Journal Article

Establishment of an indirect ELISA for detection of the novel antifibrotic peptide M10

Atsushi Noguchi, Ilia Atanelishvili, Galina S. Bogatkevich, Richard M. Silver, Tanjina Akter

2017

Overview

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. 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. 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. 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.

• Publisher: Public Library of Science (PLoS),Public Library of Science
• Subject: Amino acids / Animals / Antibodies / Antibodies - metabolism / Biology and Life Sciences / Biotechnology / c-Met protein / Calibration / Caspase / Caspase-3 / Coefficient of variation / Diagnosis / Disease / Drug dosages / Enzyme-Linked Immunosorbent Assay / Enzyme-Linked Immunosorbent Assay - methods / Enzymes / Experimental design / FDA approval / Female / Fibroblasts / Fibrosis / Genetic aspects / Growth factors / Half life / Hepatocyte growth factor / Immunoassay / Immunology / Laboratories / Life prediction / Limit of Detection / Male / Medicine / Medicine and Health Sciences / Mice, Inbred C57BL / Peptides / Peptides - analysis / Peptides - blood / Peptides - pharmacokinetics / Peptides - therapeutic use / Pharmacokinetics / Pharmacology / Physiological aspects / Plasma / Pulmonary fibrosis / Q / Quality / R / Research and Analysis Methods / Research Article / Rheumatology / Science / Sensitivity and Specificity / Signal transduction / Smad2 protein / Stem cells