ELISA法定量测定猪血清、细胞培养物上清或其它相关液体中TNF-α含量。
概述
肿瘤坏死因子(tumornecrosisfactor，TNF)是一种具有多种生物活性的细胞因子。猪TNF-α基因编码前体蛋白，其信号肽将前体蛋白固定在细胞膜上，成为具有活性的跨膜干扰素(TNF)，分子质量为26×103，经酶切去除信号肽生成分泌型TNF-α，分子质量为17×103。TNF-α细胞来源广泛，包括各种免疫细胞、内皮细胞、成纤维细胞、表皮细胞、角质细胞、平滑肌细胞、星形细胞、成骨细胞等。
TNF-α具有广泛的生物学活性，如：参与炎性反应和免疫应答，抗肿瘤，参与内毒素性休克等病理过程，引起恶病质等。其具有双重作用，,一方面在机体免疫调节，机体生理功能和抗感染等方面发挥重要作用，另一方面若持续释放或产生过多则会引起发热!、休克、恶病质等，同时TNF-α可进一步诱导IL-6、IL-8、IL-10等细胞因子的产生，这些促炎性细胞因子参与体内急性反应、发热反应、引起趋化肽释放等，还可使内皮细胞活化而导致血管通透性增加。
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TNF-α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TNF-α抗原、生物素化的抗猪TNF-α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TNF-α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 porcine TNF-α concentrations in cell culture supernates, serum, tissue homogenates and plasma.
Introduction
The prototype ligand of the TNF superfamily, TNF-α/TNFSF1A, is a pleiotropic cytokine that plays a central role in inflammation and apoptosis. It is synthesized as a 26 kDa, type II transmembrane protein that is 233 amino acids in length. It contains a 30 amino acid (aa) cytoplasmic domain, a 26 aa transmembrane segment, and a 177 aa extracellular region. TNF-αis assembled intracellularly to form a transmembrane, non-covalently-linked homotrimeric protein. The 157 aa residue soluble form of TNF-α(sTNF-αis released from the C-terminus of the transmembrane protein through the activity of TNF-α-converting enzyme (TACE), a membrane -bound disintegrin metalloproteinase. Human cells known to express TNF-αinclude B cells, colonic columnar epithelial cells, NK and CD3+CD56+ hepatic natural T cells, macrophages, monocytes and monocyte-derived dendritic cells, CD4+ and CD8+ T cells, mast cells, neutrophils, keratinocytes, plasma cells, and adipocytes.
Test principle
This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for TNF-α has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any TNF-α present is bound by the immobilized antibody. An enzyme-linked monoclonal antibody specific for TNF-α 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 TNF-α bound in the initial step. The color development is stopped and the intensity of the color is measured.
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