Bioactive Sclerostin: better analyte definition in the measurement of bone remodeling

Introduction:

Sclerostin is a 190-amino acid glycoprotein with highly flexible N- and C-terminal arms. The core consists of a cystine-knot with three loops, whereas loop two binds to the LRP5/6 receptor complex and inhibits the osteoanabolic Wnt-signaling pathway. It is not clear yet, which forms and fragments of sclerostin are circulating. There are no existing tools to detect the bioactive free receptor binding site of sclerostin, which concentration might better reflect bone remodeling in different disease states than the measurement of unknown sclerostin fragments. Therefore, we developed a defined and specific ELISA for the detection of bioactive sclerostin.

Methods:

Our sandwich immunoassay contains a monoclonal capture and a horseradish-peroxidase conjugated affinity-purified polyclonal detection antibody. Both antibodies were characterized regarding their epitopes, affinities and kinetics. The assay performance was validated according to standard guidelines. Measured bioactive sclerostin concentrations in apparently healthy individuals as well as in diseased patients were compared in other commercially available ELISAs.

Results:

Epitope mapping shows the distinct epitope within the bioactive site of sclerostin for the monoclonal capture antibody and five linear epitopes distributed throughout sclerostin for the detection antibody. All validation parameters demonstrate the robustness, accuracy and precision of the assay. The correlation between the assays was dependent on the sample type and disease state of the patients.

Discussion:

This validated ELISA is a tool for the defined detection of bioactive sclerostin in human samples and may be helpful to further investigate sclerostin as a biomarker in the diagnosis of bone remodeling disorders and in the assessment of therapeutic effectiveness.