Production of egg yolk antibody against A.fumigatus and its therapeutic potential for treating A.fumigatus keratitis

Abstract

ObjectiveTo prepare specific IgY against A. fumigatus and verify its specificity and antifungal effect on A. fumigatus keratitis. MethodLay hens were immunized with the suspension of inactivated A. fumigatus hyphae which mixed with Freund's complete adjuvant or incomplete Freund's adjuvant. The IgY protein specific for A. fumigatus was extracted by ammonium sulfate salting-out method at the fifth to eighth week after immunization. Bradford method and indirect ELISA were used to determine the concentration and titer of IgY. To verify the inhibitory effect of specific IgY on fungal growth, 1 × 105 CFU/mL A. fumigatus hyphae suspension and specific IgY of different concentrations were mixed and cultured for 24 h, 48 h and 72 h to measure the absorbance. Using specific IgY to treat A. fumigatus keratitis in mice, we observed the cornea under a slit lamp at 24 h, 72 h, and 120 h after treatment. Clinical score was used to assess the disease severity of fungal keratitis in mice cornea. The indirect ELISA method was used to determine the titer of specific IgY stored at room temperature and 4 °C for 1, 2, 3, 4, 5, 6, 7, 8, and 9 months. ResultsThe protein concentrations of specific IgY at the fifth, sixth, seventh and eighth weeks after immunization were 5.46 mg/mL, 5.79 mg/mL, 26.98 mg/mL, 28.71 mg/mL. The titer of the specific IgY of A. fumigatus can reach 1:10000, and the antifungal effect of the specific IgY is dose dependent within a certain range. Specific IgY treatment alleviated the severity of fungal keratitis of mice and reduced the clinical score. Moreover, there were no significant change in the titer of specific IgY after storage at room temperature for 2 months and storage at 4 °C for 6 months. ConclusionThe specific IgY can be successfully prepared by ammonium sulfate salting-out method. And it has excellent stability and significant antifungal effect on A. fumigatus keratitis.