A large semi-synthetic single-chain Fv phage display library based on chicken immunoglobulin genes

Wyngaardt, Wouter Van; Malatji, Teresiah; Mashau, Cordelia; Fehrsen, Jeanni; Jordaan, Frances; Miltiadou, Dubravka; Plessis, D.H. Du

doi:10.1186/1472-6750-4-6

Cited by 47 publications

(36 citation statements)

References 45 publications

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“…A chicken recombinant antibody library which had been constructed earlier expressly to provide reagents for use in developing immunotests [11] was screened by panning against HSP65, a potentially important diagnostic antigen of both M. bovis and Mycobacterium tuberculosis [5]. Sequencing of a limited number of random clones showed that at least three different scFvs which recognised the target antigen had been obtained.…”

Section: Discussionmentioning

confidence: 99%

“…Single-chain Fvs were selected by panning a phage displayed repertoire derived from the immunoglobulin genes of the chicken (Nkuku Ò library) using methods described previously [11]. Phages displaying antibody fragments that bound to HSP65 were released at high pH and used to reinfect exponentially growing TG1 host cells.…”

Section: Single-chain Fvs From the Nkuku ò Librarymentioning

confidence: 99%

“…For immunodiagnostic applications, tests based on antibodies of which the encoding sequences are known can be more easily standardised than those derived from immune serum or a potentially unstable hybridoma. Single-chain variable fragments (scFvs) based on chicken immunoglobulins are particularly suitable as a basis for developing immunotests, not only because they are serologically distinct from mammalian immunoglobulins, but because the avian antibody repertoire can be accessed [9][10][11] more readily than that of any mammal other than the camelids [12]. Recombinant chicken antibodies derived either from large ''universal'' or dedicated immune repertoires have been shown to be eminently useful in a variety of immunotests aimed at detecting either antigens or antibodies [13][14][15][16][17][18][19].…”

Section: Introductionmentioning

confidence: 99%

“…Three different scFvs were obtained by screening a large semi-synthetic antibody library based on chicken immunoglobulin genes [11]. Two of these were converted into bivalent IgY-like molecules [24].…”

Section: Introductionmentioning

confidence: 99%

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Chicken scFvs and bivalent scFv-CH fusions directed against HSP65 of Mycobacterium bovis

et al. 2010

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a b s t r a c tTwo chicken single-chain variable region antibody fragments (scFvs) that recognised the 65 kDa heatshock protein (HSP65) of Mycobacterium bovis were selected from a large semi-synthetic phage displayed library. Both recognised HSP65 in indirect enzyme-linked immunosorbent assay (ELISA) and immunoblots and retained their activity during storage. Neither, however, could function as the capture reagent in a sandwich ELISA when immobilised on polystyrene. To establish whether they could be engineered for general use in immunotests, the genes coding for these scFvs were subcloned in expression vectors that contained sequences encoding chicken IgY heavy-chain constant region domains. This resulted in larger bivalent constructs which more closely resembled IgY molecules. The engineered fragments were evaluated in ELISAs and gold-conjugated immunochromatographic tests (ICTs). In contrast to their previous behaviour as scFvs, the modified fragments (designated ''gallibodies'') could be used for immunocapture in ELISA and could be readily conjugated to colloidal gold nanoparticles. A sandwich ICT that could detect recombinant HSP65 was also devised. Although converting the recombinant single-chain monomeric antibody fragments to bivalent immunoglobulin-like molecules did not entirely 'standardise' the behaviour of the scFvs, this approach remains potentially useful for developing practical, robust, immunodiagnostic reagents.

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Section: Discussionmentioning

confidence: 99%

Section: Single-chain Fvs From the Nkuku ò Librarymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Chicken scFvs and bivalent scFv-CH fusions directed against HSP65 of Mycobacterium bovis

et al. 2010

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“…In the present study, we describe two phage-displayed scFvs that are suitable for the serotype- and serogroup-specific detection of AHSV in double antibody sandwich (DAS)-ELISAs. Lca12, a serotype-specific scFv with a detection limit of 2 ng purified AHSV-3 per well, was selected with directly immobilized, sucrose gradient-purified AHSV-3 [8] as previously described in detail [17]. The serogroup-specific scFv G7 was isolated with trapped AHSV-8.…”

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confidence: 99%

Serotype- and serogroup-specific detection of African horsesickness virus using phage displayed chicken scFvs for indirect double antibody sandwich ELISAs

et al. 2013

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There is an ongoing need for standardized, easily renewable immunoreagents for detecting African horsesickness virus (AHSV). Two phage displayed single-chain variable fragment (scFv) antibodies, selected from a semi-synthetic chicken antibody library, were used to develop double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) to detect AHSV. In the DAS-ELISAs, the scFv previously selected with directly immobilized AHSV-3 functioned as a serotype-specific reagent that recognized only AHSV-3. In contrast, the one selected with AHSV-8 captured by IgG against AHSV-3 recognized all nine AHSV serotypes but not the Bryanston strain of equine encephalosis virus. Serving as evidence for its serogroup-specificity. These two scFvs can help to rapidly confirm the presence of AHSV while additional serotype-specific scFvs may simplify AHSV serotyping.

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Yeast Surface Display in Combination with Fluorescence‐activated Cell Sorting Enables the Rapid Isolation of Antibody Fragments Derived from Immunized Chickens

Grzeschik

Yanakieva

Roth

et al. 2018

Biotechnology Journal

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Yeast surface display emerged as a viable tool for the generation of human and murine monoclonal antibodies. This platform technology enables the careful definition of selection conditions, the potential for high‐throughput screening, as well as the isolation of antibodies recognizing predefined epitopes. In this study, the applicability of yeast surface display in combination with fluorescence‐activated cell sorting (FACS) for the isolation of antigen‐specific chicken‐derived antibodies is demonstrated. To this end, yeast‐displayed recombinant antibody libraries from splenic mRNA of chickens immunized with epidermal growth factor receptor (EGFR) and human chorionic gonadotropin (hCG) were constructed as single chain variable fragments (scFv) by overlap extension polymerase chain reaction. A large number of antigen binding scFvs were readily isolated in a convenient screening process. Target‐specific scFv‐Fc molecules were produced as soluble proteins and more extensively characterized by confirming specificity, thermostability and high affinity. Essentially, we demonstrated the biotechnological applicability of binders directed against both antigens via specific cellular binding for EGFR and in the context of a lateral flow test by utilizing hCG‐binding scFvs as capturing antibodies for pregnancy detection. Altogether, the described strategy using yeast surface display expands the repertoire of display methods for the isolation of antibodies resulting from chicken immunization campaigns.

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A large semi-synthetic single-chain Fv phage display library based on chicken immunoglobulin genes

Cited by 47 publications

References 45 publications

Chicken scFvs and bivalent scFv-CH fusions directed against HSP65 of Mycobacterium bovis

Chicken scFvs and bivalent scFv-CH fusions directed against HSP65 of Mycobacterium bovis

Serotype- and serogroup-specific detection of African horsesickness virus using phage displayed chicken scFvs for indirect double antibody sandwich ELISAs

Yeast Surface Display in Combination with Fluorescence‐activated Cell Sorting Enables the Rapid Isolation of Antibody Fragments Derived from Immunized Chickens

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