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

PLoS One. 2017 Nov 27;12(11):e0188588. doi: 10.1371/journal.pone.0188588. eCollection 2017.

Abstract

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.

MeSH terms

  • Animals
  • Antibodies / metabolism
  • Calibration
  • Enzyme-Linked Immunosorbent Assay / methods*
  • Female
  • Fibrosis
  • Half-Life
  • Limit of Detection
  • Male
  • Mice, Inbred C57BL
  • Peptides / analysis*
  • Peptides / blood
  • Peptides / pharmacokinetics
  • Peptides / therapeutic use*
  • Sensitivity and Specificity

Substances

  • Antibodies
  • Peptides