Epitope identification and in silico prediction of the specificity of antibodies binding to the coat proteins of Potato Virus Y strains

Keller, H.J.H.G.; Pomp, Rikus; Bakker, J.; Schots, A.

doi:10.1007/s10658-004-6892-4

Cited by 11 publications

(17 citation statements)

References 18 publications

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“…Serological detection of PVY relies on detection of CP (virus particles) with polyclonal (PAb) or monoclonal antibodies (MAb) and is commonly carried out using the enzyme-linked immunosorbent assay (ELISA) [14] , [15] . Additionally, polymerase chain reaction (PCR)-based methods that detect viral nucleic acids are often used [16] , [17] , but they tend to be more costly, require more advanced laboratory facilities than ELISA, and may still require antibodies for immunocapture, i.e., trapping and concentrating virions from plant sap [18] – [20] .…”

Section: Introductionmentioning

confidence: 99%

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Analysis of Potato virus Y Coat Protein Epitopes Recognized by Three Commercial Monoclonal Antibodies

et al. 2014

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Background Potato virus Y (PVY, genus Potyvirus) causes substantial economic losses in solanaceous plants. Routine screening for PVY is an essential part of seed potato certification, and serological assays are often used. The commercial, commonly used monoclonal antibodies, MAb1128, MAb1129, and MAb1130, recognize the viral coat protein (CP) of PVY and distinguish PVYN strains from PVYO and PVYC strains, or detect all PVY strains, respectively. However, the minimal epitopes recognized by these antibodies have not been identified.Methodology/Principal FindingsSPOT peptide array was used to map the epitopes in CP recognized by MAb1128, MAb1129, and MAb1130. Then alanine replacement as well as N- and C-terminal deletion analysis of the identified peptide epitopes was done to determine critical amino acids for antibody recognition and the respective minimal epitopes. The epitopes of all antibodies were located within the 30 N-terminal-most residues. The minimal epitope of MAb1128 was 25NLNKEK30. Replacement of 25N or 27N with alanine weakened the recognition by MAb1128, and replacement of 26L, 29E, or 30K nearly precluded recognition. The minimal epitope for MAb1129 was 16RPEQGSIQSNP26 and the most critical residues for recognition were 22I and 23Q. The epitope of MAb1130 was defined by residues 5IDAGGS10. Mutation of residue 6D abrogated and mutation of 9G strongly reduced recognition of the peptide by MAb1130. Amino acid sequence alignment demonstrated that these epitopes are relatively conserved among PVY strains. Finally, recombinant CPs were produced to demonstrate that mutations in the variable positions of the epitope regions can affect detection with the MAbs.Conclusions/SignificanceThe epitope data acquired can be compared with data on PVY CP-encoding sequences produced by laboratories worldwide and utilized to monitor how widely the new variants of PVY can be detected with current seed potato certification schemes or during the inspection of imported seed potatoes as conducted with these MAbs.

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Section: Introductionmentioning

confidence: 99%

“…Studies on Potato virus A (PVA, genus Potyvirus ) indicate that, in potyvirus particles, CP folds such that the N-terminus is exposed at the surface of the particles [21] . Therefore, immunization of animals with potyvirus virions results in antibodies whose epitopes are mainly in the N-terminal region of CP [15] .…”

Section: Introductionmentioning

confidence: 99%

Analysis of Potato virus Y Coat Protein Epitopes Recognized by Three Commercial Monoclonal Antibodies

et al. 2014

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“…It has been reported that the antigenic epitope with the high hydrophilicity and surface probability was mainly distributed in the regions containing β-turn and coil structures. Prediction results of the computer program suggested that Nterminal amino acid; 36-40, 46-47, 67-74, 96-98, 130-133, 164-166 and 198-200, potentially participated in the formation of the epitope (Kyte and Doolittle, 1982;Keller et al, 2005).…”

Section: Discussionmentioning

confidence: 99%

Prediction of antigenic epitopes for coat protein of Potato virus Y, Egypt

Abdel-Shafi

Farouk

Taha

et al. 2019

Novel Research in Microbiology Journal

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Isolated Potato virus Y Egypt (PVY-EG) obtained from naturally infected potato plants in El-Sharkia, Egypt, was identified by biological, molecular and serological assays. The aims of this study were to predict the antigenic determinants (epitopes) of the isolated PVY using immunoinformatics; to prepare antibodies then apply them for PVY screening in potato plants in the field, as well as comparison with antibodies against coat protein. The amino acid sequence of PVY isolate was expected by DNAStar protean system; in which four parameters for epitope prediction including antigenicity, surface probability, hydrophobicity and secondary structure were used. These models were applied to PVY as a case study to predict immunogenic peptides for antibodies production. The most hydrophilic regions of PVY-coat protein (PVY-CP) were; 36-40, 46-47, 67-74, 96-98, 130-133, 164-166 and 198-200, which showed high hydrophobicity of this protein. This indicated that this protein was one of the best candidates to be immunogenic, and capable of producing antibodies that cross react with PVY. The peptide was chemically synthesized and injected into a rabbit. Obtained antibodies were evaluated using Double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), and Immune Dot-Blot assay. These antibodies positively reacted against PVY infected potato tissues.

