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

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.

SUBMITTER: Maruyama N

PROVIDER: S-EPMC5171804 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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

Maruyama Nobuhiro N Asai Tsuyoshi T Abe Chiaki C Inada Akari A Kawauchi Takeshi T Miyashita Kazuya K Maeda Masahiro M Matsuo Masafumi M Nabeshima Yo-Ichi YI

Scientific reports 20161219

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 musc ...[more]

PMID: 27991570

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Project description:IntroductionChikungunya is caused by an alpha virus transmitted to humans by an infected mosquito. Infection is generally considered to be self-limiting and non-critical. Chikungunya infection may be diagnosed by severe joint pain with fever, but it is difficult to diagnose because the symptoms of chikungunya are common to many pathogens, including dengue fever. Diagnosis mainly depends on viral culture, reverse transcriptase polymerase chain reaction (RT-PCR), and IgM ELISA. Early and accurate diagnosis of the virus can be achieved by the application of PCR methods, but the high cost and the need for a thermal cycler restrict the use of such methods. On the other hand, antibody-based IgM ELISA is considered to be inexpensive, but antibodies against chikungunya virus (CHIKV) only develop after 4 days of infection, so it has limited application in the earlier diagnosis of viral infection and the management of patients. Because of these challenges, a simple antigen-based sensitive, specific, and rapid detection method is required for the early and accurate clinical diagnosis of chikungunya.MethodsThe amino acid sequence of CHIKV ectodomain E1 and E2 proteins was analyzed using bioinformatics tools to determine the antigenic residues, particularly the B-cell epitopes and their characteristics. Recombinant E2-E1 CHIKV antigen was used for the development of polyclonal antibodies in hamsters and IgG was purified. Serological tests of 96 CHIKV patients were conducted by antigen-capture ELISA using primary antibodies raised against rCHIKV E2-E1 in hamsters and human anti-CHIKV antibodies.ResultsWe observed high specificity and sensitivity, of 100% and 95.8%, respectively, and these values demonstrate the efficiency of the test as a clinical diagnostic tool. There was no cross-reactivity with samples taken from dengue patients.DiscussionOur simple and sensitive sandwich ELISA for the early-phase detection of CHIKV infection may be used to improve the diagnosis of chikungunya.

Dataset Information

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

Publications

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

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