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The 'reverse capture' autoantibody microarray:a native antigen-based platform for autoantibody profiling

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Abstract

We have previously reported the development and the use of a 'reverse capture' autoantibody microarray for studies of antigen-autoantibody profiling. We developed the 'reverse capture' autoantibody microarray to allow the user to characterize and to compare autoantibody profiles. Based on the dual-antibody sandwich immunoassay of ELISA, our 'reverse capture' protocol facilitates the detection of autoimmunity to native host antigens. Our method has the advantage over traditional protein arrays of being able to detect autoimmunity to epitopes found on the post-translational modifications (PTMs) of native antigens. The first step of this method is to immobilize native antigens onto the monoclonal antibodies spotted on the array surface. Using the antigens captured by the microarray as 'baits,' we then incubate the array with differentially labeled IgG from test and control samples, and perform a two-slide dye-swap to normalize for dye effects. In this protocol we present a detailed description of the 'reverse capture' autoantibody microarray, a method that can be completed in 9–10 h over 1–2 d.

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Figure 1: A detailed representation of our 'reverse capture' autoantibody microarray.
Figure 2: A schematic of the dye-swap method that we use with our 'reverse capture' autoantibody microarray.
Figure 3: Consistency of the capture of our prostate tumor antigen.
Figure 4: Reproducibility of autoantibody profiles using 'swapped' dyes.
Figure 5: Reproducibility of autoantibody profiles between experiments.

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Acknowledgements

This work was supported in part by grants U01DK063665 and R01DK066020 from the US National Institutes of Health to B.C.-S.L.

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Authors and Affiliations

  1. Division of Urology, The Molecular Urology Laboratory, Brigham and Women's Hospital, Harvard Medical School, 221 Longwood Ave., LMRC-610, Boston, 02115, Massachusetts, USA

    Joshua R Ehrlich, Shuzhen Qin & Brian C-S Liu

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  1. Joshua R Ehrlich
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  2. Shuzhen Qin
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  3. Brian C-S Liu
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Correspondence to Brian C-S Liu.

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A patent is currently pending for our 'reverse capture' autoantibody microarray platform and protocols.

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Ehrlich, J., Qin, S. & Liu, BS. The 'reverse capture' autoantibody microarray:a native antigen-based platform for autoantibody profiling. Nat Protoc 1, 452–460 (2006). https://doi.org/10.1038/nprot.2006.66

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