Microplate assay for denatured collagen using collagen hybridizing peptides

Area of Science:

• Biochemistry
• Biomaterials Science
• Orthopaedic Research

Background:

• Denatured collagen is a key biomarker in various musculoskeletal conditions.
• Existing methods for quantifying denatured collagen are often time-consuming and lack high-throughput capabilities.
• There is a need for a rapid and accurate assay to measure denatured collagen.

Purpose of the Study:

• To develop and validate a novel microplate assay for the quantification of denatured collagen.
• To measure denatured collagen in response to mechanical overload in tendon fascicles and in osteoarthritic cartilage.
• To compare the new assay's performance against established methods.

Main Methods:

• Development of a fluorescence-based assay using carboxyfluorescein bound collagen hybridizing peptides (F-CHP).
• Application of Proteinase K for tissue homogenization post-staining.
• Measurement of fluorescence using a microplate reader.
• Validation against image analysis and trypsin-hydroxyproline assays.

Main Results:

• F-CHP selectively binds to denatured collagen.
• Mechanical overload of rat tail tendon fascicles demonstrated a strain-dependent increase in denatured collagen.
• Osteoarthritic cartilage exhibited higher F-CHP fluorescence compared to healthy cartilage.
• The microplate assay provided absolute quantification of denatured collagen comparable to the trypsin-hydroxyproline assay.

Conclusions:

• The developed microplate assay is a rapid, accurate, and high-throughput method for quantifying denatured collagen.
• This assay is effective across multiple collagenous tissues, including tendons and cartilage.
• The F-CHP assay offers a significant improvement in efficiency over existing methods for denatured collagen quantification.