Abstract
BackgroundSevere acute respiratory syndrome (SARS)-CoV is a newly emerging virus that causes SARS with high mortality rate in infected people. Successful control of the global SARS epidemic will require rapid and sensitive diagnostic tests to monitor its spread, as well as, the development of vaccines and new antiviral compounds including neutralizing antibodies that effectively prevent or treat this disease.MethodsThe human synthetic single-chain fragment variable (scFv) ETH-2 phage antibody library was used for the isolation of scFvs against the nucleocapsid (N) protein of SARS-CoV using a bio panning-based strategy. The selected scFvs were characterized under genetics-molecular aspects and for SARS-CoV N protein detection in ELISA, western blotting and immunocytochemistry.ResultsHuman scFv antibodies to N protein of SARS-CoV can be easily isolated by selecting the ETH-2 phage library on immunotubes coated with antigen. These in vitro selected human scFvs specifically recognize in ELISA and western blotting studies distinct epitopes in N protein domains and detect in immunohistochemistry investigations SARS-CoV particles in infected Vero cells.ConclusionThe human scFv antibodies isolated and described in this study represent useful reagents for rapid detection of N SARS-CoV protein and SARS virus particles in infected target cells.
Highlights
Severe acute respiratory syndrome (SARS)-CoV is a newly emerging virus that causes SARS with high mortality rate in infected people
Isolation of human single-chain fragment variable (scFv) antibodies To isolate specific scFv antibodies, an aliquot of the human synthetic ETH-2 library containing approximately 1 × 1012 cfu phages was introduced for panning into Maxisorp immunotubes coated with purified SARS-CoV N protein as antigen
A rodent library has proved to be very effective for the isolation of murine scFvs exhibiting high specificity against SARS-CoV E protein, which is recognized as a 31 kDa protein in western blot studies [15]
Summary
Severe acute respiratory syndrome (SARS)-CoV is a newly emerging virus that causes SARS with high mortality rate in infected people. The traditional approach to generate mAbs to SARS-CoV has presented difficulties for several reasons including safety concerns in handling SARS particles [7]. To overcome these limitations, we applied a very effective and safe in in vitro procedure based on human scFvs selection from the large synthetic ETH-2 phage antibody library [8]. We report the identification, production, and epitope characterization of human scFv antibodies recognizing distinct N protein domains These recombinant mAbs were found to bind selectively and with good affinity to distinct N protein epitopes, and were suitable to identify SARS particles in Vero infected cells
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