Department of Chemistry, University of Nebraska-Lincoln, Lincoln, Nebraska.
Curr Protoc. 2023 Aug;3(8):e867. doi: 10.1002/cpz1.867.
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 chromatography Basic Protocol 2: On-line immunoextraction by high-performance immunoaffinity chromatography Basic Protocol 3: Competitive binding chromatographic immunoassay.
免疫亲和色谱 (IAC) 是一种液相色谱，它使用固定化抗体或相关结合剂作为选择性固定相，用于样品分离或分析。抗体的强结合和高选择性使 IAC 成为许多化学物质和生物化学物质，包括蛋白质的纯化和分析的流行工具。描述了 IAC 的基本原理，涉及使用该方法进行蛋白质纯化和分析。在讨论开发新的 IAC 方法时遵循的一般策略下，还提出了该技术中要考虑的主要因素。提供了使用 IAC 的几种格式的方案，如图用人血清白蛋白 (HSA) 作为模型蛋白所示。这包括使用传统的低性能支持物（如琼脂糖）进行离线免疫萃取的 IAC 以及用于在线免疫萃取的高性能 IAC 中的支持物。还讨论并描述了 IAC 作为基于流动的或色谱免疫测定法用于蛋白质分析的情况，并用 HSA 和竞争性结合测定格式作为示例。© 2023 Wiley Periodicals LLC. 基本方案 1：传统免疫亲和色谱的离线免疫萃取 基本方案 2：高效免疫亲和色谱的在线免疫萃取 基本方案 3：竞争性结合色谱免疫测定。
