Immunodetection of Aspergillus flavus in Stored Seeds of Cowpea, Lentil and Mungbean

摘要

Polyclonal antibodies (PAbs) were raised in white male rabbits against Aspergillus flavus which was consistently isolated from stored seeds of cowpea (Cajanus cajan}, lentil (Lens culinaris) and mungbean (Vigna radiatd) and IgG was purified by ammonium sulphate precipitation and DEAE-cellulose chromatography. Initial optimization was done by agar double diffusion and ELISA. Antigens from seeds of the three pulses collected from various sources were reacted with PAb of A. flavus using PTA-ELISA and dot immune-binding assays. In ELISA, the highest absorbance values at 405 nm were obtained in reaction of A. flavus PAb with antigens from stored seeds of V. radiata, followed by C. cajan, and the least from L. culinaris. Among seeds from different localities, there were variations in ELISA values. The PAb was further used to detect the fungus in stored seeds following indirect immuno-fluoresence and immune-cytochemical staining. Localization of the fungus was confirmed either on the seed coat surface orthe parenchymatous cells just below the seed coat as evidenced by bright apple green fluorescence.