Girina N V, Karaseva E I, Metelitsa D I
Biokhimiia. 1994 Dec;59(12):1864-71.
Horseradish peroxidase (HRP) and its conjugates with IgG (HRP-IgG) and cortisol (HRP-COR) interactions with polyvalent antibodies against peroxidase (ANTI-HRP) have been studied by homogeneous enzyme immunoassay. The total catalytic activity in o-phenylenediamine oxidation for free peroxidase or its conjugates and their immunocomplexes with antibodies against the enzyme (ANTI-HRP) has been measured. Linearization of time dependencies of the initial rate for peroxidation in the ln (v-v infinity)-time coordinates, where v and v infinity are the reaction rates at a definite moment of time and after the onset of the equilibrium, as well as in the [ANTI-HRP]/(v-v infinity)-[ANTI-HRP]0 and I/[ANTI-HRP]0k1 + k-1)-[ANTI-HRP]0 made it possible to determine the dissociation constant, Kd, and rate constant for the formation and dissociation, for the immunocomplexes k1 and k-1. The Kd values were in good agreement with those determined by the classical Scatchard method.
已通过均相酶免疫测定法研究了辣根过氧化物酶(HRP)及其与IgG(HRP-IgG)和皮质醇(HRP-COR)的缀合物与抗过氧化物酶多价抗体(ANTI-HRP)的相互作用。已测量了游离过氧化物酶或其缀合物及其与抗酶抗体(ANTI-HRP)的免疫复合物在邻苯二胺氧化中的总催化活性。在ln(v-v无穷大)-时间坐标中,过氧化物初始速率的时间依赖性线性化,其中v和v无穷大是在特定时刻和平衡开始后的反应速率,以及在[ANTI-HRP]/(v-v无穷大)-[ANTI-HRP]0和I/[ANTI-HRP]0k1 + k-1)-[ANTI-HRP]0中,使得能够确定免疫复合物的解离常数Kd以及形成和解离的速率常数k1和k-1。Kd值与通过经典Scatchard方法测定的值高度一致。