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.