Our results have confirmed the high prevalence of autoantibodies to IFN-a in patients with clinically defined APS1. In addition, we have developed an assay that is relatively simple to employ and could be implemented by many independent laboratories. Our data support that this assay has high specificity (greater than 99%) and is in agreement with that of previous studies in that the antibodies to type 1 interferon are prevalent in the APS1 patients [10
Our specific immunofluorometric assay (IFMA) has several potential advantages that are worth discussing when compared to the other assays used to detect type 1 interferon autoantibodies in APS1 (). First, is the relative ease and simplicity of the europium based immunoassay. The previous studies that have looked for type 1 interferon autoantibodies in APS1 patients utilized both an ELISA-based assay and a separate neutralizing assay that requires a cell culture system to employ [10
]. In these studies, the neutralization assays showed better sensitivity and specificity than the standard ELISA without competition; however, the neutralization assay is more labor intensive because of the need for cell culture and viral infection to measure neutralization. In addition, the ELISA assay that was utilized in these studies (which involved an alkaline phosphatase-dependent optical density readout) was not completely specific or sensitive for APS1 patients. In a study by Wolff and colleagues, an ELISA assay for IFN-ω autoantibodies was positive in 17/19 APS1 patients and for IFN-α2 autoantibodies was positive in 23/29 APS1 patients.
Comparison of type 1 interferon antibody assays in APS1 subjects
In our competitive based ELISA, 6/6 APS1 patients with confirmed AIRE mutations were positive for autoantibodies to IFN-α (IFNα1). A second important feature of our assay is its high specificity. In a study by Meager et al. [12
], the ELISA-based assay for IFN-α was positive in 1/70 normal control subjects (1.4%), while in our study 0/100 normal control subjects were positive. Finally, we found that competition in our assay could be added to achieve >99% specificity. Using competition we did not detect a positive signal in any of the 100 normal controls, 71 Addison’s disease, or 141 Type 1 diabetes samples. The possible improved sensitivity and specificity of our assay is likely to be due to the europium-based fluorescence assay (or IFMA) that we used in this study to detect antibody binding. Previous studies have shown that this technique can enhance both specificity and sensitivity when compared head to head with other detection methods like that in ELISA’s or radioimmunoassays [13
]. This is due to a variety of factors and includes the large Stokes’ shift and long decay time of fluorescence that is associated with lanthanide chelates like europium and the effective labeling of detection reagents with europium. Further study using head to head comparisons, however, will be needed to determine the exact efficacy of our assay when compared to the previous published assays for type 1 interferon antibodies in APS1 subjects. A final point to comment on is the nature of the antigen that we used in our immunoassay. There are over 12 different subtypes of IFN-α and in our assay we utilized a recombinant form of IFN-α1. The previous published studies with APS1 subjects tested for IFN-α subtype 2 on ELISA and IFN-α subtypes 2, 4, 5, 6, 7, 8, 10, 14, 16, and 17 in neutralization assays [10
]. Thus, our assay results could again differ due to the slightly different nature of the antigens used in the assays. Taken together, given the relative ease of use of our assay and its high specificity for APS1 subjects, it may have certain advantages over the other published assays for type 1 interferon autoantibodies.
Given the high specificity of the assay in several independent studies, anti-IFN-α antibody testing should aid in the confirmation of the diagnosis of APS1. The diagnosis of APS1 requires a high-index of clinical suspicion and long-term follow-up, as components of the disorder develop over time in the affected individual [1
]. Therefore, in order to make the diagnosis of APS1 at any point in time, the clinician must be aware of the many manifestations of the disorder. Subjects who meet the clinical criteria for the disorder can have mutational analysis of the AIRE gene performed, however, this is labor intensive as there are many potential mutations in a given patient or family and exhaustive sequencing of the entire gene may be required. The use of an assay to detect antibodies to IFN-a may provide a quick and reliable way to screen for APS1 in subjects with autoimmunity. In a similar study on a large collection of Scandinavian APS1 patients, it was determined that anti-type 1 interferon antibodies likely develop at an early age and persist for an extended period of time [10
]. In agreement with these findings, we found evidence of these antibodies being present in a two year old with APS1 and also in a 32 year old adult who had the syndrome for over 20 years with a diagnosis at age 12. To date, the presence of IFN-α antibodies has not been reported on North American, Iranian, or Italian APS1 patients and here we also demonstrate the presence of these antibodies in samples from these APS1 patient populations. None of our subjects with Addison’s Disease or Type 1 diabetes in the absence of APS1 were positive for this autoantibody, indicating that even in subjects with “related” autoimmunity these antibodies are negative. Taken together, our results suggest that the IFN-α antibody assay is useful for helping confirm the diagnosis of APS1.
Interestingly, one of our APS1 patients did not test positive for IFN-α antibodies. This patient had chronic mucocutaneous candidiasis and hypoparathyroidism diagnosed in infancy and thus meets clinical criteria for APS1. However, we were also unable to identify a mutation in this patient’s AIRE gene on either allele after exhaustive sequencing of all coding exons and 2000 bp upstream of the first exon (data not shown). Thus, it is currently unclear to us if this patient has APS1 or a distinct disorder from APS1 that has overlapping clinical features.
What do these autoantibodies tell us about the underlying pathophysiology of APS1? The AIRE gene has been shown to be important in driving the “ectopic” expression of self-antigens within the thymus [4
]. Many self- antigens, such as insulin, whose expression is restricted to limited tissues (like the pancreatic islets in the case of insulin), have also been demonstrated to be expressed in the thymus and this expression is likely important for the development of central tolerance to self [15
]. The presence of antibodies to IFN-a in almost all patients so far reported with APS1, despite the extreme variability of their clinical phenotype suggests that for some reason the lack of interferon autoantibodies is critically dependent on AIRE. Interestingly, to date, the only other clinical syndrome associated with these autoantibodies appears to be patients with thymoma and myasthenia gravis [10
]. Perhaps it is possible that these antibodies emerge as a failure of selection against T cells with specificity for type 1 interferons given the clear link to the thymus in the two clinical syndromes (APS1 and thymoma/myasthenia gravis). Given the broad expression pattern of type 1 interferons in many cell popluations, however, it is unclear why thymic expression would be necessary to induce tolerance to them. The clinical contribution to the APS1 syndrome by these autoantibodies is also unclear. One interesting possibility is the potential link of these autoantibodies to the propensity to develop susceptibility to candida infections in these patients as suggested by Meager et al. [10
]. Clearly additional work is needed to unravel how these interesting antibodies arise and also to determine what, if any, contribution they have to the pathophysiology of APS1.
In conclusion, we have developed a novel and highly sensitive and specific assay for IFN-a antibodies that employs a competitive step to improve the test accuracy. This assay is admirably suited to clinically to screen patients for APS1 and an evaluation of large series of patients with APS1 from multiple nations still needs to be performed to refine the sensitivity and specificity of the test, Not only does our data in conjunction with recent studies show that measurement of autoantibodies to type 1 interferons can be useful in confirming the diagnosis of APS1, a rare disorder, but in a much broader content, our data shows the potential for the broad application of such competitive assays in clinical medicine.