组成图示
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传感器类型
表面等离子共振(SPR)生物传感器
检测对象
抗干扰素-α自身抗体（anti-IFN-α autoantibodies, AIAAs）；样品基质：SLE患者血清、RA患者血清、健康供体血清。
检测原理
CM5芯片表面羧基经EDC/NHS活化后，共价固定重组人IFNα4作为捕获抗原，参考通道固定人生长激素（somatropin），并用1 M乙醇胺封闭未反应位点。稀释血清流过芯片时，血清中抗IFNα IgG自身抗体（AIAA）与固定IFNα4特异性结合，使SPR响应单位（RU）增加；随后加入抗人IgG二级抗体结合AIAA，进一步放大响应。仪器记录结合曲线，净响应为IFNα4通道减去参考通道，以扣除非特异结合。AIAA浓度或亲和力越高，净RU越大；阳性判定基于健康对照均值加1.65 SD，且抗人IgG加入后至少增加100 RU。
检测灵敏度
原文未报告LOD、线性范围、灵敏度斜率或R^2。
效应效果
SPR免疫分析在49例SLE血清中检出13例（27%）AIAA阳性，25例健康对照基本无反应，25例RA中仅1例（4%）有反应性，提示对SLE相关AIAA具有较好选择性。与ELISA相比，SPR可检出更多低亲和力AIAA，文中称ELISA仅将约10%患者判为阳性，另有17%仅SPR阳性。AIAA阳性血清在体外抑制重组IFNα2活性59%–83%，健康对照约10%；也能中和PBMC来源内源性I型干扰素。AIAA阳性患者血清I型干扰素生物活性、ISM、BAFF、抗染色质/组蛋白/核糖体P抗体更低，SLEDAI更低，C3/C4更高，提示AIAA与较低疾病活动度相关。
传感器的构成
- 基底/换能器：CM5传感器芯片（金表面羧基化）与BIAcore T100 SPR换能器，提供表面结合响应。
- 化学活化层：EDC/NHS活化CM5羧基，形成氨基反应位点用于共价固定。
- 捕获抗原层：重组人IFNα4固定于flow cell 2/3，作为AIAA捕获物；flow cell 1固定人生长激素（somatropin）作参考。
- 封闭层：1 M乙醇胺封闭未反应位点，降低非特异结合。
- 样品识别层：稀释血清中的抗IFNα IgG自身抗体（AIAA）与固定IFNα4结合。
- 信号标记层：抗人IgG抗体（The Binding Site）结合AIAA，增加SPR响应。
- 读出层：BIAcore T100记录响应单位（RU），以参考通道扣除非特异结合。
中文摘要
本研究旨在表征系统性红斑狼疮（SLE）患者内源性抗干扰素-α自身抗体（AIAA）阳性者的干扰素通路活性及血清学、临床特征。采用生物传感器免疫分析检测SLE（n=49）、类风湿关节炎（n=25）和健康对照（n=25）血清中的AIAA；用U937细胞报告基因法测定血清I型干扰素生物活性及AIAA中和活性；通过微阵列分析外周血干扰素调节基因表达，并用免疫分析检测BAFF、干扰素诱导趋化因子及其他自身抗体。结果显示，生物传感器免疫分析在27%的SLE血清中检出AIAA。无监督层次聚类将患者分为IFNlow和IFNhigh两组，两组在血清I型干扰素生物活性、干扰素调节基因表达、BAFF、抗核糖体P抗体、抗染色质抗体及AIAA状态上存在差异。多数AIAA阳性患者血清I型干扰素生物活性、下游干扰素通路活性及疾病活动度低于IFNhigh组；AIAA阳性血清在体外可有效中和I型干扰素活性。结论：SLE患者常携带AIAA，AIAA阳性者I型干扰素生物活性及下游通路活性降低，AIAA可能通过削弱IFN-α效应影响SLE临床过程。
英文摘要
OBJECTIVE: Numerous observations implicate interferon-α (IFNα) in the pathophysiology of systemic lupus erythematosus (SLE); however, the potential impact of endogenous anti-IFNα autoantibodies (AIAAs) on IFN-pathway and disease activity is unclear. The aim of this study was to characterize IFN-pathway activity and the serologic and clinical profiles of AIAA-positive patients with SLE.
METHODS: Sera obtained from patients with SLE (n = 49), patients with rheumatoid arthritis (n = 25), and healthy control subjects (n = 25) were examined for the presence of AIAAs, using a biosensor immunoassay. Serum type I IFN bioactivity and the ability of AIAA-positive sera to neutralize IFNα activity were determined using U937 cells. Levels of IFN-regulated gene expression in peripheral blood were determined by microarray, and serum levels of BAFF, IFN-inducible chemokines, and other autoantibodies were measured using immunoassays.
RESULTS: AIAAs were detected in 27% of the serum samples from patients with SLE, using a biosensor immunoassay. Unsupervised hierarchical clustering analysis identified 2 subgroups of patients, IFN(low) and IFN(high) , that differed in the levels of serum type I IFN bioactivity, IFN-regulated gene expression, BAFF, anti-ribosomal P, and anti-chromatin autoantibodies, and in AIAA status. The majority of AIAA-positive patients had significantly lower levels of serum type I IFN bioactivity, reduced downstream IFN-pathway activity, and lower disease activity compared with the IFN(high) patients. AIAA-positive sera were able to effectively neutralize type I IFN activity in vitro.
CONCLUSION: Patients with SLE commonly harbor AIAAs. AIAA-positive patients have lower levels of serum type I IFN bioactivity and evidence for reduced downstream IFN-pathway and disease activity. AIAAs may influence the clinical course in SLE by blunting the effects produced by IFNα.