A faster, cheaper method to detect immune autoantibodies in whole blood

New whole-blood assay for detecting immune autoantibodies promises to revolutionize diagnostics, offering rapid, cost-effective solutions to detect inborn errors of immunity and neutralizing antibodies against type I interferons.

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

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

The study and findings

In the present study, researchers developed a simple, more affordable, quick method to detect 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 assessed 16 hours later using multiplex assays. Among the proteins tested, IFN-γ-inducible protein 10 (IP-10), encoded by C-X-C motif chemokine ligand 10 (CXCL10), had the highest levels of induction, followed by interleukin (IL)-6.

Rapid diagnostics: The new whole-blood assay delivers results within 16 hours, significantly faster than conventional tests that can take days.

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. Further, stimulation with IFN-β induced high IP-10, but stimulation with IFN-ω had a less pronounced induction of IP-10 relative to IFN-β or IFN-α2. Further, fresh PBMCs from three healthy donors were stimulated with IFN-α2, followed by bulk RNA sequencing (RNA-seq).

This confirmed that CXCL10 was among the top 20 most strongly induced transcripts. Next, the researchers examined 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. Levels of IP-10 in samples stimulated with IFN-κ or IFN-ε were similar to those in the absence of stimulation, suggesting these IFNs did not induce IP-10.

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

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 robust induction of IP-10 in samples from healthy donors. Conversely, IP-10 induction was abolished in APS-1 patients after stimulation with IFN-ω or IFN-α2, revealing the neutralizing activity of auto-Abs in patients.

Further, they tested blood from a patient with auto-Abs against type I IFNs without a genetic diagnosis, a female heterozygous for an X-linked inhibitor of nuclear factor kappa B (NFKB) kinase regulatory subunit gamma (IFBKG) allele carrying auto-Abs neutralizing IFN-ω, and a patient heterozygous for an autosomal NFKB2 allele carrying auto-Abs neutralizing IFN-ω and IFN-α2.

No complex equipment needed: Unlike traditional assays, this method uses commonly available laboratory tools, eliminating the need for expensive machinery and complex processing.

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

After stimulation with IFN-β, IFN-α2, or IFN-ω, blood from TYK2- or IFNAR1-deficient patients had IP-10 levels comparable to those observed without stimulation, implying a complete lack of response. Similarly, one IFNAR2-deficient patient showed no response, while another had detectable levels of IP-10 after stimulation with IFN-ω or IFN-β. The IRF9-deficient patient also had a weak detectable IP-10 induction.

Conclusions

Taken together, the findings illustrate the induction of IP-10 by IFN-γ, IFN-β, IFN-α2, or IFN-ω in the whole blood of healthy persons but not in patients with inborn errors of type I IFN immunity or auto-Abs against the corresponding type I IFNs. This method has numerous benefits over conventional neutralization assays; it is a whole blood-based assay and does not require blood sample processing.

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

With its practicality, cost-effectiveness, and ability to deliver rapid results, this assay has the potential to significantly impact the diagnosis and monitoring of conditions involving auto-Abs and genetic errors in type I IFN pathways.

Journal reference:

• Gervais A, Le Floc’h C, Le Voyer T, et al. A sensitive assay for measuring whole-blood responses to type I IFNs. Proceedings of the National Academy of Sciences, 2024, DOI: 10.1073/pnas.2402983121, https://www.pnas.org/doi/10.1073/pnas.2402983121