AffinityImmuno Products

Introduction

Siglec-8 is a sialic-acid-binding lectin receptor that, in humans, is expressed almost exclusively on eosinophils and mast cells, with lower levels on basophils, making it a useful marker of terminally differentiated eosinophils. It recognizes glycan structures bearing a terminal sialic acid together with an underlying 6-O-sulfated galactose, most notably 6’-sulfo-sialyl Lewis X, and this recognition can be limited by sialic acids already present on the same cell surface competing for the binding site. On the cytoplasmic side, Siglec-8 carries an immunoreceptor tyrosine-based inhibitory motif along with a second motif that recruits the SLAM-associated adaptor protein, wiring the receptor into inhibitory signaling circuits. Engaging Siglec-8, whether with natural glycan ligands or with antibodies, triggers integrin-dependent adhesion followed by reactive oxygen species generation and programmed cell death in eosinophils, and separately suppresses mast cell degranulation, properties that have made it an actively pursued target for treating eosinophil- and mast-cell-driven allergic diseases.

Its expression restricted almost entirely to eosinophils and mast cells has made Siglec-8 a clinical drug target in eosinophilic and mast-cell-driven diseases such as eosinophilic gastritis/esophagitis, asthma, and chronic urticaria, positioning this assay for biomarker and pharmacodynamic studies in that growing therapeutic area.

Principle of the Assay

This kit uses a sandwich enzyme immunoassay format to detect and quantify Human Siglec-8. Microtiter plate wells are pre-coated with a Siglec-8–specific capture antibody that binds the target analyte present in standards or samples. A biotin-conjugated detection antibody, also specific to Human Siglec-8, is then added and binds a second epitope on the captured analyte. Avidin conjugated to horseradish peroxidase (HRP) is subsequently introduced and binds to the biotin label. Upon addition of TMB substrate solution, a colorimetric signal develops only in wells where Human Siglec-8, the biotin-conjugated detection antibody, and avidin-HRP are all present. The enzymatic reaction is stopped by the addition of a sulphuric acid solution, and absorbance is measured spectrophotometrically at 450 nm (± 10 nm). Sample concentrations are determined by comparing sample optical density values against a standard curve.