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
| Autor: | Ravin Jugdaohsingh, Katarzyna S. Kopanska, Sylvaine F. A. Bruggraber, Jonathan J. Powell |
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| Rok vydání: | 2013 |
| Předmět: |
Enzyme-Linked Immunosorbent Assay
Peptide Dermatology Collagen Type I Specimen Handling Dermal fibroblast Western blot medicine Humans Cells Cultured Skin chemistry.chemical_classification Collagen type biology medicine.diagnostic_test Chemistry General Medicine Fibroblasts Serum samples Molecular biology Procollagen peptidase Cell culture Culture Media Conditioned biology.protein Antibody Filtration Procollagen |
| Zdroj: | Archives of Dermatological Research. 305:741-745 |
| ISSN: | 1432-069X 0340-3696 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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