Affinity purification of serum-derived anti-IA-2 autoantibodies in type 1 diabetes using a novel MBP-IA-2 fusion protein

Thilini Mendis; Barbora Filipova; Jing Jing Wang; Massimo Pietropaolo; Michael W Jackson

doi:10.1016/j.bbrep.2022.101413

Affinity purification of serum-derived anti-IA-2 autoantibodies in type 1 diabetes using a novel MBP-IA-2 fusion protein

Biochem Biophys Rep. 2022 Dec 16:33:101413. doi: 10.1016/j.bbrep.2022.101413. eCollection 2023 Mar.

Authors

Thilini Mendis¹, Barbora Filipova¹, Jing Jing Wang¹, Massimo Pietropaolo², Michael W Jackson¹

Affiliations

¹ Department of Immunology, Allergy & Arthritis, Flinders Medical Centre and Flinders University, Bedford Park, 5042, South Australia, Australia.
² Dept of Pathology and Immunology, Baylor College of Medicine, Houston, TX, USA.

Abstract

Autoantibodies targeting epitopes contained within the intracellular domain (IC) of the protein phosphatase-like islet antigen 2 (IA-2) are a common marker of autoimmune type 1 diabetes (T1D), however the isolation of genuine, serum derived anti-IA-2 autoantibodies has proven challenging due to a lack of suitable bioassays. In the current study, an ELISA format was developed for affinity purification of human anti-IA-2ic autoantibodies utilizing a fusion protein (FP) incorporating maltose binding protein and the full-length IA-2IC domain. Using a T1D patient cohort validated for anti-IA-2ic autoantibodies by commercial ELISA, we demonstrate the MBP-IA-2ic FP ELISA detects serum anti-IA-2IC autoantibodies from 3 of 9 IA-2 positive patients. Further to this, a multi-plate MBP-IA-2ic FP ELISA protocol specifically affinity purifies IgG enriched for anti-IA-2ic autoantibodies. Interestingly, serum derived autoantibodies immobilised on the MBP-IA-2ic FP ELISA demonstrate increased Kappa light chain usage when compared to the respective total IgG derived from donor patients, suggesting a clonally restricted repertoire of anti-IA-2ic autoantigen specific B plasma cells is responsible for autoantibodies detect by the MBP-IA-2ic FP ELISA. This study is the first to demonstrate the generation of specific, genuine human derived anti-IA-2ic autoantibodies, thereby facilitating further investigation into the origin and functional significance of IA-2 autoantibodies in T1D.

Keywords: Affinity purification; Autoantibodies; Clonal restriction; FP, fusion protein; GAD65, glutamic acid decarboxylase 65; IA-2; IA-2, islet antigen 2; IC, intracellular; OD, optical density; T1D, Type 1 Diabetes; Type 1 diabetes; β, beta cell.