The Structure Analysis and Antigenicity Study of the N Protein of SARS-CoV

Wang, Jingqiang; Ji, Jia; Ye, Jia; Zhao, Xiaoqian; Wen, Jie; Li, Wei; Hu, Jianfei; Li, Dawei; Sun, Min; Zeng, Haipan; Hu, Yongwu; Tian, Xiangjun; Tan, Xuehai; Xu, Ningzhi; Zeng, Changqing; Wang, Jian; Bi, Shengli; Yang, Huanming

Blood samples of two normal controls and nine SARS patients were provided by Beijing Plastic Surgery Hospital, Beijing Peoples’ Hospital, and Beijing Tiantan Hospital. Peroxidase-conjugated mouse anti-human IgG and peroxidase-HRP (P6782) were purchased from Sigma (New Jersey, USA). Peptides (1 μg/mL, in 0.5 M carbonate buffer, pH 9.6) were dispensed into a 96-well microplate (100 μL/well) and then incubated at 4°C overnight. After being washed with PBS containing 0.5 M Tween-20 (PBS-T), BSA (2 mg/mL) was added up and the plates were incubated at 37°C for 1 h for blocking. The patient serum sample (10 μL), diluted with 100 μL of sample buffer, was added into each well and incubated at 37°C for 30 min. After being further washed with PBS-T, 100 μL mouse anti-human IgG was added and incubated at 37°C for 20 min. Finally, the wells were washed with PBS-T. The reaction was observed by adding the TMB solution as substrate, after inculation at 37°C for 10 min. The reaction was stopped by adding 50 μL 4 M sulphuric acid, and optical density at 450 nm (ref. 630 nm) was measured with an automatic ELISA reader (Multiskan Ascent, Finland).