APPLICATION OF NEW DIAGNOSTIC REAGENTS FOR IDENTIFICATION OF DIVERSE STRAINS OF POTATO VIRUS Y
Vegetable and Forage Crops Production Research
2008 Annual Report
1a.Objectives (from AD-416)
Develop new monoclonal antibodies that can be used for rapid detection and identification of Potato virus Y (PVY) strains that can be utilized on a large scale by certification agencies, researchers, and diagnostic facilities.
1b.Approach (from AD-416)
Nucleoproteins of diverse strains of PVY will be purified using standard methods. These will be used to immunize mice for production of monoclonal antibodies (MAbs) by hybridoma technology. The MAbs will be tested for virus strain specificity using a large panel of characterized PVY isolates available to the cooperator. Documents SCA with University of Idaho.
Four PVY isolates - O (PB-Oz), N (Mont), N:O (Alt), and NTN (423-3) – were propagated in tobacco, purified by differential centrifugation, and used to immunize five BALB/c mice each. In all 20 cases, immunization led to a robust anti-PVY response, with titer exceeding 106 in direct ELISA testing. So far, one mouse was selected for a cell fusion experiment, spleen cells from this mouse were fused to myeloma cell line in vitro, initial 24 hybrid cell cultures were identified and 2 PVY-positive cultures were clonally propagated. These are being tested against various PVY strains. Another cell fusion experiment is being planned. Polyclonal mice antisera produced against different PVY isolates were cross-tested against homologous and heterologous isolates, and demonstrated to have clear specificity towards either O or N type capsid protein of PVY. In two cases, i.e. two mice, another type of specificity was noted: the one distinguishing PVY-O from all other PVY types. This finding may point to a potential for a respective monoclonal antibody of a similar specificity. All monoclonal antibodies generated so far and in the future will be screened against a large panel of PVY isolates maintained in the lab, and available from the cooperators. The key feature screened for is the ability to distinguish between PVY-O and PVY-N:O.
Progress on this project was monitored by the ADODR via phone and email correspondence with the University of Idaho's lead researcher.