

Phage Display ◽

Immunosorbent Assay ◽

Specific Binding ◽

Interleukin 2 ◽

Extracellular Domain ◽

Surface Glycoprotein ◽

Enzyme Linked Immunosorbent Assay ◽

Single Chain ◽

Single Chain Variable Fragments ◽

Selection Of

The 4-1BB is a surface glycoprotein that pertains to the tumor necrosis factor–receptor family. There is compelling evidence suggesting important roles for 4-1BB in the immune response, including cell activation and proliferation and also cytokine induction. Because of encouraging results of different agonistic monoclonal antibodies against 4-1BB in the treatment of cancer, infectious, and autoimmune diseases, 4-1BB has been suggested as an attractive target for immunotherapy. In this study, single chain variable fragment phage display libraries, Tomlinson I+J, were screened against specific synthetic oligopeptides (peptides I and II) designed from 4-1BB extracellular domain. Five rounds of panning led to selection of four 4-1BB specific single chain variable fragments (PI.12, PI.42, PII.16, and PII.29) which showed specific reaction to relevant peptides in phage enzyme-linked immunosorbent assay. The selected clones were successfully expressed in Escherichia coli Rosetta-gami 2, and their expression was confirmed by western blot analysis. Enzyme-linked immunosorbent assay experiments indicated that these antibodies were able to specifically recognize 4-1BB without any cross-reactivity with other antigens. Flow cytometry analysis demonstrated an acceptable specific binding of the single chain variable fragments to 4-1BB expressed on CCRF-CEM cells, while no binding was observed with an irrelevant antibody. Anti-4-1BB single chain variable fragments enhanced surface CD69 expression and interleukin-2 production in stimulated CCRF-CEM cells which confirmed the agonistic effect of the selected single chain variable fragments. The data from this study have provided a rationale for further experiments involving the biological functions of anti-4-1BB single chain variable fragments in future studies.

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