改良的RIHA方法定量检测PCV2 Cap抗原的研究

Study on Quantitative Detection of PCV2 Cap Antigen by Modified RIHA Method

关键词:  PCV2 Cap抗原  改良  反向间接血凝试验(RIHA)  ELISA  定量检测

Key words:  PCV2 Cap antigen  modified  reverse indirect hemagglutination assay (RIHA)  ELISA  quantitative detection

摘要:

为提高反向间接血凝试验(RIHA)定量检测PCV2 Cap抗原的稳定性和精准性,将RIHA方法改良为：1)将被检样品稀释为几个适宜稀释度,分别做RIHA检测；2)检测孔100%、75%、50%、25%、0%凝集分别记录为4、3、2、1、0分,将几个稀释度的被检样品全部检测孔的分数加和；3)同法检测参照品并将参照品分数加和；4)被检样品与参照品总分数对比计算,获得被检样品抗原含量。改良的RIHA方法做稳定性研究并与ELISA方法做比较。结果显示,改良RIHA方法的批内差为6.8％～19.4％、批间差为11.4％～23.6％、检测同一个样品变异系数为8.5%。改良RIHA方法检测纯化前和纯化后的PCV2 Cap抗原共30份,与ELISA方法检测结果比较相关系数为0.9567。说明改良的PCV2 Cap抗原RIHA定量检测方法稳定性良好,且与ELISA方法检测结果有相关性,可在PCV2 Cap抗原生产中做定量检测,也可在毒种筛选、生产工艺优化中快速定量检测PCV2 Cap抗原含量。

Abstract:

To improve the stability and accuracy of the quantitative detection of the PCV2 Cap antigen using the Reverse Indirect Hemagglutination Assay (RIHA), the RIHA method has been modified as follows: 1) Dilute the test sample into several appropriate dilutions for RIHA; 2) Record agglutination in the test wells at 100%, 75%, 50%, 25%and 0% as 4, 3, 2, 1 and 0 points respectively, and sum the scores of all test wells for each dilution of the test sample; 3) Perform the same method to test the reference standard and sum its scores; 4) Compare the total scores of the test sample and the reference standard to obtain the antigen content of the test sample. The modified RIHA method was used for stability study and compared with the ELISA method. The results showed that the intra-batch difference of the modified RIHA method was 6.8%–19.4%, the inter-batch difference was 11.4%–23.6%, and the coefficient of variation for testing the same sample was 8.5%. The modified RIHA method was used to test 30 PCV2 Cap antigen samples before and after purification, and the results were compared with those obtained from the ELISA method, yielding a correlation coefficient of 0.9567. This indicates that the modified RIHA method for quantitative detection of PCV2 Cap antigen is stable and correlates well with the ELISA method. It can be used for quantitative detection in the production of PCV2 Cap antigens and also for rapid quantitative detection of PCV2 Cap antigen content in virus seed screening and production process optimization.