title: Immunological detection of the type V collagen propeptide fragment, PVCP-1230, in connective tissue remodeling associated with liver fibrosis.
abstract: OBJECTIVE
  Liver fibrosis involves excessive remodeling and deposition of fibrillar extracellular matrix (ECM) components, which leads to malfunction of the organ, causing significant morbidity and mortality. The aim of this study was to assess whether levels of a type V collagen fragment, the propeptide CO5-1230, indicate the amount of collagen deposited during liver fibrosis.
  
  
  METHODS
  A specific competitive enzyme-linked immunosorbent assay (ELISA) was developed to measure CO5-1230 levels. The sequence TAALGDIMGH located at the start of the C-terminal propeptide between amino acid position 1230' and 1239' (CO5-1230) of the α2 chain was selected as the immunogen. Monoclonal antibodies were raised against this fragment. An assay developed using the biotin-streptavidin system was evaluated in two rat models of liver fibrosis: bile duct ligation (BDL) and carbon tetrachloride (CCl(4))-treated rats, for up to 20 weeks.
  
  
  RESULTS
  The ELISA was capable of measuring CO5-1230 in serum specifically, with an intra-assay variation of 3.46% and inter-assay variation of 5.09%. Mean CO5-1230 levels were significantly elevated in CCl(4) rats compared with controls [8 weeks: 57.4 ng/mL, controls 45.5 ng/mL (P = 0.0020); 12 weeks: 81.3 ng/mL, controls 50.2 ng/mL (P = 0.0020); 16 weeks: 85.1 ng/mL, controls 51 ng/mL (P = 0055); 20 weeks: 92 ng/mL, controls 47.8 ng/mL (P = 0.0033)]. CO5-1230 levels correlated with the total amount of collagen in sections from the injured livers, quantified from Sirius red stains (Spearman, R(2) = 0.5580). In BDL rats, serum levels of CO5-1230 were also elevated compared with controls [2 weeks: 160.1 ng/mL, controls 78.9 ng/mL (P = 0.0007); 4 weeks: 111.3 ng/mL, controls 62.2 ng/mL, (P = 0.0068)] and showed a linear correlation to the total collagen content (Spearman, R(2) = 0.3305).
  
  
  CONCLUSIONS
  Increased serum levels of CO5-1230 were associated with the extent of collagen deposition in two different models of fibrotic processes in the liver. The data indicate that formation of type V collagen may be of value as a disease-specific diagnostic biomarker that reflects the total burden of disease. The amino acid sequence selected is located in the first 10 amino acids of the C-terminal propeptide section, which is a formation-specific region.
authors: Nordic Bioscience A/S, Herlev, Denmark. eva@nordicbioscience.com
authors: Efstathios
authors: E
authors: Vassiliadis
authors: Sanne S
authors: SS
authors: Veidal
authors: Henrik
authors: H
authors: Simonsen
authors: Dorthe V
authors: DV
authors: Larsen
authors: Ben
authors: B
authors: Vainer
authors: Xiaoliang
authors: X
authors: Chen
authors: Qinlong
authors: Q
authors: Zheng
authors: Morten A
authors: MA
authors: Karsdal
authors: Diana J
authors: DJ
authors: Leeming