间接Dot—ELISA检查猪细小病毒抗原的研究

Studies on the Indirect Dot-ELISA for the Detection of PPV Antigen

本研究首次成功地建立了检测PPV抗原的间接斑点酶联免疫吸附试验(Dot—ELISA)标准化程序,在所确定的最适条件下,对纯化PPV抗原的最低检出量为2.5ng/dot,其敏感性为血凝试验(HA)的125倍,但稍低于银加强胶体金检测法(SECGA)。PPV阳性猪血清的特异性阻断试验及与猪瘟病毒,猪伪狂犬病病毒,猪巴氏杆菌的交叉反应试验证明,该方法对PPV抗原的检测具有特异性。以该方法检测PPV人工感染兔样本和自然感染猪样本,PPV抗原的阳性检出率分别为肾样本100％(17/17)、81.82％(9/11),肝样本100％(16/16)、56.52％(13/23)。20份随机样本的间接Dot—ELISA检测结果与病毒分离和鉴定结果相符。 试验证明间接Dot—ELISA对PPV感染的检测具有简单、快速、经济、敏感性高、特异性强、重复性好和便于推广应用等优点,适用于大批量抗原样本的检测,是PPV感染快速诊断和流行病学调查的有效方法。

It's the first time that a standardized procedure of indirect dot enzyme-linked immunosorbent assay (Dot-ELISA) has been establieshd for the detection of PPV antigen in this study.2.5ng per dot was the least of purified PPV antigen which the method could detect under the optimal condition,with the sensitivity of 125-times greater than the haemagglutination (HA) tests,but its sensitivity is lower than the silver-enhanced colloidal gold assay (SECGA) . Its specificity in detection of PPV antigen was well shown by specific blockage tests of PPV positive porcine serum and the cross-reaction tests with swine fever virus (SFV),pseudorabies virus (PRV) and pasteurella multocida. The positive rates of PPV antigen of the man-made infected rabbit specimens and field porcine specimens measured by indirect Dot-ELISA were separately kidney specimens 100% (17/17) and 81.8% (9/11),liver specimens 100% ( 16/1 6 ) and 55. 52 %(l3/23 ). Results of detection of 20-specimens by the indirect Dot-ELISA were consistent with those of virus isolation and identifcation. The test indicates that the indirect Dot-ELISA is a facile,rapid,low-cost sensitive,specific reproducible and easy to spreading and using method for detection of PPV antigen.It is a useful method for early diagnosis and epidemiological investigation of PPV infection.