应用ELISA测定睫状神经营养因子中卡那霉素残留量

目的: 建立ELISA检测卡那霉素残留量的方法并进行方法学验证,用于重组技术产品的质量控制。 方法: 采用间接竞争ELISA方法,样本中残留的卡那霉素将和微孔板上预包被的偶联抗原竞争抗卡那霉素抗体,加入酶标二抗后,用3,3,5,5-四甲基联苯胺(TMB)底物显色,样本吸光值与残留卡那霉素的含量成负相关,用酶标仪进行检测并用SOFT MAX分析软件进行分析。 结果: ELISA方法测定卡那霉素残留量在0.5~40.5 ng·mL^-1范围内线性良好,且符合四参数方程式:y=(A-D) / +D,r²≥0.99;板内变异小于15%,板间变异小于20%,实验重复性好,加标回收率在80%~120%之间;该方法检测与庆大霉素、盐酸四环素、链霉素和氨苄西林药物未显示交叉反应。应用该法对1批睫状神经营养因子(CNTF)原液的卡那霉素残留量进行测定,结果表明,每人用剂量CNTF(100 μg)卡那霉素残留量小于0.5 ng。 结论: 该方法具有灵敏度高、操作简便、检测迅速等特点,可用于重组技术产品中卡那霉素残留量的检定。

Objective: To establish ELISA method for determination of residual kanamycin for the quality control of recombinant products. Method: Using indirect competitive ELISA,residual kanamycin in the test sample can compete with pre-coated antigen in the microplate for antibody specific for kanamycin.An enzyme-linked antibody specific for kanamycin was added to the wells,then a substrate solution was added to the wells and color developed in negative proportion to the amount of residual kanamycin.The results were read using microplate reader and analyzed using SOFT MAX software. Results: The ELISA method for residual kanamycin had good linearity (r²≥0.99) in the concentration range of 0.5-40.5 ng·mL^-1 and complied with the 4-parameter equation:y=(A-D) / +D.The relative standard devation for intraprecision studies was less than 15%,and for interprecision studies less than 20%.Mean recoveries ranged between 80% and 120%.This method did not have the cross-reactivity with gentamicin,tetracycline hydrochloride,streptomycin and ampicillin.The method was used for determination of residual kanamycin in one batch of CNTF bulk.The results indicated that the kanamycin content was less than 0.5 ng per dose (100 μg). Conclusion: The method is sensitive,convenient and quick,and can be used for determination of residual kanamycin in recombinant products.