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  • The "reverse capture" autoantibody microarray: An innovative approach to profiling the autoantibody response to tissue-derived native antigens

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    Author(s)
    Ehrlich, Joshua R.
    Tang, Liangdan
    Caiazzo Jr., Robert J.
    Cramer, Daniel W.
    Ng, Shu-Kay
    Ng, Shu-Wing
    Liu, Brian C. S.
    Griffith University Author(s)
    Ng, Shu Kay Angus
    Year published
    2008
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    Abstract
    Recently, we reported the development and use of a "reverse capture" antibody microarray for the purpose of investigating antigen-autoantibody profiling. This platform was developed to allow researchers to characterize and compare the autoantibody profiles of normal and diseased patients. Our "reverse capture" protocol is based on the dual-antibody sandwich immunoassay of enzyme-linked immunosorbent assay (ELISA), and we have previously reported its use to detect autoimmunity to epitopes found on native antigens derived from tumor cell lines. In this protocol, we used ovarian cancer as a model system to adapt the "reverse ...
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    Recently, we reported the development and use of a "reverse capture" antibody microarray for the purpose of investigating antigen-autoantibody profiling. This platform was developed to allow researchers to characterize and compare the autoantibody profiles of normal and diseased patients. Our "reverse capture" protocol is based on the dual-antibody sandwich immunoassay of enzyme-linked immunosorbent assay (ELISA), and we have previously reported its use to detect autoimmunity to epitopes found on native antigens derived from tumor cell lines. In this protocol, we used ovarian cancer as a model system to adapt the "reverse capture" procedure for use with native antigens derived from frozen tissue samples. The use of this platform in studies of autoimmunity is valuable because it allows for the detection of autoantibody reactivity with epitopes found on the post-translational modifications (PTMs) of native antigens, a feature not present with other protein array platforms. In the first step in the "reverse capture" process, tissue-derived native antigens are immobilized onto the 500 monoclonal antibodies that are spotted in duplicate on the array surface. Using the captured antigens as "baits," we then incubate the array with labeled IgG from test and control samples, and perform a two-slide dye-swap to account for any dye effects. Here, we present a detailed description of the "reverse capture" autoantibody microarray for use with tissue-derived native antigens.
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    Book Title
    Tissue Proteomics: Pathways, Biomarkers, and Drug Discovery
    Publisher URI
    http://www.humanapress.com
    DOI
    https://doi.org/10.1007/978-1-60327-047-2_12
    Copyright Statement
    © 2008 Springer. The attached file is reproduced here in accordance with the copyright policy of the publisher. It is the author-manuscript version of the paper. Please refer to the publisher's website for further information.
    Subject
    Other Chemical Sciences
    Biochemistry and Cell Biology
    Publication URI
    http://hdl.handle.net/10072/32452
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