定量测定硫酸软骨素的双抗体夹心ELISA的建立

摘要

目的 建立定量测定硫酸软骨素(CS)的双抗体夹心酶联免疫吸附试验(ELISA),并对其应用价值进行初步评价.方法 以CS为抗原分别免疫Balb/c小鼠和日本大耳白兔,制备2种动物的多克隆抗体.用鼠抗体包被微孔板,兔抗体为第2抗体,辣根过氧化物酶(HRP)标记的羊抗兔抗体为标记抗体,建立双抗体夹心ELISA,并对线性范围、精密度、回收率和方法学比较进行了评价.结果 本研究建立的ELISA线性范围为2～500 mg/L,批内和批间精密度分别为4.48%和5.97%,回收率在99.52%～101.77%之间,与间苯三酚分光光度法测定结果具有良好的相关性(r=0.999 0,P<0.05).结论 建立的定量测定CS的双抗体夹心ELISA具有敏感性高、重现性好等优点,适合检测应用.%Objective To establish a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) to determine the quantity of chondroitin sulfate (CS), and primarily evaluate the application value. Methods 2 polyclonal antibodies were prepared by immunizing Balb/c mice and Japanese rabbits with CS as antigen. The microplate was coated by polyclonal antibody from mice, and the polyclonal antibody from rabbits was used as the second antibody.The goat anti-rabbit-antibody marked with horseradish peroxidase (HRP) was adopted as labeling antibody. A doubleantibody sandwich ELISA was developed. The performance of this method was studied, including linear range,precision, recovery rate and methodology comparison. Results The linear range of this method was 2-500 mg/L. The within-run and between-run precisions were 4.48％ and 5.97％, respectively. The recovery rate was from 99.52％ to 101.77％. With phloroglucinol spectrophotometry as reference method, its correlation was good, and the correlation coefficient (r) was 0.999 0 ( P ＜ 0.05 ). Conclusions The double-antibody sandwich ELISA to determine the quantity of CS showes high sensitivity and good repeatability. It could be applied to determine CS.