How Simoa Technology Enables Direct IFN-α2 Quantification in Pediatric Autoimmune Disease

Type I interferons (IFN-I), particularly IFN-α2, are key drivers of inflammation in autoimmune diseases such as systemic lupus erythematosus (SLE), juvenile-onset SLE (jSLE), dermatomyositis, and interferonopathies. Elevated IFN-I expression is a hallmark of these conditions, making circulating IFN-α a promising biomarker for monitoring disease severity and progression. However, direct quantification of IFN-I in blood is challenging due to its extremely low abundance. As a result, IFN-I activity has traditionally been assessed indirectly via expression profiling of interferon-stimulated genes (ISGs). 

Quanterix’s ultra-sensitive Simoa^® technology has enabled direct measurement of IFN-α. Quanterix’s IFN-α Multi-Subtype Advantage Plus assay, developed using Quanterix’s Homebrew solution, quantifies IFN-α2 protein at femtogram levels, offering attomolar sensitivity, approximately 5,000-fold higher than traditional ELISA methods.¹ Previous studies have demonstrated strong concordance between Simoa-based IFN-α quantification and ISG scores in adults with SLE¹^,². To assess whether this concordance holds in pediatric populations, where clinical heterogeneity can complicate diagnosis, a recent study compared these two approaches in a large pediatric cohort³.

Study Overview: Comparing IFN-I Biomarker Detection Methods in Pediatric Autoimmune Diseases

The study evaluated two complementary methods of assessing interferon activity in children with suspected inflammatory and autoimmune conditions: a NanoString-based IFN-I score (derived from six ISG transcripts) and direct quantification of circulating IFN-α2 using the Simoa assay. 

A total of 196 pediatric plasma samples from patients at Hospices Civils de Lyon were analyzed. Diagnoses included type I interferonopathies, SLE, dermatomyositis, and juvenile idiopathic arthritis. 

Analytical Approach: Direct IFN-I Quantification vs. ISG Scores

IFN-α2 levels were measured using the Simoa HD-1 Analyzer, with a lower limit of quantification of 16 fg/mL (detection threshold: 3.2 fg/mL; lower limit of quantification: 16 fg/mL). ISG scores were calculated based on relative gene expression, normalized to three housekeeping genes and scaled against the median values from healthy donors (HD). 

Key Findings on IFN-α2 as a Biomarker Across Interferonopathies

1. Both IFN-I score and Simoa IFN-α2 levels distinguish many IFN-I mediated diseases from controls, though the IFN-I score demonstrated stronger diagnostic performance.

Plasma IFN-α2 and transcriptomic IFN-I scores successfully differentiated type I interferonopathies from other conditions. This finding aligns with previous adult SLE studies that showed strong agreement between NanoString-based ISG profiles and Simoa IFN-α2 quantification². However, 30 of 116 samples yielded discordant results, possibly partially due to IFN-II activity, a finding later attributed to elevated IFN-II signaling, as confirmed by NanoString-based IFN-II scores, in patients who were IFN-I score–positive but IFN-α2–negative.

2. Simoa provides a direct and specific readout of IFN-I activation.

While transcriptomic IFN-I scores may reflect mixed cytokine activity, including IFN-II signaling, Simoa’s protein-based approach offers a direct and quantitative readout of IFN-α2 of IFN-I activation. 

3. Correlation between IFN-α2 and specific ISGs could help refine transcriptomic assays.

IFI27, IFI44L, and SIGLEC1 were the ISGs most strongly correlated with IFN-α2 protein levels. This supports emerging data that a reduced gene panel, particularly IFI27 and IFI44L, may be sufficient for capturing systemic IFN-I activation. 

4. IFN-II scoring complements IFN-I profiling to better distinguish disease phenotypes.

By analyzing five IFN-γ–inducible genes, researchers identified IFN-II activity in certain patients who were IFN-I score–positive but IFN-α2–negative. The combined IFN-I/II profiling approach revealed overlapping disease mechanisms and enhanced patient stratification. 

Implications for Autoimmune and Inflammatory Disease Research

The findings support the utility of Simoa cytokine assays for both research and clinical applications in autoimmune disease. While both IFN-I scores and IFN-α2 levels effectively identify IFN-I–driven disease, direct quantification offers a streamlined and biologically specific measurement. Furthermore, Simoa data may inform refinement of ISG panels, especially in distinguishing IFN-I from IFN-II pathway activation, for greater diagnostic accuracy. 

Given the reliability of circulating IFN-α2 as a biomarker of disease activity, the Simoa assay is particularly well suited for monitoring therapeutic responses to IFN-α2–targeted treatments, such as anifrolumab or JAK inhibitors. Emerging evidence also supports a broader role for type I interferons as therapeutic targets in multiple sclerosis and other inflammatory or autoimmune disorders. 

Conclusion

Simoa technology is advancing the accuracy and accessibility of IFN-α measurement. Quanterix’s IFN-α Multi-Subtype Advantage Plus assay, compatible with the fully automated Simoa HD-X Analyzer®, provides direct, ultrasensitive quantification of IFN-α2. This enables comprehensive profiling of cytokine-driven inflammation, supporting deeper biological insights and clinical translation. 

In addition to IFN-α assays, Quanterix offers a broad portfolio of cytokine assays in single- and 4-plex formats, empowering researchers with high-sensitivity tools for autoimmune and inflammatory disease studies. 

To learn more about cytokine biomarkers download the Simoa® Cytokine 4-Plex Advantage Plus sell sheet.

References

1. Rodero MP, Decalf J, Bondet V, et al. Detection of interferon alpha protein reveals differential levels and cellular sources in disease. Journal of experimental medicine. 2017 May 1;214(5):1547-55.   
2. Chasset F, Mathian A, Dorgham K, et al. Serum interferon-α levels and IFN type I-stimulated genes score perform equally to assess systemic lupus erythematosus disease activity. Annals of the rheumatic diseases. 2022 Jun 1;81(6):901-3   
3. 3. Nombel A, Perret M, Trouillet-Assant S, et al. Profiling type I and II interferon responses reveals distinct subgroups of pediatric patients with autoinflammatory disorders. Journal of Allergy and Clinical Immunology: Global. 2025 Mar 8:100450.