Distinguishing persistent insulin autoantibodies with differential risk: nonradioactive bivalent proinsulin/insulin autoantibody assay

Abstract

A subset of children develops persistent insulin autoantibodies (IAA; almost always as the only islet autoantibody) without evidence of progression to diabetes. The aim of the current study was the development and characterization of the performance of a nonradioactive fluid phase IAA assay in relation to standard IAA radioassay. We developed a nonradioactive IAA assay where bivalent IAA cross-link two insulin moieties in a fluid phase. The serum samples positive for anti-islet autoantibodies from 150 newly diagnosed patients with diabetes (Barbara Davis Center plus Diabetes Autoantibody Standardization Program [DASP] workshop) and 70 prediabetic subjects who were followed to diabetes were studied. In addition, sequential samples from 64 nondiabetic subjects who were persistently IAA(+) were analyzed. With 99th percentile of specificity, the new assay with the technology from Meso Scale Discovery Company (MSD-IAA) detects as positive 61% (61 of 100) of new-onset patients and 80% (56 of 70) of prediabetic patients compared with our current fluid phase micro-IAA radioassay (mIAA; 44 and 74%, respectively). In addition, MSD-IAA demonstrated better sensitivity than our mIAA from blinded DASP workshop (68 vs. 56% with the same 99% specificity). Of 64 IAA(+) nondiabetic subjects, 25% (8 of 32) who had only IAA and thus the low risk for progression to diabetes were positive with MSD-IAA assay. In contrast, 100% (32 of 32) high-risk children (IAA plus other islet autoantibodies) were positive with MSD-IAA. The IAA detectable by radioassay, but not MSD-IAA, were usually of lower affinity compared with the IAA of the high-risk children. These data suggest that a subset of IAA with current radioassay (not MSD-IAA) represents biologic false positives in terms of autoimmunity leading to diabetes. We hypothesize that factors related to the mechanism of loss of tolerance leading to diabetes determine high affinity and MSD-IAA reactivity.

FIG. 1.

Illustration of the bivalent plate capture MSD-IAA assay. The IAA in serum will link the Sulfo-tagged proinsulin to the biotinylated proinsulin, which will be captured on the solid phase of the streptavidin-coated plate. Detection of plate-captured Sulfo-tagged proinsulin is accomplished with electrochemiluminescence.

FIG. 2.

Both Sulfo-TAG and biotin-labeled proinsulin (Proins) were used as competitors in our standard IAA radioassay. Sulfo-TAG–labeled proinsulin (in A) and biotin-labeled proinsulin (in B) competed for binding ¹²⁵I-insulin as well as unmodified human insulin (Humulin from Eli Lilly). cpm, counts per minute. (A high-quality color representation of this figure is available in the online issue.)

FIG. 3.

Illustration of normal human serum blocking MSD-IAA signal. The MSD-IAA radioassay signals with an insulin monoclonal antibody (MoAb; A) are decreased by addition of normal human serum, compared with equal volume of PBS with partial restoration of signal with acid treatment (acid treat) of human serum. B: Illustration of MSD-IAA assay signals of 4 patient sera with and without acid treatment. (A high-quality color representation of this figure is available in the online issue.)

FIG. 4.

MSD-IAA index of sera from 100 normal control subjects, 100 newly diagnosed patients with type 1 diabetes, and 70 prediabetic patients who were followed to overt diabetes. With cutoff value of index 0.007 set at 99th percentile of 100 healthy control samples, 65% (110 of 170) of the patients were positive. (A high-quality color representation of this figure is available in the online issue.)

FIG. 5.

IAA levels from MSD-IAA assay and our current radioassay (mIAA) were compared among 100 newly diagnosed patients with type 1 diabetes (A), 70 prediabetic patients (B), and 50 blinded DASP workshop patient samples (C). The two assays were correlated (P < 0.0001), but MSD-IAA assay had higher sensitivity for all 3 groups with both assays set at 99% specificity. (A high-quality color representation of this figure is available in the online issue.)

FIG. 6.

Sera from children in the DAISY study persistently expressing IAA as a single autoantibody or with other islet autoantibodies were analyzed with the MSD-IAA assay. A: MSD-IAA were well correlated with radioassay mIAA for all five subjects (multiple follow-up positive for both assays) who were multiple islet autoantibody-positive. B: Seven and eight subjects with single islet autoantibody (mIAA only) were consistently MSD-IAA⁻. DM, diabetic subjects. (A high-quality color representation of this figure is available in the online issue.)

FIG. 7.

Sera with IAA from 13 subjects in Fig. 6 with single or multiple islet autoantibodies were incubated with different concentrations of either proinsulin (A) or insulin (B) and analyzed with our standard mIAA radioassay. IAA from subjects with single autoantibody (dotted line), compared with subjects with multiple autoantibodies (solid line), required higher concentrations of proinsulin and insulin for one-half maximal inhibition. Results were expressed as percent of signal not absorbed. (A high-quality color representation of this figure is available in the online issue.)