Microplate Assay for Denatured Collagen using CollagenHybridizing Peptides

Abstract

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 (<7.5%) exhibited minimal denature collagen, but values rapidly increased at medium strains (7.5-10.5%) and plateaued at high strains ([&ge;]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 denatured collagen in mechanically damaged rat tail tendon fascicles as reliably as the trypsin-hydroxyproline assay. Further, we achieved these results more efficiently than current methods in a rapid, high-throughput manner, with multiple types of collagenous tissue while maintaining accuracy.