The “Reverse Capture” Autoantibody Microarray:

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.