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.
Departamento de Patologia Vegetal, Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Barcelona, Spain.
J
J
Aramburu
J
J
Navas-Castillo
P
P
Moreno
M
M
Cambra
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