원문정보
초록
영어
The purpose of this study is to optimize ELISA protocol to quantify colorectal cancer antigen GA733 and Fc antibody complex protein (GA733-Fc) expressed in transgenic plant. To confirm whether GA733 was functionally fused to Fc, mAb CO17-1A recognizing GA733 was coated on the 96 well ELISA plate. Goat anti-human IgG conjugated to horseradish peroxidase was applied to detect the Fc fragment of human IgG. ELISA was conducted by variable conditions of coating, washing, blocking, and sample amount. In coating, the mammalian-derived mAb CO17-1A were diluted at 2.5, 5, 10 μg/ml (100 μl/well). In washing, 1X PBS as compared to 1X PBS-T (1X PBS+0.05% Tween 20). In blocking, two types of buffers, 5% skim milk and 3% BSA solutions were compared each others. The results showed that 3% BSA gived better blocking than 5% skim milk. For plant samples, we applied transgenic plant leaf extracts expressing antigen GA733-Fc with dilution factors 1/2, 1/5, 1/10 and 1/100. Taken together, the optimized ELISA conditions to quantify plant-drived GA733-Fc were 0.5 μg of mAb CO17-1A per well for coating 3% BSA for blocking buffer, and 1/5 leaf extract sample dilution factor.