三种方法检测口蹄疫146S抗原含量及抗原稳定性的比较研究

A Comparative Study on the Antigen Content and Antigen Stability of 146S in FMD Detected by Three Methods

关键词:  蔗糖密度梯度离心  口蹄疫146S  安捷伦Cary 100紫外分光光度计  安捷伦1260液相色谱仪  高效体积排阻色谱  全能核酸酶

Key words:  sucrose density gradient centrifugation  foot-and-mouth disease 146S  Agilent Cary 100 ultraviolet spectrophotometer  Agilent 1260 liquid chromatography  high performance size exclusion chromatography  Benzonase nuclease

摘要:

为比较三种口蹄疫146S抗原含量检测方法，分别用蔗糖密度梯度离心结合安捷伦Cary 100紫外分光光度计定量法、蔗糖密度梯度离心结合安捷伦1260液相色谱仪定量法及高效液相体积排阻色谱法三种检测方法，对口蹄疫灭活抗原中的146S含量进行检测，对三种检测方法的数据进行统计分析。将口蹄疫A型抗原和O型抗原各一株分别4℃放置12个月以及反复多次冷冻处理；口蹄疫抗原用全能核酸酶处理，然后用蔗糖密度梯度离心结合安捷伦1260液相色谱仪定量法检测处理前后的146S抗原含量变化。结果显示，三种检测方法的检测数值相关性极显著，呈正相关性；4℃放置12个月后的抗原含量没有变化，而反复多次冷冻处理后的抗原含量降解非常多；全能核酸酶处理后的抗原含杂蛋白少。本研究表明，基于临床需求，蔗糖密度梯度离心结合安捷伦1260液相色谱仪定量法更适合口蹄疫抗原检测；口蹄疫抗原适合于4℃保存；全能核酸酶能有效去除杂蛋白干扰，提高色谱纯化效率。

Abstract:

The purpose of this study is to compare and analyze three methods for the detection of 146S antigen content of FMD. The 146S content of FMD inactivated antigen was detected by sucrose density gradient centrifugation combined with Agilent Cary 100 ultraviolet spectrophotometer, sucrose density gradient centrifugation combined with Agilent 1260 liquid chromatography and high performance liquid volume exclusion chromatography, and the data of the three detection methods were analyzed statistically. One strain of FMD A antigen and one strain of FMD O antigen were placed at 4℃ for 12 months and repeatedly frozen.FMD antigen was treated with benzonase nuclease. The concentration of 146S antigen before and after treatment was determined by sucrose density gradient centrifugation and Agilent 1260 liquid chromatography. The results showed that the correlation among the three methods was significant and positive. The content of FMD antigen did not change after 12 months at 4℃. However, the antigen content degraded a lot after repeated freezing treatment. The content of heteroprotein in FMD antigen is less after treated with benzonase nuclease. This study shows that sucrose density gradient centrifugation combined with Agilent 1260 liquid chromatograph quantitative method is more suitable for FMD antigen detection based on clinical needs. FMD antigens are more suitable for preservation at 4℃. Benzonase nuclease can effectively remove the interference of heteroprotein and improve the efficiency of chromatographic purification.