scholarly journals Fusion Proteins of Single-Chain Variable Fragments Derived from Phage Display Libraries Are Effective Reagents for Routine Diagnosis of Potato Leafroll Virus Infection in Potato

1999 ◽  
Vol 89 (11) ◽  
pp. 1015-1021 ◽  
Author(s):  
R. L. Toth ◽  
K. Harper ◽  
M. A. Mayo ◽  
L. Torrance
Keyword(s):  
Phage Display ◽  
Polyclonal Antibody ◽  
Fusion Proteins ◽  
Plant Viruses ◽  
Potato Leafroll Virus ◽  
Diagnostic Tools ◽  
Single Chain ◽  
Antibody Preparation ◽  
Phage Display Libraries ◽  
Leafroll Virus

A panel of 11 different single-chain variable fragment antibodies (scFv) that bind to potato leafroll virus (PLRV) has been studied to assess each one's suitability as practical diagnostic tools. The scFv, previously obtained from naive phage display libraries, were expressed in Escherichia coli as fusion proteins. The fusion proteins comprised scFv joined to either the human light chain kappa constant domain (CL), an amphipathic helix (Zip), a combination of CL and Zip, or alkaline phosphatase (AP/S). The fusion proteins were tested for their ability to detect, or trap on enzymelinked immunosorbent assay (ELISA) plates, PLRV in extracts of infected potato leaves. The tests done with the different scFv fusion proteins were compared with a standard triple-antibody sandwich (TAS)-ELISA that employs a rabbit polyclonal antibody preparation to coat microtiter plates and a monoclonal antibody, SCR3, to detect PLRV. Of 11 scFvCL fusion proteins, 7 detected PLRV as readily as SCR3 when used as detecting antibodies in TAS-ELISA. The limit of detection of purified PLRV for the different scFvCL fusion proteins ranged from 250 to 5 ng/ml; that for SCR3 is 5 ng/ml. Of the 11 scFv, 4 cross-reacted with some other luteoviruses. Several scFvCL and scFvCLZip fusion proteins trapped PLRV from extracts of infected potato leaves as effectively as the polyclonal antibody preparation. Four scFv fusion proteins were used in a stem print assay to detect PLRV, and the results were similar to those obtained in tests using SCR3. The scFvCL fusion proteins retained activity for at least 6 months at 4°C, and all scFv fusion proteins were fully active on reconstitution after lyophilization. A fully recombinant ELISA was devised that detected PLRV in extracts of infected potato, with results comparable to those obtained using the standard TAS-ELISA. The advantages of using scFv fusion proteins for the routine detection of plant viruses include the ability to produce large quantities of reagents cheaply in bacterial fermenters and to incorporate them into standardized tests.

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

2005 ◽  
Vol 69 (3) ◽  
pp. 610-619 ◽  
Author(s):  
Yoshihito SUZUKI ◽  
Shinsaku ITO ◽  
Kaori OTSUKA ◽  
Eriko IWASAWA ◽  
Masatoshi NAKAJIMA ◽  
...  
Keyword(s):  
Phage Display ◽  
Single Chain ◽  
Single Chain Antibodies ◽  
Phage Display Libraries

Hevein-specific recombinant IgE antibodies from human single-chain antibody phage display libraries

2003 ◽  
Vol 278 (1-2) ◽  
pp. 271-281 ◽  
Author(s):  
Marja-Leena Laukkanen ◽  
Soili Mäkinen-Kiljunen ◽  
Kirsi Isoherranen ◽  
Tari Haahtela ◽  
Hans Söderlund ◽  
...  
Keyword(s):  
Phage Display ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Ige Antibodies ◽  
Phage Display Libraries ◽  
Antibody Phage ◽  
Antibody Phage Display

scholarly journals Selection of Single-Chain Variable Fragment Antibodies to Black Currant Reversion Associated Virus from a Synthetic Phage Display Library

1998 ◽  
Vol 88 (3) ◽  
pp. 230-233 ◽  
Author(s):  
Petri Susi ◽  
Angelika Ziegler ◽  
Lesley Torrance
Keyword(s):  
Alkaline Phosphatase ◽  
Phage Display ◽  
Plant Extracts ◽  
Fusion Proteins ◽  
Phage Display Library ◽  
Antibody Fragments ◽  
Healthy Plant ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Black Currant

