抗猪隐孢子虫单克隆抗体的制备及鉴定

目的筛选特异性强、亲和力高的抗猪隐孢子虫(Cryptosporidiumsuis)单克隆抗体(McAb),并探讨其生物学活性。方法将纯化的猪隐孢子虫卵囊冻融和超声波处理后免疫BALB/c小鼠,取阳性免疫鼠脾细胞与SP2/0骨髓瘤细胞融合;经间接ELISA法筛选,有限稀释法进行克隆;用间接ELISA叠加试验、间接免疫荧光试验鉴定McAbs的免疫反应活性。结果获得3株能稳定分泌抗猪隐孢子虫McAbs的杂交瘤细胞株,命名为1E1C5、1E11A5、2B7A1,分别属于Ig M、IgG3和IgG2a类;其细胞培养上清效价分别为1∶3200、1∶1600和1∶3200,小鼠腹水效价分别为1∶1.6×105、1∶1.6×105和1∶0.8×105;ELISA叠加试验表明3株单抗可识别两个不同抗原位点,1E1C5有较高的亲和力;免疫荧光试验结果显示3株单抗均能与猪隐孢子虫卵囊壁反应,但与艾美耳球虫、人隐孢子虫(C.hominis)有微弱的交叉反应。结论获得了3株可识别猪隐孢子虫卵囊壁的McAbs,为进一步开展猪隐孢子虫快速诊断研究奠定了基础。

Preparation and identification of monoclonal antibodies against Cryptosporidium suis

To reare the secific monoclonal antibodies（McAbs） against Crytosoridium suis and evaluate the bioactivities of the McAbs,Oocysts of C.suis were collected and then urified by sucrose gradient centrifugation.BALB/c mice were immunized with the urified and sonicated oocysts,and McAbs against C.suis were reared by using hybridoma technique.The characterizations of McAbs were studied by means of indirect immunofluorescent assay（IFA） and indirect ELISA.Three clones（nominated 1E1C5,1E11A5 and 2B7A1,resectively） of hybridoma cell lines secreting McAbs against oocyst antigens of C.suis were obtained,which were classified as IgM,IgG3 and IgG2a subtye,resectively.Among the three McAbs,the titers of suernatant of cell cultures and ascites fluids ranged from 1∶1 600 to 1∶3 200 and,1∶80 000 to 1∶160 000,resectively.The ELISA suerosition results showed that all McAbs could recognize two different antigenic sites and 1E1C5 demonstrated a higher affinity.The IFA results indicated that three McAbs could only conjugate the oocyst walls of C.suis,but had cross-reactions with Eimeria and C.hominis.The result indicates three McABS that could react secifically against antigens of C.suis have been obtained,which lay a foundation for further establishing raid diagnostic methods for C.suis.