Dahmus M E, Laybourn P, Borrebaeck C A
Department of Biochemistry and Biophysics, University of California, Davis 95616.
Mol Immunol. 1988 Oct;25(10):997-1003. doi: 10.1016/0161-5890(88)90006-5.
A procedure has been developed for the production of MAb against weakly immunogenic subunits of a multisubunit enzyme. This procedure takes into account the problems of insufficient antigen, single epitope immunodominance and the difficulty of mapping non-sequential determinants. Small quantities of mammalian RNA polymerase II subunits were purified by SDS-polyacrylamide gel electrophoresis and were used to immunize splenocytes in vitro. After fusion with plasmacytoma cells, the hybrid cells were cloned and screened by ELISA utilizing native RNA polymerase II. This procedure is biased towards the production of MAb directed against sequential epitopes accessible on the native enzyme. Monoclonal antibodies, produced by in vitro immunization, were shown to be useful in protein transblot analyses, to inhibit enzyme activity in vitro and to have binding affinities comparable with MAbs produced by in vivo immunization.
已开发出一种程序，用于生产针对多亚基酶弱免疫原性亚基的单克隆抗体（MAb）。该程序考虑到了抗原不足、单表位免疫显性以及绘制非连续决定簇的困难等问题。通过SDS-聚丙烯酰胺凝胶电泳纯化了少量哺乳动物RNA聚合酶II亚基，并用于体外免疫脾细胞。与浆细胞瘤细胞融合后，对杂交细胞进行克隆，并利用天然RNA聚合酶II通过酶联免疫吸附测定（ELISA）进行筛选。该程序倾向于产生针对天然酶上可及的连续表位的单克隆抗体。体外免疫产生的单克隆抗体在蛋白质转印分析中很有用，能在体外抑制酶活性，并且其结合亲和力与体内免疫产生的单克隆抗体相当。
