Archives of Dermatological Research

, Volume 305, Issue 8, pp 741–745 | Cite as

Filtration of dermal fibroblast-conditioned culture media is required for the reliable quantitation of cleaved carboxy-terminal peptide of collagen type I (CICP) by ELISA

  • Katarzyna S. Kopanska
  • Jonathan J. Powell
  • Ravin Jugdaohsingh
  • Sylvaine F. A. Bruggraber
Short Communication


Cleavage of the collagen type I carboxy-terminal peptide (CICP) from the procollagen molecule is an essential step in collagen biosynthesis. The commercial CICP ELISA (Quidel Corporation, USA), developed for quantifying CICP in serum in clinical monitoring, is often also applied to cellular studies as a measure of collagen synthesis. However, unlike in serum samples, which contain only cleaved CICP, cell-conditioned culture media also contains “uncleaved CICP”, namely procollagen, and there is no specific guidance on how to interpret the ELISA data obtained with such samples. Here we attempted to reliably quantify cleaved CICP in human dermal fibroblast-conditioned cell culture media using the CICP ELISA. CICP concentration was determined in the parent and filtered samples of culture media of dermal fibroblasts (CCD-25SK). Gel-separated samples were also subjected to protein staining or analyzed by Western blot using the anti-CICP antibodies supplied in the ELISA kit. The derived concentrations of CICP in the filtered aliquots and the parent unfiltered samples increased over time. The increase in CICP in the unfiltered samples was not proportional to the increase seen in the filtered aliquot. CICP ELISA antibodies recognized both the cleaved CICP trimer and procollagen molecule. The data presented show that (a) the commercial CICP ELISA recognizes both procollagen and cleaved CICP in cell-conditioned culture media and thus attention should be paid in interpreting data from cell culture studies using this ELISA and (b) the filtration method described herein can be used to exclusively and reliably monitor cleaved CICP.


Collagen type I carboxy-terminal peptide (CICP) Collagen type I Procollagen Dermal fibroblasts Cell culture 


  1. 1.
    Akiyama M, Nakamura M (2009) Bone regeneration and neovascularization processes in a pellet culture system for periosteal cells. Cell Transplant 18(4):443–452PubMedCrossRefGoogle Scholar
  2. 2.
    Almqvist S, Werthen M, Johansson A, Tornqvist J, Agren MS, Thomsen P (2009) Evaluation of a near-senescent human dermal fibroblast cell line and effect of amelogenin. Br J Dermatol 160(6):1163–1171. doi:10.1111/j.1365-2133.2009.09071.x PubMedCrossRefGoogle Scholar
  3. 3.
    Bigot N, Beauchef G, Hervieu M, Oddos T, Demoor M, Boumediene K, Galera P (2012) NF-kappaB accumulation associated with COL1A1 transactivators defects during chronological aging represses type I collagen expression through a −112/−61-bp region of the COL1A1 promoter in human skin fibroblasts. J Invest Dermatol 132(10):2360–2367. doi:10.1038/jid.2012.164 PubMedCrossRefGoogle Scholar
  4. 4.
    Canty EG, Kadler KE (2005) Procollagen trafficking, processing and fibrillogenesis. J Cell Sci 118(Pt 7):1341–1353. doi:10.1242/jcs.01731 PubMedCrossRefGoogle Scholar
  5. 5.
    Chesnutt BM, Yuan Y, Buddington K, Haggard WO, Bumgardner JD (2009) Composite chitosan/nano-hydroxyapatite scaffolds induce osteocalcin production by osteoblasts in vitro and support bone formation in vivo. Tissue Eng Part A 15(9):2571–2579. doi:10.1089/ten.tea.2008.0054 PubMedCrossRefGoogle Scholar
  6. 6.
    Fratzl P (2008) Collagen. Structure and mechanics. Springer, New YorkGoogle Scholar
  7. 7.
    Gelse K, Poschl E, Aigner T (2003) Collagens—structure, function, and biosynthesis. Adv Drug Deliv Rev 55(12):1531–1546 pii: S0169409X03001820PubMedCrossRefGoogle Scholar
  8. 8.
    Krishna P, Rosen CA, Branski RC, Wells A, Hebda PA (2006) Primed fibroblasts and exogenous decorin: potential treatments for subacute vocal fold scar. Otolaryngol Head Neck Surg 135(6):937–945. doi:10.1016/j.otohns.2006.07.011 PubMedCrossRefGoogle Scholar
  9. 9.
    Lundquist R, Dziegiel MH, Agren MS (2008) Bioactivity and stability of endogenous fibrogenic factors in platelet-rich fibrin. Wound Repair Regen 16(3):356–363. doi:10.1111/j.1524-475X.2007.00344.x PubMedCrossRefGoogle Scholar
  10. 10.
    Panzavolta S, Torricelli P, Bracci B, Fini M, Bigi A (2009) Alendronate and Pamidronate calcium phosphate bone cements: setting properties and in vitro response of osteoblast and osteoclast cells. J Inorg Biochem 103(1):101–106. doi:10.1016/j.jinorgbio.2008.09.012 PubMedCrossRefGoogle Scholar
  11. 11.
    Schmidmaier G, Wildemann B, Lubberstedt M, Haas NP, Raschke M (2003) IGF-I and TGF-beta 1 incorporated in a poly(d, l-lactide) implant coating stimulates osteoblast differentiation and collagen-1 production but reduces osteoblast proliferation in cell culture. J Biomed Mater Res B Appl Biomater 65(1):157–162. doi:10.1002/jbm.b.10513 PubMedCrossRefGoogle Scholar
  12. 12.
    Vester H, Wildemann B, Schmidmaier G, Stockle U, Lucke M (2010) Gentamycin delivered from a PDLLA coating of metallic implants: in vivo and in vitro characterisation for local prophylaxis of implant-related osteomyelitis. Injury 41(10):1053–1059. doi:10.1016/j.injury.2010.05.010 PubMedCrossRefGoogle Scholar
  13. 13.
    Winterbottom N, Vernon S, Freeman K, Daniloff G, Seyedin S (1993) A serum immunoassay for the C-terminal propeptide of Type-I collagen. J Bone Mine Res 8(Suppl 1):S341Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Katarzyna S. Kopanska
    • 1
  • Jonathan J. Powell
    • 1
  • Ravin Jugdaohsingh
    • 1
  • Sylvaine F. A. Bruggraber
    • 1
  1. 1.MRC Human Nutrition ResearchCambridgeUK

Personalised recommendations