Development of a Protein Array-Based Autoantibody-Screening Panel for the Diagnosis and Clinical Follow-up of Patients with Autoimmune Polyendocrine Syndrome Type 1

Presentation Number: MON 297
Date of Presentation: April 3rd, 2017

Fabian Sardh*1, Sara Ström2, Ronald Sjöberg3, Daniel Eriksson4, Sophie Bensing5, Åsa Hallgren6, Silvia Garelli7, Jan Gustafsson8, Corrado Betterle9, Eystein Sverre Husebye10, Peter Nilsson3, Olle Kämpe5 and Nils Landegren5
1Karolinska institutet, Stockholm, SWEDEN, 2Karolinska Institutet, Stockholm, SWEDEN, 3KTH - Royal Institute of Technology, Stockholm, Sweden, 4Karolinska Institutet, Uppsala, 5Karolinska Institutet, Stockholm, Sweden, 6Karolinska Institutet, Stockholm, 7University of Padua, 8Department of Women's and Children's Health, Uppsala University, Sweden, Sweden, 9University of Padua, Padua, Italy, 10University of Bergen, Bergen, Norway

Abstract

Autoimmune polyendocrine syndrome type 1 (APS1) is a rare monogenic disorder caused by mutations in the AIRE gene, and it has been instrumental in the elucidation of mechanisms underlying central immune tolerance and for the understanding of autoimmune disease development. The disease is clinically defined by three hallmark components: chronic mucocutaneous candidiasis, hypoparathyroidism and adrenal failure, of which two are required for diagnosis. Many patients also develop additional autoimmune manifestations, and the consequent variation in the disease phenotype makes the diagnosis challenging. A number of autoantigens have been defined at a molecular level, many of which are assessed as part of the routine diagnosis of APS1. Since autoantibodies typically appear before the clinical manifestation, autoantibodies can also be useful for identifying patients at risk of developing specific disease components over time. Autoantibody screening can, however, be a time consuming process as current methods usually measure only one autoantibody at the time.

In this project, we aimed to develop an antigen array that could allow for the parallel assessment of multiple autoantibodies in APS1. Purified full-length human proteins were acquired from commercial sources covering a majority of the established antigens in APS1 (n>20) and a number of candidate antigens (n>20). Proteins were deposited on coated glass slides using an array printer. The arrays were then probed with sera from APS1-patients or healthy controls followed by a fluorescence-labeled anti-human IgG. Different parameters were optimized, including the type of slide coating, printing settings and serum concentration. Autoantibody results were compared to that of in-house radio-ligand binding assays and commercial proteome arrays (ProtoArray®). Our custom array showed excellent results for many known APS1 autoantigens, including interferon alpha, interferon omega, interleukin 22, interleukin 17F, AADC and GAD65, generating elevated autoantibody signals specifically for the patient sera. The results were reproducible and consistent with that of other, independent methods. There were several clinically important autoantigens that failed to generate autoantibody signals, including 21-hydroxylase, 17-hydroxylase, side-chain cleavage enzyme, tryptophan hydroxylase, NALP5 etc. These antigens will be replaced by recombinant protein from other sources in future versions of the array, to extend the panel of informative antigens.

Our preliminary investigations indicate that the antigen array can be used for measuring multiple autoantibodies simultaneously in APS1 with excellent quality and minimal serum consumption. This screening tool may facilitate in the diagnosis, prognosis and clinical follow-up of patients with APS1.

 

Nothing to Disclose: FS, SS, RS, DE, SB, ÅH, SG, JG, CB, ESH, PN, OK, NL