Development of a luminescent immunoprecipitation system (LIPS) for improved detection of autoantibodies to the tetraspanin-7 antigen in Type 1 diabetes

Aims:Autoantibodies to islet autoantigens are important for identification of individuals at risk of Type 1 diabetes and their selection for immune intervention. Tetraspanin-7 (Tspan7) has been identified as an autoantigen in Type 1 diabetes, equivalent to the autoantibody target previously described as “Glima”. However, detection of Tspan7 antibodies by luminescent immunoprecipitation systems (LIPS) has been hampered by low sensitivity and specificity, hindering studies to determine their value in disease prediction and diagnosis. The aim of this study was to optimise performance of Tspan7 LIPS for a better understanding of the role of Tspan7 autoimmunity in disease.

Methods:Constructs representing the coding region of human Tspan7 linked to Nano-, Firefly, Renilla or Turboluciferase were expressed in mammalian cells. Freshly-prepared or frozen cell extracts were incubated with sera from Type 1 diabetes patients or nondiabetic controls. Immune complexes were captured on protein A Sepharose and bound antigen detected by luminescence. “Glima” antibodies were detected by immunoprecipitation from 35S-methionine-labelled GT1.7 cells followed by gel electrophoresis and autoradiography.

Results:LIPS assays with Nanoluciferase-tagged Tspan7 showed high non-specific binding to antibodies in some control subjects. Replacement of the tag with Firefly, Renilla or Turboluciferase abolished this non-specific binding. Immunoreactivity of autoantibodies to Tspan7 was impaired by freeze-thaw that could be recovered by addition of 50% glycerol to extracts before freezer storage. Highest sensitivity of Tspan7 antibody detection was achieved by extracting antigen with protease inhibitors and incubating with sera in buffers designed to maintain high luciferase activity. Optimized assay conditions allowed detection of Tspan7 antibodies in Glima-antibody positive patients, with low antibody levels in healthy controls.

Conclusions:Tspan7 autoantibodies bind conformational epitopes highly sensitive to freeze-thaw and proteolysis. Precautions taken to preserve conformational epitopes are required to evaluate importance of Tspan7 antibodies in diabetes prediction and diagnosis.