Development of polyclonal antibodies againstFusarium verticillioidesexoantigens

Abstract

Exoantigens from Fusarium verticillioides strains were analysed by polyacrylamide gel electrophoresis (SDS-PAGE), enzyme-linked immunosorbent assay (ELISA) and Western blot and were used for production of polyclonal antibodies. Rabbits were immunized with exoantigens from strains 97K, 162A and 113F. Polyclonal antibodies against F. verticillioides 97K exoantigen showed the highest titre in ELISA (1:12.800) and were used in further studies. The crossreactivity of anti-97K antibodies with 14 species from 7 other fungal genera was low, ranging from 4.5 Á/14.7%; the highest reactivities (51.2 Á/76.0%) were observed in Fusarium genera. The detection limit of exoantigen by indirect competitive ELISA was 0.27 mg/ml. Western blot analysis showed two immunodominant antigens (67 kDa and 113 kDa) common to all the F. verticillioides strains analysed. The results suggest that polyclonal antibodies raised against exoantigens from F. verticillioides 97K are promising for Fusarium sp. immunodetection.

Detection ofFusariumsp. contamination in corn by enzyme-linked immunosorbent assay

“…Cross-reactivities of polyclonal antibodies against exoantigens from 14 fungi species from other genera (Aspergillus , Penicillium , Cladosporium, Mucor, Alternaria, Cercospora and Colletotrichum ) were low (4.5 Á/ 14.7%). High cross-reactivities were observed for exoantigens from F. graminearum, F. subglutinans and F. sporotrichioides (51 Á/76%) (Biazon et al 2006).…”

“…and Aspergillus sp. were detected in corn samples and the ELISA developed in this study detects only Fusarium species, showing low cross-reactivity (4.54 Á/14.67%) with other fungal genera (Aspergillus , Penicillium , Cladosporium, Mucor, Alternaria, Cercospora and Colletotrichum ) (Biazon et al 2006).…”

“…The supernatants containing the exoantigens were freeze-dried, stored at (/208C and later dissolved in 0.15M phosphate-buffered saline (PBS) and dialyzed for 24 h at 48C against deionised water and PBS (two final changes). The dialyzates were used to immunize three young male New Zealand rabbits (Biazon et al 2006). Preimmunization blood samples from the rabbits were negative to F. verticillioides exoantigens in indirect ELISA.…”

“…Each rabbit was immunized subcutaneously with three injections of exoantigens (0.5 ml containing 80 mg proteins) at one-week intervals. One week after the 3rd injection, the rabbits were bled and the sera were titred by indirect ELISA (Biazon et al 2006).…”

“…Polyclonal antibody production F. verticillioides 97K was used for the production of exoantigens and polyclonal antibodies as described by Biazon et al (2006).…”