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The purpose of this study is to optimize ELISA conditions to quantify the colorectal cancer antigen GA733 linked to the Fc antibody fragment fused to KDEL, an ER retention motif (GA733-FcK) expressed in transgenic plant. Variable conditions of capture antibody, blocking buffer, and detection antibody for ELISA were optimized with application of leaf extracts from transgenic plant expressing GA733-FcK. In detection antibody, anti-EpCAM/CD362 IgG recognizing the GA733 did not detect any GA733-FcK whereas anti-human Fc IgG recognizing the human Fc existed in plant leaf extracts. For blocking buffer conditions, 3% BSA buffer clearly blocked the plate, compared to the 5% skim-milk buffer. For capture antibody, monoclonal antibody (MAb) CO17-1A was applied to coat the plate with different amounts (1, 0.5, and 0.25 μg/well). Among the amounts of the capture antibody, 1 and 0.5 μg/well (capture antibody) showed similar absorbance, whereas 0.25 μg/well of the capture antibody showed significantly less absorbance. Taken together, the optimized 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 leaf samples in Western blot. The co-efficient value R(2) between the ELISA quantified value and protein density was 0.85 (p0.01), which indicates that the optimized ELISA conditions feasibly provides quantitative information of GA733-FcK expression in transgenic plant.

A male silkworm pupae extract with herbal mixtures such as Rubus coreanus Miquel, Chinese matrimony vine Acanthopanax senticosus and tocopherol can be effectively used to recover or boost stamina. In this study, the effect of the male silkworm pupae extract on the reproductive system of Sprague–Dawley male rats was investigated. No clinical symptoms, and no dying or dead animals were found among experimental male silkworm larvae extract, male silkworm pupae extract and control rat groups during the study. No significant differences were found in body weights, feed or water consumption, or macroscopic examination among the three groups. No lesion was found upon necropsy. However, sperm in the group treated with male silkworm pupae extract were significantly more active than sperm in the control group. Sperm counts in both male groups treated with male silkworm larvae extract and male silkworm pupae extract were significantly higher than sperm counts in the control group. Analysis of phospholipid–hydroperoxide glutathione peroxidase (PHGPx) gene expression showed that the male silkworm pupae extract increased expression in testes by 26.7%. This study shows that the male silkworm pupae extract has potential to be used to enhance the function of the human reproductive system.