| Monoclonal Antibody-Based Indirect-ELISA for Early Detection and Diangnosis of Epiphytic Didymella bryoniae in Cucurbits |
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Sun Cheol Lee, Ki Soo Han, Jung Han Lee, Dong Kil Kim, Hee Kyu Kim |
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| Abstract |
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Gummy stem blight caused by Didymella bryoniae occurs exclusively in cucurbits. This fungus has been known not to produce its pycnidium in vitro unless irradiated. In this study, cultural conditions for the mass-production of pycnidiospore by Metal Halide (MH) lamp irradiation were maximized. The mycelia were cultured at 26˚C on PDA for 2 days under dark condition, and then the plate was illuminated with MH lamp continuously for 3-4 days at 26˚C. Results show that a great number of pycnidia were simultaneously formed. The pycnidiospores produced were then used as immunogen. Fusions of myeloma cell (v-653) with splenocytes from immunized mice were carried out. Two hybridoma cell lines that recognized the immunogen D. bryoniae were obtained. One monoclonal antibody (MAb), Db1, recognized the supernatant while another MAb, Db15, recognized the spore. Two clones were selected which were used to produce ascite fluid of the two MAb, Db1 and Db15, the immunotypes of which were identified as IgG1 and IgG2b, respectively. Titers of MAb Db1 and MAb Db15 were measured and the absorbance exceeded 0.5 even at a 10(-5) dilution. The MAbs reacted positively with D. bryoniae but none reacted with other viral isolates, Cucumber mosaic virus and Cucumber green mottle mosaic virus. Sensitivity of MAb was precise enough to detect spore concentration as low as 10(3)/well by indirect ELISA. Characterization of the MAbs Db1, Db15 antigen by heat and protease treatments, which suggests that the epitope recognized by these two MAbs was glycoprotein. |
| Key Words:
Didymella bryoniae, gummy stem blight, indirect ELISA, monoclonal antibody |
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