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Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography
Published on: March 9, 2010
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A proteomics-based method for identifying antigens within immune complexes.
Stephanie Menikou1, Andrew J McArdle1, Ming-Shi Li1
1Department of Infectious Disease, Section of Paediatric Infectious Disease, Imperial College London, London, United Kingdom.
Plos One
|December 28, 2020
Summary
A new method using chromatography effectively recovers and identifies immune complexes (ICs) with influenza antigens. While effective, polyethylene glycol (PEG) precipitation remains simpler for large-scale immune complex studies.
Area of Science:
- Immunology
- Analytical Chemistry
- Biochemistry
Background:
- Immune complexes (ICs) play a crucial role in immune responses and various diseases.
- Accurate recovery and identification of antigens within ICs are vital for diagnostic and research purposes.
- Current methods for IC detection and recovery have limitations in sensitivity and specificity.
Purpose of the Study:
- To develop and validate a novel chromatographic approach for the recovery and identification of immune complexes.
- To compare the performance of the novel method with the established polyethylene glycol (PEG) precipitation technique.
- To assess the capability of identifying specific antigens, such as influenza, within recovered ICs.
Main Methods:
- Immune complexes were created artificially using influenza antigen and antibodies in serum.
- Size exclusion chromatography (SEC) and affinity chromatography (HiTrap Protein G) were employed for IC purification.
- Purification was monitored using SDS-PAGE, protein staining, and Western blotting, with final antigen identification by liquid chromatography tandem mass spectrometry (LC-MS/MS).
Main Results:
- The novel chromatographic method successfully recovered and identified influenza peptides within artificially generated ICs.
- Both the novel method and PEG precipitation enabled the capture, recovery, and characterization of immunoglobulins and influenza antigens.
- LC-MS/MS confirmed the presence of influenza peptides in the recovered ICs using both techniques.
Conclusions:
- The developed SEC and affinity chromatography approach is effective for recovering and identifying antigens in immune complexes.
- While the novel method offers detailed characterization, PEG precipitation is advantageous for its simplicity and suitability for large-scale studies.
- Both methods demonstrate the ability to capture and characterize ICs, providing valuable insights into antigen-antibody interactions.
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