Microplate assay for denatured collagen using collagen hybridizing peptides
| Autor: | Jared L. Zitnay, Yang Li, Jeffrey A. Weiss, Allen H. Lin, S. Michael Yu |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
030203 arthritis & rheumatology
Mechanical overload biology Chemistry Cartilage 0206 medical engineering 02 engineering and technology Osteoarthritis medicine.disease Proteinase K 020601 biomedical engineering Fluorescence Staining Tendon Microplate Reader 03 medical and health sciences 0302 clinical medicine medicine.anatomical_structure medicine Biophysics biology.protein Orthopedics and Sports Medicine |
| Zdroj: | Journal of Orthopaedic Research®. 37:431-438 |
| ISSN: | 0736-0266 |
| DOI: | 10.1002/jor.24185 |
| Popis: | The purpose of this study was to develop a microplate assay for quantifying denatured collagen by measuring the fluorescence of carboxyfluorescein bound collagen hybridizing peptides (F-CHP). We have shown that F-CHP binds selectively with denatured collagen, and that mechanical overload of tendon fascicles causes collagen denaturation. Proteinase K was used to homogenize tissue samples after F-CHP staining, allowing fluorescence measurement using a microplate reader. We compared our new assay to our previous image analysis method and the trypsin-hydroxyproline assay, which is the only other available method to directly quantify denatured collagen. Relative quantification of denatured collagen was performed in rat tail tendon fascicles subjected to incremental tensile overload, and normal and ostoeoarthritic guinea pig cartilage. In addition, the absolute amount of denatured collagen was determined in rat tail tendon by correlating F-CHP fluorescence with percent denatured collagen as determined by the trypsin-hydroxyproline assay. Rat tail tendon fascicles stretched to low strains ( |
| Databáze: | OpenAIRE |
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