用ELISA法检测血清中抗TNF-α单克隆抗体药物浓度的方法开发与验证
Method development and validation for testing the concentration of anti-TNF-α monoclonal antibody in serum based on ELISA
胡振湘 1何丽秀 1王博 1陈曦 1刘桂丽 1秦玉敏1
作者信息
- 1. 珠海市丽珠单抗生物技术有限公司,广东珠海 519090
- 折叠
摘要
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.
Abstract
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.
关键词
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Key words
tumor necrosis factor-α/enzyme linked immunosorbent assay/monoclonal antibody/serum/drug concentration引用本文复制引用
出版年
2024
国家科技期刊平台
NSTL
万方数据