【作者】 祁小乐；

【导师】 崔保安；

【作者基本信息】 河南农业大学 ， 预防兽医学， 2004， 硕士

【摘要】 用纯化的Min-A株伪狂犬病病毒（PRV）免疫兔子和昆明鼠，制备高免血清并提纯IgG。兔抗PRV高免血清琼扩效价为1：32，纯化兔抗PRV IgG蛋白含量为7.34mg/ml；鼠抗PRV高免血清琼扩效价为1：16，纯化鼠抗PRV IgG蛋白含量为5.74mg/ml。复苏本实验室研制并保存的分泌抗伪狂犬病病毒单克隆抗体杂交瘤细胞OH6，按常规方法制备腹水、提纯IgG，并重新对其作了鉴定。该杂交瘤细胞的平均染色体数目为94条；建立了检测单抗的间接ELISA方法，杂交瘤细胞培养上清和腹水单克隆抗体的ELISA效价分别为1：1000和1：200000；该株单抗在有无补体参与下均具有中和活性，中和效价分别为1:64和1:16；该株单抗具有良好的特异性，与PRRSV、HCV、PPV不发生交叉反应。利用所制备的兔抗PRV IgG和McAb建立了检测PRV的“单克隆抗体双夹心ELISA”。兔抗PRV IgG最佳包被浓度为4.59ug/ml（1：1600倍稀释），McAb的最佳工作浓度为6.45ug/ml（1：800稀释）。该检测方法特异、灵敏、可靠、方便、快捷；与动物试验、病毒分离和中和试验符合率较高；最低病毒检出量为200ng/ml；整个操作过程仅需4.5h（不包括包被）；建立了科学的判定标准；该方法不需要特殊设备和技术，便于推广。通过对人工兔病料和自然发病猪病料的检测研究，发现扁桃体和脑是PRV最佳检测器官。更多还原

【Abstract】 Rabbits and Kunming mice were immunized with the purified PRV of Min A respectively， then the positive serum was obtained and the IgG was purified.For the rabbit-anti-PRV serum， it’s AGP titer was 1:32 ，and the protein concentration of IgG was 7.34 mg/ml.For the mice-anti-PRV serum， it’s AGP titer was 1:16 ，and the protein concentration of IgG was 5.74 mg/ml.The cell line named OH6 which could secrete McAb stably was anabiosised， then the ascites were produced and the IgG was purified by the common methods. The hydridoma cell line OH6 and the ascites were re-evaluated. The average number of chromosome of the hybridoma cell was 94.Through detection by the indirect ELISA having been established， the ELISA titer of cell supernate and ascite were 1:1000 and 1:200000.The McAb had the activity of neutralization with or without complement，the titer was 1:64 and 1:16 respectively.The McAb did not crossly react with PRRSV， HCV， PPV， which demonstrated good specificity.Based on the rabbit-anti-PRV IgG and McAb， the double sandwich enzyme-linked immunosorbent assay (ELISA) for detection of Pseudorabies virus (PRV) was developed. The best coating concentration of the rabbit-anti-PRV IgG was 4.59ug/ml. The best working concentration of McAb was 6.45ug/ml. The assay was specific， sensitive， reliable， convenient， and had a high coincidence rate with animal experiment， virus isolation and neutralization experiment. The sensitivity of the double sandwich ELISA was 200 ng/ml. The whole operation time only need 4.5h(not including coating). The scientific criterion of determination had been made. The assay did not require specific equipment and technique so that it can be extended easily.The detection of the artificially infected rabbits and naturally infected swine showed that the best tissues for the diagnosis of PRV were tonsil and brain.更多还原