均相时间分辨荧光(HTRF)法测定重组单抗含量

中国生物工程杂志

2013, Vol. 33

Issue (12): 64-68

技术与方法

均相时间分辨荧光(HTRF)法测定重组单抗含量

邓春平, 秦玉敏, 邓巧春, 王博, 彭育才

珠海市丽珠单抗生物技术有限公司珠海 519020

Establishment of Homogeneous Time-resolved Fluorescence (HTRF) Method for Measurement of Monoclonal Antibody Concentration

DENG Chun-pin, QIN Yu-min, DENG Qiao-chun, WANG Bo, PENG Yu-cai

Livzon Mabpharma Inc, Zhuhai 519020, China

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摘要：

建立了一种HTRF检测方法，可以快速、简便、稳定地测定重组单克隆抗体的含量。将检测试剂、标准品及待测样品加入96孔板，孵育后即可在酶标仪上读取荧光信号值，用软件对信号值进行四参数曲线拟合，绘制标准曲线，根据标准品四参数方程计算待测样品中抗体的含量；同时对方法进行了验证。结果表明：该方法专属性强，抗体含量测定范围为25～1000 ng/ml，线性相关系数在0.99以上，回收率范围为92.8%～103.6%，平均为98.9%；该法精密度较好，精密度的变异系数范围为0.2%～11.6%，平均为4.3%。适用于生物制药工艺开发过程中细胞培养上清、纯化产物及制剂中抗体含量的测定。

关键词： 均相时间分辨荧光; 荧光共振能量转移; 单克隆抗体

Abstract:

A rapid and simple antibody analysis method based on HTRF technology was established to measure concentration of recombinant monoclonal antibody in both cell culture medium and purified antibody solution. APC-labeled antigen,antibody samples or controls, and Eu-labeled anti-human IgG are added into 96 well plate (Black), incubate for some time, followed by fluorescence reading with microplate reader. Standard curve was generated by 4-PL model, antibody concentration in tested samples was calculated accordingly by software. The method qualification results showed that the method is antigen-antibody specific, with accuracy close to 98.9%, precision close to 4.3%, and linearity range 25～1000 ng/ml. This method can be used for cell line screening, bio-process monitoring, and quality control during antibody drug development.

Key words: Homogeneous Time-resolved Fluorescence FRET Monoclonal antibody

收稿日期: 2013-08-15 出版日期: 2013-12-25

ZTFLH:

Q819

通讯作者: 彭育才，E-mail：pengyucai@livzon.com.cn E-mail: pengyucai@livzon.com.cn

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引用本文:

邓春平, 秦玉敏, 邓巧春, 王博, 彭育才. 均相时间分辨荧光(HTRF)法测定重组单抗含量[J]. 中国生物工程杂志, 2013, 33(12): 64-68.

DENG Chun-pin, QIN Yu-min, DENG Qiao-chun, WANG Bo, PENG Yu-cai. Establishment of Homogeneous Time-resolved Fluorescence (HTRF) Method for Measurement of Monoclonal Antibody Concentration. China Biotechnology, 2013, 33(12): 64-68.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2013/V33/I12/64

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