Zhang Li, Barker Jennifer M, Babu Sunanda, Su Maureen, Stenerson Matthew, Cheng Mickie, Shum Anthony, Zamir Ehud, Badolato Raffaele, Law Adam, Eisenbarth George S, Anderson Mark S
Barbara Davis Center for Childhood Diabetes, University of Colorado Health Sciences Center, Box B140 Building M20, 1775 N. Ursula Street, Aurora, CO 80045-6511, USA.
Clin Immunol. 2007 Nov;125(2):131-7. doi: 10.1016/j.clim.2007.07.015. Epub 2007 Sep 6.
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-alpha 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-alpha incubated with sera with or without competition with fluid phase IFN-alpha, followed by anti-IgG biotinylated antibody and detection with streptavidin-europium. The index of IFN-alpha 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.
For normal controls (n=100) CPS without competition were 31,237+/-17,328 CPS while after subtracting the competition value, the results were -6563+/-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-alpha (K(d) of 0.5 nM). The CPS, delta, and index for 6/7 APS1 patients were 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 > or =86% or greater and a specificity of >99.5%.
最近有报道称，在芬兰和挪威携带AIRE基因突变的自身免疫性多内分泌腺综合征1型（APS1）患者中，针对1型干扰素的高滴度抗体具有高度特异性。这些研究采用了复杂的中和试验来鉴定1型干扰素自身抗体。在此，我们建立了一种用于检测IFN-α自身抗体的竞争性铕时间分辨荧光测定法，并检测了APS1患者、APS1患者的一级亲属、艾迪生病患者或1型糖尿病患者的血清。基于铕的免疫测定法是将包被在板上的人IFN-α与血清一起孵育，血清可与液相IFN-α竞争或不竞争，然后加入生物素化抗IgG抗体，并用链霉亲和素 - 铕进行检测。IFN-α抗体指数的计算方法为（无竞争时的每秒计数（CPS） - 有竞争时的CPS）/（无竞争时阳性标准血清的CPS - 有竞争时阳性标准血清的CPS）。报告了原始CPS和指数的结果，并在不同受试者之间进行了比较。
对于正常对照组（n = 100），无竞争时的CPS为31,237±17,328 CPS，减去竞争值后的结果为 - 6563±10,303 CPS。最初用于创建指数为1.0的APS1患者，无竞争时的CPS为394,063 CPS，有竞争时的差值为363,662±31,587 CPS。对该患者样本进行Scatchard图分析显示，其对IFN-α具有高亲和力（解离常数K(d)为0.5 nM）。6/7例APS1患者的CPS、差值和指数均呈强阳性，比正常对照组高出3个标准差或更多。以高于正常对照组2个标准差为临界值，APS1患者的亲属、71例艾迪生病（非APS1）患者和141例1型糖尿病患者的I型干扰素自身抗体均为阴性。这种简单的高通量竞争性铕时间分辨荧光测定法的灵敏度≥86%或更高，特异性>99.5%。
