预期应用
ELISA法定量测定兔血清、细胞培养物上清或其它相关液体中TM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TM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TM抗原、生物素化的抗兔TM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TM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 rabbit TM concentrations in cell culture supernates, serum, tissue homogenates and plasma.
Introduction
Thrombomodulin (TM) is the cell surface receptor for thrombin. When occupied, thrombomodulin converts thrombin from a procoagulant protein into the activator of Protein C. Once activated Protein C (APC) has been generated, thrombomodulin acts as a major anticoagulant through its ability to inactivate various blood factors (Va, VΙΙΙa, Xa and XΙΙΙa). In competing for thrombin binding, thrombomodulin inhibits the proteolytic effect of thrombin in its clotting of fibrinogen, the inactivation of Protein S and the induction of platelet aggregation.
TM is an integral membrane glycoprotein resembling in structure the low-density lipoprotein (LDL) receptor. TM possesses several EGF repeats, of which numbers five and six are responsible for the high affinity binding of thrombin (Kd = 0.5 nM). In addition, the B chain of thrombin possesses a domain, distinct from the active catalytic site, termed anion-binding Exosite Ι, which is involved in the binding of thrombin to thrombomodulin. Also, TM contains a chondroitin sulfate (glycosoaminoglycan) which accelerates the inactivation of thrombin by anti-thrombin ΙΙΙ. On SDS-polyacrylamide gels, rabbit thrombomodulin appears as a single band at Mr 75,000 D under nonreducing conditions and shows a band at approximately Mr 110,000 D following reduction of its disulfide bonds.
Test principle
This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for TM has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any TM present is bound by the immobilized antibody. An enzyme-linked monoclonal antibody specific for TM 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 TM bound in the initial step. The color development is stopped and the intensity of the color is measured.
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