Immunoaffinity Chromatography for Protein Purification and Analysis

Abstract

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

Comparison of different extraction methods of active ingredients of Chinese medicine and natural products

“…"ISE" has obvious biological recognition effects by combining antigen and antibody selectively. Recently, "MIPE" [114], "LCE" [115], and "SPE" [116] have been reported to recognize biological macromolecules so they can all have clear biological roles. "ISE" and "LCE" contain each other, for example, immunoaffinity chromatography is a cross-cutting method that combines immunological and chromatographic techniques [117].…”