本试剂盒应用双抗体夹心酶标免疫分析法测定标本中乙酰肝素酶水平。用纯化的抗体包被微孔板，制成固相抗体，往包被抗体的微孔中依次加入乙酰肝素酶抗原、生物素化的抗人乙酰肝素酶抗体、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乙酰肝素酶呈正相关。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 heparanase,HPA concentrations in cell culture supernates, serum, and plasma.
Introduction
Heparanase is an endo-β-D-glucuronidase that catalyzes the hydrolytic cleavage of the β-1,4-glycosidic bond between a D-glucuronate and a D-glucosamine in heparan sulfate. The physiological role of heparanase is simply to degrade heparan sulfate but this activity can take many turns.
The reaction mechanism probably involves general acid/base catalysis by Glu225 and nucleophilic catalysis by Glu343:Glu225 protonates the oxygen of the glycosidic bond, and Glu343 performs a nucleophilic attack on the anomeric carbon to break the glycosidic bond and form an unstable ester intermediate. The ester is hydrolyzed by an incoming water molecule and Glu225 now acts as a general base to activate the water and regain a proton.
It is known that the antithrombin binding motif found in heparin is a substrate for heparanase, but since this structure is very rare in the natural substrate heparan sulfate, there is still much to find out about the substrate specificity of heparanase. Which O-sulfation patterns promote or inhibit catalysis? And which N-substituents.
Test principle
This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal
antibody specific for heparanase has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any heparanase present is bound by the immobilized antibody. An enzyme-linked polyclonal antibody specific for heparanase 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 heparanase bound in the initial step. The color development is stopped and the intensity of the color is measured.
