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. 2017 May 26;7:2446. doi: 10.1038/s41598-017-02719-8

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Diagram of workflow for affinity chromatography column generation and validation for antigen identification. Column IgG capture following serum loading was assessed using serum protein electrophoresis (SPE). Capture and cross-linking efficiency of IgG antibodies was determined using rabbit IgG specific ELISA. Specificity of antigen capture and non-specific protein binding were assessed using silver stained gels and Western blots with post-immunization rabbit serum (D84), for protein run-through from columns eluted at pH 5, 4 and 2.9. Antigen identifications were made using LC-MS/MS analysis of specifically bound proteins eluted from D0 versus D84 columns.