Cartilage Turnover Reflected by Metabolic Processing of Type II Collagen: A Novel Marker of Anabolic Function in Chondrocytes
Abstract
:1. Introduction
2. Results
2.1. Specificity of the Monoclonal Antibody
2.2. Technical Performance of the Pro-C2 ELISA
Assay Specifications | Pro-C2 |
---|---|
Slope of standard curve | 1.03 |
IC50, nM | 7.82 |
Intra-assay CV% | 10.8 |
Inter-assay CV% | 9.2 |
Lower limit of detection, nM | 0.96 |
Quantifiable range, nM | 2.5–19 |
2.3. PIIBNP Levels Measured in Serum of Humans and Other Species
2.4. PIIBNP Release in Bovine Cartilage Explants
2.5. Size of the Detected Fragments in Supernatant
2.6. PIIBNP Measured in Human Cartilage Explants
3. Discussion
Species | PIIBNP (nM) |
---|---|
Human (adult) serum | 1.5 |
Amniotic fluid | 175 |
Fetal bovine serum | 875 |
Bovine serum | 9 |
Fetal horse serum | 415 |
Chicken serum | 0.3 |
Rabbit serum | 0.2 |
4. Experimental Section
4.1. Immunogen Identification, Alignment and Selection
Species | Sequence | NCBI Reference |
---|---|---|
Human Col2α1, isoform 2 (target) | QDVRQPG | NP_149162.2 |
Human Col2α1, isoform 1 | QDV G | NP_001835.3 |
Bovine Col2α1, isoform 1 | QDV G | NP_001001135.2 |
Bovine Col2α1, isoform 2 | QDVRQPG | NP_001106695.1 |
Mouse Col2α1, isoform 1 | QD G | NP_112440.2 |
Mouse Col2α1, isoform 2 | QD G | NP_001106987.2 |
Rat Col2α1 | QD G | NP_037061.1 |
Rabbit Col2α1 | QDV G | NP_001182600 |
Horse Col2α1 | QDVRQPG | NP_001075233.1 |
Chicken Col2α1 | RQPG | NP_989757.1 |
4.2. Generation of the Monoclonal Antibody
4.3. Assay Development
4.4. Bovine and Human Cartilage Explants Culture
4.5. Western Blotting of BEX Supernatant
4.6. Statistics
5. Conclusions
Acknowledgments
Author Contributions
Abbreviations
AGG-C1 | A biomarker of an aggrecan specific neoepitope [19] |
AGNX-II | A biomarker of an aggrecan specific neoepitope [18] |
BEX | Bovine cartilage explants |
Bio | Biotinylated |
C2C | A biomarker of a collagen type II specific neoepitope |
CIIM | A biomarker of a collagen type II specific neoepitope [13] |
CIINE | A biomarker of a collagen type II specific neoepitope [16] |
COMP | A biomarker for cartilage oligomeric matrix protein [20] |
CTX-II | A biomarker of a type II collagen C-terminal telopeptide [14] |
CV | Coefficient of variation |
DJD | Degenerative joint disease |
DMARD | Disease-modifying anti-rheumatic drugs |
DMEM | Dulbecco’s modified Eagle’s medium |
DMOARD | Disease modifying osteoarthritis drug |
ECL | Enhanced chemiluminescence |
ECM | Extra cellular matrix |
FBS | Fetal bovine serum |
FGF | Fibroblastic growth factor |
HAT | Hyposanthine/aminoptrein/thymidine |
HEX | Human cartilage explants |
IGF | Insulin like growth factor |
LLOD | Lower limit of detection |
LLOQ | lower limit of quantification |
MRI | Magnetic resonance imaging |
OA | Osteoarthritis |
PBS | Phosphate buffered saline |
PIIANP | Isotype I of type II procollagen |
PIIBNP | Isotype II of type II procollagen |
PIICP | C-terminal propeptide of type II collagen |
PIINP | N-terminal propeptide of type II collagen |
RA | Rheumatoid arthritis |
SD | Standard deviation |
TBS | Tris-buffered Saline |
TGF | Transforming growth factor |
TIINE | A biomarker of a type II collagen inter-helical fragment [15] |
TMB | Tetramethyl benzinidine |
ULOQ | Upper limit of quantification |
WO | without treatment |
Conflicts of Interest
References
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Gudmann, N.S.; Wang, J.; Hoielt, S.; Chen, P.; Siebuhr, A.S.; He, Y.; Christiansen, T.G.; Karsdal, M.A.; Bay-Jensen, A.C. Cartilage Turnover Reflected by Metabolic Processing of Type II Collagen: A Novel Marker of Anabolic Function in Chondrocytes. Int. J. Mol. Sci. 2014, 15, 18789-18803. https://doi.org/10.3390/ijms151018789
Gudmann NS, Wang J, Hoielt S, Chen P, Siebuhr AS, He Y, Christiansen TG, Karsdal MA, Bay-Jensen AC. Cartilage Turnover Reflected by Metabolic Processing of Type II Collagen: A Novel Marker of Anabolic Function in Chondrocytes. International Journal of Molecular Sciences. 2014; 15(10):18789-18803. https://doi.org/10.3390/ijms151018789
Chicago/Turabian StyleGudmann, Natasja Stæhr, Jianxia Wang, Sabine Hoielt, Pingping Chen, Anne Sofie Siebuhr, Yi He, Thorbjørn G. Christiansen, Morten Asser Karsdal, and Anne Christine Bay-Jensen. 2014. "Cartilage Turnover Reflected by Metabolic Processing of Type II Collagen: A Novel Marker of Anabolic Function in Chondrocytes" International Journal of Molecular Sciences 15, no. 10: 18789-18803. https://doi.org/10.3390/ijms151018789