A Novel In Vitro Assay Correlates Insulin Receptor Autoantibodies With Fasting Insulin in Type B Insulin Resistance

Abstract

Context: Severe insulin resistance (IR) in the presence of insulin receptor autoantibodies (InsR-aAb) is known as type B insulin resistance (TBIR). Considerable progress in therapy has been achieved, but diagnosis and monitoring of InsR-aAb remains a challenge.

Objective: This work aimed to establish a robust in vitro method for InsR-Ab quantification.

Methods: Longitudinal serum samples from patients with TBIR at the National Institutes of Health were collected. A bridge-assay for InsR-aAb detection was established using recombinant human insulin receptor as bait and detector. Monoclonal antibodies served as positive controls for validation.

Results: The novel assay proved sensitive, robust, and passed quality control. The measured InsR-aAb from TBIR patients was associated with disease severity, decreased on treatment, and inhibited insulin signaling in vitro. Titers of InsR-aAb correlated positively to fasting insulin in patients.

Conclusion: Quantification of InsR-aAb from serum samples via the novel in vitro assay enables identification of TBIR and monitoring of successful therapy.

Keywords: acanthosis nigricans; assays; hyperglycemia; hypoglycemia; in vitro diagnostics; uncontrolled diabetes.

Figure 1.

Establishment of a novel assay for the quantification of antibodies to the insulin receptor. A, Detection principle by a bridge format; the fusion protein of insulin receptor with luciferase (1) is used as detector, the antibodies (Abs) serve as a bridge (2) between detector and the catcher antigen (3) that is a tagged insulin receptor immobilized to the support via a precoated tag-specific monoclonal Ab (mAb) (4). B, Dose-response curve of luciferase signals (RLU; relative light units) in response to increasing concentrations of Abs (unrelated SELENBP1-mAb1 as negative control, vs 2 InsR-specific mAb as positive controls) under the standardized test conditions for InsR-aAb.

Figure 2.

Analysis of autoantibodies to the insulin receptor in patients with insulin resistance (IR). A, InsR-aAb concentrations of type B insulin resistance (TBIR) patients at serial visits mirror disease state (color coded: red; active/severe, orange; active/mild, blue; hypoglycemic, black; in remission). B, Patients with IR unrelated to TBIR (gray symbols) showed no InsR-aAb (binding index of 1.0 equals the average noise from negative controls), whereas the titers of InsR-aAb in patients with TBIR are elevated vs controls in all disease states. C, Within-patient comparison of InsR-aAb at different visits indicates declining titers with amelioration of symptoms. D, Concentration of InsR-aAb correlates positively with fasting insulin (Spearman correlation coefficient r = 0.825 and P < .001; antibody titers during visits in the hypoglycemic state were not included in this analysis). For Figs. B and C, antibody titer at only the first visit in any disease state is used for each individual.

Figure 3.

Biological activity of insulin receptor autoantibodies on insulin signaling in vitro. Human hepatic HepG2 cells were incubated with immunoglobulins (Igs) isolated from individual serum samples from InsR-aAb–negative individuals (control) or patients with type B insulin resistance (TBIR). Phosphorylation of the insulin receptor at Tyr1361 (P-Y1361-InsR) was determined by Western blot as a biomarker of insulin signaling. A, The degree of insulin receptor phosphorylation at Y1361 was not different in cells incubated with Ig from control or TBIR serum samples. B) In the presence of insulin (1 ng/mL), insulin receptor phosphorylation at Y1361 was stronger in cells incubated with Ig from control serum in comparison to cells incubated with Ig from InsR-aAb–positive serum samples from patients with TBIR. C, Quantification of the band intensities of P-Y1361-InsR (arrow) by ImageJ analysis supports this notion and highlights the extent of inhibition. The results indicate an antagonistic activity of InsR-aAb in TBIR on insulin-mediated receptor activation.