斑点酶联免疫吸附试验检测猪瘟血清抗体最佳条件的研究

以硝酸纤维膜为固相载体,以猪瘟病毒为抗原,确定了Dot-PPA-ELISA检测猪瘟抗体的最佳反应条件。试验表明,猪瘟兔化弱毒经仔猪睾丸原代细胞培养纯化,以0.05mol/LpH9.5CBS缓冲液稀释至浓度为0.98～1.15mg/mL制备诊检抗原膜片;采用4%蛋清蛋白0.05mol/LpH9.5CBS封闭;检测反应稀释液应用0.01MPPBST(含0.1%PEG、0.05Tween-20),酶结合物工作浓度以1∶30,底物选取4-氯1-萘酚等为检测猪瘟血清抗体的最佳反应条件。重复试验证明了其检测稳定性好。交叉试验、阻断试验证明了其特异性强。

Studies on the Optimum Condition for Detecting Antibody against Hog Cholera Virus with Dot-PPA-ELISA

The assay was developed in order to explore optimum conditiones of detection for antibody against Hog Cholera Virus with Dot-PPA-ELISA and the optimum conditiones was determined through a series of experiments.The result was showed by the following lists:the antigen was made from the supernatant of primary new-born piglet kidney cell vaccinated with rabbit-adapted strain(C strain) of HCV and its concentration was 0.98～0.15 mg/mL;the best antigen diluent buffer,antibody diluent,coating buffer,the concentration of HRP-labelled SPA conjugate,substrate were 0.05M carbonate buffer (pH9.6),0.01M PPBST(containing 0.1%PEG,0.05%Tween-20),4% egg white protein (diluted with 0.05M pH9.6CBS),1:30,4-chloro-1-naphthol respectively.Its good stability and specificity were showed by repeat examination,crossing test and blocking test respectively,So the optimum conditiones were determined with nitrocellulose membrane (as the solid phase carrier)and partially purified HCV antigen.