Development and evaluation of C-telopeptide enzyme-linked immunoassay for measurement of bone resorption in mouse serum

Abstract

The mouse is increasingly being used as an animal model for the study of skeletal phenotypes in humans, mainly because of the ease of genetic manipulation. Biochemical markers of bone metabolism provide a valuable parameter for the assessment of skeletal metabolism. In the mouse model, assays for bone formation have been available for a long time; however, little is known about bone resorption markers. The present study describes the development of a serum C-telopeptide enzyme-linked immunoassay (ELISA), which measures degradation products of type I collagen that are generated by osteoclastic bone resorption. The C-telopeptide ELISA uses affinity-purified antibodies generated against human sequence DFSFLPQPPQEKAHDGGR. The epitope involves an amino acid sequence, which is identical in the mouse and human C-terminal peptide of type I collagen (alpha1 chain). Sensitivity of the ELISA used was <0.1 ng/mL. The average intra- (n = 10) and interassay (n = 8) coefficient of variation for two controls was <12%. The average dilution and spike recovery rates were 98% and 97%, respectively. Application of the ELISA to measure C-telopeptide in 3-4-week postovariectomized (ovx) C57BL/6J (B6) mice (n = 9 or 10) showed a 45% higher C-telopeptide concentration than the sham-operated mice. Treatment of ovx mice with estradiol (400 microg/kg body weight) or alendronate (1.0 mg/kg body weight) resulted in a 20%-50% decrease in C-telopeptide levels compared to the vehicle-treated ovx group. In addition, B6 mice fed a calcium-deficient diet (0.01% calcium) showed a 50% higher C-telopeptide concentration compared to the B6 mice receiving a normal diet (0.6% calcium). In conclusion, the C-telopeptide ELISA exhibited acceptable analytical performance and sufficient discriminatory power to show expected directional changes in the rate of bone resorption following ovariectomy, ovx plus estradiol or alendronate treatment, and administration of a calcium-deficient diet. Therefore, the ELISA developed in this study could be used for measuring bone resorption in the mouse model.