Mouse monoclonal antibody with specificity for human interferon gamma.
Mouse monoclonal antibody with specificity for human interferon gamma.
复制标题
对人干扰素 γ 具有特异性的小鼠单克隆抗体。
作者：
E. Oleszak;H. Feickert;I. Mécs;F. Fox
我们开发了一种单克隆抗体（MAb）特异性的人干扰素γ（HuIFN-γ）的杂交细胞从NS-1骨髓瘤细胞系与脾淋巴细胞从BALB/c小鼠免疫与部分纯化的HuIFN-γ。使用感染脑心肌炎病毒（EMC）的人成纤维细胞，通过测定核酸合成抑制试验（INAS）的方法筛选杂交瘤培养物上清液中HuIFN-γ的抗病毒活性的中和作用。将表现出中和HuIFN-γ的抗病毒活性的克隆再克隆，再测试，并选择具有最大中和活性的MAb。该单抗为IgM亚类，对HuIFN-γ具有特异性。人白细胞衍生的干扰素（HuIFN-α）或人成纤维细胞衍生的干扰素（HuIFN-β）的抗病毒活性均不受该单克隆抗体的影响，如通过INAS试验测定的。通过间接免疫沉淀法进一步证实MAb对HuIFN-γ的特异性，其中用兔抗小鼠免疫球蛋白免疫沉淀单克隆抗体-HuIFN-γ复合物，并通过病毒产率降低测定法测定上清液中剩余的IFN活性。与IFN-γ存在下获得的病毒产量相比，硫酸铵沉淀的该MAb制剂能够显著增加（范围为230- 1300倍）病毒产量。SDS-PAGE分析表明，单克隆抗体免疫沉淀分子的Mr = 47 kD的非还原条件下。在还原条件下，观察到Mr = 26 kD（主带）和Mr = 21 kD（次带）的两个额外条带。使用该MAb构建琼脂糖亲和柱，并且能够保留约60%的所应用的部分纯化的HuIFN-γ制剂。通过增加离子强度和降低pH洗脱显著量的HuIFN-γ。HuIFN-α和HuIFN-β不被该柱保留。
We developed a monoclonal antibody (MAb) specific for human interferon gamma (HuIFN-gamma) by hybridizing cells from the NS-1 myeloma cell line with spleen lymphocytes from BALB/c mice immunized with partially purified HuIFN-gamma. Hybridoma culture supernatants were screened for neutralization of antiviral activity of HuIFN-gamma by the method determining the inhibition of nucleic acid synthesis assay (INAS), employing human fibroblasts infected with encephalomyocarditis virus (EMC). Clones exhibiting neutralization of antiviral activity of HuIFN-gamma were recloned, retested and an MAb with maximum neutralization activity was selected. This MAb was of IgM subclass and was specific for HuIFN-gamma. Antiviral activities either of human leukocyte-derived (HuIFN-alpha) or human fibroblast-derived interferon (HuIFN-beta) were not affected by this monoclonal antibody as determined by the INAS test. The specificity of the MAb for HuIFN-gamma was further confirmed by an indirect immunoprecipitation method, where monoclonal antibody-HuIFN-gamma complexes were immunoprecipitated with rabbit anti-mouse immunoglobulin and remaining IFN activity in the supernatants was determined by virus yield reduction assay. Ammonium sulfate precipitated preparations of this MAb were able to significantly increase (range of 230- to 1300-fold) the virus yield when compared with that obtained in the presence of IFN-gamma. SDS-PAGE analysis revealed that the MAb immunoprecipitates a molecule of Mr = 47 kD under nonreducing conditions. Under reducing conditions, two additional bands of Mr = 26 kD (major band) and Mr = 21 kD (minor band) were observed. A sepharose affinity column was constructed using this MAb and was able to retain approximately 60% of the partially purified HuIFN-gamma preparation applied. Significant amounts of HuIFN-gamma were eluted by increasing the ionic strength and decreasing the pH. HuIFN-alpha and HuIFN-beta were not retained by this column.
影响因子：
56.9
作者：
Y. Yip;B. Barrowclough;C. Urban;J. Vilček
通讯作者：
Y. Yip;B. Barrowclough;C. Urban;J. Vilček