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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。
Ferritin (FER) was used as the material to prepare nano-europium core particles, and competitive FER immunoassay was established. After dissociation of FER subunit with dilute hydrochloric acid, the free iron is purified and neutralized, and then the protein shell is reconstituted by the acid-base neutralization method. At the same time, europium ions are encapsulated in deferron protein to prepare europium ferritin. Optimized by the reaction conditions, to determine pH 2.0 FER optimum acidity. When the FER shell is reorganized, the maximum capture efficiency can be obtained when the addition amount of the europium reagent is about 10,000 times of the FER mass. The time-resolved fluorescence immunoassay of europium nuclear -FER was established. The sensitivity was 0.576 ng / mL and the IC 50 was 13.15 ng / mL. The coefficient of variation (CV) between batches was less than 15% and the nonspecific binding rate was low. The competition between TRFIA and FER sandwich assay kit was constructed by the preparation of europium nuclear -FER, the correlation coefficient between the two was 0.966. By means of the above method, nano-molecules with good immunocompetence and high europium content are obtained, which simplifies the traditional method of europium labeling and establishes a new method for constructing nano-europium ferritin.