Novel sandwich ELISAs for rat DMP1: age-related decrease of circulatory DMP1 levels in male rats

Area of Science:

• Biochemistry
• Bone Biology
• Assay Development

Background:

• Dentin matrix protein 1 (DMP1) is crucial for bone mineralization and phosphate homeostasis, produced by osteocytes.
• Current limitations in measuring circulating DMP1 hinder understanding its biological roles.
• DMP1 undergoes cleavage into N- and C-terminal fragments during secretion.

Purpose of the Study:

• To develop and validate sensitive sandwich ELISAs for quantifying N- and C-terminal rat DMP1 fragments.
• To investigate the presence and levels of circulating DMP1 in rats across different ages.
• To explore correlations between circulating DMP1 and other bone turnover markers.

Main Methods:

• Development of two sandwich ELISAs (ELISA 1-2 for N-terminal, ELISA 4-3 for C-terminal DMP1) using specific polyclonal antibodies.
• Characterization of antibodies via immunohistochemistry and LC-MS/MS.
• Measurement of circulating DMP1 and bone markers (osteocalcin, Trap5b, Dkk-1, SOST) in Wistar rats aged 2-96 weeks.

Main Results:

• Immunohistochemistry confirmed DMP1 expression in osteocytes and matrix; LC-MS/MS detected DMP1 in rat plasma.
• Both ELISAs demonstrated high specificity, reproducibility, and accuracy.
• Circulating DMP1 levels, measured by both ELISAs, significantly decreased with age.
• ELISA 4-3 showed higher DMP1 levels than ELISA 1-2 during rapid growth, with levels converging in older rats.
• DMP1 levels positively correlated with Dkk-1 and osteocalcin, and less so with Trap5b and SOST.

Conclusions:

• Novel, highly specific sandwich ELISAs enable precise measurement of circulating rat DMP1 fragments.
• Circulating DMP1 levels decline with age, reflecting changes in bone turnover.
• These ELISAs offer a valuable tool for studying DMP1's role and its potential as a biomarker for osteocyte-mediated bone remodeling.