METHODS: We developed an in-house ELISA to assess the reactivity against the MDA5 domains (conformational epitopes) in plasma (n = 8) and serum (n = 24) samples from MDA5(+) patients with varying clinical manifestations and disease outcomes. The reactivities were also assessed using western blot (linearized epitopes). An ELISA-based depletion assay was developed to assess cross-reactivity among the different MDA5 domains.
RESULTS: All eight plasma samples consistently showed reactivity towards conformational and linearized epitopes on the helicase domains of the MDA5 protein. The ELISA-based depletion assay suggests that anti-MDA5 autoantibodies specifically target each of the three helicase domains. Twenty-two of the 24 serum samples showed reactivity in the in-house ELISA and all 22 displayed reactivity towards the helicase domains of the MDA5 protein.
CONCLUSIONS: Our data revealed that the main immunogenic targets of anti-MDA5 autoantibodies from MDA5(+) patients are the helicase domains. Considering that the helicase domains are responsible for the enzymatic activity and subsequent triggering of an inflammatory response, our findings suggest that binding of anti-MDA5 autoantibodies could alter the canonical activity of the MDA5 protein and potentially affect the downstream induction of a pro-inflammatory cascade.
方法:我们开发了一种内部ELISA来评估MDA5(+)患者的血浆(n=8)和血清(n=24)样品中针对MDA5结构域(构象表位)的反应性具有不同的临床表现和疾病结果。还使用Western印迹(线性化表位)评估反应性。开发了基于ELISA的耗竭测定以评估不同MDA5结构域之间的交叉反应性。
结果:所有八个血浆样品一致显示对MDA5蛋白的解旋酶结构域上的构象和线性化表位的反应性。基于ELISA的耗竭测定表明抗MDA5自身抗体特异性靶向三个解旋酶结构域中的每一个。24个血清样品中的22个在内部ELISA中显示出反应性,并且所有22个都显示出针对MDA5蛋白的解旋酶结构域的反应性。
结论:我们的数据显示,MDA5(+)患者抗MDA5自身抗体的主要免疫原性靶标是解旋酶结构域。考虑到解旋酶结构域负责酶活性和随后的炎症反应的触发,我们的研究结果表明,抗MDA5自身抗体的结合可能改变MDA5蛋白的典型活性,并可能影响促炎级联的下游诱导.