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A robust immunoassay for anti-interferon autoantibodies that is highly specific for patients with Autoimmune Polyglandular Syndrome Type 1

机译:一种强大的抗干扰素自身抗体免疫测定方法对患有1型自身免疫性多腺综合征的患者具有高度特异性

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摘要

High titer antibodies to type 1 interferons have been recently reported as being highly specific for patients with autoimmune polyglandular syndrome type 1 (APS1) in Finnish and Norwegian patients with mutations in the AIRE gene. Those studies employed a complex neutralization assay to define the type 1 interferon autoantibodies. Here we have established a competitive europium time resolved fluorescence assay for IFN-a autoantibodies and measured sera from subjects with APS1, first degree relatives of APS1 patients, patients with Addison’s disease or Type 1 diabetes. The europium-based immunoassay utilizes plate bound human IFN-a incubated with sera with or without competition with fluid phase IFN-a, followed by anti-IgG biotinylated antibody and detection with streptavidin-europium. The index of IFN-a Ab was calculated as (CPS (Counts per second) without competition-CPS with competition)/(CPS positive standard sera without competition-CPS positive standard sera with competition). Results are reported for raw CPS and indices and are compared across the different subjects.ResultsFor normal controls (n=100) CPS without competition were 31,237±17,328 CPS while after subtracting the competition value, the results were −6,563±10,303 CPS. The initial APS1 patient (used to create the index as 1.0) gave 394,063 CPS without competition and a delta of 363,662±31,587 CPS with competition. Scatchard plot analysis of this patient sample revealed a high avidity for IFN-a (Kd of 0.5 nM). The CPS, delta, and index for 6/7 APS1 patients was strongly positive and 3 standard deviations or more above that of the normal controls. Using a cut–off of 2 standard deviations above normal controls, relatives of APS1 patients were negative for type I interferon autoantibodies as were 71 patients with Addison’s disease (non-APS1) and 141 Type 1 diabetes patients. This simple high throughput competitive europium time resolved fluorescence assay had a sensitivity of =86% or greater and a specificity of > 99.5%.
机译:最近有报道称,在芬兰和挪威的AIRE基因突变患者中,针对1型干扰素的高滴度抗体对自身免疫性多腺综合征1型(APS1)患者具有高度特异性。这些研究采用复杂的中和测定法来确定1型干扰素自身抗体。在这里,我们建立了一种竞争性的time时间分辨荧光测定IFN-α自身抗体的方法,并测量了APS1患者,APS1患者的一级亲属,Addison病或1型糖尿病患者的血清。基于immuno的免疫测定法利用与血清孵育的结合板的人IFN-α与或不与液相IFN-α竞争,然后使用抗IgG生物素化抗体,并用链霉亲和素-eur进行检测。 IFN-αAb的指数计算为(无竞争的CPS-有竞争的CPS)/(无竞争的CPS阳性标准血清-有竞争的CPS阳性标准血清)。报告原始CPS和指标的结果,并在不同受试者之间进行比较。结果对于正常对照组(n = 100),无竞争的CPS为31,237±17,328 CPS,而减去竞争值后,结果为-6,563±10,303 CPS。最初的APS1患者(用于创建指数为1.0)在没有竞争的情况下给出了394,063 CPS,在有竞争的情况下得出了363,662±31,587 CPS。该患者样品的Scatchard图分析显示了对IFN-α的高亲和力(Kd为0.5 nM)。 6/7 APS1患者的CPS,delta和指数均为强阳性,并且比正常对照组高3个标准差或更多。使用比正常对照组高2个标准差的标准,APS1患者的亲属对I型干扰素自身抗体阴性,71例Addison病(非APS1)患者和141例1型糖尿病患者。这种简单的高通量竞争性time时间分辨荧光测定法的灵敏度为= 86%或更高,特异性> 99.5%。

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