Developing Custom Chinese Hamster Ovary-host Cell Protein Assays using Acoustic Membrane Microparticle Technology
Developing Custom Chinese Hamster Ovary-host Cell Protein Assays using Acoustic Membrane Microparticle Technology
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DOI：
10.3791/2493
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发表时间：
2011-02-01
影响因子：
1.2
通讯作者：
Madison, Lara
中科院分区：
文献类型：
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作者：
Dickerson, Matthew;Leong, Kristen;Madison, Lara
由于在不同平台上开发的复杂性，对独特蛋白质的定制分析通常仅限于耗时的手动检测和定量技术，如ELISA或Western blots。BioScale专有的声膜微粒(AMMP)技术使夹心免疫分析可以轻松开发用于VIBE平台，从而提供更好的灵敏度、重复性和自动化操作。作为一个例子，本方案概述了建立一种定制的中国仓鼠卵巢宿主细胞蛋白(CHO-HCP)分析的程序。这里概述的一般原理可用于开发各种免疫分析方法。AMMP分析通过测量微粒与传感器表面结合而引起的振荡频率的变化来计算抗原浓度。它由四个主要部分组成：(1)含有功能化八传感器芯片的盒(2)标记磁性微粒的抗体，(3)结合到功能化芯片表面的半抗原标记抗体(4)含有目的抗原的样品。BioScale的生物传感器是一种共振装置，它包含八个单独的膜，具有不同的流体路径。膜随着表面质量的积累而改变振荡频率，这种频率变化被用来定量增加的质量的量。为了便于在各种免疫分析中使用，传感器被抗半抗原抗体功能化。通过半抗原标签与一种抗体和生物素与另一种抗体的共价结合来修饰检测特异性抗体。生物素标记被用来将抗体结合到链霉亲和素偶联磁珠上，这些磁珠与半抗原标记的抗体结合在一起，用于捕获三明治中的分析物。该复合体通过抗半抗原/半抗原相互作用与芯片结合。在每次分析运行结束时，传感器用稀酸清洗，从而能够对96孔板上的柱子进行顺序分析。这里，我们介绍了一种用于生物过程开发的定制CHO-HCP AMMP分析方法。开发AMMP分析方法或将现有的分析方法修改为AMMP分析方法可以在复杂样品中提供更好的性能(重复性、灵敏度)，并减少操作人员的时间。该协议显示了开发步骤，讨论部分回顾了具有代表性的结果。有关测试优化和定制参数的更深入解释，请联系BioScale。该试剂盒提供通用生物过程开发分析，如残留蛋白A、产品效价和CHO-HCP。
Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to the complexity of development on alternate platforms. BioScale's proprietary Acoustic Membrane MicroParticle (AMMP) technology allows sandwich immunoassays to be easily developed for use on the ViBE platform, providing better sensitivity, reproducibility, and automated operation. Provided as an example, this protocol outlines the procedure for developing a custom Chinese Hamster Ovary-Host Cell Protein (CHO-HCP) assay. The general principles outlined here can be followed for the development of a wide variety of immunoassays.An AMMP assay measures antigen concentration by measuring changes in oscillation frequency caused by the binding of microparticles to the sensor surface to calculate. It consists of four major components: (1) a cartridge that contains a functionalized eight sensor chip (2) antibody labeled magnetic microparticles, (3) hapten tagged antibody that binds to the surface of the functionalized chip (4) samples containing the antigen of interest. BioScale's biosensor is a resonant device that contains eight individual membranes with separate fluidic paths. The membranes change oscillation frequency in response to mass accumulating on the surface and this frequency change is used to quantitate the amount of added mass.To facilitate use in a wide variety of immunoassays the sensor is functionalized with an anti-hapten antibody. Assay specific antibodies are modified through the covalent conjugation of a hapten tag to one antibody and biotin to the other. The biotin label is used to bind the antibody to streptavidin coupled magnetic beads which, in combination with the hapten-tagged antibody, are used to capture the analyte in a sandwich. The complex binds to the chip through the anti-hapten/hapten interaction. At the end of each assay run the sensors are cleaned with a dilute acid enabling the sequential analysis of columns from a 96-well plate.Here, we present the method for developing a custom CHO-HCP AMMP assay for bioprocess development. Developing AMMP assays or modifying existing assays into AMMP assays can provide better performance (reproducibility, sensitivity) in complex samples and reduced operator time. The protocol shows the steps for development and the discussion section reviews representative results. For a more in-depth explanation of assay optimization and customization parameters contact BioScale. This kit offers generic bioprocess development assays such as Residual Protein A, Product titer, and CHO-HCP.