Routine Detection of Citrus Tristeza Virusby Direct Immunoprinting-ELISA Method Using Specific Monoclonal andRecombinant Antibodies

Abstract

Extract preparation is the most limiting factor for large-scale plant virus testing. Direct tissue print of fresh cross sections of tender shoots or leaf petioles on cellulose membranes, allows the collection of samples and testing of a large number of plants (1,250 plants per team of two workers per day). The printed membranes can be analyzed in the field, mailed, or kept for several months before testing. The analysis is performed by a simple and fast (3 h) direct ELISA protocol using a mixture of citrus tristeza virus (CTV)-specific, alkaline phosphatase conjugated monoclonal antibodies 3DF1 and 3CA5 or using a mixture of 3DF1 and 3CA5 scFv-AP/S recombi-nant antibodies expressed in E. coli as a fusion protein with the alkaline phosphatase enzyme. The sensitivity of immunoprinting-ELISA method was the same as immunocapture-PCR but it was more reliable. A kit has been designed and evaluated under nursery conditions. This kit has been successfully used by nurserymen to test more than 600,000 plants over the last five years.