Detection Method:Sandwich
Test principle:This assay employs a two-site sandwich ELISA to quantitate SIGLEC8 in samples. An antibody specific for SIGLEC8 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and anySIGLEC8 present is bound by the immobilized antibody. After removing any unbound substances, a biotin-conjugated antibody specific for SIGLEC8 is added to the wells. After washing, Streptavidin conjugated Horseradish Peroxidase (HRP) is added to the wells. Following a wash to remove any unbound avidin-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of SIGLEC8 bound in the initial step. The color development is stopped and the intensity of the color is measured.
Product Overview:Floyd et al. (2000) identified a cDNA from an eosinophil cDNA library encoding SIGLEC8. Sequence analysis predicted that like other SIGLECs, the 431-amino acid, type 1 transmembrane protein contains a signal peptide, an N-terminal V-set domain, and 2 C2-set domains, as well as 3 potential N-linked glycosylation sites and a transmembrane region; however, SIGLEC8 has a truncated 47-residue cytoplasmic tail lacking the conserved tyrosine-based motifs.
Binding analysis confirmed that SIGLEC8 binds to red blood cell sialic acids with a preference for 3-prime over 6-prime sialyllactose-conjugated polyacrylamide. FACS and immunoprecipitation analyses demonstrated SIGLEC8 expression on eosinophils but not other leukocytes as a 45- and 89-kD dimer.
