Development of Rabbit Monoclonal Antibodies for Detection of Alpha-Dystroglycan in Normal and Dystrophic Tissue

Abstract

Alpha-dystroglycan requires a rare O-mannose glycan modification to form its binding epitope for extracellular matrix proteins such as laminin. This functional glycan is disrupted in a cohort of muscular dystrophies, the secondary dystroglycanopathies, and is abnormal in some metastatic cancers. The most commonly used reagent for detection of alpha-dystroglycan is mouse monoclonal antibody IIH6, but it requires the functional O-mannose structure for recognition. Therefore, the ability to detect alpha-dystroglycan protein in disease states where it lacks the full O-mannose glycan has been limited. To overcome this hurdle, rabbit monoclonal antibodies against the alpha-dystroglycan C-terminus were generated. The new antibodies, named 5–2, 29–5, and 45–3, detect alpha-dystroglycan from mouse, rat and pig skeletal muscle by Western blot and immunofluorescence. In a mouse model of fukutin-deficient dystroglycanopathy, all antibodies detected low molecular weight alpha-dystroglycan in disease samples demonstrating a loss of functional glycosylation. Alternately, in a porcine model of Becker muscular dystrophy, relative abundance of alpha-dystroglycan was decreased, consistent with a reduction in expression of the dystrophin-glycoprotein complex in affected muscle. Therefore, these new rabbit monoclonal antibodies are suitable reagents for alpha-dystroglycan core protein detection and will enhance dystroglycan-related studies.

Mitochondrial maintenance via autophagy contributes to functional skeletal muscle regeneration and remodeling

“…During functional testing, the testing platform was heated to 37°C to maintain the animal's body temperature. Torque as a function of stimulation frequency was then measured during nine isometric contractions at varying stimulation frequencies (5,10,20,40,60,80,100,150,and 200 Hz). Finally, muscle fatigability was assessed by 120 isometric contractions, one elicited every second using a 300-ms, 60-Hz stimulation.…”

“…HM550, Thermo Scientific) and mounted on microscope slides. Tissues were stained for immunofluorescence according to standard protocols (2,10). Briefly, slides were moistened with phosphatebuffered saline (PBS) and blocked with 5% donkey serum (Jackson ImmunoResearch) in PBS.…”

From rabbit antibody repertoires to rabbit monoclonal antibodies

“…18 Correspondingly, in many IHC studies that compared rabbit and mouse mAbs to the same human antigens, rabbit mAbs consistently revealed higher sensitivity. 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62 In a recent study, 63 1410 rabbit mAbs raised against 15-amino-acid peptides representing 100 different antigens revealed a typical affinity range of 20–200 p M (median 66 p M ), as determined by high-throughput surface plasmon resonance. A fraction of rabbit mAbs even revealed higher affinities near the detection limit of 1 p M .…”

LARGE2-dependent glycosylation confers laminin-binding ability on proteoglycans

“…Antibodies to the α-DG glycan epitope (IIH6), core α-DG protein (5–2), LARGE1 and LARGE2 have been described previously (Michele et al 2002; Kanagawa et al 2004; Inamori et al 2013; Fortunato et al 2014). Anti-myc antibody 9E10 was purchased from the Developmental Studies Hybridoma Bank at the University of Iowa (Iowa City, IA).…”