抗猪流行性腹泻病毒单克隆抗体杂交瘤细胞株的建立及初步应用

Establishment and Preliminary Application of Hybridoma cell Lines Producing Monoclonal Antibodiets to Porcine Epidemic Diarrhea Virus

应用提纯的猪流行性腹泻(PED)病毒抗原,建立了1D_8、5D_7、4B_1、2B_8、1A_4 5株抗PED病毒的单克隆抗体杂交瘤细胞林。用ELISA对此5株单抗的培养上清及诱生的腹水测定,培养上清效价为1D_8 1:1280、5D_7 1:640、4B_1 1:320、2B_8 1:320、1A_4 1:640;腹水效价为1D_8 10^(-7)、5D_7 10(-7)、4B_1 10(-7)、2B_8 10(-5)、1A_4 10(-4)_8应用兔抗鼠IgG和IgM标准抗血清做免疫双扩散试验,5株单抗中有1株属IgG类4株属IgM类。将单抗提纯后,应用夹心间接法ELISA及间接免疫荧光试验(IFA)进行特异性鉴定,初步证明,5株单抗除与PED病毒反应外,不与猪传染性胃肠炎病毒(TGEV)、禽传染性支气管炎病毒(IBV)、犬冠状病毒(CCV)、人肠道冠状病毒(HCV)和熊冠状病毒发生交叉反应。取效价高而稳定的1D_8、5D_7、4B_1细胞株分泌的McAb,以间接免疫荧光试验及直接ELISA检测PED病毒,初步证明MCAb在PED的诊断上具有敏感性高、特异性强的优点,可以作为PED病毒检测的一种新试剂。

The fusion of NS-1 myeloma cells with spleen cells frommice immunized with purified PEDV resulted in the cloning of 5hybridoma cell lines (1D_8, 5D_7, 4B_1, 2B_8, 1A_4) producing monoclonaantibodies to PEDV. The titres of the monoclonal antibodies in ascites and supernatants of hybridoma cell cultures were assayed with indirectELISA. Determination of antibody class was done via Ouchterlany analysis.It proved that the 4 of 5 monoclonal antibodies are mouse IgM, the otherone is IgG. After purification they were identified with indirect ELISA andIFA. The results preliminarily showed that they did not react with othermembers of coronavirus such as TGEV, AIBV, CCV and HCV but did withPEDV.