目的:建立报告基因法(reporter gene assay,RGA)检测重组人白细胞介素-1受体拮抗剂(IL-1Ra)生物学活性。方法:通过IL-β刺激D10G4.1-NFκB-Luc细胞产生荧光,IL-1Ra抑制此反应,且抑制的效果与其用量具有线性关系测定其生物学活性。对细胞密度、刺激性物质IL-1β的量、拮抗物质IL-1Ra的量、稀释梯度及孵育时间等进行摸索后建立方法,对方法的准确度、线性范围、专属性和耐用性进行考察;用建立的方法与通用法:A375.S2细胞活性法进行比对。结果:成功建立了RGA检测IL-1Ra生物学活性,该方法在效价水平范围50%~160%检测结果的相对偏差均在±12%范围内,回归方程的斜率为1.001 3,相关系数为0.999 8,对不同细胞代次、细胞密度及培养时间考察耐用性的几何变异(GCV)为4.0%。方法耗时较短,检测结果与A375.S2细胞活性法一致,且精密度及准确性都具有显著提高。结论:成功建立了报告基因法检测IL-1Ra生物学活性,并通过了方法验证,可替代A375.S2细胞活性法用于产品的质量控制。
Objective: To establish a reporter gene assay (RGA) for detecting the biological activity of recombinant human interleukin-1 receptor antagonists(IL-1Ra). Methods: D10G4.1-NFκB-Luc cells were stimulated by IL-β to produce fluorescence, the IL-1Ra inhibited this reaction, and the effect of inhibition had a linear relationship with its dosage to determine its biological activity. The method was established after exploring cell density, the amount of irritant IL-1β, the amount of antagonist IL-1Ra, dilution gradient and incubation time, and the accuracy, linear range, specificity and durability of the method were investigated. The established method was compared with the A375.S2 cell viability method. Results: The biological activity of IL-1Ra was successfully established by RGA, and the relative deviation of the detection results in the range of 50%-160% in the titer level range was within the range of ±12%, the slope of the regression equation was 1.001 3, the correlation coefficient was 0.9998, and the geometric variation (GCV) of durability for different cell generations, cell densities and culture time was 4.0%. The method took a short time, the detection results were consistent with the A375.S2 cell viability method, and the precision and accuracy were significantly improved. Conclusion: The reporter gene assay has been successfully established to detect the biological activity of IL-1Ra, and has been validated, which can replace the A375.S2 cell activity method for product quality control.
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