Selection of a monoclonal antibody to detect pvyn and its use in elisa and diba assays

Three monoclonal and four polyclonal antibodies were evaluated for the specific detection of PVYN (tobacco veinal necrosis strain of potato virus Y) of North American origin. All polyclonal antibodies reacted with PVY isolates, irrespective of the type of strains. Monoclonal antibodies (Mabs) Bioreba and 4E7 perform better in DAS (double antibody sandwich) ELISA, whereas Mab VN 295.5 does so in TAS (triple antibody sandwich). Mab Bioreba cross-reacted with several PVY0 (common strain of PVY) isolates, but Mab 4E7 did not. Mab 4E7 detected PVYN infection in composite samples of 1 infected: 199 healthy sprouts and in a 1:320 dilution of potato leaf sap. To obtain a high absorbance reading, overnight (16 h) enzymatic hydrolysis of substrate was needed for Mab 4E7. Most samples (94.8%) that gave a positive serological reaction to Mab 4E7 produced PVYN symptoms in tobacco bioassay. Other methods of immunoassay, like TAS-ELISA and dot immunobinding assay (DIBA), were evaluated with Mabs 4E7 and VN 295.5. Mab VN 295.5 reacted strongly in TAS-ELISA, but did not detect all PVYN isolates. Mab 4E7 reacted strongly in TAS-ELISA, but there were cross-reactions with some PVY isolates. In DIBA, titres of Mabs 4E7 and VN 295.5 were found to be over 1:1 x 106. With Mab 4E7, sensitivity of PVYN detection increased to 1:640 dilution of leaf sap. The lower limit of detection on various filter papers or hand towel paper as solid supports for spotting virus samples in DIBA was 10 ng, in contrast to 170 pg for nitrocellulose and nylon membranes.