Development of sensitive polyclonal antibodies against dominant stored wheat grain fungus for its immunological detection: Presentation

Fungal infestation causes deterioration of stored food grains. Most fungal species produce secondary metabolites like aflatoxins which are highly toxic to animals and humans. Aspergillus flavus has been found to be the predominant contaminant in stored wheat grains collected from the godowns of Food Corporation of India, West Bengal. The present study focuses on the development of sensitive polyclonal antibodies (PAbs) for molecular immunological detection of dominant toxigenic fungus. Pure A. flavus isolate was cultured on coconut agar media and its spores were harvested and inactivated by 4% formaldehyde. The inactivated spores were injected into a rabbit along with Freund’s complete/incomplete adjuvant for the development of PAbs. Specificity of the raised antibodies in rabbit serum was examined by enzyme-linked immunosorbent assay (ELISA) using spore proteins as antigen obtained by bead beating method. Out of several proteins (ranging from 10 to 200 kDa present in spore, only two prominent proteins of around 76 kDa and 100 kDa were detected by western blot analysis using raised polyclonal antiserum. The PAbs were purified with protein A column followed by spore proteins conjugated CNBr activated sepharose column for its use in the detection of fungal antigens. This highly purified raised antibody can be used for the development of rapid, sensitive, and accurate techniques (such as dot blot/ELISA) for the detection of toxigenic fungi present in stored wheat grains.Fungal infestation causes deterioration of stored food grains. Most fungal species produce secondary metabolites like aflatoxins which are highly toxic to animals and humans. Aspergillus flavus has been found to be the predominant contaminant in stored wheat grains collected from the godowns of Food Corporation of India, West Bengal. The present study focuses on the development of sensitive polyclonal antibodies (PAbs) for molecular immunological detection of dominant toxigenic fungus. Pure A. flavus isolate was cultured on coconut agar media and its spores were harvested and inactivated by 4% formaldehyde. The inactivated spores were injected into a rabbit along with Freund’s complete/incomplete adjuvant for the development of PAbs. Specificity of the raised antibodies in rabbit serum was examined by enzyme-linked immunosorbent assay (ELISA) using spore proteins as antigen obtained by bead beating method. Out of several proteins (ranging from 10 to 200 kDa present in spore, only two prominent proteins of around 76 kDa and 100 kDa were detected by western blot analysis using raised polyclonal antiserum. The PAbs were purified with protein A column followed by spore proteins conjugated CNBr activated sepharose column for its use in the detection of fungal antigens. This highly purified raised antibody can be used for the development of rapid, sensitive, and accurate techniques (such as dot blot/ELISA) for the detection of toxigenic fungi present in stored wheat grains