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为进一步研究牛IFN-γ(BoIFN-γ)单克隆抗体在牛免疫学以及牛疫病诊断中的应用,用原核表达的重组牛IFN-γ(rHis-BoIFN-γ)免疫6周龄雌性BALB/c小鼠,免疫3次后取其脾细胞和骨髓瘤细胞SP2/0进行融合。用建立的间接ELISA方法筛选阳性杂交瘤细胞,经3～5次亚克隆,最终获得12株能稳定分泌抗体的阳性杂交瘤细胞株。Ig亚类鉴定结果显示这12株杂交瘤细胞均为IgG,其中5株为IgG1,3株为IgG2a,4株为IgG2b。这12株杂交瘤细胞在体外具有稳定分泌抗牛IFN-γ单克隆抗体的能力。Western-blot及间接ELISA证明这12株单克隆抗体均可特异性地结合rBoIFN-γ蛋白。间接免疫荧光试验显示,这12株单抗均与真核细胞BHK-21表达的rBoIFN-γ产生荧光反应,证明这些单抗能够与接近天然的BoIFN-γ反应,证实了单抗的特异性。ELISA叠加试验表明,3C2与1H4、1G6与1G4针对不同表位,1A10、1B2、2B9、3C4四株和1C1与1G6、1G4与3C2、1A10与1G8针对相同或相近的表位。
To further investigate the application of bovine IFN-γ (BoIFN-γ) monoclonal antibody in the diagnosis of bovine immunology and cattle epidemics, BALB / c mice were immunized with rHis-BoIFN- c mice were immunized 3 times after the spleen cells and myeloma cells were SP2 / 0 fusion. The positive hybridoma cells were screened by indirect ELISA and subcloned 3 to 5 times to obtain 12 positive hybridoma cell lines that can stably secrete antibodies. Ig subclass identification results showed that these 12 hybridoma cells were IgG, of which 5 were IgG1, 3 strains were IgG2a, 4 strains were IgG2b. These 12 hybridoma cells have the ability to stably secrete anti-bovine IFN-γ monoclonal antibodies in vitro. Western-blot and indirect ELISA proved that these 12 monoclonal antibodies could specifically bind to rBoIFN-γ protein. Indirect immunofluorescence assay showed that these 12 McAbs reacted fluorescently with rBoIFN-γ expressed by eukaryotic cells BHK-21, demonstrating that the McAbs reacted with the native BoIFN-γ, confirming the specificity of the mAb. ELISA superposition experiments showed that 3C2 and 1H4, 1G6 and 1G4 against different epitopes, 1A10,1B2,2B9,3C4 four strains and 1C1 and 1G6, 1G4 and 3C2, 1A10 and 1G8 against the same or similar epitope.