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A single chain Fv antibody displayed on phage surface recognises conformational group-specific epitope of bluetongue virus.

ABSTRACT: A single chain fragment variable (scFv) antibody gene was isolated from hybridoma cell line secreting monoclonal antibody (MAb) 20E9 that recognises bluetongue virus (BTV) VP7. DNA fragments encoding variable regions of heavy and light chains were amplified by RT-PCR and library of scFv was constructed in phage vector. Two scFv clones that were selected showed specific reactivity with conformational epitope VP7. The N-terminal 22 amino acid residues of 20E9 light chain were identical to that deduced from scFv DNA sequence. An in-frame TAG stop codon was found in the coding sequence and its potential role in regulating the expression and stability of scFv in phage is discussed.

SUBMITTER: Nagesha HS

PROVIDER: S-EPMC7172176 | biostudies-literature | 2001 Feb

REPOSITORIES: biostudies-literature

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A single chain Fv antibody displayed on phage surface recognises conformational group-specific epitope of bluetongue virus.

Nagesha H S HS Wang L F LF Shiell B B Beddome G G White J R JR Irving R A RA

Journal of virological methods 20010201 2

A single chain fragment variable (scFv) antibody gene was isolated from hybridoma cell line secreting monoclonal antibody (MAb) 20E9 that recognises bluetongue virus (BTV) VP7. DNA fragments encoding variable regions of heavy and light chains were amplified by RT-PCR and library of scFv was constructed in phage vector. Two scFv clones that were selected showed specific reactivity with conformational epitope VP7. The N-terminal 22 amino acid residues of 20E9 light chain were identical to that ded ...[more]

PMID: 11164502

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Project description:BackgroundMost Yersinia pestis strains are known to express a capsule-like antigen, fraction 1 (F1)(.) F1 is encoded by the caf1 gene located on the large 100-kb pFra plasmid, which is found in Y. pestis but not in closely related species such as Yersinia enterocolytica and Yersinia pseudotuberculosis. In order to find antibodies specifically binding to Y. pestis we screened a large single chain Fv antibody fragment (scFv) phage display library using purified F1 antigen as a selection target. Different forms of the selected antibodies were used to establish assays for recombinant F1 antigen and Y. pestis detection.MethodsPhage antibody panning was performed against F1 in an automated fashion using the Kingfisher magnetic bead system. Selected scFvs were screened for F1-binding specificity by one-step alkaline phosphatase enzyme linked immunosorbant assay (ELISA), and assayed for binding to recombinant antigen and/or Y. pestis by flow cytometry and whole-cell ELISA.ResultsSeven of the eight selected scFvs were shown to specifically bind both recombinant F1 and a panel of F1-positive Yersinia cells. The majority of the soluble scFvs were found to be difficult to purify, unstable and prone to cross-reactivity with F1-negative Yersinia strains, whereas phage displayed scFvs were found to be easy to purify/label and remarkably stable. Furthermore direct fluorescent labeling of phage displaying scFv allowed for an easy one-step flow cytometry assay. Slight cross-reactivity was observed when fixed cells were used in ELISA.ConclusionsOur high throughput methods of selection and screening allowed for time and cost effective discovery of seven scFvs specifically binding Y. pestis F1 antigen. We describe implementation of different methods for phage-based immunoassay. Based on the success of these methods and the proven stability of phage, we indicate that the use of phage-displayed, rather than phage-free proteins, might generally overcome the shortcomings of scFv antibodies.

Project description:Single chain Fvs (scFvs) are widely applied in research, diagnostics and therapeutic settings. Display and selection from combinatorial libraries is the main route to their discovery and many factors influence the success of this process. They exhibit low thermodynamic stability, resulting in low levels of premature cytosolic folding or aggregation which facilitates sec YEG-mediated translocation and phage in E. coli. However, there is little data analysing how this is related to and influenced by scFv protein expression.We characterised the relationship between overall scFv expression and display propensity for a panel of 15 anti-tetanus toxin scFvs and found a strong positive correlation (Rho = 0.88, p < 0.005) between the two parameters. Display propensity, overall expression and soluble localisation to the periplasm and extracellular fractions were clone specific characteristics which varied despite high levels of sequence homology. There was no correlation between display of scFv or its expression in non-fused (free) form with soluble scFv localisation to the periplasm or culture supernatant. This suggests that divergence in the fate of scFv-pIII and non-fused scFv after translocation to the periplasm accounts for the observed disparity. Differential degrees of periplasmic aggregation of non-fused scFv between clones may affect the partitioning of scFv in the periplasm and culture supernatant abrogating any correlation. We suggest that these factors do not apply to the scFv-pIII fusion since it remains anchored to the bacterial inner membrane as part of the innate phage packaging and budding process.We conclude that in the absence of premature cytosolic aggregation or folding, the propensity of a scFv to be displayed on phage is directly related to its overall expression level and is thus indirectly influenced by factors such as codon bias, mRNA abundance or putative DNA motifs affecting expression. This suggests that scFvs capable of high overall expression and display levels may not produce high yields of non phage-fused soluble protein in either the periplasmic or extracellular fractions of E. coli. This should be considered when screening clones selected from combinatorial libraries for further study.

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A single chain Fv antibody displayed on phage surface recognises conformational group-specific epitope of bluetongue virus.

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A single chain Fv antibody displayed on phage surface recognises conformational group-specific epitope of bluetongue virus.

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