Establishment of a highly sensitive sandwich ELISA for the N-terminal fragment of titin in urine.

Nobuhiro Maruyama,Takeshi Kawauchi,Masahiro Maeda,Tsuyoshi Asai,Chiaki Abe,Yo-Ichi Nabeshima,Kazuya Miyashita,Masafumi Matsuo,Akari Inada

doi:10.1038/srep39375

Abstract

Muscle damage and loss of muscle mass are triggered by immobilization, loss of appetite, dystrophies and chronic wasting diseases. In addition, physical exercise causes muscle damage. In damaged muscle, the N-terminal and C-terminal regions of titin, a giant sarcomere protein, are cleaved by calpain-3, and the resulting fragments are excreted into the urine via glomerular filtration. Therefore, we considered titin fragments as promising candidates for reliable and non-invasive biomarkers of muscle injury. Here, we established a sandwich ELISA that can measure the titin N-terminal fragment over a biologically relevant range of concentrations, including those in urine samples from older, non-ambulatory Duchenne muscular dystrophy patients and from healthy donors under everyday life conditions and after exercise. Our results indicate that the established ELISA could be a useful tool for the screening of muscular dystrophies and also for monitoring the progression of muscle disease, evaluating the efficacy of therapeutic approaches, and investigating exercise-related sarcomeric disruption and repair processes.

Highlights

Muscle damage and loss of muscle mass are triggered by immobilization, loss of appetite, dystrophies and chronic wasting diseases
Titin fragments were detected by mass spectrometric analysis in serums collected from young Duchenne muscular dystrophy (DMD) patients between the ages of 3 and 4 years old[8], and comprehensive proteome studies of serum[9] and urine[10] of DMD patients and healthy donors revealed that titin showed the highest fold-change between healthy subjects and DMD patients
To prepare a suitable immunogen, a vector encoding a fusion protein consisting of glutathione S-transferase (GST) peptide, the N-terminal 200 residues of titin (Titin-N) and a His-tag was designed, and the recombinant protein was synthesized in E. coli

Summary

Introduction

Muscle damage and loss of muscle mass are triggered by immobilization, loss of appetite, dystrophies and chronic wasting diseases. We established a sandwich ELISA that can measure the titin N-terminal fragment over a biologically relevant range of concentrations, including those in urine samples from older, non-ambulatory Duchenne muscular dystrophy patients and from healthy donors under everyday life conditions and after exercise. Our results indicate that the established ELISA could be a useful tool for the screening of muscular dystrophies and for monitoring the progression of muscle disease, evaluating the efficacy of therapeutic approaches, and investigating exercise-related sarcomeric disruption and repair processes. The results suggest that it could be suitable for screening purposes and for evaluation of disease-stage, outcomes of therapeutic treatment(s), and investigations of exercise-related muscle damage and repair processes

Methods

Results

Conclusion