Selection of beet necrotic yellow vein virus specific single-chain Fv antibodies from a semi-synthetic combinatorial antibody library

Methods for the generation of monoclonal antibodies against plant viruses are limited because current hybridoma techniques do not allow efficient exploitation of the immune repertoire. Moreover, the immunization procedures often lead to a bias towards an immunodominant contaminant in the immunogen preparation and not to the plant virus itself. The selection of six different single-chain antibody variable fragments (scFv) against beet necrotic yellow vein virus from a semi-synthetic human combinatorial antibody library showed the feasibility of the phage display system. No bias towards minor contaminants in the purified virus preparation was observed in ELISA, as all the selected scFvs reacted only with beet necrotic yellow vein virus infected plant homogenates. In addition, two of the isolated beet necrotic yellow vein virus-specific scFvs could be produced in E. coli as a scFv fusion protein with alkaline phosphatase, and were applied in ELISA as specific ready to use antibody-enzyme conjugates. Because of their specificity, these antibodies have potential to be used as reagents in sensitive diagnostic assays for routine testing for beet necrotic yellow vein virus in sugar beets

Selection of beet necrotic yellow vein virus specific single-chain Fv antibodies from a semi-synthetic combinatorial antibody library

Methods for the generation of monoclonal antibodies against plant viruses are limited because current hybridoma techniques do not allow efficient exploitation of the immune repertoire. Moreover, the immunization procedures often lead to a bias towards an immunodominant contaminant in the immunogen preparation and not to the plant virus itself. The selection of six different single-chain antibody variable fragments (scFv) against beet necrotic yellow vein virus from a semi-synthetic human combinatorial antibody library showed the feasibility of the phage display system. No bias towards minor contaminants in the purified virus preparation was observed in ELISA, as all the selected scFvs reacted only with beet necrotic yellow vein virus infected plant homogenates. In addition, two of the isolated beet necrotic yellow vein virus-specific scFvs could be produced in E. coli as a scFv fusion protein with alkaline phosphatase, and were applied in ELISA as specific ready to use antibody-enzyme conjugates. Because of their specificity, these antibodies have potential to be used as reagents in sensitive diagnostic assays for routine testing for beet necrotic yellow vein virus in sugar beets

Fluobodies : green fluorescent single-chain Fv fusion proteins

An expression system (pSKGFP), which permits the expression of single-chain variable fragments as fusion proteins with modified green fluorescent proteins, was designed. This expression system is comparable to frequently used phage display vectors and allows single-step characterization of the selected recombinant antibodies by flow cytometry or fluorescent cell staining. Two different single-chain variable fragment antibodies, both directed against the lipopolysaccharide of the bacterium Ralstonia solanacearum have been genetically fused to a red-shifted green fluorescent protein and the produced fusion protein tested for usefulness. These fluobodies can be produced in cultures of bacterial cells and purified using immobilized metal affinity chromatography. They function well in flow cytometry and immunofluorescent cell staining, are specific for their target antigens and, unlike FITC-conjugated antibodies, they do not fade upon illumination

Structural dynamics of green fluorescent protein alone and fused with a single chain Fv protein

Structural information on intracellular fusions of the green fluorescent protein (GFP) of the jellyfish Aequorea victoria with endogenous proteins is required as they are increasingly used in cell biology and biochemistry. We have investigated the dynamic properties of GFP alone and fused to a single chain antibody raised against lipopolysaccharide of the outer cell wall of Gram-negative bacteria (abbreviated as scFv-GFP). The scFv moiety was functional as was proven in binding assays, which involved the use of both fluorescence correlation spectroscopy observing the binding of scFv-GFP to Gram-negative bacteria and a surface plasmon resonance cell containing adsorbed lipopolysaccharide antigen. The rotational motion of scFv-GFP has been investigated with time-resolved fluorescence anisotropy. However, the rotational correlation time of scFv-GFP is too short to account for globular rotation of the whole protein. This result can only be explained by assuming a fast hinge motion between the two fused proteins. A modeled structure of scFv-GFP supports this observatio

Application of phage display in selecting Tomato spotted wilt virus - specific single-chain antibodies (scFvs) for sensitive diagnosis in ELISA

A panel of recombinant single-chain antibodies (scFvs) against structural proteins of Tomato spotted wilt virus (TSWV) was retrieved from a human combinatorial scFv antibody library using the novel phage display technique. After subcloning the encoding DNA sequences in the expression vector pSKAP/S, which allowed the scFvs to be expressed as alkaline phosphatase fusion proteins, 17 different scFv antibodies were obtained. Of these, 12 scFvs were directed against the nucleoprotein (N) and 5, putatively, against the glycoproteins (G1 and G2). Five of the N-specific antibodies cross-reacted with two other tospoviruses (Tomato chlorotic spot virus and Groundnut ringspot virus), but none recognized the more distantly related tospoviruses Impatiens necrotic spot virus, Watermelon silverleaf mottle virus, Iris yellow spot virus, or Physalis severe mottle virus. The successful use of one of the antibodies as coating and detection reagent in a double-antibody sandwich enzyme-linked immunosorbent assay showed the potential of the phage display system in obtaining antibodies for routine TSWV diagnosis