Purification and production of antiserum against abaca mosaic potyvirus

Abaca mosaic potyvirus (AbaMV) in field samples was purified following a modification of the procedure of Thomas et al. (1997). Rate zonal centrifugation in CsCl gradient showed a major light-scattering band with a UV absorption spectrum (220nm-320nm) typical of the putative AbaMV nucleoprotein. This light-scattering band was absent in the extract of healthy abaca plants. SDS-PAGE analysis of the aliquot revealed two different profiles, with the one prepared from relatively fresh samples showing approximately 31, 34, and 39 kDa and the other prepared from samples kept at -20 C for 3-4 wk or longer showing 29 and 31 kDa protein bands. These bands are likely to be the coat proteins of AbaMV as they reacted to maize dwarf mosaic virus strain B (MDMV-B) antiserum but not to banana bract mosaic potyvirus (BBrMV) antiserum in western blot. The reaction of MDMV-B antiserum to the AbaMV-dissociated proteins in western blot indicates that AbaMV is serologically related to MDMV-B, a virus classified as sugarcane mosaic potyvirus (SCMV). When the purified AbaMV was used as antigen to produce polyclonal antiserum in rabbit, the resulting antiserum reacted specifically to abaca plants infected with AbaMV in indirect ELISA but not to extracts of abaca infected with bunchy top nanovirus (BTV) and bract mosaic virus (BrMV). The antiserum detected the same bands corresponding to the dissociated virions of AbaMV in western blot, indicating a comparable reaction with MDMV-B antiserum. This study demonstrates successful purification of the AbaMV from field samples and the production of polyclonal antibodies. This reagent now enhances the detection of AbaMV in abaca samples, whether from the field or abaca plantlets produced through tissue culture, and serological diagnosis of abaca virus diseases in general.