Electrochemiluminescence assays for insulin and glutamic acid decarboxylase autoantibodies improve prediction of type 1 diabetes risk

Abstract

We recently developed new electrochemiluminescence (ECL) insulin autoantibody (IAA) and glutamic acid decarboxylase 65 autoantibody (GADA) assays that discriminate high-affinity, high-risk diabetes-specific autoantibodies from low-affinity, low-risk islet autoantibodies (iAbs) detected by radioassay (RAD). Here, we report a further validation of the ECL-IAA and -GADA assays in 3,484 TrialNet study participants. The ECL assay and RAD were congruent in those with prediabetes and in subjects with multiple autoantibodies, but only 24% (P<0.0001) of single RAD-IAA-positive and 46% (P<0.0001) of single RAD-GADA-positive were confirmed by the ECL-IAA and -GADA assays, respectively. During a follow-up (mean, 2.4 years), 51% of RAD-IAA-positive and 63% of RAD-GADA-positive subjects not confirmed by ECL became iAb negative, compared with only 17% of RAD-IAA-positive (P<0.0001) and 15% of RAD-GADA-positive (P<0.0001) subjects confirmed by ECL assays. Among subjects with multiple iAbs, diabetes-free survival was significantly shorter if IAA or GADA was positive by ECL and negative by RAD than if IAA or GADA was negative by ECL and positive by RAD (P<0.019 and P<0.0001, respectively). Both positive and negative predictive values in terms of progression to type 1 diabetes mellitus were superior for ECL-IAA and ECL-GADA, compared with RADs. The prevalence of the high-risk human leukocyte antigen-DR3/4, DQB1*0302 genotype was significantly higher in subjects with RAD-IAA or RAD-GADA confirmed by ECL. In conclusion, both ECL-IAA and -GADA are more disease-specific and better able to predict the risk of progression to type 1 diabetes mellitus than the current standard RADs.

FIG. 1.

Comparison of islet autoantibody levels between electrochemiluminescence (ECL) and radioassay (RAD) among 384 TrialNet prediabetes subjects who were prospectively followed up to clinical type 1 diabetes mellitus. The dotted lines represent the assay cutoffs. All assays were set at 99% specificity. (A) ECL-insulin autoantibody (IAA) versus RAD-IAA with correlation R²=0.5480 and P<0.0001. (B) ECL-glutamic acid decarboxylase 65 autoantibody (GADA) versus RAD-GADA with correlation R²=0.7047 and P<0.0001. (Color graphics available online at www.liebertonline.com/dia)

FIG. 2.

Electrochemiluminescence-insulin autoantibody (IAA) results for all 3,484 TrialNet subjects: all four islet autoantibodies negative (Ab–) (n=802); islet autoantibodies positive with radioassay (RAD)-IAA negative (n=1,581), subdivided into single islet autoantibody (1Ab) positive (n=1,306) and multiple islet autoantibodies (2Ab) positive (n=275); islet autoantibodies positive with RAD-IAA-positive (n=717), subdivided into 1Ab positive (IAA+ only; n=421) and 2Ab positive (n=296); and pre-diabetes mellitus (DM) (n=384). (Color graphics available online at www.liebertonline.com/dia)

FIG. 3.

Electrochemiluminescence-glutamic acid decarboxylase 65 autoantibody (GADA) results for all 3,484 TrialNet subjects: all four islet autoantibodies negative (Ab–) (n=802); islet autoantibodies positive with radioassay (RAD)-GADA negative (n=627), subdivided into single islet autoantibody positive (1Ab) (n=558) and multiple islet autoantibodies positive (2Ab) (n=69); islet autoantibodies positive with RAD-GADA positive (n=1,671), subdivided into 1Ab positive (GADA+ only; n=1,169) and 2Ab positive (n=502); and pre-diabetes mellitus (DM) (n=384). (Color graphics available online at www.liebertonline.com/dia)

FIG. 4.

Prospective clinical follow-up on the subjects who were single islet antbody positive for their most recent status of development of islet authoantibodies and progression to type 1 diabetes mellitus (T1D). (A) Follow-up study on the subjects with radioassay-insulin autoantibodies positive only (n=338), subdivided into electrochemiluminescence (ECL)-insulin autoantibodies positive (n=86) and ECL-insulin autoantibodies negative (n=252). (B) Follow-up study on the subjects with radioassay-glutamic acid decarboxylase autoantibodies positive only (n=938), subdivided into ECL-glutamic acid decarboxylase autoantibodies positive (n=436) and ECL-glutamic acid decarboxylase autoantibodies negative (n=502). Ab–, antibody negative; 1Ab, single islet autoantibody; 2Ab, multiple islet autoantibodies. (Color graphics available online at www.liebertonline.com/dia)

FIG. 5.

Type 1 diabetes mellitus survival distribution analysis for the subjects with multiple (m) islet autoantibodies (AB): (A) between electrochemiluminescence (ECL)-insulin autoantibodies (IAA) positive plus other islet AB with radioassay-IAA negative (n=122) and radioassay-IAA positive plus other islet AB with ECL-IAA negative (n=45) (P=0.019) and (B) between ECL-glutamic acid decarboxylase (GAD) 65 autoantibodies (GADA) positive plus other islet AB with radioassay-GADA negative (n=45) and radioassay-GADA positive plus other islet AB with ECL-GADA negative (n=82) (P<0.001).

FIG. 6.

Comparison of predictive values between electrochemiluminescence (ECL) and radioassay (RAD) on 2,944 subjects for their very first initial screening samples in TrialNet Pathway to Prevention Study: comparison of (A) positive predictive values between RAD-insulin autoantibodies (IAA) and ECL-IAA (P<0.0001) and between RAD-glutamic acid decarboxylase 65 autoantibodies (GADA) and ECL-GADA (P<0.0001) and (B) negative predictive values between RAD-IAA and ECL-IAA (P<0.05) and between RAD-GADA and ECL-GADA (P=0.007). (Color graphics available online at www.liebertonline.com/dia)

FIG. 7.

(Left) Comparison of percentage of subjects with high-risk human leukocyte antigen DR3/4 (DQB1*0302 genotype) in subgroups of islet autoantibody (Ab) negative (0Ab), single islet Ab (1Ab), multiple islet Abs (≥2Ab), and pre-diabetes mellitus (DM). (Right) It was also compared among radioassay (RAD)-insulin Abs (IAA)-positive or RAD-glutamic acid decarboxylase 65 Abs (GADA)-positive status subdivided by their corresponding electrochemiluminescence assay positive versus negative finding in the all positivity group and the single islet Ab (iAb) group. n.s., difference not significant. (Color graphics available online at www.liebertonline.com/dia)

FIG. 8.

(Left) Comparison of mean age of subjects in subgroups of islet autoantibody (Ab) negative (0Ab), single islet Ab (1Ab), multiple islet Abs (≥2Ab), and pre-diabetes mellitus (DM). (Right) It was also compared among radioassay (RAD)-insulin autoantibody (IAA)-positive or RAD-glutamic acid decarboxylase 65 acid (GADA)-positive status subdivided by their corresponding electrochemiluminescence assay positive versus negative finding in the all positivity group and the single islet Ab (iAb) group. (Color graphics available online at www.liebertonline.com/dia)