Use of capillary Western immunoassay (Wes) for quantification of dystrophin levels in skeletal muscle of healthy controls and individuals with Becker and Duchenne muscular dystrophy

Abstract

Duchenne muscular dystrophy (DMD) is a neuromuscular disease characterized by progressive weakness of the skeletal and cardiac muscles. This X-linked disorder is caused by open reading frame disrupting mutations in the DMD gene, resulting in strong reduction or complete absence of dystrophin protein. In order to use dystrophin as a supportive or even surrogate biomarker in clinical studies on investigational drugs aiming at correcting the primary cause of the disease, the ability to reliably quantify dystrophin expression in muscle biopsies of DMD patients pre- and post-treatment is essential. Here we demonstrate the application of the ProteinSimple capillary immunoassay (Wes) method, a gel- and blot-free method requiring less sample, antibody and time to run than conventional Western blot assay. We optimized dystrophin quantification by Wes using 2 different antibodies and found it to be highly sensitive, reproducible and quantitative over a large dynamic range. Using a healthy control muscle sample as a reference and α-actinin as a protein loading/muscle content control, a panel of skeletal muscle samples consisting of 31 healthy controls, 25 Becker Muscle dystrophy (BMD) and 17 DMD samples was subjected to Wes analysis. In healthy controls dystrophin levels varied 3 to 5-fold between the highest and lowest muscle samples, with the reference sample representing the average of all 31 samples. In BMD muscle samples dystrophin levels ranged from 10% to 90%, with an average of 33% of the healthy muscle average, while for the DMD samples the average dystrophin level was 1.3%, ranging from 0.7% to 7% of the healthy muscle average. In conclusion, Wes is a suitable, efficient and reliable method for quantification of dystrophin expression as a biomarker in DMD clinical drug development.

Effects of salt‐loading on supraoptic vasopressin neurones assessed by ClopHensorN chloride imaging

“…Capillary electrophoresis‐based Protein Simple Wes was used in the present study because of its ability to analyse very low concentrations of protein and also because of its automation . This technology allowed us to show that KCC2 and β‐actin expression is found in the neurones that responded to muscimol during chloride imaging.…”

Duchenne Muscular Dystrophy (DMD) Diagnosis: Past and Present Perspectives

“…In some cases, muscle biopsy is required to fully characterize the phenotypic effect of the mutation. The muscle tissue is used in immunoassays, using different antibodies targeting different regions of dystrophin protein (C-terminal, Rod and N-terminal domains), such as western blotting [81][82][83] or immunohistochemistry [83]. Uchino et al [83] developed a multiplex western blotting assay to analyze the expression of other muscle proteins like dysferlin, merosin, different forms of sarcoglycan (alpha, beta, gamma, delta), and calpain in addition to dystrophin protein, due to the frequent epigenetic changes incited in these proteins as a consequence to the alteration in dystrophin expression.…”

Absolute quantification of dystrophin protein in human muscle biopsies using parallel reaction monitoring (PRM)

“…Indeed, recent studies conducted on the same muscle biopsies in different laboratories and even within the same laboratory showed great variation in the results with CVs ranging from 23% to 223% . More recently, an emerging western blot capillary technique showed a reliable and reproducible data with excellent linearity ( r 2 = .99) and only required a small amount (~1 μg) of total protein extract . But both this method and the gel‐based western blot method and immunofluorescence cannot determine the absolute amount of dystrophin because of the lack of a pure recombinant dystrophin protein to use as a calibration standard.…”

“…17 More recently, an emerging western blot capillary technique showed a reliable and reproducible data with excellent linearity (r 2 = .99) and only required a small amount (~1 μg) of total protein extract. 18 But both this method and the gel-based western blot method and immunofluorescence cannot determine the absolute amount of dystrophin because of the lack of a pure recombinant dystrophin protein to use as a calibration standard. The dystrophin gene is one of the largest genes in humans and is very challenging to clone in order to produce a pure recombinant dystrophin protein.…”