用MTT比色法建立B型CpGODN活性检测标准

Determination of optimized MTT colorimetric assay as the identification method for the activity of B type CpG ODN BW006

目的:B型CpG ODN,BW006作为疫苗佐剂已进入临床试验阶段,所以需要建立稳定、安全、简便易行的活性检测方法以控制不同批次BW006的生产质量。方法:根据BW006具有刺激人PBMC和小鼠脾细胞增生的特点,选择无放射性污染的MTT比色法作为首选检测方案。用BW006刺激BALB/c小鼠脾细胞作为筛选平台,确定如下反应条件:脾细胞用量、培养板形状、BW006浓度、培养液体积和培养时间以及如何优化MTT检测过程以提高反应灵敏度。结果:用3 mg/LBW006与96孔平底板中含100 mL/LFBS的200μL无酚红RPMI1640培养的6×105个小鼠脾细胞在37℃含50 mL/L CO2孵箱中共孵育36 h。随后每孔弃100μL上清,加入MTT(5 g/L)10μL,37℃避光孵育4 h使各孔充分形成甲瓒颗粒。每孔加入DMSO 150μL,经平板振荡器振荡20 min至颗粒完全溶解,在酶标仪上检测A578值即可。结论:建立了无污染、造价低、可操作性强的BW006活性检测方法。

AIM： BW006, B type CpG ODN, will be approved as vaccine adjuvant for human use. So it is necessary to determine a stable, safety and easy-to-operate method for activity identification of BW006 with different lots. METHODS： According to the characteristic of BW006 to stimulate the proliferation of PBMC or mice splenocytes, MTT assay was preferentially selected to test the characteristic of BW006. The splenocytes of female BALB/c mice was stimulated by BW006, and different experiment parameters including splenocyte numbers, the shape of plate, BW006 concentration, culture medium, culture time and optimized MTT conditions were determined in these courses. RESULTS： the whole experiment scheme was defined as following： 6 × 10^5 splenocytes were co-cultured with 3 mg/L BW006 in 200 microliter RPMI1640（without phenol red） supplemented with 100 mL/L FBS in a 96 well square plate. PBS, the solvent of BW006 was used as negative control, and culture medium without cells was used as blank. After being co-cultured for 36 hours at 37℃; in humidified incubator with 50 mL/L CO2, 100 microliter supernatants was aspired out and 10 microliter MIF （5 g/L） was added into each well. The plate was further incubated in dark at 37℃ for 4 hours that is sufficient for the formation of formazan. Afterward, 150 microliter DMSO was directly added into each well. The plate was shaken on plate shaker for nearly 20 minutes until the formazan was completely dissolved and detected A578 value at at ELISA reader. CONCLUSION： optimized MTT assay, which is non-radioactive contamination, low-price and simple operation, could be used as activity identification of BW006 during large-scale production.