制备醛固酮多克隆抗体建立检测血浆醛固酮的化学发光免疫分析法

Preparation of polyclonal antibody and establishment of chemiluminescence immunoassay for aldosterone

目的:制备可用于免疫分析的醛固酮(ALD)多克隆抗体,并建立基于生物素-链霉亲和素放大系统的ALD化学发光免疫分析方法,用于测定人血液中的ALD含量。方法:对ALD进行化学改造,制备醛固酮肟,再与BSA偶联制备免疫原,免疫兔,制备抗ALD多克隆抗体。以生物素-链霉亲和素放大系统采用竞争法建立ALD化学发光免疫分析方法。结果:经检测免疫的3号兔获得的抗ALD抗体灵敏度最高,50%抑制率(IC50)ALD浓度是268 pg/ml。用该抗体建立的化学发光免疫分析法的检测范围为62.5~2 000 pg/ml,灵敏度为23.7 pg/ml,批内变异系数为6.9%~9.5%,批间变异系数为8.5%~12.7%,回收率范围为93.1%~104.1%,稀释实验测定值与理论值呈线性相关,相关系数为r=0.996,与放射免疫分析试剂盒的相关性方程分别为y=0.932x+4.596,相关系数r=0.948(n=95)。结论:建立的检测ALD的化学发光免疫分析法符合临床应用的基本要求。

Objective： The polyclonal antibody of aldosterone（ ALD） for immunoassay was developed. And a chemilu-minescence immunoassay（ CLIA） for the determination of ALD in human blood was established. Methods： Aldosterone oxime was prepared by chemical modification and then conjugated with BSA to prepare immunogen. Rabbit anti ALD polyclonal antibody was prepared by immunizing rabbits with the ALD-BSA. The CLIA of ALD was performed using biotin streptavidin amplification system and competition method. Results： After identification,rabbit No. 3 received the highest sensitivity to ALD antibody,and the 50% binding inhibition（ IC50） value for ALD concentration was 268 pg/ml. The measuring range of CLIA method using the antibody was 62. 5-2 000 pg/ml. The assay sensitivity was 23. 7 pg/ml. The intra-and inter-assay coefficients of variation were 6. 9%-9. 5% and 8. 5%-12. 7%,respectively. Analytical recovery rate was in the range of 93. 1%-104. 1%. The correlation coefficient between measured and expected values were 0. 996 after serial dilution. Compared with radioimmunoassay kit,the correlative equation was y = 0. 932 x ＋ 4. 596,the correlation coefficient was 0. 948（ n = 95）. Conclusion： The result of methodological identification shows that it was in line with the basic requirements of clinical application.