Journal of Virological Methods, 33 (1991) l-l 1 0 1991 Elsevier Science Publishers B.V. / 0168-8510/91/$03.50 AD0N1S0168851091001716 VIRMET 01166 Detection of double-stranded RNA by ELISA and dot immunobinding assay using an antiserum to synthetic polynucleotides Jose Aramburu’, Jestis Navas-Castillo*, Pedro Moreno’ and Mariano Cambra ‘Departamento de Patologia Vegetal, Institut de Recerca i Tecnologia Agroaliment&ies (IRTA). Barcelona, Spain and ‘Institute Valenciano de Investigac'iones Agrarias (IVIA). Moncada, Valencia. Spain (Accepted 7 January 199 1) Summary An antiserum against polyinosinic-polycytidylic acid (In-Cn) was used to detect double-stranded RNA (dsRNA) by several serological techniques. DsRNA was read- ily detected by indirect ELISA (ELBA-I) and dot immunobinding assay (DIA). Addition of the antigen to poly-L-lysine-precoated plates and blocking with uncrea- med milk powder allowed detection levels of 100 pgm-’ In-Cn by ELISA-I. Concen- trations as low as 1 ng.mlli were detected by DIA using polyvinyliden difluoride (PVDF) membranes, Detection capacity with nitrocellulose membranes was lo00 times lower than with PVDF. ELISA-I and DIA enabled detection of dsRNA in enriched fractions from cucumber mosaic virus (CMV)- and citrus tristeza virus (CTV)-infected plants and from virus-infected Penicillium chrysogenum mycelium. These techniques showed similar or higher sensitivity for detection of dsRNA than separation by polyacrylamide gel electrophoresis and silver staining. Double-stranded RNA; Dot immunobinding assay; ELISA; Serological detection Correspondence to: J. Aramburu, Departamento de Patologia Vegetal, IRTA, Ctra. de Cabrils s/n, 08348 Cabrils, Barcelona, Spain.