Articles
SEROLOGICAL COMPARISON OF TOSPOVIRUS ISOLATES USING POLYCLONAL AND MONOCLONAL ANTIBODIES
Article number
431_11
Pages
135 – 158
Language
Abstract
A test was conducted to compare tospovirus isolates using different poly- and monoclonal antibodies.
All isolates and antibodies were compared under identical conditions.
From 130 tospovirus isolates, which were obtained from all over the world and included well-characterized isolates from all four serogroups, 96 were chosen because they could be recovered after inoculation to Nicotiana benthamiana and N. rustica. Antigen extracts were tested at 1:100 and 1:1000 in parallel in two different enzyme-linked immunosorbent assay (ELISA) protocols: double antibody sandwich ELISA (DAS-ELISA) with 10 polyclonal antisera and triple antibody sandwich ELISA (TAS-ELISA) with 33 monoclonal antibodies (MAbs). According to both tests, 47 isolates belonged to serogroup 1 (TSWV); 8 belonged to serogroup II (2 TCSV, 2 GRSV, and 4 of uncertain classification); 8 belonged to serogroup III (INSV); and 6 belonged to serogroup IV. Among the remaining 27 isolates, 16 did not react with any of the different antibodies; whereas, 11 were detected by only a few antibodies that related them either to serogroup I or II. It can be assumed that they include hitherto unknown tospovirus isolates.
DAS-ELISA revealed that polyclonal antisera, prepared against complete virions, had a much broader reactivity than nucleocapsid (N)-specific antisera; however, in this comparison they, were only available for TSWV. TAS-ELISA included both N-and glycoprotein (G)-specific MAbs against TSWV and revealed a high degree of variability, reflecting the presence of several different epitopes on both proteins.
In addition, a polymerase chain reaction (PCR) detection method was tested with three different primer sets that were designed to detect tospoviruses in general as well as specifically.
Two different primer sets were able to detect tospoviruses from all four serogroups.
All isolates and antibodies were compared under identical conditions.
From 130 tospovirus isolates, which were obtained from all over the world and included well-characterized isolates from all four serogroups, 96 were chosen because they could be recovered after inoculation to Nicotiana benthamiana and N. rustica. Antigen extracts were tested at 1:100 and 1:1000 in parallel in two different enzyme-linked immunosorbent assay (ELISA) protocols: double antibody sandwich ELISA (DAS-ELISA) with 10 polyclonal antisera and triple antibody sandwich ELISA (TAS-ELISA) with 33 monoclonal antibodies (MAbs). According to both tests, 47 isolates belonged to serogroup 1 (TSWV); 8 belonged to serogroup II (2 TCSV, 2 GRSV, and 4 of uncertain classification); 8 belonged to serogroup III (INSV); and 6 belonged to serogroup IV. Among the remaining 27 isolates, 16 did not react with any of the different antibodies; whereas, 11 were detected by only a few antibodies that related them either to serogroup I or II. It can be assumed that they include hitherto unknown tospovirus isolates.
DAS-ELISA revealed that polyclonal antisera, prepared against complete virions, had a much broader reactivity than nucleocapsid (N)-specific antisera; however, in this comparison they, were only available for TSWV. TAS-ELISA included both N-and glycoprotein (G)-specific MAbs against TSWV and revealed a high degree of variability, reflecting the presence of several different epitopes on both proteins.
In addition, a polymerase chain reaction (PCR) detection method was tested with three different primer sets that were designed to detect tospoviruses in general as well as specifically.
Two different primer sets were able to detect tospoviruses from all four serogroups.
Authors
G. Adam, D. Peters, R.W. Goldbach
Keywords
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