Epitope identification and in silico prediction of the specificity of antibodies binding to the coat proteins of Potato Virus Y strains

A phage library containing 2.7 × 10(9) randomly expressed peptides was used to determine the epitopes of three monoclonal antibodies that bind to the coat protein of Potato Virus Y. Construction of the consensus sequences for the peptides obtained after three selection rounds indicated that each antibody recognized a different epitope located within the first 50 N-terminal amino acids of the coat protein. The location of the epitopes was confirmed by heterologous expression of the N-terminal part of the coat protein in Escherichia coli, and, subsequently, by performing an immunological test with the three antibodies. The accuracy of the phage library was demonstrated by predicting in silico the cross-reactivity of the three antibodies with other potyvirus family members. ELISA and in silico predictions revealed the same results in almost every case. The potential of peptide phage libraries to optimize the use of antibodies in plant virology is discusse

Towards sorting of biolibraries using single-molecule fluorescence detection techniques

The selection of specific binding molecules like peptides and proteins from biolibraries using, for instance, phage display methods can be quite time-consuming. It is therefore desirable to develop a strategy that is much faster in selection and sorting of potential binders out of a biolibrary. In this contribution we separately discuss the current achievements in generation or biolibraries, single-molecule detection techniques and rnicrofluidic devices. A high-throughput microfluidic platform is then proposed that combines the propulsion of liquid containing fluorescent components of the biolibrary through microchannels, single-molecule fluorescence photon burst detection and real-time sorting of positive hits

Isolation of recombinant antibodies (scFvs) to grapevine virus B

A panel of 15 recombinant single chain antibodies (scFv) specific to grapevine virus B (GVB) were recovered from a human combinatorial scFv antibody library using the phage display technique against purified virus particles. Two selected scFv-encoding genes were expressed in recombinant Escherichia coli cells as dimeric antibodies. Successful detection of GVB in tissues of herbaceous hosts and grapevine was obtained in a direct binding assay using dimeric scFvs. This reagent was also shown to substitute efficiently for a GVB polyclonal serum in standard DAS-ELISA test used routinely for diagnosis

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