目的 建立检测一种测定血清中抗肿瘤坏死因子α(TNF-α)单克隆抗体浓度的间接酶联免疫吸附测定(ELISA)方法.方法 对TNF-α包被浓度及酶标抗体种类、存放稳定性和浓度等进行考察,确定关键试验参数.考察优化后方法的专属性、精密度与回收率、标准曲线与定量下限.结果 TNF-α包被浓度为400 ng·mL-1,选择Sigma辣根过氧化物酶标记羊抗人免疫球蛋白G,以1∶3.0×105倍进行稀释;稀释后的酶标抗体4℃保存3 d稳定性良好.同时确认了方法专属性良好,回收率为84.00％～106.82％,不同浓度样品精密度的变异系数均≤10％,方法线性良好,曲线方程为y=(-8.37 × 103-2.37 × 106)/[1+(x/29.80)106]+2.37 ×106(R2=0.999),定量下限:1 ng·mL-1,均符合要求.结论 本研究建立了一种准确、可靠的ELISA法测定血清中抗TNF-α单克隆抗体药物的浓度.
Objective To establish an indirect enzyme-linked immunosorbent assay(ELISA)method for testing the concentration of a monoclonal antibody target tumor necrosis factor-α(TNF-α)in animal serum.Methods The critical parameters of the method including coating concentration of human TNF-α,source,concentration and stability of HRP-labeled goat anti-human immunoglobulin G(IgG)were investigated.The specificity,accuracy,precision,linearity and Limited of Determination of the method were investigated.Results The critical parameters of the method were confirmed as below:TNF-α was coated at 400 ng·mL-1;HRP labeled goat anti-human IgG antibody was diluted at 1:3.0 ×105;the diluted horseradish peroxidase labeled goat anti-human IgG antibody is well stored at 4 ℃ for 3 days.Meanwhile the method was confirmed to have good specificity,the recovery rate ranged from 84.00％to 106.82％,the coefficient of variation of different antibody concentration levels were no more than 10％;the method had a good linearity and the standard curve was y=(-8.37×103-2.37 × 106)/[1+(x/29.80)106]+2.37 × 106(R2=0.999);the limit of quantification was 1 ng·mL-1,all of which met the requirements.Conclusion A accurate and robust ELISA method was developed to test the concentration of anti-TNF-α monoclonal antibody in serum.
胡振湘;何丽秀;王博;陈曦;刘桂丽;秦玉敏
珠海市丽珠单抗生物技术有限公司,广东珠海 519090
药学
肿瘤坏死因子α;酶联免疫吸附测定法;单克隆抗体;血清;药物浓度
tumor necrosis factor-α;enzyme linked immunosorbent assay;monoclonal antibody;serum;drug concentration
《中国临床药理学杂志》 2024 (011)
1642-1645 / 4
10.13699/j.cnki.1001-6821.2024.11.021
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