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“…The variable termini, especially the large N terminus, establish the immunodominant regions, whereas the trypsin-resistant core region is highly conserved and contains potyvirus group-specific epitopes (49). Therefore, many antibodies prepared from intact potyvirus particles only specifically recognize the surface virusspecific epitopes at the N or C terminus of CPs (50,51). Nevertheless, certain MAbs that are also raised against native virions such as TEV (44) or JGMV (42) cross-react with other potyviruses.…”

Section: Discussionmentioning

confidence: 99%

A Strategy for Generating a Broad-Spectrum Monoclonal Antibody and Soluble Single-Chain Variable Fragments against Plant Potyviruses

Liu

Lin

et al. 2015

Appl Environ Microbiol

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Potyviruses are major pathogens that often cause mixed infection in calla lilies. To reduce the time and cost of virus indexing, a detection method for the simultaneous targeting of multiple potyviruses was developed by generating a broad-spectrum monoclonal antibody (MAb) for detecting the greatest possible number of potyviruses. The conserved 121-amino-acid core regions of the capsid proteins of Dasheen mosaic potyvirus (DsMV), Konjak mosaic potyvirus (KoMV), and Zantedeschia mild mosaic potyvirus (ZaMMV) were sequentially concatenated and expressed as a recombinant protein for immunization. After hybridoma cell fusion and selection, one stable cell line that secreted a group-specific antibody, named C4 MAb, was selected. In the reaction spectrum test, the C4 MAb detected at least 14 potyviruses by indirect enzyme-linked immunosorbent assay (I-ELISA) and Western blot analysis. Furthermore, the variable regions of the heavy (V H ) and light (V L ) chains of the C4 MAb were separately cloned and constructed as single-chain variable fragments (scFvs) for expression in Escherichia coli. Moreover, the pectate lyase E (PelE) signal peptide of Erwinia chrysanthemi S3-1 was added to promote the secretion of C4 scFvs into the medium. According to Western blot analysis and I-ELISA, the soluble C4 scFv (V L -V H ) fragment showed a binding specificity similar to that of the C4 MAb. Our results demonstrate that a recombinant protein derived from fusion of the conserved regions of viral proteins has the potential to produce a broad-spectrum MAb against a large group of viruses and that the PelE signal peptide can improve the secretion of scFvs in E. coli. The genus Potyvirus, one of the largest groups of plant viruses, includes 158 species, many of which are economically important, such as Potato virus Y (PVY) and Plum pox virus (1, 2). Most potyviruses infect various host plants and are spread worldwide by aphid vectors and mechanical inoculation (3). Potyviruses have a single-stranded, positive-sense RNA genome of approximately 10 kb in length that encodes a polyprotein translated from a large open reading frame (ORF) (3) and a trans-frame movement-related protein (P3N-PIPO) encoded from the 5=-terminal portion of P3 and the PIPO cistrons (4, 5). During proteolytic processing, the large polyprotein can be separated into 10 mature proteins that are involved in virus replication, movement, and virion assembly (3). Potyviral capsid proteins (CPs) not only form the viral particle but also participate in cell-to-cell and systemic movement (6, 7). The variable N-and C-terminal regions of the CPs exposed on the virion surface are required for long-distance transport, are involved in aphid transmission (DAG motif), and may also assist in cell-to-cell spreading (7-10), whereas the conserved core regions of CPs are necessary for virion assembly and the cell-to-cell movement of potyviruses (6, 7).In Taiwan, the viral disease caused by potyviruses is one of the limiting factors for production of calla lilies. At least five potyvirus...

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Epitope identification and in silico prediction of the specificity of antibodies binding to the coat proteins of Potato Virus Y strains

Cited by 11 publications

References 18 publications

Analysis of Potato virus Y Coat Protein Epitopes Recognized by Three Commercial Monoclonal Antibodies

Analysis of Potato virus Y Coat Protein Epitopes Recognized by Three Commercial Monoclonal Antibodies

Prediction of antigenic epitopes for coat protein of Potato virus Y, Egypt

A Strategy for Generating a Broad-Spectrum Monoclonal Antibody and Soluble Single-Chain Variable Fragments against Plant Potyviruses

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