J. Phytopathology 132, 46—56 (1991) © 1991 Paul Parey Scientific Publishers, Berlin and Hamburg ISSN 0931-1785 Department of Virology, Agricultural University, Wageningen, the Netherlands Comparison of Polyclonal Antisera in the Detection of Tomato Spotted Wilt Virus Using the Double Antibody Sandwich and Cocktail ELISA R. DE O. RESENDE, A. C. DE AVILA, R. W . GOLDBACH and D. PETERS Authors' address: Department of Virology, Agricultural University, Bitmenhaven 11, 6709 PD Wageningen, the Netherlands. 'With 3 figures Received July 30, 1990; accepted November 20, 1990 Abstract Different polyclonal antisera and enzyme-linked immunosorbent assay (ELISA) procedures have been tested for their potential to detect tomato spotted wilt virus (TSWV). The virus could efficiently be detected in high dilutions of sap from infected plants, and at low concentrations of purified virus and nucleocapsid protein preparations in the cocktail ELISA and the double antibody sandwich ELISA (DAS-ELISA). Amounts of 1 to 3 ng of virus protein still gave positive readings using purified preparations, while sap could be diluted approximately 100,000 times. Differences in the detection level were observed using nucleocapsid protein antiserum (anti-N-serum) and the antiserum against intact vims particles (anti-TSWV-serum), but both antisera showed to be powerful sera for the detection of TSWV. Using anti-N-senim, TSWV could be detected in highly diluted extracts of different hosts, and also in leaf extracts or intact tissues stored for 30 days under different conditions. These results indicate that the TSWV nucleocapsid protein remains antigenic for long periods. Zusammenfassung Vergleich der polyklonaien Antisera bei der Feststellung des tomato spotted wilt virus durch die Anwendung des double antibody sandwich und cocktail ELISA Untersucht wurden verschiedene polyklonale Antisera und enzymgekoppelte Immunosorbent- assay(ELISA)-Verfahren auf ihr Vermogen, das tomato spotted wilt virus (TSWV) zu ermineln. Mit dem cocktail ELISA und dem double antibody sandwich ELISA (DAS-ELISA) konnte das Virus im hoch verdunnten Saft aus infizierten Pflanzen sowie bei niedrigen Konzentrationen des gereinigten Virus und in Nucleocapsidproteinpraparaten sehr gut ermittelt werden. Vinisproteinmengen um 1 bis 3 ng ergaben positive Messungen, wenn gereinigte Aufbereitungen verwendet wurden, der Pfianzen- saft konnte sogar ca. 100 OOOfach verdiinm werden. Unterschiede in der Ermittlungsgrenze wurden beobachtet, abhangig von dem angewandten Antiserum — Nucleocapsidproteinantiserum (anti-N- serum) oder dem Antiserum gegen intakte Viruspartikeb (anti-TSWV-serum). Beide Antisera zeigten U.S. Copyri^t aearance Center Code Sutement: 0931-1785/91/3201-0046$02.50/0