A rapid, inexpensive method for detecting immune autoantibodies in whole blood

A new whole blood test for the detection of immune autoantibodies promises to revolutionize diagnostics, providing a rapid, cost-effective solution for detecting inborn errors of immunity and neutralizing antibodies against type I interferons.

Study: A sensitive assay for measuring whole blood responses to type I IFNs. ​​​​​​​Image credit: nobeastsofierce / Shutterstock

A recent study published in the journal Proceedings of the National Academy of Sciences reported a novel, cost-effective whole blood test to measure responses to type I interferons (IFNs) and to detect autoantibodies (auto-Abs) against these IFNs.

Autoantibodies (auto-Abs) against type I IFNs were first described in the early 1980s in a patient with disseminated shingles. They were long thought to be clinically silent, and their discovery in patients with autoimmune polyendocrine syndrome type 1 (APS-1) led to their use as a diagnostic marker for APS-1. Currently, in vitro cell-based assays are available to detect auto-Abs against type I IFNs. While these assays are sensitive and robust, they are expensive, time-consuming, and labor-intensive, which restricts their widespread use.

Studies and findings

In the current study, the researchers developed a simpler, more economical, rapid method for detecting auto-Abs against type I IFNs. First, whole blood from three healthy donors was stimulated with recombinant human IFN-α2. The production of 25 chemokines and cytokines was evaluated 16 hours later using a multiplex assay. Among the proteins tested, IFN-γ-inducible protein 10 (IP-10), encoded by C-X-C motif chemokine ligand 10 (CXCL10), had the highest level of induction, followed by interleukin (IL)-6.

The other tested molecules had poor induction. At the transcriptomic level, bulk RNA sequencing (RNA-seq) confirmed strong induction of CXCL10 expression in fresh peripheral blood mononuclear cells (PBMCs) tested six hours after stimulation. Furthermore, stimulation with IFN-β induced higher IP-10, but stimulation with IFN-ω had a less pronounced induction of IP-10 relative to IFN-β or IFN-α2. In addition, fresh PBMCs from three healthy donors were stimulated with IFN-α2, followed by bulk RNA sequencing (RNA-seq).

This confirmed that CXCL10 was one of the top 20 most strongly induced transcripts. Next, the researchers investigated whether stimulating fresh whole blood samples with tissue-restricted IFNs, such as IFN-ε (restricted to the female reproductive system) and IFN-κ (restricted to the skin), could induce IP-10. Stimulation with IFN-γ (a type II IFN) served as a control. IP-10 levels in samples stimulated with IFN-κ or IFN-ε were similar to those in the absence of stimulation, indicating that these IFNs do not induce IP-10.

In contrast, stimulation with IFN-γ-induced IP-10 to levels comparable to those induced by IFN-α2, IFN-β, or IFN-ω. These findings indicated that IP-10 was a suitable target for the detection of auto-Abs against type I or II IFNs. Further experiments suggested that for optimal results, blood should be collected in tubes containing lithium heparin and stimulated immediately after sampling, preferably within 24 hours, ideally 14 to 16 hours, or for at least six hours.

Next, the researchers collected blood samples from five APS-1 patients and nine healthy individuals and stimulated them with IFN-α2, IFN-β, or IFN-ω. IP-10 levels were measured 16 hours after stimulation. The team observed a strong induction of IP-10 in samples from healthy donors. In contrast, IP-10 induction was abolished in APS-1 patients after stimulation with IFN-ω or IFN-α2, suggesting aberrant activity of auto-Abs in patients.

In addition, they tested the blood of a patient with auto-Abs against type I IFNs with no genetic diagnosis, tested the blood of a female heterozygous for an X-linked inhibitor of nuclear factor kappa B (NFKB) kinase regulatory subunit gamma (IFBKG) allele who had auto-Abs that neutralized IFN-ω, and tested the blood of a patient heterozygous for an autosomal NFKB2 allele who had auto-Abs that neutralized IFN-ω and IFN-α2.

The previously observed neutralization data from these subjects were replicated using the whole blood IP-10 assay. Finally, the researchers examined the effect of genetic deficiency on the type I IFN response. For this purpose, fresh blood from patients with complete IFN regulatory factor 9 (IRF9), tyrosine kinase 2 (TYK2), IFN-α/β receptor 1 (IFNAR1), or IFNAR2 deficiency was stimulated.

After stimulation with IFN-β, IFN-α2, or IFN-ω, IP-10 levels in the blood of TYK2- or IFNAR1-deficient patients were comparable to the levels seen without stimulation, implying a complete lack of response. Similarly, one IFNAR2-deficient patient showed no response, whereas another had detectable levels of IP-10 after stimulation with IFN-ω or IFN-β. The IRF9-deficient patient also had weakly detectable IP-10 induction.

conclusion

Overall, the findings demonstrate the induction of IP-10 by IFN-γ, IFN-β, IFN-α2, or IFN-ω in whole blood of healthy individuals, but not in patients with inborn errors of type I IFN immunity or auto-Abs against the respective type I IFNs. This method has several advantages over conventional neutralization assays; it is a whole blood-based assay and does not require blood sampling processing.

Furthermore, the stimulation step is efficient, with overnight stimulation (or at least six hours). Furthermore, this assay does not require expensive or sophisticated machines, and the reagents and materials required are commonly used in most clinical laboratories. The cost per sample is estimated to be between $3 and $5, making it accessible for widespread clinical use. Overall, this assay is a robust and sensitive tool for detecting auto-antibodies against IFNs and inborn errors of the type I IFN response pathways.

Because of its practicality, cost-effectiveness, and ability to provide rapid results, this test has the potential to have a significant impact on the diagnosis and monitoring of conditions associated with genetic errors in the auto-Abs and type I IFN pathways.