新颖抗猪流行性腹泻免疫制剂

COMPREHENSIVE REPORT ON NEW IMMUNIZING AGENT AGAINST PORCINE EPIDEMIC DIARRHEA VIRUS

以新生乳猪增殖猪流行性腹泻病毒（PEDV），通过蔗糖密度梯度离心纯化，获得结构较为完整的病毒颗粒，病毒ELISA效价为1：3×105，蛋白含量为3．96mg／mL．将提纯的PEDV免疫Balb／C小鼠，取其脾细胞与SP2／0骨髓瘤细胞进行融合．融合率为95．6％。用间接ELISA筛选并进行3次再克隆，阳性率达100％，共获用15株能稳定分泌特异性抗PEDV单克隆抗体（McAb）的杂交瘤细胞．其染色体数平均为90～100．所分泌抗体具有高度特异性，腹水ELISA效价为1：103～1：106。用混合单抗对新生猪的保护试验证明，这些单抗不论在体内或体外．对PEDV均有中和作用．试验绪可以得到抗体的保护耐过不死，而病毒对照组的猪只全部死亡。将混合单抗用于PED的发病场，保护率达91％．建立了PPA－ELISA、McAb阻断ELISA及MCAb免疫荧光试验等快速诊断方法．

Porcine epidemic diarrhea virus (PEDV)was multiplied in newborn colostrumdeprived piglets. The PEDV antigen preparation was purified by PEG-6000 precipitation,differential centrifugation and sucrose density gradient centrifugation with an ELISA titer of 1: 3×105 and protein content of 3. 96 mg/mL. The myeloma cell line SP2/0 fused with the spleen cells of Balb/C mice conventionally immunized three days early with purified PEDV with the fusion rate of 95. 6%. Fifteen hybridoma cell lines steadily secreting monoclonal antibodies(McAb)highly specific for PEDV by ELISA blocking and cross tests were screened through three clonings. The numbers of chromosomes of hybridoma cell lines were 90~ 110,with an average of 95. The ELlSA titers of ascites were 1: 103 ~ 1: 106. Three of these McAbs (A4C7、E10 G5, F4D10 ) were mixed and applied in the laboratory prevcntion and treatment tests, the results showed that they could neutralize PEDV and inhibit the virus infection on pigtets. Their elementary use on sick piglets in infected pig farms proved that they had a very obvious curative effect with a protection rate of 91%,but the protection rate in the treatment with hyperimmune serum was 83 %,while almost all of the control untreated sick piglets died. The results above indicate that the mixture of these McAbs can alleviate the symptoms of the disease and reduce the mortality of sick piglets. The development of mixed McAbs opens a new field for the prevention and therapy of the disease. Additionally, several rapid diagnostic methods were established with the McAbs. Among them, PPA-ELlSA and McAb-ELISA blocking tests were used for detection of PEDV antibodies, and McAb immuno-fluorescent assay for diagnosis of PEDV antigen.