ELISA法定量测定小鼠血清、血浆、尿液、细胞培养物上清或其它相关液体中LTB₄含量。

本试剂盒应用双抗体夹心酶标免疫分析法测定标本中白三烯B4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白三烯B4抗原、生物素化的抗小鼠白三烯B4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白三烯B4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。

This immunoassay kit allows for the specific measurement of mouse LTB₄ concentrations in cell culture supernates, serum, urine, and plasma.

 

Leukotriene B4 (LTB₄) is a potent pro-inflammatory molecule that belongs to a family of eicosanoid lipid mediators. It is synthesized from arachidonic acid that is generated from nuclear membrane phospholipid. In general, and upon cell activation, phospholipase A2 is directed to the nuclear membrane. Here, it generates arachidonic acid that is bound by one 18 kDa membrane-bound protein termed Five-Lipooxygenase Activating Protein (FLAP). FLAP-bound arachidonic acid is subjected to two distinct, sequential actions by 80 kDa 5-lipooxygenase (5-LO). The first 5-LO action generates 5(S)-HETE, an oxygenated form of arachidonic acid. The newly generated 5(S)-HETE is then acted on again by 5-LO to generate LTA4. The LTB₄ synthetic pathway has two key checkpoints. The first is at the level of arachidonic acid formation, where COX enzymes offer an alternative to 5-LO in the generation of prostaglandins. The second is at the level of LTA₄, where 69 kDa LTA4 hydrolase activity will generate LTB₄ (a pro-inflammatory molecule), and 18 kDa LTC₄ synthetase activity will generate LTC/D/E4 (smooth muscle contractants). The key enzyme in LTB4 synthesis is 5-LO, which has limited cell expression. Cells known to express 5-LO include B cells, macrophages, monocytes, mast cells, neutrophils, neurons, eosinophils and dendritic cells. Notably, all cells are potential sources for LTB₄ production. This is due to the fact that LTA4 hydrolase is ubiquitously expressed, and excess LTA₄ generated by 5-LO expressing cells can diffuse into neighboring LTA₄ hydrolase-containing cells for conversion into LTB₄.

 

This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for LTB₄ has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any LTB₄ present is bound by the immobilized antibody. An enzyme-linked monoclonal antibody specific for LTB₄ 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 LTB₄ bound in the initial step. The color development is stopped and the intensity of the color is measured.