Fig 1.
Schematic of sample-sparing ADAP assay for detection of multiple islet autoantibodies and the analytical characterization.
(A) The workflow of ADAP is consisted of three steps. First, 1μL of serum is incubated with islet antigen conjugate probes harboring distinct DNA barcode pairs for 30 min. The multivalency of target autoantibodies agglutinates the cognate antigen-DNA conjugate pairs into close proximity. Secondly, the addition of a ligase and a bridge oligonucleotide reunites the two-separated barcode pairs into a full length amplicon. Finally, the ligated product is PCR amplified and then quantified with distinct primer pairs in the RT-PCR. Since each antigen-DNA conjugate only has one primer binding site and thus not PCR amplifiable on its own, no washing or centrifugation step is needed to remove unreacted probes. (B) The multiplex ADAP assay detected cognate antibodies without cross-reactivity. From left to right, antibodies from immunized animals against GAD, IA-2 and insulin were serially diluted and assayed by ADAP. The x-axis displays the quantities of the antibodies in the sample. The y-axis is ΔCt calculated by the difference of Ct value between the sample and a blank (S7 Fig). Signals for GAD, IA-2 and insulin antibodies are color coded in blue, orange and green respectively. Error bars represent standard deviation from triplicate, but for many data points are too small to be visualized. (C) Tolerance of ADAP for common blood contaminant was investigated by spiking hemoglobin at various concentration in T1D and healthy serum. No interference is observed up to 500 mg/dL of hemoglobin.
Fig 2.
Clinical performance of ADAP assay in an evaluation cohort of T1D and healthy control.
(A) The ROC curves of ADAP showed AUC of 0.94 (95%CI: 0.89–0.99), 0.82 (95%CI: 0.71–0.93) and 0.95 (95%CI: 0.90–1.00) for GAD, IA-2 and insulin antibodies/autoantibodies respectively. The samples were also analyzed by radioassay and showed corresponding AUC of 0.92 (95%CI: 0.85–0.99), 0.80 (95%CI: 0.68–0.92) and 0.95 (95%CI: 0.88–1.00). (B) Comparison plots of ADAP and radioassay signals. The x-axis displays radioassay signals in logarithm scales. The y-axis shows ADAP signal in ΔCt. The use of logarithm was necessary as ΔCt is a logarithmic parameter. (For instance, consider a sample of ΔCt value 2 and another sample of ΔCt of 4, their amplicon quantities differ by 4 fold (24/22) rather than 2 fold). The horizontal and the vertical dash lines denote ADAP and radioassay cutoff thresholds respectively. T1D sample data is shown in blue circle, whereas health serum signal is shown in red square. A total of 30 T1D and 39 control was analyzed without blinding.
Table 1.
The ADAP assay performance in IASP 2018 study.
Fig 3.
Validation of ADAP performance in a cohort of T1D and T2D samples.
(A) The ROC curves of ADAP showed AUC of 0.92 (95%CI: 0.83–1.00), 0.83 (95%CI: 0.70–0.95) and 0.97 (95%CI: 0.92–1.00) for GAD, IA-2 and insulin antibodies/autoantibodies respectively. The samples were also analyzed by radioassay and showed corresponding AUC of 0.91 (95%CI: 0.82–1.00), 0.72 (95%CI: 0.56–0.87) and 0.90 (95%CI: 0.79–1.00). (B) Comparison plots of ADAP and radioassay signals. The x-axis displays radioassay signals in logarithm scales. The y-axis shows ADAP signal in ΔCt. The horizontal and the vertical dash lines denote ADAP and radioassay cutoff thresholds respectively. T1D sample data is shown in blue circle, whereas health serum signal is shown in red square. This cohort included 20 T1D and 30 T2D, and was analyzed with blinding.
Table 2.
Analysis of a challenging sample cohort with ADAP assay.
Fig 4.
Heatmap of islet autoantibody patterns in at-risk T1D.
Serum samples (n = 39) were analyzed by ADAP (left) and radioassay (right). ADAP and radioassay data was divided by corresponding cutoffs and plotted according to the color key at the bottom. Patients positive for two or more autoantibodies are at high-risk of progression to clinical onset of T1D. Radioassays identified 20 high-risk individuals, and 19 of those were also positive by ADAP, indicating ADAP’s ability for risk identification using 1μL of serum sample. This cohort was analyzed with blinding.
Table 3.
Number of people found autoantibody positivity by the multiplex ADAP and radioassay.