Monoclonal antibodies against tomato spotted wilt virus: Characterisation and application*

Abstract

Mouse hybridoma cells, secreting monoclonal antibodies (MCA) against tomato spotted wilt virus, were produced and screened for virus specificity by an indirect triple antibody ELISA, using a rabbit polyclonal antiserum for antigen trapping. A Bulgarian virus isolate from tobacco was used for immunisation of mice and rabbits. One fusion eventually led to 10 stable hybridoma cell lines, all of which produced antibodies of IgG-type though of different subgroups. Since none of the MCAs reacted with TSWV structural proteins after electrophoresis and transfer to nitrocellulose, other methods were chosen to examine their protein specificity. Purified viral cores and detergent-solubilised envelope proteins were used as antigens for ELISA, or, alternatively, glycosylated viral envelope proteins were trapped onto microtitre plates coated with lectins in order to detect MCAs specific for them. Both methods, independently, led to the identification of two MCAs that were specific for envelope proteins of TSWV. Only these two antibodies reacted with intact TSWV particles when examined by immunogold labelling in the electron microscope. The reaction of all MCAs with 11 different TSWV isolates eventually led to the selection of one core-and one envelope-specific antibody for routine use. Core-specific MCAs revealed serological differences between isolates belonging to the common serotype ( = lettuce serotype), but did not react with the serotype TSWV-I. When comparing different ELISA procedures, broadest reactivity and highest sensitivity with different isolates were obtained in an indirect test procedure, using goat anti-mouse antibody conjugates.

Cloning and sequencing of the S RNA from a Bulgarian isolate of tomato spotted wilt virus

“…The antibodies specifically stained a protein band of Mr 79000 which is in good agreement with the calculated value of 76000 for the expected fusion protein (results not shown). This result further verifies that the 29K ORF codes for the N protein, as has been reported by de Haan et al (1990) for TSWV CNPH1 and emphasizes the close serological relationship between the two TSWV isolates reported by Adam et aL (1990).…”

“…The variability among different TSWV isolates has been estimated by differences in symptom expression (Best, 1954) and confirmed by serological comparisons using either polyclonal (Wang & Gonsalves, 1990) or monoclonal antibodies (Adam et al, 1990;de .Avila et al, 1990).…”

“…In order to compare the nucleotide and amino acid sequences of TSWV isolates from different geographical locations, we have started to sequence the S RNA from the Bulgarian isolate TSWV-L3 that is described in more detail by Adam et al (1990). The TSWV S RNA sequence obtained for a Brazilian isolate, TSWV CNPH1 (de Haan et al, 1990), was used for comparison.…”

“…We assumed that the protein encoded by this ORF was the N protein of TSWV-L3, for which an apparent Mr of 28000 was estimated by SDS-PAGE (Adam et al, 1990). To prove this, we inserted pTSWV-L3/308 into the expression vector pEX2 to give pEXcpTSWV-L3 This construct was expressed in E. coli pop 2136 cells (Maiss et al, 1990) and the resulting fusion protein was analysed after SDS-PAGE, transfer to nitrocellulose (Adam et al, 1987) and incubation with TSWV N-specific antibodies (Loewe Biochemicals).…”

“…The virus isolate TSWV-L3 and its purification and core preparation are described in detail by Adam et al (1990). RNA from purified cores was isolated according to Maiss et al (1988).…”