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Analysis of two ELISA formats and antigen preparations using polyclonal antibodies against Phomopsis longicolla



Analysis of two ELISA formats and antigen preparations using polyclonal antibodies against Phomopsis longicolla



Phytopathology 84(2): 173-179



Polyclonal antibodies (PAbs) were produced in New Zealand white rabbits with culture filtrate and mycelial extract immunogen preparations from the soybean (Glycine max) fungal pathogen Phomopsis longicolla. The PAbs were purified to the immunoglobin fraction and tested in indirect enzyme-linked immunosorbent assay (ELISA) and in double antibody sandwich-ELISA (DAS-ELISA). The PAbs raised to culture filtrate were more specific but less active in binding to members of the DiaporthePhomopsis complex than were those raised to the mycelial extract immunogen preparation. DAS-ELISA was more specific and 100-fold more sensitive in detecting members of the complex than was indirect ELISA. Variability in specificity between different PAbs was lower in DAS-ELISA compared with indirect ELISA. Immunization of one rabbit with culture filtrate over an extended time resulted in maximum anti-P. longicolla activity after three immunizations, and the activity became constant against most members of the complex at the same time. Reactivity to some cultures of P. longicolla was undetectable following the fourth and fifth immunizations, whereas reactivity to all of our other cultures of the complex remained high.

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