Autoantibodies are one important characteristic of autoimmune disease and can be used as diagnostic biomarkers. Protein arrays, containing thousands of full-length proteins, have successfully been used to identify antigens targeted by autoantibodies in human autoimmune disease but has not previously been used in dogs. Autoantibodies are often directed against proteins that are well conserved between species and we hypothesized that the human protein arrays could be used to identify novel autoantigens in dogs with autoimmune disease.

Sera from 21 Nova Scotia Duck Tolling Retrievers (NSDTRs), a dog breed predisposed to autoimmune disease, were included. Twelve of the dogs had clinical signs consistent with immune-mediated rheumatic disease (IMRD), an autoimmune disease characterized by chronic pain and stiffness from multiple joints, and nine were healthy controls. All the IMRD dogs were positive on the indirect immunofluorescence (IIF) antinuclear antibody (ANA) test, which is the standard test used to identify autoantibodies. Nine displayed a speckled ANA pattern on IIF and three a homogenous pattern.

Human proteins arrays (HuProt^TM v2 Human Proteome Arrays, Cambridge Protein Arrays, Cambridge, UK) were incubated with sera from the IMRD affected and healthy dogs, followed by incubation with Alexa Fluorâ 647 conjugated rabbit anti-dog IgG. Signal intensities, representing autoantibody levels, were measured in a microarray scanner. Potential autoantigens were identified by introducing a cut off based on the healthy controls (mean + 3SD, log-transformed data).

Interleukin enhancer binding factor 2 (ILF2) was identified as the most promising autoantigen. ILF2 is a transcription factor involved in regulation of T-cells, and is particularly important for expression of IL-2, a cytokine involved in immune response and immune regulation. ILF2 antibodies were present in seven of the twelve IMRD dogs. The seven dogs displayed a speckled staining pattern on IIF.

To validate the findings, presence of autoantibodies against ILF2 was investigated in a larger group of NSDTRs as well as in dogs of other breeds, using a radioligand binding assay. ILF2 autoantibodies were present in 27 out of 29 IMRD affected NSDTRs displaying a speckled ANA pattern, and in various dogs of other breeds with a speckled ANA pattern. ILF2 antibodies were not present in any sera from 141 control dogs.

This study describes the use of human protein arrays for autoantigen discovery in dogs, and also identifies autoantibodies against ILF2 as a promising biomarker candidate in diagnostics development for autoimmune disease in dogs.

Disclosures

Disclosures to report.

The study was founded by the Swedish Research Council FORMAS, the Swedish Kennel Club, and Agria Research Foundation. Hanna Bremer has been awarded a travel grant from Knut and Alice Wallenberg Foundation.