Monoclonal Antibody-Based Indirect-ELISA for Early Detection and Diangnosis of Epiphytic Didymella bryoniae in Cucurbits

다국어 초록 (Multilingual Abstract)

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 26oC on PDA for 2 days under dark
condition, and then the plate was illuminated with MH
lamp continuously for 3-4 days at 26oC. 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 103/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.