A novel, high-performance, low-volume, rapid luciferase immunoprecipitation system (LIPS) assay to detect autoantibodies to zinc transporter 8

Abstract

Background: Zinc transporter 8 autoantibodies (ZnT8A) are thought to appear close to type 1 diabetes (T1D) onset and can identify high-risk multiple (≥2) autoantibody positive individuals. Radiobinding assays (RBA) are widely used for ZnT8A measurement but have limited sustainability. We sought to develop a novel, high-performance, non-radioactive luciferase immunoprecipitation system (LIPS) assay to replace RBA.

Methods: A custom dual C-terminal ZnT8 (aa268-369; R325/W325) heterodimeric antigen, tagged with a NanoluciferaseTM (Nluc-ZnT8) reporter, and LIPS assay was developed. Assay performance was evaluated by testing sera from new onset T1D (n = 573), healthy schoolchildren (n = 521), and selected first-degree relatives (FDRs) from the Bart's Oxford family study (n = 617; 164 progressed to diabetes).

Results: In new-onset T1D, ZnT8A levels by LIPS strongly correlated with RBA (Spearman's r = 0.89; P < 0.0001), and positivity was highly concordant (94.3%). At a high specificity (95%), LIPS and RBA had comparable assay performance [LIPS pROC-AUC(95) 0.032 (95% CI: 0.029-0.036); RBA pROC-AUC(95) 0.031 (95% CI: 0.028-0.034); P = 0.376]. Overall, FDRs found positive by LIPS or RBA had a comparable 20-year diabetes risk (52.6% and 59.7%, respectively), but LIPS positivity further stratified T1D risk in FDRs positive for at least one other islet autoantibody detected by RBA (P = 0.0346).

Conclusion: This novel, high-performance, cheaper, quicker, higher throughput, low blood volume Nluc-ZnT8 LIPS assay is a safe, non-radioactive alternative to RBA with enhanced sensitivity and ability to discriminate T1D progressors. This method offers an advanced approach to current strategies to screen the general population for T1D risk for immunotherapy trials and to reduce rates of diabetic ketoacidosis at diagnosis.

Keywords: LIPS; autoantibodies; immunoassay; method development; risk prediction; type 1 diabetes.

Graphical Abstract

Figure 1.

Nluc-ZnT8 LIPS and RBA were highly correlated and concordant, and at high specificities, assay sensitivity was comparable. Scatterplots comparing levels of ZnT8A binding (arbitrary units; AU) between Nluc-ZnT8 LIPS and monomeric RBAs in 573 newly diagnosed T1D patients (A) and 521 healthy schoolchildren (B) show that levels of ZnT8A detected by both assays were strongly correlated and ZnT8A positivity was highly concordant. The maximum AU obtained by monomeric RBA (ZnT8-R325/ZnT8-W325) was utilized for analysis as this improved the correlation between the two assays. (C) Receiver operator characteristic (ROC) analysis comparing new-diagnosed T1D and healthy schoolchildren cohorts indicated that the area under the curve (AUC) and assay sensitivity at 95% specificity was slightly improved in Nluc-ZnT8 LIPS [AUC 0.82 (95% CI 0.80–0.85, P < 0.0001); sensitivity 71.2%] compared to RBA [AUC 0.79 (95% CI 0.76–0.82); sensitivity 68.6%] (P = 0.0007 between methods). However, pAUC-ROC was comparable between the methods [Nluc-LIPS pROC-AUC 0.032 (95% CI: 0.029–0.036); RBA pROC-AUC 0.031 (95% CI: 0.028–0.034); P = 0.376]

Figure 2.

Measurement of ZnT8A by Nluc-ZnT8 LIPS or RBA has comparable diabetes risk in FDRs. § Reference category for Mantel–Cox log-rank tests. ****P < 0.0001. Positivity for ZnT8A measured by Nluc-ZnT8 LIPS (A) or RBA (B) had a much higher diabetes risk than those found ZnT8A negative by either method (P < 0.0001). Overall diabetes risk is comparable in FDRs found positive or negative by either Nluc-ZnT8 LIPS or RBA however, Nluc-ZnT8 LIPS identified a greater number of at-risk individuals at first sampling or first ZnT8A positive sample (67 vs. 53)

Figure 3.

Nluc-ZnT8 LIPS ZnT8A status does not identify FDRs with higher diabetes risk when stratified by RBA ZnT8A status. § Reference category for Mantel–Cox log-rank tests. NS: not significant. (A) Nluc-ZnT8 LIPS status in FDRs found positive by RBA. FDRs identified as positive in both Nluc-ZnT8 LIPS and RBA had the highest 20-year risk at 59.8%. Only four FDRs found positive by RBA were found negative by Nluc-ZnT8 LIPS but only two of these (50%) progressed to diabetes within 20 years of follow-up. Due to limited numbers, these two curves could not be robustly compared. (B) Nluc-ZnT8 LIPS status in FDRs found negative by RBA. Nluc-ZnT8 LIPS identified a small subset of additional FDRs that progressed to diabetes than RBA (20-year diabetes risk of 32.2%), but this was of comparable risk to relatives found negative by both methods (20-year diabetes risk of 25.9.%)

Figure 4.

Positivity of ZnT8A by Nluc-ZnT8 LIPS further stratifies diabetes risk in FDRs positive for other islet autoantibodies. § Reference category for Mantel–Cox log-rank tests. *P < 0.05; ****P < 0.0001. FDRs classified as single or multiple autoantibody positive considering GADA/IA-2A/IAA islet autoantibodies determined by RBA stratified according to Nluc-ZnT8 LIPS status. Nluc-ZnT8 LIPS positivity compared to negativity confers a higher 20-year diabetes risk in single autoantibody positive FDRs (28.6% versus 14.6%, P = 0.0346). Similarly, Nluc-ZnT8 LIPS positivity in multiple autoantibody FDRs conferred the highest 20-year diabetes risk (64.9%) however, this was not significantly different to FDRs found multiple autoantibody positive considering other islet autoantibodies (56.2%) (P > 0.05). Of the 7 single and 26 multiple autoantibody positive individuals that were additionally identified by Nluc-ZnT8 LIPS, a total of 2/7 (28.6%) and 11/26 (42.3%), respectively, rapidly progressed to T1D within 5 years of sampling