A luciferase-based immunoprecipitation system assay for IAA using Nluc-proinsulin shows improved discrimination of diabetes risk in children and young adults

Background: Insulin autoantibodies (IAA) are a key early marker of islet autoimmunity, particularly prevalent in young children who progress rapidly to Type 1 diabetes. Radiobinding assays (RBAs) are used to measure IAA but have limited specificity and sustainability. We evaluated the performance of novel Luciferase-based Immunoprecipitation System (LIPS) assays to measure IAA.

Methods: Luciferase-tagged proinsulin (Nluc-PI) was evaluated in 150 new-onset Type 1 diabetes patients (median age 10.4 years, range 1.3-20.7yrs) and 609 first-degree relatives (FDRs) (median follow-up 16.4 years, 0-31.6yrs), including 83 who progressed to diabetes and/or had multiple islet autoantibodies and 435 IAA negative FDRs. Thresholds were set at the 97.5^th percentile of 267 healthy schoolchildren. Affinity measurements were available for 112 FDRs found IAA positive by RBA.

Results:  The sensitivity of Nluc-PIAA in new-onset patients (76%) was comparable to that of 125I-IAA (72%, p=0.17). The partial (>90%) area under the receiver operator characteristics curve was 0.057 for 125I-IAA and 0.075 for NLuc-PIAA (p=0.004). Of 22 125I-IAA positive FDRs who developed diabetes under 35 years of age, 86% were PIAA positive (p>0.05) compared with only 48% of the 25 125I-IAA positive FDRs who developed diabetes over 35 years (p=0.0009). The LIPS assay was more specific as only 16% of 90 single 125I-IAA positive FDRs who have not progressed to diabetes were Nluc-PIAA positive. Of 73 FDRs with high/moderate affinity 125I-IAA, 64% were Nluc-PIAA positive compared with only 18% of 39 FDRs with low affinity 125I-IAA (p<0.0001). In 125I-IAA positive FDRs, the 10-year diabetes-risk was 35% if Nluc-PIAA positive and 11% if Nluc-PIAA negative (p<0.0001).

Conclusion:  The high-throughput LIPS assay uses one fifth the serum of RBAs, with shorter assay duration. Nluc-PIAA LIPS has improved disease specificity with comparable sensitivity to the RBA in patients, identifies relatives at highest diabetes risk and should allow improved targeting of therapeutic interventions.