Development of 4-hydroxyproline analysis kit and its application to collagen quantification

Abstract

4-Hydroxyproline (4-Hyp) is a specific amino acid of collagen and widely used as a factor to estimate the collagen content in biological specimens. The authors have developed an analysis kit with the ability to detect collagen on microwells. The method includes chromophore formation without solvent transfers, that allows the analysis of multiple specimens with excellent sensitivity, high specificity at low cost with shorter analysis time. The calibration curve of 4-HYP kit exhibiting a high positive relationship ( R 2 = 0.999) while showing a very low detection limit of (1.0 μg/ml). Specificity of 4-HYP kit was decreased with increasing hydrolysed non-collagenous biomolecules (HNCB), however this was negligible since only a few collagen specimens have a high amount of HNCB. The 4-HYP kit was successfully applied to commercial collagen quantification, measuring the collagen content of connective tissue and collagen synthesis of fibroblast with high satisfactory results; therefore, this is a more suitable alternative to previous analysis methods.