From the Department of Neuroscience (G.S., S.F., A.E.), Università Cattolica del Sacro Cuore, Rome, Italy; German Center for Neurodegenerative Diseases (DZNE) Berlin (G.S.), Berlin, Germany; Nuffield Department of Clinical Neurosciences (A.V., B.S., L.W.J.), University of Oxford; Fluidic Analytics Ltd (S.D.), The Paddocks Business Centre, Cambridge, United Kingdom; and Department of Neurosciences (V.D.), Drugs and Child Health, University of Florence, Italy.
Neurol Neuroimmunol Neuroinflamm. 2024 Nov;11(6):e200313. doi: 10.1212/NXI.0000000000200313. Epub 2024 Sep 9.
In this retrospective longitudinal study, we aimed at exploring the role of (a) MuSK-immunoglobulin G (IgG) levels, (b) predominant MuSK-IgG subclasses, and (c) antibody affinity as candidate biomarkers of severity and outcomes in MuSK-MG, using and comparing different antibody testing techniques.
Total MuSK-IgGs were quantified with radioimmunoassay (RIA), ELISA, flow cytometry, and cell-based assay (CBA) serial dilutions using HEK293 cells transfected with MuSK-eGFP. MuSK-IgG subclasses were measured by flow cytometry. SAffCon assay was used for determining MuSK-IgG affinity.
Forty-three serum samples were obtained at different time points from 20 patients with MuSK-MG (median age at onset: 48 years, interquartile range = 27.5-72.5; women, 16/20), with 9 of 20 (45%) treated with rituximab. A strong correlation between MuSK-IgG levels measured by flow cytometry and RIA titers was found (r = 0.74, 95% CI 0.41-0.89, = 0.0003), as well as a moderate correlation between CBA end-point titers and RIA titers (r = 0.47, 95% CI 0.01-0.77, = 0.0414). A significant correlation was found between MuSK-IgG flow cytometry levels and disease severity (r = 0.39, 95% CI 0.06-0.64, = 0.0175; mixed-effects model estimate: 2.296e-06, std. error: 1.024e-06, t = 2.243, = 0.032). In individual patients, clinical improvement was associated with decrease in MuSK-IgG levels, as measured by either flow cytometry or CBA end-point titration. In all samples, MuSK-IgG4 was the most frequent isotype (mean ± SD: 90.95% ± 13.89). A significant reduction of MuSK-IgG4 and, to a lesser extent, of MuSK-IgG2, was seen in patients with favorable clinical outcomes. A similar trend was confirmed in the subgroup of rituximab-treated patients. In a single patient, MuSK-IgG affinity increased during symptom exacerbation ( values: 62 nM vs 0.6 nM) while total MuSK-IgG and IgG4 levels remained stable, suggesting that affinity maturation may be a driver of clinical worsening.
Our data support the quantification of MuSK antibodies by flow cytometry. Through a multimodal investigational approach, we showed that total MuSK-IgG levels, MuSK-IgG4 and MuSK-IgG2 levels, and MuSK-IgG affinity may represent promising biomarkers of disease outcomes in MuSK-MG.
在这项回顾性纵向研究中，我们旨在使用和比较不同的抗体检测技术，探索（a）MuSK-免疫球蛋白 G（IgG）水平、（b）主要 MuSK-IgG 亚类和（c）抗体亲和力作为 MuSK-MG 严重程度和结局的候选生物标志物的作用。
使用转染 MuSK-eGFP 的 HEK293 细胞进行血清稀释，用放射免疫分析（RIA）、酶联免疫吸附测定（ELISA）、流式细胞术和基于细胞的测定（CBA）分别定量总 MuSK-IgG。通过流式细胞术测量 MuSK-IgG 亚类。使用 SAffCon 测定法测定 MuSK-IgG 亲和力。
从 20 名 MuSK-MG 患者（发病中位年龄：48 岁，四分位距 = 27.5-72.5；女性 16/20）不同时间点获得了 43 份血清样本，其中 9 名（45%）接受了利妥昔单抗治疗。发现流式细胞术和 RIA 滴度测定的 MuSK-IgG 水平之间存在很强的相关性（r = 0.74，95%CI 0.41-0.89， = 0.0003），CBA 终点滴度和 RIA 滴度之间也存在中度相关性（r = 0.47，95%CI 0.01-0.77， = 0.0414）。发现 MuSK-IgG 流式细胞术水平与疾病严重程度之间存在显著相关性（r = 0.39，95%CI 0.06-0.64， = 0.0175；混合效应模型估计：2.296e-06，标准误差：1.024e-06，t = 2.243， = 0.032）。在个体患者中，MuSK-IgG 水平的降低与临床改善相关，这可以通过流式细胞术或 CBA 终点滴度来衡量。在所有样本中，MuSK-IgG4 是最常见的同种型（平均值 ± 标准差：90.95% ± 13.89）。在具有良好临床结局的患者中，观察到 MuSK-IgG4 和 MuSK-IgG2 的显著减少，程度较轻。在利妥昔单抗治疗患者的亚组中也证实了类似的趋势。在一名患者中，MuSK-IgG 亲和力在症状恶化期间增加（ 值：62 nM 与 0.6 nM），而总 MuSK-IgG 和 IgG4 水平保持稳定，表明亲和力成熟可能是临床恶化的驱动因素。
我们的数据支持通过流式细胞术定量 MuSK 抗体。通过多模态研究方法，我们表明总 MuSK-IgG 水平、MuSK-IgG4 和 MuSK-IgG2 水平以及 MuSK-IgG 亲和力可能是 MuSK-MG 疾病结局的有前途的生物标志物。
