Use of superparamagnetic beads for the isolation of a peptide with specificity to cymbidium mosaic virus

Abstract

A modified method for the rapid isolation of specific ligands to whole virus particles is described. Biopanning against cymbidium mosaic virus was carried out with a commercial 12-mer random peptide display library. A solution phase panning method was devised using streptavidin-coated superparamagnetic beads. The solution based panning method was more efficient than conventional immobilized target panning when using whole viral particles of cymbidium mosaic virus as a target. Enzyme-linked immunosorbent assay of cymbidium mosaic virus-binding peptides isolated from the library identified seven peptides with affinity for cymbidium mosaic virus and one peptide which was specific to cymbidium mosaic virus and had no significant binding to odontoglossum ringspot virus. This method should have broad application for the screening of whole viral particles towards the rapid development of diagnostic reagents without the requirement for cloning and expression of single antigens.

Introduction

Orchids are among the world's most popular flowers and their trade as both cut flowers and potted plants plays a significant role in the economy of many Asean countries (Hew, 1994, Laws, 1995, Eun and Wong, 1999). Over 25 viruses are known to occur in orchids (Vejaratpimol et al., 1999). Of these, Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) are among the most common (Hu et al., 1998, Wong et al., 1994). These pathogens are prevalent in cultivated orchids throughout the world, are easily spread by mechanical means and are often asymptomatic during early stages of infection (Jensen, 1950, Lawson and Brannigan, 1986). Crop production losses attributed to plant viruses can be enormous, especially when high-value cash crops, such as ornamentals are at stake (Webster et al., 2004). The control of plant viruses is limited largely to agronomic practices and although some viral diseases can be diagnosed quickly by visual examination of symptoms, others require molecular tests for early diagnosis (Webster et al., 2004). Among the possible molecular tests available, antibody and other ligand-based tests are often preferred as they have the advantages of being relatively inexpensive, easy for lay-men to perform and where dip-stick types tests can be developed, can be performed on site. For this reason, there is much interest in the isolation of ligands with specificity to emerging and important pathogens for the development of inexpensive diagnostic kits (Willats, 2002, Ziegler and Torrance, 2002).

The development of phage display technology and its use for display of combinatorial libraries such as antibody fragments (Hoogenboom et al., 1998, Johns et al., 2000, McCafferty et al., 1990) and peptides (Cabilly, 1999, Cortese et al., 1995, Cwirla et al., 1990; Devlin et al., 1990) have made it much more rapid and straightforward to isolate ligands with specificity for target antigens such as viral coat proteins (Barbas et al., 2000, Gough et al., 1999, Hong and Boulanger, 1995). The practicality of this method has led to its increasing importance in plant pathology (Ziegler and Torrance, 2002) as well as for the development of human therapeutics (Udo and Jurgen, 2005). Conventionally, screening of such libraries involves the binding of the target antigen to a solid support such as the surface of a well in a microtitre plate (McCafferty and Johnson, 1996). However, this may limit the amount of antigen surface available to the ligand library during panning due to low binding affinity of the antigen for the surface and the limited surface area available. A method was devised to allow the use of whole viral particles as the antigen and to allow a much larger antigen surface area per volume ratio, by the use of superparamagnetic beads in solution, for antigen display.