预期应用
ELISA法定量测定人血清、血浆或其它相关液体中可溶性凋亡相关因子(sFAS/Apo-1)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sFAS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sFAS抗原、生物素化的抗人sFAS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sFAS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 human soluble Factor-related Apoptosis ,sFAS/Apo-1 concentrations in cell culture supernates, serum, and plasma.
Instruction
Fas (APO-1 or CD95) is a cell-surface receptor that transduces apoptotic signals from Fas ligand (FasL). It is a glycoprotein with a mass estimated at 43 to 48 kDa . Fas is a member of the Tumor Necrosis Factor Receptor Superfamily (TNFRSF), and it shares a cytoplasmic motif with TNF RI, referred to as the death domain, that binds cytoplasm signaling molecules to trigger the cytoplasmic apoptotic signal . Fas is expressed to a large extent on activated T and B lymphocytes, and on malignant lymphoid cells. To a lesser extent, Fas is expressed on cells from liver, heart, kidney, ovaries, and on many other malignant cells.
FasL, the physiological agonist for Fas, is also a transmembrane protein with homology to the TNF family in its extracellular domain. FasL is expressed primarily by activated T lymphocytes and by cells of the small intestine and lung. Mice with mutations in either Fas or FasL exhibit accumulation of activated lymphocytes and classical autoimmune symptoms, suggesting that a major function of Fas-mediated apoptosis is the elimination of activated immune cells from the peripheral circulation. Similarly, humans with autoimmune lymphoproliferative syndrome have mutations in Fas.
Fas and FasL have been observed as soluble molecules in addition to their membrane-associated forms, suggesting additional complexity to regulation of this apoptotic mechanism. Soluble Fas (sFas) arises from alternatively spliced mRNA, leading to proteins with deletion or disruption of the single membrane-spanning domain. Five alternatively spliced Fas mRNAs have been described, each protein detected in the supernatant solution of cultures of peripheral blood mononuclear cells or certain tumor cell lines. Each sFas inhibited apoptosis induced by FasL, and tumor-cell lines resistant to anti-Fas were shown to produce alternatively spliced Fas, thereby making them less sensitive to FasL. In addition, plasma Fas can arise by exfoliation of membrane vesicles, which also inhibit FasL-induced apoptosis. Serum Fas has been reported to be elevated in cancer patients , possibly originating in the tumor cell itself, and in autoimmune diseases .
Test principle
This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal
antibody specific for sFAS has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any sFAS present is bound by the immobilized antibody. An enzyme-linked polyclonal antibody specific for sFAS 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 sFAS bound in the initial step. The color development is stopped and the intensity of the color is measured.
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