预期应用
ELISA法定量测定大鼠血清、血浆、细胞培养物上清或其它相关液体中 P-selectin含量。
实验原理
本试剂盒应用双抗体夹心酶标免疫分析法测定标本中P-selectin水平。用纯化的抗体包被微孔板，制成固相抗体，往包被单抗的微孔中依次加入P-selectin抗原、生物素化的抗大鼠P-selectin抗体、HRP标记的亲和素，经过彻底洗涤后用底物TMB显色。TMB在过氧化物酶的催化下转化成蓝色，并在酸的作用下转化成最终的黄色。颜色的深浅和样品中的P-selectin呈正相关。用酶标仪在450nm波长下测定吸光度（OD值），计算样品浓度。
Intended use
This immunoassay kit allows for the specific measurement of rat P-selectin concentrations in cell culture supernates, serum, and plasma.
Introduction
GMP-140 (P-selectin), also known as CD62 or PADGEM, is a 140 kDa integral membrane glycoprotein functionally categorized as a member of the LEC-CAM family (lectin-epidermal growth factor-complement binding cell adhesion molecules), or the selectin family. In non-activated conditions, GMP-140 is present in the α-granules of platelets or in the Weibel Palade bodies of endothelial cells. However, when activated by stimulators such as thrombin or by inflammatory or hemostatic mediators during intravascular inflammation, GMP-140 is rapidly secreted to the plasma membrane to promote adhesion of cells to vascular endothelia or to neutrophils and monocytes at the site of tissue injury. Previous studies suggested that GMP-140 is indeed an useful marker of platelet and endothelium activation in experimental inflammation models, and potentially in clinical situations. GMP-140 is also known to interact with leukocytes by a Ca2+-dependent lectin-like mechanism. GMP-140 has recently been cloned from endothelia and is found to be highly homologous to two other LEC-CAM adhesion molecules, the ELAM-1 and the Mel-14 antigen, both of which are involved in various aspects of leukocyte adhesion. Analysis of the endothelium cDNA suggests that there are three variants of GMP-140 resulting from alternative splicing: two transmembrane forms with different numbers of complimentbinding consensus repeats, and one putative cytoplasmic lacks the hydrophobic transmembrane domain. Recently it was shown that GMP-140 molecules extracted by Triton X-100 from platelet membranes polymerize into soluble tetramers when returned to detergent-free conditions, and that this tetramer strongly inhibits the adhesion of activated neutrophils to endothelia. This is evidence that GMP-140- mediated adhesion works via a saturable GMP-140 receptor on the surface of myeloid cells. This also suggests that if free GMP-140 molecules are at all present in the circulation, they may serve to maintain the non-adhesiveness of neutrophils and prevent the development of inflammatory responses.
Test principle
This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for P-selectin has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any P-selectin present is bound by the immobilized antibody. An enzyme-linked monoclonal antibody specific for P-selectin is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of P-selectin bound in the initial step. The color development is stopped and the intensity of the color is measured.
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