earticle

영어

The purpose of this study is to optimize sandwich ELISA conditions to quantify the colorectal cancer antigen GA733 linked to the Fc antibody fragment complex protein fused to KDEL, an ER retention signal (GA733-FcK) expressed in the transgenic plant. Variable conditions of capture antibody, blocking buffer, and detection antibody for ELISA were optimized with application of leaf extracts from the transgenic plant, expressing GA733-FcK. In detection antibody, anti-EpCAM/CD362 IgG conjugated to HRP recognizing the GA733 did not detect any GA733-FcK whereas anti-human Fc IgG conjugated to HRP recognizing the human Fc detected GA733-FcK distinguish the presence of GA733-FcK in leaf extract. For blocking buffer conditions, 3 % BSA buffer properly clearly blocked the ELISA plate, compared to the 5 % skim milk buffer. For the capture antibody, mAb CO17-1A was applied to coat the 96 well ELISA plate with different amounts (1, 0.5, and 0.25 μg /well). Among the different amounts of the capture antibody, 1 and 0.5 μg/well of the capture antibody showed similar absorbance; whereas, 0.25 μg/well of the capture antibody showed significantly less absorbance. Taken together, the optimized sandwich ELISA conditions to quantify plant-derived GA733-FcK were 0.5 μg/well of mAb CO17-1A per well for the capture antibody, 3 % BSA for blocking buffer, and anti-human Fc conjugated HRP. To confirm the optimized ELISA conditions, correlation analysis was conducted between the quantified amount of GA733-FcK in ELISA and its protein density values of different transgenic plant leaf samples in Western blot. The coefficient value R2 between the ELISA quantified expression value and protein density was 0.85 (p<0.01), which indicates that the optimized sandwich ELISA conditions feasibly provides quantitative information of GA733-FcK expression in the transgenic plant leaves.