小鼠腹水中抗人食管癌单克隆抗体的纯化

研究目的从小鼠腹水中纯化抗人食管癌单克隆抗体。处理方法小鼠腹水通过羟基磷灰石柱层析后(层析环境温度15℃,起始缓冲液pH6.8,0.01MPB。流速1ml/min,线性梯度洗脱液500ml,0.1～0.5MPB)。由酶联免疫过滤法(EIF)鉴别出抗体活性部分。然后以SDS-PAGE鉴定所得抗体的分子量及其纯度(10％聚丙烯酰胺凝胶,电极缓冲液pH8.3,上样量约50/μg,电流3.5mA/管,考马斯亮兰R-250染色)。研究结果层析洗脱液经紫外监测A_(280nm)得出三个吸收峰;由EIF作抗体活性鉴定,确证第3个峰为活性部分;SDS-PAGE中于分子量约75KD及30KD处显示两个单一色带,相当于该单抗的重链和轻链。结论本法纯化所得抗人食管癌单克隆抗体纯度良好。

The purification of anti-human esophagus carcinoma monoclonal antibody from mice ascites by hydroxylapatite column chromatography

Study Objective To pruify anti- human esophagus carcinoma monoclonal antibody from mice ascites.Interventions After the hydroxylapatite chromatography, the column fractions were assayed for antibody activity using an enzyme linked immunosorption filtering assay (EIF) , and the antibody purity was analyzed by SDS PAGE. The chromaiography conditions: the temperature at 15℃ ; begining buffer pH 6.8, 0.01 M PB; flow rate 60 ml / h; linear gradient buffer a 500 ml 0.01-0.5 M PB.Results Three peaks were resolved on the OD280nm profile by ultraviolete absorbtion spectrum of the column fractions. It was confirmed that the activity fractions were confined to the peak 3 by the EIF. There are two only bends at MW75KDand30KDonSDS PAGE.Conclusions It is successful in pruification of anti-human esophagus carcinoma monoclonal antibody from mice ascites.