用纯化的抗体包被微孔板，制成固相载体，往包被抗Protein S抗体的微孔中依次加入标本或标准品、生物素化的抗Protein S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Protein S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 use in vitro quantitative determination of human Protein S concentrations in cell culture supernates, serum, plasma and other biological fluids.
Introduction
Protein S is a vitamin K-dependent plasma glycoprotein synthesized in the liver. In the circulation, Protein S exists in two forms: a free form and a complex form bound to complement protein C4b.
The best characterized function of Protein S is its role in the anti coagulation pathway, it functions as a cofactor to Protein C in the inactivation of Factors Va and VIIIa. Only the free form has cofactor activity.
Protein S can bind to negatively charged phospholipids via the carboxylated GLA domain. This property allows Protein S to function in the removal of cells which are undergoing apoptosis. Apoptosis is a form of cell death that is used by the body to remove unwanted or damaged cells from tissues. Cells which are apoptotic (ie. in the process of apoptosis) no longer actively manage the distribution of phospholipids in their outer membrane and hence begin to display negatively charged phospholipids, such as phosphatidyl serine, on the cell surface. In healthy cells, an ATP (Adenosine triphosphate)-dependent enzyme removes these from the outer leaflet of the cell membrane. These negatively charged phospholipids are recognized by phagocytes such as macrophages. Protein S can bind to the negatively charged phospholipids and function as a bridging molecule between the apoptotic cell and the phagocyte. The bridging property of Protein S enhances the phagocytosis of the apoptotic cell, allowing it to be removed cleanly without any symptoms of tissue damage such as inflammation occurring.
Test principle
The microtiter plate provided in this kit has been pre-coated with an antibody specific to Protein S. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for Protein S and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB substrate solution is added to each well. Only those wells that contain Protein S, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of Protein S in the samples is then determined by comparing the O.D. of the samples to the standard curve.
