抗猪流行性腹泻病毒单克隆抗体的研制及其特性鉴定

Development and Characterization of Monoclonal Antibodies 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将其纯化，获得较为完整的病毒颗粒。病毒ＥＬＩＳＡ效价为１∶３×１０５，蛋白含量为３．９６ｇ／Ｌ。将提纯的ＰＥＤＶ免疫ＢＡＬＢ／ｃ小鼠，取免疫鼠脾细胞与ＳＰ２／０骨髓瘤细胞进行融合，融合率为９５．６％。用间接ＥＬＩＳＡ进行筛选，共检出３８个阳性孔，阳性率为２８．８％。对其中１５个阳性值较高的孔进行３次再克隆，阳性率达１００％，最后获得１５株稳定分泌特异性抗ＰＥＤＶ单抗（ＭｃＡｂ）的杂交瘤细胞。ＥＬＩＳＡ阻断试验与交叉试验证明，其分泌的抗体具有高度特异性。腹水和细胞培养上清的ＥＬＩＳＡ效价分别为１∶１０３～１∶１０６和１∶１２８～１∶５１２。经鉴定，１５株ＭｃＡｂ的分子类型均为ＩｇＧ，均无免疫沉淀特性。杂交瘤细胞的染色体数目为９０～１１０，平均９５。尤为重要的是，这些杂交瘤细胞所分泌的抗体，有的对ＰＥＤＶ具有中和作用，能够阻断病毒对猪只的侵害作用。

Porcine epidemic diarrhea virus (PEDV) was multiplied in newborn colostrum deprived piglets. The PEDV antigen preparation was purified by PEG 6000 precipitation, differential centrifugation and sucrose density gradient centrifugation with the ELISA titer and protein content being 1∶3×10 5 and 3.96 g/L. 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%. Thirty eight positive reactions (up to 28.8%) were detected by indirect ELISA. Of them, fifteen hybridoma cell lines steadily secreting monoclonal antibody (McAb) highly specific for PEDV by ELISA block and cross tests were screened through three clonings. These fifteen monoclonal antibodies without precipitation activity were identified as IgG. The ELISA titers of ascites and cell supernatant were 1∶10 3 to 1∶10 6 and 1∶128 to 1∶152, respectively. The number of chromosome of hybridoma cell lines was 90 to 110, at an average of 95. Especially, some of these cell lines secreted neutralization antibody against PEDV and would be able to inhibit the virus infection on piglets.