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

Joshua R Ehrlich1, Shuzhen Qin, Brian C-S Liu

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

Nature Protocols
|April 5, 2007
PubMed
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This study details a novel reverse capture autoantibody microarray for profiling autoantibodies against native antigens. This method efficiently detects autoimmunity, including to post-translational modifications, in under two days.

Area of Science:

  • Immunology
  • Biotechnology
  • Analytical Chemistry

Background:

  • Autoantibody profiling is crucial for understanding autoimmune diseases.
  • Existing protein arrays have limitations in detecting autoantibodies against native antigens and post-translational modifications (PTMs).

Purpose of the Study:

  • To describe a detailed protocol for the 'reverse capture' autoantibody microarray.
  • To enable characterization and comparison of autoantibody profiles against native antigens.

Main Methods:

  • Development of a 'reverse capture' autoantibody microarray based on ELISA principles.
  • Immobilization of native antigens onto monoclonal antibodies on an array surface.
  • Incubation with labeled IgG from test/control samples and a two-slide dye-swap for normalization.

Related Experiment Videos

Main Results:

  • The 'reverse capture' method allows detection of autoimmunity to native antigens.
  • This technique can identify autoantibodies targeting epitopes on PTMs of native antigens.
  • The protocol is efficient, completable within 9-10 hours over 1-2 days.

Conclusions:

  • The 'reverse capture' autoantibody microarray is a valuable tool for comprehensive autoantibody profiling.
  • This method offers advantages over traditional arrays for detecting autoimmunity to native antigens and their modifications.
  • The described protocol provides a robust and efficient approach for immunological studies.