目的:在这项回顾性纵向研究中,我们旨在探索(a)MuSK-免疫球蛋白G(IgG)水平的作用,(b)主要的MuSK-IgG亚类,和(c)抗体亲和力作为MuSK-MG严重程度和结果的候选生物标志物,使用和比较不同的抗体检测技术。
方法:用放射免疫分析法(RIA)定量总的MuSK-IgG,ELISA,流式细胞术,和使用用MuSK-eGFP转染的HEK293细胞的基于细胞的测定(CBA)系列稀释。通过流式细胞术测量MuSK-IgG亚类。SAffCon测定用于测定MuSK-IgG亲和力。
结果:在不同时间点从20例MuSK-MG患者中获得43份血清样本(发病年龄中位数:48岁,四分位间距=27.5-72.5;女性,16/20),20个中的9个(45%)用利妥昔单抗治疗。通过流式细胞术测量的MuSK-IgG水平与RIA滴度之间存在强相关性(rs=0.74,95%CI0.41-0.89,p=0.0003),以及CBA终点滴度和RIA滴度之间的中等相关性(rs=0.47,95%CI0.01-0.77,p=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着相关性(rs=0.39,95%CI0.06-0.64,p=0.0175;混合效应模型估计:2.296e-06,std。误差:1.024e-06,t=2.243,p=0.032)。在个别患者中,临床改善与MuSK-IgG水平降低相关,通过流式细胞术或CBA终点滴定测量。在所有样品中,MuSK-IgG4是最常见的同种型(平均值±SD:90.95%±13.89)。MuSK-IgG4和显著减少,在较小程度上,在临床预后良好的患者中观察到MuSK-IgG2。在利妥昔单抗治疗的患者亚组中证实了类似的趋势。在一个病人身上,MuSK-IgG亲和力在症状加重期间增加(KD值:62nMvs0.6nM),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。
结论:我们的数据支持通过流式细胞术对MuSK抗体进行定量。通过多模态研究方法,我们发现总的MuSK-IgG水平,MuSK-IgG4和MuSK-IgG2水平,和MuSK-IgG亲和力可能代表MuSK-MG疾病结局的有希望的生物标志物。
OBJECTIVE: 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.
METHODS: 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.
RESULTS: 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 (rs = 0.74, 95% CI 0.41-0.89, p = 0.0003), as well as a moderate correlation between CBA end-point titers and RIA titers (rs = 0.47, 95% CI 0.01-0.77, p = 0.0414). A significant correlation was found between MuSK-IgG flow cytometry levels and disease severity (rs = 0.39, 95% CI 0.06-0.64, p = 0.0175; mixed-effects model estimate: 2.296e-06, std. error: 1.024e-06, t = 2.243, p = 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 (KD 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.
CONCLUSIONS: 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.