Generation and characterization of recombinant single chain Fv antibody that recognizes platelet glycoprotein Ibα

Thrombosis and Hemostasis Research Unit, Jiangsu Institute of Hematology, The First Affiliated Hospital of Suzhou University, Suzhou 215006, China.
Thrombosis Research (Impact Factor: 2.45). 02/2003; 109(2-3):137-44. DOI: 10.1016/S0049-3848(03)00152-X
Source: PubMed


A recombinant single chain Fv (scFv) fragment with specific activity against platelet glycoprotein (GP) Ibalpha was developed and characterized. The scFv was generated from the SZ-2 hybridoma, which produced an anti-platelet antibody reactive to GPIbalpha. VH and VL gene segments were generated from the SZ-2 hybridoma by reverse transcribed-polymerase chain reaction (RT-PCR). After cloning into pUCm-T vector, the DNA sequences of both VH and VL genes were analyzed from two different clones, respectively, the same results were obtained. Comparison of SZ-2 variable region to the Kabat database showed that VH belonged to the mouse Ig heavy family XV while VL belonged to the mouse Ig kappa family XXVI. For assembly of the SZ-2 scFv, VH and VL fragments were cloned into pSW1-scFv successively. The scFv was arranged in VH-VL orientation, being joined together with a 15-amino-acid (Gly(4)Ser)(3) linker. The scFv encoding sequence was amplified and cloned into pET22b vector in-frame with a pel B leader sequence to direct secretion of the protein. Escherichia coli strain BL-21(DE3)PlysS was transformed with the recombinant plasmid, and expression of the scFv was induced using isopropyl-beta-D-thiogalactopyranoside (IPTG). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the recombinant antibody revealed a protein with apparent molecular weight of approximately 31,000. By comparing band intensity on a Coomassie brilliant blue-stained SDS-PAGE, the production yield of SZ-2 scFv was about 25% of the total cellular proteins. The recombinant SZ-2 scFv antibody was successfully purified using Ni-NTA affinity chromatography with a yield of 120 mg/l. The SZ-2 scFv antibody could bind to platelets demonstrated by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Analyzed by Western blot, it could bind to platelet GPIb. It retained the binding capacity of its parental SZ-2 monoclonal antibody (MoAb). In functional studies, SZ-2 scFv inhibited platelet agglutination and aggregation induced by ristocetin and thrombin, respectively, but had no effect on ADP-induced platelet aggregation. Therefore, SZ-2 scFv has the potential to be used as an antithrombotic agent.

12 Reads
  • Source
    • "In the scFv (single-chain fragment variable) construction, the order of the domains can be either VH-linker-VL or VL-linker-VH and both orientations have been applied [46–48]. Even though Luo et al. [49] have shown that the expression of scFv (single-chain fragment variable) of Pichia pastoris system is VL-linker-VH orientation-dependent; most of the scFv (single-chain fragment variable) are constructed in a VH-linker-VL orientation. "
    [Show abstract] [Hide abstract]
    ABSTRACT: To date, generation of single-chain fragment variable (scFv) has become an established technique used to produce a completely functional antigen-binding fragment in bacterial systems. The advances in antibody engineering have now facilitated a more efficient and generally applicable method to produce Fv fragments. Basically, scFv antibodies produced from phage display can be genetically fused to the marker proteins, such as fluorescent proteins or alkaline phosphatase. These bifunctional proteins having both antigen-binding capacity and marker activity can be obtained from transformed bacteria and used for one-step immunodetection of biological agents. Alternatively, antibody fragments could also be applied in the construction of immunotoxins, therapeutic gene delivery, and anticancer intrabodies for therapeutic purposes. This paper provides an overview of the current studies on the principle, generation, and application of scFv. The potential of scFv in breast cancer research is also discussed in this paper.
    Clinical and Developmental Immunology 03/2012; 2012(8):980250. DOI:10.1155/2012/980250 · 2.93 Impact Factor
  • Source
    • "In the decade since the original publications describing the recombinant scFv-HIV reagent, there have been significant advances in protein expression systems. scFvs have been produced in bacterial, mammalian, yeast and insect cell systems with varying success in terms of yield and function (Ridder et al., 1995; Kretzschmar et al., 1996; Sanchez et al., 1999; Fernandez et al., 2000; Freyre et al., 2000; Dai et al., 2003). Increasing numbers of recombinant therapeutics are being manufactured using mammalian expression systems (Wurm, 2004). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The autologous red cell agglutination assay reagent consists of an antibody or antibody fragment of a human erythrocyte-specific monoclonal antibody (mAb) conjugated to an antigen of interest. This bi-functional reagent causes the agglutination of the patient's erythrocytes in the presence of the antigen-specific antibodies in the patient's serum. Previously, such reagents have been produced either by chemical conjugation or recombinant expression in bacteria. These protocols required laborious processes for purification and refolding. The aim of the work reported in this article was to explore the production of the agglutination assay reagent as both a single chain Fv (scFv) antibody fragment and recombinant full-length mAb, expressed in a secreted form in commonly used mammalian cell lines. The DNA encoding the anti-erythrocyte antibodies was linked to that of a diagnostic peptide from West Nile virus, which requires glycosylation for recognition by antibodies present in the sera of infected horses. The expression vectors were designed to allow the rapid directional insertion of DNA encoding other immunogenic peptides to mediate the secretion of agglutinating scFv and full-length mAb reagents from transfected mammalian cells. Stable cell lines were produced for the expression of most, but not all of the constructs. The recombinant reagents could be used directly from the cell culture media after a simple concentration step. The results indicate that further modifications to increase the yield of recombinant protein will enable the direct use of culture supernatant in diagnostic assays without further processing.
    Journal of virological methods 09/2010; 168(1-2):177-90. DOI:10.1016/j.jviromet.2010.05.012 · 1.78 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Recombinant antibodies are important tools for biomedical research and are increasingly being used as clinical diagnostic/therapeutic reagents. In this article, a background to humanized antibodies is given, together with details of the generation of antibody fragments--for example, single chain Fv fragments. Phage antibody fragments are fast becoming popular and can be generated by simple established methods of affinity enrichment from libraries derived from immune cells. Phage display methodology can also be used for the affinity enrichment of existing antibody fragments to provide a reagent with a higher affinity. Here, phage antibodies are demystified to provide a greater understanding of the potential of these reagents and to engage clinicians and biomedical scientists alike to think about potential applications in pathology and clinical settings.
    Journal of Clinical Pathology 10/2004; 57(9):912-7. DOI:10.1136/jcp.2003.014407 · 2.92 Impact Factor
Show more

Similar Publications