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目的 应用本组制备的白细胞介素 2受体 (IL- 2 R) ,建立一种既安全又简便 ,并能定量检测白细胞介素2 (IL- 2 )生物活性的方法。方法 采用 Southern印迹杂交方法 ,检测重组分泌型 IL- 2 Rα亚基 (rs IL- 2 Rα)基因在高效表达的转染细胞基因组中的稳定整合 ;以 Western免疫印迹法测定 rs IL- 2 Rα的相对分子质量 ,建立定量检测人 IL- 2生物活性的受体与抗体夹心酶联免疫吸附测定 (r EL ISA)方法。结果 (1) Southern印迹分析表明 ,rs IL- 2 Rα基因已稳定整合到高表达的转染 CHO细胞基因组中 ;(2 ) rs IL- 2 Rα的相对分子质量为 40 0 0 0左右 ;(3)应用建立的r EL ISA方法测定人 IL- 2标准溶液结果表明 ,IL- 2的标准曲线范围为 31.2 5～ 5 0 0 .0 0 U (r=0 .9995 ) ;预先将山羊抗人 IL- 2 Ig G与 IL- 2保温后 ,所得标准曲线的斜率明显降低 (P<0 .0 5 )。结论 与传统测定 IL- 2的方法相比 ,用rs IL- 2 Rα建立的受体 -抗体夹心 r EL ISA方法不仅具有准确、特异性强及操作简便的特点 ,而且 ,所得结果直接反映了被测样品中具有结合 IL- 2 R活性的 IL- 2的水平
Objective To establish a safe and simple method for the quantitative determination of the biological activity of interleukin-2 (IL-2) using the interleukin-2 receptor (IL-2R) prepared in this study. Methods Southern blot hybridization was used to detect the stable integration of recombinant IL-2 Rα subunit (rs IL-2 Rα) in the highly expressed transfected cells. The level of rs IL-2 Rα Relative molecular mass, a method for quantitatively detecting the biological activity of human IL-2 by sandwich enzyme-linked immunosorbent assay (rELISA) was established. Results (1) Southern blot analysis showed that the rs IL-2 Rα gene was stably integrated into the highly expressed CHO cell genome; (2) the relative molecular mass of rs IL-2 Rα was about 40 000; (3) ) The results of the r EL ISA assay established by human IL-2 standard solution showed that the standard curve of IL-2 ranged from 31.2 to 5.000 U (r = 0.9995). The goat anti-human IL - 2 Ig G After incubation with IL-2, the slope of the resulting standard curve was significantly reduced (P <0.05). Conclusion Compared with the traditional method of measuring IL-2, the receptor-antibody sandwich ELISA ELISA method established by rs IL-2 Rα is not only accurate, specific and easy to operate, but also the results directly reflect the The level of IL-2 binding to IL-2R activity is measured in the sample