NMM抗肿瘤治疗性DNA疫苗中卡那霉素残留量检测方法的建立与验证

Establishment and Verification of the Residual Kanamycin in NMM anti-Tumor DNA Vaccine

建立并验证检测NMM抗肿瘤DNA疫苗中卡那霉素残留量的方法。采用间接竞争ELISA方法检测卡那霉素残留量,采用四参数Logstic曲线进行回归分析,并对本方法的专属性、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,验证方法有效后对多批次样品进行检测。使用该方法检测卡那霉素残留量时,样品无需稀释,辅料、质粒DNA对卡那霉素的检测无干扰;本方法检测限为0.15 ng/mL;定量限为0.5 ng/mL;在0.5~40.5 ng/mL范围内线性关系良好,且符合四参数方程式:y=D+(A-D)/[1+(x/C)^(B)],R^(2)≥0.999;在检测线性范围内加入不同浓度的卡那霉素对照溶液,回收率均值在95%~115%之间(n=12,RSD=6.20%);本方法重复性试验中卡那霉素含量RSD值为4.22%(n=6),中间精密度试验中DNA疫苗中卡那霉素含量RSD值为10.95%。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、不同酶标仪),对测定结果的影响在可接受范围内。应用该方法对7批样品中卡那霉素残留量进行检测,结果表明,每个人用剂量(0.3 mg DNA)卡那霉素的残留量均小于0.5 ng。本方法操作简便、准确可靠、专属性强,可用于NMM抗肿瘤DNA疫苗卡那霉素残留量的检定。

To establish and validate an enzyme-linked immunosorbent assay(ELISA)for detecting kanamycin residues in naked plasmid DNA vaccine(NMM),indirect competitive ELISA was used to detect kanamycin residues and four-parameter logstic curve was used for regression analysis.The specificity,detection limits,quantitative limits,linearity range,precision,accuracy and durability of the method were verified.After the validation,the samples of NMM anti-Tumor DNA vaccine were tested.When using this method to detect kanamycin residues,the DNA vaccine does not need to be diluted,the ingredients and DNA vaccine had no influences to detect the kanamycin,the specificity of the method was good.The detection limit of kanamycin was 0.15 ng/mL and the limit of quantification was 0.5 ng/mL.The detection range of kanamycin residues by this method was 0.5~40.5 ng/mL and it fit for the four-parameter equation:y=D+(A-D)/[1+(x/C)^(B)],R^(2)≥0.999.The average recovery rate of adding different concentrations kanamycin control solution to DNA vaccine solution ranged from 95% to 115%(n=12,RSD=6.20%),the RSD of the kanamycin content in the repeat tests was 4.22%,the RSD of inntermediate precision test of the DNA vaccine kanamycin was 10.95%,the precision of the method were good.When the test conditions were slightly change with different personnel,different batches of kits and different enzyme-labeled instruments,the results were all within an acceptable range.The method was applied to detect the residual kanamycin in 7 batches of DNA vaccine,the result displaied that the residual kanamycin were less than 0.5 ng per dose(0.3 mg DNA).The ELISA detection method is simple,accurate,reliable and specific.This method can be used for the determination of kanamycin residues in NMM anti-Tumor DNA vaccine.