AffinityImmuno Products

Introduction

Siglec-15 is a sialic-acid-recognizing receptor expressed at the cell surface, where its charged transmembrane segment allows it to pair with the signaling adaptors DAP10 or DAP12 rather than signaling on its own. Its expression becomes concentrated on osteoclast precursors once they are stimulated with RANKL and CSF-1, and through the DAP12 partnership it shapes PI3K/Akt and ERK signaling that governs how these precursors fuse and mature into active, bone-resorbing osteoclasts. Consistent with this role, mice lacking Siglec-15 develop a mild osteopetrosis-like bone phenotype marked by reduced osteoclast activity, and monoclonal antibodies that block Siglec-15 can similarly suppress osteoclast maturation and bone resorption in experimental models. Because of this role, Siglec-15 has drawn interest as a therapeutic target for osteoporosis and other diseases marked by excessive osteoclast-driven bone loss.

Beyond its established role in osteoclast maturation and bone resorption, Siglec-15 has more recently emerged as a PD-L1-independent immune checkpoint expressed on tumor-associated macrophages, drawing interest as a target in cancers that respond poorly to PD-1/PD-L1 blockade — making this assay useful for both bone-biology and tumor-immunology research.

Principle of the Assay

This kit uses a sandwich enzyme immunoassay format in which the microtiter plate wells are pre-coated with a capture antibody specific to Human Siglec-15. Standards or samples are added to the appropriate wells together with a biotin-conjugated detection antibody that also recognizes Human Siglec-15. Avidin conjugated to horseradish peroxidase (HRP) is then added to each well and allowed to incubate. Upon addition of TMB substrate solution, a colorimetric signal develops exclusively in wells containing Human Siglec-15, the biotin-conjugated detection antibody, and the avidin-HRP conjugate. The enzymatic reaction is stopped by addition of a sulphuric acid solution, and absorbance is measured spectrophotometrically at 450 nm ± 10 nm. Human Siglec-15 concentrations in the samples are determined by comparing sample optical density values against a standard curve.