Figure 5.

Establishment and validation of a sandwich ELISA for PICP quantification. (a) Schematic representation of the developed sandwich ELISA (see text for details). (b) Standard curve of the developed sandwich ELISA for quantification of recombinant human PICP. The data was fitted into a 4PL model to assess the correlationship between absorbance and the increasing concentration of recombinant PICP (r ² = 0.99). (c) Western blotting to monitor the levels of PICP, type 1 procollagen, and phosphorylated Smad2 in LX-2 cells. For western blot analysis, an equal quantity of LX-2 cellular lysate was loaded using β-actin as a loading control. The number below the panel indicates the relative value of band intensity of the proteins compared with that of control after normalization of the band intensity to that of β-actin for each sample. The corresponding full-length blots are shown in Supplementary Fig. 7. (d) Quantification of PICP levels in the cellular extracts and culture supernatants of TGF-β stimulated LX-2 cells using the developed sandwich ELISA and the commercially available Takara ELISA kit (MK101). Error bars, ±SD (n = 3). (c,d) LX-2 cells were treated with medium (control) or the indicated concentrations of TGF-β for 12 h, and then the soluble cellular extracts and culture supernatants were subjected to the analyses. (e) Measurements of PICP levels in a normal human serum diluted at the indicated rate using the developed sandwich ELISA kit and the commercially available Takara ELISA kit (MK101). Error bars, ±SD (n = 3). Inset shows the equation for the fitted line and the correlation coefficient value (r ²).