Single-chain variable fragment (scFv) antibodies that bind to black currant reversion associated virus (BRAV) were obtained from a synthetic phage display antibody gene library without recourse to animal immunizations. Several different BRAV-specific phage scFv were obtained quickly, after only three rounds of selection against immobilized virus antigen. The phage scFv gave enzyme-linked immunosorbent assay (ELISA) absorbance values that were greater than seven times the control healthy plant extracts. In contrast, comparative tests using a rabbit antiserum failed, because unacceptably high background values were obtained with healthy plant extracts. Two of the scFv were subcloned into the pDAP2 vector for the rapid and efficient production of scFv-alkaline phosphatase fusion proteins. Functional fusion proteins were obtained after expression in Escherichia coli, and preparations from periplasmic extracts detected BRAV in ELISA. The results demonstrate that antibody fragments obtained from a synthetic phage display library are useful research tools, and they proved to be a viable practical alternative when traditional antisera failed to detect BRAV, a weak immunogen. Furthermore, the genetic fusion of antibody fragments to alkaline phosphatase obviates the need for further chemical coupling procedures, and the fusion proteins can be obtained cheaply.

Characterization of anti-mouse FcγRII single-chain Fv fragments derived from human phage display libraries

1998 ◽  
Vol 4 (1) ◽  
pp. 71-87 ◽  
Author(s):  
A.M McCall ◽  
A.R Amoroso ◽  
C Sautès ◽  
J.D Marks ◽  
L.M Weiner
Keyword(s):  
Phage Display ◽  
Single Chain ◽  
Single Chain Fv ◽  
Phage Display Libraries

scholarly journals Phage display and kinetic selection of antibodies that specifically inhibit amyloid self-replication

2017 ◽  
Vol 114 (25) ◽  
pp. 6444-6449 ◽  
Author(s):  
Anna Munke ◽  
Jonas Persson ◽  
Tanja Weiffert ◽  
Erwin De Genst ◽  
Georg Meisl ◽  
...  
Keyword(s):  
Phage Display ◽  
Amyloid Fibrils ◽  
Amyloid Β ◽  
Amyloid Β Peptide ◽  
Nucleation Process ◽  
Single Chain ◽  
Secondary Nucleation ◽  
Single Chain Antibody ◽  
Phage Display Libraries ◽  
Selection Of

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.

Isolation and Characterization of Neutralizing Single-Chain Antibodies againstXenopusMitogen-Activated Protein Kinase Kinase from Phage Display Libraries†

Biochemistry ◽  
1996 ◽  
Vol 35 (40) ◽  
pp. 13212-13221 ◽  
Author(s):  
Hidetaka Kosako ◽  
Yoshiko Akamatsu ◽  
Naoya Tsurushita ◽  
Kyung-Kwon Lee ◽  
Yukiko Gotoh ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Phage Display ◽  
Single Chain ◽  
Single Chain Antibodies ◽  
Isolation And Characterization ◽  
Phage Display Libraries ◽  
Protein Kinase Kinase

scholarly journals Inhibition of potato leafroll virus multiplication and systemic translocation by siRNA constructs against putative ATPase fold of movement protein

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Priyanka Kumari ◽  
Jitesh Kumar ◽  
Ravi Ranjan Kumar ◽  
Mohammad Ansar ◽  
Kumari Rajani ◽  
...  
Keyword(s):  
Gene Expression ◽  
Movement Protein ◽  
Control Plant ◽  
Plant Viruses ◽  
Potato Leafroll Virus ◽  
Plant Diseases ◽  
World Population ◽  
Genome Replication ◽  
Potato Plants ◽  
Leafroll Virus

AbstractViruses cause many severe plant diseases, resulting in immense losses of crop yield worldwide. Therefore, developing novel approaches to control plant viruses is crucial to meet the demands of a growing world population. Recently, RNA interference (RNAi) has been widely used to develop virus-resistant plants. Once genome replication and assembly of virion particles is completed inside the host plant, mature virions or sometimes naked viral genomes spread cell-to-cell through plasmodesmata by interacting with the virus-encoded movement protein (MP). We used the RNAi approach to suppress MP gene expression, which in turn prevented potato leafroll virus (PLRV) systemic infection in Solanum tuberosum cv. Khufri Ashoka. Potato plants agroinfiltrated with MP siRNA constructs exhibited no rolling symptoms upon PLRV infection, indicating that the silencing of MP gene expression is an efficient method for generating PLRV-resistant potato plants. Further, we identified novel ATPase motifs in MP that may be involved in DNA binding and translocation through plasmodesmata. We also showed that the ATPase activity of MP was stimulated in the presence of DNA/RNA. Overall, our findings provide a robust technology to generate PLRV-resistant potato plants, which can be extended to other species. Moreover, this approach also contributes to the study of genome translocation mechanisms of plant viruses.

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