Preparation of Monoclonal Antibodies Against Alternaria tenuissima and Development and Application of Colloidal Carbon Immunochromatographic Test Strip
JIN Yi-Mei, PAN Xin-Yi, YUAN Shu-Xian, ZHAO Wei-Chun*
School of Life Sciences, Zhejiang Chinese Medical University, Hangzhou 310053, China
Abstract：Alternaria tenuissima is one of the pathogenic fungi that causes black spot disease of Fritillaria thunbergii. In order to solve the problem of rapid detection of F. thunbergii black spot disease in the field, this study prepared the monoclonal antibodies (McAbs) that against A. tenuissima and the colloidal carbon immunochromatographic test strip. The cell strains secreting specific McAbs against A. tenuissima were obtained by hybridoma technique using the mixture of hypha and spores of A. tenuissima as immunogen. The titer, sensitivities and specificities of the McAbs were determined by indirect ELISA. The McAbs binding protein was detected by Western blot. The colloidal carbon immunochromatographic test strip was prepared by double antibody sandwich method. Finally, five cell strains were obtained and named as AtA1, AtA8, AtD2, AtG7 and AtH9, respectively. The McAbs did not cross-react with the common pathogenic fungi of F. thunbergii, such as A. alternata, Fusarium solani, F. incarnatum, F. equiseti, F. oxysporum, Botrytis cinerea, Phomopsis oblonga, Phoma sp. and so on. The titer of the McAbs were all higher than 105 times. The relative molecular weight of McAbs binding protein were 30, 30, 43, 148 and 50 kD, respectively. The types of antibodies are IgG2a, IgG3, IgM, IgG2a and IgM, among which the detection sensitivities of AtA1, AtA8 and AtG7 were 12.21 ng/mL, and the detection sensitivities of AtD2 and AtH9 were 24.41 ng/mL. The protein of F. thunbergii had no effect on the detection sensitivity of the 5 McAbs. The test strips that based on AtA1 and AtH9 were prepared for the detection of A. tenuissima and the black spot disease of F. thunbergii infected by A. tenuissima. The strip has the advantages of high sensitivity, strong specificity, simple operation, fast and economical, suitable for large-scale test and so on, which provides technical support for early detection and prevention of black spot disease of F. thunbergii.
金怡美, 潘心怡, 袁淑贤, 赵伟春. 细极链格孢菌单克隆抗体的制备和胶体碳免疫层析试纸条的研制及应用[J]. 农业生物技术学报, 2023, 31(2): 436-444.
JIN Yi-Mei, PAN Xin-Yi, YUAN Shu-Xian, ZHAO Wei-Chun. Preparation of Monoclonal Antibodies Against Alternaria tenuissima and Development and Application of Colloidal Carbon Immunochromatographic Test Strip. 农业生物技术学报, 2023, 31(2): 436-444.
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