Abstract
A novel approach to recover and identify immune complexes (ICs) was developed using size exclusion chromatography (SEC) and affinity chromatography on immunoglobulin binding columns (HiTrap Protein G). The purification process was monitored by 1D SDS-PAGE, protein staining, Western blotting and, finally, liquid chromatography tandem mass spectrometry (LC MS/MS) was used to identify the recovered antigens. This approach was applied to serum with artificially created immune complexes (ICs) comprising vaccine antigen (influenza) and antibody, which led to recovery and identification of influenza peptides within the recovered ICs. This approach was compared with the established method for IC detection and recovery, polyethylene glycol (PEG) precipitation, followed by LC MS/MS. Both approaches successfully enabled capture, recovery and characterization of immunoglobulins and influenza antigen(s) in complex with the immunoglobulins. However, PEG precipitation has the advantage of simplicity and is more suited for large scale studies.
Highlights
Immune complexes (ICs) are formed when antibodies bind to antigens
SDS-PAGE followed by Western blotting for IgG and IgM detection showed that the HMW1 peak mainly contained IgM, and the HMW2 contained IgG
A pool of fractions from size exclusion chromatography (SEC) was generated that corresponded to the HMW1 and HMW2 peaks and each pool was applied to a HiTrap Protein G column (Fig 2)
Summary
Immune complexes (ICs) are formed when antibodies bind to antigens. Antigen(s) may be endogenous as occurs in autoimmune disorders or exogenous (i.e. drugs, toxins or infectious agents). The binding of ICs to FcγRs may lead to either activation or suppression of inflammatory cells depending on the type of FCGR gene expressed [2], and IC-induced cellular activation or suppression has been documented in many disease processes [3, 4]. ICs may precipitate in tissues, leading to local inflammation or organ damage, influx of inflammatory cells and complement activation [5]. Their importance is well established in renal diseases [6]; inflammatory disorders such as rheumatoid arthritis [7] and systemic lupus erythematosus; infectious diseases such as dengue, sclerosing panencephalitis, hepatitis and malignancies [5, 8]. Many studies report identification of ICs during disease processes, only a few have reported identification of antigens within ICs [9,10,11,12]
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