Detection of Sclerotinia sclerotiorum Using a Monomeric and Dimeric Single-Chain Fragment Variable (scFv) Antibody

Abstract

Sclerotinia sclerotiorum (Lib.) de Bary is a phytopathogenic fungus capable of causing significant yield losses in numerous crops, including canola, in which the fungus causes sclerotinia stem rot. Immunological detection methods to rapidly determine the presence of S. sclerotiorum on plants may provide growers with a viable diagnostic tool to aid with fungicide use decisions. This paper discusses the generation of a monomeric and dimeric single-chain, variable fragment (scFv) antibody with affinity for S. sclerotiorum using phage display technology. The bacterially expressed and purified scFv is shown to bind S. sclerotiorum with some cross-reactivity with the closely related phytopathogen Botrytis cinerea (Pers.:Fr.). The dimeric scFv displayed improved binding to the fungus as compared to the monomer and could detect the presence of mycelia in inoculated canola petals. To the authors' knowledge, this is the first report of a scFv dimer with affinity for S. sclerotiorum that has the potential for use in the development of a new diagnostic test.

Isolation of Single Chain Variable Fragments against Six Esters of Pyrethrins by Subtractive Phage Display

“…The heavy chain CDR3, critical for antigen binding, showed a greater diversity than the other regions. This conclusion is in agreement with that obtained by other researches, 27,36) and the high mutation rates in these variable genes suggest an antigendriven response in mouse immunization. According to the IMGT, the scFv gene of the a12 clone showed that the VH sequence exhibited 88-95% homology to the variable fragments of known mouse antibodies and shared high sequence similarity with the mouse IGHV1-9 Ã 01 gene (AJ235948) scoring of 1327, and the IGHV1-63 Ã 02 gene (D14633) scoring of 1156, and that the VL genes showed about 90% sequence similarity to the known mouse monoclonal genes IGKV4-74 Ã 01(AJ2231217) and IGKV4-57-1 Ã 01(AC1586723).…”

“…23,34) In instances where, a total of 40 clones with an antibody activity to pyrethrins were obtained, and individual clones with different affinity and number have been isolated against six esters of pyrethrins. As in reports associated with scFv detection, 35,36) the dimeric scFv displayed improved binding to pyrethrin I and II as compared to the monomer (Figs. 7 and 8).…”

Expression of anti-sclerotinia scFv in transgenic Brassica napus enhances tolerance against stem rot

“…Generation of scFv construct for plant transformation As described in [13] the scFv antibody fragment used in this study was isolated by phage display technology using mRNA purified from antibody-producing spleen cells extracted from mice. The antibody library from which the scFv was isolated was derived from mice immunized with whole S. sclerotiorum mycelial antigen.…”

“…The generation of an scFv with affinity for S. sclerotiorum has recently been reported [13]. It was hypothesized that generating transgenic plants expressing this scFv might confer increased levels of tolerance to infection by S. sclerotiorum.…”

Determination of Aspergillus pathogens in agricultural products by a specific nanobody-polyclonal antibody sandwich ELISA

“…And sandwich ELISA has especially advantageous: better capture of antigens, not susceptible to impurities in the sample, and can obtain reliable quantitative relationships 8 . For the past 30 years, immunoassays have been developed for detection of Aspergillus contamination by using polyclonal antisera 3, 7, 9 , monoclonal antibodies 10, 11 or single-chain variable fragment (scFv) antibodies 12–14 . For A. flavus pathogens, however, only monoclonal antibody have been used, with a detection limit in PBS of 1~2 μg mL −1 by ELISA 10 and 1 μg g −1 in maize or peanut by a developed scFv antibody fused to AP 14 .…”