Abstract：
Duchenne muscular dystrophy (DMD) is a genetic disease characterized by progressive muscle loss caused by mutations in dystrophin, resulting in decreased dystrophin levels. Dystrophin protein expression is a biomarker used to evaluate treatments that restore patient dystrophin levels. Currently, a semiquantitative assay using western blotting, which normalizes dystrophin expression to that of a control population, is used for regulatory filing. However, the current methods are limited in terms of sensitivity, quantification, and reproducibility. To address this, a highly sensitive and quantitative sandwich immune assay using Single Molecule Counting technology was established, with recombinant dystrophin protein as the calibrator. Capture and detection antibodies were selected to detect full-length dystrophin. Using this optimized assay, dystrophin levels in muscle samples from Myotonic Dystrophy (n = 9) and DMD (n = 8) subjects were 93.2 ± 31.9 (range: 49.4-145.3) and 14.5 ± 6.8 (range: 6.18-22.6) fmol/total protein mg, respectively. The lowest concentration of dystrophin measured in the DMD samples was 5 times higher than that in the lower limit of quantitation, a level not detected by western blotting. These data indicate that this assay accurately and sensitively measured dystrophin protein and may be useful in clinical trials assessing dystrophin restoration therapies.
摘要：
Duchenne型肌营养不良症(DMD)是一种遗传性疾病，其特征是由肌营养不良蛋白突变引起的进行性肌肉损失。导致肌养蛋白水平降低。肌营养不良蛋白表达是用于评估恢复患者肌营养不良蛋白水平的治疗的生物标志物。目前,使用蛋白质印迹的半定量分析，将肌营养不良蛋白的表达标准化为对照群体的表达，用于监管备案。然而,目前的方法在灵敏度方面受到限制，量化，和再现性。为了解决这个问题，建立了一种使用单分子计数技术的高度灵敏和定量的夹心免疫测定法，重组肌营养不良蛋白作为校准物。选择捕获和检测抗体以检测全长肌养蛋白。使用这种优化的测定法，肌强直性营养不良（n=9）和DMD（n=8）受试者的肌肉样本中的肌营养不良蛋白水平为93.2±31.9（范围：49.4-145.3）和14.5±6.8（范围：6.18-22.6）fmol/总蛋白mg，分别。DMD样品中测得的肌营养不良蛋白的最低浓度比定量下限高5倍，蛋白质印迹法未检测到的水平。这些数据表明该测定准确且灵敏地测量了肌营养不良蛋白,并且可用于评估肌营养不良蛋白恢复疗法的临床试验。
