基于对羟基桂皮醇的新型酶联荧光免疫传感系统测定布氏杆菌抗体

An enzyme-linked fluoroimmunosensing system for Brucella melitensis antibody detection based on a novel substrate 4-hydroxycinnamic alcohol for HRP

以一种纯天然产物对羟基桂皮醇(4-hydroxycinnamic alcohol,HCA)为辣根过氧化物酶(HRP)底物,建立对羟基桂皮醇-辣根过氧化物酶-过氧化氢新体系。对羟基桂皮醇本身只有极弱的荧光,在HRP催化下可被H2O2氧化成二聚体产物,该二聚体在315nm的激发光下能发射波长为467nm的强荧光,并且反应体系荧光强度增加值与HRP量在一定浓度范围内呈线性相关。根据此关系和竞争型免疫定量原理,建立兔布氏杆菌抗体测定的荧光酶联免疫传感体系,并对免疫测定条件如pH值、HRP-BrAb用量、BSA和流速等进行优化。运用制备传感体系测定兔布氏杆菌抗体的质量浓度线性范围为2.7～90μg/L,检测限为2.7μg/L,相对标准偏差为4.6%;对羟基桂皮醇在空气中较稳定,对人体无毒害,在临床上可代替传统HRP底物。

A natural product,4-hydroxycinnamic alcohol （HCA） was used as a substrate for HRP in enzyme-linked fluoroimmunoassay. In enzymatic reaction procedure,HRP-Brucella melitensis antibody conjugate （HRP-BrAb） catalyze the polymerization of HCA by H2O2,and the HCA is partly converted to polymers,a fluorescent species. The fluorescence increase of the HRP-enzymatic product at emission of 467 nm （excitation at 315 nm） is proportional to the concentration of HRP-BrAb binding to the Brucella melitensis antigens,which were entrapped in cellulose-paraffin matrix. The linear range of determination for BrAb is 2.7-90 μg/L with the relative standard deviation of 4.6%. The detection limit is 2.7 μg/L. HCA is stable in air and non-toxic to human health. The proposed method can be used for analysis of commercial formulation and plasma sample with satisfactory results.