Immunoaffinity Chromatography for Protein Purification and Analysis
Immunoaffinity Chromatography for Protein Purification and Analysis
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DOI：
10.1002/cpz1.867
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发表时间：
2023-08-01
期刊：
影响因子：
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通讯作者：
Hage，David S.
中科院分区：
文献类型：
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作者：
Sharmeen，Sadia;Suh，Kyungah;Hage，David S.
免疫亲和层析(IAC)是一种使用固定化抗体或相关结合剂作为选择性固定相来分离或分析样品的液相色谱技术。抗体的强结合和高选择性使IAC成为包括蛋白质在内的许多化学品和生物化学品的提纯和分析的流行工具。IAC的基本原理被描述为与使用这种方法进行蛋白质纯化和分析有关。在讨论了在开发新的IAC方法时应遵循的一般策略的同时，还提出了在这项技术中要考虑的主要因素。如使用人血清白蛋白(HSA)作为模型蛋白所示，提供了几种形式的IAC的使用方案。这既包括将IAC与传统的低性能载体(如用于离线免疫提取的琼脂糖)一起使用，也包括将IAC用于高性能IAC用于在线免疫提取。还讨论了IAC作为流动免疫分析或层析免疫分析的使用，并以人血清白蛋白和竞争结合分析格式为例进行了描述。2023 Wiley期刊LLC基本方案1：传统免疫亲和层析离线免疫提取基本方案2：高效免疫亲和层析在线免疫提取基本方案3：竞争结合层析免疫分析
Immunoaffinity chromatography (IAC) is a type of liquid chromatography that uses immobilized antibodies or related binding agents as selective stationary phases for sample separation or analysis. The strong binding and high selectivity of antibodies have made IAC a popular tool for the purification and analysis of many chemicals and biochemicals, including proteins. The basic principles of IAC are described as related to the use of this method for protein purification and analysis. The main factors to consider in this technique are also presented under a discussion of the general strategy to follow during the development of a new IAC method. Protocols, as illustrated using human serum albumin (HSA) as a model protein, are provided for the use of IAC in several formats. This includes both the use of IAC with traditional low‐performance supports such as agarose for off‐line immunoextraction and supports used in high‐performance IAC for on‐line immunoextraction. The use of IAC for protein analysis as a flow‐based or chromatographic immunoassay is also discussed and described using HSA and a competitive binding assay format as an example. © 2023 Wiley Periodicals LLC.Basic Protocol 1: Off‐line immunoextraction by traditional immunoaffinity chromatographyBasic Protocol 2: On‐line immunoextraction by high‐performance immunoaffinity chromatographyBasic Protocol 3: Competitive binding chromatographic immunoassay