Discovery of Native Autoantigens via Antigen Surrogate Technology: Application to Type 1 Diabetes

Abstract

A fundamental goal in understanding the mechanisms of autoimmune disease is the characterization of autoantigens that are targeted by autoreactive antibodies and T cells. Unfortunately, the identification of autoantigens is a difficult problem. We have begun to explore a novel route to the discovery of autoantibody/autoantigen pairs that involves comparative screening of combinatorial libraries of unnatural, synthetic molecules for compounds that bind antibodies present at much higher levels in the serum of individuals with a given autoimmune disease than in the serum of control individuals. We have shown that this approach can yield “antigen surrogates” capable of capturing disease-specific autoantibodies from serum. In this report, we demonstrate that the synthetic antigen surrogates can be used to affinity purify the autoantibodies from serum and that these antibodies can then be used to identify their cognate autoantigen in an appropriate tissue lysate. Specifically, we report the discovery of a peptoid able to bind autoantibodies present in about one-third of nonobese diabetic (NOD) mice. The peptoid-binding autoantibodies were highly enriched through peptoid affinity chromatography and employed to probe mouse pancreatic and brain lysates. This resulted in identification of murine GAD65 as the native autoantigen. GAD65 is a known humoral autoantigen in human type 1 diabetes mellitus (T1DM), but its existence in mice had been controversial. This study demonstrates the potential of this chemical approach for the unbiased identification of autoantigen/autoantibody complexes.

Serology in the 21st century: the molecular-level analysis of the serum antibody repertoire

“…In an alternative approach very high density printed arrays of synthetic random peptide arrays were used for immunosignature discovery and were reported to exhibit higher accuracy than tiled peptide epitope arrays [37,38]. Since peptides may not be able to capture antibodies that bind to post-translational modifications and conformational epitopes Kodadek and coworkers pioneered the use of libraries of unnatural molecules and specifically peptoids (N-substituted oligoglycine polymers) for the discovery of surrogate antigen biomarkers from patient sera [29,39]. Other powerful methods that are currently used for antigen discovery include antigen microarrays [40,41], nucleic acid programmable protein arrays (NAPPAs) produced using in-vitro transcription/translation[42], peptide arrays [43] or finally arrays based on a different targets such as DNA, peptides and recombinant/native proteins [44].…”

Reliable diagnosis of murine type 1 diabetes using a panel of autoantigens and “antigen surrogates” mounted onto a liquid array

“…In a recent study, we identified GAD65 as a humoral autoantigen in the NOD mouse 10 and demonstrated that a GST-GAD65 fusion protein can be immobilized on glutathione-coated TentaGel beads without obstructing epitopes in GAD65 critical for autoantibody binding. We also have reported the isolation of a different antigen surrogate, compound 3 (Fig.…”

High-throughput Identification of DNA-Encoded IgG Ligands that Distinguish Active and Latent Mycobacterium tuberculosis Infections

“…Notably, however, the immobilized antigen identifies an entirely different population of ATB patients; neither discovery nor test ATB patient sera that were positive for 2-B binding respond positively in the immobilized Ag85B ELISA. We have observed previously that antigen immobilization on an ELISA plate can occlude the epitope that the small-molecule is mimicking 14 . This does not rule out Ag85B as a diagnostic antigen or viable target for mimicry as a surrogate.…”