Production of polyclonal antisera to manganese superoxide dismutase expressed in downy mildew resistant pearl millet and its application for immunodiagnosis

Level of superoxide dismutase (SOD) in the seedlings of downy mildew resistant pearl millet genotype (IP 18293) increased 3-fold upon inoculation with Sclerospora graminicola. Mn-SOD was purified from pearl millet by ion-exchange and gel filtration chromatography. Polyclonal antibody was raised in rabbit against Mn-SOD of pearl millet. Anti-Mn SOD had an antibody titer of 1:20,000. ELISA results revealed a 1.5-fold increase in pure Mn-SOD preparation compared to the crude preparation. The antibody reactivity as detected by ELISA revealed that Mn-SOD is higher at 4 hrs post-inoculation in resistant pearl millet seedlings. The reactivity also showed the Mn-SOD is more in roots of inoculated resistant seedlings. SOD reactivity was also determined by DIBA (Dot-immunobinding assay). The results suggested that the Mn-SOD antibody reacted more strongly with crude SOD of the inoculated resistant seedlings than inoculated resistant seedlings. Western blot analysis revealed the induction of 35 kDa SOD protein in the resistant genotype. Its appearance on western blot coincided with high SOD activity. Immunolocalization experiments showed that SOD protein was abundant in the vascular bundles. The antibody produced to Mn-SOD was specific as judged by ELISA, DIBA, western blot and immunofluorescence assays. The work described here investigates the possibility of using the serological techniques to assess the reactivity of antibody with the SOD antigen.

Keywords
DIBA, downy mildew, ELISA, immunofluorescence, manganese superoxide dismutase, pearl millet, Sclerospora graminicola, Western blot.