基于纳米铕核铁蛋白时间分辨荧光免疫方法的建立

A ferritin time-resolved immunoassay based on nanometer nuropium core-ferritin

以人铁蛋白(ferritin,FER)为材料,制备纳米铕核微粒,并建立竞争型FER免疫检测方法。用稀盐酸解离FER的亚基后,纯化脱去游离铁,再通过酸碱中和方法重组蛋白外壳,同时将铕离子包裹于去铁蛋白内,制备铕核铁蛋白。经反应条件优化,确定pH 2.0为FER最适解离酸度。FER外壳重组时,铕试剂的添加量约为FER物质量的10 000倍时能获得最大捕获效率。以此建立了铕核-FER时间分辨荧光免疫检测方法,其灵敏度为0.576ng/mL,IC_(50)为13.15ng/mL,批间变异系数CV%<15%,非特异性结合率低。用制备成的铕核-FER构建的竞争法TRFIA与FER夹心法试剂盒对照,两者相关系数达0.966。通过上述方法获得免疫活性好、铕含量高的标记用纳米分子,简化了传统的铕标记方法,建立了构建纳米铕核铁蛋白的新方法。

Nanometer europium core-ferritin is prepared from human ferritin and used to develop a competitive immunoassay for determination of serum ferritin. Amount of ferritin was dissociated by diluted hydrochloric acid, followed by purification to remove iron ion. The apoferritin captured Europium （Eu） atoms and self-assembled by acid-basis neutralization. After that, Eu core-ferritin was formed. The reaction conditions were under estimated. The dissociation of acidic conditions was pH 2.0. Ten thousand times of EuCl3 vs ferritin （mol ： tool） was used to compound the Eu eore-ferritin effectively. Afterwards, the competitive method of ferritin was developed with Eu core-ferritin by time resolved fluoroimmunoassay. The sensitivity of assay was 0. 576ng/ml and IC50 was 13.15 ng/ml. The inter-batch coefficient of variation was below 15% with a very low rate of nonespecific bounding. The results obtained from the sera samples compared a good correlation, 0.966, with those of sandwich TRFIA kits. This method declared a new approach of preparation of Eu core-ferritin, which simplified the Eu labeling procedure with high immunological activity of ferritin molecular assembled high content of Eu atom.