ISHS Acta Horticulturae 657: XIX International Symposium on Virus and Virus-like Diseases of Temperate Fruit Crops - Fruit Tree Diseases
PRODUCTION OF MONOCLONAL ANTIBODIES FOR THE SEROLOGICAL IDENTIFICATION AND RELIABLE DETECTION OF APPLE STEM PITTING AND PEAR YELLOW VEIN VIRUSES IN APPLE AND PEAR |
| Authors: | P. Gugerli, M.E. Ramel |
| Keywords: | Monoclonal antibodies, apple stem pitting virus ASPV, pear yellow vein virus PYVV, quince, ELISA, detection |
| Abstract:
Monoclonal antibodies (Mabs) to apple stem pitting (ASPV) and pear yellow vein virus (PYVV) were developed in order to allow fast, large scale testing of apple and pear propagation material. Enzyme-linked immunosorbent assay (ELISA), western blots and electron microscopy were used for their evaluation. Seven Mabs reacted in western blots to multiple virus specific polypeptides of 28 up to 48 kDa. Four of these Mabs strongly decorated ASPV virions. Mabs 4-0, 4-1, 4-7, 5-4 and 6-1 were evaluated in various combinations in double-antibody-sandwich ELISA (DAS-ELISA) as primary and enzyme conjugated secondary antibodies. The virus specific signal to noise ratio in homologous DAS-ELISA was significantly superior to that obtained with heterologous DAS-ELISA based on a polyclonal primary rabbit antibody and enzyme conjugated secondary Mabs. The new Mabs were also evaluated by means of 1,2-dioxetane chemiluminescent (Dropix CDP-StarTM / Sapphire IITM) substrate in a otherwise unchanged DAS-ELISA. Chemiluminescence rendered ELISA at least one magnitude more sensitive and also significantly faster. The new Mabs reacted well to a wide range of ASPV and PVYV isolates maintained on Nicotiana occidentalis as well as on apple and pear. Both viruses, ASPV and PYVV, were well detected by ELISA in older and intermediate leaves from apple and pear. ASPV could also be detected in apple seeds. The new Mabs allowed us to detect ASPV and PYVV in crude extracts of wood scrapings of dormant material in winter. At a larger scale, Mab 4-7 was evaluated in homologous DAS-ELISA, with samples from wood scrapings of 142 nuclear stock candidate trees. Mab based ELISA exceeded biological indexing, demonstrating that DAS-ELISA, based on these new Mabs, makes large scale detection of ASPV and PYVV possible. | |
Download Adobe Acrobat Reader (free software to read PDF files) | |
