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A cell-based assay for the detection of pathogenic anti-voltage-gated calcium channel autoantibodies in immunoglobulin G from patients with type 1 diabetes.

Journal of immunological methods (2018-06-26)
Ahmed M Alshehri, Thilini Mendis, Michael W Jackson
ABSTRACT

Recent studies have postulated the presence of functional autoantibodies (Abs) against L-type voltage gated calcium channels (VGCCs) in the serum of patients with type 1 diabetes, with various proposed physiological consequences, both islet cell associated and extra-glandular. Arguably, the most potentially damaging effect reported for these Abs is induction of apoptosis in pancreatic beta (β) cells, yet a convincing pathogenic mechanism remains to be demonstrated. In the current study, we report an assay of reactive oxygen species (ROS) stress induction in the rat insulinoma cell line Rin A12, as determined by 2', 7'-Dichlorofluorescein diacetate (DCF-DA) fluorescence detection by flow cytometry. We demonstrate that incubation of Rin A12 cells with immunoglobulin G (IgG) containing anti-VGCC activity from patients with T1D mediates a significant increase in ROS, with subsequent induction of apoptosis, as determined by positivity for annexin V expression. Neither T1D patient-derived IgG lacking anti-VGCC activity or IgG from healthy donors altered ROS or annexin V expression, indicating the new assay is specific for the detection of functional anti-VGCC Abs. Subsequent screening of IgG samples derived from individual patients indicated a prevalence of approximately 75% in a cohort of 20 patients with T1D. The new cell-based assay provides, for the first time, experimental evidence supporting a plausible pathophysiological mechanism underlying anti-VGCC Ab-mediated apoptosis induction in β cells. Additionally, the assay is a considerable advance on previously published methods for detecting and characterising the functional activity of anti-VGCC Abs in patient-derived samples.

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Hanks′ Balanced Salt solution, Modified, with sodium bicarbonate, without phenol red, calcium chloride and magnesium sulfate, liquid, sterile-filtered, suitable for cell culture