Use of nitrocellulose films for affinity-directed mass spectrometry for the analysis of antibody/antigen interactions.
Combination of affinity extraction procedures with mass spectrometric analyses is termed affinity-directed mass spectrometry, a technique that has gained broad interest in immunology and is extended here with several improvements from methods used in previous studies. A monoclonal antibody was immobilized on a nitrocellulose (NC) membrane, allowing the corresponding antigen to be selectively captured from a complex solution for analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). This method was also used to rapidly determine the approximate binding region responsible for the antibody/antigen interaction. The tryptic fragments of antigen protein in buffer were applied to the antibody immobilized on NC film and allowed to interact. The NC film was then washed to remove salts and other unbound components, and subjected to analysis by MALDI-TOFMS. Using interferon-alpha(2a) and anti-interferon-alpha(2a) monoclonal antibody IgG as a model system, we successfully extracted the antigen protein and determined the approximate binding region for the antigen/antibody interaction (i.e., the tryptic fragment responsible).
Sun
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Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China.
Mo
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W
Ji
Y
Y
Liu
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S
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry; D000911:Antibodies, Monoclonal / Q000737:chemistry; D000937:Antigen-Antibody Reactions; D000941:Antigens / Q000737:chemistry; D001665:Binding Sites; D003101:Collodion; D006801:Humans; D006868:Hydrolysis; D007074:Immunoglobulin G / Q000737:chemistry; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D008567:Membranes, Artificial; D051379:Mice; D008969:Molecular Sequence Data; D011994:Recombinant Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin
D000906:Antibodies; D000911:Antibodies, Monoclonal; D000941:Antigens; D007074:Immunoglobulin G; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D008567:Membranes, Artificial; D011994:Recombinant Proteins; D003101:Collodion; D014357:Trypsin