分泌抗猪流感病毒（河南株）单克隆抗体杂交瘤细胞株的制备、鉴定和初步应用
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摘要
用纯化的猪流感病毒（SIV）免疫BALB/C小鼠，取其脾细胞与SP2/0骨髓瘤细胞融合，经间接ELISA和红细胞凝集抑制实验筛选，经过4次有限稀释法克隆，获得了1株能稳定分泌抗猪流感病毒单克隆抗体杂交瘤细胞株，命名为5B4。经鉴定该单抗为IgG1亚类，杂交瘤细胞的平均染色体数目为96.8条，杂交瘤细胞培养上清及小鼠腹水单克隆抗体的ELISA效价分别为1:1024和1:51200。5B4株单克隆抗体与猪伪狂犬病毒、猪繁殖-呼吸综合征病毒、猪瘟病毒、猪细小病毒均不发生交叉反应，显示良好的特异性。连续培养21代，5B4杂交瘤细胞株能稳定分泌抗体。利用5B4杂交瘤细胞株的小鼠腹水单克隆抗体建立了检测SIV的双夹心ELISA的检测方法。经测定，单克隆抗体的最适包被浓度为2.95μg/ml，兔抗SIV 高免血清的最适工作浓度为1:800。其检测灵敏度为2.43μg/ml。用该方法对自然发病死亡猪的气管、支气管、肺、扁桃体、淋巴结等的组织研磨物进行检测，结果发现在气管和支气管检出率最高，同时对于发病猪的鼻腔拭子擦拭物，不经特殊处理就可以用建立的双夹心ELISA检测出SIV。并且与病毒分离对比试验有较高的符合率。双夹心ELISA的建立为生产上提供了一种更简单、快速、灵敏、高效、实用的SIV检测方法。
Hybridomas secreting monoclonal antibodies (McAbs)specific for Swine influenza virus were produced by fusion of SP2/0 myelomas cells with spleen cells from BALB/C mice immunized with purified SIV.Wells containing hybridomas secreting immunoglobulin G (IgG1) antibodies against SIVwere specifically identified by an indiect enzyme-linked immunosorbent assay(ELISA),and one ELISA-positive hybridomas were selected and cloned forth by limiting dilution. and named 5B4,which could secrete McAb stably.The isotype of the McAb was IgG1,and the average number of chromosome of the hybridoma cell lines was 96.8.The ELISA titer of cell supernate and ascites were respectively 1:1024and 1:51200.The McAb did n’t cross-react with PRV、PRRSV、HCV、PPV et al,this demonstrated it has good specifity.The 5B4cell lines can steadly secreted McAb after subcultured twenty-one times .Indirect –ELISA and HI test for detecting SIV were developed by using 5B4 strain monoclonal antibody. The best optimal coating concentration of McAb was 2.95μg/ml,and working concentration of rabbit-anti-SIV positive serum was 1:400.The sensitivity of the double decker Sandwhich-ELISA was 2.43μg/ml. No cross-reaction were dected with major pathogenic microbe,and the high positive coincidence rate with virus isolation assay .The results demonstrated that positive rates sequence is bronchia，trachea，lung，tonsil, scrofula of pigs infected by SIV. And the dipping things obtained by nasal cavity swab that can be dected under no any special disposed, by the double decker sandwhich-ELISA .
Hybridomas secreting monoclonal antibodies (McAbs)specific for Swine influenza virus were produced by fusion of SP2/0 myelomas cells with spleen cells from BALB/C mice immunized with purified SIV.Wells containing hybridomas secreting immunoglobulin G (IgG1) antibodies against SIVwere specifically identified by an indiect enzyme-linked immunosorbent assay(ELISA),and one ELISA-positive hybridomas were selected and cloned forth by limiting dilution. and named 5B4,which could secrete McAb stably.The isotype of the McAb was IgG1,and the average number of chromosome of the hybridoma cell lines was 96.8.The ELISA titer of cell supernate and ascites were respectively 1:1024and 1:51200.The McAb did n’t cross-react with PRV、PRRSV、HCV、PPV et al,this demonstrated it has good specifity.The 5B4cell lines can steadly secreted McAb after subcultured twenty-one times .Indirect –ELISA and HI test for detecting SIV were developed by using 5B4 strain monoclonal antibody. The best optimal coating concentration of McAb was 2.95μg/ml,and working concentration of rabbit-anti-SIV positive serum was 1:400.The sensitivity of the double decker Sandwhich-ELISA was 2.43μg/ml. No cross-reaction were dected with major pathogenic microbe,and the high positive coincidence rate with virus isolation assay .The results demonstrated that positive rates sequence is bronchia，trachea，lung，tonsil, scrofula of pigs infected by SIV. And the dipping things obtained by nasal cavity swab that can be dected under no any special disposed, by the double decker sandwhich-ELISA .
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