Detection of double-stranded RNA by ELISA and dot immunobinding assay using an antiserum to synthetic polynucleotides.
An antiserum against polyinosinic-polycytidylic acid (In-Cn) was used to detect double-stranded RNA (dsRNA) by several serological techniques. DsRNA was readily detected by indirect ELISA (ELISA-I) and dot immunobinding assay (DIA). Addition of the antigen to poly-L-lysine-precoated plates and blocking with uncreamed milk powder allowed detection levels of 100 pg.ml-1 In-Cn by ELISA-I. Concentrations as low as 1 ng.ml-1 were detected by DIA using polyvinyliden difluoride (PVDF) membranes. Detection capacity with nitrocellulose membranes was 1000 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.
Aramburu
J
J
Departamento de Patologia Vegetal, Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Barcelona, Spain.
Navas-Castillo
J
J
Moreno
P
P
Cambra
M
M
D000906:Antibodies; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005069:Evaluation Studies as Topic; D015151:Immunoblotting / Q000379:methods*; D010942:Plant Viruses / Q000302:isolation & purification; D011070:Poly I-C / Q000276:immunology; D011145:Polyvinyls; D012330:RNA, Double-Stranded / Q000276:immunology / Q000302:isolation & purification*; D012367:RNA, Viral / Q000276:immunology / Q000302:isolation & purification*; D014773:Virology / Q000379:methods; D005465:Fluorocarbon Polymers
D000906:Antibodies; D011070:Poly I-C; D011145:Polyvinyls; D012330:RNA, Double-Stranded; D012367:RNA, Viral; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers