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应用常规方法制备了4株能稳定分泌抗人重组γ-干扰素单克隆抗体(McAb)的小鼠杂交瘤细胞系:3D3、3D12、3F9和3B8.经免疫琼脂双扩散法鉴定,其分泌的McAb的亚类分别为:IgG2b、IgG3、IgG1和1gG1.相对亲和常数前两株McAb为6.25×10-11、后两株McAb为6.25×10-13.问接ELISA法测培养上清的效价为2×102~1.28×105.由小鼠腹水提取的4株McAb与天然γ-干扰素均产生交叉反应,对重组的人γ-干扰素均具有中和活性.其中3F9和3D3培养上清对重组人γ-干扰素亦有明显的中和活性。用3F9McAb与溴化氢活化的SePharose4B偶联制备了亲和层析柱,用其纯化粗提的γ-干扰素纯度达到95%以上,比活性达1.9×107IU/mg蛋白.
Four mouse hybridoma cell lines stably secreting anti-human recombinant γ-interferon monoclonal antibody (McAb) were prepared by conventional methods: 3D3, 3D12, 3F9 and 3B8. Identified by immunogold agar double diffusion method, the subclasses of McAb secreted were IgG2b, IgG3, IgG1 and IgG1 respectively. Relative affinity constant before the two McAb 6.25 × 10-11, the latter two McAb 6.25 × 10-13. The titer of the culture supernatant was 2 × 102 ~ 1.28 × 105. The 4 McAbs extracted from mouse ascites produced cross-reaction with natural γ-interferon and had neutralizing activity on recombinant human γ-interferon. The 3F9 and 3D3 culture supernatants also showed significant neutralizing activity on recombinant human interferon-γ. Affinity chromatography was carried out by coupling 3F9McAb with hydrogen bromide-activated SePharose4B. Purified crude interferon-γ reached more than 95% purity with a specific activity of 1.9 × 107IU / mg protein.