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Preparation of human single chain Fv antibody against hepatitis C virus E2 protein and its identification in immunohistochemistry.

ABSTRACT: AIM:To screen human single chain Fv antibody (scFv) against hepatitis C virus E2 antigen and identify its application in immunohistochemistry. METHODS:The phage antibody library was panned by HCV E2 antigen, which was coated in microtiter plate. After five rounds of biopanning,56 phage clones were identified specific to HCV E2 antigen. The selected scFv clones were digested by SfiI/NotI and DNA was sequenced. Then it was subcloned into the vector pCANTAB5E for expression as E-tagged soluble scFv. The liver tissue sections from normal person and patients with chronic hepatitis B and chronic hepatitis C were immunostained with HCV E2 scFv antibody. RESULTS:The data of scFv-E2 DNA digestion and DNA sequencing showed that the scFv gene is composed of 750 bp. ELISA and immunohistochemistry demonstrated that the human single chain Fv antibody against hepatitis C E2 antigen has a specific binding character with hepatitis virus E2 antigen and paraffin-embedded tissue, but did not react with liver tissues from healthy persons or patients with chronic hepatitis B. CONCLUSION:We have successfully screened and identified HCV E2 scFv and the scFv could be used in the immunostaining of liver tissue sections from patients with chronic hepatitis C.

SUBMITTER: Zhong YW

PROVIDER: S-EPMC4656576 | biostudies-literature | 2002 Oct

REPOSITORIES: biostudies-literature

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Preparation of human single chain Fv antibody against hepatitis C virus E2 protein and its identification in immunohistochemistry.

Zhong Yan-Wei YW Cheng Jun J Wang Gang G Shi Shuang-Shuang SS Li Li L Zhang Ling-Xia LX Chen Ju-Mei JM

World journal of gastroenterology 20021001 5

<h4>Aim</h4>To screen human single chain Fv antibody (scFv) against hepatitis C virus E2 antigen and identify its application in immunohistochemistry.<h4>Methods</h4>The phage antibody library was panned by HCV E2 antigen, which was coated in microtiter plate. After five rounds of biopanning,56 phage clones were identified specific to HCV E2 antigen. The selected scFv clones were digested by SfiI/NotI and DNA was sequenced. Then it was subcloned into the vector pCANTAB5E for expression as E-tagg ...[more]

PMID: 12378631

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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.

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Preparation of human single chain Fv antibody against hepatitis C virus E2 protein and its identification in immunohistochemistry.

Publications

Preparation of human single chain Fv antibody against hepatitis C virus E2 protein and its identification in immunohistochemistry.

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