Open AccessEstablishment of an indirect ELISA for detection of the novel antifibrotic peptide M10
Author(s) -
Tanjina Akter,
Ilia Atanelishvili,
Atsushi Noguchi,
Richard M. Silver,
Galina S. Bogatkevich
Publication year - 2017
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0188588
Subject(s) - peptide , detection limit , immunoassay , coefficient of variation , chemistry , in vivo , microbiology and biotechnology , chromatography , antibody , biochemistry , medicine , biology , immunology
Objective M10 is a ten amino acid peptide generated from the intracellular cytoplasmic tail of the hepatocyte growth factor (HGF) receptor c-Met following cleavage by caspase-3. Recently we reported that M10 interacts with Smad2 and demonstrates antifibrotic properties in vitro and in vivo and can be advanced into a novel antifibrotic remedy. The current study was undertaken to develop an immunoassay to measure M10 concentration in biological specimens. Experimental design An Indirect Enzyme-Linked Immunosorbent Assay (ELISA) for detection of M10 in biological fluids was developed using pharmaceutical grade synthetic M10 as a calibrator and commercially available anti-c-Met C12 antibody. Results M10 ELISA specifically detected in plasma M10, but not a scrambled peptide, following a single intraperitoneal administration of M10 (1mg/kg) to mice. The detection limit was 9.6 ng/ml, and the measuring limit was between 15 ng/ml and 200 ng/ml. The recovery limits of M10 were between 80% and 120%; intra-assay coefficient of variation was between 5.3% and 6.3%; inter-assay coefficient of variation was between 5.0% and 8.0% over the buffer concentration tested in the range from 15 ng /ml to 250 ng /ml. The peak of M10 concentration following a single intraperitoneal injection (1mg/kg) was achieved within 6 hours and declined to minimal levels by 48 hours. The experimentally obtained half-life for M10 was comparable to the theoretically predicted half-life for M10. Conclusions We have established a highly sensitive ELISA to detect the antifibrotic peptide M10 in plasma samples, which should prove to be a novel tool to study the pharmacokinetics and efficacy of M10 in the treatment of fibroproliferative disorders.