Data comparing the plasma levels of procollagen C-proteinase enhancer 1 (PCPE-1) in healthy individuals and liver fibrosis patients

This article provides a protocol for determination of human procollagen C-proteinase enhancer 1 (PCPE-1) concentrations by ELISA. The inter-assay and intra-assay coefficients of variability are given and so are the average plasma concentrations of PCPE-1 in healthy (control) individuals and liver fibrosis patients

Correction: Procollagen C-Proteinase Enhancer 1 (PCPE-1) as a Plasma Marker of Muscle and Liver Fibrosis in Mice.

[This corrects the article DOI: 10.1371/journal.pone.0159606.]

Sandwich ELISA for quantification of mPCPE-1.

<p>(A) Standard curve. Increasing amounts of purified mPCPE-1 were adsorbed to wells pre-coated with a rat monoclonal antibody to mPCPE-1. Bound mPCPE-1 was detected using a goat antibody to mPCPE-1 and quantified using an APA-conjugated rabbit anti goat IgG antibody. Each value represents mean ± standard deviation (SD); n = 2. mOD, Optical Density expressed in milli units; R², coefficient of determination. (B) The ELISA is specific and permits determination of PCPE-1 concentration in mouse plasma. Plasma samples from a six weeks old C57/BL/6 mouse were subjected to IP with either goat antibody to mPCPE-1 (black) or a rabbit antibody to <i>Pseudomonas</i> elastase (negative control; grey). PCPE-1 concentrations in untreated plasma (white) and in plasma samples that underwent IP were determined using the sandwich ELISA. Values are mean ± SD (n = 4). Results are from a representative experiment out of two. <b>IP</b>, immunoprecipitation; <b>Ab</b>, antibody.</p

Plasma concentrations of PCPE-1 reflect the progression of liver fibrosis in CCl₄-treated mice.

<p>C57/BL/6 mice were given CCl₄ for 6 weeks and then remained untreated for 7 additional weeks. Liver fibrosis was assessed at the indicated times by Sirius red staining, immunoblotting, and immunofluorescence analyses. Plasma concentrations of PCPE-1 were determined by the sandwich ELISA at the indicated time points. <b>(A</b>) Sirius Red staining of liver sections from mice euthanized at weeks 6, 10 and 13. Each panel shows results for a single randomly selected mouse from the indicated group. <b>Red</b>, collagen; <b>Green</b>, non-collagenous proteins. <b>(B</b>)Immunofluorescence for type I collagen and PCPE-1 in liver sections from CCl₄-treated and control mice at different times (given in brackets as the number of weeks). Each panel displays results for a single randomly selected mouse from the indicated group. Collagen type I (red) and PCPE-1 (green) were detected using Rhodamine- and Cy2-conjugated secondary antibodies at 488 and 580 nm, respectively. <b>(C)</b> Plasma concentrations of PCPE-1 after 6 weeks of CCl₄ administration are significantly higher than those of control mice (n = 12). <b>(D</b>) Plasma concentrations of PCPE-1 in CCl₄-treated (▲) and control (●) mice as a function of time. <b>Arrow,</b> termination of CCl₄ treatment. Results are presented as mean ± SD. (n at weeks 0 to 6 = 12; n at weeks 7–10 = 9; n at week 13 = 6). <b>(E)</b> Immunoblot evaluating collagen I and PCPE-1 content in liver extracts from CCl₄-treated and control mice at weeks 6, 10 and 13. Samples from a single control mouse and two CCl₄-treated mice (all randomly selected) were analyzed. The amounts of protein applied on each lane were 4 and 36 μg for collagen and PCPE-1 detection, respectively.</p

Plasma concentrations of PCPE-1 in <i>mdx</i> mice are higher than in the respective controls and the increase is comparable to that of PIIINP.

<p><b>(A)</b> Plasma concentrations of PCPE-1 were determined using the current sandwich ELISA. Results are presented as the mean ± SD (n = 8). (<b>B)</b> Plasma concentrations of PIIINP were determined using a commercial kit. Each plasma sample was diluted 10 fold in blocking buffer and PIIINP concentrations were determined in duplicates. The results are presented as mean ± SD (n = 4 rather than 8 as in the case of PCPE-1 determination). This has to do with the high cost of the PIIINP kit. Nonetheless, and importantly, the results derived from four plasma samples were statistically valid.</p

CCl₄-induced liver fibrosis in mice is accompanied by increased PCPE-1 plasma concentration.

<p>CCl₄ was given to C57/BL/6 mice for 6 weeks as detailed in Methods. <b>(A)</b> Sirius red staining of liver sections from randomly selected control and CCl₄-treated mice shows increased collagen expression in the liver of the CCl₄-treated mouse. <b>Red</b>, collagen; <b>Green</b>, non-collagenous proteins. <b>(B)</b> Immunoblot showing increased amounts of collagen type I and PCPE-1 in livers from CCl₄-treated mice as compared to control mice. Each lane represents liver extract from a single randomly selected mouse of the indicated group. The amount of protein applied on each lane was 5 and 24 μg for collagen type I and PCPE-1 detection, respectively. Samples from 4 (out of 8) mice were analyzed per group. α1 (I), alpha 1 chain of type I collagen. (<b>C)</b> Plasma concentrations of PCPE-1 in CCl₄-treated mice are significantly higher than those of control mice (n = 8).</p

Diaphragms from <i>mdx</i> mice show increased collagen type I and PCPE-1 levels (A,B) and both are co-localized in the diaphragm tissue (C).

<p><b>(A)</b> Sirius red staining of diaphragm sections from 4 and 8.5 months old <i>mdx</i> mice and age-matched C57/BL/6 (control, wt) (one mouse per group). <b>Red</b>, collagen; <b>Green</b>, non-collagenous proteins. (<b>B)</b> Immunoblot showing increased amounts of collagen type I and PCPE-1 in protein extracts of diaphragms from <i>mdx</i> mice relatively to corresponding wild type (wt) controls (one mouse per group). The amounts of protein applied on each lane were 1 and 25 μg for collagen type I and PCPE-1 detection, respectively. α1 (I), alpha 1 chain of type I collagen. <i>kDa</i>, kilo Dalton. (<b>C)</b> Immunofluorescence analysis for type I collagen and PCPE-1 in diaphragms from a four months old C57/BL/6 (wt) and an aged matched <i>mdx</i> mouse. Collagen type I (red) was detected using Rhodamine-conjugated secondary antibody and PCPE-1 (green) was visualized using a Cy2-conjugated secondary antibody. Light emission was examined at 488 and 580 nm for Rhodamine and Cy2, respectively. Results displayed in each panel are from a representative randomly selected mouse from each group.</p