Precision of the M10 ELISA.

<p>Precision of the M10 ELISA.</p

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

<div><p>Objective</p><p>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.</p><p>Experimental design</p><p>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.</p><p>Results</p><p>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.</p><p>Conclusions</p><p>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.</p></div

The pharmacokinetic parameters of M10.

<p>The pharmacokinetic parameters of M10.</p

Effects of primary antibody concentrations on sensitivity in M10 ELISA.

<p>A series of primary antibody dilutions (1:500, 1:600, 1:700, 1:800, 1:900 and 1:1000) in PBS were tested against 1:10,000 (0.2 μg /ml) concentration of detection antibody. Each data point represents the mean value of the corresponding triplicates.</p

The calibration curves of M10.

<p>Individual lines represent independent experiment and each data point represents the mean value of the corresponding triplicates. Absorbance of standards was measured at 450 nm and graph was generated by using 4-parametric logistic regression model.</p

Analytical recovery of M10.

<p>Analytical recovery of M10.</p

Specification of M10 ELISA.

<p>Specification of M10 ELISA.</p

Pharmacokinetic profile of M10 in mouse plasma.

<p>Plasma was collected over a period of 15 minutes—48 hours after M10 injection and measured by indirect M10 ELISA. Plot represents the experimentally estimated M10 concentration over time course to model prediction following IP injection in mice.</p

Coefficient of determination (R²) values corresponding to antibodies, concentrations and condition.

<p>Coefficient of determination (R²) values corresponding to antibodies, concentrations and condition.</p

The predicted half-life of M10 in plasma was estimated by ProtLifePred software.

<p>Experimental half-life of M10 (6.8 ± 0.7 h) in mouse plasma complies with predicted half-life of M10 (7.2 h).</p