Approach to a general tospovirus assay using antibodies to purified tomato spotted wilt tospovirus G proteins 1

Abstract

The generally assumed presence of common epitopes on the glycoproteins (G proteins) of different tospovirus species should make possible an ELISA with broad reactivity. We have isolated the native G proteins of a type isolate of tomato spotted wilt tospovirus (TSWV) from purified complete virus particles and used them to raise monoclonal and polyclonal antibodies. Purification of the G proteins was achieved by isoelectric focussing in a Rotofor apparatus after solubilization of the lipid envelope with non‐ionic detergent and removal of the nucleoprotein complexes by ultracentrifugation. After isoelectric focussing, three fractions predominantly contained proteins that reacted with antisera against complete virus and with a monoclonal antibody against the G1 protein. These fractions were used to immunize rabbits and mice. The polyclonal antibodies obtained reacted well in DAS‐ELISA with all TSWV and impatiens necrotic spot tospovirus (INSV) isolates tested and also reacted with groundnut bud necrosis tospovirus (GBNV). Several positively reacting hybridoma mother clones were obtained. One of these was cloned and the resulting monoclonal antibody was tested for the detection of all available tospovirus isolates using TAS‐ELISA. Except for GBNV, all isolates were detected with equal efficiency, supporting the view that most tospoviruses share common epitopes in their G proteins. Seventy additional clones await intensive screening and it is hoped that at least one of these may possess even broader reactivity.