Microplate Assay for Denatured Collagen using Collagen Hybridizing Peptides

ABSTRACTThe 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 com pared 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 tensil e 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 (<7.5%) exhibited minimal denature collagen, but values rapidly increased at medium strains (7.5 ‐10.5%) and plateaued at high strains (≥12%). Osteoarthritic cartilage had higher F‐CHP fluorescence than healthy cartilage. Both of these outcomes are consistent with previous studies. With the calibration curve, the microplate assay was able to absolutely quantify denat...
Source: Journal of Orthopaedic Research - Category: Orthopaedics Authors: Tags: Research Article Source Type: research