Department of Ophthalmology, The Affiliated Hospital of Qingdao University, Qingdao, China; Tianjin Key Laboratory of Retinal Funtions and Diseases, Tianjin Branch of National Clinical Research Center for Ocular Disease, Eye Institute and School of Optometry, Tianjin Medical University Eye Hospital, Tianjin, China.
Department of Ophthalmology, The Affiliated Hospital of Qingdao University, Qingdao, China.
Microb Pathog. 2021 Sep;158:105081. doi: 10.1016/j.micpath.2021.105081. Epub 2021 Jul 8.
To prepare specific IgY against A. fumigatus and verify its specificity and antifungal effect on A. fumigatus keratitis.
Lay 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 × 10 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.
The 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.
The 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.
制备针对烟曲霉的特异性 IgY 并验证其对烟曲霉角膜炎的特异性和抗真菌作用。
用弗氏完全佐剂或弗氏不完全佐剂混合灭活烟曲霉菌丝悬液免疫产蛋母鸡，于免疫后第 5 至 8 周采用硫酸铵盐析法提取针对烟曲霉的 IgY 蛋白。采用 Bradford 法和间接 ELISA 法测定 IgY 的浓度和效价。为验证特异性 IgY 对真菌生长的抑制作用，将 1×10 CFU/mL 烟曲霉菌丝悬液与不同浓度的特异性 IgY 混合培养 24、48 和 72 h，测量吸光度。用特异性 IgY 处理烟曲霉菌性角膜炎小鼠，于治疗后 24、72 和 120 h 在裂隙灯显微镜下观察角膜。采用临床评分评估小鼠角膜真菌病的严重程度。采用间接 ELISA 法测定在室温下和 4℃下储存 1、2、3、4、5、6、7、8 和 9 个月时特异性 IgY 的效价。
免疫后第 5、6、7 和 8 周特异性 IgY 的蛋白浓度分别为 5.46、5.79、26.98、28.71 mg/mL，特异性 IgY 对烟曲霉的效价可达 1∶10000，且在一定范围内特异性 IgY 的抗真菌作用呈剂量依赖性。特异性 IgY 治疗可减轻小鼠烟曲霉菌性角膜炎的严重程度，降低临床评分。而且，在室温下储存 2 个月和在 4℃下储存 6 个月后，特异性 IgY 的效价无明显变化。
采用硫酸铵盐析法可成功制备特异性 IgY，其对烟曲霉角膜炎具有良好的稳定性和显著的抗真菌作用。
