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Am J Transl Res 2013;5(3):303-315
Original Article
The neo-epitope specific PRO-C3 ELISA measures true formation of
type III collagen associated with liver and muscle parameters
Mette J Nielsen, Anders F Nedergaard, Shu Sun, Sanne S Veidal, Lise Larsen, Qinlong Zheng, Charlotte Suetta,
Kim Henriksen, Claus Christiansen, Morten A Karsdal, Diana J Leeming
Nordic Bioscience, Fibrosis Biology and Biomarkers, Herlev, Denmark; Nordic Bioscience, Musculoskeletal
Diseases, Herlev, Denmark; Institute of Sports Medicine Copenhagen, Bispebjerg Hospital, Copenhagen,
Denmark; Nordic Bioscience Beijing, Beijing, China; Department of Diagnostics, Glostrup Hospital, University of
Copenhagen, Denmark
Received February 19, 2013; Accepted March 23, 2013; Epub April 19, 2013; Published April 30, 2013
Abstract: Aim: The present study describes the assessment of true formation of type III collagen in different
pathologies using a neo-epitope specific competitive Enzyme-linked immunosorbent assay (ELISA) towards the
N-terminal propeptide of type III collagen (PRO-C3). Methods: The monoclonal antibody was raised against the
N-protease mediated cleavage site of the N-terminal propeptide of type III collagen and a competitive ELISA was
developed using the selected antibody. The assay was evaluated in relation to neo-epitope specificity, technical
performance, and as a marker for liver fibrosis and muscle mass using the rat carbon tetrachloride (CCl4) model
and a study of immobilization induced muscle loss in humans, respectively. Results: The ELISA was neo-epitope
specific, technically stable and can be assessed in serum and plasma samples. In the CCl4 liver fibrosis model
it was observed that serum PRO-C3 were significantly elevated in rats with liver fibrosis as seen by histology
(56% elevated in the highest quartile of total hepatic collagen compared to control rats, p<0.001) and correlated
significantly to total hepatic collagen in the diseased rats (r=0.46, p<0.01) and not in control rats, suggesting the
pathological origin of the epitope. Human plasma PRO-C3 correlated significantly to muscle mass at baseline
(R2=0.44, p=0.036). Conclusion: The developed neo-epitope specific serum ELISA for type III procollagen
(PRO-C3) reflects true formation as it is specific for the propeptide cleaved off the intact collagen molecule. In a
clinical and in a rodent study we showed that this marker was highly related to liver fibrosis and muscle mass.
(AJTR1302010).
Keywords: Biochemical markers, type III collagen, formation, neo-epitope, liver fibrosis, muscle mass
Address correspondence to: Mette J Nielsen, Nordic Bioscience A/S, Herlev Hovedgade 207, DK-2730 Herlev,
Denmark. Tel: +45 4452 5203; Fax: +45 44525251; E-mail: mju@nordicbioscience.com
Original Article
The neo-epitope specific PRO-C3 ELISA measures true formation of
type III collagen associated with liver and muscle parameters
Mette J Nielsen, Anders F Nedergaard, Shu Sun, Sanne S Veidal, Lise Larsen, Qinlong Zheng, Charlotte Suetta,
Kim Henriksen, Claus Christiansen, Morten A Karsdal, Diana J Leeming
Nordic Bioscience, Fibrosis Biology and Biomarkers, Herlev, Denmark; Nordic Bioscience, Musculoskeletal
Diseases, Herlev, Denmark; Institute of Sports Medicine Copenhagen, Bispebjerg Hospital, Copenhagen,
Denmark; Nordic Bioscience Beijing, Beijing, China; Department of Diagnostics, Glostrup Hospital, University of
Copenhagen, Denmark
Received February 19, 2013; Accepted March 23, 2013; Epub April 19, 2013; Published April 30, 2013
Abstract: Aim: The present study describes the assessment of true formation of type III collagen in different
pathologies using a neo-epitope specific competitive Enzyme-linked immunosorbent assay (ELISA) towards the
N-terminal propeptide of type III collagen (PRO-C3). Methods: The monoclonal antibody was raised against the
N-protease mediated cleavage site of the N-terminal propeptide of type III collagen and a competitive ELISA was
developed using the selected antibody. The assay was evaluated in relation to neo-epitope specificity, technical
performance, and as a marker for liver fibrosis and muscle mass using the rat carbon tetrachloride (CCl4) model
and a study of immobilization induced muscle loss in humans, respectively. Results: The ELISA was neo-epitope
specific, technically stable and can be assessed in serum and plasma samples. In the CCl4 liver fibrosis model
it was observed that serum PRO-C3 were significantly elevated in rats with liver fibrosis as seen by histology
(56% elevated in the highest quartile of total hepatic collagen compared to control rats, p<0.001) and correlated
significantly to total hepatic collagen in the diseased rats (r=0.46, p<0.01) and not in control rats, suggesting the
pathological origin of the epitope. Human plasma PRO-C3 correlated significantly to muscle mass at baseline
(R2=0.44, p=0.036). Conclusion: The developed neo-epitope specific serum ELISA for type III procollagen
(PRO-C3) reflects true formation as it is specific for the propeptide cleaved off the intact collagen molecule. In a
clinical and in a rodent study we showed that this marker was highly related to liver fibrosis and muscle mass.
(AJTR1302010).
Keywords: Biochemical markers, type III collagen, formation, neo-epitope, liver fibrosis, muscle mass
Address correspondence to: Mette J Nielsen, Nordic Bioscience A/S, Herlev Hovedgade 207, DK-2730 Herlev,
Denmark. Tel: +45 4452 5203; Fax: +45 44525251; E-mail: mju@nordicbioscience.com
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