NMM抗肿瘤DNA疫苗原液大肠杆菌菌体蛋白质残留量检测方法的建立与验证  

Establishment and verification of a method for detection of E.coli proteins residues in NMM antitumor DNA vaccine stock solution

作　　者：郭润姿 伊君梅 孙澳 田园 车亦伟 邱创钧 王宇 GUO Runzi;YI Junmei;SUN Ao;TIAN Yuan;CHE Yiwei;QIU Chuangjun;WANG Yu(Gu'an Dingtai Haigui Biotechnology Co.,Ltd.,Hebei,Langfang 065500,China)

机构地区：[1]固安鼎泰海规生物科技有限公司,河北廊坊065500

出　　处：《中国医药科学》2023年第9期85-89,共5页China Medicine And Pharmacy

基　　金：河北省重点研发计划项目(20372404D)。

摘　　要：目的建立并验证检测NMM抗肿瘤DNA疫苗原液中大肠杆菌菌体蛋白质残留量的双抗体夹心ELISA试验方法。方法使用双抗体夹心ELISA试验方法检测NMM抗肿瘤DNA疫苗原液中大肠杆菌菌体蛋白残留量,采用四参数logstic曲线进行回归分析,并对该方法的专属性、检测限、定量限、线性与范围、精密度、准确度、耐用性等进行验证,验证通过对5批样品进行检测。结果采用双抗体夹心ELISA试验方法进行大肠杆菌蛋白质残留量检测时,DNA疫苗原液无需进行稀释。NMM抗肿瘤DNA疫苗原液对大肠杆菌菌体蛋白质的检测无干扰及抑制作用,方法的专属性良好;本法检测限为0.41 ng/ml;定量限为0.98 ng/ml;该方法测定宿主菌蛋白质残留量在0~100 ng/ml范围内线性良好,R^(2)≥0.9999;本方法重复性试验中宿主菌蛋白质含量RSD值均低于15%,中间精密度实验中样品吸光值RSD值低于10%,精密度结果良好。在检测线性范围内加入高、中、低3种浓度的大肠杆菌菌体蛋白,回收率均值为100.35%(n=9,RSD=3.58%);本方法检测实验条件发生微小变动时(不同人员、不同批次试剂盒),对测定结果的影响在可接受范围内。应用该方法对5批NMM抗肿瘤DNA疫苗原液中大肠杆菌蛋白质残留量进行检测,大肠杆菌菌体蛋白残留量均在1 ng/mg左右,远低于相关指导原则中规定的不高于1μg/mg的质量标准。结论该方法可用于NMM抗肿瘤DNA疫苗原液中宿主菌蛋白质含量的检测。Objective To establish and validate a double-antibody sandwich enzyme-linked immunosorbent assay(ELISA)method for detecting Escherichia coli bacteriophage protein(E.coli proteins)residues in NMM antitumor DNA vaccine stock solution.Methods The E.coli proteins residues in NMM antitumor DNA vaccine stock solution was detected by using the double-antibody sandwich ELISA method,and a regression analysis was performed using the four-parameter Logistic curve.Besides,the specificity,limit of detection,limit of quantification,linearity and range,precision,accuracy and robustness of the method were validated by testing five batches of samples.Results No dilution of the DNA vaccine stock solution was required for the E.coli proteins residues detection with double-antibody sandwich ELISA method.The NMM antitumor DNA vaccine stock solution did not interfere with or inhibit the detection of E.coli proteins,suggesting a good specificity of the method.The limit of detection of the method was 0.41 ng/ml,and the limit of quantification was 0.98 ng/ml.The Escherichia coli protein(E.coli P)residues detected by the method showed a good linearity in the range of 0-100 ng/ml,with R^(2)≥0.9999.The relative standard deviation(RSD)values of E.coli P in repeatability tests were all lower than 15%,and the RSD values of absorbance of samples in intermediate precision tests were all lower than 10%,suggesting a good precision of the method.The mean recovery was 100.35%(n=9,RSD=3.58%)for three concentrations(high,medium,and low)of E.coli proteins in the linear range of detection.A slight change of the experimental conditions(different personnel,different batches of kits)of this method had minor effect on the detection result,which was acceptable.The E.coli proteins residues of five batches of NMM antitumor DNA vaccine stock solution detected by the method were all around 1 ng/mg,which was much lower than the quality standard of no more than 1μg/mg specified in relevant guidelines.Conclusion The method can be used for detecting E.coli P cont

关 键 词：抗肿瘤DNA疫苗 重组生物制品 双抗原夹心ELISA法 大肠杆菌菌体蛋白残留 质量控制 

分 类 号：R446.6[医药卫生—诊断学]