Inter- and intra-assay variation for the C3C assay.

<p>As quality controls (QC1-5), human sera was used. The controls (CO1 and -2) included in every run of the C3C ELISA were also included. The variation was calculated as the mean variation between 10 individual runs of each sample run in double determination.</p

Patient demographics.

<p>Patient demographics.</p

Products used for cleavage analysis.

<p>Products used for cleavage analysis.</p

Analyte stability in human serum.

<p>The serum samples were either subjected to four freeze/thaw cycles or stored at 4 or 20°C for 0, 2, 4 and 24 hours. All data are shown as mean percent recovery (RE%) compared to baseline (ie, 1 freeze/thaw cycle and storage time 0 hours, respectively).</p

C3C ELISA runs showing typical standard curves and native reactivity against human serum, heparin and EDTA plasma.

<p>(A) Standard curve and inhibition of the competitive ELISA signal using healthy human serum, (B) human heparin plasma and (C) human EDTA plasma. The native material was run from undiluted and up to 8-fold diluted as indicated. (D) Neo-epitope specificity of the C3C ELISA was shown by comparing reactivity towards an elongated peptide, i.e. a peptide with an additional amino acid at the N-terminal generated by cleavage, a non-sense peptide, i.e. a peptide with a different sequence, and the standard peptide. The standard peptide (i.e. standard curve), elongated peptide, and non-sense peptide were diluted 2-fold from 100 ng/mL. The data is presented as percent (%) of background absorbance, which is the absorbance with only assay buffer present, as a function of peptide concentration.</p

C3C levels in COPD patients and healthy controls.

<p>(A) Levels of C3C were measured in serum of patients with COPD (n = 68) and compared to levels in healthy controls (n = 11). Results are shown as geometric mean with 95% confidence intervals. Statistical significance between healthy controls and COPD patients was assessed using the two-tailed Mann-Whitney unpaired t-test. (B) The correlation of the C3C fragment levels in serum and heparin plasma was tested in the cohort of COPD patients (n = 68) by the use of linear regression analysis. Correlation between age and levels of C3C in the healthy controls (C) and COPD patients (D) was analyzed using nonparametric spearman rank analysis. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.</p

Sequence alignment between the alpha-1 chain of type III collagen in humans, rats and mice.

<p>The antibody recognizes the neo-epitope starting from residue 642 to 651 in the human protein (ie, GLPGTGGPPG). 90% homology was observed for the human and mouse sequence due to an amino acid change at the second residue in the target sequence (L → I). 80% sequence homology was observed between the human and rat sequence due to two amino acid changes at the second (L → I) and sixth (G → S) position of the target sequence.</p

Generation of C3C by cleavage analysis of type III collagen.

<p>No reactivity was seen for either of the buffers or intact type III collagen (COL3). The only proteases generating C3C were the cathepsins B, L, S and K. Data are presented as mean ± SEM. Results below the detection limit were given the value of LLOQ. Statistical significance was determined by ANOVA with multiple comparison testing using the Tukey test with type III collagen, without any protease present (COL3), as a reference; ****p<0.0001.</p

Percentage dilution recovery for the C3C assay using human serum and plasma samples.

<p>Samples were measured from undiluted to 1:4 diluted and linearity was assessed.</p

Spiking recovery of standard peptide in human serum, and high serum in low serum.

<p>The recovery (RE%) was calculated as the percentage recovery of the measured amount in the sample alone. The experiment was performed for three separate healthy human control sera. The standard peptide was added in 2-fold dilutions starting from 50 ng/mL (StdB) and high serum was added in 2-fold dilution starting from 1:2.</p