Detection and identification of plant viruses by ELISA using crude sap extracts and unfractionated antisera.
A simple and rapid procedure of enzyme immunoassay (PTA-ELISA) was used to detect and identify viruses in individual plants. Virus antigen in crude leaf extracts was adsorbed directly to a solid-phase support, allowed to react with unfractionated antiserum and the antigen-antibody complex detected with a general purpose conjugate of protein A and enzyme. Viral antigens were trapped most effectively by high bonding polystyrene microtitre plates loaded with leaf extracts prepared in carbonate buffer at pH 9.6. With protein A-alkaline phosphatase conjugate and the substrate p-nitrophenyl phosphate as the antibody-detection system, 18 plant viruses in 8 virus groups were detected reliably and nonspecific reactions did not occur. However, when the substrate 3,3',5,-tetramethyl benzidine was used in conjunction with protein A-horseradish peroxidase conjugate, nonspecific reactions were given by leaf extracts from some uninfected or virus-infected plant species. Where less sensitivity is required than is provided by versions of ELISA that rely on antibody-captured antigen, this method provides a simple and rapid means of detecting and identifying viruses in crude sap extracts with the aid of unfractionated antisera.
D000469:Alkaline Phosphatase; D000936:Antigen-Antibody Complex / Q000032:analysis; D000956:Antigens, Viral / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay; D006735:Horseradish Peroxidase; D007106:Immune Sera / Q000276:immunology; D009029:Mosaic Viruses / Q000276:immunology / Q000302:isolation & purification; D010936:Plant Extracts; D010942:Plant Viruses / Q000276:immunology / Q000302:isolation & purification*; D010944:Plants / Q000382:microbiology*; D013045:Species Specificity; D013205:Staphylococcal Protein A
D000936:Antigen-Antibody Complex; D000956:Antigens, Viral; D007106:Immune Sera; D010936:Plant Extracts; D013205:Staphylococcal Protein A; D006735:Horseradish Peroxidase; D000469:Alkaline Phosphatase